WOUND HEALING SKIN TISSUE ENGINEERING

Siti Julaiha Gruebner

Pasca Sarjana Teknologi Biomedis

Universitas Indonesia

Siti Julaiha

Desember 2010 i Page

DAFTAR ISI
UNDERSTANDING OF TISSUE .
EPITHELIAL TISSUE ..................... ............... 1.1. CHARACTERISTIC ............... ............... 1.2. CLASSIFICATION 1.2.1. Simple Squamous... 1.2.2. Simple Squamous.. 1.2.3. Non Keratinized Stratified Squamous . . 1.2.4. Simple Cuboidal Epithelium .. ... 1.2.5. Simple Columnar Epithelium.. .. 1.2.6. Stratified Cuboidal Epithelium . 1.2.7. Stratified Columnar Epithelium... .. . .. 1.2.8. Pseudostratified Ciliated Columnar Epithelium ... .. .... 1.2.9. Transitional Epithelium ..... . .... 2. CONNECTIVE TISSUE ............. 2.1. COMPOSITION ... 2.1.1. Cells ............. 2.1.2. Extracellular Matrix.. ... ............. ... ............. ... 2.2. CLASSIFICATION... ......................... ... 2.2.1. Connective/Fiber Tissue Proper.. .. 2.2.2. Fluid Connective Tissue.. . 2.2.3. Supporting Connective Tissue.. .. . .. 3. NERVOUS TISSUE . 4. MUSCULAR TISSUE. . 4.1. CLASSIFICATION. . 4.1.1. Skeletal muscle. . 4.1.2. Smooth Muscle. . 4.1.3. Cardiac muscle. .. INTEGUMENTARY SYSTEM: SKIN 1. 2. 3. GENERAL FUNCTION.. ORGAN SYSTEM INTEGRATION. SKIN. 3.1. FUNCTIONS OF SKIN. 3.2. ANATOMY OF SKIN.. 3.3. THE EPIDERMIS. 3.3.1. Cells 3.3.2. Layers. ......... ... ......... ... ......... .... ......... ... ......... ... ......... ... ......... ... ......... .. 3.3.3. The Dermoepidermal Junction.. .. . 3.4. THE DERMIS.. .... 3.4.1. Collagen Tissue. .... 3.4.2. Elastic Tissue. .... .... .... .... 3.4.3. The diffuse and filamentous Dermal Matrix. . 3.4.4. Cells of The Dermis.. .... .... 3.4.5. Organization of The Dermis Major 1. 1 2 3 3 4 4 4 5 5 5 5

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... .. ... ............. .. 3.7. THE NERVES AND SENSORY RECEPTORS OF THE SKIN ... ... 3.7.1. Somatic Sensory .... .... 3.7.2. Sympathetis Autonomic Fibers. .. 3.7.3. Musculocutaneous Nerves. . .. . 3.7.4. Free Nerve Endings.. .... 3.7.5. Penicillate Fibers .... .... .... 3.7.6. Papillary Nerve Endings. .. 3.7.7. Corpuscular Receptors... . 3.7.8. Mucocutaneous End Organs. .. 3.8. THE MUSCLES OF THE SKIN.. . .. . .. . 3.8.1. Smooth Muscles .... 3.8.2. Striated Muscle.. .... .... .... 3.9. THE HYPODERMIS OR THE SUBCUTANEOUS TISSUE/FATTY LAYER.. . 3.10.ORIGIN AND DEVELOPMENT OF THE SKIN .. 3.11.SKIN COLOR.. .... 3.12.SKIN APPENDAGES .... .... .... TISSUE ENGINEERING....... 3.5. THE CUTANEOUS VASCULATURE 3.6. THE CUTANEOUS LYMPHATICS

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1. APPROACHING TISSUE ENGINEERING .. . 1.1. REPLACEMENT CELL .... 1.2. MATERIAL REQUIREMENT.. .... 1.3. DELIVERY METHODE .... .... .... .... 1.4. CLASSIFICATION TRANSPLATION/DELIVER METHODE.. 1.5. MODELS for TISSUE ENGINEERING .. . 1.6. VARIABLES NEEDED.. .... .... PROBLEMS OF TRANSPLANTATION. .. THE FUTURE OF TISSUE ENGINEERING. . .. . 3.1. SIDE OF IMPROVEMENT .... 3.2. CHALLENGES .... 3.3. APPLICATIONS. .... .... .... ....

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WOUND HEALING AND REPAIR... ...

1. 2. 3. 4. HISTORY. INTRODUCTION. TYPE OF WOUND. 3.1. CLOSED WOUND. 3.2. OPENED WOUND. REGENERATION PROCESS.. 4.1. BODY CELLS 4.1.1. Labile Cells. 4.1.2. Stable Cells. 4.1.3. Permanent Cells REPAIR PROCESS PHASES OF WOUND HEALING. .... .... .... .... .... .... .... .... .... .... .... .... ....

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.... .... .... .... .... .... .... .... .... .... .... 6.1. HEMOSTATIS AND INFLAMMATION PHASE.. ... 6.1.1. Vascular Events. ........ 6.1.2. Cellular Events.. .... 6.2. PROLIFERATION PHASE.. ........ .... ........ .... 6.2.1. Angiogenesis (Vascular and Lymphatic Proliferation).. 6.2.2. Fibroplasia and granulation tissue formation. . . 6.2.3. Extracellular Matrix ........ 6.2.4. Epithelialization. .... 6.2.5. Contraction. ........ .... .... .... 6.3. MATURATION AND REMODELLING PHASE 6.4. SUMMARIZE OF WOUND HEALING PHASE ... .. 7. NATURE OF WOUND HEALING.. .. 7.1. HEALING BY PRIMARY INTENTION. . .. . 7.1. HEALING BY SECONDARY INTENTION. .. . 8. FACTORS INFLUENCING HEALING.. .. . 8.1. LOCAL FACTORS. ........ 8.2. SYSTEMIC FACTORS. ... ........ ... 9. GROWTH FACTORS INVOLVED IN HEALING. ... 10. COMPLICATION OF WOUND HEALING .. . 11. RESEARCH IN WOUND HEALING ........ ...

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SKIN TISSUE ENGINEERING. ...

1. 2. 3. OBJECTIVES OF SKIN SUBSTITUTES. HISTORY OF SKIN REPLACEMENT.. .. . .. . DESIGN PRINCIPLES PERMANENT SKIN REPLACEMENT 3.1. GENERAL DESIGN PROPERTIES. .. 3.2. STAGE 1 DESIGN PARAMETERS.. . 3.3. STAGE 2 .. .. CURRENT TREATMENT OF SKIN LOSS .. . .. . 4.1. NATURAL AND SYNTHETIC POLYMER .. . 4.2. HUMAN HOMO/ALLOGRAFTS ANIMALS HETERO/XENOGRAFTS. . 4.3. AUTOLOGOUS CELLS.. ENGINEERING SKIN TISSUE . 5.1. DESIGN CONSIDERATIONS 5.2. EPIDERMAL REGENERATION..

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 5.4. COMPOSITE SKIN GRAFTS.. 5.5. COMBINED DERMAL AND EPIDERMAL SUBSTITUTES. 5.6. EPIDERMAL AUTOGRAFTS AND ALLOGRAFTS.. 5.7. SKIN EQUIVALENT, SE.. 5.8. ACELLULAR SKIN SUBSTITUTE.. 5.9. ALLOGENEIC CELLULAR SKIN SUBSTITUTE.. 5.10.AUTOLOGOUS CELLULAR SKIN SUBSTITUTE 5.10.1. Cultured Epithelial Autografts, CEA. 5.10.2. The Meshed Autografts. 5.11.CELL COMMUNICATION AND REGULATION IN SKIN 6. COMERSIAL ARTIFICIAL SKIN... 6.1. ACELLULAR SKIN SUBSTITUTE.. 6.2. ALLOGENEIC CELLULAR SKIN SUBSTITUTE.. 6.3. AUTOLOGOUS CELLULAR SKIN SUBSTITUTE. 7. CLINICAL CONSIDERATIONS 7.1. ASSESSMENT........................................... 7.2. REGULATORY ISSUE. 8. FUTURE DIRECTIONS 9. STEM CELLS APPLICATION IN SKIN TECHNOLOGY. . .... 10. RESEARCH IN ARTIFICIAL SKIN .... ....

5.3. DERMAL REPLACEMENT..

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UNDERSTANDING OF TISSUE
Tissue a cellular organizational level intermediate between cells and a complete organism. Hence, a tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. Multicellular (large) organisms function more efficiently if cells become specialized for specific functions. The study of tissue is known as histology or, in connection with disease, histopathology. Tissues have diverse functions in the body, which include protection, support, transport, movement, storage, and control. Because organs are made of several tissue types, an understanding of tissue structure and function will provide an appropriate foundation for a more thorough understanding of organs and organ systems, and therefore the human body.

Types of Tissue
There are types of tissues found : Connective Tissue, Muscular Tissue, Nervous Tissue, Epithelial Tissue and Cell Junctions. While also the system in Organism : Integumentary System, Organ System, Homeostasis. Here is the diagram of Animal Tissue classification which is similar with Human:

Here are the description each of Tissue type.

Cell Junctions
Between the tissue, there are Cell junctions that aid tissues in their functions by joining cells together either by tight junctions, adhesion junctions or gap junctions. TIGHT JUNCTIONS: Cell layers become resilient creating a tough barrier by the joining of plasma membrane proteins. ADHESION JUNCTIONS: Cells cytoskeleton fibers attached to one another. Found in tissues which stretch, like skin. GAP JUNCTIONS: A junction formed by two neighboring plasma membranes, allowing molecules/ions to circulate through channels.

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EPITHELIAL TISSUE
Epithelial tissues cover body surfaces, line most internal cavities and organs, and are the major components of glands. It is made up of cells that are closely packed and are composed of one or more layers. As a boundary between different environments, epithelial tissues have several different functions, including:

a. Provide physical protection, abrasion, dehydration, form the covering or lining of all internal and
external body surfaces, such as Skin, linings of body cavities, linings of any body opening, and linings of internal passageways. b. From a barrier for permeability Can give selective Absorption (movement of substances from a cavity into the blood stream), or Secretion (movement of substances from the blood stream into a cavity), form glands from secretory structures from epithelium (Glandular epithelium may be scattered in other epithelium). Can be regulated & modified by stimuli, I.e., changed with need a. Filtration, Excretion. c. Provide sensations: Epithelial cells deeply innervated w. sensory nerves Detect changes in environment & relay to body For example, the epithelium of skin protects underlying tissues from mechanical and chemical damage, and from bacterial invasion. In contrast, epithelial tissue lining the small intestine is designed to absorb ingested nutrients, whereas the epithelium of glands secrete products, such as saliva or digestive enzymes. Some epithelial tissues in the kidneys are designed to filter blood, and others selectivelyabsorb and secrete substances in the filtrate in order to produce urine. Epithelial tissue that occurs on surfaces on the interior of the body is known as endothelium. Epithelia also form secretory parts of glands, structures that are specialized to produce and release specific substances. Glands that secrete their products into ducts opening onto external surfaces or into internal body cavities are called exocrine glands. Examples of exocrine glands include salivary glands, sweat glands, pancreatic glands, mammary glands, and sebaceous glands. In contrast, glands that secrete their products (hormones) into tissue fluids or the blood stream are called endocrine glands. These include the thyroid gland, adrenal glands, and pituitary gland. The glands of the endocrine system, pictured here, are

composed of epithelial tissue. Specifically, the epithelial cells compose the secretory parts of the glands, allowing them to secrete their products (hormones) throughout the body

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CHARACTERISTIC
Epithelial tissues have unique characteristics :

Cellularity:
Epithelial cells are also tightly packed, with little intercellular space and material between them. In fact, adjacent cells are usually bound together at many points by special contacts found in the plasma membrane. It is this tight packing of cells that makes epithelia effective barriers. because epithelia cover surfaces, they always have one free side; that is, a side exposed to the outside of the body or an internal cavity. This exposed side is called the apical surface. The opposite side, or basal surface, is anchored to underlying tissues by a non-living substance called the basement membrane

Polarity:
Interestingly, the two cell surfaces (apical and basal) have different properties, they are uneven distribution of materials in the cells. One side always faces an opening; and one side always attached to a basement membrane to adjacent tissue resulting from different peripheral and integral proteins in their membranes. As a result, cells with distinct basal and apical surfaces are said to be polarized. It also is common for the apical surface of epithelial cells to possess microvilli, fingerlike extensions of the plasma membrane that tremendously increase surface area.

Attachment:
The basement membrane located underneath the basal surface is actually composed of two distinct layers. Its outer layer, the basal lamina, is secreted by epithelial cells, whereas the inner reticular lamina is made by cells of the underlying connective tissue. Together these two layers provide support and attachment for epithelial tissues.

Avascularity:
As a general rule, they are avascular, meaning they lack or No blood vessels in epithelium. They therefore obtain necessary substances by diffusion from blood vessels located in underlying connective tissues. Nourished by diffusion or absorption

Regeneration:
Surface cells replaced continually by mitotic cells below meaning epithelial cells have the capacity to reproduce readily. For instance, the inner lining of the small intestine is replaced about every five days. In addition, injuries to an epithelium heal quickly as new cells replace lost or damaged ones. This explains in part how an abrasion or cut in the skin heals. This figure is a diagrammatic view of the basement membrane, an

extracellular layer that defines an epithelial boundary and also helps reinforce epithelial sheets, resisting stretching and tearing. The basement membrane is actually two layers. The outer layer, the basal lamina, is secreted by epithelial cells, whereas the reticular lamina is formed by underlying connective tissue (indicated by the two arrows).

CLASSIFICATION
Epithelial tissues are classified according to the shape of their cells and the number of cells they contain. However, sometimes a single layer of cells can appear multilayered because some of the cells do not extend all the way to the apical surface. Flat, scale-like cells are called squamous, whereas cubeshaped cells are considered cuboidal. Cells that are shaped like a column or cylinder are termed columnar. Transitional cells change their shape as the particular tissue they are located in stretches. Classified according to the number of cell layers they contain, such as epithelia composed of a single layer of cells are considered simple, whereas those made of multiple layers are called stratified. This arrangement describes pseudostratified epithelia.

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Simple Squamous
Simple squamous epithelium consists of a single layer of flattened, scale-like cells, which allow substances to easily pass through. That is why this tissue type is a common site for diffusion and filtration. For instance, simple squamous epithelia lines the air sacs (alveoli) of the lungs and also the inside walls of blood capillaries. It is also bin protected regions for absorption such as Respiratory alveoli for gas exchange, and in the regions to reduce friction, Linings of blood vessels & heart = endothelium, Serous membranes lining body cavities such as Mesothelium which is a simple squamous epithelium lining ventral body cavities.

Keratinized Stratified Squamous

Stratified squamous epithelium contains several layers of cells, with those in the outermost layers being squamous. However, cells in the deeper layers may be more cuboidal or columnar in shape. This type of tissue lines surfaces of the mouth, throat, vagina, and anal canal. It also forms the outer layer of the skin (the epidermis). Multilayered epithelium covered with layer of compact, dead squamous cells packed with protein keratin Retards water loss & prevents penetration of organisms Forms epidermal layer of skin

Non Keratinized Stratified Squamous

Multilayered epithelium that lacks surface layer of dead cells forming abrasion-resistant,
moist, slippery layer. Found on tongue & vagina

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Simple Cuboidal Epithelium

forms a single layer of cube-shaped cells, often with microvilli. It covers structures in the ovaries and lines tubules within the kidneys and many glands, such as salivary glands, the thyroid gland, and the pancreas.

Absorption & secretion; produces mucus.

Mammary and salivary glands.

Simple Columnar Epithelium

is a single layer of elongated cells and is found in the uterus and most organs of the digestive tract, including the stomach and small intestine. It is a Single row of tall, narrow cells ; vertically oriented, oval nuclei in basal half of cell Absorption & secretion; secretion of mucus Inner lining of GI tract

Stratified Cuboidal Epithelium

Two or more layers of cells; surface cells square Secretes sweat; ovarian hormones & produces sperm Found ovarian follicles & seminiferous tubules

Stratified Columnar Epithelium

Pseudostratified Ciliated Columnar Epithelium

Pseudostratified columnar epithelium appears stratified or layered, but is not (although all of the cells are anchored to the basement membrane, not all of them reach the apical surface, giving the tissue a multilayerd appearance). These cells line the respiratory passages and reproductive

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systems, and are often ciliated. In the respiratory system, their cilia move mucus and trapped particles, such as dust and microorganisms, away from the lungs.

Transitional Epithelium
Transitional epithelium consists of several layers of cells, which vary in appearance from cuboidal to squamous depending on the degree to which the tissue is stretched. This tissue type is found in the urinary bladder and parts of the uterus and urethra. Multilayered epithelium with rounded surface cells that flatten when the tissue is stretched Stretches to allow filling of urinary tract Found in urinary tract -- urinary bladder

The summary of all the types of Epithelial Tissue is below :

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CONNECTIVE TISSUE
Connective tissues are the most widely distributed and abundant of the four tissue types. They also have numerous and varied functions, which include binding structures, providing support and protection, serving as a framework, filling spaces, storing fat, producing blood cells, protecting against infection, and helping repair tissue damage. Connective tissues also vary widely regarding their degree of vascularization. Cartilage, for example, is essentially avascular, and ligaments and tendons are poorly vascularized. In contrast, bone is vascularized, and adipose tissue has a rich supply of blood vessels. Although connective tissues display a tremendous amount of morphological and functional diversity, they all share some common properties. For example, all connective tissues have a common embryological origin. In addition, they are all surrounded by a non-living extracellular matrix, secreted by the connective tissue cells. Thus, unlike all other primary tissue types that are composed mostly of cells, connective tissues are largely composed of nonliving matrix, which may widely separate living cells. However, it is this matrix that provides most connective tissues with the ability to withstand great tension and physical trauma. It is a tissue that is characterized by the abundance of extracellular components (such as fibers and intercellular substances). The tissue derives its name from its function in connecting, supporting, surrounding or binding cells and tissues. It is called also as Fibrous tissue. Connective tissue helps attach materials together through fibrous, supportive, bone and fluid connective tissues. Connective tissue have Mechanical properties that making the function of Binding together, Compartmentalization , Support , Physical , Immunologic protection & Storage.

Pictures are from http://en.wikipedia.org/wiki/Fibrous_connective_tissue

COMPOSITION
Connective tissue is composed of:

Cells

widely space in the extracellular matrix, with several types classification which are : Younger or Undifferentiated cell substance Macrophages- and other phagocytic cells Mast cells- with function to secrete histamine & heparin which mediate inflammatory process Plasma cells- with function to secrete antibodies Eosinophils, Neutrophils, etc.

Fibrocytes- most numerous, synthesis and turnover of both fibres and non fibrillar ground

Adipocytes- with function to stores fat

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Extracellular Matrix
is a special feature that distinguishes connective tissue from the other tissues of the body. This matrix may be jelly-like, fluid, dense or rigid. The nature of matrix differs according to the function of that particular connective tissue. Mesenchymal cells are found in an embryo and are the source of all connective tissues, which secrete an interstitial (extracellular) matrix that contains ground substance and protein fibers Bladder and parts of the uterus and urethra. There are common Components of Extracellular matrix which are a. WATER b. GROUND SUBSTANCE is chemical substances that saturates space in between cells and fibers such as: semisolid gel containing Tissue fluids, salts & Glycoconjugates two types Proteoglycans core protein GAG (Glycoproteins) is attached five Chondrotin sulphate, dermatan sulphate, keratan sulphate & heparan sulphate. Glycoproteins shorter branched oligosaccharides- Fibronectin & laminin. Ground substance serves as connective tissue glue, filling the space between cells and containing protein fibers. Depending on the type of connective tissue, ground substance can be liquid (blood), semisolid or gel-like (cartilage), or very hard (bone). c. PROTEIN FIBERS The fibers provide physical support. There are three different types of fibers:

Collagen Fibers (white fibers). The strongest and most common type of fiber in ECM.

Collagen fibers are found in structures that resist pulling forces, such as tendons, which are used to connect muscles to bones. They are dominant constructed of a protein (collagen) main type is tropocollagen type III which aids in flexibility and durability, and other types are different tropocollagen types (named type I to XX), Types I, II and III are the major fibreforming tropocollagens, and Tropocollagen type IV is component of the basal lamina. This fibers are widely distributed. Unbranched, and its function - strength to the connective tissue. This thickness of the fibres varies. Each of these fibrils is composed of microfibrils.

Reticular Fibers. Similar to or consist of collagen fibers, branch outward to form thin

support systems. These typical as fine collagen fibers found in spleen/Lymphoid organ and liver. Some of the functions are Delicate, Forming fine networks such as scaffolding for support resident cells, supporting network of an organ/gland (Stroma), Characterized the gland/organ for specific cells (principal Parenchymal cells- lymphocytes).

Elastic Fibers (yellow fibres) found in the lungs, or blood vessels. They are composed of
the protein elastin. They have a greater ability to stretch than collagen fibers, and also have a great tolerance to repeated bending. Elastic cartilage is found in the external ear, vocal cords, and epiglottis (the flap that prevents food from entering the respiratory passages). It is a protein known as Elastin which makes up this fiber to offer flexibility.

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Elastic fibers embedded in an amorphous matrix which is composed of the protein Elastin. Its an Individual Microfibrils. Fibres are branched and its branches end in hook like fashion. Normally it is thinner than the collagen fibers.

CLASSIFICATION
A Classification of Connective Tissues

Connective/Fiber Tissue Proper

Connective tissue profer is classified as Loose or Dense Tissue as below : a. LOOSE TISSUE This Tissue have more cells and fewer fibers than any other type of connective tissue, except for blood. Such as Embryonic mesenchyme, mucous connective tissues

Areolar tissue or called Loose aerolar tissue. Siti Julaiha Page 10

The most widely distributed loose connective tissue. It forms delicate, thin membranes throughout the body and acts as a packing material and glue. For example, it binds skin to underlying organs and also fills spaces between muscles. In addition, it wraps small blood vessels and nerves. Because of the loose and fluid nature of its extracellular matrix, it provides a reservoir of water and salts for surrounding tissues. In fact, virtually all body cells obtain nutrients from the matrix of areolar tissue (and they also release waste products into it). When a body region becomes inflamed, such as from an infection, areolar tissue takes up excess fluid like a sponge, causing the area to swell (a condition called edema).Loose fibrous contain of Fibroblast cells which forms protective layer over organs and aids the function of epithelium. It have numerous cells and blood vessels and abundant ground substance, flexible, not resistant to stress. Mostly found under epithelial surfaces, around blood vessel and glands. These tissue is classified again into Dense Regular and irregular tissue where Fibroblast cells created from closely bundled collagen fibers. There tissue often found in tendons/ligaments connections to forming into it. The 5 primary components of the superficial fascia (loose irregular areolar connective tissue) are: Fibroblasts Collagen Fibers Elastic Fibers Tissue Fluid Fat

Adipose tissue Adipose tissue is a type of loose connective tissue that is designed to store fat in droplets within the cytoplasm of its cells. It helps forms the tissue layer beneath skin (subcutaneous layer), where it insulates the body from temperature changes. Adipose tissue also cushions and protects some organs, such as the kidneys, heart, and eyeballs. In addition, adipose stores fat in the abdominal membranes and hips as an energy reserve. Adipose tissue can be used to cushion certain organs, helping hold them in place. This function becomes readily apparent in individuals who are severely malnourished and emaciated, as can happen with the eating disorder anorexia nervosa. If there is insufficient dietary intake of calories, the fatty encasement around the kidneys can diminish, which may cause the organs to lower their position. This, in turn, can kink the ureters, blocking urine flow to the bladder. As a result, urine backs up into the kidneys, causing severe damage and ultimately renal failure. Adipose (White tissue) is kind of loose connective tissue where cells swell to store fat, an aggregation of fat cells (adipocytes). This typical Tissue protects organs, insulates organs, and is used for energy. Contains a large droplet of fat that fills it. Nucleus is pushed against the plasma membrane & is flattened. Cells are supported by reticular fibres. Adipose (Brown tissue) is Adipocytes that have nucleus centrally placed. Its Cytoplasm has a frothy appearance and Cell borders are not clear while Capillaries are very frequent.

Reticular tissue

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b. DENSE TISSUE
This tissue contains many tightly woven fibers of collagen, which are produced by fibroblasts. It is found in structures that are designed to act as strapping material, such as tendons and ligaments (structures that connect bone to bone). It also makes up the dermis, the lower layers of skin. The fibroblasts that compose dense (and loose) connective tissue also help repair tears in body tissues. For example, when skin is cut, fibroblasts move to the area of the wound and produce collagen fibers that help close the wound and provide a surface upon which the outer layer of skin can grow.

Dense regular Tissue Dense regular connective tissue found in areas of tension, tendons, ligaments & aponeuroses, stroma of spleen & lymph nodes. Coarse collagen fibres are aligned with each other with only very narrow opens spaces between them. Like in most other connective tissues, there will be only a few cells between the fibres. Their cytoplasm is difficult to identify but the nuclei can be seen scattered among the collagen fibres. Nuclei are often elongated, and their long axis runs parallel to the course of the collagen fibres. Dense irregular Tissue Dense irregular connective tissue forms the dermis of the skin. In contrast to the overlying dermis and the underlying deep fascia, the superficial fascia may be distinguished by the presence of fat. These tissues are very extensive. It contains few cells and High density of collagen fibres in their irregular distribution. The Dark spots scattered between the collagen fibres represent the nuclei of the cells.

Fluid Connective Tissue

Contains a distinctive cell population Watery ground substance with dissolved proteins Two types : Blood is a special connective tissue consisting of a liquid matrix called plasma, with formed elements (red and white blood cells, and platelets). Bloods duties include carrying oxygen and nutrients throughout the body to other tissues fluid and circulates heat. In this case, the fibers

a. BLOOD

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of the extracellular matrix are soluble proteins in the plasma that become visible during blood clotting. Blood is mainly designed to transport substances within the body, including nutrients from digested food, waste products, respiratory gases, hormones, and antibodies. In addition, blood flow also can be used for temperature regulation by distributing heat throughout the body. This is accomplished in part by altering its flow patterns, which occurs by changing the diameter of vessell when it is necessary to dissipate heat to the external environment. Increasing the diameter of blood vessels refers to Vasodilation, which increases blood flow. Vasodilation of vessels under the skin surface occurs when it is necessary to release heat to the external environment. This explains why skin looks flushed when someone is overheated. In contrast, vasoconstriction of skin blood vessels conserves body heat by restricting blood flow to deep body areas, virtually bypassing skin. Because skin is separated from deeper organs by an insulating layer of adipose tissue, heat loss through the outer layer is reduced in this case. Restricting blood flow to the skin for short periods of time does not pose a problem. However, restriction of blood flow for extended periods of time can lead to frostbite. This is because the temperature of the outer body layer approaches that of the external environment, which can cause skin to freeze. In addition, skin cells die if they are deprived of oxygen and nutrients for too long.

RED BLOOD CELLS (Erythrocytes) transport oxygen throughout the body by loosely
binding the oxygen with the cells and also in carrying some carbon dioxide away.. Red blood cells do not contain a nucleus. HEMOGLOBIN, an iron containing structure.

WHITE BLOOD CELLS (Leukocytes) contain a nucleus and are larger in size. Also, they PLATELETS are present in bone marrow where they aid in the reconstruction of broken
blood vessels. Platelets are pieces of cells. Platelets are involved with blood clotting.

have a more translucent appearance. White blood cells help to fight infections by either consuming the pathogens through phagocytosis or creating antibodies to fight infections.

b. LYMPH
Lymph: Yellowish fluid containing white blood cells. Lymph originates from tissue fluid and is cleansed in the LYMPH NODES which is lymphatic tissue on a lymphatic vessel.

Supporting Connective Tissue

It is also called Skeletal Tissue, which aids in the formation of bone and cartilage. Due to its solid matrix, this tissue cells usually occupy small cavities known as LACUNAE. This tissue have characteristic which are: Less diverse cell population Dense ground substance

Closely packed fibers Tissue is classified into tow types which are Bone and Cartilage.

a. BONE
is the most rigid connective tissue. Its living cells, reside in cavities called lacunae. The lacunae are surrounded by an extracellular matrix deposited in layers arranged in concentric circles known as lamellae, which together form the basic structural unit of bone called an osteon. Many osteons glued together form a large part of the substance of bone. The hardness of bone is due to mineral salts deposited in the extracellular matrix. The matrix also contains a significant amount of collagen, which keeps the bone from becoming brittle (without collagen, bone would have a consistency similar to chalk. On the other hand, with only collagen, bone would be more like a garden hose). Bone is used to support body structures, protect vital organs (skull and ribs), provide attachment sites for muscles, and store minerals, such as calcium and phosphate. In addition, bone marrow produces blood cells.

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Bone could be found as compact or spongy bone, are the firmest connective tissue which is constructed of a hard matrix and collagen fibers. Bones get some of their structure from OSSEOUS TISSUE, which lends to the cylindrical shape of them (OSTEONS). Long and dense COMPACT BONES have rings of hard matrix in the osteons. At the ends of long bones is a lighter structure known as SPONGY BONE. This material has a separated formation permitting space for marrow and blood vessels.

b. CARTILAGE
is a flexible tissue, consisting of cells called chondrocytes that also sit within lacunae. Cartilage is avascular, and tendons and ligaments are poorly vascularized. In addition, older chondrocytes lose their ability to divide. This explains why these three types of connective tissues heal very slowly when injured. In addition, later in life cartilage tends to calcify, making its matrix resemble that of bone, and its ability to heal virtually impossible. There are three different types of cartilage: Hylane Cartilage is the most abundant in Cartilage which constructed of a whitish matrix with thin collagen fibers. This type of cartilage is found at the end of bones, in the trachea, and in the nose. It is designed to provide support and flexibility. For instance, it is used to attach ribs to the breastbone (sternum), form the voice box (larynx), and cover the ends of bones where they form joints. It also is found in the soft part of the nose. Elastic Cartilage similar to Hyaline, contains more elastic fibers which yields more flexibility. Elastic cartilage is found in the outer ear area, the voice box (larynx), and the epiglottis. Fibro Cartilage consists of very durable collagen fibers which can endure pressure/weight and absorb shock. It is forms cushionlike disks between the vertebrae of the backbone and also between bones in the knee joint and is found in the joint of the pubic bones, spinal disks,

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Pictures sources are from http://en.wikipedia.org/wiki/Fibrocartilage, http://www.victoriacollege.edu/dept/, Basic%20Tissues/Connective%20Tissue.html#Elastic%20CT.

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NERVOUS TISSUE
Nervous tissue makes up the brain, spinal cord, and peripheral nerves, which coordinate, regulate, and integrate many body functions. Nervous tissue consists of two major cell types:neurons and neuroglia. Neurons are the cells that generate and conduct electrical impulses, sometimes over substantial distances. These impulses influence other neurons, muscles, and glands. Some neurons have the ability to convert external stimuli, such as light, heat, or sound, into electrical signals that can be recognized by the brain. Most neurons have a cell body, which contains a nucleus and most organelles. Dendrites are highly branched processes of the cell body that are designed to receive information. Impulses are carried away from the cell body by a single, long process called an axon (Figure 6.5). Neuroglia do not generate and conduct nerve impulses as do neurons. However, they are essential for normal neuronal function. Some neuroglia wrap themselves repeatedly around an axon, forming layers of membrane called a myelin sheath. This sheath acts as electrical insulation, which increases the rate at which impulses are conducted (over 100 meters per second for some neurons). Other neuroglial cells are phagocytic, whereas some provide neurons with nutrients by connecting them to blood vessels. All living cells have the ability to react to stimuli. Nervous tissue is specialised to react to stimuli and to conduct impulses to various organs in the body which bring about a response to the stimulus. Nerve tissue (as in the brain, spinal cord and peripheral nerves that branch throughout the body) are all made up of specialised nerve cells called neurons. Neurons have many different shapes and sizes. However, a typical neuron in a human consists of four major regions: a cell body, dendrites, an axon, and synaptic terminals. Like all cells, the entire neuron is surrounded by a cell membrane. The cell body is the enlarged portion of a neuron that most closely resembles other cells. It contains the nucleus and other organelles (for example, the mitochondria and endoplasmic reticulum). The dendrites and axon are thin cytoplasmic extensions of the neuron. The dendrites, which branch out in treelike fashion from the cell body, are specialized to receive signals and transmit them toward the cell body. The single long axon carries signals away from the cell body. In humans, a single axon may be as long as 1 meter (about 3 feet). Some neurons that have cell bodies in the spinal cord have axons that extend all the way down to the toes.

A nerve is an enclosed, cable-like bundle of axons (the long, slender projections of neurons). A nerve provides a common pathway for the electrochemical nerve impulses that are transmitted along each of the axons. Nerve tissue consists of Neurons and Neuroglia.

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MUSCULAR TISSUE
Muscle tissues are highly cellular, well vascularized, and have the ability to generate force by contracting.

CLASSIFICATION
There are three different types of muscle tissue: skeletal, smooth, and cardiac.

Skeletal muscle
is attached to bones and is consciously controlled. Under a microscope, its cells are cylindrical in shape, and reveal alternating light and dark patterns called striations (that is why this tissue type is sometimes called striated muscle). Skeletal muscle is responsible for generating movements of the limbs, trunk and head, as well as allowing us to make facial expressions, talk, chew, swallow, breathe, and write. In addition, skeletal muscle helps maintain posture, stabilizes joints, and generates heat (shivering refers to involuntary contractions of skeletal muscle).

Smooth Muscle
The cells of smooth muscle lack striations, and this tissue type is considered involuntary. However, disciplined individuals who practice yoga or biofeedback can develop the ability to control some smooth muscle action. Smooth muscle is found in the walls of blood vessels (except capillaries) and the airways (bronchioles), where its contraction reduces flow of blood or air, respectively. Smooth muscle also is located in the walls of hollow organs, such as the stomach, intestines, uterus, and urinary bladder, where it aids in propelling contents.

Cardiac muscle
is only found in the walls of the heart. Its contractions are responsible for pumping blood. Like skeletal muscle, its cells have a striated appearance when observed with a microscope.However, similar to smooth muscle, the contractions of this tissue are generally considered involuntary. A unique anatomical property of cardiac muscle is a specialized junction that electrically connects heart cells, thereby allowing a rapid conduction of impulses throughout the heart muscle. This junction is called an intercalated disk.

Muscles of the body are made up of elongated muscle cells also known as muscle fibre. The movement of the body is brought about by the contraction and relaxation of contractile protein present in muscle cells. These contractile proteins are actin and myosin.

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INTEGUMENTARY SYSTEM : SKIN

Two or more tissue types may be organized into more complex structures called organs, which perform specific functions for the body. Many organs, such as skin, are composed of all four tissue types. Although skin is often referred to as the cutaneous membrane, it is by definition an organ. In fact, skin, is one of the largest organs of the body. In an average adult, it weighs 4 5 kilograms (911 pounds), accounting for about 7% of total body weight. Along with its derivatives (sweat glands, oil glands, hair, and nails) and accessory structures (blood vessels and nerves), Skin is part of the integumentary system (the word integument refers to a covering). The integumentary system includes a number of diverse structures derived from the epidermis of skin. These appendages include hair, nails, oil glands, and sweat glands. Each has a unique role in helping maintain homeostasis.

GENERAL FUNCTION
General Function of the integumentary system Protection Excretion Maintenance of normal body temperature as blood flow conserve or release heat

Thermoreceptors and

Alterations in cutaneous

Neural control (primary determining factor) Local control Synthesis of vitamin D3

Storage of lipids Detection of touch, pressure, pain and temperature stimuli Accessible and Varied in function Underappreciated The integument does not only function in isolation. There are also: extensive network of blood vessels sensory receptors (touch, pressure, temperature & pain)

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hypodermis / subcutaneous layer : separates the integument from the deep fascia around other organs

ORGAN SYSTEM INTEGRATION

Homeostasis is the single most important concept of physiology, a branch of biology that deals with the functions and vital processes of living organisms or their parts. In fact, the concept of homeostasis is used as a central paradigm (model) to explain the complex processes of animal physiology. During the late nineteenth century, the French physiologist Claude Bernard wrote all the vital mechanisms, however varied they may be, have only one object, that of preserving constant the condition of life in the inner environment. In the early twentieth century, the American physiologist Walter Cannon coined the word homeostasis (stable condition) to describe Bernards concept of the inner environment. Thinking in terms of homeostasis provides a clearer understanding of how and why the human body functions the way it does. In other words, all the cells, tissues, organs, and systems are designed to work together to maintain a relatively constant internal environment., as shown in the table below. No system in the body acts independently of the others. In fact, all systems must be integrated in order to maintain homeostasis, and the integumentary system is no exception. Recall that skin is able to synthesize vitamin D when exposed to sunlight. However, this form of vitamin D does not have significant biological activity. Rather, it must be metabolized into an active hormone, first by a chemical modification (hydroxylation reaction) in the liver, followed by a second hydroxylation in the kidneys. Vitamin D promotes absorption of dietary calcium by the small intestine. In turn, calcium is necessary for the proper formation of bones and teeth, as well as for blood clotting and the normal function of nerve and muscle tissues. Thus, by providing the body with vitamin D, the integumentary system is linked to the activity of the digestive, skeletal, muscular, nervous, cardiovascular, and renal systems. The integumentary system also contains receptors sensitive to touch, pressure, temperature, and pain. These receptors provide the nervous system with important information about our external environment. The nervous system in return controls the activity of sweat glands and blood flow, thereby using the skin as a means to help regulate body temperature.

The integumentary system affects other systems in the body

The integumentary system is affected by other systems in the body

NERVOUS SYSTEM Nerve endings in skin and subcutaneous tissue Control blood flow and sweat gland activity for provide input to the brain for touch, pressure, thermoregulation thermal and pain sensations RESPIRATORY SYSTEM Hairs in nose filter dust particles from inhaled Provide oxygen to skin cells and eliminates air carbon dioxide via gas exchange with blood Stimulation of pain nerve endings in skin may alter breathing rate ENDOCRINE SYSTEM Keratinocytes in skin help activate 7 Sex hormones for Stimulate sebaceous gland dehydrocholesterol to cholecalciferol(vit D3), activity, Influence growth, distribution of later on vit D3 will be changed into calcitriol in subcutaneous fat, and apocrine sweat gland kidney, a hormone that aids absorption of activity dietary calcium and phosphorus Adrenal hormone by Altering dermal blood flow and helping mobilize lipids from adipocytes DIGESTIVE SYSTEM Helps activate vitamin D to the hormone Provides nutrients for all cells and lipids for calcitriol, which promotes absorption of dietary storage by adipocytes calcium and phosphorus in the small intestine CARDIOVASCULAR SYSTEM Local chemical changes in dermis cause Provides oxygen and nutrients for the skin; widening and narrowing of skin blood vessels, delivers hormones and cells of immune system which help adjust blood flow to the skin to the skin; provide substances needed by skin glands to make their secretions Prevents fluid loss of the body Carries away carbon dioxide, waste products Serves as blood reservoir and toxins from the skin Provides heat to maintain normal skin temperature URINARY SYSTEM Assists in excretion of water and solutes in Maintains normal pH and ion composition of sweat body fluids Keratinized epidermis limits fluid loss through skin Excretes waste products REPRODUCTIVE SYSTEM

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 Nerve endings in skin and subcutaneous tissue

respond to erotic stimuli,contributing to sexual pleasure Mammary glands (modified sweat glands) produce milk and Suckling of a baby stimulates nerve endings in skin leading to milk ejection Skin stretches during pregnancy as fetus enlarges

Sex hormones affect hair distribution, adipose tissue distribution in subcutaneous layer, and mammary gland development

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1. SKIN
The integumentary system includes a number of diverse structures derived from the epidermis of skin, including hair, nails, oil glands, and sweat glands. Hair is an outgrowth of skin unique to mammals. It protects the scalp from ultraviolet rays and mechanical bumps, keeps foreign particles out of the respiratory tract, and has a sensory role. Each terminal hair consists of a central core called the medulla, which is surrounded by the cortex and enclosed by a cuticle. Hair is embedded in a group of cells called a follicle, which has an inner layer of epithelial cells responsible for producing hair and an outer layer of dermal connective tissue that provides blood vessels and physical reinforcement. Sebaceous glands secrete sebum, an oily substance that lubricates hair and inhibits the growth of bacteria. Eccrine glands produce sweat, which helps regulate body temperature through the evaporation of water on the skin surface. Apocrine glands are a type of sweat gland, mainly located in the armpits and pubic region. They do not function until puberty, and are assumed to be analogous to the sexual scent glands of other animals. Ceruminous glands are modified apocrine glands found in the lining of the external ear canal that produce earwax, whereas mammary glands are modified sweat glands specialized to secrete milk.

FUNCTIONS OF SKIN
Skin is absolutely essential for homeostasis, the ability to maintain a relatively constant environment within the body. A primary function of skin are:

Protect the entire body from mechanical injury: bumps , abrasions, and cuts Protect underlying tissue from chemical damaged such as acids and bases. Shields the body from biological damaged, Prevent entrance of foreign bodies: microorganisms such as a continual bacterial invasion or an ultraviolet radiation in sunlight. Play an important role in temperature regulation. This is accomplished with its rich blood supply and sweat glands, which are controlled by the nervous system. For example, during strenuous exercise, excess heat may be eliminated through dilated surface blood vessels and by activating sweat glands. Because sweat contains water, salts, and urea, the integumentary system technically has an excretory function. Prevent excess water loss or protects the body from dehydration. In fact, the biggest threat to survival for terrestrial animals is dehydration, and the waterproof nature of skin keeps fluids and other important substances inside.

Serve as a reservoir for food and water: adipose tissue Assist in the process of excretion: H20, Salt, Urea, Lactic Acid. Serve as a sense organ for cutaneous senses: pain, Skin contains components of the nervous system that detect temperature, touch, pressure, and pain stimuli. As a result, skin provides us with a great deal of information about our external environment.heat, cold, pressure, touch.

Prevent entrance of foreign bodies: microorganisms. Serve as a seat of origin for Vitamin D.

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The synthesizes vitamin D from modified cholesterol molecules when exposed to ultraviolet radiation (actually vitamin D is not a true vitamin because individuals with adequate exposure to sunlight do not require dietary supplementation). Vitamin D is necessary for the small intestine to absorb dietary calcium. That is why a lack of this vitamin can lead to the disease rickets, a disorder characterized byinadequate mineralization of bones. Symptoms include bowed legs and deformities of the pelvis, skull, and rib cage.

ANATOMY OF SKIN
The skin has a surface area of 2 sq.meter. It accounts for 16-20% of the total body weight. It is the largest organ of the body It is composed of epidermis, dermis and the subcutaneous tissue (the hypodermis). Full thickness skin is 1.5 - 5 mm. Human skin is of 2 types. They are nonhairy (glabrous skin) and hairy skin. The glabrous skin is marked by a series of ridges ad grooves with a configuration unique to each individual , known as dermatoglyphics.

Skin has two tissue layers: the epidermis and dermis. The outer epidermis is composed of stratified squamous epithelium. In contrast, the thicker dermis is made of connective tissue. As is true for all epithelial tissues, blood vessels are absent in the epidermis (i.e., it is avascular), but present in the dermis. Although the two skin layers are firmly connected, a burn or friction can cause them to separate, forming a blister. The skins subcutaneous tissue or hypodermis is technically not part of skin. However, it shares many of the skins protective functions. The hypodermis consists mostly of adipose tissue and some areolar connective tissue. It helps anchor skin, stores fat, and acts as thermal and mechanical insulation. Because of its extensive adipose, hypodermis can thicken when one gains weight, which occurs in a genderspecific manner in adults. For instance, females tend to accumulate excess fat in the thighs, hips, and breasts, whereas men first increase adipose in the abdomen. Describing of this skin components are below:

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THE EPIDERMIS
The epidermis is made of stratified squamous epithelium. It gives rise to derivate structures ( pilosebaceous units, nails and sweat glands) called appendages. The epidermis thickness is approximately 0.4 1.5 mm.

Cells
The epidermis is composed of four different cell types, Keratinocytes and the immigrant cells of the epidermis which are Melanocytes, Langerhans Cells and Merkel Cell. Other cels like the lymphocytes are extremely rare in normal skin. The epidermis is organized also into four or five layers called strata. And this tissue rests on and is attached to a basal lamina that seperates it with the dermis.

a. KERATINOCYTE
The most abundant cell type is the keratinocyte. It is an ectodermally derived cell that constitutes 80% of the epidermis. All keratinocytes have cytoplasmic keratin intermediate filaments and desmosomes It produces a fibrous protein called keratin that gives the epidermis its protective properties. Keratinocytes arise in the deepest layer of the epidermis by mitosis and are gradually pushed outwards towards the skin surface. During this migration, these cells flatten, fill with keratin, and die. That means the outer layer of skin is actually composed of dead cells, and millions of dead keratinocytes are rubbed off every day (it has been estimated that we lose about 40 pounds of skin cells in an average lifetime). The total life span of a keratinocyte, from its formation to being rubbed off, is 25 to 45 days. In addition, persistent friction can increase the rate of cell production and keratin formation, leading to a thickening of the epidermis called a callus. Keratin filaments are a hallmark of keratinocytes and other epithelial cells. They are the cytoskeleton. More than 30 different keratins are present ------- 20 epithelial and 10 hair keratins, all within the range of 40-70kDa. The keratins are saperated into acidic (type I; cytokeratins K10 to K20) and basic to neutral (type II; cytokeratins K1 to K9). Keratins are obligate hetero polymers meaning that a member of each family (acidic and basic) must be coexpressed for a filament structure. The end point of keratinization is a terminally differentiated, dead keratinocyte (corneocyte) that contains keratin filaments, matrix proteins and protein reinforced plasma membrane with surface associated lipids. KERATINIZATION is a genetically programmed, carefully regulated, complex series of morphologic and metabolic events that occur in postmitotic keratinocytes and involve in : Increase in cell size and flattening of the cell shape Appearance of new cellular organelles and the structural reorganization of those present

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 A change from a generalized cellular metabolism to a more focused metabolism associated with the synthesis and modification of molecules (structural proteins and lipids) related to keratinization. Alteration of the properties of the plasma membrane, cell surface antigens and receptors Eventual degradation of cellular organelles with chromatin fragmentation, a characteristic of apoptosis. Dehydration.

b. MELANOCYTES Are cells located near the base of the epidermis. They produce a pigment called melanin, which influences skin color and also absorbs
ultraviolet radiation.

Melanin is released from melanocytes and then transferred to keratinocytes, where it

accumulates over the nucleus forming a pigment shield. It is a DENDRITIC, pigment synthesizing cell It is a Neural Crest derived cell. It is confined to Basal Layer. Melanocyte processes contact keratinocytes in basal and the more superficial layers BUT DO NOT FORM JUNCTIONS WITH THEM AT ANY LEVEL. Melanocytes are recognized light microscopically by their pale staining cytoplasm, ovoid nucleus and the intrinsic color of the pigment containing melanosomes. Differentiation of the melanocyte correlates with the acquisition of its primary functions: melanogenesis, arborization and transfer of the pigment to keratinocytes. The melanosome is a distinct organelle of the melanocytes. It is an ovoid membrane bound structure where melanin is produced 4 different stages (I to IV) of the melanosome are EUMELANOSOMES: Melanosomes that are involved in the synthesis of brown or black eumelanin are ELLIPTICAL AND HAVE AN INTERNAL ORGANISATION OF LONGITUDINALLY ORIENTED CONCENTRIC LAMELLAE. PHEOMELANOSOMES: Melanosomes that synthesize red or yellow pheomelanin pigments have a spheroidal shape and a microvesicular internal structure. EPIDERMAL MELANIN UNIT: Approximately 36 basal and suprabasal keratinocytes are thought to coexist functionally with each melanocyte in an epidermal melanin unit. Within the unit, the melanocytes transfer pigment to associated keratinocytes. As a result, pigment is distributed throughout the basal layers and to a lesser extent, the more superficial layers where it protects the skin by absorbing and scattering potentially harmful radiation. Once within the keratinocytes, melanosomes exist either individually or in membrane bound aggregates (melanosome complex). Melanocytes within the keratinocytes are degraded by the lysosomal enzymes as the cells differentiate and move upwards. CONTROL OF MELANIN PRODUCTION: Keratinocytes produce growth factors that are mitogenic for the melanocytes ( eg. bFGF & TGF alpha), but also produce growth inhibitory factors ( IL-1, IL-6, TGF- beta). Proliferation of the melanocytes and their dendrites, melanogenesis as well as transfer of pigment also relies on hormonal control (melanocyte stimulating hormone, sex hormone), inflammatory mediators and vitamin D3 synthesized within the epidermis.

c. LANGERHANS CELLS The Langerhans cells are dendritic cells. Belong to a group of cells called macrophages
(white blood cells capable of phagocytosis). They originate in bone marrow and migrate to the epidermis, where they recognize and ingest foreign substances, such as bacteria. In this regard, they play a role in immunityThey account for 2-8% of the total epidermal cell population. Langerhans cells are pale staining with a convoluted nucleus. The cytoplasm of the Langerhans cells as seen by EM contains dispersed VIMENTIN INTERMEDIATE FILAMENTS AND SMALL ROD OR RACKET SHAPED STRUCTURES CALLED LANGERHANS CELL GRANULES OR BIRBECK GRANULES. It is a cup shaped disc which forms when membrane bound antigen is internalized by endocytosis. Phagolysosomes are common in the cytoplasm of the Langerhans cells They are distributed in the basal, spinous and granular layers showing preference for suprabasal position They migrate from the bone marrow to the circulation into the epidermis early in embryonic development and continue to repopulate the epidermis throughout life. They are antigen processing and presenting cells that are involved in a variety of T cell responses.

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They are implicated in the pathologic mechanisms underlying ALLERGIC CONTACT DERMATITIS, CUTANEOUS LEISHMANIASIS AND HIV INFECTION. They are reduced in the epidermis of patients with certain skin diseases like psoriasis, sarcoidosis. It is found in the other squamous epithelia including the oral cavity, esophagus, and vagina and also in lymphoid organs such as the spleen, thymus and lymph nodes. It is also present in the dermis. It is characterized by 2 distinct stages. The Langerhans cells in the epidermis can ingest and process antigens efficiently but are weak stimulators of unprimed T cells. Activated Langerhans cells that have been induced to migrate after contact with antigen are not phagocytic but are potent stimulators of nave T cells. They are characteristic of other cells of the monocyte- macrophage lineage. They are impaired functionally by the UVR. Following UVR, there is a decrease in the ability of Langerhans cells to present antigens, a decreased production of cytokines by keratinocytes and depletion in Langerhans cell numbers. Thus UVB irradiation results in overall diminished capacity for immune surveillance. Because of their effectiveness in antigen presentation and lymphocyte stimulation, dendritic cells and Langerhans cells have become prospective vehicles for tumor therapy and tumor vaccines.

d. MERKEL CELLS Present at the epidermal-dermal junction, are associated with sensory nerve endings,
forming a Merkel disk. These structures function as sensory receptors for touch. present in outer root sheath of large hair follicles. Also

They are slowly adapting type I mechanoreceptors. They are located in sites of high tactile sensitivity like the digits, lips, regions of the oral
cavity and the ORS of the hair follicle keratinocytes.

They are present among basal keratinocytes and extend cytoplasmic spines towards the Merkel cells receive stimuli as the keratinocytes are deformed. Changes in epidermal cell
shape during keratinisation

They have a pale staining cytoplasm and lobulated nucleus. Immunohistochemical markers of the Merkel cells include -certain keratin peptides.
KERATIN 20 is restricted to Merkel cells in the skin and thus may be the most reliable marker. Merkel cells make synaptic contacts with nerve endings to form the Merkel cell- neurite complex. In the ultra structural levels, membrane bounded dense core granules that contain neurotransmitter like substances are metenkephalin, VIP, neuron specific enolase and synaptophysin. Clusters of Merkel cellneurite complexes in glabrous skin = touch corpuscles (Tastscheiben), and in hairy skin =tactile hair discs. Detect vertical, shearing, or other directional deformations (continuous touch), and direction of hair movement

Layers

a. BASAL LAYER or STRATUM BASALE

The deepest epidermal layer is the stratum basale, which is firmly attached to the dermis. It mainly consists of a single row of cuboidal-shaped cells capable of rapid cell division. About one-fifth of the cells in this layer are melanocytes. There is also an occasional Merkel

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cell in this stratum. Sometimes invasion by a papilloma virus causes a dramatic increase in the rate of cell division. This, in turn, causes a wart to form, which is a type of benign tumor.

Consists of 1-2 layers of wide layer o cubodial or columnar cells in contact with the dermis.

Contains mitotically active, columnar shaped keratinocytes. It has a large nucleus and organelles Provides the germinal cells necessary for the regeneration of the layers of the epidermis. These germinal cells are separated from the dermis by a thin layer of basement membrane. Continuous mitotic activity The K5 and K14 pair of keratins are expressed in the basal layer of the epidermis and other stratifying epithelia. Other keratins like K15 and K19 are present in putative stem cells. Microfilaments and microtubules are other cytoskeletal elements present in basal cells. Some microfilaments maintain important links with the external environment via their association with integrin receptors of the cell surface. Integrins are a large family of cell surface molecules involved in cell- cell and cell matrix interactions and INITIATION OF TERMINAL DIFFERENTIATION.

Not all basal cells display equal proliferative properties. Based on cell kinetics, 3
populations coexist in this layer are stem cells, transient amplifying cells and postmitotic cells. Stem cells are present in the bulge region of the hair follicle and also in the basal layer. These cells happen to be clonogenic, progress rapidly through the S phase of the cell cycle and divide infrequently during stable self renewal. However, under conditions requiring rapid proliferative activity ( wound healing) or after exposure to exogenous growth factor. The stem cells undergo rapid multiple cell divisions. Transient amplifying cells are a subset of daughter cells produced by the stem cells. These transient amplifying cells are needed for stable self renewal and are the most common cell type of the Basal Layer. The postmitotic cells arise from the TAC after they undergo several cell divisions. It is the post mitotic cells that undergo terminal differentiation, detaching from the basal lamina and migrating superficially. Cell divisions in stratum corneum occur every 18-19 days. The normal transit time of a basal cell, from the time it detaches from the basal lamina to the time it reaches the stratum corneum is at least 14 days. Transit through the stratum corneum and desquamation require another 14 days.

b. SPINOUS LAYER or STRATUM SPINOSUM (spiny layer).

It contains 8-10 layers of cells The cells that divide in the statum germinativum soon begin to accumulate many desmosomes on their outer surface. These provide the characteristic prickles of the stratum spinosum (SS), which is called the prickle-cell layer. They are named for the spine like appearance of the cell margins in histologic sections. The spines are the abundant desmosomes, calcium dependant cell surface modifications that promote adhesion of epidermal cells and resistance to mechanical stresses. It contains several layers of cuboidal cells, with scattered melanin granules and Langerhans cells. Cells of stratum spinosum are consisting of several rows of Polyhedral cell with a Rounded nucleus. Cells of the upper spinous layers are larger, more flattened and contain organelles called lamellar granules.

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 KERATIN PAIRS IN STRATUM SPINOSUM: Keratin filaments are present as usual. K5,

K14 of the basal cells are present but in addition a new pair K1/K10 also occurs in the spinous layers. This new pair is characteristic of epidermal pattern of differentiation and thus referred to as differentiation or keratinization specific keratins. In hyperproliferative states ( psoriasis, actinic keratoses), the sprabasal keratiocytes switch to an alternative pathway of differentiation in which the synthesis of K1/K10 is down regulated and K6/K16 is favored. Correlated with this change in keratin expression is a loss of normal phenotypic differentiation in granular and cornified cell layer. DESMOSOMES: Within each cell is a desmosomal plaque associated with the internal surface of the plasma membrane and is composed of 6 polypeptides such as PLAKOGLOBIN, DESMOPLAKIN I & II, KERATOCALMIN, DESMOYOKIN AND BAND 6 PROTEIN (plakophillin). Transmembrane glycoprotein of the cadherin family provide the adhesive properties on the external surface or core of the desmosome. These glycoproteins are ----- desmogleins 1 &3 and desmocollin I & II. The extracellular domains of these proteins form part of the core. The intracellular domains insert into the plaque, linking then to the KIF. GAP JUNCTIONS: gap junctions between keratinocytes are the sites of physiologic communication. Gap junctions are more abundant in more differentiated keratinocytes. Communications with these junctions are important in the regulation of cell metabolism, growth and differentiation. LAMELLAR GRANULES: Deliver precursors of stratum corneum lipids into the intercellular space. The granules are first evident in the cytoplasm of the upper statum spinosum. Their primary site of action is the granular cornified layer interface. They are 0.2-0.3 micrometer in diameter, membrane bound, secretory organelles that contain a series of alternating thick and thin lamellae. They contain GLYCOPROTEINS, GLYCOLIPIDS, PHOSPHOLIPIDS, FREE STEROLS AND A NUMBER OF ACID HYDROLASES INCLUDING LIPASES, PROTEASES, ACID PHOSPHATASE AND GLYCOSIDASES. GLUCOSYLCERAMIDES, the precursors to ceramide and the dominant component of the stratum corneum lipids, are found in the lamellar granules. Roles of lamellar granule in providing the epidermal lipids responsible for the barrier properties of the stratum corneum, synthesis and storage of cholesterol and adhesion/ desquamation of cornified cells have been hypothesized. KERATOHYALIN GRANULES: This is so called because of the presence of intracellular BASOPHILIC keratohyaline granules. KERATOHYALIN GRANULES are composed primarily of an electron dense protein, profillagrin. Loricin, a protein of the cornified cell envelope is also found in the granules. Profillagrin is a high molecular mass histidine rich phosphorylated intermediate filament associated protein. it is made up of tandem repeats of filaggrin monomers joined by small linker peptides. Conversion of profilaggrin precursor to filaggrin subunits occurs stepwise during the transition of granular cell to cornified cell by proteolysis and by dephosphorylation. Final proteolysis to free amino acids occurs in the outer layers of the stratum corneum. This is important for the regulation of Envelope are : Involucrin, it is an insoluble, cysteine rich protein, first synthesized in the cytoplasm of spinous cells. It becomes cross linked by transglutaminases in the granular layer into an insoluble cell boundary that is resistant to denaturing and reducing chemicals. Keratolinin Loricin is a highly insoluble, sulfer- and glycine/serine rich protein . it is a later differentiation product and is the major CE protein comparising 75% of the total CE protein mass. The cytoplasmic surface of the CE is composed primarily of loricin. Small proline rich proteins ( cornifin, SPR 1 & SPR-2) Serine proteinase inhibitor elafin Filaggrin linker segment peptide. Envoplakin --- links CE to the desmosomes and KF. Although the envelope precursors are first synthesized in the spinous and/or granular layers where they can be recognized biochemically and immunohistochemically, the CE is evident morphologically only in cornified cells. The calcium requiring transglutaminases are present in all stratified squamous epithelia and in hair follicles. There are 3 transglutaminases in the epidermis : o TGase 1 (keratinocyte TGase) : Exists as membrane bound activated forms responsible for most of the activity in differentiating keratinocytes. Some activity is present in the basal cells but the enzyme activity is especially prevalent in the granular layer of the epidermis. Mutations occur in lamellar ichthyosis. o TGase 2 is present in the fetal epidermis and in the basal layer of the adult. It appears to play a role in apoptosis. It is similar to tissue transglutaminase. It is an autoantigen reacting in DH. o TGase 3 ( epidermal TGase) is expressed after early differentiation marker ( K1 & K10 ) and is coincident with loricin and filaggrin.

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c. STRATUM GRANULOSUM or GRANULAR LAYER

Consists of two to five layers of polygonal cells that gradually become flattened cells and

the cytoplasm contains contains keratohyalin granules, a substance that contributes to the formation of keratin. It turns out all the cells above this layer die because they are too far from dermal capillaries to obtain adequate nutrients. The progressive maturation of a keratinocyte is characterized by the accumulation of keratin, called keratinization. The cells of the stratum granulosum (SGR) accumulate dense basophilic Keratohyalin granules These granules contain lipids, which along with the desmosomal connections, help to form a waterproof barrier Action of Lamellar Granules starts in the Granular Corneal Interface. The lamellar granules act in the interface between the granular and cornified cell layers. In this position they aggregate into clusters, fuse with the plasma membrane and release their contents into the intercellular space. The extruded material is stacked into discs, similar to the internal organization of the granule, and is rearranged in various stages, leading to the formation of sheets. The hydrolytic enzymes are released with the lipids and are involved in the reorganization and the subsequent assembly into intercellular lamellae. Thus the probarrier lipids (glycolipids, free sterols and phospholipids) are converted to barrier lipids eg. sphingolipids that create a seal at the interface of the stratum granulosum and stratum corneum. SELF DESTRUCTION OF CELLULAR ORGANELLES IN THIS LAYER: The granular layer also plays a role in its own programmed destruction . the change involves the loss of nucleus and virtually all cell components with the exception of the KERATIN FILAMENTS AND FILAGGRIN MATRIX. DNAase, RNAase, acid hydrolases, esterases, phosphatases, proteases and plasminogen activator are implicated in the destruction.

d. STRATUM LUCIDUM or CLEAR LAYER or Eosinophilic

Contains three to four thin layers of transparent flattened dead cells. This layer is only found

in the palms of the hands and soles of the feet, areas known as thick skin. The stratum lucidum is normally only well seen in thick epidermis and represents a transition from stratum granulosum to stratum corneum Epidermis varies in thickness depending mainly on frictional forces and is thickest on the palms and soles. e. STRATUM CORNEUM or HORNY LAYER

It is the outermost layer. It consists of 20 to 30 rows of squamous and extermelly flattended, dead cells completely filled with keratin, having lost their nuclei and other organelles. It is this layer that prevents water loss and protects from us from biological, chemical, and physical insults. Dandruff occurs when dry patches of epidermal cells flake off the scalp.

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This affliction is most common in middle age, and also is associated with stress and a high fat diet. This layer is composed of 20-25 layers of cells. The stratum corneum barrier is formed by a two compartment system. The flattened polyhedral horny cell is the largest cell of the epidermis. They contain keratins and fillagrin. High molecular mass keratins stabilized by intermolecular disulfide bonds account for upto 80% of the cornified cell. In SC a keratinocyte gradually migrates to the surface and is sloughed off in a process called desquamation. The remainder of the cell contains electron dense matrix material, probably filaggrin surrounding the filaments. The lipid enrichment results from the deposition of lamellar body contents. Three key lipid types are cholesterol, ceramides and FFA , they form the lipid bilayers. Changes in the structure, composition and function of cornified cells accompany their movement towards the outer surface of the skin. Cells of the lower SC , sometimes called the stratum compactum, are thicker and have more densely packed organized parallel arrays of KF, a more fragile cornified CE and modified desmosomes. They have less capacity for water binding than the mid and upper regions. The outer SC cells called the stratum disjunctum are more prone to desquamation. Cells in the mid SC have the highest concentration of free AA and therefore are able to bind water with greater efficacy. The CE becomes rigid. The desmosomes undergo proteolytic degradation is shedding. Lipid extraction and metabolic imbalances such as FA deficiency perturb the barrier function as well as resulting in epidermal hyperproliferation, scaling and inflammation.

The Dermoepidermal Junction

It forms an extensive interface between the epidermis and the dermis and also at the junctions between epidermal appendages and the dermis. It is not visible in H&E stain. On staining with PAS, it is seen as 0.5-1.0 micrometer thick homogenous band. It can be divided into 3 supramolecular network , which are : The hemidesmosome-anchoring filament complex o The hemidesmosome achoring filament complex binds the basal keratinocytes to the basement membrane. o The hemidesmosome has cytoplasmic plaque, transmembranous and extracellular components. o KIF insert into the cytoplasmic plaque portion which consists of BP230 antigen and plectin The basement membrane itself o The transmembrane component consists of BP180 ( collagen XVII) and a6b4 integrin . The extracellular domain of BP180 has been localised to the extracellular space beneath the hemidesmosome ---- the lamina lucida. The intracellular domain of BP180 is localised in the hemidesmosomal plaque. o The extracellular matrix components of the hemidesmosome are the subbasal dense plate and the anchoring filaments. o Anchoring filaments originate from the hemidesmosome and insert into the lamina densa. The major component of the anchoring filaments is LAMININ 5 which is localized mainly in the lamina densa and the lower lamina lucida. It is associated with a6b4 integrin of the hemidesmosome. o The lamina lucida is the primary location of several noncollagenous glycoproteins ------- laminins, entactin/nidogen and fibronectin. Because these molecules self aggregate, bind to other matrix molecules and to the cells, they are all important in promoting the adhesion between the epidermis and the lamina densa. THE LAMINA LUCIDA APPEARS TO BE THE WEAKEST ZONE OF THE DEJ. o Type IV collagen is the primary component of the LAMINA DENSA. It is a nonbanded network forming collagen synthesized by the keratinocytes that provides structural support and flexibility to this layer. Type V collagen is codistributed with type IV collagen. Sulfated proteoglycans in this layer probably assist in regulating permeability by restricting the passage of cationic molecules. LAMININ is also present in this layer. Anchoring fibrils o ANCHORING FIBRILS are broad (20-60nm), elongated (200-800 nm) flexible, banded, fibrillar structures that originate at the lamina densa and extend into the dermis. The fibrillar portions of anchoring fibrils have the morphologic appearance of collagen fibrils and have been shown to be composed of parallel bundles of type VII collagen molecules. These are synthesized by the epidermal keratinocytes.

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They arise from the subbasal dense plaque and pass through the lamina lucida to the lamina densa where they loop around and merge to the lamina densa or terminate in the papillary dermis (where they interweave with type III collagen fibres). The anchoring fibrils penetrate the deepest zone of the DEJ, the sublamina densa region or the reticular lamina. Interstitial collagens and procollagens are present in this zone. In addition the first fibres of the elastic fibre system are organized and probably form part of the complex that anchors the epidermis and the dermis through the basement membrane zone. The oxytalan fibres are bundles of the microfibrillary components of the elastic fibres. They originate in the lamina densa and insert into the planar network of elastic fibres at the junction of the papillary and reticular dermis. The oxytalan fibres are coated with soluble elastin: they are flexible integrating elements that accommodate deformation of the skin without compromise to structural integrity. These fibres exert tension on the epidermis.

THE DERMIS
The dermis varies in thickness from 1mm in the eyelids to 5 mm on the back. The dermis is the mesodermally derived layer of dense irregular collagenous connective tissue that underlies and interdigitates with the epidermis. The dermis is the mid layer of skin, thick inner layer of skin, which comprises blood vessels, connective tissue, nerves, lymph vessels, sweat glands and hair shafts. The upper layer for touch, pain and heat, which communicate with the central nervous system and is responsible for the folds of the fingerprints. The lower layer made of dense elastic fibers that house the hair follicles, nerves, gland, and gives the skin most of its stretchiness and strength . The dermis functions are : o Provides pliability, elasticity and tensile strength. o It protects the body from mechanical injury, o Binds water, o Aids in thermal regulation and o Supports the vascular network to supply the avasular epidermis. o Includes receptors of sensory stimuli. The dermis interacts with the epidermis in maintaining the properties of both the tissue and collaborates during morphogenesis of the DEJ and epidermal appendages (teeth, pilosebaceous units and sweat glands) and interacts in the repairing and remodeling the skin as wounds are healed. The dermis forms the main bulk of the skin. It consists of : o o o o o o o A supporting matrix, Several protein fibres like the collagen, elastin and A number of cells like the fibroblasts, macrophages and mast cells. Vessels, Lymphatics and Sensory and motor nerve endings. Dermis also present are immune cells that are involved in defense against foreign invaders passing through the epidermis.

The superficial one tenth part of the dermis is called the papillary dermis and the lower nine tenth part is called the reticular dermis. The papillary dermis and the periadnexal dermis together are also called the adventitial dermis. Unlike the epidermis no differentiation occurs in the dermis but the matrix and the connective tissue undergo restructuring and remodeling in response to external stimuli. The connective thick tissue matrix of dermis are containing Fibrous Connective tissue with Collagen and Elastic connective tissue, and Non Fibrous Connective tissue with Filamentous glycoproteins and the proteoglycans and the glycosaminoglycans (GAGs). Thick fibrous (collagen and elastic) tissue under the epidermis. Allows movement and flexibility without tearing. Blood vessels deliver nutrients to the skin while regulating body temperature. Dermis has two main layers which is typically subdivided into two zones, a papillary dermis and a reticular layer.

Collagen Tissue
Collagen fibres are soft and flexible but strong and inelastic. They provide tensile strength.

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 They are the major components of the dermis accounting for 75% of the dry weight of the

skin. They are of different types. Approximately 80-90% collagen fibres in the dermis are type I,8-10% are type III and less than 5%type V. Type I fibres are larger in diameter forming large and coarser bundles present mainly in the reticular dermis. Type III fibrils are smaller and form small and fine network which are particularly concentrated in the papillary dermis and around blood vessels and adnexa. Type V is also present in the papillary dermis. Type VI is abundant throughout the dermis and is associated with the interfibrillar space. On light microscopy they are seen as 12-15 micrometer unbranched fibres either as finely woven network or as thick bundles. On electron microscopy, they are seen as regular cross striated fibrils with a 60-70nm periodicity WHICH IS THE CHARCATERISTIC BANDING PATTERN OF COLLAGEN FIBRILS. EM also reveals triple helical structure of the collagen fibres. The triple helix formation is initiated by the spontaneous association of 3 individual C- terminal peptides (procollagens) in the fibroblasts. This is followed by the ordered helical binding from the C- to the Nterminus of the 3 alpha chains.

Elastic Tissue
It forms a continuous network from the lamina densa of DEJ to the CT of the subcutis. Elastic fibres are also present in the walls of the blood vessels and lymphatics. Function and special properties: Because of their rubbery recoil, they maintain the normal configuration (elasticity) of the skin. The sequence of elastogenesis is initiated with the synthesis and deposition of microfibrils . Elastin is then deposited in variable amounts on the microfibrillar network. It is resistant to chemical cleavage and extremely insoluble. This insoluble elastin is formed from a soluble precursor, tropoelastin, by the action of lysyl- 6- oxidase which forms cross linkages. Mature elastic fibres contain as much as 90% elastin; microfibrils are embedded within and collected on the surface of the elastin matrix. Elaunin fibres have an intermediate amount of insoluble cross linked elastin. Type of elastic fibres are Oxytalan, elaunin and mature elastic fibres occur in order progressively beginning at the DEJ. The Oxytalan fibres extend perpendicularly from the DEJ to the junction between the papillary and reticular dermis where they merge with the horizontal network of Elaunin fibres. Elaunin fibres are flexible and in turn evolve into the network of mature elastic fibres that extend throughout the reticular dermis. Elastic fibres are positioned between the collagen fibres. Fibrillin is Elastic fibres have microfibrillar and amorphous matrix components. Several glycoproteins have been identified as constituents of the microfibrils. Among the most characterized of these molecules is FIBRILLIN, a 350 kDa molecule. Appearance on light microscopy is elastic fibres are seen as darkly stained, unbranched fibrils that occur both separately and intermingled with collagen fibrils and matrix. Appearance on transmission electron microscopy is they show distinctive sheet like agglomerates. The central speckled amorphous element of the elastic fibrils contains elastin whereas the peripheral part contains fibrillin and a heterogenous collection of other glycoproteins.

The diffuse and filamentous Dermal Matrix

They are present between the fibrous structures and make up the ground substance. The main components are the PROTEOGLYCANS ( PGs) and GLYCOSAMINOGLYCANS( GAGs). The PGs and GAGs can bind upto 1000 times their own volume and thus regulate the water binding capabilities of the dermis and influence the dermal volume and compressibility. They also bind growth factors ( eg bFGF) and link cells with the fibrillar and filamentous matrix, thereby influencing proliferation, differentiation , tissue repair and morphogenesis. Other matrix glycoproteins are

Fibronectin is insoluble filamentous glycoprotein synthesized in the skin by both epithelial

and mesenchymal cells; it ensheathes collagen and elastin fibres, the surfaces of cells is mediate cell matrix adhesion. Laminin Thrombospondin Vitronectin

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 Tenascin

Cells of The Dermis

Fibroblasts, macrophages and mast cells are the regular residents of the dermis. They are found in greatest density in normal skin in the papillary region and surrounding vessels of the subpapillary plexus, but they also occur in the reticular dermis where they are found in the interstices between the collagen fibre bundles. Small numbers of lymphocytes collect around the blood vessels in normal skin and at the site of inflammation, the lymphocytes and leucocytes from the blood are prominent. Pericytes and veil cells ensheath the walls of blood vessels. Schwann cells encompass nerve fibres.

a. THE FIBROBLAST
It is a mesenchymally derived cell. They appear as bipolar cells with ovoid nucleus. It is responsible for the synthesis and degradation of fibrous and nonfibrous connective tissue matrix proteins and a anumber of soluble factors Thus they provide the ECM framework as well as interact with the epidermis and dermis via soluble mediators. The cytoplasm of the fibroblast contains RER and Golgi complexes and suggesting active synthetic activity. Human fibroblast cell lines are not identical and are a highly diverse population. Resting and proliferating fibroblasts respond to immune mediators including IL-1 and . Fibroblasts from hypertrophic scars appear abnormally sensitive to TGF- whereas interferon 1 alpha can decrease their proliferation and collagen production.

b. THE MACROPHAGES

The macrophages, monocytes and dermal dendrocytes are a heterogenous collection of

cells that constitute the MONONUCLEAR PHAGOCYTIC SYSTEM OF THE SKIN.

Macrophages are bone marrow derived. They are difficult to distinguish from fibroblasts except that macrophages have lysosomes

and phagocytic vacuoles. Several antigenic markers characterize the macrophages and distinguish them from fibroblasts ---- MAC387, RFD7, KiM8, RFDR. They have an extensive list of functions. They are phagocytic, they process and present antigens to immunocompetant lymphoid cells. They are microbicidal, tumoricidal, secretory ( growth factors, cytokines and other immunomodulatory molecules) and hematopoetic.

c. MAST CELLS They are special secretory cells distributed in the connective tissue throughout the body,

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 They occur typically at sites adjacent to the interface of an organ and the environment. In the skin, the mast cells are present in greatest density in the papillary dermis, near the DEJ, in the sheaths of epidermal appendages and around blood vessels and nerves of subpapillary plexus. They are also common in the SC tissue. They are easily identified histologically as oval or spindle shaped cells with round or oval nucleus and abundant cytoplasmic granules that do not stain with H&E but stain metachromatically with methylene blue. At the ultrastructural level the granules are membrane bound can be separated into Secretory and lysosomal granules. Mast cells originate in the bone marrow from CD34+ stem cells. Differentiation towards mast cells occurs in the tissues under the influence of other cells and ECM matrix. Like basophils they contain metachromatic granules and have IgE antibodies on their surface. Differentiation towards mast cells occurs in the tissues under the influence of other cells and ECM matrix. Mast cells produce many mediators. Some of them are preformed and stored --- Histamine, Heparin, Tryptase, Chymase, Carboxypeptidase, Neutrophillic cationic factor and Eosinophilic cationic factor. The mast cells synthesize and release other substance without storage Growth factors, Cytokines ( IL-1,3,4,5,GM-CSF,TNF-), Leukotreins and PAFs. Lysosomal granules ----- Acid hydrolases that degrade GAGs, PGs, and complex glycolipids intracellularly. These take part in the repair process and degradation of foreign material.

d. THE DERMAL DENDOCRYTE

It is a stellate, dendritic or sometimes spindle shaped It is a highly phagocytic cell in the dermis of the normal skin. They are APCs. They originate in the bone marrow. They are particularly abundant in the papillary dermis and upper reticular dermis frequently in the proximity of vessels of the subpapillary plexus. Dermal dendrocytes are also present around the vessels in the reticular dermis and in the subcutaneous tissue.

Organization of The Dermis Major

The dermis is organized into papillary and reticular dermis; the distinction of the two zones is based largely on their difference in connective tissue organization, cell density, and nerve and vascular patterns.

a. PAPPILARY DERMIS It is characterized by small bundles of small diameter collagen fibrils and oxytalan elastic
fibres.

The thinner outer papillary layer consists of loose (areolar) connective tissue with

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collagen and elastic fibers. Its outer surface forms obvious folds, where the vasculature interdigitates in area is called dermal papillae. (DP). Many of these papillae contain receptors for touch and pain. In addition, these folds reach up to the epidermis, causing ridges on the surface of skin that increase friction, thereby enhancing the gripping ability of hands andfeet. The specific patterns of papillary folds are genetically determined. Because the ridges on the fingertips have a rich supply of sweat pores, they may leave unique fingerprints, essentially outlines of sweat on the surfaces they touch. Papillary layer contains vascular networks that have two functions: support the avascular epidermis with nutrients and provide a network for thermoregulation. The vasculature by increasing or decreasing blood flow, can conserve or dissipate heat.

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 PD also contains free sensory nerve endings and Meissners corpuscles

Superficial thin layer of loose connective tissue beneath the epithelium Mature elastic fibres are usually not found in the normal papillary dermis but they are common in the skin of patients with certain inherited connective tissue diseases (e.g. dominantly inherited forms of EHLER DANLOS SYNDROME), in aging skin, and in actinically damaged skin. Large dense elasic fibers with abnormal structure are the hallmark of sun damaged skin. The structural characteristics of the matrix in the papillary dermis permit the skin to accommodate to impact. The papillary dermis also has a high density of fibroblastic cells that proliferate more rapidly, have a higher rate of metabolic activity and SYNTHESIZE DIFFERENT SPECIES OF PROSTAGLANDINS as compared to those of reticular dermis. Capillaries from the subpapillary plexus project into the epidermis within the dermal papillae. The epidermis and the proximal dermis exchange a number of cytokines and growth factors, and matrix components of the dermis are linked to the cytoskeleton of the epidermis through transmembrane receptors. It is possible therefore that the organization and composition of the papillary dermis reflect the zone of influence of epidermis through its soluble and diffusible factors. The sublamina densa region or the reticular lamina can be identified as a subdivision of the papillary dermis. It stains selectively for type I procollagen and is characterized by fine but dense organization of fibrils. Because of this, the structure is sometimes called a COMPACT ZONE. It is a region that is rich in cells that bear receptors for growth factors produced by the epidermis (e.g. PDGF) and has abundance of molecules that have adhesive properties (e.g. tenascin). This region is more prominent and thickened in pathologic skin. b. RETICULAR DERMIS

The deeper and thicker reticular layer is composed of dense connective tissue. It is composed primarily of a combination of large diameter collagen fibrils and elastic fibers organized into large, interwoven bundles in the extracellular matrix, which allow skin to strect and then return to its original shape. Mature elastic fibres form a superstructure around the collagen fibre bundles. This gives a strong and resilient mechanical property. In normal individuals, the elastic fibres and collagen bundles of the reticular dermis increase in size progressively toward the hypodermis. Substantial body weight gain, as with pregnancy or obesity, can tear the dermis, resulting in visible lines called stretch marks. Also, the resilience of skin decreases with age, as collagen fibers stiffen and elastic fibers lose their elasticity. These effects, along with a reduction in the ability of the dermis to hold moisture, produce wrinkles and sagging skin, which usually first become apparent by the late forties. The reticular layer also contains blood vessels, sweat and oil glands, and receptors for the sensation of deep pressure.

THE CUTANEOUS VASCULATURE

The skin is richly supplied with a vascular network consisting of distributing and collecting channels and horizontal plexuses located at boundaries within the dermis and supplying the epidermal appendages. The microcirculatory beds in the skin contain arterioles, precapillary sphincters, arterial and venous capillaries, postcapillary venules and collecting venules. By comparison with the vasculature of other organs, the vessels of the skin have a thick wall supported with connective tissue and smooth muscle cells. This structure is advantageous to an organ that is regularly subjected to shearing forces. The vessels that supply the dermis are small branches from the musculo cutaneous arteries that penetrate the subcutaneous tissue and enter the deep reticular dermis where they are organized into a deep horizontal arteriolar plexus. Ascending arterioles extend vertically from the plexus toward the epidermis. At the junction of the papillary and reticular dermis, these terminal arterioles form the superficial or subpapillary plexus. From here the capillary loops ascend into the papillary dermis, one capillary for each dermal papilla. The postcapillary descending limb of the loop is the venous capillary which drains into the subpapillary venous plexus and desending venous channels to the deep horizontal plexus and drains into larger deep veins. Structure and ultrastructure The ascending arterioles: They have o Two layers of smooth muscle cells, o A discontinous internal elastic lamina,

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Pericytes and veil cells outside the vessel wall.

The anastomosing arterioles have single layer of smooth muscle and lack elastic fibres.
The ascending limb of the capillary loop has a continuous endothelium, basal lamina and few pericytes. At the apex of the loop, both the endothelium and the basal lamina are attenuated to allow transport of the material out of the capillary. The descending post capillary limb and the descending venule have multiple layers of basal lamina and a loose sheath of pericytes and veil cells. These descending venules develop gaps between the adjacent endothelial cells allowing extravasation of fluid and escape of cells from the lumen. The endothelial cells on an ultrastructural level demonstrate many common cytoplasmic organelles like the Golgi apparatus, RER, SER, mitochondria and lysosomes. In addition they have many pinocytic vesicles, intermediate filaments containing vimentin and dense rod shaped bodies, the Weibel- palade bodies. These WP bodies are associated with actin like filaments which are 5-6 micrometer in diameter. Laminin and type IV collagen is present in the vascular basal lamina. GLOMUS BODIES: Direct connections exist between arterial and venous circulation in certain regions of the skin (e.g. fingerpads, nail beds, palms and soles) without the interposition of capillaries. They serve as alternative bypass routes that shunt blood around congested capillary beds. These sites consist of an ascending arteriole (called a glomus body) which is modified by 3-6 layers of smooth muscle cells and has associated sympathetic nerve fibres and venules. The glomus can close completely when the blood pressure is below a critical level. Glomus bodies are associated with temperature regulation.

THE CUTANEOUS LYMPHATICS

The cutaneous lymphatics regulate the interstitial fluid by resorption of leaked fluid. They also clear the tissue of unwanted proteins, lipids, organisms, cells and degraded material. Arrangement is like the blood vessels. Bicuspid valves prevent backflow and stasis of lymphatic fluid. Lymphatic vessels have a larger lumen as compared to blood vessels but a thinner vessel wall consisting of flattened endothelial cells, discontinous basal lamina and elastic fibres. Smooth muscle fibres in the lymphatic vessel wall are present only at the level of SC tissue. On electon microscopy, lymphatic endothelial cells contain cytoplasmic filaments that represent vimentin filaments.

THE NERVES AND SENSORY RECEPTORS OF THE SKIN

The nerve networks of the skin contain

Somatic Sensory
The sensory fibres alone (free nerve endings) or in conjunction with specialized structures (corpuscular receptors) function at every point of the body as receptors of touch, pain, temperature, itch, and Mehcanical stimuli. The density and types of receptors are regionally variable and specific. Receptors are particularly dense in the hairless areas such as the AREOLA, LABIA AND GLANS PENIS.

Sympathetis Autonomic Fibers

Sympathetic fibres are codistributed with the sensory nerves in the dermis until they branch to innervate the sweat glands, vascular smooth muscle, the arrector pili muscle of the hair follicles and the sebaceous glands.

Musculocutaneous Nerves
The skin is innervated by the large myelinated cutaneous branches of the musculocutaneous nerves that arise segmentally from the spinal nerves. Small branches that enter the deep dermis are surrounded by the epineural sheath. Perineural sheath cover the fibre bundles and endoneural sheath cover individual fibres respectively. The pattern of nerve fibres of the skin is similar to the vascular pattern. The sensory nerves, in general, supply the skin segmentally (dermatomes), but the boundaries are imprecise and there is overlapping innervation to any given area. Autonomic innervation does not follow exactly the same pattern because the post ganglionic fibres distributed in the skin originate in the sympathetic chain ganglia where pre ganglionic fibres of several spinal nerves synapse.

Free Nerve Endings

Free Nerve is Rapidly Adapating. They are the most widespread and the most important sensory receptors of the body. They are always unsheathed by Schwann cells and a basal lamina. They are particularly common in the papillary dermis just beneath the epidermis.

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Penicillate Fibers
They are the primary nerve fibres found subepidermally in the hairy skin. Separate, unmyelinated branches from one or more myelinated stem axons are unsheathed collectively by the processes of a Schwann cell. They are further surrounded by the basal lamina and collagen fibrils. They are rapidly adapting receptors and function in the perception of touch, temperature, pain, and itch. In areas such as the palms and soles, they have a precise distribution and project into the dermal papilla individually and vertically. In other areas they are not so precisely distributed.

Papillary Nerve Endings

They are found at the follicular orifice. They are branches of nerves that innervate the
deeper portion of the follicle. They have more mitochondria and vesicles and are particulary receptive to cold sensation.. Other free nerve endings: They are associated with specific structures such as hair follicles (slow adapting that respond to bending or movement of hair), eccrine sweat gland (cholinergic sympathetic), arrector pili muscle (adrenergic and cholinergic fibres) Free nerve endings are associated with individual Merkel cells.

Corpuscular Receptors
a. MEISSNERS CORPUSCLES

It is an elongated or ovoid mechanoreceptor located in the dermal papillae of digital skin

and oriented vertically towards the epidermal surface and in contact wit basal cells. 1-6 myelinated axons enter the base of the corpuscle, lose their myelin coverings, ramify extensively and terminate in bulboid endings that are surrounded by lamellae. Elongated ovoid bodies found in the dermal papillae. They are rapidly adapting mechanoreceptors that subserve discriminative touch sensations. Most numoreous in the inger tips, palm and sole, lips and nipple.

b. PACICIANS CORPUSCLES is rapidly adapting Mecahnoreceptors: Theser are located deep in the hypodermis. Their fungtion is for deep pressure reception, detection of vibration and mediate deep pressure sensation.

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 It looks like sliced onion-like lamellar structure located in the deep dermis and subcutaneous tissue. They lie in the deep dermis and SC tissue of the skin that covers the weight bearing areas of the body. The perineurium (capsule) is organized into 30 or more concentric layers of cells and fibrous connective tissue. There is a subcapsular zone of collagen fibres and fibroblasts and an inner core that consists of Schwann cell derived hemilamellae around the nerve fibre. Usually one axon from a large nerve extends to the corpuscle without branching.

Mucocutaneous End Organs

They have a capsule and an inner core and contain both neural and nonneural components. The capsule is the continuation of the perineurium and the core contains both preterminal and terminal portions of the fibre surrounded by the lamellated wrappings of Schwann cells. Remodeling of the corpuscular receptors occur throughout life. They express neuron specific enolase, neurofilaments, calbindin D28K and other calcium binding proteins, S-100 and p75 neurotrophin receptor. Mucocutaneous end organs have the same structure as Meissners corpuscles and are located at the junctions of skin and mucous membrane like the lips, eyelids, perianal region, glans, prepuce, clitoris and labia minora.

THE MUSCLES OF THE SKIN

Smooth Muscles
The smooth or involuntary muscles of the skin occur as ARRECTOR PILI, TUNICA DARTOS AND IN THE AREOLA OF NIPPLES. Smooth muscles lack striations and contain a central nucleus with a cigar shaped nucleolus. The muscle fibres of the arrector pili arise in the connective tissue and get attached to the hair follicle at an obtuse angle. When they contract, the hair follicle is pulled into a vertical position. EM demonstrates myofilaments in the smooth muscle cells and the narrow spaces between the cells are occupied by collagen fibres and Schwann cells. They contain vimentin and desmin intermediate filaments.

Striated Muscle
The striate or voluntary muscles are present in the skin of the neck as PLATYSMA and the MUSCLES OF FACIAL EXPRESSION. These muscles originate in the fascia or periosteum and extend to the lower dermis through the SC tissue.

THE HYPODERMIS OR THE SUBCUTANEOUS TISSUE/FATTY LAYER

There is an abrupt transition from dermis to the Subcutaneous tissue. Both are functionally integrated via vessels, nerves and epidermal appendages. Actively growing hair follicles extend into the Subcutaneous fat, and the apocrine and eccrine sweat glands are normally confined to this depth of the skin.

The Subcutaneous fat consists of lobules composed of adipocytes separated by tin fibrous

septa through which nerves, vessels and lymphatics pass. Constructed of adipose and loose connective tissue. Offers protective layer against external abuse Adipocytes are the primary cells of the Subcutaneous tissue. It is a mesenchymally derived cell. It has a cytoplasm with membrane bound lipids that displace the nucleus eccentrically.

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 Provides insulation, shock absorption, energy storage, and the ability of skin to slide over It unction also as Anchors skin to underlying tissues and shook absorber and insulator.
joints. Contains the major blood vessels of the skin.

Lipids dissolve in routinely processed specimens giving the appearance of empty spaces within the cell. But lipids are visible in glutaraldehyde fixed plastic embedded specimens. Subcutaneous fat deposits begin to form in the midtrimester fetus and are already well developed in the newborn infant. The synthesis and storage of fat continues throughout life by enhanced accumulation of lipid within fat cells, proliferation of adipocytes and by recruitment of new cells from undifferentiated mesenchyme. The hormone LEPTIN secreted by adipocytes appears to provide feedback control regulating fat mass. Hypodermis contains of : Arrector Pili muscle, An obliquely arranged small bundle of smooth muscle attached to the lower portion of hair follicle.. If a person is emotionally upset or cold, nerve impulses may stimulate this muscle to contract, causing it to pull on a follicle, thereby decreasing its angle with the skin surface. This, in turn, generates goose bumps. Although this action does not play a significant role in humans, it does keep other mammals warm in cold weather by increasing the thickness of their insulation. Contraction of arrector pili muscles also is used for body language signals. For instance, a scared cat looks larger when its fur stands on end, and a dog sends a clear message that it should not be touched when it raises the hair on the back of its neck while baring its teeth.

ORIGIN AND DEVELOPMENT OF THE SKIN

The Prospective epidermis originating from two major embryological elemen is the gastrula 8th-11th week = a middle layer forms and a few microvlli appear on the surface of the
and the prospective mesoderm, leads to the development of the skin at about the third week of fetal life. 3rd week = epidermis is a single layer of glycogen filled cells 6th week = the epidermis consists of 2 layers, the epithelial layer and the germinative layer.

epidermis 12th-16th week = more intermediate layers and many microvilli are formed. The cells of the intermediate layers contain mitochondria, golgi complexes and tonofilaments within and around them. 16th- 26th week = the intermediate layers increase in number with keratohyalin granules being present in the upper layers by the 21st week. 24th week = cells are shed which along with lanugo and sebum constitute vernix caseosa. Most of the dermis develops from migrating mesenchymal cells and a small part from the ventrolateral part of the somite (dermatome). The dermis develops at about 4 weeks and is at first very cellular with collagen fibres detectable at about 4 weeks and elastic fibres at 22 weeks. Between 8-14 weeks, cells in the dermis also include stellate cells, phagocytes, melanoblasts and mast cells. From 14-22 weeks, fibroblasts, perineural cells, pericytes, merkel cells and mast cells can be demonstrated. SC tissue is primarily developed from mesenchymal cells starting at 14 weeks with lipoblasts appearing close to the developing blood vessels. These lipoblasts then develop into lipocytes.

SKIN COLOR
Two main factors contribute to skin color: the quantity and distribution of pigments
(melanin and carotene) in the skin, and blood flow.

Melanin is a skin pigment made of amino acids. It comes in two forms: yellow to red
(pheomelanin) and the more common brown to black (eumelanin).

Although melanin is only produced by melanocytes, it is continually released by exocytosis

from these cells. Surrounding cells subsequently accumulate the pigment by endocytosis. Interestingly, all people have roughly the same number of melanocytes. That means variations in skin color are due to differences in the form and amount of melanin produced, and in the way it is dispersed. predisposition: that is, the particular characteristics inherited from the parents. Also, melanocytes are stimulated by exposure of skin to sunlight, causing them to increase their production of melanin. This response helps protect DNA when there is an increased exposure to ultraviolet radiation. It also is responsible for the development of a tan. However, excessive exposure to sunlight causes clumping of elastic fibers, which leads to

The most important factor in determining melanin production is a persons genetic

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wrinkles and leathery-looking skin. More importantly, excessive exposure to ultraviolet light temporarily suppresses the immune system and also can alter DNA enough to cause skin cancer. The protective nature of melanin is illustrated by the observation that black people seldom have skin cancer, whereas this disease is much more common in fair-skinned individuals.

Freckles and moles are local accumulations of melanin. Albinism is an inherited disorder in which melanocytes are incapable of producing melanin. Carotene is a yellow-orange pigment that can influence skin color. It is found in many food
items, such as carrots, apricots, and oranges. It tends to accumulate in the stratum corneum and in fatty tissues of the dermis. Its color is most obvious in the palms of the hands and soles of the feet, and is most intense when a large amount of carotene-rich food has been consumed. lungs to body tissues. The pinkish hue of fair skin is due to the reddish color of oxygenated hemoglobin in blood circulating through dermal capillaries. Because Caucasian skin contains relatively small amounts of melanin, the almost transparent epidermis allows the color of underlying hemoglobin to show through. Specific circulation patterns of blood flow also can influence skin color. For instance, embarrassment increases blood flow to the skin, particularly in the face and neck regions. This is what leads to blushing. An increase in blood flow also can be caused by high blood pressure (hypertension), inflammation, or an allergic response. In contrast, a sudden fright or anger can cause a rapid drop in blood flow to the skin, causing its color to blanch. Pale skin also can indicate low blood pressure (hypotension), anemia (low red blood cell count), or impaired blood flow. In addition, hemoglobin changes its color when it releases oxygen. known as cyanosis. Melanin masks the appearance of cyanosis in darkskinned people. However, it can still be detected by looking at the color of fingernail beds. Cyanosis is common during heart failure or extreme breathing disorders. body tissues. Bilirubin is formed by the liver during the breakdown of worn-out or damaged red blood cells. Thus, jaundice usually indicates a problem with the liver. However, jaundice is common in newborns (called physiological jaundice), usually appearing two or three days after birth in over 50% of babies. This occurs because fetal red blood cells are short-lived, and break down rapidly following birth so they can be replaced with adult red blood cells. Frequently, an infants liver is unable to process the resulting bilirubin fast enough to prevent its accumulation in blood. Usually physiological jaundice in babies is not harmful and disappears by 1 to 2 weeks of age. In most cases, an increase in the supply of breast milk or formula is recommended. However, high levels of bilirubin can cause deafness or brain damage in some babies. These complications can be prevented by lowering bilirubin using phototherapy for a few days (blue light helps break down bilirubin in the skin).

Hemoglobin is a pigment found in red blood cells that is used to carry oxygen from the

Consequently, poorly oxygenated blood causes skin to take on a bluish hue, a condition

A yellow color (jaundice) occurs when bile pigments, such as bilirubin, are deposited in

SKIN APPENDAGES
The skin contains a variety of appendages:

1.1.1 Hair or Hair shaft

Hair is an outgrowth of skin that is unique to mammals. Its main function is to provide thermal insulation. In this capacity, however, the hairs scattered over the human body are essentially useless. Nonetheless, human hair does provide some important functions. For instance, it protects the scalp from ultraviolet rays and mechanical bumps. Eyelashes shield the eyes, and cause a reflex blinking when unexpectedly touched. Hair lining the respiratory tract and ear canals keep out foreign particles. In addition, hair has a significant sensory role because receptors associated with follicles are sensitive to touch. For humans, hair is present on all skin surfaces, except the palms of the hands, soles of the feet, lips, nipples, and parts of the external genitalia. Humans have three different types of hair: lanugo, vellus, and terminal. Lanugo is the soft, fine hair that covers a fetus beginning around the third or fourth month after conception. It falls off about a month before birth, and is replaced by a second coat that is shed a few months after birth. Vellus hair also is soft and fine. However, unlike lanugo, it grows and persists throughout life, covering most of the body surface. Terminal hair is thick and strong. It forms eyebrows and eyelashes, and is found on the scalp. During adolescence, in response to changing hormone levels, many vellus hairs of the armpits and pubic area are replaced with terminal hairs. In males, the same is true for the face, chest, legs, forearms, back, and shoulders. Each terminal hair consists of a central core called the medulla (fine hair lacks a medulla). The medulla is surrounded by the cortex, which in turn is enclosed by a cuticle. The cuticle is formed by a single layer of cells that overlap one another like shingles on a roof. This arrangement helps keep hairs from matting or

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tangling with each other. The cuticle can wear away with continual exposure to the elements and abrasion, allowing the underlying cortex to frizz, forming split ends. The cuticle also can be damaged by exposure to chlorinated water in swimming pools. Hair has both a shaft and a root. The shaft projects above the surface of the skin. In contrast, the root extends below the surface into the dermis, where it is embedded in a group of cells called a hair follicle. The follicle is really a compound structure, meaning it is composed of several parts. Its inner layer is a flexible sheath composed of epithelial cells that are responsible for producing hair. The outer layer is composed of dermal connective tissue. It provides blood vessels and physical reinforcement. The inferior end (bottom) of a hair follicle is enlarged, forming a structure called the hair bulb. Each hair bulb is wrapped by a knot of sensory nerve endings, the hair root plexus. This anatomical arrangement allows hair to act as a sensitive touch receptor permitting us, for instance, to feel insects crawling on our skin (hopefully before they have a chance to sting or bite us). Hair is formed by division of cells in the matrix, a growth zone located in the hair bulb. The matrix displays active mitosis because it is continuous with the stratum basale, the epidermal layer capable of cell division. The newly formed cells are nourished by blood vessels located in the papilla, which is an indentation of the dermal connective tissue at the base of the follicle. As daughter cells continue to divide, they are pushed farther away from the growing region and also become keratinized. Shortly thereafter they die. Thus, the bulk of hair is composed of non-living cells. Hair growth depends on several factors, including nutritional status, gender, and age, as well as circulating levels of some hormones. For instance, poor nutrition will result in poor hair growth. In addition, the hormone testosterone encourages growth of hair. Although scalp hair typically grows an average of 2 millimeters per week, each follicle goes through a series of growth cycles. Initially, there is an active phase, which generally lasts 2 to 6 years. This is followed by a resting phase, where the follicle is inactive for several months. Following the resting phase, the matrix proliferates again, forming a new hair that will replace the old one, which has already fallen out or will be pushed out. Because follicles generally spend more time in an active phase, we only shed about 90 hairs from our scalp each day. Interestingly, the active phase for eyebrow hair is only three to four months long. This explains why eyebrow hair is much shorter than scalp hair. Hair growth is generally fastest from the teen years to the forties. However, after that it slows down, and hairs are not replaced as fast as they are shed. This leads to hair thinning, which occurs in both sexes. However, true baldness, usually known as male pattern baldness, is a genetically determined condition influenced by the presence of male hormones. That is, true baldness is caused by a delayed action gene, which is turned on in adulthood, thereby changing the response of hair follicles to circulating levels of testosterone. As a result, hair follicles shrink, and the length of time spent in a growth cycle decreases. In fact, growth cycles can become so short, hair does not have a chance to emerge before it is shed. In addition, thick terminal hairs are replaced by soft, fine vellus hairs. This change occurs in a characteristic pattern, beginning at the forehead and temple, and eventually reaching the crown. Interestingly, a drug originally used to treat high blood pressure (minoxidil) was accidentally found to stimulate hair growth in some individuals. The lotion appears to work by increasing blood flow to the scalp, thereby stimulating the activity of existing follicles. Elongated keratinized structure derived from invagination of epidermal epithelium At the base of the hair follicle is a single layer of mitotic cells derived from the stratum basale. This is the hair matrix. All the cells of the hair are derived from the hair matrix. Just beneath the hair matrix is an obvious dermal papilla called the hair papilla. It contains the blood vessels that nourish the matrix and the cells of the hair follicle.

1.1.2 Hair Follicle

A tubular invagination of the epidermis which extends down into the dermis The hair follicle surrounds much of the hair root. It contains an outer connective tissue sheath and an inner epithelial root sheath. Wrapped around the bulb of the follicle is a network of sensory nerve endings known as the hair root plexus. Allow the hairs to serve a sensory function. Attached to each hair is a bundle of smooth muscle known as an arrector pili muscle. In times of fright or cold, these muscles contract and cause the hair to stand on end and produces goose bumps. Increases airflow in mammals with significant hair (i.e., not humans) and increases the apparent size of an animal with significant hair. Vestigial in humans Wrapped around the bulb of the follicle is a network of sensory nerve endings known as the hair root plexus. Allow the hairs to serve a sensory function. Attached to each hair is a bundle of smooth muscle known as an arrector pili muscle. In times of fright or cold, these muscles contract and cause the hair to stand on end and produces goose bumps. Increases airflow in mammals with significant hair (i.e., not humans) and increases the apparent size of an animal with significant hair.

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Structure of a hair and hair follicle. The region of a hair that projects from the skin is the shaft, whereas the embedded portion is called the root. The hair follicle extends from the epidermis into the dermis, and its deep end is expanded, forming a hair bulb (enlarged in the diagram). Hair is produced in the bulb by active cell division in a single layer of epidermal cells called the matrix, which is nourished by a knot of capillaries in the dermal papilla. The hair shaft has a central core, the medulla (consists of large cells and air spaces), which is surrounded by the cortex (several layers of flattened cells). The outermost cuticle is formed from a single layer of cells.

1.1.3 Cutaneous Glands

The cutaneous glands are all exocrine glands that secrete to the skin surface via ducts. There are two main types: sebaceous glands and sweat glands. Both reside almost entirely in the dermis, but are formed by cells of the stratum basale (an epidermal layer).

1.1.4 Oil or Sebaceous Glands

Oil glands or sebaceous glands are found all over the body, except the palms of the hands and soles of the feet. Although they are an epidermal derivative, the secretory part of the gland is located in the dermis. In some cases, the glands open directly onto the skin surface. However, in most instances they open into hair follicles.

Sebaceous glands secrete an oily substance called sebum, which is made of fats,

cholesterol, protein, and salts. Sebum is used to lubricate hair and skin. It also protects skin against desiccation. In addition, sebum contains anti-bacterial chemicals, which help prevent bacteria normally present on the skin surface from invading deeper regions. Unfortunately, the ducts of oil glands can become blocked, allowing sebum and bacteria to accumulate Connected to the hair follicle, compound of polygonal cells with clear cytoplasm and centrally located nucleus which are all embryologically epidermal in origin. Simple alveolar glands found everywhere except palms of the hands and soles of the feet. Secrete an oily, lipid-rich secretion called sebum. Lanolin is actually sheep sebum Sebum is typically secreted into a hair follicle or occasionally onto the body surface. Sebum softens and lubricates the skin. It also decreases the skins permeability to water and is quite bactericidal.

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Hair follicles with well developed sebaceous glands and their ducts.

1.1.5 Sweat Glands

Sweat glands or sudoriferous glands are widely distributed numoreous in the skin of the forehead, palms, and soles, except nipples lips, eardrums, nail beds and portions of the external genitalia. Each person has about 2.5 million cells. There are two main types: eccrine/mecorine and apocrine. a. ECRINE or MECORINE GLANDS

Produce their secretions in coiled structures located in the dermis, and then dump their

contents (sweat) directly on the skin surface via a pore. Simple, coiled, tubular glands, Duct bound by cuboidal cells empties into a funnel-shaped pore at skin surface. Major function of merocrine sweating is to cool the body thermoregulation. Merocrine sweat is a dilute watery solution of some salts (including NaCl), vitamin C, antibodies, small amounts of nitrogenous wastes (urea, uric acid, and ammonia), and lactic acid. pH of sweat is 4-6 creating a film on the body known as the acid mantle. Such an acidic environment is bacteriostatic prevents bacterial reproduction and growth. Sweat is mostly composed of water. It also contains some salts, lactic acid, vitamin C, and metabolic wastes, such as urea and ammonia. The principal function of sweat is to help regulate body temperature through the evaporation of water on the skin surface. In fact, on a hot day one can easily lose several liters of body water in this way. In addition, the slightly acidic pH of sweat inhibits growth of bacteria.

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b. APOCRINE GLANDS Apocrine glands are a type of sweat gland, mainly located in the armpits and pubic region. They are usually larger than eccrine glands, and empty their contents into hair follicles. Although their secretion contains all the substances present in eccrine sweat, they also contain additional fatty acids and proteins. These substances make the secretion more viscous (thick), and also gives it a whitish-yellowish color. Apocrine secretion is typically odorless. However, bacterial action on the skin surface converts its proteins and fats into compounds that release an unpleasant odor. In fact, antiperspirants are designed to inhibit such secretions, whereas deodorants mask their odor. Apocrine glands do not function until puberty, at which time they are stimulated by a rise in sex hormones (testosterone and estrogen). Although their exact function is not known, they generally become most active when a person is emotionally upset or excited, such as when frightened, in pain, or sexually aroused. Apocrine glands also enlarge and shrink with the phases of a womans menstrual cycle. It is therefore unlikely these glands play a significant role in temperature regulation. Instead, it is generally assumed that apocrine glands are analogous to the sexual scent glands of other animals, and they also may play a scent role during a fight or flight response. It has been suggested that pubic and axillary (under arm) hair help disperse the odor of apocrine secretions; that is, a way of enhancing the spread of ones scent. c. CERUMINOUS GLANDS Ceruminous glands are modified apocrine glands found in the lining of the external ear canal. They secrete a thick, sticky substance called cerumen or earwax. Along with tiny hairs in the ear canal, this substance deters insects and blocks the entry of foreign substances. Modified sweat glands specialized to secrete milk are called mammary glands.Although they are present in both genders, mammary glands normally only function in females. In nonpregnant women, the glandular structure is largely undeveloped and the duct system is rudimentary. However, under stimulation of the hormone prolactin, which is secreted from the pituitary gland during pregnancy, the glandular tissue develops the ability to form milk.

1.1.6 Nails
Nails are protective coverings on the ends of the fingers and toes. Like hair, nails are modified skin tissue (stratified squamous epithelium), which has been hardened by the protein keratin. However, nails differ from hair in that they grow continuously. Also, compared to hair, nail growth is relatively slow. Whereas hair can grow 5 to 6 inches per year, fingernails grow about 1.5 inches per year, and toenails about 0.5 inches. Nail cells form in a region called the nail root, which is embedded in skin. The growing region is the lunula, the whitish, crescent-moon-shaped area at the base of a nail. As a nail develops, it slides forward over a layer of epithelium, the nail bed, which is continuous with the stratum basale. The free edge of a nail extends over the tip of a finger or toe and is the part we trim. The border of a nail is overlapped with skin folds, and the proximal nail fold is called the cuticle. Most of a nail appears pink due to the influence of blood vessels in the dermis below. The lunula, however, looks white because it has a thickened matrix, which obscures underlying tissue.

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TISSUE ENGINEERING
Tissue engineering is the process of creating living, physiological 3D tissues and organs. Bringing together the fields of medicine, biology, engineering and biotechnology. Creation of a functional biological substitute using living cells and a matrix to maintain, improve or restore damage to tissues and organs (Atala,AEngineering tissues,organs-cells 2007 J Tissue Eng Regen Med 1:83-96) The process starts with a source of cells derived from a patient or from a donor. The cells may be immature cells, in the stem cell stage, or cells that are already capable of carrying out tissue functions; often, a mixture of different cell types (e.g., liver cells and blood vessel cells) and cell maturity levels is needed. Many therapeutic applications of tissue engineering involve disease processes that might be prevented or treated if better drugs were available or if the processes could be better understood .

APPROACHING TISSUE ENGINEERING

REPLACEMENT CELL
There are three main approaches to tissue engineering: To use isolated cells or cell substitutes as cellular replacement parts; To use acellular materials capable of inducing tissue regeneration; and To use a combination of cells and materials (typically in the form of scaffolds and this approach be categorized into two categories: Open and closed systems. These systems are distinguished based on the exposure of the cells to the immune system upon implantation.

MATERIAL REQUIREMENT
Material for Tissue Engineering have some requirement : Biocompatible Made with the patients own cells Should not elicit immune or inflammatory response Engineered to fill the exact role required Degradation rate Composition Size Mechanical properties

Off-the-shelf availability Functional Adequate mechanical and hemodynamic function, Mature ECM, Durability Living Growth and remodeling capabilities of the construct should mimic the native structure

DELIVERY METHODE
a. Injectable stem cells Cells or cell-polymer mix Less invasive

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 Adopt shape of environment Controlled growth factor release b. Solid scaffold manufacturing Computer-aided design Match defect shape

CLASSIFICATION TRANSPLATION/DELIVER METHODE

The need for Tissue Engineering are for Transplation of Failing tissues and organs and Additing tissue in the healing process. In the Laboratory, Tissue Engineering are needed to observe immunological, pathological and healing changes in human tissue without harming patiens. Drug therapies is one of the reason usage of Tissue Engineering to efficacy and side effects observation of drugs. Shortfalls of current options classification transpalation are

1.1

Autograft / Autolog
A tissue transferred from one part of the body to another in the same individual. The ideal option are Biocompatible. Due to genetic homology of the tissue, the immune system does not respoint to it and it run
as Natural.

Decreasing availability of healthy tissue from patients with disease.

Vascular small diameter vessels

1.2

Homograft / Allograft / Allogeneic

A tissue transferred from a genetically different individual of the same species, from human to human or from cadaver to human As there are more and more people every year waiting for donor organs and tissues, allografting transplantation has become quite common. Allografting transplantation has many applications. More practical than autologous harvest Severe shortage of donors Increased immune response to foreign material Immunosuppressant drugs required (Unpleasant side effects, Expensive)

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1.3

Xenograft
Potentially readily available Immune response from host to foreign material Risk of disease transmission from animal to human e.g. prions and PERVs Significant ethical considerations

A graft transferred from an individual of one species to an individual of another species, such as from animals

1.4

Synthetic Materials
The materials used for tissue engineering are either synthetic biodegradable materials such as polylactic acid (PLA), polyglycolic acid (PGA), poly lactic-glycolic acid (PLGA), polypropylene fumarate, poly ethylene glycol (PEG) and polyarylates) or

Natural materials such as collagen, hydroxyapatite, calcium carbonate, and alginate. Natural materials are typically more favorable to cell adherence, whereas the properties of
synthetic materials such as degradation rate, mechanical properties, structure, and porosity can be better controlled Expansion of a population ex vivo prior to transplantation into the host, Ex vivo recreation of a tissue or organ for transplantation, and Current approaches for tissue engineering using tissue (postnatal) stem cells:

Design of substances and/or devices for in vivo activation of stem cells, either local or
distant, to induce appropriate tissue repair

MODELS for TISSUE ENGINEERING

In Vitro Differentiation
o Construct tissues outside body before transplantation Ultimate goal are Most economical and Least waiting time Host remodeling of environment

o
o

In Situ Methodology Ex Vivo Approach

Excision and remodeling in culture

VARIABLES NEEDED
1.6.1 Delivery
a. Tissue-like contructs / A scaffold

The scaffold refers to the tissue model construct to allow growth and differentiation of cells
Structural integrity to support cell attachment, growth and differentiation Correct pore size Mechanical strength to withstand in vivo compression Can be natural, synthetic or combined

A sheet of small intestinal submucosal scaffold . http: //www.rcsed.ac.uk/ journal/ Biomimetic Scaffold Fabrication. bms.dent.umich.edu/research/malab.html

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b. Cells of the relevant type and number Able to cause change and affect structure and function of a graft

Stem cells much potential but difficult to direct differentiation and achieve sufficient cell
numbers Examples: Skin model: fibroblasts and keratinocytes Vascular construct: smooth muscle and endothelial cells Cartilage: chondrocytes Ideally autologous Difficulties in isolating cells from diseased tissue Stem cells o Adult o Embryonic Immunoprivileged Potentially could differentiate into a wide variety of cell types Potentially teratogenic/carcinogenic Ethically, legally and financially questionable

Cell Sourcing

1.6.2 Chemical Property

Optimum conditions for cell growth/Signal a. Growth factors, Directing cellular activity b. Degradation particles c. ECM surface

1.6.3 Physical Property

a. Bioreactors
Designed to expose cells to physical stimuli and/or maintain desired conditioin example Bladder urothelium.

b. Structure c. Topography d. Rigidity

e. Mechanical Loading

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PROBLEMS OF TRANSPLANTATION
There are not enough organs patients in industrially developed countries badly need donor organs and tissues. People die because of the lack of available organs for transplant each day. In immune system rejection, Often a transplanted organ is not identified by the immune system as the tissue of the organism or detect foreign graft tissue . It can be attacked and destroyed resulting in tissue rejection. . Against this effect the patient has to swallow Immunesuppressiva which cause symptoms like suffering from AIDS. In 15-20 minutes the organ dies, unable to withstand the immune system attack.

THE FUTURE OF TISSUE ENGINEERING

SIDE OF IMPROVEMENT
Some tissues already in clinical use Improvements needed to increase availability and safety For widespread use, reduced cost is essential Further work should focus on:

Vascularisation of new tissue; maintaining nutrient supply to cells in matrix with

increasing size Achieving full potential of stem cells to differentiate into desired cell types

CHALLENGES
Unforeseen hurdles in the creation of multicellular constructs Creation of collagen matrices Containing fibroblasts Seeded with endothelial cells Incorporation of fibrin Novel imaging techniques of tissue-engineered constructs

Challenges occur at the most basic level In the laboratory Supply of nutrients
o o o Removal of waste Size Mechanical stability

In the patient o Availability o o o Evidence of efficacy Safety; graft rejection Cost

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APPLICATIONS
Autologous de novo cartilage formed on Skelite tissue engineering scaffold (grown in vitro), illustrating the configuration of the implant that provides functional cartilage tissue at the articular surface. The presence of functional cartilage tissue represents a major advance over current cell therapy techniques. Cell therapy involves the implantation of cells that still have to make new cartilage in vivo at the defect site under very challenging conditions. The histology image on the right shows that cells are healthy and growing, while attaching themselves to the Skelite and beginning to differentiate into mature cartilage.

www.millenium-biologix.com/Html/00_ScientificInformationCartiGraft.htm Foetal lamb tracheal defects

Fuchs et al. Fetal tracheal augmentation with cartilage engineered from bone marrowderived mesenchymal progenitor cells. (2003) J Pediatr Surg 38: 984987

Heart valves - decellularised

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Construction of engineered bladder. Scaffold seeded with cells (A) and engineered bladder anastamosed to native bladder with running 40 polyglycolic sutures (B). Implant covered with fibrin glue and omentum (C). Atala et al. Tissue-engineered autologous bladders for patients needing cystoplasty. (2006) Lancet. 367(9518):1241

Kidney

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Lanza et. al. Generation of histocompatible tissues using nuclear transplantation(2002)Nat Biotechnol. 20(7):689-696.

Decellularised Porcine Ureter Mr Chris Derham

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WOUND HEALING AND REPAIR

Wound Definition : -Interruption of continuity of tissue resulting from a certain injury especially external physical trauma -A disruption of normal anatomic relations as a result of injury intentional or unintentional. Wound Healing is a natural spontaneous response for restoration of tissue continuity after injury. Healing is the interaction of a complex cascade of cellular events that generates Reconstitution and Resurfacing, Restoration of the tensile strength of injured skin. Sometimes, tissue has been disrupted so severely that it cannot heal naturally. Regardless of causation or tissue type, wound healing presents with identical biochemical and physiologic processes, though wound healing may vary in timing and intensity. WOUND HEALING TERM Repair Vs Regeneration (speed Vs accuracy) Acute (orderly and timely) Vs Chronic (stalled in inflammatory phase)

HISTORY
The earliest accounts of wound healing date back to about 2000 B.C

Galen of Pergamum emphasized the importance of maintaining a moist environment to

ensure adequate healing. Ambriose Par found that simply dressed gunshot wounds heal faster and are less painful than when treated with boiling oil, the previously accepted method. Ignaz Philipp Semmelweis advocated need for washing hands Joseph Lister began soaking his instruments in phenol and spraying the operating rooms, reducing the mortality rates from 50 to 15%.

INTRODUCTION
Wound healing is the effort of tissues to restore normal function and structure after injury with purpose: -to reform barriers to fluid loss and infection, -limit further entry of foreign organisms and material, -re-establish normal blood and lymphatic flow patterns, -restore the mechanical integrity of the injured system

The repair of tissue damage broadly separated into two processes, regeneration and
healing Regeneration refers to growth of cells and tissues to replace lost structures.

TYPE OF WOUND
CLOSED WOUND
Closed Wound is occurred when skin surface intact without loss of skin a. Abrasions is also called scrapes, they occur when the skin is rubbed away by friction against another rough surface (e.g. rope burns and skinned knees).

b. Avulsions is occurred when an entire structure or part of it is forcibly pulled away, such as
the loss of a permanent tooth or an ear lobe. Animal bites may cause avulsions.

c. Contusions are also called bruises and are the result of a forceful trauma that injures an
internal structure without breaking the skin. Blows to the chest, abdomen, or head with a blunt instrument (e.g. a football or a fist) can cause contusions.

OPENED WOUND
Opened Wound is occurred when skin surface interrupted or loss of skin.

a. Crush wound is occurred when a heavy object falls onto a person, splitting the skin,
shattering or tearing underlying structures.

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b. Cuts is slicing wounds made with a sharp instrument, leaving even edges. They may be as
minimal as a paper cut or as significant as a surgical incision.

c. Fish-hook wound is an injury caused by a fish-hook becoming embedded in soft tissue. d. Incised wound is any sharp cut in which the tissues are not severed; a clean cut caused by
a keen cutting instrument. The wound may either be aseptic or infected, depending on the circumstances.

e. Lacerations. is also called tears, these are separating wounds that produce ragged edges.
They are produced by a tremendous force against the body, either from an internal source as in childbirth, or from an external source like a punch.

f. Penetrating wound
broken glass.

is a wound in which the skin is broken and the agent causing wound enters subcutaneous tissue or a deep lying structure or cavity, e.g. nails, splinters, spikes etc.

g. Punctures is a deep, narrow wounds produced by sharp objects such as nails, knives, and

REGENERATION PROCESS
Regeneration means proliferation of the parenchymal cells resulting in complete restoration of the original tissues. It requires cell proliferation which is largely regulated by micro environment that can either stimulate or inhibit cell growth. Regeneration is restitution of lost tissue which are: Tissue with high proliferative capacity = labile tissue (e.g hematopoietic cells, epithelial cells of skin and gastrointestinal tract regenerate from stem cells) Quiescent tissues = stable tissue , which normally have low levels of replication, however can undergo rapid cell division when stimulate (e.g pancreas, kidney, parenchymal cells of liver, ; mesenchymal cells as lymphocytes, fibroblasts, smooth muscle , endothelial cells) Regeneration requires : Presence of stem cells for renewal or tissue cells that are capable to divide in response to growth factors Intact tissue scaffold Most of the processes that are referred to as regeneration in mammalian organs are actually compensatory growth processes that involve cell hypertrophy and hyperplasia (e.g liver regeneration)

BODY CELLS
Cell proliferation depends on the cell growth cycle which consists of 4 unequal phases: M (mitotic) phase G1 (pre synthetic) phase S (DNA synthesis) phase

G2 (pre mitotic) phase Quiescent or resting cells will be in a physiologic state called G0. Cells of the body are usually classified into three groups depending on their capacity for regeneration:

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Labile Cells
Also called continuously dividing cells Follow the cell cycle from one mitosis to other Continue to proliferate throughout life, replacing tissues that are continuously destroyed Can easily regenerate after injury Contain a pool of stem cells

These include: surface epithelial cells of epidermis, alimentary tract, respiratory tract,

cervix,urinary tract, vagina,uterine endometrium,haematopoietic cells of bone marrow and cells of lymph node and spleen.

Stable Cells
Also called quiescent cells Limited ability to proliferate and to regenerate (except liver) Normally in G0, but can proliferate if injured These cells lose or decrease their capacity to proliferate after adoloscene but retain the capacity to multiply in response to stimuli throughout adult life. These include: parenchymal cells of organs like liver, pancreas, kidney, adrenal and thyroid; mesenchymal cells of smooth muscle cells ,fibroblasts,vascular endothelium,bone and cartilage cells.

Permanent Cells
Also called non dividing cells. Have left the cell cycle and cannot undergo mitotic division in post natal life (cells can not proliferate and regenerate). The injury always lead to scar. These include: neurons of nervous system, skeletal muscle and cardiac muscle cells.

Regeneration occurs all the time in labile tissues where cells are constantly being lost and replaced. If demand increases, supply increases easily. Regeneration occurs in limited form in stable tissues such as remove one kidney: the other one undergoes hypertrophy and hyperplasia. Remove half of the liver: it will grow back. Regeneration only occurs if residual tissue is intact.

REPAIR PROCESS
If injury is severe, regeneration cant happen. So, fibrosis (a scar) replaces the injured tissue by fibrous tissue. The Processes involved in repair are Granulation tissue formation and Contraction of wounds.

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Four components to this process: o New vessel formation (angiogenesis) o o o Fibroblast proliferation Synthesis of collagen (scar formation) Remodeling of scar

By 24 hours: Endothelial cells start proliferating and Fibroblasts emigrate By 3-5 days: Granulation tissue present Weeks later: Dense fibrosis (scar) and scar is remodeled over time. Repair may restore original structures but results in collagen deposition and scar formation in tissue where scaffold is disrupted or damage occurs in non dividing = permanent tissue (e.g central nervous system, skeletal and cardiac muscle).

PHASES OF WOUND HEALING

Normal wound healing follows a predictable pattern that can be divided into overlapping phases defined by characteristic cellular populations and biochemical activities, which are : Hemostasis & inflammation Proliferation Maturation and Remodeling

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HEMOSTATIS AND INFLAMMATION PHASE

In the inflammatory phase clotting takes place in order to obtain hemostasis, or stop blood loss, and various factors are released to attract cells that phagocytise debris, bacteria, and damaged tissue and release factors that initiate the proliferative phase of wound healing. The Aim of this phase is Translation of Mechanical injury into biochemical signals. Sign of Inflammation

Vasodilatation (more persistent) Increase capillary engorgement ,Increase the capillary permeability, and blood flow under effect of histamine and bradykinin, serotonin, prostaglandins from platelets and mast cells flow of the necessary inflammatory cells and factors that fight infection and deriding the wound. This period is the event responsible for the erythema, edema, and heat observed after tissue injury

Alterations in pH (secondary to tissue and bacterial degradation), The increase fluid tension in the area causes swelling proses and further press of the nerve endings, and tissue hypoxemia at the injury site contribute to the sensation of wound pain. The events can be divided into Vascular events and Cellular events. This starts by:

Changes the charge on the surface of collagen molecule. Platelets aggregation and extravasated plasma contact with the extravascular tissue proteins leads to activation of Hageman's factors (factor XII) and platelets. Substrate or reactive phase, immediate typically days 1-10

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Response to limit and prevent further injury, inflammation, hemostasis, sealing surface, removing necrotic tissue and debris, migration of cells into wound by chemotaxis, cytokines, and growth factors Initial intense local vasoconstriction of arterioles and capillaries followed by vasodilation and vascular permeability A clot forms stop bleeding , continue with Vasodilatation of WBCs Cells of inflammation defending and debridment of injured tissue Defending is migration of inflammatory cells & Chemoattraction to WBCs

PNL & Lymphocytes invade the wound and fibrin network within 3 hours

defending and lysis with their lysosomes. release inflammatory mediators and bactericidal oxygen-free radicals. Lymphocytes also play a role in cellular immunity and antibody production. O2 is essential for the optimistic results of this defending process

Vascular Events
Earliest manifestation is vasodilatation, it follows a transient constriction of arterioles lasting a few seconds Vascular reaction start with activation of clotting factors cascade, continue to Platelet aggregation and Clot formation (The scab) which temporarily closes the wound consists mainly of fibrin mesh trapped other blood cells, and hemostasis. Temporary constricting of small blood vessels (few minutes) leading to temporary blanching. Activation of complement system, a chemotaxis, causes degranulation of mast cells and cytolysis. Platelets then accumulate and release alpha granules that containing Vasoactive agents, chemotactic factors, and growth factors. Alpha granules (growth factors) initiator proliferative phase by activating the local mesenchymal and epidermal cells. Alpha granules (growth factors) initiator proliferative phase by activating the local mesenchymal and epidermal cells. Wounding disrupts tissue integrity and direct exposure of extracellular matrix to platelets Initial contact between platelets and collagen requires the von Willebrand factor (vWF) Binding results in changes in platelet conformation, triggering intracellular signal transduction pathways that result in platelet activation and the release of biologically active proteins. Platelet granules are storage organelles that contain

Platelet-derived growth factor (PDGF),, chemoattractant

Epidermal growth factor (EGF),

Transforming growth factor-(TGF-), a key component tissue repair Transforming growth factor-(TGF-)
Insulin-like growth factor (IGF)-1, Vascular endothelial growth factor (VEGF) Fibronectin, Fibrinogen, Thrombospondin, vWF.

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 The dense granules/ bodies contain the vasoactive amines, such as adenosine, calcium, and

serotonin, which cause vasodilation and increased vascular permeability. Other factors released: TXA, Platelet activate factor, Transform. growth factor alpha, Fibroblast growth factor, Beta lysin (antimicrobial), PGE2 and PGI2 (vasodilate) and PGF2 (vasoconstrict). The clotting cascade is initiated through both the intrinsic and the extrinsic pathways. Vasodilation is followed by increased permeability of microvasculature, followed by stasis, which leads to accumulation of leucocytes along vascular endothelium which then migrate through vascular wall into interstitial tissue. Increased permeability is due to formation of endothelial gaps in venules, direct endothelial injury, delayed prolonged leakage, leucocyte mediated endothelial injury, increased transcytosis and leakage from new vessels The combination of intense vasodilation and increased vascular permeability leads to clinical findings of inflammation are rubor (redness), tumor (swelling),calor (heat), and dolor (pain).

Platelets Successful hemostasis is dependent on platelet adhesion and aggregation. Platelets first adhere to interstitial connective tissue, then aggregate. In the process of aggregation, platelets release many mediators, including ADP, and express several clotting factors on their membrane surface. Together these platelet products facilitate coagulation and further platelet activation. When activated platelets discharge their=granules, several adhesive proteins, including fibrinogen, fibronectin, thrombospondin, and von Willebrand factor VIII, are released. The first three act as ligands for platelet aggregation whereas von Willebrand factor VIII mediates platelet adhesion to fibrillar collagens and their subsequent activation (Ruggeri, 1993). Platelet adhesion to all four adhesive proteins is mediated through the platelet glycoprotein (GPIIb/IIIa; integrin _IIb_3) surface receptor (Ginsberg et al., 1992). Platelet fibrinogen, once converted to fibrin by thrombin, adds to the fibrin clot. In addition, platelets release chemotactic factors for blood leukocytes (Weksler, 1992), and growth factors such as platelet-derived growth factor (PDGF) (Heldin, 1992), transforming growth factor=(TGF-) (Nanney and King, 1996), and TGF- (Roberts and Sporn, 1996), which promote new tissuegeneration.

Cellular Events
a. Polymorphonuclear cells (PMNs) or Neutrophils

First infiltrating cells to enter the wound site, peaking at 24 to 48 hours

Neutrophil migration is stimulated by Increase vascular permeability, Release local prostaglandin and

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 The presence of chemotactic substances, such as complement factors, interleukin-1 (IL

1), tumor necrosis factor alpha (TNF-), TGF , platelet factor 4, or bacterial products. Chemotaxins attract after extravasation. Migrate through the ECM by transient interaction with integrins PMNs scavenge, present antigens, provide cytotoxicity-free radicals (H2O2), Migration PMNs stops with wound contamination control usually a few days. Persistant contaminant continuous influx PMNs and tissue destruction, necrosis, abscess, & systemic infection Neutrophils are known to produce VEGF (Vascular Endothelial Growth Factor) PMNs are also a major source of cytokines early during inflammation, especially TNF-, also release proteases such as collagenases. Following functional activation neutrophils scavenge necrotic debris, foreign material, and bacteria. Stimulated neutrophils generate free oxygen radicals with electrons donated by the reduced form of nicotinamide adenine dinucleotide phosphate, (NADPH). The electrons are transported across the membrane into lysosomes where superoxide anion (O2-) is formed. This very potent free radical is bactericidal, but it is also toxic to neutrophils and surrounding viable tissues. Migration of PMNs stops when wound contamination has been controlled, usually within the first few days after injury. PMNs do not survive longer than 24 hours. If wound contamination persists or secondary infection occurs, continuous activation of the complement system and other pathways provides a steady supply of chemotactic factors, resulting in a sustained influx of PMNs into the wound. PMNs are not essential to wound healing because their role in phagocytosis and antimicrobial defense may be taken over by macrophages. Sterile incisions will heal normally without the presence of PMNs.

b. Macrophages Second population of inflammatory cells that invades the wound. Macrophage is the one cell that is truly central to wound healing, serving to orchestrate the release of cytokines and stimulate many of the subsequent processes of wound healing. Proinflammatory cytokines are considered to be wound healing mediators. Derived from circulating monocytes, achieve significant numbers in the wound by 48 to 96 hours post injury and remain present until wound healing is complete. Macrophages (monocytes) enter the wound from the 2nd after wounding and present until the reparative process is complete. Along time macrophages continue phagocytose & cleaning the wound site of bacteria, debris, F.B and necrotic matter, producing the activation growth and chemotactic factors similar to those of platelets (complete the function of platelets). Monocytes migrate & activate are Macrophages. This migrate similar to PMNs secrete enzyme to degrade and alter ECM instigate fibroblast. Fibronectin attract more phages too. Chemotactic factors specific for monocytes include bacterial products, fibronectin, complement degradation products (C5a), collagen, thrombin, TGF-

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 Macrophages induce PMN apoptosis

Macrophages have specific receptors for IgG (Fc-receptor), C3b (CR1 and CR3), and fibronectin (integrin receptors), which permit surface recognition of opsonized pathogens and facilitate phagocytosis. Activated wound macrophages also produce nitric oxide which has antimicrobial properties.

Phospholipase is induced, causing enzymatic degradation of the cell membrane

phospholipids, releasing thromboxane A2 and prostaglandin F2. Releases leukotriene B4, a potent neutrophil chemo attractant, and C4 and 15- and 5 hydroxyeicosatetraenoic acid. MMP-9) which degrade the ECM and are crucial for removing foreign material, promoting cell movement through tissue spaces, and regulating ECM turnover. Growth factors that stimulate fibroblast, endothelial cell, and keratinocyte proliferation (PDGF, EGF, TGF- and TGF- in addition to insulin-like growth factor (IGF) and fibronectin (scaffold/anchor for fibroblasts) and activate Fibroblasts, endothelial and epithelial cells to form Gran.

Release proteinases, including matrix metalloproteinases (MMP-1,MMP-2, MMP-3, and Release of enzymes (collagenase, elestase), PGEs, cytokines (IL-1, TNF alpha, IFN),

c. T Lymphocytes Another population of inflammatory/immune cells that routinely invades the wound.

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 Less numerous than macrophages, numbers peak at about 1 week post injury Bridge the transition from the inflammatory to the proliferative phase of healing

Depletion of most wound T lymphocytes decreases wound strength and collagen content

Also exert a down regulating effect on fibroblast collagen synthesis by cell-associated interferon-, TNF-, and IL-1.

PROLIFERATION PHASE
Second phase of wound healing and roughly spans days 4 through 21 It is during this phase
that tissue continuity is re-established. Fibroblasts and endothelial cells are the last cell populations to infiltrate the healing wound, and the Fibroblasts are secrete or stimulated by strongest chemotactic factor/growth factor (FGF) and PDGF to invade the wound site and will produce extracellular matrix (ECM) components such as collagen, glycosaminoglycancs, alstin, to generate the granualiton tissue. Growth factors Tgf beta, PDGF & cytokines IL1, TNF alpha as well as thrombin, elastin,leukotriene b4, complement trigger fibroblast formation and migration from wound edge (do not arrive by diapedesis) FGF with the VEGF secreted by platelets and neutrophils, act as an angiogenic factor to stimulate or activation endothelial cell proliferation, that line blood vessel walls, and migration and thus promote vascularization at the healing site. Recruited fibroblasts first need to proliferate, and then become activated, to carry out their primary function of matrix synthesis remodeling. The proliferative phase is also called the reconstruction phase. The events in this stage are subdivide into: Angiogenesis Fibroplasia and granulation tissue formation Epithelization Contraction

Angiogenesis (Vascular and Lymphatic Proliferation)

Also called neovascularization, the process of angiogenesis occurs concurrently with fibroblast proliferation when endothelial cells migrate to the area of the wound. Because the activity of fibroblasts and epithelial cells requires oxygen and nutrients, angiogenesis is imperative for other stages in wound healing, like epidermal and fibroblast migration. The tissue in which angiogenesis has occurred typically looks red (is erythematous) due to the presence of capillaries. Stem cells of endothelial cells, originating from parts of uninjured blood vessels, develop pseudopodia and push through the ECM into the wound site to establish new blood vessels.

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 Endothelial cells are attracted to the wound area by fibronectin found on the fibrin scab and chemotactically by angiogenic factors released by other cells, e.g. from macrophages and platelets when in a low-oxygen environment. Endothelial growth and proliferation is also directly stimulated by hypoxia, and presence of lactic acid in the wound. To migrate, endothelial cells need collagenases and plasminogen activator to degrade the clot and part of the ECM. Zinc-dependent metalloproteinases digest basement membrane and ECM to allow cell migration, proliferation and angiogenesis When macrophages and other growth factor-producing cells are no longer in a hypoxic, lactic acid-filled environment, they stop producing angiogenic factors. Thus, when tissue is adequately perfused, migration and proliferation of endothelial cells is reduced. Eventually blood vessels that are no longer needed die by apoptosis. The macrophage growth factors stimulates angiogenesis . New capillaries bud from endothelial cells in capillary near the wound edges appear, proliferation occure and a new network of capillaries is formed inside the granulation tissues causing red granulations. Angiogenesis indicate growth of new blood vessels. Angiogenesis occurs in the healthy body for healing wounds and for restoring blood flow after tissue injury. Healthy angiogenesis is tightly controlled by a serious of on and off switches (Angiogenic growth factors versus angiogenesis inhibitors). In many serious diseases the body loses control over angiogenesis and angiogenesis-related diseases occur when new blood vessels grow excessively or insufficiently. Angiogenesis / Neovascularization is critical to chronic inflammation and fibrosis,tumor growth and vascularization of ischemic tissue. VEGF and Angiopoietins are the most important angiogenic factors

Fibroplasia and granulation tissue formation

Fibroblast is a critical component of granulation tissue. Fibroplasia begins from surrounding
mesenchymal cells 3-5 days after injury and may last as long as 14 days. responsible for the production of collagen, elastin, ground substance.

Fibroblasts migrate and proliferate in response to platelets growth factors. Fibroblasts are Simultaneously with angiogenesis, fibroblasts begin accumulating in the wound site.
Fibroblasts begin entering the wound site two to five days after wounding as the inflammatory phase is ending, and their numbers peak at one to two weeks postwounding.By the end of the first week, fibroblasts are the main cells in the wound. Fibroplasia ends two to four weeks after wounding. This steps includes Inflammatory cells, Fibroblasts and collagen, ground substance (Hyaluronic acid/GAG - glycosaminoglycan) and Vascular and lymphatic proliferation (Capillary ingrowth), and Macrophages. In the first two or three days after injury, fibroblasts mainly proliferate and migrate, while later, they are the main cells that lay down the collagen matrix in the wound site. Fibroblasts from normal tissue migrate into the wound area from its margins. Initially fibroblasts use the fibrin scab formed in the inflammatory phase to migrate across, adhering to fibronectin. Fibroblasts then deposit ground substance into the wound bed, and later collagen, which they can adhere to for migration

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 Granulation tissue functions as rudimentary tissue, and begins to appear in the wound already during the inflammatory phase, two to five days post wounding, and continues growing until the wound bed is covered. Granulation tissue consists of new blood vessels, fibroblasts, inflammatory cells, endothelial cells, myofibroblasts, and the components of a new, provisional extracellular matrix (ECM). The presence of fibroblasts in dermal substitutes helps wound healing by providing factors of the FGF family by the secretion of the ECM responsible for its mechanical properties and by the structuring effect they have on the formation of capillaries during angiogenesis. Growth factors (PDGF, TGF-) and fibronectin encourage proliferation, migration to the wound bed, and production of ECM molecules by fibroblasts. Fibroblasts also secrete growth factors that attract epithelial cells to the wound site. Hypoxia also contributes to fibroblast proliferation and excretion of growth factors, though too little oxygen will inhibit their growth and deposition of ECM components, and can lead to excessive, fibrotic scarring.

Extracellular Matrix
The wound is strengthened by proliferation of fibroblasts and myofibroblasts which get structural support from the extracellular matrix (ECM). The process in this matrix are :

Mast cell Histamine and platelet serotonin increases capillary vascular permeability Complement factors C5a and leukotreine B4 promote neutrophil chemoattraction As do IL1 and TNF alpha (from endothelial cells & macrophages) Increase chemotactic factors

and spillage of intravascular plasma into interstitial fluid aid diapedesis of neutrophils . IL1, TNF alpha proinflam mediators 1st enothelial then by macrophages Neutrophils release elastase and proteases, further vasc. dilation and permeability causes inflammation: rubor, tumor, calor, and dolor

ECM has five main components: collagen, adhesive glycoprotein, basement membrane, elastic fibres and proteoglycans. a. Collagen deposition

One of fibroblasts' most important duties is the production of collagen. Fibroblasts begin
secreting appreciable collagen by the second or third post-wounding day, and its deposition peaks at one to three weeks. Collagen production continues rapidly for two to four weeks, after which its destruction matches its production and so its growth levels off.

Collagen deposition is important because it increases the strength of the wound; before it is laid down, the only thing holding the wound closed is the fibrin-fibronectin clot, which does not provide much resistance to traumatic injury. Also, cells involved in inflammation, angiogenesis, and connective tissue construction attach to, grow and differentiate on the collagen matrix laid down by fibroblasts.

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 Collagen synthesis, as well as post translational modifications, highly dependent on systemic factors as: an adequate oxygen supply the presence of sufficient nutrients (amino acids and carbohydrates) cofactors (vitamins and trace metals) the local wound environment (vascular supply and lack of infection). Collagen synthesis, produce collagen fibers which is essential for bridging the wound gap , supporting the growing vessels and wound strength. The process of collagen synthesis start on the 3rd day, the peak reaches by the 5-7 days and may extend to 6 m to 1 year. This active metabolic process depends mainly on Vitamins: B, ascorbic acid, O2 , amino acids and Elements: zinc, iron, copper. And Collagen formation decreased by decrease of vit C. and steroids (high dose). In this phase the Protein starvation occurred. weak salt solution. It is laid down irregularly and haphazardly then polymerization occurs by cross linked to the collagen molecules. The Thick strong less soluble collagen [I] become more regular and perpendicular on the Proline and glycine triple stranded helical structure. Collagen fibrils secreted into ECM and bundled into collagen fibers. Synthesa on membran bound ribosomes enter E.Reticulum as proalpha chains, within lumen ER some proline and lysine hydroxylated [Alpha-ketoglutarate, vitamin C, oxygen, and iron REQ] forming a stable triple helix thru hydrogen bonds, proalpha chain combines with 2 others forming procollagen (Vit C def prevents hydroxylation) then procollagen secreted into ECM and cleaved into collagen :

Fibroblasts and Collagen III held together by weak electrostatic forces and is soluble in

o o o

Type III predominant collagen synthesis days 1-2 Type I days 3-4 Type III replaced by Type I in 3 weeks

b. Adhesive glycoprotein

Fibronectin is the best characterised glycoprotein in ECM and has binding properties to
other cells and ECM. It is of 2 types : Plasma fibronectin, synthesized by the liver cells and is trapped and is trapped in basement membrane such as in filtration through the renal glomerulus.

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 Tissue fibronectin, is formed by fibroblasts , endothelial cells and other mesenchymal cells. It is responsible for the primitive matrix in wound healing.

Tenascin or cytotactin is the glycoprotein associated with fibroblasts and appears in

wound after 48 hours of injury. It disappears from mature scar tissue.

Thrombospondin is mainly synthesised by granules of platelets. Function as adhesive

protein for keratinocytes and platelets but is inhibitory to attachment of fibroblasts and endothelial cells.

c. Basement membrane Provide the resilience to allow for recoil after transient stretch. Elastin is composed of hydrophobic and alanine and lysine-rich -helical segments that alternate along the polypeptide chain Elastic fibers consist of an elastin core covered with a sheath of microfibrils, which are composed of several distinct glycoproteins, such as fibrillin. Microfibrils appear before elastin in developing tissues and seem to form a scaffold on which the secreted elastin molecules are deposited. d. Elastic fibers Flexible, thin (40- to 120-nm thick) mats of specialized ECM that separate cells and epithelia from the underlying or surrounding connective tissue. In the skin, the basal lamina is tethered to the underlying connective tissue by specialized anchoring fibrils This composite of basal lamina and collagen is the basement membrane.

Most mature basal laminae contain type IV collagen and the glycoproteins laminin. The basal lamina serves numerous functions Provide tissue with the ability to recoil
Elastins are found in large vessels, uterus, skin and ligaments Fibrillins form a scaffolding for the deposition of elastins Marfan syndrome is an inherited autosomal dominant defect in fibrillin synthesis. Without the structural support provided by fibrillin, many tissues are weakened, which can have severe consequences, for example, ruptures in the walls of major arteries. As a molecular filter, preventing passage of macromolecules (i.e., in kidney glomerulus) As a selective barrier to certain cells (i.e., the lamina beneath the epithelium prevents fibroblasts from contacting epithelial cells, but does not stop macrophages or lymphocytes) As a scaffold for regenerating cells to migrate is important in tissue regeneration where the basal lamina survives.

e. Proteoglycans These are agroup of molecules having 2 components- an essential carbohydrate polymer (called glycosaminoglycan), and a protein bound to it, and hence the name proteoglycan. Various proteoglycans are distributed in different tissues as under: Chondrointin sulphate- abundant in cartilage , dermis Heperan sulphate- in basement membranes Dermatan sulphate- in dermis Keratan sulphate- in cartilage Hyaluronic acid- in cartilage , dermis In wound healing the deposition of proteoglycans precedes collagen laying. Proteoglycans (mucoproteins) are formed of glucosaminoglycans (GAGs) covalently attached to core proteins and are highly negatively charged.

Biophysical functions due to ability to fill space, bind and organize water molecules and

repel negatively charges molecules. They are ideal lubricating fluids in the joint due to high viscosity and low compressibility. Biochemical functions are mediated by specific binding of GAGs to other macromolecules e.g Antithrombin III (AT III) binds tightly to heparin and heparan sulfates and inactivates factor II, IXa and XIa thus controlling blood coagulation Proteoglycans (such as Syndecan) act as reservoirs for growth factors secreted into the ECM by binding the latter. Role of extracellular matrix in wound healing and scar formation

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Extracellular matrix (ECM) is formed by specific secreted macromolecules that form a network on which cells grow and migrate along ECM is secreted locally and forms a significant proportion of the tissue volume ECM o o o ECM o sequesters water that provides turgor to soft tissues and minerals that provides rigidity to skeletal muscles Forms a reservoir for growth factors proteins assemble into two general organizations Interstitial matrix (present between cells) o Basement membrane [BM] (produced by epithelial and mesenchymal cells and is closely associated with the cell surface) ECM is the network that surrounds cells o Two forms: interstitial matrix and basement membrane o Sequesters water and minerals o Gives cells a scaffold to adhere to Stores growth factors (Growth Factors is Very important in tissue repair. Actions: o stimulate cell division and proliferation o promote cell survival Bottom line: ECM regulates proliferation, movement, and differentiation of the cells living in it.

Epithelialization
The formation of granulation tissue in an open wound allows the reepithelialization phase to take place, as epithelial cells migrate across the new tissue to form a barrier between the wound and the environment. Basal keratinocytes from the wound edges and dermal appendages such as hair follicles, sweat glands and sebaceous (oil) glands are the main cells responsible for the epithelialization phase of wound healing. They advance in a sheet across the wound site and proliferate at its edges, ceasing movement when they meet in the middle.

Keratinocytes migrate without first proliferating. Migration can begin as early as a few hours
after wounding. Stats within hours by mitosis of the basal cell layer.

However, epithelial cells require viable tissue to migrate across, so if the wound is deep it

must first be filled with granulation tissue. Thus the time of onset of migration is variable and may occur about one day after wounding. Cells on the wound margins proliferate on the second and third day post-wounding in order to provide more cells for migration.

If the basement membrane is not breached, epithelial cells are replaced within three days by division and upward migration of cells in the stratum basale in the same fashion that occurs in uninjured skin. However, if membrane is ruined at the wound site, reepithelization must occur from the wound margins and from skin appendages such as hair follicles and sweat-oil glands that enter the dermis that are lined with viable keratinocytes. If the wound is very deep, skin appendages may also be ruined and migration can only occur from wound edges. Migration of keratinocytes over wound site is stimulated by lack of contact inhibition and by chemicals such as nitric oxide. Before they begin to migrate, cells must dissolve their desmosomes and hemi desmosomes, which normally anchor the cells by intermediate

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filaments in their cytoskeleton to other cells and to the ECM. Transmembrane receptor proteins called integrins, which are made of glycoproteins and normally anchor the cell to the basement membrane by its cytoskeleton, are released from the cell's intermediate filaments and relocate to actin filaments to serve as attachments to the ECM for pseudopodia during migration. Thus keratinocytes detach from the basement membrane and are able to enter the wound bed. Keratinocytes change shape, becoming longer and flatter and extending cellular processes like lamellipodia and wide processes that look like ruffles. Actin filaments and pseudopodia form. During migration, integrins on the pseudopod attach to the ECM, and the actin filaments in the projection pull the cell along. Molecules interaction in the ECM through integrins further promotes the formation of actin filaments, lamellipodia, and filopodia. Epithelial cells climb over one another in order to migrate. This growing sheet of epithelial cells is often called the epithelial tongue. The first cells to attach to the basement membrane form the stratum basale. These basal cells continue to migrate across the wound bed, and epithelial cells above them slide along as well. The more quickly this migration occurs, the less of a scar there will be.

Fibrin, collagen, and fibronectin in the ECM may further signal cells to divide and migrate. Like fibroblasts, migrating keratinocytes use the fibronectin cross-linked with fibrin that was deposited in inflammation as an attachment site to crawl across. As keratinocytes migrate, they move over granulation tissue but underneath the scab (if one was formed), separating it from the underlying tissue . Epithelial cells have the ability to phagocytize debris such as dead tissue and bacterial matter that would obstruct their path because they must dissolve any scab that forms. Keratinocyte migration is best enhanced by a moist environment, since a dry one leads to formation of a bigger-tougher scab. To make their way along the tissue, keratinocytes must dissolve the clot,debris& parts of the ECM in order to get through . They secrete plasminogen activator, which activates plasminogen, turning it into plasmin to dissolve the scab. Cells can only migrate over living tissue , so they must excrete collagenases and proteases like matrix metalloproteinases (MMPs) to dissolve damaged parts of the ECM in their way, particularly at the front of the migrating sheet . Keratinocytes also dissolve the basement membrane, using instead the new ECM laid down by fibroblasts to crawl across . As keratinocytes continue migrating, new epithelial cells must be formed at the wound edges to replace them and to provide more cells for the advancing sheet.

Proliferation behind migrating keratinocytes normally begins a few days after wounding and occurs at a rate that is 17 times higher in this stage of epithelialization than in normal tissues. Until the entire wound area is resurfaced, the only epithelial cells to proliferate are at the wound edges. Growth factors, stimulated by integrins and MMPs, cause cells to proliferate at the wound edges. Keratinocytes themselves also produce and secrete factors, including growth factors and basement membrane proteins, which aid both in epithelialization and in other phases of healing. Growth factors are also important for the innate immune defense of skin wounds by stimulation of the production of antimicrobial peptides in keratinocytes. Keratinocytes continue migrate across the wound bed until cells from either side meet in the middle, at which point contact inhibition causes them to stop migrating. When they have finished migrating, the keratinocytes secrete proteins that form new basement membrane.

Cells reverse morphological changes by underwent in order to begin migrating; they reestablish desmosomes and hemidesmosomes and become anchored once again to the basement membrane. Basal cells begin to divide and differentiate in the same manner as do in normal skin to re-establish the strata found in reepithelialized skin.

The epidermal cells advanced from the edges and creep across the wound surface in a favorable plane dissecting the wound between the living and dead tissue. Migration stops when it meets the opposite advanced epithelium. The new epithelium is thin non-pigmented. Incisional wounds are epithelized within 24-48 hours after injury (distance of less than 1 mm). This epithelial layer provides a seal between the underlying wound and the environment. In open wounds: if the wound is moist well oxygenated with viable moist surface and epithelization rapid (few days) and cell migrate over the surface of the wound.

However, The process is more slower if the wound dry. The cells burring under the eschar and slowly separating the mobile from the immobile tissue. This explain why the epithelial is more rapid in intact blister than after the blister has been debride and the base of the blister allowed to dry.

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In sutured wounds: epithelium may invade the lining of the suture tracks. It usually degenerate with early removal of sutures. However, prolonged sutures causing ugly punctuate scars. This may be avoided by adhesions taps for better cosmoses Epithelization as Physical Barrier with Growth Factors ((PDGF, TGF, and EGF) stimulate Mitosis of epithelial cells. Epiboly leapfrog like motion until contact inhibition reestablished . An early tensile strength for: blood vessel growth, epithelialization, protein (fibrin) aggregation, later collagen formation.

Contraction
Contraction is a key phase of wound healing. If contraction continues for too long, it can lead disfigurement and loss of function. Thus there is a great interest in understanding the biology of wound contraction, which can be modelled in vitro using the collagen gel contraction assay or the dermal equivalent model. Contraction commences average a week after wounding, when fibroblasts differentiated into myo-fibroblasts. In full thickness wounds, contraction peaks at 5 to 15 days post wounding. Contraction can last for several weeks and continues even after the wound is completely reepithelialized. A large wound can become 40 to 80% smaller after contraction. Wounds can contract at a speed of up to 0.75 mm/day, depending on how loose the tissue in the wounded area is. Contraction usually does not occur symmetrically (most wounds have an 'axis of contraction') which allows greater organization and alignment of cells with collagen. At first, contraction occurs without myofibroblast involvement. Later, fibroblasts stimulated by growth factors, differentiate into myofibroblasts. Myofibroblasts, which are similar to smooth muscle cells, are responsible for contraction. Myofibroblasts contain same kind of actin as found in smooth muscle cells.

The myofibroblast is considered to be the major cell responsible for contraction and is easily distinguished from normal fibroblasts by the presence of -smooth muscle actin (-SMA). SMA protein expression is undetectable until day 6 after wounding, after which it increases gradually for 15 days in the healing process and fades after 4 weeks [15] . Although wound contraction is a normal part of the healing process, it is desirable that it remains limited to avoid contractures. It has been reported that wound contraction and scar formation in an in vivo model could be prevented with a substitute dermal matrix seeded with skin cells, but not with unseeded similar matrices.

Myofibroblasts are attracted by fibronectin and growth factors and they move along fibronectin linked to fibrin in the provisional ECM in order to reach the wound edges . They form connections to the ECM at the wound edges, and attach to each other and to the wound edges by desmosomes. Also, at an adhesion called the fibronexus, actin in myofibroblast is linked across cell membrane to molecules in the extracellular matrix like fibronectin and collagen. Myofibroblasts have many such adhesions, which allow them to pull ECM when they contract, reducing the wound size. In this part of contraction, closure occurs more quickly than in the first, myofibroblast-independent part . As the actin in myofibroblasts contracts, the wound edges are pulled together. Fibroblasts lay down collagen to reinforce the wound as myofibroblasts contract The contraction stage

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in proliferation ends as myofibroblasts stop contracting and commit apoptosis. The breakdown of provisional matrix leads to a decrease in hyaluronic acid and an increase in chondroitin sulfate, which gradually triggers fibroblasts to stop migrating and proliferating. These events signal the onset of the maturation stage of wound healing. As summarize :

maturation myofibroblasts centripetal movement of wound edges contraction decrease wound size facilitates closure of a
Fibroblasts in the peripheral granulations defect. Lag period 2-3 days (with collagen synthesis) with Maximum rapid contraction 3-14 days (The maximal rate of contraction is 0.75 mm/d). It specially occurs at the back of the neck, trunk and face where the skin is loose. Contraction must be distinguished from contracture. Contraction is decreased by : x-ray, steroids, grafting with dermis, Burns. If prevented slow healing large fibrous tissue - ugly scar and cicatrisation & complications. Contraction: centripetal movement of the whole thickness of surrounding skin reducing scar. Myofibroblasts: special Fibroblasts express smooth muscle and bundles of actin connected through cellular fibronexus to ECM fibronectin, communicate via gap junctions to pull edges of the wound. Contracture: the physical constriction or limitation of function as the result of Contraction (scars across joints, mouth, eyelid).

MATURATION AND REMODELLING PHASE

The last step of normal acute wound healing is remodeling, also called maturation, during which the ECM components will be modified by the balanced mechanisms of proteolysis and new matrix secretion, and during which the wound becomes gradually less vascularized. The initial granulation tissue is weak but will gain in strength over time because of remodeling effects such as the gradual replacement of immature type III collagen by mature type I collagen. Wound contraction starts taking place around 6 days after injury or when the levels of collagen production and degradation equalize, the maturation phase of tissue repair is said to have begun. The maturation phase can last for a year or longer after the epithelialization phase, depending on the size of the wound and various factors that can affect the healing process such as it was initially closed or left open.

The phases of wound healing progress in a predictable, timely manner; if they do not, healing may progress inappropriately either a chronic wound such as a venous ulcer/pathological scarring such a keloid scar. Type I replaces Type III Collagen: net amount doesnt change after 6 weeks, organization & crosslinking. Decreased vascularity, less fibroblasts & hyaluronic acid. Peripheral nerves regenerate @ 1mm/day . Accelerated Wound Healing: reopening results in quicker healing 2nd time around. Collagen forms tight cross-links to other collagen and with protein molecules. Increasing the tensile strength of the scar. During maturation, type III collagen, which is prevalent during proliferation, is gradually degraded and the stronger type I collagen is laid down in its place.

Originally disorganized collagen fibers are rearranged, cross-linked, and aligned along tension lines. As the phase progresses, the tensile strength of the wound increases, with the strength approaching 50% that of normal tissue by three months after injury and ultimately becoming as much as 80% as strong as normal tissue. Since activity at the wound site is reduced, the scar loses its red appearance as blood vessels that are no longer needed are removed by apoptosis. This Phase includes: Devascularization Collagen remodeling Cicatrisation Devascularization is when the granulation tissue is gradually replaced by a scare tissue which is relatively acellular and avascular tissue and causing pale scare tissue. The extracellular tissue change its contents. Water is resorbed from the scar. Collagen remodeling during the maturation phase depends on continued collagen synthesis in the presence of collagen destruction under effect of collagenase. The ratio of the collagen type [I] increase. New collagen is formed in more orderly fashion along the lines of tension in the scare. Facilitating collagen fibers cross-linking and ultimately decreasing scar thickness and increasing wound bursting strength. From 4 to 12 weeks, a pale red thick strong scare tend to contract is formed. Excessive contracture of the scare tissue is named cicatrisation. [contracture] a pathologic process of excessive fibrosis that limits motion of the underlying tissues and is typically caused by the

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application of excessive stress to the wound. From 12 to 40 weeks, soft white scare tend to relax. Hyalinization, calcification and even ossification may sometimes occur. Closure of Wounds Primary: 1st intention immediately sealed with suturing, skin graft, flap closure (tensile strength) Secondary: Spontaneous involves no active intent to seal wound, gen. For highly contaminated wound, closes by reepithelialization and contraction of the wound (epithelial integrity) Tertiary: delayed primary closure of contaminated wound initially treated to control infection (repeated debridement, abx, wnd vac) then closed by suturing, skin graft, flap design, strip-strip etc.

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SUMMARIZE OF WOUND HEALING PHASE

Injury leads to accumulation of platelets and coagulation factors. Coagulation results in fibrin formation and release of PDGF and TGF-band other inflammatory mediators by activated platelets. This leads to more Neutrophil recruitment which signals the beginning of inflammation (24 h). After 48 h macrophages replace neutrophils. Neutrophils and macrophages are responsible for removal of cellular debris and release growth factors to reorganize the cellular matrix. At 72 hours the proliferation phase begins as recruited fibroblasts stimulated by FGF and TFG-b begin to synthesize collagen. Previously formed fibrin forms initial matrix for fibroblasts Collagen cross-linking and reorganization occurs following months after injury in the remodeling phase of repair. Wound contraction follows in large surface wounds and is facilitated by actin-containing fibroblasts (myofibroblasts)

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NATURE OF WOUND HEALING

Accomplished by one of the following two ways:

HEALING BY PRIMARY INTENTION

Healing by primary intention is the healing of wounds with characteristics clean and uninfected, surgically incised, without much loss of cells and tissue, and edges of wound are approximated by surgical sutures. The Sequence of events involved are : Initial Haemorrrhage is the events when a space between approximated surfaces of the incised wound is filled with blood which than clots and seals the wound against dehydration and infection.

Acute inflammatory response is even within 24 hours that appearance of polymorphs from the margins of incision. By the 3rd day polymorphs are replaced by macrophages. Epithelial changes is event when Basal cells of epidermis from both the cut margins start proliferating and migrating towards incisional space in the form of epithelial spurs. The migrated epithelial cells separate the underlying viable dermis from the overlying necrotic material and clot, forming scab which is cast off. Basal cells from the margins continue to proliferate and by the 5th day a multilayered new epidermis is formed. Organisation is even by the 3rd fibroblasts that also invade the wound area. By 5th day new collagen fibrils start forming which dominate till healing is completed. In 4 weeks, the scar tissue with scanty cellular and vascular elements, a few inflammatory cells and epithelialised surface is formed. Suture tracks is when each suture track is a separate wound and incites the same phenomenon as in healing of primary wound. When the sutures are removed around 7 th day, much of epithelialised suture track is avulsed and the remaining epithelial tissue in the track is absorbed. Summarize in First Intention healing are occuring in small wounds that close easily. Epithelial regeneration predominates over fibrosis. Healing is fast, with minimal scarring/infection such as Paper cuts or Well-approximated surgical incisions. Healing by First Intention Timeline are By 24 hours (with clot forms, neutrophils come in, and epithelium begins to regenerate ), By 3-7 days (with macrophages come in, granulation tissue is formed, new blood vessels, fibroblasts, collagen begins to bridge incision and epithelium increases in thickness), and by Weeks later (with granulation tissue gone, collagen is remodelled, epidermis full, mature but without dermal appendages, eventually scar forms).

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HEALING BY SECONDARY INTENTION

Healing by secondary intention is the healing of a wound with characteristic of a tissue defect, at times infected, having extensive loss of cells and tissues, and the wound is not approximated by surgical sutures but is left open, occurs in larger wounds that have gaps between wound margins. Fibrosis predominates over epithelial regeneration, Healing is slower, with more inflammation and granulation tissue formation, and more scarring such as Infarction, Large burns and ulcers, and Extraction sockets. The Differences from healing by first intention are more inflammation, more granulation tissue and wound contraction.

The Sequence of events involved are:

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 Initial haemorrhage, As a result of injury, the wound is filled with blood and fibrin clot which dries. Inflammatory phase, is an initial acute inflammatory response followed by the appearance of macrophages which clear off the debris as in primary union Epithelial changes The epidermal cells from both the margins of wound proliferate and migrate into the wound in the form of epithelial spurs till they meet in the middle and re-epithelialise the gap completely . However the proliferating epithelial cells do not cover the surface fully until granulation tissue from the base has started filling the wound space.In this way, pre existing viable connective tissue is seperated from the necrotic material and clot on the surface, forming scab which is cast off. In time the regenerated epidermis becomes stratified and keratinised. Granulation tissue Main bulk of secondary healing is by granulations. Granulation tissue is formed by proliferation of fibroblasts and neovascularisation from the adjoining viable elements. The newly formed granulation tissue us deep red, granular and very fragile .With time the scar on maturation becomes pale and white due to increase in collagen and decrease in vascularity. Specialised structures of skin like hair follicles and sweat glands are not replaced unless their viable residues remain which may regenerate Wound contraction Due to the action of myofibroblasts present in granulation tissue, the wound contracts to one-third of its original size. Wound contraction occurs at a time when active granulation tissue is being formed. Presence of infection Bacterial contamination of open wounds delays th process of healing due to release of bacterial toxins that provoke necrosis, suppuration and thrombosis. Surgical removal of dead and necrosed tissue, debridement , helps in preventing the bacterial infection of open wounds.

FACTORS INFLUENCING HEALING

LOCAL FACTORS
Infection single most important reason for delayed wound healing Foreign bodies such as suture material, bone and wood splinters . and interfere with
healing and cause inflammatory reaction and infection. Mechanical factors are early movement, Pressure, and the delays healing process Ionising radiation is delays granulation tissue formation Ultra violate light is facilitates healing Type size and location of injury determines whether healing takes place by resolution or organization Intraoperative Surgical Factors Oksigen Tension Inadequate blood supply Wound Tension Extend of tissue loss, slough and F.B Age of wound Multiple dressing and Movement Drying

SYSTEMIC FACTORS
Malnutrition such as Protein deficiency delays wound healing and deficiency Vitamin C
inhibit collagen synthesis.

Metabolic status such as Diabetes mellitus

inflammation and collagen synthesis veins

and Cortisone treatment which inhibits

Circulatory status such a iInadequate blood supply due to arteriosclerosis and Varicose Siti Julaiha Page 76

 Age : Older patient at higher risk of poor wound healing Medication is anti-inflammatory (aspirin), cytotoxic, immunosuppressive, steroids and

anticoagulant drugs all reduce healing rates by interrupting cell division or the clotting process. Hematologic abnormalities like defect of neutropjil functions and neutropenia and bleeding disorders slow the process of wound healing. Smoking Fluids electrolyte imbalance Hormones Temperature Chronic diseases : Anemia, Uremia, Jaundice, Diabetes.

GROWTH FACTORS INVOLVED IN HEALING

Factors Involved in Wound Healing

Following are the main growth factors involved in wound healing: Growth factor Abbreviati on Main origins Effects

Activated
Epidermal factor growth EGF PDEGF

macrophages

Salivary glands Keratinocytes Activated

macrophages

Keratinocyte and fibroblast mitogen Keratinocyte migration Granulation tissue formation Migration mitosis of Epidermal (Epithelization)

cell

Transforming growth factor-

TGF-

T-lymphocytes Keratinocytes Mesenchymal cells Mesenchymal cells

Platelets Macrophages Endothelial cells Smooth muscle cells

Hepatocyte and epithelial cell proliferation Expression of antimicrobial peptides Epithelial and endothelial cell proliferation Hepatocyte motility Vascular permeability Endothelial cell proliferation Granulocyte, macrophage, fibroblast and
smooth muscle cell chemotaxis activation

Hepatocyte growth HGF factor Vascular endothelial growth VEGF factor Platelet derived PDGF growth factor

Granulocyte, macrophage and fibroblast

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 Fibroblast, endothelial cell and smooth Keratinocytes

muscle cell proliferation Matrix metalloproteinase, fibronectin and hyaluronan production Angiogenesis Wound remodeling Integrin expression regulation

Platelet derived angiogenesis PDAF factor Platelets factor 4 PF-4

Capillary
cells

Endhothelial

Chemoattraction
Cheomattractant for Neutrophil Fibroblast chemotaxis Fibroblast and keratinocyte proliferation Keratinocyte migration Angiogenesis Wound contraction matrix deposition Granulocyte, macrophage, lymphocyte, fibroblast and smooth muscle cell chemotaxis TIMP synthesis Angiogenesis Fibroplasia Matrix metalloproteinase production inhibition Keratinocyte proliferation Chemoattraction for monocytes inhibit endothelial cell mitosis stimulate collagen synthesis by fibroblasts. differentiation

Neutrophil
Macrophages Mast cells T-lymphocytes Endothelial cells Fibroblasts

Fibroblast growth FGF-1, -2 factor 1 and 2

Transforming growth factor-

TGF-

Platelets T-lymphocytes Macrophages Endothelial cells Keratinocytes Smooth muscle cells Fibroblasts

Keratinocyte growth factor

KGF

Keratinocytes

Keratinocyte migration, proliferation and

Importance of Growth Factors in a Dermal Replacement

Wound healing is a complicated process that involves a number of dynamic cellular and molecular events. Factors regulating wound healing are many and originate from different sources. Key parameters, including cell migration, proliferation, angiogenesis, matrix deposition and degradation, and immune modulation, are essential for this process (Arbiser, 1996; Bennett and Schultz, 1993; Folkman and Klagsbrun, 1987; Marks et al., 1991; Moulin, 1995; Pettet et al., 1996; Raghow, 1994; Schaffer and Nanney, 1996). Improvement of cell motility (e.g., dermal fibroblasts, keratinocytes, melanocytes), angiogenesis (formation of vessels; migration and motility of endothelial cells), and modification of matrix turnover all have impact on wound healing. A tissue-engineered human dermal replacement can exploit the fact that the skin fibroblast is one of the most important and predominant cell types in the tissue repair process. This cell type appears at an early stage after injury, increasing in number as healing progresses (Arbiser, 1996; Bennett and Schultz, 1993; Folkman and Klagsbrun, 1987; Marks et al., 1991; Moulin, 1995; Pettet et al., 1996; Raghow, 1994). The cell is known to play a central role in the regulation of matrix deposition and degradation in the wound. Fibroblasts, however, also synthesize a rich array of cytokines, regulators of tissue repair in wounds, including interleukins, growth factors, and angiogenic factors.

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COMPLICATION OF WOUND HEALING

Bleeding - shock - anemia. Injury of Imp. structures. Infection Dehiscence (bursting wound; Infection, Weak scare due to continuous strain (coughing vomiting) or stretch, Decrease the bursting strength, Rapid absorbed catgut, Poor surgical technique, General conditions poor wound healing, Decrease nutrition (Proteins) and vitamins (vit c). Implantation or epidermoid cyst Hypertrophic Scar: confined within ; Excess collagen deposit causing raised scar remains within the original wound confines, Darker pigmented skin & flexor surfaces of upper torso, Often occurs in burns or wounds that take a long time to heal, sometimes preventable, Can regress spontaneously, Tx: steroids, silicone, pressure garments Keloid formation ; Excess Deposition of Collagen Causes Scar Growth Beyond the Border of the Original wound , Keloid also contain a greater amount of type III collagen than a mature scar, which suggests a failure in scar maturation. The collagen is loose disorganized wavy pattern of irregularly shaped fibers with a lower content of collagen cross-links compared to normal skin. Keloid and hypertrophic scars have rich blood supply, high mesenchymal density, and a thick epidermal layer. Pigmentation tattooing Painful scare local or reformed neuroma. Cicatrization : burns deformity stricture and stenosis in tubes. Neoplasia: sq cell ca. on scare tissue. F.b. retained Maggots.

Impediments to Wound Healing

Bacteria>105/cm2 : Decreased O2 content, collagen lysis, prolonged inflammation

Devitalized Tissue & Foreign Body: Retards Granulation Tissue formation and healing Cytotoxic drugs: 5FU, MTX, Cyclosporine, FK-506 can impair wound healing. D-Penicillamineinhibit collagen x-linking Chemotherapy: no effect after 14 days Radiation: Collagen synthesis abnormal, fibrosis of vessel Diabetes: impedes the early phase response

Malnurishment: Albumin<3.0, Vit-C Smoking: vasoconstriction, atherosclerosis, carboxyhemoglobin, decreased O2 delivery Steroids: inhibit macrophages, PMNs, Fibroblast collagen synthesis, cytokines, and decreased
wound tensile strength -Vit A (25,000 IU QD) counteracts effect of steroids DENERVATION has NO EFFECT on Wound Healing

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RESEARCH IN WOUND HEALING

RESEARCH IN WOUND CONTRACTION AND EXTRACELLULAR Smart Matrix for Wound Healing
An effort in laboratory has taken an indirect approach to dermal repair, by creating smart (a trademark name) matrix constructs that will provide a reparative dressing or packing material for acute and chronic wounds and in addition facilitate granulation tissue formation (patent issued). The first construct will be a recombinant fibronectin mutant composed of the three cell-binding domains (described below) conjugated to a matrix backbone of hyaluronan. Using a testing system (patent issued) wherein fibroblasts move from a collagen gel onto a surface coated with protein, various combinations of recombinant fibronectin fragments (Barkalow and Schwarzbauer, 1994), as well as the enzymatically derived 120-kDa fibronectin fragment, have been tested for their ability to permit fibroblast movement. The 120-kDa enzymatically derived fragment, which contains cell-binding peptides limited to the classic RGD domain (Pierschbacher and Ruoslahti, 1984) and the PHSRN synergy site (Obara et al., 1988), does not permit fibroblast movement. Furthermore, recombinant proteins containing the Hep II domain (Heparin II domain; HV0) have little, if any activity. However, combinations of the 120-kDa fragment with the Hep II domain do permit fibroblast motility. A recombinant protein containing both the classic cell-binding domain and the Hep II domain also allows movement, but no better than the combination of the 120-kDa fragment and the Hep II domain. These studies demonstrate that the classic cell-binding domain plus the Hep II domain are necessary and sufficient to permit fibroblast movement, but the movement is submaximal compared to intact fibronectin. Maximum movement, however, is attained when the IIICS domain is present along with the 120-kDa fragment and Hep II domain. A recombinant protein containing the classic cell-binding domain, the Hep II domain, and the IIICS domain also promotes maximal migration. Thus, the classic cell-binding domain plus the Hep II domain plus the IIICS domain are necessary and sufficient to allow maximal fibroblast movement compared to intact fibronectin. In experiments in which synthetic CS1, CS5, and HI through HV fibronectin peptides were added to the fibrin gel, we found that CS1 and CS5, when added separately to the assay, enhanced PDGFstimulated fibroblast movement from collagen gels into the fibrin gel, whereas HI, HII, HIV, and HV, but not HIII, inhibited this movement. In contrast, when CS1 and CS5 were added to the in vitro assay together, they inhibited fibroblast migration. Thus, HI, HII, HIV, HV, and either CS1 or CS5, together with RGDS and possibly PHSRN, are necessary for mesenchymal cell migration.

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Composites will be tested for their ability to permit or promote fibroblast transmigration from a collagen organotypic construct into the interstices of the composites. All composites that have the capacity to permit or promote fibroblast migration in vitro will be tested for their capacity to promote cutaneous wound repair in vivo (McClain et al., 1996). The matrix will be biocompatible and absorbable over a several-week period.

Research in Matrix Reorganization

During the second and third week of healing, fibroblasts begin to assume a myofibroblast phenotype characterized by large bundles of actin-containing microfilaments disposed along the cytoplasmic face of the plasma membrane and the establishment of cellcell and cellmatrix linkages (Welch et al., 1990). In some (Darby and Gabbiani, 1990), but not all (Welch et al., 1990), wound situations myofibroblasts express smooth muscle actin. Interestingly, TGF-_ can induce cultured human fibroblasts to express smooth muscle actin (Desmouliere et al., 1993) and may also be responsible for its expression in vivo. The appearance of the myofibroblasts corresponds to the commencement of connective tissue compaction and the contraction of the wound. Fibroblasts link to the extracellular fibronectin matrix through 51 (Welch et al., 1990), to collagen matrix through 11 and 21 collagen receptors (Ignatius et al., 1990), and to each other through direct adherens junctions (Welch et al.1990). Fibroblast 21 receptors are markedly up-regulated in 7-day-old wounds (Xu and Clark, 1996), a time when new collagenous matrix is accumulating and fibroblasts are beginning to align with collagenous fibrils through cellmatrix connections (Welch et al., 1990). New collagen bundles in turn have the capacity to join end to end with collagen bundles at the wound edge and ultimately to form covalent cross-links among themselves and with the collagen bundles of the adjacent dermis (Yamauchi et al., 1987; Birk et al., 1989). These cellcell, cellmatrix, and matrix matrix links provide a network across the wound whereby the traction of fibroblasts on their pericellular matrix can be transmitted across the wound (Singer et al., 1984). Cultured fibroblasts dispersed within a hydrated collagen gel provide a functional in vitro model of tissue contraction (Bell et al., 1979). When serum is added to the admixture, contraction of the collagen matrix occurs over the course of a few days. When observed with time-lapse microphotography, collagen condensation appears to result from a collection of collagen bundles executed by fibroblasts as they extend and retract pseudopodia attached to collagen fibers (Bell et al., 1983). The transmission of these traction forces across the in vitro collagen matrix depends on two linkage events: fibroblast attachment to the collagen matrix through the 21 integrin receptor (Schiro et al., 1991) and cross-links between the individual collagen bundles (Woodley et al., 1991). This linkage system probably plays a significant role in the in vivo situation of wound contraction as well. In addition, cellcell adhesions and cellfibronectin linkages appear to provide an additional means by which the traction forces of the myofibroblast may be transmitted across the wound matrix. F-Actin bundle arrays, cellcell and cellmatrix linkages, and collagen cross-links are all facets of the biomechanics of extracellular matrix contraction. The contraction process, however, needs a cytokine signal. In fact, cultured fibroblasts mixed in a collagen gel contract the collagen matrix only if serum is added to the medium (Bell et al., 1979). PDGF, the major fibroblast mitogen serum, in fact, stimulates fibroblast contraction of the collagen matrix (Clark et al., 1989). PDGF is abundant in wounds (Ansel et al., 1993), thus it may also provide the signal for wound contraction. TGF- has also been shown to stimulate fibroblast-driven collagen gel contraction (Reed et al., 1994). Because this factor persists in wound fibroblasts during the time of tissue contraction (Clark et al., 1995), it is an additional candidate for the stimulus of contraction. Perhaps both PDGF and TGF- signal wound contractionone more example of the many redundancies observed in the critical processes of wound healing. In summary, wound contraction represents a complex and masterfully orchestrated interaction of cells, extracellular matrix, and cytokines. Collagen remodeling during the transition from granulation tissue to scar is dependent on continued collagen synthesis and collagen catabolism. The degradation of wound collagen is controlled by a variety of collagenase enzymes from macrophages, epidermal cells, and fibroblasts. These collagenases are specific for particular types of collagens but most cells probably contain two or more different types of these enzymes (Hasty et al., 1986). Three matrix metalloproteinases have been described that have the ability to cleave native collagen: MMP-1, or classic interstitial collagenase, which cleaves types, I, II, III, XIII, and X collagens (Grant et al., 1987); neutrophil collagenase (Hasty et al., 1990); and a novel collagenase produced by breast carcinomas and prominent in chronic wounds (Vaalamo et al., 1997). It is not known whether interstitial collagenases, other than MMP-1, are active in the remodeling stage of human wound repair (Saarialho-Kere et al., 1993; Stricklin and Nanney, 1994). Other MMPs potentially important in wound repair include two gelatinases, 72-kDa gelatinase A (MMP-2) and 92-kDa gelatinase B (MMP-9), with identical substrate specificity to degrade denatured collagens of all types as well as native types V and XI collagens (Hibbs et al., 1987; Stetler-Stevenson et al., 1989); and stromelysin-1, -2, and -3, which degrade a wide variety of substrates, including types III, IV, V, VII, and IX collagens as well as proteoglycans and

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glycoproteins (Saus et al., 1988; Murphy et al., 1991). Although gelatinase A and B are expressed in early wounds (Salo et al., 1994), they may play a lesser role in later tissue remodeling. Gelatinase A is produced constitutively by many cells and is up-regulated only by TGF-_ (Overall et al., 1991), whereas gelatinase B is subject it to regulation by a variety of physiologic signals, because its promoter contains two AP-1 regulatory elements (Huhtala et al., 1991). Two other metalloproteinases that do not belong to the above subgroups are macrophage metalloelastase (Shapiro et al., 1993) and a novel transmembrane metalloproteinase that can activate secreted progelatinase A (Sato et al., 1994). MMP enzymatic activities are controlled by various inhibitor counterparts called tissue inhibitors of metalloproteinases (TIMPs), which are finely regulated during wound repair (Madlener et al., 1998). Cytokines such as TGF-, PDGF, and IL-1 plus the extracellular matrix clearly play an important role in the modulation of collagenase and TIMP expression in vivo (Circolo et al., 1991; Sporn and Roberts, 1992; Huhtala et al., 1995). Interestingly, type I collagen induces MMP-1 expression through the _2_1 collagen receptor while suppressing collagen synthesis through the 11 collagen receptor (Langholz et al., 1996). Type I collagen also induces expression of 21 receptors (Klein et al., 1991; Xu and Clark, 1996), thus collagen can induce the receptor that signals a collagen degradationremodeling phenotype. Such dynamic, reciprocal cellmatrix interactions probably occur generally during tissue formation and remodeling processes such as morphogenesis, tumor growth, and wound healing, to name a few. Wounds gain only about 20% of their final strength by the third week, during which time fibrillar collagen has accumulated relatively rapidly and has been remodeled by myofibroblast contraction of the wound. Thereafter the rate at which wounds gain tensile strength is slow, reflecting a much slower rate of collagen accumulation. In fact, the gradual gain in tensile strength has less to do with new collagen deposition than with further collagen remodeling, with formation of larger collagen bundles and an accumulation of intermolecular cross-links (Bailey 1975). Nevertheless, wounds fail to attain the same breaking strength as uninjured skin. At maximum strength, a scar is only 70% as strong as intact skin (Levenson et al., 1965).

Therapies Designed to Reduce Scarring

Fetal healing is characterized by lack of scarring. The relative lack of TGF-1, a profibrotic cytokine, in fetal skin may help explain why the fetus heals without scarring (Adzick and Lorenz, 1994). Studies using neutralizing antibodies to TGF-1 and TGF-2, as well as using TGF-3, which down-regulates the other TGF-_ isoforms, have resulted in reduced scarring, supporting the central role of TGF-1 in scar formation (Shah et al., 1995). Several strategies are being used to block TGF1 and TGF-2 in wounds in an attempt to attenuate scar formation.

Research in Immunology and the Skin

The first stage in the induction of a primary immune response in skin is the processing of antigen by dendritic cells (Langerhans cells with dermal dendritic cells), the antigen-presenting cells in skin. These cells process antigen and migrate out of the skin to the draining lymph node, where they can recruit and activate T cells. The interaction of keratinocytes and fibroblasts with cells has important implications for the use of allogeneic cells in tissue engineering. Under normal conditions, keratinocytes and fibroblasts do not express major histocompatability complex (MHC) class II antigens; they can be induced, however, by interferon-_ to express MHC- class II molecules and thereby acquire the ability to present antigen to T cells. Because the keratinocytes and fibroblasts are deficient in the necessary costimulatory molecules (Nickoloff and Turka, 1994; Phipps et al., 1989), antigen presentation by keratinocytes and fibroblasts does not result in T cell activation. Instead, this antigen presentation can result in T cell nonresponsiveness (Gaspari and Katz, 1988; Bal et al., 1990) or T cell anergy (Gaspari and Katz, 1991). Therefore, the primary mode of skin rejection is likely mediated via an attack on the vasculature present in a normal skin graft (Pober et al., 1986; Moulon et al., 1999). Autologous skin grafts avoid issues of immunogenicity, of course, but autologous grafts have significant limitations. Growing graft tissues from biopsy takes several weeks, the donor site creates another wound, and in some patients (e.g., severe burn patients) there may be no appropriate donor site. Reproducibly making complex HSE constructs to order from autologous cells would be technically difficult, time consuming, and very costly. Therefore, our ability to effectively use allogeneic human cells is a key element in the success of engineered skin replacements.

Research in The Process of Wound Healing

The immediate tissue response to wounding is clot formation to stop bleeding. Simultaneously, there is a release of inflammatory cytokines that regulate blood flow to the area, recruit lymphocytes and macrophages to fight infection, and later stimulate angiogenesis and collagen depo- sition (Williams and Kupper, 1996). These latter processes result in the formation of granulation tissue, a highly vascularized and cellular wound connective tissue. Fibroblasts rich in actin, called myofibroblasts (Desmouliere and Gabbiani, 1996), are recruited through the action of factors such as platelet-derived growth factor (PDGF) and transforming growth factor=(TGF). Granulation tissue forms in the wound bed, stimulated by factors such as PDGF. This tissue is gradually replaced by scar tissue through the action of the myofibroblasts and factors such as

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TGF. Keratinocytes are stimulated to proliferate and to migrate into the wound bed to restore epidermal coverage. From our preclinical observations using both full-thickness HSE and dermal matrice coverage by the epidermis appears to play a key role in the regulation of the underlying inflammatory response. Providing a noninflammatory living connective tissue implant in the wound defect also appears to be beneficial in directing the granulation response. As this brief description shows, wound healing involves the interaction of many tissue factors and elements. The poor healing response in chronic wounds has been attributed to an imbalance of factors rather than to an insufficiency of any particular factor (Parenteau et al., 1997). However, most, if not all, factor-based approaches have had marginal success. Identification of putative wound healing factors has led to several attempts to speed wound healing by local application of one or more factors that promote cell attachment and migration. Transforming growth factor-_, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor have been candidates for this purpose (McKay and Leigh, 1991; Martin et al., 1992; Abraham and Klagsbrun, 1996; Nanney and King, 1996; Roberts and Sporn, 1996). Of these, only PDGF has shown efficacy in clinical trials and is approved for clinical use (Regranex, Ortho McNeil, Inc., Raritan, NJ). The arginineglycineaspartic acid (RGD) matrix peptide sequence has been found to promote the migration of connective tissue cells, and thus stimulate production of a dermal scaffold within the wound bed. This approach has been shown to accelerate healing of sickle cell leg ulcers (Wethers et al., 1994) and diabetic ulcers (Steed et al., 1995), compared with placebo, but not when compared with standard care. In addition, complex cell extracts have been used in hopes of providing the appropriate mixture of elements. These include the use of platelet extract to provide primarily platelet-derived growth factor (Knighton et al., 1989), and the use of keratinocyte extracts to provide a complex mixture of elements of rapidly growing keratinocytes (Duinslaiger et al., 1994); again, effects have been marginal, in part due to the complex nature of the wound healing response (Nathan and Sporn, 1991). In addition, the use of factors is not a sufficient approach, in and of itself, in situations where there is severe or massive loss of skin tissue. Why should living tissue be different in its response? The epidermal and dermal responses are regulated by inflammatory cytokines and by autocrine and paracrine factors produced by the dermal fibroblasts and epidermal keratinocytes (Ansel et al., 1990; McKay and Leigh, 1991). These factors regulate growth and differentiation of keratinocytes, proinflammatory reaction, angiogenesis, and deposition of extracellular matrix. Living tissue created through tissue engineering can provide complex temporal control of factor delivery and effect and can be used to provide the needed combination of chemical, structural, and, last but not least, normal cellular elements (Marks et al., 1991; Sabolinski et al., 1996).

Stimulation of angiogenesis in the chick chorioallantoic membrane

Ten-day-old chicken embryos were obtained from McIntyre Farms (Lakeside, CA) and incubated at 37_C. Eggs were candled to locate and mark a target area void of large vessels. Two small holes were made in the shell with a needle, directly over the air sac and over the target area. Suction was applied to the first hole, causing the chorioallantoic membrane (CAM) to drop away from the marked area. Using a Dremel Moto-Tool, the eggshell was removed from the target area to create a window. A 4-mm-diameter circular sample (fibroblast-based engineered tissue or control) was then placed on the membrane near, but not on top of, a large blood vessel. The hole was covered with a piece of clear adhesive tape and the eggs were incubated for 72 hr at 37_C to allow blood vessel growth. The treated section of the membrane was then removed, photographed, and fixed in methanol. The number of fine blood vessel branch points in the region of the sample was counted. Biopsy samples were fixed in methanol and sections stained with Massons Trichrome. The three-dimensional fibroblast-based tissues induced vessel development in the CAM to a greater extent than control, including both fine capillary development and evidence for increased permeability. The development of capillary blood vessels in CAMs treated with threedimensional fibroblast cultures was also clearly visible by histology. This type of capillary development is characteristic of vascular endothelial growth factor (VEGF)-induced angiogenesis. It differs from that observed using basic fibroblast growth factor (FGF) stimulation, whereby the vesselsshow a larger diameter with little or no increase in permeability. When the number of vessels per sample in the CAM was counted, there was a statistically significant difference between the scaffold and the three-dimensional cultures. VEGF stimulated angiogenesis in a dosedependent manner. The angiogenic activity of the three-dimensional culture was reduced by _90% by preincubation with anti-VEGFs neutralizing antibody prior to placement on the CAM.

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Dermagraft-stimulated angiogenesis in the chorioallantoic membrane assay. Top two panels show the macroscopic view, bottom two panels show histology. Left panels are of scaffold alone (top) or nonviable Dermagraft (bottom); the panels to the right are treated with Dermagraft.

Effect of Dermagraft (Dg) on capillary blood vessel formation in the CAM.Bars present 95% confidence intervals.

Recombinant Growth Factors for Wound Healing

With the development of recombinant DNA technology the production of large quantities of growth factors is now possible, leading to an increased interest in their potential therapeutic role in wound healing. The four families of growth factors that have shown the greatest potential for enhancing wound repair include the EGF family, the FGF family, the PDGF family, and the TGF- family. The EGF family of growth factors includes EGF, TGF-, and HB-EGF. As mentioned in the previous discussion of epithelialization, these growth factors are potent keratinocyte mitogens and motogens (promote cellular motility) that are released at the site of injury (Feliciani et al., 1996). Attempts to enhance healing of burns by application of EGF or TGF- in animals have led to conflicting results. Although some have noted enhancement (Brown et al., 1989), other studies have failed to demonstrate consistent and significant biologic effects when EGF was applied to either partial- or full-thickness burns in pigs (Danilenko et al., 1995). Limited acceleration of healing of chronic venous stasis ulcers has similarly been demonstrated in humans with EGF (Brown et al., 1991; Falanga et al., 1992). The family of FGF includes multiple polypeptides that stimulate proliferation of both keratinocytes and fibroblasts. In excisional wound animal models, basic FGF (FGF-2) significantly increases both granulation tissue formation and reepithelialization (Pierce et al., 1992). FGF has been shown to enhance the healing of chronic pressure sores in a limited number of paraplegics (Robson et al.,

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1992a). Larger studies sponsored by the pharmaceutical industry, however, failed to show clinical benefit. The superfamily of PDGF includes PDGF and VEGF. PDGF occurs in three isoforms PDGF-AA, -AB, and -BB (Heldin, 1992). The latter two isoforms are the most potent and are secreted by platelets (PDGF-AB), macrophages (PDGF-BB), and epidermal cells (PDGF-BB) in response to injury or other perturbations and cause substantial proliferation of fibroblasts (Heldin and Westermark, 1996). Application of PDGF to incisional wounds has resulted in more rapid healing. PDGF has also stimulated reepithelialization and granulation tissue formation in animal excisional wound models and burns (Pierce et al., 1992; Danilenko et al., 1995). Clinical trials using PDGF for the treatment of pressure sores and diabetic ulcers have been encouraging, demonstrating more rapid and complete healing in wounds treated with PDGF (Robson et al., 1992b; Steed, 1995). Recombinant PDGF-BB, in fact, is the first recombinant growth factor to gain approval by the Food and Drug Administration (FDA) with an indication to enhance healing of diabetic foot ulcers. This factor has been placed on the market as Regranex by OrthoMcNeil Pharmaceuticals. VEGF is secreted by keratinocytes and macrophages and induces vascular permeability as well as angiogenesis (Iruela-Arispe and Dvorak, 1997). It has been shown to enhance healing of gastric and duodenal ulcers (Folkman, 1995) and is currently under investigation for use in the treatment of ischemic tissues (Isner, 1997). Other isoforms of VEGF have been described (Veikkola and Alitalo, 999), but no animal or clinical data are available at this time. Within another large family, TGF-_ has three isoforms that are chemotactic for macrophages and are potent stimulators of procollagen type I and fibronectin (Roberts and Sporn,1996). Multiple animal studies have demonstrated the ability of TGF-1 and TGF-2 to enhance wound healing (Davidson et al. 1985; Mustoe et al. 1987; Ksander 1990). Preliminary human studies suggest that TGF-2 may be of benefit in the treatment of venous stasis ulcers (Robson et al., 1995). Both TGF-1 and TGF-2 have been shown to increase scar formation in wound models (Border and Noble, 1994), whereas application of neutralizing antibodies has been shown to reduce scarring (Shah,1994). Interestingly, TGF-3 has also been shown to reduce scar formation in rat incisions (Shah,1995), but this finding is under debate.

Research in Other Cytokines

Growth hormone has been studied and found to be of benefit in the treatment of donor sites in both children and adults with extensive burns (Herndon et al., 1990, 1995). Granulocyte macrophage colony-stimulating factor (GM-CSF) and monocyte colony-stimulating factor (MCSF) are potent stimulators of monocyte activation and chemotaxis and have been shown to accelerate healing of wounds in rats (Middleton and Aukerman, 1991; Jyung et al., 1994). In contrast, granulocyte colony-stimulating factor (G-CSF) has not had any beneficial effects on wound healing. Interleukin-1, which is chemotactic for neutrophils and macrophages, has been evaluated in the management of pressure sores (Robson et al., 1994). Although clinical experience with growth factors and other mediators is limited, overall results have been discouraging. This is not surprising when one considers that wound repair is the result of a complex set of interactions between soluble cytokines, formed blood elements, extracellular matrix, and cells. It is possible that combinations of various mediators given at precisely timed intervals will be more efficacious at promoting wound healing. Indeed, synergistic effects on wound healing have been demonstrated for the following combinations: IGF-1 and PDGF (Lynch et al., 1989; Greenhalgh et al., 1993), TGF- and PDFG (Brown et al., 1994), and KGF and PDGF (Danilenko et al., 1995).

Stimulation of endothelial cell proliferation

Endothelial cell proliferation is a critical component of angiogenesis. The ability of fibroblast- based engineered tissue to stimulate this activity was determined by [3H]thymidine incorporation. Various growth factors and concentrated conditioned medium samples were assessed for their influence on the proliferation of human vascular endothelial cells (HUVECs). Confluent cultureswere detached and resuspended in HUVEC growth medium to a final concentration of 2.5X 104 cells/ml. Attachment Factor Solution (Cell Applications, Inc.) was used to pretreat 24-well plates and cells were added, 1 ml of cell suspension per well. Cells were allowed to settle and attach and then were switched to Endothelial Serum Free medium (Cell Applications, Inc.), supplemented with fibroblast culture medium or medium conditioned by monolayer or three-dimensional fibroblast cultures. On day 2, the cells received fresh serum-free medium supplemented as appropriate with 1 micron Curie/ml [3H]thymidine. On day 3, medium was removed, cells were washed three times with phosphate-buffered saline (PBS), and 250 micron l of 2.3% sodium dodecyl sulfate (SDS) solution was added to solubilize the cells. After 30 min, the SDS extract and 1 ml of a PBS wash were transferred to a scintillation vial. Then 5 ml of ScintiVerse (Fair Lawn, NJ) was added to vials and radioactivity was determined using a Beckman LS6500 Scintillation Counter (Fullerton, CA). Medium conditioned by incubation with three-dimensional fibroblast cultures stimulated [3H]thymidine incorporation in cultures of endothelial cells (Fig. 63.7). The proliferative effect is dose dependent.

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Stimulation of endothelial cell proliferation by Dermagraft (Dg).

Stimulation of endothelial cell chemokinesis

The ability of our fibroblast-based engineered tissue to stimulate endothelial cell migration was tested in two assays. The first was a chemokinesis assay that determines the stimulation of cell movement without any directional definition. The second measured cell migration toward a stimulation source. The chemokinesis assay was performed and reported by Martin and colleagues (1998). Endothelial cells were grown on Cytodex-2 beads. The assay estimates the dissociation of cells from the beads and reassociation with a culture plate. The cells on the plate were stained and counted. Coculture of the cells with the fibroblast-based engineered tissue gave a marked increase in transfer of cells from bead to plate (p = 0.0003). The activity of the three-dimensional fibroblast cultures was inhibited about 60% using anti-HGF neutralizing antibody.

Stimulation of endothelial cell motility by Dermagraft (Dg).

Stimulation of endothelial cell chemotaxis by Dermagraft.

Stimulation of endothelial cell chemotaxis

Cell migration was analyzed with an endothelial cell chemotaxis assay utilizing a Neuro Probe 48well Boyden chemotaxis chamber (Neuro Probe, Inc.). Polycarbonate membrane filters (Poretics Corporation; 25 X 80 mm) were soaked in 0.5 M acetic acid overnight, washed three times for 1 hr with water, incubated in a solution of 0.01% calf skin gelatin type III (Sigma; St. Louis, MO) for 12 16 hr, and air dried. HUVECs were detached and resuspended in HUVEC growth medium at a final concentration of 1.0 X 105 cells/ml. The Boyden chamber was assembled as follows: 30 l/well of sample or standard was added to the bottom wells, the gelatin-coated membrane was placed on top, and 50 l of cell suspension was added to the upper wells. The chamber was incubated at 37_C for 3 hr. Membranes were then carefully removed from the chamber and the cell side was rinsed in PBS and drawn across a wiper blade to remove nonmigrated cells. The membranes were stained with Wrights Geimsa stain and either the number of cells counted or the density of staining was reported against a standard curve generated with 20, 10, 5.0, and 0 ng/ml purified VEGF. Medium conditioned by the fibroblast-based engineered tissue greatly stimulated cell migration in a dose-dependent manner (Fig. 63.9). The three-dimensional fibroblast-

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conditioned medium stimulated HUVEC migration to a greater extent than the positive control using VEGF, even at 50 ng/ml. Anti-VEGF antibody inhibited migration stimulated by threedimensional fibroblast- culture-conditioned medium by 50%.

Research in Epithelialization
If the basement membrane is destroyed by injury, epidermal cells migrate over a provisional matrix of fibrin(ogen), fibronectin, tenasin, and vitronectin as well as stromal type I collagen (Clark, 1996b). Fibrinogen is interspersed with stromal type I collagen at the wound margin and interwoven with the fibrin clot in the wound space (Clark et al., 1999). Wound keratinocytes express cell surface receptors for fibronectin, tenasin, and vitronectin (Clark, 1990; Juhasz et al., 1993; Larjava et al., 1993; Clark et al., 1996a). These specialized cell membrane receptors are of the integrin superfamily (Hynes, 1992; Yamada et al., 1996). In addition, the integrin 21 collagen receptors, which are normally disposed along the lateral sides of basal keratinocytes, redistribute to the basal membrane of wound keratinocytes as they come in contact with type I collagen fibers of the dermis. The migrating wound epidermis does not simply transit over a wound coated with provisional matrix but rather dissects through the wound, separating desiccated or otherwise nonviable tissue from viable tissue. The path of dissection appears to be determined by the array of integrins that the migrating epidermal cells express on their cell membranes, as described above. In addition, v3, the receptor for fibrinogen/fibrin (Cheresh et al., 1989) and denatured collagen (Davis, 1992), is not expressed on keratinocytes in vitro (Adams and Watt, 1991) or in vivo (Gailit et al., 1994). Therefore, keratinocytes do not have the capacity to interact with these matrix proteins (Kubo et al., 1999). Furthermore, in the presence of fibrinogen or fibrin, epidermal cells fail to bind fibronectin (Kubo et al., 1999), yet can bind type I collagen. Hence migrating wound epidermis avoids the fibrin-, fibronectin-rich clot while migrating over a bed of fibrinogen, fibronectin, and type I collagen. Extracellular matrix degradation is clearly required for the dissection of migrating wound epidermis between the collagenous dermis, the fibrin eschar (Bugge et al., 1996), and probably depends on epidermal cell production of both collagenase (Woodley et al., 1986) and plasminogen activator (Grondahl-Hansen et al., 1988) and perhaps stromelysin (Saarialho-Kere et al., 1994). Plasminogen activator activates collagenase as well as plasminogen (Mignatti et al., 1996) and exclusive of the former, are release of autocrine or paracrine growth factors that induce epidermal migration and proliferation and/or increased expression of growth factor receptors. Leading contenders include the epidermal growth factor (EGF) family (Nanney and King, 1996), especially TGF-(Barrandon and Green, 1987) and heparin-binding epidermal growth factor (HB-EGF) Higashiyama et al., 1991); the FGF family (Abraham and Klagsbrun, 1996), especially keratinocyte growth factor (KGF) (Werner, 1998); insulin-like growth factor (Galiano et al., 1996); and TGF- (Zambruno et al., 1995). Although some growth factors, such as IGF, may derive from the circulation and thereby act as a hormone, other growth factors, such as HB-EGF and KGF, derive from macrophages and dermal parenchymal cells, respectively, and act on epidermal cells through a paracrine pathway (Werner, 1998). In contrast, TGF-and TGF-, originate from keratinocytes and act directly on the producer cell or adjacent epidermal cells in an autocrine or juxtacrine fashion (Coffey et al., 1987; Brachmann et al., 1989). Many of these growth factors have been shown to stimulate reepithelialization in animal models (Brown et al., 1989; Lynch et al., 1989; Hebda et al., 1990; Staino-Coico et al., 1993) or to lack receptors in models of deficient reepithelialization (Werner et al., 1994), supporting the hypothesis that they are active during normal wound repair. As reepithelialization ensues, basement membrane proteins reappear in a very ordered sequence from the margin of the wound inward in a zipperlike fashion (Clark, 1996b). Epidermal cells revert to their normal phenotype, once again firmly attaching to reestablished basement membrane through two hemidesmosomal proteins, 64 integrin and 180-kDa bullous pemphigoid antigen (Gipson et al., 1993), and to the underlying neodermis through type VII collagen fibrils (Gipson et al., 1988).

Recombinant Growth Factors for Epidermal Wounds

Epithelial-targeted growth factors, such as EGF and more recently KGF, have been investigated as potential treatment for nonhealing epidermal wounds and/or acute large-defect epidermal wounds. EGF was studied in patients with thermal burns and it was shown to accelerate the healing of donor graft sites by 1.5 days (Brown et al., 1989). Although initial clinical investigations of EGF appeared significant (G. L. Brown et al., 1989; G. Brown et al., 1991), later large clinical trails sponsored by the biotechnology and pharmaceutical industries did not confirm therapeutic benefit. Keratinocyte growth factors are the newest members of the FGF family (Werner, 1998). KGF-1 and -2 are secreted by fibroblasts in response to injury and have both mitogenic and motogenic paracrine effects on keratinocytes, particularly those in hair follicles and sebaceaous and sweat glands (Pierce et al., 1994; Igarashi et al., 1998). Although exogenous administration of KGF-1 has been shown to stimulate keratinocyte proliferation and reepithelialization in partialthickness porcine burns, it had no effect on reepithelization of full-thickness burns (Danilenko et al., 1995). Likewise, KGF-1 stimulated reepithelialization of partial-thickness, but not full-thickness, excisional

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porcine wounds; however, the thickness of the neoepidermis in both partial- and full-thickness wounds was significantly increased (Staino-Coico et al., 1993). Animal studies with KGF-2 are promising (Jimenez and Rampy, 1999) and clinical investigations with this factor are now in progress. Clinical investigations with recombinant growth factors that target dermal components of the skin, as well as epidermis, will be discussed at the conclusion of the following section on granulation tissue.

Research in Granulation Tissue

New stroma, often called granulation tissue, begins to form approximately 4 days after injury. The name derives from the granular appearance of newly forming tissue when it is incised and visually examined. Numerous new capillaries endow the neostroma with its granular appearance. Macrophages, fibroblasts, and blood vessels move into the wound space as a unit (Hunt, 1989), which correlates well with the proposed biologic interdependence of these cells during tissue repair. Macrophages provide a continuing source of cytokines necessary to stimulate fibroplasia and angiogenesis, fibroblasts construct new extracellular matrix necessary to support cell ingrowth, and blood vessels carry oxygen and nutrients necessary to sustain cell metabolism. The quantity and quality of granulation tissue depend on biologic modifiers present, the activity level of target cells, and the extracellular matrix environment (Sporn and Roberts, 1986; Juliano and Haskill, 1992). As mentioned previously in the discussion on inflammation, the arrival of peripheral blood monocytes and their activation to macrophages establish conditions for continual synthesis and release of growth factors. In addition, injured and activated parenchymal cells can synthesize and secrete growth factors. The provisional extracellular matrix also promotes granulation tissue formation. Once fibroblasts and endothelial cells express the proper integrin receptors, they invade the fibrin/fibronectin-rich wound space.

Research in Fibroplasia
Components of granulation tissue derived from fibroblasts, including the fibroblast cells and the extracellular matrix, are collectively known as fibroplasia. Cytokines, especially PDGF and TGF- (Heldin and Westermark, 1996; Roberts and Sporn, 1996), in concert with the provisional matrix molecules (Brown et al., 1993;Gray et al., 1993; Xu and Clark, 1996), presumably stimulate fibroblasts of the periwound tissue to proliferate, express appropriate integrin receptors, and migrate into the wound space. Many of these growth factors are released from platelets and macrophages; however, fibroblasts can produce growth factors, to which they respond in an autocrine fashion (Leof et al., 1986; Raines et al., 1989). Multiple complex, interactive biologic phenomena occur within fibroblasts as they respond to wound cytokines, including the induction of additional cytokines (Leof et al., 1986; Raines et al., 1989) and modulation of cytokine receptor number or affinity) Oppenheimer et al., 1983; Assoian , 1984). In vivo studies support the hypothesis that growth factors are active in wound repair fibroplasia. Several studies have demonstrated that PDGF, PDGF-like peptides, TGF-, TGF-, HB-EGF, and FGF family members are present at sites of tissue repair (Matsuoka and Grotendorst, 1989; Pierce 1992; Werner., 1992; Ansel 1993; Levine 1993; Marikovsky 1993; Frank, 1996). Furthermore, purified and recombinantderived growth factors have been shown to stimulate wound granulation tissue in normal and compromised animals (Sporn 1983; Lynch, 1989; Greenhalgh 1990; Mustoe 1991; Steed 1992; Shah 1995), and a single growth factor may work both directly and indirectly by inducing the production of other growth factors in situ (Mustoe,1991).

Provisional matrix integrin expression is the rate-limiting step in granulation tissue induction. Once the appropriate integrins are expressed on periwound endothelial cells and fibroblasts on day 3, the cells invade the wound space shortly thereafter (on days 4 and 5). Fibroblasts and endothelial cells express the fibrinogen/fibrin receptor v3 and therefore are able to invade the fibrin clot; the epidermis, however, does not express v3 and therefore dissects under the clot. Ultimately the clot that has not been transformed into granulation tissue by invading fibroblasts and endothelial cells is dissected free of the wound and sloughed as eschar.

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Structural molecules of the early extracellular matrix, termed the provisional matrix (Clark et al., 1982), contribute to tissue formation by providing a scaffold or conduit for cell migration (fibronectin) (Greiling and Clark, 1997), low impedance for cell mobility (hyaluronic acid) (Toole, 1991), a reservoir for cytokines (Nathan and Sporn, 1991), and direct signals to the cells through integrin receptors (Damsky and Werb, 1992). Fibronectin appearance in the periwound environment as well as the expression of fibronectin receptors appear to be critical rate-limiting steps in granulation tissue formation (Clark et al., 1996b; Gailit et al., 1996; McClain et al., 1996; Xu and Clark, 1996; Greiling and Clark, 1997). In addition, a dynamic reciprocity between fibroblasts and their surrounding extracellular matrix creates further complexity. That is, fibroblasts affect the extracellular matrix through new synthesis, deposition, and remodeling of the extracellular matrix (Kurkinen et al., 1980; Welch et al., 1990), whereas the extracellular matrix affects fibroblasts by regulating their function, including their ability to synthesize, deposit, remodel, and generally interact with the extracellular matrix (Mauch et al., 1988; Grinnell, 1994; Clark et al., 1995; Xu and Clark, 1996). Thus the interactions between extracellular matrix and fibroblasts dynamically evolve during granulation tissue development. As fibroblasts migrate into the wound space, they initially penetrate the blood clot composed of fibrin and lesser amounts of fibronectin and vitronectin. Fibroblasts presumably require fibronectin in vivo for movement from the periwound collagenous matrix into the fibrin/fibronectin- laden wound space, as they do in vitro for migration from a three-dimensional collagen gel into a fibrin gel (Greiling and Clark, 1997). Fibroblasts bind to fibronectin through receptors of the integrin superfamily (Hynes, 1992; Yamada et al., 1996). The Arg-Gly-Asp-Ser (RGDS) tetrapeptide within the cell-binding domain of these proteins is critical for binding to the integrin receptors31, 51, v3, v1, and v5. In addition, the CSIII domain of fibronectin provides a second binding site for human dermal fibroblasts via the 41 integrin receptor (Gailit et al., 1993). In vivo studies have shown that the RGD-dependent, fibronectin receptors 31 and 51 are up-regulated on periwound fibroblasts the day prior to granulation tissue formation and on early granulation tissue fibroblasts as they infiltrate the provisional matrix- laden wound (Xu and Clark, 1996). In contrast, the non-RGD-binding 11 and 21 collagen receptors on these fibroblasts were either suppressed or did not appear to change appreciably (Gailit ., 1996; Xu and Clark, 1996). Both PDGF and TGF- can stimulate fibroblasts to migrate (Seppa et al., 1982; Senior et al., 1985; Postlethwaite et al., 1987) and can up-regulate integrin receptors (Heino et al., 1989; Ahlen and Rubin, 1994; Gailit et al., 1996). Therefore these growth factors may be partially responsible for inducing a migrating-fibroblast phenotype. Interestingly, PDGF increases 31 and 51 while decreasing 11 in cultured human dermal fibroblasts (Gailit et al., 1996). Furthermore, fibronectin- or fibrin-rich environments promote the ability of PDGF to increase 31 and 51, but not 21, mRNA steady-state levels by increasing the stability of these mRNA moieties (Xu and Clark, 1996). This suggests a positive feedback loop between the extracellular matrix and extracellular matrix receptors. In vitro fibroblast migration has also been observed in response to a variety of chemoattractants, including fragments of the fifth component of complement (Senior et al., 1988); types I, II, and III collagen-derived peptides (Postlethwaite and Kang, 1976); a fibronectin fragment (Postlethwaite et al., 1981); elastin-derived peptides (Senior et al., 1980); and interleukin-4 (Postlethwaite and Seyer, 1991). Movement into a cross-linked fibrin blood clot or any tightly woven extracellular matrix may also necessitate an active proteolytic system that can cleave a path for migration. A variety of fibroblast- derived enzymes in conjunction with serum-derived plasmin are potential candidates for this task, including plasminogen activator, interstitial collagenase-1 and -3 (MMP-1 and MMP- 13, respectively), the 72-kDa gelatinase A (MMP-2), and stromelysin (MMP-3) (Mignatti et al., 1996; Vaalamo et al., 1997). In fact, high levels of immunoreactive MMP-1 have been localized to fibroblasts at the interface of granulation tissue with eschar in burn wounds (Stricklin and Nanney, 1994) and many stromal cells stain for MMP-1 and MMP-13 in chronic ulcers (Vaalamo 1997). Although TGF- down-regulates proteinase activity (Laiho , 1987; Overall,1989), PDGF stimulates the production and secretion of these proteinases (Circol., 1991). Once the fibroblasts have migrated into the wound they gradually switch their major function to protein synthesis (Welch ,1990). Ultimately the migratory phenotype is completely supplanted by a profibrotic phenotype characterized by decreased 31 and 51 provisional matrix receptor expression, increased 21 collagen receptor expression, abundant rough endoplasmic recticulum, and Golgi apparatus filled with new collagen protein (Welch 1990; Xu and Clark, 1996). The fibronectin-rich provisional matrix is gradually supplanted with a collagenous matrix (Welc, 1990; Clark, 1995). Under these conditions, PDGF, which is still abundant in these wounds (Pierce, 1995), stimulates extremely high levels of 21 collagen receptor, but not 31 and 51 provisional matrix receptors, supporting the contention that the extracellular matrix provides a positive feedback for integrin expression (Xu and Clark, 1996).TGF- _, which is observed in wound fibroblasts at this time (Clark,1995), can induce fibroblasts to produce great quantities of collagen (Ignotz and Massague, 1986; Roberts, 1986). IL-4 also can induce a modest increase in types I and III collagen production (Postlethwaite,1992). Because IL-4-producing mast cells are present in healing wounds, as well as fibrotic tissue, they may contribute to collagenous matrix accumulation in these sites.

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Once an abundant collagen matrix is deposited in the wound, fibroblasts cease collagen production despite the continuing presence of TGF- (Clark et al., 1995). The stimuli responsible for fibroblast proliferation and matrix synthesis during wound repair were originally extrapolated from many in vitro investigations over the past two decades and then confirmed by in vivo manipulation of wounds within the past 10 years (Sprugel et al., 1987; Pierce et al., 1991; Schultz et al., 1991). Less attention had been directed toward elucidating the signals responsible for downregulating fibroblast proliferation and matrix synthesis. Both in vitro and in vivo studies suggest that interferon may be one such factor (Duncan and Berman, 1985;Granstein et al., 1987). In addition, collagen matrix can suppress both fibroblast proliferation and fibroblast collagen synthesis (Grinnell, 1994; Clark et al., 1995). In contrast, a fibrin or fibronectin matrix has little or no suppressive effect on the mitogenic or synthetic potential of fibroblasts (Clark et al., 1995; Tuan et al., 1996). Although the attenuated fibroblast activity in collagen gels is not associated with cell death, many fibroblasts in day 10 healing wounds develop pyknotic nuclei (Desmouliere et al., 1995), a cytological marker for apoptosis or programmed cell death (Williams, 1991), as well as other signs of apoptosis. These results in studies of cutaneous wounds and other results in studies of lungs and kidney suggest that apoptosis is the mechanism responsible for the transition from a fibroblastrich granulation tissue to a relatively acellular scar (Desmouliere et al., 1997). The signal(s) for wound fibroblast apoptosis have not been elucidated. Thus fibroplasia in wound repair is tightly regulated, whereas, in fibrotic diseases such as keloid formation, morphea, and scleroderma, these processes become dysregulated.

Research in Neovascularization
Fibroplasia would halt if neovascularization failed to accompany the newly forming complex of fibroblasts and extracellular matrix. The process of new blood vessel formation is called angiogenesis (Madri et al., 1996), and has been extensively studied in the chick chorioallantoic membrane and the cornea (Folkman and Shing, 1992). The soluble factors that can stimulate angiogenesis in wound repair are gradually being elucidated (Roesel and Nanney, 1995), but the factors that do stimulate wound angiogenesis are less clear. Angiogenic activity can be recovered from activated macrophages as well as various tissues, including the epidermis, soft tissue wounds, and solid tumors. Some years ago acidic or basic fibroblast growth factor (aFGF and bFGF) appeared to be responsible for most of these activities (Folkman and Klagsbrun, 1987), but other molecules have now also been shown to have angiogenic activity. These include vascular endothelial growth factor (VEGF) (Keck et al., 1989), TGF- (Yang and Moses, 1990), TGF- (Schreiber et al., 1986), tumor necrosis factor=(TNF- (Leibovich et al., 1987), platelet factor 4 (PF4) (Montrucchio 1994), angiogenin (Vallee and Riordan, 1997), angiotropin (Hockel et al., 1988), angiopoietin (Suri et al., 1996), interleukin-8 (Koch et al., 1992), PDGF (Battegay et al., 1994), thrombospondin (Nicosia and Tuszynski, 1994), low-molecular-weight substances, including the peptide KGHK (Lane et al., 1994), low oxygen tension, biogenic amines, and lactic acid (Folkman and Shing, 1992), and nitric oxide (NO) (Montrucchio,1997). Some of these factors, however, are intermediaries in a single angiogenesis pathway, for example TNF- induces PF4, which stimulates angiogenesis through NO (Montrucchio et al., 1997). Angiogenesis cannot be directly related to proliferation of cultured endothelial cells. In fact, Folkman (Folkman and Shing, 1992) has postulated that endothelial cell migration can induce proliferation. If this is true, endothelial cell chemotactic factors may be critical for angiogenesis. Some factors, of course, may have both mitogenic and chemotactic activities. For example, PDGF (Senior et al., 1985) and EGF (Chen et al., 1994) can be either chemotactic factors or mitogenic factors for dermal fibroblasts. Besides growth factors and chemotactic factors, an appropriate extracellular matrix is also necessary for angiogenesis. Three-dimensional gels of extracellular matrix proteins have more pronounced effects on cultured endothelial cells than do monolayer protein coats (Feng et al., 1999a), as has been observed with smooth muscle cells (Koyama et al., 1996). Rat epididymal microvascular cells cultured in type I collagen gels with TGF- produce capillary-like structures within 1 week (Madri , 1988). Omission of TGF- markedly reduces the effect. In contrast, laminincontaining gels in the absence of growth factors induce human umbilical vein and dermal microvascular cells to produce capillary-like structures within 24 hr of plating (Kubota et al., 1988). Matrix-bound thrombospondin also promotes angiogenesis (Nicosia and Tuszynski, 1994), possibly through its ability to activate TGF- (Schultz-Cherry, Murphy-Ullrich, 1993). We have developed a sprouting angiogenesis assay using neonatal human dermal microvascular endothelial cells (HDMECs) and have shown that fibrin, but not collagen, gels support sprouting angiogenesis (Feng, 1999b). These studies support the hypothesis that the extracellular matrix plays an important role in angiogenesis. Consonant with this hypothesis, angiogenesis in the chick chorioallantoic membrane is dependent on the expression of v3, an integrin that recognizes fibrin and fibronectin, as well as vitronectin (Brooks, 1994a). Furthermore, in porcine cutaneous wounds v3 is expressed only on capillary sprouts as they invade the fibrin clot (Clark, 1996b). In vitro studies in fact demonstrate that v3 can promote endothelial cell migration on provisional matrix proteins (Leavesley, 1993).

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Given the information outlined above, a series of events leading to angiogenesis can be hypothesized. Substantial injury causes tissue cell destruction and hypoxia. Potent angiogenesis factors such as FGF-1 and FGF-2 are released secondary to cell disruption whereas VEGF is induced by hypoxia. Proteolytic enzymes released into the connective tissue degrade extracellular matrix proteins. Specific fragments from collagen, fibronectin, and elastin, as well as many phylogistic agents, recruit peripheral blood monocytes to the injured site, where these cells become activated macrophages that release more angiogenesis factors. Certain angiogenic factors, such as FGF-2, stimulate endothelial cells to release plasminogen activator and procollagenase. Plasminogen activator converts plasminogen to plasmin and procollagenase to active collagenase and in concert these two proteases digest basement membrane constituents. The fragmentation of the basement membrane allows endothelial cells to migrate into the injured site in response to FGF, fibronectin fragments, heparin released from disrupted mast cells, and other endothelial cell chemoattractants. To migrate into the fibrin/fibronectin-rich wound, endothelial cells express v3 integrin. The newly forming blood vessels first deposit a provisional matrix containing fibronectin and proteoglycans, but ultimately form basement membrane. TGF- may induce endothelial cells to produce the fibronectin and proteoglycan provisional matrix as well as assume the correct phenotype for capillary tube formation. FGF and other mitogens such as VEGF stimulate endothelial cell proliferation, resulting in a continual supply of endothelial cells for capillary extension. Capillary sprouts eventually branch at their tips and join to form capillary loops through which blood flow begins. New sprouts then extend from these loops to form a capillary plexus. Within a day or two after removal of angiogenic stimuli, capillaries undergo regression as characterized by mitochondria swelling in the endothelial cells at the distal tips of the capillaries, platelet adherence to degenerating endothelial cells, vascular stasis, endothelial cell necrosis, and ingestion of the effete capillaries by macrophages. Although v3 has been shown to regulate apoptosis of endothelial cells in culture and in tumors (Brooks et al., 1994b), v3 is not present on wound endothelial cells as they undergo programmed cell death, indicating another pathway of apoptosis in healing wound blood vessels (X. Feng, R.A.F. Clark, and M. G. Tonnesen, unpublished observations). Thrombospondin appears to be a good candidate for this phenomenon (DiPietro et al., 1996).

Research Angigenic properties of Demograft

Tissue-engineered fibroblast-based tissues are capable of inducing rapid endothelialization and vascularization. Providing such biologically active natural materials has been clinically shown to induce new capillary formation and reduce inflammation in the wound bed of patients with diabetic foot ulcers ( Jiang and Harding, 1998; Newton 1999). The tissue-engineered implants secrete a variety of growth factors known to be critical to tissue regeneration and angiogenesis . The angiogenic properties of our fibroblast-based engineered tissue have been investigated using a range of techniques, including the chick chorioallantoic membrane assay, the rat aortic ring assay, stimulation of endothelial cell proliferation, chemokinesis, chemotaxis, and the inhibition of apoptosis. These assays cover a wide range of the important individual steps in angiogenesis as well as the overall process. Analysis of our tissue-engineered human matrix has shown it to contain several components that may be beneficial in neovascularization. Fibronectin present in the matrix has been shown to stimulate the proliferation of endothelial cells, and the denatured collagen has been proved to be a favorable substrate for human endothelial cell attachment. Bound growth factors in the matrix include transforming growth factor (TGF-_) and hepatocyte growth factor (HGF), which are important in stimulating new capillary formation and endothelialization. Finally, the matrix contains laminin-1, which can serve to inhibit initial hyperplasia via the YIGSR peptide. The combination of these matrix proteins along with naturally secreted growth factors offers a physiologic solution to promoting the in vivo induction of angiogenesis.

Therapeutics that modulate this balance may be beneficial in patients suffering from fibrotic diseases. Drugs that directly inhibit TGF-b1 and IL-13 might prove the safest and most effective approach.

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TH-2 cytokines IL-4 and IL-13 lead to alternative activation of macrophages

Macrophages differentiate into at least two functionally distinct populations depending on whether they are exposed to TH-1 or TH-2 cytokines TH-1 cytokine activate NOS2 in classically activated macrophages whereas TH-2 cytokines IL-4 and IL-13 preferentially stimulate Arginase-1 (ARG1) leading to an alternative activation pathway ARG1 promotes the generation of polyamines and L-proline via metabolism of L-arginine to L-ornithine and activation of ODC and OAT Polyamines are crucial for cell growth and L-proline is a substrate for collagen synthesis A balance between TH-2 and TH-1 cytokines is necessary to promote healing but inhibit excessive fibrotic tissue remodeling.

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SKIN TISSUE ENGINEERING

Normal human skin performs a wide variety of function, which are perceptive, provide a viable interface with the individuals environment, protection from water and electrolyte loss, infection, protection from heat and cold, mechanical friction, chemicals, UV radiation, responsible for a substantial part of the thermoregulatory and communication that help the body maintain homeostasis, including the transduction of signals from the environment such as touch, pressure, and temperature, transmits important emotional signals to the environment, such as paleness or blushing of the face and the mission of scents (pheromones), being a passive membrane that keeps the internal organs in shape. Normal Skin contain : Schematic view skin which highlights epidermis, the basement The Epidermis, The outermost layer of the skin, a the membrane interleaved between 0.1-mm thick sheet is comprised : - Ten layers of keratinocytes, which provide the barrier epidermis and dermis, and the dermis underneath. Only a small fraction of function of the skin. the thickness of the dermis is shown. - Other epidermal cells and structures include : Adnexal cells that comprise (Redrawn with permission from J. Darnell, J. Lodish, and D. Baltimore, the glands, hair, and nails; Melanocytes, which Molecular Cell Biology, Scientific American Books, New York, Chapter 5, contribute pigment; Langerhans cells of the Fig. 552, 1986.) immune system; Sensory structures of nerves. The epidermis contains only : Very small amounts of extracellular matrix, predominantly carbohydrate polymers and stratum corneum lipids that form a barrier to permeability of aqueous fluids. The Dermis, consists mainly of extracellular matrix molecules, a 2- to 5-mm-thick layer of vascularized and innervated connective tissue with very few cells, Dermis is a massive tissue, accounting for 15 to 20% of total body weight. contain Collagens, Elastin,Polysaccharides,and Reticulin that give mechanical strength to the skin. Dermal cells include fibroblasts, which synthesize collagen and Other matrix components; Vascular components, such as Endothelial cells and smooth muscle cells Nerve cells; and Mast cells of the immune system. o Interleaved between epidermis and dermis is the basement membrane, approximately 20-nmthick multilayered. o A fourth layer, the subcutis, underneath the dermis and 0.4- to 4-mm in thickness, comprises primarily fat tissue. o Skin contains appendages (adnexa),including hair follicles, sweat and sebaceous glands.

1. OBJECTIVES OF SKIN SUBSTITUTES

A substantial deficit in the integrity of skin leaves the individual unprotected either from shock, the result of excessive loss of water and electrolytes, or from sepsis, the result of a massive systemic infection. The use of skin substitutes may be suitable as an adjunctive therapy in cases without other alternatives, such as very large burns or chronic wounds that failed to respond to conventional treatments. Ideally, skin replacements should promote permanent engraftment without the need for regrafting; allow rapid healing to replace both dermal and epidermal layers; be ready to use when needed; achieve acceptable functional and cosmetic outcome; and be free from risk of disease transmission and immunological reaction . These many goals have not yet been satisfied by any skin substitute, but ongoing research in biomedical and genetic engineering may someday make an off-the-shelf skin replacement a reality. To be useful in a clinical setting, the primary goal for any skin substitute is restoration of skin barrier, normally a function of the epidermis, which helps to minimize protein and fluid loss and prevent infection. There are many products that meet this need, but most provide only temporary wound coverage or partial skin replacement. Currently, limitations of tissue engineering aritificial skin, compared with native skin grafts, include: Reduced rates of engraftment,

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Increased microbial contamination, Mechanical fragility, Increased time to healing, Increased requirement for regrafting, and Very high cost.

HISTORY OF SKIN REPLACEMENT

Thirty years ago, burn surgeons determined that badly burned skin should be removed as quickly as possible, followed by immediate and permanent replacement of the lost skin. Doctors are able to take a postage stamp-sized piece of skin from the patient and grow the skin under special tissue culture conditions. From small piece of skin, technicians can grow enough skin to cover nearly the entire body in just 3 weeks Although attempts to cover wounds and treat severe burns is cited as far back as 1500 B.C., it has only been in the past few centuries that a significant number of solutions have emerged. The bulk of these solutions involve using skin grafts from humans (allografts) or animals (xenografts), or using membranes fabricated from natural or synthetic polymers. The first synthetic skin was invented by John F. Burke, V. Yannas, at the Massachusetts Institute of Technology. 1979 Burke and Yannas used their artificial skin on first patient, a woman whose burns covered over half her body Integra is the first FDA approved product for burn and reconstructive surgery. Patented on August 14, 1990 When skin is damaged or lost due to severe injury or burns, bacteria and other microorganisms have easy access to warm, nutrient-rich body fluids. To treat a severe burn, surgeons first remove the burned skin and then quickly cover the underlying tissue, usually with a combination of laboratory-grown skin cells and artificial skin.

A Literary Review
Wayne R. Fischer.ME 597 Introduction to Solid Biomechanics Boise State University. May 8th, 2003 The best material for wound closure is the o The donor site is a new wound. patients own skin; however autografting has o Scarring and pigmentation changes several disadvantages (Schulz, 2000) occur. o Dermis is not replaced. o Donor site is a potential site for infection. o Donor site is not unlimited. o Extensive burns makes it impossible Cadaver Skin: Allograft as The annual national requirement for cadaver skin is estimated to be only a Temporary Skin 3000 m2. Substitute Yet only 14% to 19% of human skin needed is being recovered. Xenografts, particularly porcine skin grafts, are commercially available and are an effective means of short-term wound closure (Yannas, 1980). Xenograft is normally removed on the third or fourth day of use before extensive adhesion onto the wound bed sets in, thereby necessitating its traumatic excision prior to drying and sloughing off (Yannas, 1980). Observations from designing dermal replacements (Schulz, 2000) o The thicker the dermal layer of a split-thickness skin graft, the less the graft contracts. o Partial-thickness wounds with superficial dermal loss heal with less hypertrophic scarring. o Full-thickness skin grafts contract minimally. o The length of illness in burn cases is essentially restricted to the length of time the burn wound is open. o Full-thickness dermal injuries heal by contraction and hypertonic scarring, producing subepithelial scar tissue that is nothing like the original dermis. Research topics of Dr. Yannas at Dept. of Engineering, MIT Study the mechanical behavior of artificial skin as a function of processing variables. Study the surface tension of artificial skin. Study the stress relaxation rate of artificial skin in standardized solutions of tissue enzymes. Study the design of novel processes for the inexpensive and reproducible fabrication of artificial skin. Study the pore structure of artificial skin by scanning electron microscopy. Study the moisture permeability of artificial skin. Synthetic o The use of synthetic polymers has not so far led to the solution of the problem of a Polymers skin substitute. (Yannas, o A high incidence of infection and a relatively low capacity for inducing vascularisation

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1980)

and epithelialisation are frequently reported. o However, useful insights into the requirements for a satisfactory skin replacement have been discovered through the use of synthetic polymers.

Literary Review Up to 1990s (Beele, 2002) o Antiquity: Indian description of using autologous soft tissue flaps. o Greeks used dressings for skin wounds. o Renaissance: Amboise-Pare provide wound healing foundation. o 1850s: Reverdin and Thiersch use autologous skin grafts. o 1914: Kreibich was the first person to cultivate keratinocytes in vitro. o 1948: Medawar autotransplanted keratinocytes. o 1960s: Yannas and Burke begin their work using materials science mechanics. o 1975: Rheinwald & Green describe a technique to cultivate human keratinocytes. o 1980s: Yannas and Burke describe a bilaminate collagen-glycosaminoglycan matrix with a silicon surface. After take of the matrix,silicon surface is removed and can be replaced with autologous cultured epidermal cells. o 1981: Bell constructs first living skin equivalent with collagen fibroblast gel with keratinocytes cultured on top of contracted gel. o 1983: Helton used cultured allografts in burn patients o 1985: Boyce and Ham introduce an alternative culturing method. o 1989: Possible to cryo-preserve keratinocyte sheets. (Buras, 1989) The actual biological elements and events being critically tested in mechanical studies are only guessed at, and analysis can rarely go beyond the science of mechanics. There are promising possibilities: Pulsed ultrasound techniques may soon provide accurate imaging of skin structures as well as measurements of blood flow in the skin. The multifrequency shear wave method may be able to resolve mechanical properties of the epidermal tissues discretely.

DESIGN PRINCIPLES PERMANENT SKIN REPLACEMENT

Controlled loss of epidermis in a laboratory experiment with an animal can result from the repeated use of adhesive tape to peel off the keratinocyte layers. In either case, the long-term outcome is an apparently faithful regeneration of the epidermis by migration of epithelial cells from the wound edge, and from roots of hair follicles, over the underlying basement membrane and dermis. Epidermis can regenerate spontaneously provided a dermal substrate over which epithelial migration and eventual anchoring to the underlying connective tissue can occur. The depth of skin loss is a critical parameter in the design of a treatment for a patient who has a skin deficit. In the treatment of burns, physicians distinguish among : A first-degree burn (loss of epidermis alone) A second-degree burn (loss of epidermis and A fraction of the thickness of the dermis), and a third-degree burn (loss of the epidermis and the entire dermis down to muscle tissue). The analysis of the plight of the patient who has suffered extensive skin loss leads logically to a wound cover which treats the problem in two stages. Stage 1 is the early phase of the clinical experience, one in which protection against severe fluid loss and against massive infection are defined as the major design objectives. Stage 2 is the ensuing phase, one in which the patient needs protection principally against disfiguring scars and crippling contractures. The sequential utilization of features inherent in stages 1 and 2 in a single device can be ensured by designing the graft as a bilayer membrane. In this approach, the top layer incorporates the features of a stage 1 device, while the bottom layer delivers the performance expected from a stage 2 device. The top layer is subject to disposal after a period of about 10 to 15 days, during which time the bottom layer has already induced substantial synthesis of new dermis. Following removal of the top layer, the epidermal cover is provided either by covering with a thin epidermal graft or by modifying the device (cell seeding) so that an epidermis forms spontaneously by about 2 weeks after grafting.

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Schematic of the bilayer membrane which has become known as the artificial skin. The top layer is a silicone film which controls moisture flux through the wound bed to nearly physiologic levels, controls infection of the wound bed by airborne bacteria, and is strong enough to be sutured on the wound bed. The bottom layer is the skin regeneration template, which consists of a graft copolymer of type I collagen and chondroitin 6-sulfate, with critically controlled porosity and degradation rate. About 14 days after grafting, the silicone layer is removed and replaced with a thin epidermal autograft. The bottom layer induces synthesis of a nearly physiologic dermis and eventually is removed completely by biodegradation. (From Yannas IV, Burke J.F., Orgill D.P., et al. 1982. Science215: 74.)

3.1 GENERAL DESIGN PROPERTIES

Essential Design Properties "The dermal replacement should provide both the information necessary to control the inflammatory and contractile processes and also the information necessary to evoke ordered recreation of autologous tissue in the form of a neodermis" (Schulz, 2000). "The initial replacement material should provide immediate physiologic wound closure and be eliminated once it has provided sufficient information for neodermis reconstitution of " (Schulz). It should protect the wound by providing a barrier to the outside (Beele, 2002) It should control water evaporation and protein and electrolyte loss and It should limit excessive heat loss (Beele) It should decrease pain and allow early mobilization and It should provide an environment for accelerated wound healing (Beele) and The risk of infection must be taken into account (Beele) Physical Characteristics It should be easy to manipulate the product, i.e. easy to place and dress the skin substitute effectively (Beele) It should improve the cosmetic appearance of the scar (Beele) Availability It should be readily available off the shelf and custom made. Cost Cost should not preclude the use of the device. Biological skin substitutes The epidermis injuries are healed by regeneration of the epidermis The migration of keratinocytes from the periphery of the wound and the proliferation would lead to the total healing without scars However if dermis is injured recovery is harder since the dermis cannot regenerate In order to shorten the healing process or abolish the side effects, skin substitutes should be used temporarily or permanently. Adhere to the substrate Be durable and sufficiently elastic to tolerate some deformation Allow evaporative water loss at the rate typical of the external layer Have optimal water permeability to prevent either desiccation of the wound or fluid accumulation under the covering Skin substitutes for wound closure Skin substitutes for wound cover Wound closure requires a material to restore the epidermal barrier function and become incorporated into the healing wound Wound cover necessitates a material which relies in the in growth of granulation for adhesion. They are used in superficial burns Table Design Criteria for Tissue-Engineered Bilayer Skin Substitute

Desired properties for skin substitutes

Categories of skin substitutes

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STAGE 1 DESIGN PARAMETERS

The overriding design requirement at this stage is based on the observation that air pockets (dead space) at the graft-wound bed interface readily become sites of bacterial proliferation. Such sites can be prevented from forming if the graft surface wets, in the physicochemical sense, the surface of the wound bed on contact and thereby displaces the air from the graft-tissue interface. It follows that the physicochemical properties of the graft must be designed to ensure that this leading requirement is met, not only when the graft is placed on the wound bed but for several days thereafter, until the function of the graft has moved clearly into its stage 2, in which case the graft-wound bed interface has been synthesized de novo and the threat of dead space has been thereby eliminated indefinitely. (a) The graft (cross-hatched) does not displace air pockets (arrows) efficiently from the graft-wound bed interface. (b) Flexural rigidity of the graft is excessive. The graft does not deform sufficiently, under its own weight and the action of surface forces, to make good contact with depressions on the surface of the wound bed; as a result, air pockets form (arrows). (c) Shear stresses t (arrows) cause buckling of the graft, rupture of the graft-wound bed bond and formation of an air pocket. (d) Peeling force P lifts the graft away from the Certain physicochemical and mechanical wound bed. requirements in the design of an effective (e) Excessively high moisture flux rate J through closure for awound bed with full-thickness skin the graft causes dehydration and development of loss. (From Yannas I.V. and Burke J.F. 1980. J. shrinkage stresses at the edges (arrows), which Biomed. Mater. Res. 14: 65.) cause lift-off away from the wound bed. (f) Very low moisture flux J causes fluid accumulation (edema) at the graft-wound bed interface and peeling off (arrows). First, the flexural rigidity of the graft, that is, the product of Youngs modulus and moment of inertia of a model elastic beam, must be sufficiently low to provide for a flexible graft which drapes intimately over a geometrically non uniform wound bed surface and thus ensures that the two surfaces will be closely apposed. In practice, these requirements can be met simply by adjusting both the stiffness in tension and the thickness of the graft to appropriately low value. Second, the graft will wet the wound bed if the surface energy of the graft-wound bed interface is lower than that of the air-wound bed surface, so that The graft can adequately displace air pockets from the air-wound bed surface. Although the measurement of a credible value of the surface energy is not a simple matter when the graft is based on certain natural polymers in the form of a hydrated gel, the requirement of adequate adhesion can be met empirically by chemical modification of the surface or by proper use of structural features such as porosity. Third, the moisture flux through the graft must be maintained within bounds which are set by the following considerations. The upper bound to the moisture flux must be kept below the level where excessive dehydration of the graft occurs, thereby leading to alteration of the surface energy of the graft wound bed interface and loss of the adhesive bond between graft and wound bed. Further, when the moisture flux exceeds the desired level, the graft is desiccated, and shrinkage stesses develop which pull the graft away from the wound bed. An estimate of the maximum normal stress sm can be obtained by modeling the desiccating graft in one dimension as a shrinking elastic beam bonded to a rigid surface m = 0.45(V2-V1)E . is the coefficient of expansion of a graft which swells in water, V1 and V2 are initial and final values of the volume fraction of moisture in the graft, and E is Youngs modulus of the graft averaged over the range V1 to V2, the latter range being presumed to be narrow. If, by contrast, the moisture flux through the graft is lower than the desired low bound, water accumulates between the graft and the wound bed, and edema results with accompanying loss of the adhesive bond between the two surfaces.

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STAGE 2 RATIO TIME CONSTANT

The leading design objectives in this stage are two: synthesis of new, physiologic skin and the eventual disposal of the graft. The lifetime of the graft, expressed as the time constant of biodegradation tb, was modeled in relation to the time constant for normal healing of a skin incision th,is about 25 days. In preliminary studies with animals, it was observed that when matrices were synthesized to degrade at a very rapid rate, amounting to tb << th , the initially insoluble matrix was reduced early to a liquidlike state, which was incompatible with an effective wound closure. At the other extreme, matrices were synthesized which degraded with exceptional difficulty within 3 to 4 weeks, compatible with tb<< th. In these preliminary studies it was observed that a highly intractable matrix, corresponding to the latter condition, led to formation of a dense fibrotic tissue underneath the graft which eventually led to loss of the bond between graft and wound bed. Accordingly, it was hypothesized that a rule of isomorphous matrix replacement, equivalent to assuming a graft degradation rate of order of magnitude similar to the synthesis rate for new tissue, and represented by the relation (tb / th) = 1, would be optimal. Control of tb is possible by adjustment of the crosslink density of the matrix. Equationis the defining equation for a biodegradable scaffold which is coupledwith, and therefore interacts with, the inflammatory process in a wound. The ratio of the time constant of biodegradation to the time constant for normal healing of a skin incision ideally must be unity Adequate Moisture Flux

Migration of cells into the matrix is necessary for synthesis of new tissue. Such migration can proceed very slowly, defeating Equation, when fibroblasts and other cells recruited belowthewound surface are required to wait until degradation of a potentially solid-like matrix has progressed sufficiently. An easier pathway to migrating cells can be provided by modifying a solid-like matrix into one which has an abundance of pore channels, where the average pore is at least as large as one cell diameter (about 10 m) for ready access. Although this rationale is supported by experiment, results with animal studies have shown that not only is there a lower limit to the average pore diameter, but there is also an upper limit. Migration of cells into the porous graft can proceed only if nutrients are available to these cells. Two general mechanisms are available for transport of nutrients to the migrating cells, namely, diffusion from the wound bed and transport along capillaries which may have sprouted within the matrix (angiogenesis). Since capillaries would not be expected to form for at least a few days, it is necessary to consider whether a purely diffusional mode of transport of nutrients from the wound bed surface into the graft could immediately supply the metabolic needs of the invading cells adequately. The cell has been modled as a reactor which consumes a critical nutrient with a rate r, in units of mol/cm3/sec; the nutrient is transported from the wound bed to the cell by diffusion over a distance l, the nutrient concentration at or near the surface of the wound bed is c 0, in units of mole/cm3, and the diffusivity of the nutrient is D in cm2/sec. The appropriate conditionswere expressed in terms of a dimensionless number S, the cell lifeline number, which expresses the relative importance of reaction rate for consumption of the nutrient by the cell to rate of transport of the nutrient by diffusion alone: S = rlc2/D c0 . This equation suggests that when S = 1, the critical value of the path length, lc, corresponds to the maximum distance along which cells can migrate inside the graft without requiring angiogenesis (vascularization) for nutrient transport. The value of lc defines the maximum thickness of graft that can be populated with cells within a few hours after grafting, before angiogenesis has had time to occur. These conceptual objectives have been partially met by designing the graft as an analog of extracellular matrix (ECM) which possesses morphogenetic activity since it leads to partial regeneration of dermis. The discovery of the specific ECM analog that possesses this activity has been based on the empirical observation that, whereas the vast majority of ECM analogs apparently do not inhibit wound contraction almost at all, one of the analogs does. The activity of this analog, for which the term regeneration template has been coined, is conveniently detected as a significant delay in the onset of wound contraction.When seeded with (uncultured) autologous keratinocytes, an active regeneration template is capable of inducing simultaneous synthesis both of a dermis and an epidermis in the guinea pig and in the swine (Yorkshire pig). The regeneration is almost complete; however, hair follicles and other skin adnexa are not formed. The resulting integument performs the two vital functions of skin, that is, control of infection and moisture loss, while also poviding physiologic mechanical protection to the internal organs and, additionally, providing a cosmetic effect almost identical to that of intact skin. The morphogenetic specificity of the dermis regeneration template depends sensitively on retention of certain structural characteristics. The overall structure is that of an insoluble, three-dimensional covalently crosslinked network. The primary structure can be described as that of a graft-copolymer of type I collagen and a glycosaminoglycan (GAG) in the approximate ratio 98/2. The GAG can be either chondroitin 6-sulfate or dermatan sulfate; other GAGs appear capable of contributing approximately

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equal increments to morphogenetic specificity. The collagen fibers lack banding almost completely although the integrity of the triple helical structure is retained through the network. The resistance of the network to collagenase degradation is such that approximately two-thirds of the mass of the network becomes solubilized in vivo within about 2 weeks. The structure of the network is highly porous. The pore volume fraction exceeds 95% while the average pore diameter is maintained in the range 20 to 125 m. The regeneration template loses its activity rapidly when these structural features are flawed deliberately in control studies. The dermis regeneration template, a porous matrix unseeded with cells, induces synthesis of a new dermis and solves this old surgical problem. Simultaneous synthesis of a new, confluent epidermis occurs by migration of epithelial cell sheets from the wound edges, over the newly synthesized dermal bed. With wounds of relatively small characteristic dimension, for example, 1 cm, epithelial cells migrating at speeds of about 0.5 mm/day from each wound edge can provide a confluent epidermis within 10 days. In such cases, the unseeded template fulfills all the design specifications set above. However, the wounds incurred by a massively burned patient are typically of characteristic dimension of several centimeters, oftenmore than 20 to 30 cm. These wounds are large enough to preclude formation of a new epidermis by cell migration alonewithin a clinically acceptable timeframe, say 2weeks.Wounds of that magnitude can be treated by seeding the porous collagen-GAG template, before grafting, with at lest 5 104 keratinocytes per square centimeter wound area. These uncultured, autologous cells are extracted by applying a cell separation procedure, based on controlled trypsinization, to a small epidermal biopsy. Details of the synthesis of the dermis regeneration template, as well as of other templates which regenerate peripheral nerves and the knee meniscus, are presented elsewhere in this handbook. The dermis regeneration template was first reported as a synthetic skin and as an artificial skin.

CURRENT TREATMENT OF SKIN LOSS

There are two different types of wounds stimulated the development of various tissue engineered skin substitutes: - The burn wounds represent an acute injury to the skin. Partial-thickness wounds have the capacity to heal without the need for tissue replacement from stem cells present in skin appendages. However, full-thickness burn wounds require grafting of skin to replace the destroyed tissue. The grafting of splitthickness autologous skin has been the prevailing standard for permanent closure of excised fullthickness burn wounds accomplished in patients with relatively small wounds, but in patients with massive burns involving a large total body surface area (TBSA), permanent wound closure is problematic because of the lack of donor sites for skin autografting. Delayed wound coverage increases the likelihood of infection and sepsis, causes of burn mortality.

- The chronic wounds is tend to involve a relatively small area of skin, but represent a major medical

need because they affect a large population of patients. Chronic wounds occur when the normal healing process cannot proceed at its proper pace and reach its conclusion. Various genetic or acquired conditions, such as epidermolysis bullosa, old age, diabetes and bacterial burden, can lead to effects such as prolongation of the inflammatory stage, slower cell proliferation, impaired cell migration, imbalance of matrix metalloproteinases relative to their inhibitors, reduced growth factor availability and ensuing reduced cellular response. Such wounds thus present special problems for treatment that can go beyond the capacity of acellular skin substitutes for optimal healing. The most common chronic wounds include pressure ulcers and leg ulcers. In some patients, wound closure can be enhanced with topical agents, such as growth factors to stimulate healing and antimicrobial agents to minimize infection. However, a large percentage of patients require grafting for permanent closure of chronic wounds. Autograft may not be a feasible option in these patients due to underlying deficiencies in wound healing, which compromise healing of donor sites. The need for timely wound closure in these diverse clinical settings has led to the development of skin substitutes as alternatives to split-thickness or full-thickness autograft. Although none of the skin substitutes currently available can replace all of the structures and functions of native skin, they can be used to provide wound coverage and facilitate healing of acute and chronic wounds. Further, the technologies that have yielded skin substitutes for grafting have also been used to produce materials for in vitro irritancy and toxicology studies.

The comparison number of approaches to treat chronic nonhealing wounds resulted in the conclusion that bilayer skin substitutes are efficacious for chronic wounds and especially so for venous leg ulcers. Also, the crucial role of keratinocytes for wound closure was pointed out. Rapid replacement of the epidermal layer is important to restore, as fast as possible, control over fluid loss, body temperature and protection against bacteria. Epidermal or bilayer substitutes that do not provide autologous keratinocytes or immunocompatible living tissue, such as cadaveric skin, xenografts and inert membranes like silicone, can only provide temporary coverage. They are indicated if availability an expected healing speed make them the best or the only option at the beginning of the treatment. However, live keratinocytes have to be present to achieve permanent wound coverage and restore the epidermal barrier. With epidermal substitutes, the presence of autologous living cells is thus necessary

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for proper healing of extensive areas where spontaneous epithelialization is impossible or would take too long to complete.

NATURAL AND SYNTHETIC POLYMER

The treatment of skin loss has traditionally focused on the design of a temporary wound closure include is membranes or sheets fabricated from Natural and synthetic polymers. Polymeric membranes which lack specific biologic activity, such as synthetic polymeric hydrogels, have to be removed after several days due to incidence of infection and lack of formation of physiologic structures.

HUMAN HOMO/ALLOGRAFTS ANIMALS HETERO/XENOGRAFTS

Skin grafts from human cadavers (homografts, or allografts) or from animals (heterografts, or xenografts). Allograft is a human cadaver skin, which is frequently stored in frozen state in a skin bank. It is a temporary cover. If left on the wound longer than about 2 weeks, allograft is rejected by the severely burned patient, even though the latter is in an immune compromised condition. When rejection is allowed to occur, the wound bed is temporarily ungraftable and is subject to infection. In a modification of this basic use of the allograft, the latter has been used as a dermal equivalent prior to grafting with cultured epithelia. Since the allograft is rejected if allowed to remain on the wound long enough for the epithelia to spread accoss the wound bed, the allograft has been treated in a variety of media in an effort to eliminate its immunogenicity. o Have greater similarity to native skin at grafting, considered as temporary skin substitutes. Eventually replaced by host-derived cells. Secrete extracellular matrix and growth factors that improve healing, and can provide wound coverage until autograft is available. o Advantages for small wounds which stimulate healing from the wound bed and margins without further grafting and immediate availability. o Patients with cadaver allografts and xenografts are frequently immunosuppressed to avoid rejection; It is a stop-gap operation which is eventually terminated by removal of the graft after several days. o Temporary dressings use to delay the time at which a permanent graft autograft, is necessary and are invaluable aids in the management of the massively injured patient. o The result of treatment of a third-degree burn with a split-thickness autograft is an almost fully functional skin which has become incorporated into the patients body and will remain functional over a lifetime. o Autografts usually lack hair follicles and certain adnexa as well. However, the major price paid is the removal of the split thickness graft from an intact area of the patients body. o The remaining dermis eventually becomes epithelialized but not without synthesis of scar over entire area of trauma (donor site). To alleviate the problem with the limited availability, surgeons have resorted to meshing, a procedure in which the sheet autograft is passed through an apparatus which cuts slits into the sheet autograft, allowing expansion of the graft by several times and thereby extending greatly the area of use. o An inevitable long-term result of use meshed autografts is scar synthesis in areas coinciding with the open slits of the meshed graft and a resulting pattern of scar which greatly reduces value of resulting new organ. o An important aspect of the use of the autograft is the requirement for early excision of dead tissue and the provision, thereby, of a viable wound bed to take the autograft.

AUTOLOGOUS CELLS
Skin grafts from contain Autologous cells Act as permanent skin for permanent closure of large wounds once engraftment is achieved. A disadvantage is the increased time required for cell culture and graft preparation. This increased time may be offset by a reduction in the number of surgical procedures required for permanent wound closure. By increasing availability of skin grafts, skin substitutes can provide several advantages over conventional therapy, including reduced donor site area required to close wounds permanently, decreased number of surgical procedures and hospitalization time, and reduced scarring.

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ENGINEERING SKIN TISSUE

FLAP is Any tissue used for reconstruction or wound closure that retains all or part of its original blood supply after the tissue has been moved to the recipient location. It maintains original blood supply. GRAFT is A skin graft is a tissue of epidermis and varying amounts of dermis that is detached from its own blood supply and placed in a new area with a new blood supply. Does not maintain original blood supply.

Grafts are typically described in terms of thickness or depth. directly related to the thickness of dermis in the graft.

The amount of primary contraction is

Split thickness contains 100% of the epidermis and a portion of the dermis. Split thickness grafts are further classified as thin or thick. Used when cosmetic appearance is not a primary issue or when the size of the wound is too large to use a full thickness graft, such as chronic ulcers, temporary coverage, correction of pigmentation disorders, and burns. Full thickness contains 100% of the epidermis and dermis. Indications for full thickness skin grafts include adjacent tissue which has premalignant or malignant lesions and precludes the use of a flap.

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Specific locations that lend themselves well to FTSGs include the nasal tip, helical rim, forehead, eyelids, medial canthus, concha, and digits. The processing phase duration is Phase 1 (0-48h) is phase when plasmatic Imbibition diffusion of nutrition from the recipient bed. Phase 2 is phase of inosculation, Vessels in graft connect with those in recipient bed. Phase 3 (day 3-5) is phase of Neovascular Ingrowth . Graft revascularized by ingrowth of new vessels into bed. Requirements for Survival besides bed must be well vascularized. The contact between graft and recipient must be fully immobile. As also Low bacterial count at the site. The routine treatment of skin wounds using engineered skin grafts has become a reality thanks to a firm scientific foundation established over the past 25 years, a readily available cell source (infant foreskin tissue), and the hard work and dedication of many individuals. Although the epidermis has an enormous capacity to heal, there are situations in which it is necessary to replace large areas of epidermis or in which normal regeneration is deficient. The dermis has very little capacity to regenerate. The scar tissue that forms in the absence of dermis lacks the elasticity, flexibility, and strength of normal dermis. Consequently, scar tissue limits movement, causes pain, and is cosmetically undesirable. Engineered tissues that not only close wounds but also stimulate the regeneration of dermis would provide a significant benefit in human wound healing.

DESIGN CONSIDERATIONS
Tissue engineering has not focused on regenerating certain skin structures, such as hair follicles or sebaceous glands, the loss of which is clinically less significant than the loss of dermis and epidermis, which are needed to cover and protect the underlying tissues. There is some preliminary evidence that sebaceous glands may be possible in the HSE (Wilkins et al., 1994), although the development of functioning adnexal structures is likely to be years away. There has also been little need to stimulate extraneously the regeneration of other dermal components (e.g., blood vessels and cells of the immune system) through tissue engineering methods because these components have the ability to repopulate quickly and to normalize the area of a wound. Langerhans cells, for example, have been shown to migrate and repopulate effectively within months (Desmouliere and Gabbiani, 1996). Control of vascularization is dependent on the makeup of the extracellular matrix and the degree of inflammation present in the wound. Whether modification of vascularization through the use of exogenous factors will be of additional benefit for certain wounds remains to be determined. Although it is technically possible to add melanocytes to HSE for pigmentation (L. Wilkins, personal communication), clinical studies using HSE, which essentially lacks melanocytes, have shown repigmentation of the grafted areas through repopulation of the area with host melanocytes, resulting in normal skin color for each individual. Therefore, tissue engineering approaches have primarily focused on providing or imitating structural and biologic characteristics of dermis, epidermis, or both. Some technologies, such as the HSE, have sought to reproduce living, full-thickness tissue for transplantation. The key features to be replicated in an engineered skin construct are as follows: 1. A dermal or mesenchymal element capable of aiding appropriate dermal repair and epidermal support. 2. An epidermis capable of easily achieving biologic wound closure. 3. An epidermis capable of rapid reestablishment of barrier properties. 4. A permissive milieu for the components of the immune system, nervous system, and vasculature. 5. A tissue capable of achieving normalization of structure and additional function such as reduction of long-term scarring and reestablishment of pigmentation.

EPIDERMAL REGENERATION
Reepithelialization of the wound is a paramount concern. Without epithelial coverage, no defense exists against contamination of the exposed underlying tissue or loss of fluid. The approaches to reestablishing epidermis are numerous, ranging from the use of cell suspensions to full-thickness skin equivalents possessing a differentiated epidermis. Silicone membranes have been used as temporary coverings in conjunction with dermal templates (Heimbach et al., 1988), but living epidermal keratinocytes are necessary to achieve permanent, biologic wound closure. Green et al. (1979; Phillips et al., 1989) developed techniques for growing human epidermal keratinocytes from small patient biopsy samples using coculture methods (Rheinwald and Green, 1975). The mouse 3T3 fibroblast feeder cell system allows substantial expansion of epidermal keratinocytes and can be used to generate enough thin, multilayered epidermal sheets to resurface the body of a severely burned patient (Gallico et al., 1984). Once transplanted, the epidermal sheets quickly form epidermis and reestablish epidermal coverage (Compton, 1993). With time, the cultured epithelial autograft (CEA) stimulates formation of new connective tissue (neodermis) immediately beneath the epidermis (Compton et al., 1989), but scarring and wound contraction remain significant problems (Sheridan and Tompkins, 1995). Studies have shown that grafting of CEA onto pregrafted cadaver dermis greatly improves graft take (Odessey, 1992). Cultured epithelial autografts (Epicel) have been available since the late 1980s.

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DERMAL REPLACEMENT
Human cadaver allograft skin has been used when autologous skin grafts are not possible. Problems associated with human cadaver allografts include the possibility of an immune rejection reaction, potential for infection, and problems of supply and variability in the quality of the material. Decellularized dermal tissue has also been used in an attempt to recapitulate as much of the normal architecture as possible while providing a natural scaffold for reepithelialization (Middelkoop et al., 1995; Langdon et al., 1988; Livesey et al., 1995; Cuono et al., 1986). Cadaver allograft dermis can be processed to make an immunologically inert, acellular dermal matrix with an intact basement membrane to aid the take and healing of ultrathin autografts (AlloDerm) (Lattari et al., 1997). Currently, only the upper papillary layer of dermis is used clinically (Allo- Derm). One limitation to this approach is that deep dermis and the more superficial papillary layer differ in architecture. The deep reticular dermis is needed to prevent wound contraction. Work is being done to develop a similar type of implant derived from deeper reticular dermis. Providing an appropriate scaffold for deep dermal repair remains a challenge for groups investigating native as well as synthetic matrices. Tissue engineering has investigated the possibility of redirecting granulation tissue formation through the use of scaffolds and livings cells. In one of the earliest tissue engineering approaches to improving dermal healing. Yannas et al. (1982) designed a collagenglycosaminoglycan sponge to serve as a scaffold or template for dermal extracellular matrix. The goal was to promote fibroblast repopulation in a controlled way that would decrease scarring and wound contraction. A commercial version of this material composed of bovine collagen and chondtroitin sulfate, with a silicone membrane covering (Integra, Integra Life Sciences, Plainsboro, NJ) is currently approved for use in burns (Burke et al., 1981; Heimbach et al., 1988). The dermal layer is slowly resorbed, and the silicone membrane is eventually removed, to be replaced by a thin autograft (Lorenz et al., 1997). Several variations on the collagen sponge have been studied. Efforts have been made to improve fibroblast infiltration and collagen persistence by collagen cross-linking (Middelkoop et al., 1995; vanLuyn et al., 1995; Cooper et al., 1996), by inclusion of other matrix proteins (Hansbrough et al., 1989; Ansel et al., 1990; de Vries et al., 1993; Murashita et al., 1996), with hyaluronic acid (Cooper et al., 1996), and by modifying porosity of the scaffold (Hansbrough et al., 1989; Yannas et al., 1989). Although matrix scaffolds have shown some improvement in scar morphology, no acellular matrix has yet been shown to lead to true dermal regeneration. This may be due in part to limits in cell repopulation, the type of fibroblast repopulating the graft (J. Gross, personal communication), and control of the inflammatory and remodeling processes (i.e., the ability of the cells to degrade old matrix while synthesizing new matrix). The inflammatory response must be controlled in dermal repair in order to avoid the formation of scar tissue. Therefore, dermal scaffolds must not be inflammatory and must not stimulate a foreign-body reaction. This has been a problem in the past for some glutaraldehyde crosslinked collagen substrates, for example (de Vries et al., 1993). The ability of the matrix to persist long enough to redirect tissue formation must be balanced with effects of the matrix on inflammatory processes. One way to achieve this is to form a biologic tissue that is recognized as living tissue, not a foreign substance. There have been advances in the design of artificially grown dermal tissues using human neonatal fibroblasts grown on rectangular sheets of biodegradable mesh (Dermagraft). The fibroblasts propagate among the degrading fibers, producing extracellular matrix in the interstices of the mesh (Hansbrough et al., 1992). Clinical trials of this material are ongoing and the material is commercially available in some European countries and Canada. A related product is a nonviable temporary covering for burns. In this case a nylon mesh, coated with porcine collagen, layered with a nonpermeable silicone membrane (Biobrane, Dow Hickam, Sugarland, TX), serves as a platform for deposition of human matrix proteins and associated factors by the human dermal fibroblast (Transcyte, Dermagraft-TC) (Hansbrough et al., 1997). The material is then frozen to preserve the matrix and factors produced by the fibroblasts. The temporary covering must be removed prior to autografting. It is commercially available for the treatment of second- and thirddegree burns (Hansbrough, 1997)

COMPOSITE SKIN GRAFTS

Human skin autograft has been the gold standard for resurfacing the body and closing wounds that are difficult to heal. Cultured epidermal grafts are more likely to take when the dermal bed is relatively intact, probably because dermal factors influence epithelial migration, differentiation, attachment, and growth (Cuono et al., 1987; Phillips et al., 1990; Clark, 1993; Greiling and Clark, 1997). The epidermis and dermis act synergistically to maintain homeostasis (Leary et al., 1992; Parenteau et al., 1997). Boyce et al. have modified the approach first proposed by Yannas et al. to form a bilayered composite skin made using a modified collagenglycosaminoglycan substrate seeded with fibroblasts and overlaid with epidermal keratinocytes (Boyce and Hansbrough, 1988). An autologous form of this composite skin construct has been used to treat severe burns with some success (Hansbrough et al., 1989). An allogeneic form of the construct showed improved healing in a pilot study in chronic wounds (Boyce et al., 1995). A similar technology has been studied in the treatment of patients with genetic blistering diseases (Eisenberg and Llewelyn, 1998). One of the first attempts to replicate a full-thickness skin graft was by Bell et al. (1981), who described a bilayered skin equivalent. The dermal component consisted of a lattice of type I collagen contracted by

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tractional forces of rat dermal fibroblasts trapped within the gelled collagen. This contracted lattice was then used alone or as a substrate for rat epidermal keratinocytes. Bells group demonstrated the ability of these primitive skin equivalents to take as a skin graft in rats. This technology has now advanced to enable the production of large amounts of human graft HSE from a single donor (Wilkins et al., 1994). Using methods of organotypic culture, which provides a three-dimensional culture environment that is permissive for proper tissue differentiation, the resulting HSE develops many of the structural, biochemical, and functional properties of human skin (Parenteau et al., 1992; Bilbo et al., 1993; Nolte et al., 1993). The process for formation of the HSE has been covered in detail many times (Parenteau, 1994; Wilkins et al., 1994) and will not be detailed here. However, there are points to be made about the approach to these procedures. The culture of the HSE proceeds best with minimal intervention. Normal cell populations seem to have an intrinsic ability to reexpress their differentiated program in vitro to an extent that is now only beginning to be appreciated (Parenteau, 1999). A medium that supplies adequate amounts of nutrients, lipid precursors, vitamins, and minerals may be all that is required (Wilkins et al., 1994). Another element is the environmental stimulus provided by culture at the airliquid interface, which promotes differentiation and formation of the epidermal barrier (Nolte et al., 1993). The immunology of allogeneic tissue-engineered skin grafts is poorly understood. Inconsistencies in the literature are due, in part, to the complexity of biologic and immunologic factors, which determine the ability of a graft to take and to persist over time. Among the properties that determine the immunogenicity of an engineered allogeneic graft are purity of cell populations, antigen-presenting capabilities of graft cells, and the vascularity of the graft. Purity of cell populations is critical. Differences in cell purity between laboratories may contribute to the conflicting results found in murine and human studies (Hefton et al., 1983; Thivolet et al., 1986; Geilen et al., 1987; Aubock et al., 1988; Cairns et al., 1993). Both culture condition and passage number will affect the purity of cell populations. Very early passages of keratinocyte and fibroblast cultures might be expected to contain contaminating cell populations. The antigen-presenting capabilities of keratinocytes and fibroblasts are also critical to determining the immunogenicity of the HSE grafts. Fibroblasts and keratinocytes are not professional antigen-presenting cells and fail to stimulate the proliferation of allogeneic T cells (Nickoloff et al., 1986; Niederwieser et al., 1988; Gaspari and Katz, 1991; Theobald et al., 1993). Both of these cell populations inherently do not express HLA class II molecules (Nickoloff et al., 1985) or costimulatory molecules such as B7-1 (Nickoloff et al., 1993). The inability of keratinocytes and fibroblasts to induce proliferation of allogeneic T cells is primarily due to the lack of expression of costimulatory molecules, even though aberrant antigen processing and invariant chain expression may also contribute (Nickoloff and Turka, 1994). The ability to utilize allogeneic cells rather than autologous cells, as in CEA therapy, enables the reproducible manufacture of consistent graf HSE (Wilkins et al., 1994). The inability of epidermal keratinocytes and dermal fibroblasts to stimulate a T cell response, discussed above, permits their use in allogeneic applications. Studies in athymic mice also indicate that the use of a differentiated tissue also beneficially affects its ability to engraft successfully (Nolte et al., 1994; Parenteau et al., 1996). Severe combined immunodeficient (SCID) mice lack a functioning immune system and can be successfully transplanted with a functioning human immune system without risk of rejection. SCID mice transplanted with human leukocytes were used as an in vivo model to assess the immunogenicity of allogeneic skin grafts (Moulon et al., 1999). In these studies, human skin was rejected but allogeneic tissue-engineered skin graft survival was 100%. Falanga et al. (1998) tested the safety, efficacy, and immunologic impact of HSE (Graftskin) in the treatment of venous leg ulcers. There were no signs of rejection or bovine collagen- specific immune responses or response to alloantigens expressed on keratinocytes or fibroblasts. HSE (Graftskin) has now been studied clinically in a number of applications, including chronic wounds (Sabolinski et al., 1996), dermatologic excisions, and burns. Its effectiveness in the treatment of venous leg ulcers has been attributed to its ability to interact with the wound in multiple ways (Sabolinski et al., 1996). The integrated tissue architecture of graf HSE provides a robustness needed for clinical manipulation. The pivotal study by Falanga et al. (1998) of graf HSE for the treatment of venous leg ulcers showed graf HSE to be safe and effective, healing more ulcers in shorter time when compared to conventional therapy. The living epidermal keratinocytes and the dermal fibroblasts produce cytokines, which serve to regulate themselves, each other, and the cells of the patient. The living dermal extracellular matrix contains collagen and glucosaminoglycans, which serve as noninflammatory living tissue implants and scaffolds for tissue remodeling. The stratum corneum provides physical protection and maintains the proper physical and biologic environment for wound closure. Application of graf HSE (Graftskin) can trigger a cascade of events that lead to normal healing even in the absence of graft take. In this regard, the HSE serves as a smart material, providing the appropriate factors as needed, responding to the dynamics of the wound healing process, and beneficially changing the condition of the wound, even though the HSE may not always persist as a graft. Our clinical observations and results lead us to believe that all factors are important contributors to the wound healing process (Sabolinskiet al., 1996).

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COMBINED DERMAL AND EPIDERMAL SUBSTITUTES

Bell was the first to describe a bilayered model of skin consisting of a collagen lattice with dermal fibroblasts that was covered with epidermal cells (Bell et al., 1981). Modification of this human skin equivalent (HSE) composite consisting of type I bovine collagen and live allogeneic human skin fibroblasts and keratinocytes has been developed (Apligraf; Organogenesis; Canton, MA). It has been used successfully in surgical wounds (Eaglstein et al., 1995) and venous ulcers (Sabolinski et al., 1996). In a large multicenter trial this product resulted in accelerated healing of chronic nonhealing venous stasis ulcers when compared to standard compressive therapy (Falanga et al., 1998). Several other composite skin substitutes combining dermal and epidermal elements have been developed. Composite cultured skin (CCS; Ortec International Inc.; New York, NY) is composed of both neonatal keratinocytes and fibroblasts embedded in distinct layers of bovine type I collagen. This product is being evaluated in clinical trials for the treatment of burns and in patients with epidermolysis bullosa. More extensive comparative data of the various biologic dressings have been published (Phillips, 1998).

EPIDERMAL AUTOGRAFTS AND ALLOGRAFTS

Immediate wound coverage, whether permanent or temporary, is one of the cornerstones of wound management. When the epidermis fails to heal normally or when a large surface area has been destroyed, supplemental epidermal coverage can be beneficial. Ideally, wound coverage would be achieved using autologous skin or material having qualities similar to those of autologous skin. Use of skin grafts, however, often requires extensive, invasive harvesting and is of limited quantity. Autologous cultured keratinocyte grafts were first used in humans by OConner et al. (1981). Subsequently there has been extensive experience with cultured epidermal grafts for the treatment of burns as well as other acute and chronic wounds (Gallico et al., 1984; Odessey, 1992; Munster, 1996). The major advantage of this technique is the ability to provide autologous grafts capable of covering large areas with reasonable cosmetic results. Another significant advantage of autologous grafts is their ability to serve as permanent wound coverage, because the host does not reject them. Disadvantages include the time interval of 23 weeks required before sufficient quantities of keratinocytes are available, the need for an invasive and painful procedure to obtain autologous donor cells, and the large costs, estimated at $13,000 per 1% total body surface area covered (Rue et al., 1993). Furthermore, graft take is widely variable based on wound status, general host status, and operator experience. Cultured keratinocyte allografts were developed to help overcome the need for a biopsy and a separate cultivation for each patient to produce autologous grafts and to ameliorate the long lag period between epidermal harvest and graft product. Successful keratinocyte allografting was first reported in burns using keratinocytes obtained from cadaver skin (Hefton et al., 1983). Since then, cultured epidermal cells from both cadavers and unrelated adult donors have been used for the treatment of burns (Madden et al., 1986), for skin grafts (Thiovlet et al., 1986), and for treating chronic leg ulcers (Leigh et al., 1987). Importantly, these allografts can result in accelerated and sustained wound healing without any evidence of rejection. This tolerance is probably secondary to the inability of cultured keratinocytes to express major histocompatability complex class II human leukocyte (HLA-DR) antigens (Hefton et al., 1983), and the absence of Langerhans cells, the major antigen-presenting cells (APCs) of the epidermis (Thiovlet et al., 1986). Nevertheless, these keratinocyte allografts are eventually replaced by recipient cells. Current allografts utilize neonatal foreskin keratinocytes, which are more responsive than adult cells to mitogens. In addition, they release more growth factors that stimulate adjacent keratinocytes (Gilchrest et al., 1983). Cultured neonatal foreskin allografts promoted accelerated healing and prompt pain relief in a variety of acute and chronic skin ulcers (Phillips et al., 1989). Another major advance was the development of cryopreserved allografts that gave results comparable to those with fresh allografts (Teepe et al., 1990; De Luca et al., 1992). Cryopreserved allografts have been used to cover large wounds with exposed bone and cartilage after Mohs micrographic surgery at a cost of $150 per application (Kolenik and Leffell, 1995). It is hoped that cryopreservation will allow mass allograft production and wide availability.

SKIN EQUIVALENT, SE
Skin equivalent (SE) refers to a collagen lattice which has been prepared by contraction of a collagen gel by heterologous fibroblasts (dermal equivalent or DE) and has subsequently been overlayed with a keratinocyte culture to induce formation of a mature, cornified epidermis in vitro prior to grafting of skin wounds. Clinical studies of the SE have been limited. In an early study (1988), the SE was used to cover partially full-thickness burn wounds covering over 15% of body surface area on eight patients. In every patient grafted with SE, an extensive lysis of the SE grafts was observed at the first dressing (48 h). In one patient only, a significant percentage of take (40%) was observed 14 days after grafting. It was concluded that the SE was not completely appropriate to serve routinely as a substitute for the autograft. In a later study (1995),the wounds treated were acute, mostly the result of excision of skin cancers. Twelve patients had clinical takes at the time of grafting and there was no evidence of rejection or toxicity following grafting with the SE.

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The wounds grafted with SE contracted by 10 to 15%, an extent larger than that observed following grafting with full-thickness skin. Biopsies of the grafted sites showed formation of scar tissue. The authors hypothesized that the SE was eventually replaced by host tissue. Recent studies of the SE have focused on patients with venous ulcers; these studies are in progress.

ACELLULAR SKIN SUBSTITUTE

Some of the acellular materials have components that incorporate into the wound bed and may become populated with dermal cells from the host. These dermal substitutes replace the dermal component of the skin, but may require grafting of autologous epidermis in a second surgical procedure. An acellular biocomposite dressing, has been used for over two decades for treatment of partial thickness burn wounds. It has been shown to control pain and reduce frequency of dressing changes, decrease the length of hospitalization, and improve healing times. A synthetic acellular skin replacement, but one that combines both temporary and permanent components. It consists of a crosslinked collagenglycosaminoglycan membrane as a dermal matrix, and a silastic coating that provides a synthetic epidermal structure for barrier replacement. It has been widely used for coverage of excised burn wounds. The artificial dermis does not contain cells at the time of grafting, but within a few weeks it becomes populated with cells from the wound bed and is vascularized. Thus, the dermal component incorporates into the wound, generating a neodermis; the temporary silastic coating can then be removed and replaced with a thin sheet of split-thickness skin. The option available to treat full-thickness wounds is bilayer skin substitutes. A bilayer temporary skin substitute, which has been used for coverage of partial-thickness wounds during re-epithelialization, or for full-thickness wounds prior to autograft placement. It is comprised of a synthetic semipermeable epidermal layer, and a dermal nylon mesh layer seeded with allogeneic human fibroblasts. The fibroblasts are allowed to proliferate within the synthetic dermal construct, where they secrete extracellular matrix components and growth factors that can facilitate wound healing. An acellular dermal matrix derived from donated human skin. The skin is processed to remove cells, circumventing tissue rejection but preserving much of the dermiss three-dimensional dermal structure. Vascular channels with basement membrane are present, even though the cells have been destroyed, facilitating graft vascularization. It has been most useful for soft tissue replacement, such as abdominal wall reconstruction. For treatment of burn wounds, it has been used in conjunction with split-thickness autograft to yield results similar to split-thickness grafts of greater thickness.

ALLOGENEIC CELLULAR SKIN SUBSTITUTE

The cells are allogeneic, typically isolated from donated human neonatal foreskin, these products are considered temporary skin substitutes rather than permanent skin replacements. Usullay this tissue substitute contain allogeneic keratinocytes and fibroblasts, coupled with a biopolymer consisting of either a bovine type I collagen gel or collagen sponge, respectively. Grafts that contain allogeneic cells provide wound coverage and supply growth factors that facilitate wound repair, but the allogeneic cells do not persist on the patient after healing is complete. It is generally believed that allogeneic cells are replaced within 1 to 6 weeks after grafting by the patients own cells. Cultured skin models containing allogeneic cells have been developed for in vitro testing purposes. Bilayer substitutes that contain both living keratinocytes and fibroblasts should benefit from the higher amount of inflammatory/ angiogenic mediators secreted by those 2 cell types when they are both present and influence each other.

AUTOLOGOUS CELLULAR SKIN SUBSTITUTE

Autologous keratinocytes and fibroblasts have generally been derived from biopsies of the patients uninjured skin. Recently, keratinocytes have been isolated from the outer root sheath of plucked hair follicles. The cells can be expanded in culture and used to populate skin substitutes in vitro. Grafted as epithelial sheets or as composites of biopolymers and cells, these are theoretically permanent skin substitutes once engraftment is achieved. Relatively few commercial skin substitutes contain autologous cells. Autologous living cells can also be desirable to improve the speed and/or quality of the healing in wounds that would otherwise be small enough to epithelialize on their own. Furthermore, evidence indicates that in order to achieve successful permanent healing, epidermal substitutes must not only contain differentiated keratinocytes but also epidermal stem cells that will provide the necessary self-renewal capacity needed for long-term maintenance of the new epidermal layer .

Cultured Epithelial Autografts, CEA

Cultured epithelial autografts (CEA) consist of a mature, cornified epidermis which has been produced by culturing keratinocytes in vitro, prior to grafting on skin wounds. The major goal of these treatments has been to replace definitively the use of the autograft in the treatment of patients with massive skin loss.

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The use of cultured epithelial autografts has been studied clinically. In this approach autologous epidermal cells are removed by biopsy and are then cultured in vitro for about 3 weeks until a mature keratinizing epidermis has formed; the epidermis is then grafted onto the patient. The epithelial cells spread and cover the dermal substrate, eventually covering the entire wound. Later studies showed that the take of CEA was very good on partial thickness wounds but was questionable in full thickness wounds. In particular, blisters formed within 2 weeks in areas grafted by CEA, a problem which has recurred persistently in clinical studies. The mechanical fragility of the resulting integument resulting fromuse of CEA has been traced to lack of three structural features which are required for formation of a physiological dermalepidermal junction at the grafted site, namely, the 7-S domain of type IV collagen, anchoring fibrils, and rete ridges. Early studies of the connective tissue underlying the CEA grafts have shown lack of a convincing dermal architecture as well as lack of elastin fibers. In another development, a skin equivalent has been prepared by populating a collagen lattice with heterlologous fibroblasts, observing the contraction of the lattice by the cells and finally seeding the surface of the lattice with a suspension of epidermal cells from an autologous source or from cell banks. The latter attach, proliferate, and differentiate to formamultilayered epidermis in 7 to 10 days of exposure to the atmosphere and the resulting skin equivalent is then grafted on wounds. Comparison between treatment with the meshed autograft (R) and treatment with the artificial skin (L). Autograft is usually meshed before grafting; scar forms in areas coinciding with the open slits of the autograft. The artificial skin treatment consists of grafting the excised wound bed with a skin regeneration template, followed by grafting on about day 14 with a very thin epidermal autograft. (Photo courtesy of J.F. Burke.)

5.10.1 The Meshed Autografts

An attempt has been made to correct the erratic cover provided by split-thickness autografts; the latter are normally applied in a meshed form (meshed autograft) and, consequently, fail to cover the entire wound bed with a dermal layer. The attempted improvement consisted in grafting underneath the meshed autograft a living dermal tissue replacement, consisting of a synthetic polymeric mesh (polyglactin-910) which had been cultured in vitro over a period of 2 to 3 weeks with fibroblasts isolated from neonatal foreskin. Seventeen patients with full-thickness burn wounds were included in a preliminary clinical trial. Epithelialization of the interstices of the meshed autograft led to complete wound closure in 14 days in sites where the dermal living replacement had been grafted underneath the meshed autograft (experimental sites) and in those where it was omitted (control sites). Take of the meshed autograft was slightly reduced when the living dermal tissue replacement was underneath. Basement membrane structures developed both in control and experimental sites. Elastic fibers (elastin) were not observed in neodermal tissue either in control or experimental sites at periods up to one year after grafting. A subsequent clinical study explored the use of this device for the temporary closure of excised burn wounds. Patient multicenter trial showed that the biosynthetic skin replacement was equivalent or superior to cadaver skin graft (frozen human cadaver allograft) with respect to its ability to prepare wounds for eventual closing with autograft. The dermal layer is essential for better elasticity and mechanical resistance, the absence of epithelialmesenchymal communication increases the frequency of developing fibrotic conditions and evidence indicates its presence allows highly complex ex vivo function of epidermal cells. This supports the hypothesis that the dermal ECM not only serves as a destination scaffold for the cells, but also seems to have the ability to instruct positioning of specific cell types and influence some functions or differentiation processes, as was shown by the correct cellular repopulation of a decellularized whole cardiac scaffold. Dermal reconstruction should thus be an essential part of the treatment in cases where none of the original dermis is left on the wound bed prepared for grafting. The presence of living fibroblasts is not a necessity in dermal substitutes. Dermal substitutes provide a crucial component to help repair full-thickness damage, but they do not create a barrier effect and need to be covered with an epidermal substitute to that end. This epidermal substitute can be temporary or, as a much more desirable option, can incorporate autologous keratinocytes over the reconstructed dermal layer .

CELL COMMUNICATION AND REGULATION IN SKIN

Regulation of its own function is an essential requirement of skin. Epidermis, for example produces parathyroid hormone-related protein (PTHrP), and plays a role in the regulation of keratinocyte growth and differentiation (Blomme et al., 1998). Keratinocytes produce a large variety of

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polypeptide growth factors and cytokines; these act as signals between cells, regulate keratinocyte migration and proliferation, and stimulate dermal cells in various ways (e.g., to promote matrix deposition and neovascularization) (McKay and Leigh, 1991; Parenteau et al., 1997). Cytokines produced by keratinocytes are thought to regulate Langerhans cell migration and differentiation (Lappin et al., 1996). Keratinocytes also translate a variety of stimuli into cytokine signals, which are transmitted to the other cells of the skin immune system. Dermal cells produce and respond to cytokines and growth factors to regulate numerous processes critical to skin function (Williams and Kupper, 1996). Approaches to dermal repair and regeneration center around control of fibroblast repopulation and collagen biosynthesis to limit scar tissue formation. One of the keys to improving dermal repair is control, or redirection, of the wound healing response so that scar tissue does not form. One promising observation is that fetal wounds heal without scarring (Mast et al., 1992). TGF-1, which is not expressed in the fetus, is a potent stimulator of collagen biosynthesis by fibroblasts in the adult and is thought to be an important inducer of scar formation. The matrix composition of the fetal dermis is also significantly different than that of adult dermis or granulation tissue, therefore providing a better matrix environment for dermal cell repopulation, which may also be key in controlling scar formation. An engineered skin graft should incorporate as many of these factors as possible: (1) the extracellular matrix, (2) dermal fibroblasts, (3) the epidermis, and (4) a naturally occurring semipermeable membrane, the stratum corneum. These components may act alone, but more importantly they should act synergistically as part of a fully integrated tissue to protect the underlying tissues of a wound bed and to direct healing of the wound (Sabolinski et al., 1996). Dermis containing fibroblasts may be necessary for the maintenance of the epidermal cell population (Lazarus et al., 1994). In turn, the epidermis is necessary for the formation of the so-called neodermis, in the absence of a dermal layer (Compton et al., 1989), and can dramatically influence underlying connective tissue response. The formation of the epidermal barrier also likely influences these processes through control of water loss and its influence on epidermal physiology (Parenteau et al., 1996).

COMERSIAL ARTIFICIAL SKIN

Artificial Skin available vary in complexity, ranging from temporary synthetic wound dressings to permanent skin replacements, either with or without incorporation of cultured skin cells. The term artificial skin used to describe a cell-free membrane comprising a highly porous graft copolymer of type I collagen and chondroitin 6-sulfate which degrades at a specific rate in the wound and regenerates the dermis in dermis-free wounds in animal models and patients (dermis regeneration template, DRT).

Dermis Regeneration Template - porous matrix seeded with cells, induces synthesis of a new dermis, simultaneously synthesis of a new epidermis occurs by migration of epithelial cell. The depth of tissue loss must be known as epithelial cells cannot migrate if loss of tissue is high.

ACELLULAR SKIN SUBSTITUTE

a. Biobrane Biobrane is composed of a collagen peptide-coated nylon mesh material attached to a semipermeable silicone membrane. The nylon mesh is coated with peptides to aid adherence. It is applied to clean, debrided wounds with the mesh side down; the mesh adheres to the wound, and the material is removed after the underlying tissue has healed.

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b. Integra Because of its acellular composition, Integra TM is a readily available product that does not impose the delay necessary for autologous keratinocyte cultures, which is the major drawback of products that use autologous cells. It is an off-theshelf product that provides a dermal substitute with a temporary silicone epidermal-like component. But if Integra is used to cover large body areas, it must be followed with skin grafts or living epidermal substitutes to achieve permanent wound closure.

c. TransCyte Prior to grafting, the material is frozen, without cryoprotection of the cells. Thus, at the time of grafting, Transcyte does not contain any viable cells. The difference between trancyte and biobrane is the seeding the neonatal fibroblast on to the collagen coated nylon membrane. Since nylon is not biodegradable, it cannot be used as a dermal substitute. The removal process is more successful because of less bleeding.

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d. Oasis and Matriderm TM Sufficient to provide a scaffold that will be repopulated, revascularized and remodeled with the patients own fibroblasts and endothelial cells. However, although acellular dermal substitutes have their use, studies with Dermagraft TM , a product that contains viable cells, yielded excellent results for chronic diabetic foot ulcers, venous leg ulcers and recessive dystrophic epidermolysis bullosa e. AlloDerm Soft tissue replacement, used in conjunction with split-thickness autograft to yield results similar to split-thickness grafts of greater thickness.

ALLOGENEIC CELLULAR SKIN SUBSTITUTE

a. Dermagraft Composed of a polymer mesh scaffold seeded with allogeneic fibroblasts. Cyropreserved human fibroblast derived dermal substitute on polyglactin-910 mesh scaffold. Enhances healing by stimulating the ingrowth of firbovascular tissue from the wound bed. Used in chronic lesions.

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Histologic evaluations of wound site biopsies of patients before and after implantationwith Dermagraft show a marked reduction in inflammatory cell infiltration and an increase in blood vessels. Similar results were not evident in the tissue of placebotreated patients. Magnification, 40X. Previous studies have shown that fibroblasts implanted clinically in the Dermagraft product persist for at least 6 months in vivo after implantation. The persistence of these cells clearly shows the uniqueness of a tissue-engineered product in wound healing: persistent cells are capable of secreting growth factors and normal matrix proteins long term into the wound bed, thus promoting an on-going physiological healing environment.

Dermagraft secretes a variety of growth factors and proteins that have been shown to be important in reducing inflammation and enhancing wound bed regeneration and reepithelialization

b. Apligraf, OrCel, and TissueTech TM autograft system,

Contain allogeneic keratinocytes and fibroblasts, coupled with a biopolymer consisting of either a bovine type I collagen gel or collagen sponge, respectively. Bilayer substitutes that contain both living keratinocytes and fibroblasts, such as OrCel, Apligraf and TissueTech TM autograft system. The presence of allogeneic cells in Or- Cel and Apligraf might limit the duration of the higher amount of inflammatory/ angiogenic mediators secreted by those 2 cell types.

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http://www.cbte.group.shef.ac.uk/research http://www.myskininfo.com/index.php Skin collagen matrix seeded with fibroblasts or keratinocytes

c. SkinEthic Laboratories

Developed a Reconstituted Human Epidermis model, comprised of stratified epidermal keratinocytes supplied on cell culture inserts, for in vitro test applications. In addition, several tissue-specific models of Reconstructed Human Epithelium have been prepared. These include models of oral, vaginal, corneal, and alveolar epithelium.

d. EpiDerm A similar models designed as SkinEthic for in vitro toxicology testing, a differentiated model of human epidermis, and Melanoderm, a stratified coculture of human melanocytes and keratinocytes. e. EpiDermFT (EpiDermFull Thickness) Comprised of neonatal foreskin-derived fibroblasts and keratinocytes grown on cell culture inserts. These skin models closely parallel human skin at the ultrastructural level, and can be reproducibly manufactured, offering attractive alternatives to in vivo animal testing for irritancy, toxicology, and gene expression studies.

AUTOLOGOUS CELLULAR SKIN SUBSTITUTE

In cases where the dermal layer is still at least partially present even after debridement, epidermal substitutes that provide living autologous keratinocytes, such as Epicel TM , Epidex TM , Laserskin TM , Myskin TM and CellSpray TM , can be applied directly on the wound bed and eventually achieve permanent wound closure. However, in cases where the dermal layer has been destroyed, the application of even the best epidermal substitute is not enough to ensure optimal healing.

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a. Epicel The first commercial product comprised of cultured autologous cells for wound transplantation. It is indicated for use in burn patients with very large deep partial-thickness or full-thickness wounds and in congenital nevi patients. Also referred to as cultured epithelial autograft (CEA), Epicel consists of a sheet of autologous keratinocytes that are grown in culture and transplanted to patients with a petrolatum gauze backing. The keratinocytes are isolated from a full-thickness biopsy of the patients uninjured skin, and grafts are generally ready within 3 weeks time. Epicel can be grafted on top of vascularized allogeneic dermis or directly onto debrided wounds; however, engraftment is higher when used in conjunction with allodermis. A major problem encountered with Epicel has been epidermal blistering. Small blisters may resolve spontaneously, but larger blisters result in graft failure. This phenomenon has been attributed to absence of dermalepidermal junction components at grafting. Despite this limitation, the availability of Epicel has provided a much needed treatment option for patients with massive burns where donor skin for autograft is extremely limited. b. EpiDex A similar product, is a cultured epidermal sheet graft that is indicated for the treatment of small chronic wounds. The unique aspect of EpiDex is that the keratinocytes are not cultured from skin biopsies, but from scalp hair follicles. For preparation of grafts, hair is plucked from the patient and the outer root sheath cells are placed in explant culture. The keratinocytes that are cultured in this fashion are highly proliferative, apparently regardless of donor age. After approximately 5 weeks of cell expansion, a secondary culture is initiated for preparation of epithelial sheet grafts. These are transplanted to wounds as discs measuring approximately 1 cm diameter, attached to a silicone backing to facilitate handling. Early clinical results have been favorable, though the wound areas treated with EpiDex are relatively small. A limitation of these autologous skin replacements is that they contain only keratinocytes, and thus they supply only an epidermal layer. For large full-thicknesswounds, such as burns, replacement of both dermal and epidermal layers is beneficial, to reduce scarring and improve the functional and cosmetic outcome. This can be accomplished by combining dermal substitutes and epidermal skin replacements, but this generally requires multiple surgical procedures.

c. A composite cultured skin substitute (CSS) Siti Julaiha Page 113

Contains both autologous keratinocytes and fibroblasts in vitro is currently in clinical trials. CSS are comprised of bovine collagen-glycosaminoglycan sponges that are populated with autologous fibroblasts and keratinocytes. The most extensive experience with autologous CSS has been in the treatment of patients with burns affecting greater than 50% TBSA. In these patients, donor sites for autografting are extremely limited and adjunctive treatments for wound coverage are required. For preparation of CSS, primary cultures of keratinocytes and fibroblasts are isolated using standard techniques from a small split-thickness skin biopsy that is usually taken during a patients first autografting procedure. Selective in vitro culture of keratinocytes and fibroblasts stimulates exponential increases in cell numbers, resulting in very large populations of cells in only 2 to 3 weeks of culture. Grafting to patients can generally be performed within 2 weeks of inoculation of CSS, which corresponds to 4 to 5 weeks after the initial patient biopsy. Because the CSS contains both dermal and epidermal layers and develops a functional basement membrane in vitro, grafting of CSS can replace both skin layers in a single surgical procedure. Culture of CSS at the airliquid interface promotes development of a stratified epidermal layer with functional barrier properties in vitro, providing protection of the wound immediately after grafting. CSS have been used with favorable results as an adjunctive treatment for the healing of large burn wounds, and have been shown to significantly reduce the requirement for autograft and shorten the number of surgical procedures needed for definitive wound closure. CSS have also been used to a limited extent for treatment of chronic wounds and congenital giant nevi. Advantages and Disadvantages of Temporary Skin Substitutes

Advantages and Disadvantages of Permanent Skin Substitutes

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Figure Possible timeline for advances in clinical skin substitutes. Times are not exact, but show many of the contributions to this field.

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CLINICAL CONSIDERATIONS
Multiple clinical factors can determine whether treatment of wounds with engineered skin substitutes will result in skin repair. Modifications of care protocols for wounds must be used to compensate for the anatomic and physiologic deficiencies in engineered skin. Currently available skin substitutes are avascular, tend to heal more slowly than skin autograft, and may be mechanically fragile. Factors that affect the clinical outcome with bioengineered skin replacements include, but are not limited to: Composition at the time of grafting; Wound bed preparation; Control of microbial contamination; Dressings and nursing care; and Survival of transplanted cells during vascularization of grafts. Attachment of cultured epithelium to a dermal substitute in vitro is advantageous because both epidermal and dermal components can be applied in a single procedure, similar to skin autograft. Culture conditions can be optimized to promote deposition of basement membrane proteins at the dermal epidermal junction prior to grafting, thereby eliminating the problem of blistering that is frequently observed after grafting of epithelial sheets. Alternatively, dermal and epidermal components of skin substitutes may be applied in two stages: First, application of a dermal substitute followed by vascularization; and Second, grafting of an autologous epidermal substitute.

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This two-step approach increases the density of blood vessels and extracellular matrix in the wound bed, and has been reported to improve engraftment of cultured keratinocyte sheets. However, it requires two surgical procedures to achieve permanent wound closure. The lack of a vascular plexus is a major limitation of all skin replacements currently available. Split thickness skin contains a vascular plexus and adheres to debrided wounds by coagulum. Inosculation of vessels in the graft to vessels in the wound occurs within 2 to 3 days. In the absence of microbial contamination or mechanical damage, autograft skin is generally engrafted and reperfused within 1 week after transplantation. In contrast, current clinical models of engineered skin substitutes are avascular, requiring reperfusion from de novo angiogenesis. The time required for perfusion is proportional to the thickness of the dermal component of the skin substitute, and is longer than perfusion of split-thickness skin. Vascularization can be accelerated by secretion of angiogenic factors from engineered skin containing keratinocytes and fibroblasts, but growth factors alone cannot compensate for the lack of a vascular plexus prior to grafting. The additional time required for vascularization may contribute to epithelial loss from microbial destruction and nutrient deprivation. Due to the delayed vascularization of skin replacements, control of contamination is critical for engraftment. Topical antimicrobials are more effective for control of wound contamination than parenteral agents. Topical treatments must provide effective coverage of a broad spectrumof microorganisms, but must have low cytotoxicity to allow healing to proceed. It is also important to avoid overlap of topical agents with parenteral drugs used for treatment of sepsis, which could facilitate development of resistant organisms. Several studies have identified individual agents, and mixtures of multiple agents, which are effective against common wound organisms but are not inhibitory to proliferation of keratinocytes and fibroblasts. An additional limitation of engineered skin substitutes is mechanical fragility, which contributes to graft failure due to shear and maceration. For delicate grafts, a backing material can facilitate handling and attachment to the wound. For example, CEA are routinely attached to petrolatum-impregnated gauze for surgical application. However, this material may not be compatible with wet dressings used to manage infection. CSS may be handled and stapled to wounds with a backing of N-Terface, a relatively strong, nonadherent, highly porous material. Porous dressings do not interfere with the delivery of topical solutions and permit drainage of wound exudate. Expense can become a limiting factor for treatment of very large wounds, such as those seen in severely burned patients. Unfortunately, these are the patients most in need of skin substitutes. Although the use of skin substitutes can theoretically reduce the number of surgeries required to heal large burns, which should decrease the total time of hospitalization, there are currently no studies that clearly demonstrate a decrease in costs by use of skin substitutes of any kind. Engineered skin grafts remain an important adjunct to conventional skin grafting, particularly in the treatment of burns, but cannot be used as a primary modality of wound closure except in the most extreme cases. Meshing of the silicone layer of the artificial skin, without affecting the continuity of the collagen-GAG layer, could lead to improved take and probably to reduced incidence of infection. The healing time for donor sites associated with use of the artificial skin was shorter than for donor sites that were used to harvest autograft. An even shorter healing time for donor sites for artificial skin can be realized by reducing the thickness of the epidermal graft which is required to close the dermal bed. Increasing familiarity of surgeons with the procedure for harvesting these thin epidermal grafts is expected to lead to harvesting of thinner grafts in future studies. The importance of harvesting a thin graft cannot be overestimated, since the healing time of the donor site decreases rapidly with decreasing thickness of harvested graft. . Not only does the time to heal increase, but the incidence of hypertrophic scarring at a donor site also increases with the thickness of the harvested graft. This observation explains the higher incidence of hypertrophic scarring in donor sites associated with harvesting of autografts. An additional advantage associated with use of a thin epidermal graft is the opportunity to reharvest (recropping) within a few days; this reflects the ability of epithelial tissues to regenerate spontaneously provided there is an underlying dermal bed. When frequent recropping of donor graft is possible, the surface area of a patient that can be grafted within a clinically acceptable period increases rapidly. In the long term rarely did a patient or a physician in this clinical study prefer the new skin provided by the autograft to that provided by the artificial skin treatment. This result is clearly related to the use of meshed autografts, a standard procedure in the treatment of massively burned patients. Meshing increases the wound area which can by autografted by between 1.5 and 6 times, thereby alleviating a serious resource problem. However, meshing destroys the dermis as well as the epidermis; although the epidermis regenerates spontaneously and fills in the defects, the dermis does not. The long-term result is a skin site with the meshed pattern permanently embossed on it. The artificial skin is a device that, in principle, is available in unlimited quantity; It has been established that the artificial skin regenerates the dermis and, therefore, its use leads to complete inhibition of scar formation in full-thickness skin wounds. The regeneration is partial because skin adenexa (hair follicles, sweat glands) are not recovered. The results of studies of the mechanism by which the artificial skin regenerates the dermis in full-thickness skin wounds in animal models have been described elsewhere. The artificial skin leads to a new skin which appears closer to the patients intact skin than does the meshed autograft. Take of the artificial skin is as good as all comparative materials except for the unmeshed autograft, which is superior in this respect. Donor sites associated with the artificial skin

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treatment heal faster, can be recropped much more frequently, and eventually heal to produce sites that look closer to the patients intact skin than do donor sites harvested for the purpose of autografting. In comparison to the allograft, the artificial skin is easier to use, has the same take, does not get rejected, and is free of the risk of viral infection associated with use of allograft.

ASSESSMENT
The Vancouver Scale is used for assessment of burn scar by trained clinicians, and provides an ordinal score for properties of skin including pigmentation, vascularity, pliability, and scar height. Such scales assign quantitative values to qualitative measurements and can provide a relative comparison for evaluation, but they are inherently subjective and dependent on the examiner. Objectivity may be increased by use of noninvasive instruments to measure biophysical properties of skin, including vascular perfusion, epidermal barrier, pliability, color, and surface pH. Quantitative assessment of skin substitutes can highlight deficiencies compared to normal skin or splitthickness autograft, or can be used to assess the advantage of skin substitutes to the patient without interfering with recovery. Although no single biophysical property is definitive,multiple measurements can provide a general assessment for evaluation of outcome. For example, measurements of surface electrical capacitance (SEC) can be used to define the degree of skin barrier development. SEC is measured using a dermal phase meter, an instrument that is easily used in a clinical setting, with minimal pain or discomfort for the patient . Pigmentation of grafted wounds treated with engineered skin substitutes can be measured using a chromameter. Multiple parameters of skin function must be measured to quantify overall benefit from treatment with skin substitutes.

REGULATORY ISSUE
Safety considerations for engineered skin substitutes must take several factors into account, including media composition, tissue acquisition, graft fabrication and storage, and sterility testing of the final product. For example, Cell culture media must be of the highest purity and free from toxic contaminants. Cells derived from allogeneic donors must test negative for transmissible pathogens. Autologous cells must be handled carefully aswell. Because autologous tissues are not routinely screened for pathogens, universal precautions to protect laboratory personnel must be practiced. Xenogeneic components, such as bovine collagen, must not only be free from pathogens that can cross species boundaries, but must also be nonimmunogenic. If xenogeneic cells, such as irradiated 3T3 mouse fibroblasts, are used to facilitate initiation of keratinocytes cultures, compliance with safety standards for xenogeneic transplantationmust be assured. Although these common cells are generally thought to be free from risk of disease transmission, unknown risks may exist, and hence patients are excluded from future donation of blood or body parts.

FUTURE DIRECTIONS
Despite encouraging clinical results with bioengineered skin for the adjunctive treatment of burns, chronic wounds, and other skin deficiencies, skin substitutes containing just two cell types are limited by anatomic and physiologic deficiencies compared to split-thickness skin autograft. Several areas for further engineering are

Increase homology to native human skin. These include the incorporation of additional cell types to improve functional and cosmetic outcome, and the use of genetically modified skin cells to enhance performance after grafting. Iimprove wound healing for extensive wounds that annot heal spontaneously because they are deep and affect a high percentage of the total body surface and for smaller wounds that cannot heal because of underlying factors such as those mentioned above. Faster and/or a better quality of healing for skin substitutes indicated for wounds that would heal of their own accord. Unless there is severe arterial hemorrhage, hemostasis spontaneously begins immediately after the injury. It involves platelet aggregation, fibrin clot formation and activation of the coagulation pathways.

Used skin substitutes for hemostatis as the first aspects of wound treatment involve cleaning and debriding the wound. However, it should be noted that chitosan dressings have been demonstrated to efficiently improve hemostasis compared to standard dressings, even with significant arterial hemorrhage. Polysaccharide is currently present only in dressings and not in any commercially available skin substitute.

Improve coagulation might be an interesting improvement of skin substitutes that are intended for application immediately after aggressive debridement. Efect on coagulation with dermal substitutes that contained chitosan.

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Initiate adverse antigenic reaction, includes steps meant to make them less immunologically reactive. The technique most used to minimize such immune reaction is to decellularize the material, as done for Alloderm TM , Oasis TM , EZ-Derm TM , Repliform TM , Cymetra TM and Cryoskin TM . Other products, such as OrCel TM , Apligraf TM and ICX-SKN TM , rely on the hypothesis that neonatal cells are less prone to elicit a severe immunogenic response than adult cells and, thus, incorporate human neonatal foreskin fibroblasts and/or keratinocytes to provide substitutes that contain such living cells.

Another analysis with the presence of living dermal fibroblasts in dermal substitutes that resulted in better wound healing with less myofibroblast activity and thus lower contractile properties . The same work showed that autologous cells were preferable to allogeneic ones and that, comparing autologous sources, better results were obtained from dermal than adipose tissue fibroblasts.

Chances of optimal wound healing. Based on this information, it would seem that dermal and bilayer substitutes that incorporate living autologous dermal fibroblasts, such as Hyalograft 3D TM , PermaDerm TM , VCT01 TM and the LOEX self-assembled bilayer skin substitute, would offer better chances of optimal wound healing.

Promising research involves the genetic modification of cells in cultured skin grafts to reduce or eliminate immune rejection. Preclinical studies have shown that reduced expression of major histocompatibility complex (MHC) class I and II antigens can prolong engraftment of skin grafts in mouse allograft models. In one study, fetal skin was used because it exhibited substantially reduced MHC class I and II expression compared with neonatal skin. In another study, keratinocytes were genetically modified to overexpress indoleamine 2,3-deoxygenase (IDO), a tryptophan-catalyzing enzyme that functions to prevent fetal rejection during pregnancy . Increased IDO expression in keratinocytes led to a down-regulation of MHC class I expression. These studies suggest a possible mechanism for preparation of allogeneic skin substitutes that would escape immune rejection, and may someday lead to universal donor cultured skin grafts.

Improvement in research in term of Pigmentation. Normal skin pigmentation results from the appropriate epidermal distribution and function of melanocytes. The most critical function of melanocytes is protection from ultraviolet irradiation, but they have psychological importance aswell, as a patients body image and personal identity can impact recovery from massive skin injury. Pigmentation of cultured skin may result from transplantation of passenger melanocytes, which may persist in selective cultures of epidermal keratinocytes . Melanocytes can survive under conditions used for keratinocyte culture, though they proliferate at slower rates and are depleted upon serial passage or cryopreservation. In CSS grafted to excised burns, pigmented areas resulting from passenger melanocytes have been observed as individual foci within two months after transplantation. By 1 to 2 years after healing, the foci increase in area, occasionally fusing together to form larger pigmented regions. Uniform pigmentation was demonstrated in preclinical studies with CSS deliberately populated with selectively cultured human melanocytes. Future studies will be needed to address regulation of the level of pigmentation in uniformly pigmented cultured skin.

Improvement research in Vitro Angiogenesis. The absence of a vascular plexus in bioengineered skin necessitates vascularization to occur de novo, rather than through inosculation of the graft with the wound, increasing the time of nutrient deprivation and susceptibility to microbial contamination after grafting. This limitation can be indirectly addressed in a clinical setting by irrigating the graft with solutions of nutrients and antimicrobial agents for several days after transplantation.Adirect approach would be to initiate angiogenesis in the skin substitutes in vitro, prior to grafting. This would permit vascularization to occur through both inosculation of existing vessels and also neovascularization, as occurs for grafted split-thickness skin. Initiation of angiogenesis in vitro requires the addition of endothelial cells to the engineered skin. Endothelial cells may organize into vascular structures in culture with the aid of biomaterial supports and coculture with accessory cells. For example, engineered blood vessels have been constructed in vitro using mixed cultures of fibroblasts, human umbilical vein endothelial cells (HUVEC), and vascular smooth muscle cells in a collagen matrix. In preclinical studies, transplantation of engineered blood vessels constructed by culture of HUVEC in three-dimensional collagen/fibronectin gels has been reported. More recently, a composite cultured skin containing HUVEC and keratinocytes in a human dermal matrix was reported, which displayed evidence of perfusion after grafting to mice. To do the feasibility of grafting synthetic vessels in cultured skin, overexpression of Bcl-2 through retroviral modification was required to promote survival of the transplanted endothelial cells. In addition, another potential limitation of these studies that will impede their clinical application is the reliance on nondermal or nonautologous endothelial cells. Ideally,multiple cell types (keratinocytes, fibroblasts, and human dermal microvascular endothelial cells or HDMEC) could be derived froma single autologous skin sample. Transplantation of HDMEC in a composite skin substitute containing isogenic keratinocytes and fibroblasts was demonstrated in an athymic mouse model, though perfusion was not observed. The transplantation of HDMEC in a clinically relevant cultured skin model showed the feasibility of preparing autologous cultured skin containing HDMEC,but future studiesmust demonstrate inosculation of vessels in the graft with vessels in the wound bed to yield improved performance after grafting.

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Cutaneous Gene Therapy Improvement Keratinocytes and fibroblasts are amenable to genetic modification in vitro by a variety of methods. Genetically modified cells can be used to populate engineered skin substitutes. This is termed ex vivo gene therapy because cells are removed from the body and genetically modified in culture before being transplanted back to the recipient. There has been a great deal of interest in the use of genetically modified skin substitutes for the treatment of cutaneous diseases. For example, preclinical studies suggest that ex vivo gene therapy can be useful for treatment of lamellar ichthyosis, a condition characterized by defective epidermal barrier, and the blistering skin disease junctional epidermolysis bullosa (JEB). Theoretically, genetically modified keratinocytes can be transplanted for secretion of circulating factors to treat systemic diseases. Keratinocytes have been genetically modified to secrete human growth hormone and clotting factor IX, but therapeutic protein levels have been difficult to obtain after grafting. Another application of cutaneous gene therapy is the regulation of wound healing. Hypothetically, genetic modification may be used to overcome anatomic limitations or to enhance their biological activity. For example, keratinocytes modified to overexpress the mesenchymal cell mitogen Platelet Derived Growth Factor-A (PGDF-A), seeded on an acellular dermal matrix, showed increased cellularity, vascularization, and collagen deposition after grafting to mice, suggesting improved function due to PDGF-A overexpression. In other studies, keratinocytes were genetically modified by retroviral transduction to overexpress the angiogenic cytokine Vascular Endothelial Growth Factor (VEGF). After transplantation to athymic mice, skin substitutes containing fibroblasts andV EGFmodified keratinocytes showed enhanced and accelerated vascularization, decreased contraction, and increased engraftment compared to control grafts containing unmodified cells. Thus, genetic modification of keratinocytes can hypothetically be used to overcome the lack of a vascular plexus in engineered skin grafts. Bioengineered skin substitutes may be regulated as either devices or biologics, depending on their composition and primary mode of action. Skin substitutes consisting of autologous cells only, or an acellular human tissue matrix, may not require collection of effectiveness data for regulatory approval. Living autologous cell populations intended for structural repair are considered to be inherently efficacious. However, if no effectiveness data are collected, no claims of effectiveness can be made. Skin substitutes that combine cells with biopolymers are currently considered class III (significant risk) devices that require demonstration of effectiveness in addition to safety. Technological advances in the fabrication of biomaterials and the culture of skin cells have permitted the production of bioengineered skin substitutes. These have provided improved therapeutic options for patients suffering from acute or chronic wounds, and offer the promise of new treatments for inherited cutaneous diseases. Continued research will be needed to identify more efficient methods to utilize precious autologous tissue, which will provide greater amounts of skin substitutes for grafting as well as shorten the time required for their preparation. Increasing the complexity of skin substitutes, from acellular biopolymers to composite materials with multiple cell types, will result in continued improvements in anatomy and physiology, working toward greater homology to native human skin. These improvements will lead to enhanced performance of engineered skin grafts, greater clinical efficacy, and reduction of morbidity and mortality for patients with wounds or cutaneous disease.

STEM CELLS APPLICATION IN SKIN TECHNOLOGY

STEM CELLS ARE : are undifferentiated cells that do not yet have a specific function. can replicate for a long period of time and give rise to differentiated cells. In every cell division one cell retains its self renewing capacity while the other cell can undergo differentiation (asymmetric replication)

Two types of stem cells

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 Embryonic stem cells derived from the inner cell mass of a blastocyst from in vitro fertilized eggs
are pluripotent and can generate all tissues

Adult (somatic) stem cells

they are present in small numbers in various tissues of the adult body are typically programmed to form different cell types of their own tissue and are therefore multipotent in tissues with high turn over (hematopoietc system, epithelial lining of the gut and skin) they are instrumental in renewal although present in a variety of permanent non-dividing tissues they are not very active

Bone marrow contains two different types of adult stem cells:

The hematopoietic stem cell and the bone marrow stromal cell

Potential plasticity of hematopoietic stem cells

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Epidermal stem cells located in the bulge area of the hair follicle serve as a stem cells for the hair follicle and the epidermis. Differentiation pathways for pluripotent bone marrow stromal cells. Activation of key regulatory proteins by growthfactors,cytokines,or matrix components leads to commitment of stem cells to differentiate into specific cellular lineages.

Therapic Cloning

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RESEARCH IN ARTIFICIAL SKIN

Preparing Artificial Skin with Cultured skin cells , polyglycolic fabric , collage gels and glycosaminoglycans are incorporated. Rapid Keratinocyte cultures are obtained by growing the same on a feeder layer of irradiated fibroblasts. Also , neonatal fibroblasts are being used (Paed Medical Center , Munster Germany )

1. 2. 3. 4. 5. 6.

Vascular Response Blood coagulation Inflammation Formation of new tissue Epithelialisation Contraction & Remodeling

The graft is a bilayer membrane. In this approach, the top layer , a Silicone Layer incorporates the features of moisture control. While the bottom layer delivers the performance of sealing the skin breach and preventing scarring. The top layer is removed after a period of about 1015 days. Following removal of the top layer, the epidermal cover is provided by covering with a thin epidermal graft.

CELL TESTING AND ESTABLISHMENT OF MANUFACTURING CELL BANKS

Tissue-engineered products offer advantages over cadaveric materials in that the cells utilized, as well as the final manufactured product, are highly tested for a variety of viruses, contaminants, and pathogens. The human fibroblast cell strains used to produce the human dermal equivalent, Dermagraft (Dg) (Advanced Tissue Sciences, Inc., La Jolla, CA), are established from neonatalforeskin (discarded after surgical circumcision) (Kruse and Patterson, 1973; Jakoby and Pastan, 1979) and cultured by standard

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methods. Maternal blood samples are tested for exposure to infectious diseases, including human immunodeficiency virus (HIV), human T cell lymphototropic virus (HTLV), herpes simplex virus (HSV), cytomegalovirus (CMV), and hepatitis virus. An initial screen is made of the cultured cells for sterility, mycoplasma, and for the eight human viruses: adeno-associated virus, HSV-1 and -2, CMV, HIV-1 and -2, and HTLV-I and -II. Master cell banks (MCBs) and manufacturers working cell banks (MWCBs) are created and tested accordingto applicable sections of the U.S. Food and Drug Administration (FDA) points to consider (1993) and the guidelines from the European Union Committee for Proprietary Medicinal Products (CPMP) (1989). This testing provides a more uniform and highly tested product thanis available from cadaveric donors (tissue banks) (Blood et al., 1979; White et al., 1991).

A transmission electron micrograph of Dermagraft bundles and have normal banding periodicity.

Human collagen fibers are arranged in parallel

Tissue Expansion Properties of Dermagraft

Jiang and Harding (1998) evaluated the ability of Dermagraft to stimulate tissue growth in an ex vivo tissue expansion assay. Extracellular matrix gel was used as a support of tissue in order to test the effect of factors on the tissue expansion from patients with chronic and acute wounds. Briefly, Matrigel was dissolved in culture medium at 500 g/ml. This was added to a 24-well tissue culture plate. Materials and the complete system were kept ice cold at any given time until the next step. Wound tissue was washed vigorously in culture medium and finely minced with a dissecting scalpel and scissors (approximately 0.5 mm in diameter), then further washed and transferred to cool Matrigel solution in a culture well. The temperature for the system was then quickly raised to 37_C to form an irreversible gel, thus allowing the wound tissue to be embedded in the Matrigel. Coculture inserts with Dermagraft were then placed on top of the well for coculturing. The system was monitored over a 4-week period using a Panasonic chargecoupled device (CCD) camera and the distance between the wound edge and the leading front of the expansion was calculated using image analysis software (Optimas; Optimas Ltd., UK). In Matrigel, wound tissue was able to expand in a three-dimensional manner. Viable tissue obtained from patients with acute and chronic wounds revealed various patterns of cell migration. Briefly, after embedding, polymorphonuclear cells began to migrate out of the wound tissue. However, a large-scale migration of other cells (fibroblasts and endothelial cells) were seen from day 3 onward. The distance between the leading front of the area and the edge of the wound became quantifiable from day 7. Images from these cocultured tissues were then taken with a digital camera (UltraBix; Cambridge

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Scientific Instrument; Cambridge, UK). The relative distance between this leading edge and wound tissue, termed the expansion index in this report, was then quantified from these images with image analysis softward (Optimas). When Dermagraft was included, there was a marked expansion of the wound tissue when compared with tissue without the Dermagraft ( p=0.0015). At a higher magnification, the expansion areas represents a mixture of cells and structures. Most cells are fibroblast-like cells and endothelial cells. After a prolonged culture (over 2 weeks), a vessellike structure was clearly seen ( Jiang and Harding, 1998). A number of studies have been performed to correlate growth factor expression and secretion with in vitro and in vivo wound healing characteristics of Dermagraft ( Jiang and Harding, 1998; Mansbridge et al., 1998; Naughton et al., 1997; Newton et al., 1999). The angiogenic-promoting properties of this human dermal replacement have been well characterized and provide a unique tool for wound bed vascularization and promotion of healing.

Artificial Skin From Hair Roots

Fraunhofer - Gesellschaft (2008, January 4). Growing Artificial Skin From Hair Roots Euroderm and the Fraunhofer Institute for Cell Therapy and Immunology in Leipzig have been granted approval to produce artificial skin from patients own cells. Few hairs off the back of the patients head are pulled Adult stem cells from the roots are extracted, Proliferated in a cell culture for about two weeks.

ICX-SKN - Mimicking nature

Paul Kemp and colleagues at British biotech company Intercytex Fully and consistently integrates into the human body No need for further grafting .

Skin cells genetically engineered to be resistant to bacteria

Scientists at the Cincinnati Shriners Hospital for Children have engineered bacteria resistant skin cells. Due to delay in angiogenesis, the skin is vulnerable to bacteria as there are no circulating macrophages. incorporating anti-bacterial factors like Human Beta Defensin 4, will help void bacteria at an initial stage

Hence

The Tissue Culture laboratory Siti Julaiha Page 126

Grows keratinocyte cells into epithelial grafts for burn patients in hospitals. From a small piece (2 x 2cm) of the patients own skin, it can be can grew enough epithelial grafts to cover a whole person in 3 weeks. The individual grafts are typically 10 x 7cm in size and are multi-layered, very much like normal epidermis. At the base of the graft is the basal cell layer. As the cells move through each layer of the skin, they become increasingly differentiated. Once the epithelial graft is placed on the patient and exposed to air, the top layer takes on the protective role of the skin by becoming cornified. One of medium culture for skin growing in the lab. Consist of insulin, epidermal growth factor (EGF), hydrocortisone, Penicillin /streptomycin and fungizone. Cultured epithelium(H&E stain, magnification approx.

Normal epidermis x350)

Detached cultured epithelial graft

In the Near Future Overview- Integumentary Development

A totally new epidermis is present every 25 to 45 days. Melanocytes create melanin, the substance that gives our skin color. These cells are found deep in the epidermis layer. Accumulations of melanin are packaged in melanosomes (membrane-bound granules). These granules form a pigment shield against UV radiation for the keratinocyte nuclei.

For chronic skin ulcer treatment

It has been established a keratinocyte cell line from neonatal foreskin. These cells are free of contamination by HIV, Hepatitis B & C and CMV and have been used to produce cultured epithelial allografts for the successful treatment of chronic leg ulcers. Cryo-preserved allografts are available as biological dressings for immediate use on request.

Self Healing Artificial Skin

http://www.mvac.uiuc.edu

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Microvascular Autonomic Composites Initiative (VAC) is creating materials with a microvascular network, capable of pumping self-healing polymers to repair sites of skin breech Skin capable of healing, even though only to a certain degree, could prove incredibly useful for the robotics industry Microvascular Autonomic Composites Initiative (VAC) is for Robotic Skin. Imagine the same with our Artificial Skin Skin that regenerates when breeched accidentally or intentionally

FILM Skin For robots, Artificial Limbs

Flexible, Integrated, Lightweight, Multifunctional skin Oak Ridge National Laboratory's Nanomaterials Synthesis and Properties Group Carbon Nanotubes are being used electrical conductor, or as part of a polymer material with mechanical and thermal properties similar to those of human skin.

The material can be designed to behave as both a temperature and pressure sensor, as a flexible

Embryology of skin
Ectoderm forms the surface epidermis and the associated glands. Mesoderm forms the underlying connective tissue of dermis and hypodermis

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EMBRYONIC WOUND HEALING

Classically, adult wound closure is described as being composed of two main components; one being reepithelialization, whereby front row keratinocytes crawl forward by means of lamellipodia; and the other being a contractile process occurring in the granulation tissue of the newly repairing mesenchyme. In contrast, it has been demonstrated that, although embryonic wound healing involves both epithelial and mesenchymal elements, which take place in the primitive tissue layers already described, the nature of these movements is markedly different from those occurring in an adult wound. Additionally, the extent of the acute inflammatory reaction provoked by wounding is significantly reduced, or even absent, in embryonic wounds in comparison with adult wounds.

The Embryonic Epithelial Wound Is Closed by an Actin Purse-String

In 1977, England and Cowper described how incisional wounds to the endoderm of chick embryos close by a process whereby the endoderm sweeps over the underlying mesoderm without apparen formation of lamellipodia. Stanisstreet et al. demonstrated a similar mechanism occurring at the wound edge in neurula- stage frog embryos. Clearly, the lamellipodial crawling that drives reepithelialization of adult wounds is not essential for embryonic repair. Martin and Lewis, observing excisional wounds in 4-day-old chick embryos, also found no evidence of lamellipodial crawling by wound edge epithelial cells. In addition, unlike adult cells with lamellipodia, these embryonic cells remained adherent to the underlying basement membrane. In fact, the wounds created in the chick wing buds were seen to rapidly form a smooth edge, and by 24 hr, the majority of these wounds were closed. By marking the boundary mesenchyme with a lipophillic dye, DiI, it was seen that the epidermal cells moved independently of and over the underlying mesenchyme. Fluorescently labeled phalloidin, which binds filamentous actin, revealed the rapid assembly of a cable of actin in the basal epithelial cells of the leading wound edge. This cable appeared continuous from one cell to the next and apparently acted as a pursestring to draw the epithelial wound margins closed. A similar actin purse-string was seen in tissue culture by Bement et al. after wounding a confluent monolayer of the gut epithelial cell line Caco-2BBe. Additionally, it seems that various morphogenic processes, most clearly dorsal closure in the fruitfly Drosophila, which occurs about 12 hr after egglaying, might also be driven by contraction of a similar actin purse-string Further work by McCluskey and Martin has shown that an actin cable is also rapidly assembled in excisional wounds in mouse embryos. Importantly, addition of cytochalasin D, which prevents new polymerization of actin, results in complete failure of reepithelialization of the wound, providing good evidence that the actin cable is required for reepithelialization of embryo wounds.

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Figure (a) Scanning electron micrograph (SEM) of whole mouse embryo at embryonic day 11.5. Arrow marks wound formed by amputation of hindlimb bud. (b) SEM of wound 12 hours postamputation; e=epithelium, m=mesenchyme. (c) High-power magnification of b showing elongation of wound margin cells (arrows). (d) By 24 h, the wound is closed leaving only a central clumping of debris marking the original wound site (arrow). (e) Transmission electron micrograph (TEM) of epidermal wound margin showing the blunt face of the leading edge cell and no evidence of lamellipodial extension. (From Ref. 4. Copyright Springer-Verlag GmbH & Co.)

AntiTransforming Growth Factor-

Fetal wounds heal without scarring and have a lower inflammatory and cytokine response compared with adults. Administration of TGF-to fetal wounds induces scarring . Suppression of TGF-in adults by using antibodies, which target TGF-has been proposed as a possible therapy to reduce scar formation. Shah et al. used an adult rat dermal wound model to demonstrate that antiTGF-1,2 administration at the time of wounding or shortly after resulted in a dose-dependent reduction in scarring. The wounds treated with anti TGF- 1,2 had fewer macrophages, monocytes, and blood vessels than control wounds. The anti TGF- 1,2 wounds also had reduced type I and type III collagen and fibronectin levels, but retained the same tensile strength as controls. The similarity in wound strength in the anti TGF- 1,2 wounds, despite the lower collagen content, was considered to be due to the regular arrangement of the fibrils in these wounds compared with abnormally oriented collagen fibrils in wounds treated with TGF- irrelevant antibody or no injection. The authors suggest that the reduction in TGF- immediately after wounding helps prevent scarring by decreasing the recruitment of immune cells. It may also alter levels of PDGF, bFGF, as well as the autocrine induction of TGF- .Early administration of antiTGF- may also decrease the synthesis of PAI-1 and increase the synthesis of plasminogen and plasmin, which aid in fibrinolysis as ECM production ensues. This may result in a more organized pattern of the ECM proteins.

Mannose 6-Phosphate
Another potential therapy for the prevention of fibroproliferative disorders is exogenous mannose 6phosphate. As previously discussed, latent TGF- -binds to the M6P/IGF-2 receptor, and its activation is inhibited by the addition of M6P or antibodies directed against this receptor. It has not been determined whether cell surfaceassociated plasmin alone activates latent TGF- after it binds to the M6P/IGF-2 receptor, or whether latent TGF- is internalized and the low pH in the endosomal compartment is responsible for activation. Although further investigation is required to determine potential side effects of administration, it is reasonable to consider M6P as a therapy for excessive scarring.

FIBROGENIC GROWTH FACTORS Siti Julaiha Page 130

Cells communicate with each other through the specific binding of cytokines and growth factors with protein receptors on their cell membranes. The functions of growth factors are diverse and include stimulation or inhibition of cell proliferation, differentiation, migration, or gene expression, depending on the cell type involved. Of the many growth factors and cytokines potentially involved in HSc and keloids, transforming growth factor- is certainly one of the most complex and pleiotropic. Because of the many functions of this growth factor, its regulation is considered crucial in the control of normal wound healing.

Transforming Growth Factor-1

Transforming growth factor- belongs to a supergene family consisting of three groups, the TGF- s, the activins, and the bone morphogenic proteins (BMPs). Five isoforms of TGF-have been identified to date; TGF- 1, - 2, - 3, - 4, and -5. Of these, TGF- 1, 2, and - 3 are found in mammals. Transforming growth factor-is released from platelets into the wound environment following injury and acts as a chemotactic agent for neutrophils, T lymphocytes, monocytes, and fibroblasts. Although TGF-is essential for normal wound healing , overexpression or persistent expression of this growth factor may lead to fibrosis as seen in HSc and keloids. Transforming growth factor- 1 is implicated in the formation of HSc and keloids because of its ability to elicit an overproduction of ECM proteins. This is achieved both by up-regulation of collagen synthesis and down-regulation of collagenase production. It has been reported that TGF- 1 mRNA expression is greater in postburn HSc relative to that of normal tissue obtained from the same patients. It has also been shown that TGF- 1 is capable of upregulating its own receptor expression and stimulating the differentiation of fibroblasts into myofibroblasts. Transforming growth factor- 1 is secreted as a small latent complex (LTGF- 1) consisting of a 25-kDa dimeric mature protein and an N-terminal pro-protein called the latency-associated peptide (LAP) (98). Important features of the LAP are the presence of three N-linked oligosaccharides, two of which include mannose 6-phosphate (M6P) (99). In cells such as fibroblasts, platelets, and bone cells, the LTGF- 1 complex may form a large latent complex with latent TGF-1 binding protein (LTBP), a 125- to 205-kDa glycoprotein that is required for the secretion and targeting of TGF-1 in some cells . The bind ing of LTBP masks the M6P moieties on LAP and prevents the uptake of LTGF-into lysosomes. After its release from degranulating platelets, TGF-1 can exist as the small latent complex and be sequestered in the ECM, or as the large latent complex and either be released into the serum or be bound to the ECM, where it can be released by proteolytic cleavage. It is generally believed that either a conformational change of the latent complex or dissociation of LAP is required for activation of TGF-1 as the TGF-receptors do not recognize LTGF-1. Wakefield et al. studied the tissue distribution of both recombinant latent and active TGF-1 in rats. Active TGF-1 was shown to accumulate in the lungs, liver, and kidney, which is similar to the tissue distribution of macroglobulin , which is a carrier molecule involved in the clearance of 2active TGF-1. Conversely, latent TGF-1 did not accumulate in any one organ, instead, it was present in low levels in all organs. The authors suggested that the LAP may extend the half-life of TGF-1 in circulation by preventing it from complexing with 2-macroglobulin. Thus, while active TGF- 1 may act locally in an autocrine or paracrine fashion, latent TGF-in circulation may have endocrine activity. Dickson et al. used [125I]-TGF-1 to demonstrate the distribution of administered active TGF-in mice and rats. The investigators showed that the microvascular endothelium was the major site of TGFbinding. In response to tissue injury, TGF-up-regulates adhesion molecules and has chemotactic properties. However, it has previously been suggested that a major function of systemic TGF-may be to reduce adhesiveness of endothelial cells for immune cells by inhibiting E-selectin expression. This function is perhaps best demonstrated by MRL/1pr mice, a murine autoimmune model used to study diseases such as systemic lupus erythematosus (SLE). In these mice, the TGF-1 gene is disrupted and an inflammatory response results in death 2 to 3 weeks after birth. Whereas increased local production of TGF- 1 may result in fibrotic disorders by activating fibroblasts, endocrine TGF- 1 interacts mainly with endothelial cells and, to a lesser degree, fibroblasts and macrophages . Chronic inflammation can lead to excessive systemic TGF-as a control mechanism to dampen the immune response. Use of TGF-as an immunosuppressant has been suggested; however, excessive TGF-may lead to an unresponsive immune system, resulting in life-threatening bacterial infections. Elevated endocrine TGF-has been noted in conditions that result in immunosuppression, such as SLE, human immunodeficiency virus (HIV), and arthritis . The mechanisms by which TGF-1 activation occurs in vivo have not been fully elucidated. Plasmin is capable of activating TGF-1 by cleaving LAP. Plasmin is also the major fibrinolytic enzyme involved in wound healing and activated from its precursor form, plasminogen, by urokinase-type plas minogen activator (uPA) and tissue-type plasminogen activator (tPA). Activation of plasmin is inhibited by plasminogen activator inhibitor-1 (PAI-1). Transforming growth factor-1 itself is capable of regulating plasmin activation, and thus, potentially at least, of controlling its own activation, by up-regulating PAI-1. Tuan et al. reported a decrease in uPA and an increase in PAI-1 levels in keloid fibroblasts versus normal fibroblasts, suggesting a decrease in the role of keloid fibroblasts in fibrinolysis. This same pattern was shown after treating normal fibroblasts with TGF-1. Another function of plasmin is the activation of matrix metalloproteases, such as collagenase, which is crucial in wound remodeling. Transforming growth factor-1 also has a role in the

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regulation of matrix metalloproteinases by stimulating the synthesis of tissue inhibitor of metalloproteinases- 1 (TIMP-1) and inhibiting collagenase mRNA. Mast cell chymase, in contrast to plasmin, is released as an active heparinbound enzyme that is not easily inhibited by protease inhibitors. Chymase releases TGF-1 as a large latent complex 10-fold more efficiently than plasmin, but is not directly involved in TGF-1 activation. However, chymase does allow for exposure and subsequent activation of latent TGF-by other factors. The mannose 6-phosphate/insulin-like growth factor-2 (M6P/IGF-2) receptor may be involved in the activation of LTGF-1. Dennis and Rifkin (133) have demonstrated the binding of the small latent complex to the M6P/IGF-2 receptor via the two mannose 6-phosphate moieties on the LAP. Exogenous M6P and anti-M6P receptor were able to inhibit the activation of LTGF-1 in bovine aortic endothelial and smooth muscle cells in coculture. However, neither M6P nor anti-M6P had any effect on basal cell migration, the activity of exogenously added TGF-1, the activation of LTGF-1 by plasmin, or the release of LTGF-1 from cells. Ghahary et al. (134) have studied the mechanism of TGF-1 activation via the M6P/IGF-2 receptor in a coculture system, and found that latent TGF-1 released from genetically modified keratinocytes is capable of increasing collagen expression from dermal fibroblasts. This effect was inhibited in a dose-dependent manner by the addition of mannose 6-phosphate. This study also suggested that activation of TGF-1 is due to a conformational change rather than due to cleavage of LAP from mature TGF-1. Isolated fibroblast cell membranes were incubated with either latent TGF-1 or latent TGF-1 and recombinant active TGF-1. Using the mink lung epithelial cell growth inhibition assay, a standard assay for demonstrating TGF-1 bioactivity, it was shown that after centrifugation, supernatants from latent TGF-1 alone did not significantly inhibit cell growth compared with those incubated with active TGF-1. These results suggest that interaction of latent TGF-1 with the M6P/IGF-2 may not result in cleavage of LAP from mature TGF-1. Although the precise mechanism by which M6P/ IGF-2 receptors are involved in LTGF-1 activation is unknown, activation does require PA and plasmin. It has been proposed that the effective concenntrations of both enzyme and substrate are increased by binding to the cell surface, thus facilitating the activation and release of LTGF-1. In vitro studies have suggested a role for retinoids in the activation of TGF-1 through their ability to increase plasminogen and plasmin levels and to increase the expression of cellular type II transglutaminase . Transglutaminase has been shown to be required for TGF-1 activation, possibly by concentrating plasminogen activator to the extracellular matrix by cross-linking it to fibronectin . Thrombospondin is a glycoprotein that is also capable of activating both the large and small latent complexes of TGF-but without proteolytic cleavage of LAP from TGF-1. Instead, it may work by inducing a change in conformation. Similar to thrombospondin, it has been suggested that IgG may also be capable of activating TGFindependent of proteases. Active TGF-1 in MRL/1pr mice was found complexed to IgG in B cells and plasma cells. This complex was shown to strongly inhibit neutrophil function by inhibiting the adhesion and subsequent uptake of bacteria to activated neutrophils. The IgGTGF-complex was shown to be 500 times more potent than recombinant active TGF-in suppressing neutrophil function. This may be due to a more efficient presentation of active TGF-1 to neutrophil TGF-receptors by IgG or because IgG functions as a carrier molecule, thus extending the half-life of active TGF-in circulation. Once TGF-1 is activated, it is capable of binding to heteromeric receptor complexes consisting of type I (RI) and type II (RII) receptors. Each of these receptors possesses a different serine/threonine kinase and both receptors are required for signal transduction following TGF-1 binding. Receptor type II is necessary for the recruitment and activation of RI, and RI is responsible for the propagation of the signal to downstream targets. In normal human skin, RI and RII are present in the epidermis, epidermal appendages, and in vascular cells. Schmid et al. reported that in granulation tissue, the expression of both receptors increased and, as remodeling proceeded, the levels decreased. However, in HSc, the levels of both RI and RII remained high for up to 20 months after injury. It was proposed that the failure to clear high receptorexpressing fibroblasts during remodeling induced a positive feedback loop for the autoinduction of TGF-1. TGF-1 is capable of autoinducing TGF-1 mRNA transcription via activation of the AP-1 complex consisting of c-jun and c-fos proto-oncogene proteins. High levels of the cytokine may thus persist long after the initiating stimulus and this may contribute to the development of fibroproliferative disorders. The downstream molecules responsible for TGF-1 signal transduction are able to produce diverse cellular responses following TGF- 1 binding to its receptor. Transforming growth factor-1 is both a stimulatory and an inhibitory molecule. It is a chemoattractant for monocytes, neutrophils, and fibroblasts, and induces the release of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor- (TNF-), and bFGF from these cells. The effect of TGF-1 on target cells depends on many factors, including cell origin, the state of differentiation local concentrations of activating and inhibiting molecules, and the presence of other growth factors and cytokines. The half-life of active TGF-is approximately 2 to 3 minutes and yet physiological levels are maintained at about 5 ng/ml in normal humans, which indicates that carrier proteins may be involved in transporting TGF-in the plasma.

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Thrombospondin, IgG, or 2-macroglobulin may act as carrier molecules for latent TGF-. Once activated, regulation of TGF-1 appears to occur by its binding molecules, such as the proteoglycan decorin, in the ECM and 2-macroglobulin in the circulation. Mast cells may enhance the levels of TGF- 1, as heparin is capable of releasing active TGF-1 from 2-macroglobulin. It has been reported that patients with HSc and keloids have a statistically significant increase in allergy symptoms, which are often associated with an increase in IgE levels and mast cells counts. The decrease in decorin content in HSc may be in part due to TGF-1. Scott et al. used normal and HSc fibroblasts from the same patients to show that decorin synthesis was lower in HSc compared with normal fibroblasts, and following TGF-1 treatment, decorin was further reduced in all 6 strains of HSc and in 5 of the 6 strains of normal fibroblasts. After removal of TGF-1 and passaging cells, decorin synthesis was no longer suppressed. The decrease in decorin following TGF-1 treatment is in agreement with the results of Kahari et al. (149) who treated normal human skin and gingival fibroblasts with TGF-1. Proteoglycans may normally function to control cell proliferation by regulating growth factors, such as TGF-1 and bFGF, in the ECM, or conversely, downregulation of decorin expression in HSc by TGF-1 may be associated with the increased cell numbers involved. The localization of decorin, versican, biglycan, and TGF-has been demonstrated in normal skin, mature scars, and HSc. In normal skin, decorin was present throughout the dermis, versican and biglycan were present in very low levels, and TGF-1 was not detected. In HSc, decorin was present in the deep dermis and a narrow zone under the epidermis but was absent in the ultrastructural nodules typical of HSc, whereas TGF-1 was localized to the nodules and the deep dermis. Scott et al proposed that the colocalization of TGF-1 and decorin in the deep dermis may be important in the resolution of the scar, as staining for both was quite intense in this region in the mature scars.

Insulin-Like Growth Factor-1

Insulin-like growth factor-1 is another growth factor that may promote excessive matrix deposition in HSc and keloids due to its mitogenic effects and its ability to stimulate synthesis of certain PGs and collagen by fibroblasts. Insulin-like growth factors are expressed in most tissues at various stages in development and may function as autocrine, paracrine, or endocrine factors. In the uterus, IGF-1 is mainly regulated by estrogen. Estrogen is involved in the proliferation of many uterine cells, such as stromal and epithelial cells resulting in uterine growth. It has been shown that IGF-1 mRNA and the IGF-1 receptor expression are increased following estrogen treatment. Estrogen has also been shown to down-regulate insulin-like growth factor binding protein (IGFBP)-1, a binding protein capable of inhibiting the growth-promoting effects of IGF-1. There may be an estrogen-responsive element in the IGF-1 gene, which interacts with an activated estrogen receptor.In rats, estrogen has been shown to inhibit the expression of IGF-1 mRNA in tissues such as kidney, lung, and liver. However, recent studies in humans and primates indicate that low doses of estrogen may stimulate growth in other tissues, perhaps through enhanced growth hormone secretion.Insulin-like growth factor-1 in the serum is bound to specific binding proteins, which protect it from proteolytic degradation. Type III collagen and fibronectin are capable of binding IGFBP-3 and -5, so that IGF-1 released from immune and epithelial cells may associate with the ECM. Insulinlike growth factor-1 may contribute to the development of HSc due to its ability to increase mRNAs for type I and type III procollagens and downregulate collagenase activity. Ghahary et al.have demonstrated an approximately 2-fold increase in IGF-1 mRNA in HSc compared with normal dermis from the same patients. Treating dermal fibroblasts with IGF1 was associated with a 150% increase in pro-1(I) mRNA and a 170% increase in pro-1(III) mRNA. Insulin-like growth factor-1 levels in HSc could be increased by the disruption of sweat and sebaceous glands following injury. In normal skin, IGF-1 is localized to the epithelial cells located in the superficial epidermal layer, sweat and sebaceous glands, and in the deep dermis. However, in HSc, these structures are disrupted. Reepithelialization is dependent upon deep dermal epithelial cells migrating from the residual sweat and sebaceous elements where they are able to secrete IGF-1 in the presence of dermal fibroblasts. As these cells contribute to reepithelialization and to the healing of sweat and sebaceous glands in the skin, the fibroblasts may no longer be exposed to IGF-1. This could facilitate the resolution of HSc. Interestingly, animals such as the rat, rabbit, mouse, and pig lack sweat glands similar to those seen in humans and do not develop keloids or HSc. Insulin-like growth factor-1 may also be capable of inducing TGF-1 expression in dermal fibroblasts, thus augmenting the fibrotic environment. These growth factors are coexpressed in several physiological and pathological conditions by different cell types, such as fibroblasts, platelets , and activated macrophages. Ghahary et al.reported that treatment of fibroblasts with IGF-1 caused an increase in transcription of TGF-and proteinproduction, and this effect persisted for at least 48 hr after withdrawing IGF-1. It was proposed that IGF-1 may stimulate the expression of TGF-1 mRNA in dermal fibroblasts through activation of the AP-1 complex. Transforming growth factor-1 may then act as an autocrine factor and induce its own further expression.

Induction of integrin v3 Siti Julaiha Page 133

The v3integrin has been shown to play an important role in angiogenesis, and neutralizing antibodies directed at it are capable of blocking capillary blood vessel formation. Its expression is induced by VEGF and it is thought to play a critical role in endothelial cell migration. The presence of integrins and cell surface receptors was determined by flow cytometry on a FACStar (Cytometry Research Services; San Diego, CA). Cells were prepared for analysis as follows: HUVECs were trypsinized and the cells resuspended at 1 X 106 cells/ml. Volumes of 250 500 l of the cell suspensions were washed three times with Hanks Balanced Salt Solution (HBSS; GibcoBRL; Grand Island, NY) and finally resuspended in 10% FBS in HBSS. The cells were incubated for 30 min with primary antibodies diluted to 1 g/ml in 10% FBS in HBSS, washed three times with HBSS, incubated for 30 min with secondary antibodies diluted to 1 g/ml in 10% FBS in HBSS, washed three times with HBSS, and fixed in 200 l of 10% formalin (Baxter; Deerfield, IL) at a density of 106 cells/ml. The presence of v3 integrin on the surface of endothelial cells was analyzed by flow cytometry after treatment with medium conditioned by fibroblast-based engineered tissue. Cultured HUVECs display substantial surface expression of v3 integrin under normal culture conditions. Nonetheless, medium conditioned by the fibroblast-based engineered tissue stimulated a significant increase in expression of this integrin.

Integrins in early (three-dimensional) reepithelialization. The migrating epidermis in contact with type I dermal collagen expresses a gene set including the collagen receptor 21, urokinase plasminogen activator (uPA), and interstitial collagenase (MMP-1). With this armamentarium the epidermis can dissect a path between viable dermis and nonviable clot and denature stroma, ultimately leading to slough of the eschar.

Expression of angiogenic growth factors

Several of the angiogenic properties of fibroblast-based engineered tissue described above were demonstrated to be sensitive to neutralizing antibodies against specific growth factors, notably vascular endothelial growth factor and hepatocyte growth factor/scatter factor. Accordingly, experiments were performed to examine the expression of these and other angiogenic factors by the fibroblast- based tissue. Growth factor expression was examined both by estimation of mRNA levels by polymerase chain reaction methods and estimation of the free protein by enzyme-linked immunosorption assay. Specific messenger RNAs were estimated by quantitative reverse transcriptase and polymerase chain reaction (RT-PCR) using the ABI TaqMan method (Perkin-Elmer; Foster City, CA). Total RNA was extracted from the cells using a Rapid RNA Purification Kit (Amresco; Solon, OH). The RNA was reverse transcribed using Superscript II (Life Technologies; Grand Island, NY) with random hexamer primers (Sigma; St. Louis MO). Amplification of samples of cDNA containing 200 ng of total RNA was detected in real time and compared with the amplification of plasmid-derived standards for specific mRNA sequences using a copy number over a range of five orders of magnitude within 404,000,000/reaction. In purification and the efficiency of reverse transcription, mRNA sequences for platelet-derived growth factor (PDGF) B chain, VEGF, or TGF-1 were added to RNA isolations, and their yield measured by the TaqMan procedure. The control mRNA sequences were obtained by T7 RNA polymerase transcription of plasmids containing the

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corresponding sequence. The values were normalized using glyceraldehyde-3-phosphate dehydrogenase as a control.

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References
Biomedical Engineering Handbook - J.D.Bronzino Successful Use of a Physiologically Acceptable Artificial Skin in the Treatment of Extensive Burn Injury John F Burke , MD Tissue Engineering Concepts and Strategies , Amulya K Saxena

Journal of US-China Medical Science , Jul. 2007, Volume 4, No.7 (Serial No.32)

http://www.nigms.nih.gov/Publications/Factsheet_ArtificialSkin.htm http://www.seattlepi.com/local/burn231.shtml http://www.discoveriesinmedicine.com/images/mdis_0000_0001_0_img0032.jpg http://www.scielo.org.za/img/revistas/sajs/v104n11-12/a30fig02.gif http://pubs.acs.org/subscribe/archive/mdd/v07/i09/html/904feature_willis1.html http://www.integra-ls.com/products/default.aspx?product=46#Product%20Description http://www.nlm.nih.gov/medlineplus/ency/article/002363.htm http://www.webmd.com/skin-problems-and-treatments/guide/skin-biopsies http://www.burnsurvivor.com/skin_substitutes.html

Bladder: Kerr et al. The bladder as a bioreactor: urothelium production and secretion of growth hormone into urine.(1998) Nature Biotechnology 16(1):75-9 Vascular: Engbers-Buijtenhuijs et al. Biological characterisation of vascular grafts cultured in a bioreactor. (2006) Biomaterials. 27(11):2390-7 Medical School, and Burn Center, Brigham and Womens, Hospital, Boston, Massachusetts.

In Clinical Use of Skin Substitutes, Dennis P. Orgill, Christine Park, and Robert Demling, Harvard Skin Substitutes and Wound Healing, F.A. Auger D. Lacroix L. Germain, Laboratoire
dOrganognse Exprimentale (LOEX), Hpital du Saint-Sacrement du CHA and Department of Surgery, Laval University, Qubec, Qu. , Canada, Skin Pharmacol Physiol 2009;22:94102, Published online: February 4, 2009.

Scarless Wound Healing, Hari G. Garg, Harvard Medical School at Massachusetts General Hospital

Charlestown, Massachusetts Michael T. Longaker. New York University School of Medicine New York, New York, Copyright 2000 by Marcel Dekker. In Clinical Use of Skin Substitutes Dennis P. Orgill, Christine Park, and Robert Demling, Harvard Medical School, and Burn Center, Brigham and Womens, Hospital, Boston, Massachusetts. Massachusetts Institute of Technology, Cambridge, Massachusetts.

Facts and Models of Induced Organ Regeneration: Skin and Peripheral Nerves, Ioannis V. Yannas, Characteristics of Fetal Wound Repair, Gyu S. Chin, Eric J. Stelnicki, George K. Gittes, and Michael
T. Longaker New York University School of Medicine, New York, New York . Surgery and Burns, Broomfield Hospital, Chelmsford, Essex, England.

Recent Advances in Embryonic Wound Healing, Alison M. Shaw, St. Andrews Centre for Plastic The Role of Transforming Growth FactorsBeta in Cutaneous Scarring, Mamta Shah, Patricia
Rorison, and Mark W. J. Ferguson, University of Manchester, Manchester, England.

Molecular and Cellular Biology of Dermal Fibroproliferative Disorders, Barbara S. Bauer, Edward E.
Tredget, Paul G. Scott, and Aziz Ghahary, University of Alberta, Edmonton, Alberta, Canada.

YOUR BODY How It Works Douglas Light, Introduction by Denton A. Cooley, M.D., President and

Surgeon-in-Chiefof the Texas Heart Institute Clinical Professor of Surgery at the University of Texas Medical School, Houston, Texas Chelsea House, An imprint of Infobase Publishing, 132 West 31st Street, New York NY 10001. CRC Press, London, 2007 .

Tissue Engineering - John P. Fisher, University of Maryland, Antonios G.Mikos, Rice University, Bioengineering of Human Skin Substitutes, Dorothy M. Supp, Shriners Hospital for Children
Cincinnati Burns Hospital , Steven T. Boyce University of Cincinnati. Tissue Engineering Second Edition Robert P. Lanza.

Wound Repair: Basic Biology to Tissue Engineering, Richard A. F. Clark and Adam J. Singer, in Tissue
Engineering and Transplant Medicine Advanced Cell Technology, Worcester, Massachusetts, Robert Langer Department of Chemical and Biomedical Engineering, HarvardMIT Division of Health, Sciences and Technology Massachusetts Institute of Technology Cambridge,

Siti Julaiha

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Massachusetts, Joseph Vacanti Harvard Medical School and Massachusetts General Hospital Boston, Massachusetts, Academic Press , 2000.

Skin Nancy L. Parenteau, Janet Hardin-Young, and Robert N. Ross , Wound Repair: Basic Biology
to Tissue Engineering , Richard A. F. Clark and Adam J. Singer Dermal Equivalents, Gail K. Naughton

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