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1. Introduction
The tissue repair process is a complex cascade of biological events controlled by numerous
cytokines and growth factors that provides local signals at sites of injury. Just after a tissue injury
occurs, a large number of intercellular and intracellular pathways are activated and coordinated
[1]
with the aim of restoring tissue integrity and homeostasis . Cellular and humeral components,
the inflammatory pathways and the blood coagulation cascade are activated in addition.
A wide range of cells from the injured tissue and adjacent locations undergo marked changes
[2,3]
in gene expression and phenotype, leading to cell proliferation, differentiation and migration .
The development of a new microvasculature and microcirculation is also critical for the
homeostasis and correct tissue repair and regeneration. In fact, the absence of a suitable
vasculature transporting oxygen, nutrients, soluble growth factors and biologically active proteins
and numerous cell types to the injured tissue would imply the degeneration of the latter.
One pivotal discovery that has fuel the research in regenerative medicine and tissue
engineering has been the role that cytokine and growth factors play in the process of tissue repair
[5,6]
. These molecules provide signals at local injury sites, regulating the mechanisms and
pathways that govern wound healing and tissue regeneration [7]. Determining the roles that growth
factors play in tissue repair and regeneration is as important as designing, developing and
applying suitable formulations that release them with a spatiotemporal control. This is especially
important as providing the injured tissue a milieu of biological signals may be desirable for the
[8–11]
functional and accelerated repair of the tissue .This knowledge has stimulated the rapid
progress of tissue engineering. This field aims at repairing and restoring damaged tissue function
employing three fundamental ―tools‖, namely cells, scaffolds and growth factors. [12,13].
The success of this approach relies on the delicate and dynamic interplay among these three
[14,15]
components . It has been reported that future generation of scaffolds will have to provide
adequate mechanical and structural support, be biocompatible and bio-resorbable at a controllable
degradation and resorption rate as well as actively guide and control cell attachment, migration,
proliferation and differentiation. This may be achieved if the functions of scaffold are extended to
supply biological signals able to guide and direct cell function through a combination of cue
exposition and cytokine and growth factor controlled delivery [16].
In this review, we summarize the most interesting delivery technologies designed to permit
the local and controlled spatiotemporal release of growth factors for tissue repair and regeneration.
Especially, the progress that has been accomplished in the field of endogenous regenerative
medicine will be described in detail. The use of patient's own growth factors and scaffolds for
regenerative medicine will be highlighted, as some of the most interesting therapeutic applications
of this technology including the treatment of chronic ulcers, bone and soft tissue regeneration in
oral and maxillofacial surgery and oral implantology, the treatment of musculoskeletal injuries
and disorders, and the development of tissue-engineered approaches among others.
2. HISTORY:-
While transplant medicine made rapid progress, cell biologists were reporting similarly
important Advances, although the clinical significance of these had yet to be identified. From
initial research on cell and tissue culture back in the 1960s, where scientists were looking at ways
of keeping pieces of tissue alive outside of the body, work progressed to cell biology, where
different cell types were purified out of tissue samples to produce monolayer cultures of a single
cell type. Once scientists were able to do this, and began to understand what different cell types
made, the role of different metabolites and signals and how each cell behaved, tissue biology
emerged. Researchers started to look at ‗organotypic cultures‘, whereby two or more cell types
were grown together and their interactions examined.
From there, it was a short intellectual step to tissue engineering, a term introduced by
YC Fung of the University of California, San Diego (CA, USA), in 1985 [17].
Definitions of tissue engineering vary. In 1993, Langer and Vacanti described it as ‗an
interdisciplinary field that applies the principles of engineering and life sciences toward
the development of biological substitutes that restore, maintain, or Improve tissue function
or a whole organ‘ [18].
MacArthur and Oreffo defined tissue engineering as ‗understanding the principles of tissue
[19]
growth, and applying this to produce functional replacement tissue for clinical use‘ .
Tissue engineering, subsequently identified as a subset of the overarching field of
regenerative medicine, was in effect born in Massachusetts as a result of that serendipitous
combination of circumstances that sometimes occurs in science. Much of the initial work in
the field began in four independent laboratories at the Massachusetts Institute of
Technology (MIT) in Cambridge (MA, USA), run respectively by Green, Bell, annas and
Vacanti (who collaborated with Naughton out of the Hunter College School of Health
Sciences, NY, USA) [20].
Each laboratory pursued a somewhat different strategy and each strategy eventually resulted in
approved medical products that are available today.
Harvard Medical School in Boston (MA, USA), WL Chick was working to develop a bio-
artificial pancreas [21]. His alternative approach, to encapsulate islet cells in order to isolate them
from immune attack, led to a variety of bio-hybrid organ projects some of which are showing
clinical promise.
[22]
By 1993, the first major review publication on tissue engineering appeared in Science
and, in 1998, the US FDA granted its first approval for an allogeneic tissue engineered product in
the form of Apligraf®, which was described originally as a living skin equivalent by
Organogenesis, a company spawned by Eugene Bell‘s laboratory. The launch of Apligraf ®
followed the autologous burns treatment, Epicel® which originated from Green‘s laboratory and
was, at first, unregulated by the FDA and treated as a tissue graft. Epicel® was originally
commercialized by Biosurface Technology and later by Genzyme Biosurgery after its purchase of
Green‘s company.
The cell thrives in the porous material and secretes various amount of extracellular matrix
depending on their nature. (Developed by Robert Langer’s group MIT).
There have been countless modifications and additions to the different types of synthetic materials
used over the last decade in this approach.
The obvious advantage of the scaffold approach is the immediate creation of a three-
dimensional structure that already has significant structural properties.
Drawback: The intrinsic nature of most of these polymers, which are suture materials,
entails slow degradation with an ensuing lowering of pH of surrounding tissues. This leads
to a slow but rather protracted low-level inflammatory process.
Many groups are therefore attempting to alter the chemical nature of these biomaterials to
inhibit this inflammatory process.
In this approach various types of cells, mostly of mesencymal origin, are grown in such a
fashion within a culture flask that they literally embedded themselves in their very oven
extracellular matrix.
Among many factors, the addition of sodium ascorbate allows the significant appearance
of the various components of the extracellular matrix.
These sheets are than either stacked or rolled to obtain various tissue substitutes.
The main advantage: The absence of extraneous collagens and any synthetic material.
There are approximately 500,000 surgical procedures performed every year in the U.S. which
require bone substitutes. Currently available bone substitutes, including autografts, allografts ,
and synthetic materials, are the most implanted materials second only to transfused blood
products.
3.2 Autograft:-
A graft or portion of living tissue, taken from one part of the body and placed in another site on
the same individual.
3.3 Allograft:-
Grafts between two or more individuals allogenic (genetically different although belonging to or
obtained from the same species) at one or more loci. A bone graft is a piece of bone
transplanted to another part of the skeleton where it is needed to improve function or strengthen
the structure of the area. Sometimes a bone graft is taken from a cadaver, but usually it is
harvested from the patient for which it will be used. Bone grafts are typically harvested from
the patient's iliac crest (top of the hip bone), ribs, or fibula in the lower leg. This can be quite
painful and the complication rate can be high. Approximately 40% of spine fusion patients
complain of pain at the harvest site for as long as five years after surgery.
4.2 Tissues:-
Tissue is 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. Organs are then formed by the functional
grouping together of multiple tissues.
The study of tissue is known as histology or, in connection with disease, histopathology.
The classical tools for studying tissues are the paraffin block in which tissue is embedded
and then sectioned, the histological stain, and the optical microscope.
Transplanted cells:-
Mature, functional cells
Modified cell human cells
Tran differentiated own patient‘s cells
Non-human cells (XENOTRANSPLANTATION)
Stem cells (autologous or allogeneic)
6. What is Scaffold-cell?
The scaffold is three-dimensional and is composed of hollow or solid fibers of a
biocompatible, synthetic polymer which is biodegradable or non-biodegradable. The fibers of the
scaffold may have a branched configuration extending outwardly from a central stem. Fibers of
the scaffold are spaced apart such that the maximum distance over which diffusion of nutrients
and gases must occur through a mass of cells attached to the fibers is between 200 and 300
microns. The diffusion provides free exchange of nutrients, gases and waste to and from cells
proliferating throughout the scaffold in an amount effective to maintain cell viability throughout
the scaffold in the absence of vascularization. Cells derived from vascularized organ tissue are
attached in vitro to the surface of the fibers uniformly throughout the scaffold in an amount
effective to produce functional vascularized organ tissue in vivo, the cells are grown on the
scaffold in a nutrient solution in vitro to form the cell-scaffold composition which is implanted in
a host at a location having adequate vascularization to allow growth of blood vessels into the cell-
scaffold composition. Growth factors, compounds stimulating angiogenesis and
immunomodulators may be provided in the cell-scaffold composition and the fibers may have a
coating to enhance cell attachment. Combinations of cell-scaffold compositions containing
different cell populations may be implanted.
6.1.1 Scaffold:-
• Allow cell attachment and migration .
• Deliver and retain cells and biochemical factors .
• Enable diffusion of vital cell nutrients and expressed products ..
• Exert certain mechanical and biological influences to modify cell behaviour.
problems for patients and healthcare professionals alike. Transplantation of the patient‘s own skin by
way of a split-thickness skin graft remains the ‗gold standard‘ in the treatment of major skin loss.
However, this incurs significant donor site morbidity and harvest sites are limited. Advances in tissue
engineering mean that there is now a limitless supply of skin through the development of innovative
products such as Integra®, a matrix designed to provide immediate wound closure and permanent
regeneration of the dermis.
In the absence of underlying disease, almost every full-thickness wound will heal with minimal
intervention, however, the process can be enhanced by judicious wound management. The first
clinical decision to be made is whether to repair the wound or to allow it to heal by secondary
intention (Rivera and Spencer, 2009). There are four phases to the healing process:
1. The inflammatory phase involves bleeding, immediate narrowing of the blood vessels
and clot formation
2. The proliferative phase is where new skin cells and blood vessels are formed
3. The remodeling phase takes place after 2–3 weeks and involves the replacement of the
collagen layer
4. Finally, the skin forms a protective barrier (epithelial cells) between the outer environment
and the body.
Advances in wound care have resulted in a vast range of products that can accelerate healing,
regenerate the dermis and reduce bacterial inflammation. Furthermore, in complex full-
®
thickness wounds, the use of skin substitutes such as Integra Dermal Regeneration Template
(Integra) (Figure 1) is gaining popularity. Dermal regeneration templates have been used for
three decades in the management and reconstruction of complicated wounds (Yannas et al,
1982)
3 months
In this surgery Professor Macchiarini and colleagues removed a trachea from a human
cadaver, decellularized it (i.e., removed all cellular material leaving the extra cellular matrix
scaffold behind) and, using the bioreactor, recellularized the scaffold with adult stem cells
cultured from a biopsy of the patient's own tissue. After four days in the bioreactor the tissue
engineered airway was implanted in the patient replacing her defective left main bronchus. The
patient made a full recovery, showed a substantial improvement in quality of life and showed no
immune rejection of the new airway.
The eyeball is covered by the cornea — the eye‘s most important light-refracting structure
(Fig. 1a). The cornea produces the initial image and casts this onto the lens behind it.The clearness
of the cornea is essential to visual acuity and depends on both the integrity of the corneal
epithelium covering the eye‘s surface and a lack of blood vessels in the underlying support tissue
(the stroma). At its margins, the corneal epithelium is attached to the delicate mucous
(conjunctival) epithelium that covers the whites of the eye (sclera) and the internal part of the
eyelids. The narrow zone between the cornea and the conjunctiva is known as the limbus.
Experimental and clinical evidence indicates that the limbus is the source of corneal epithelial
stem cells in humans.The limbus can be destroyed by ocular burns or infection, causing corneal
stem-cell deficiency. But, in one of nature‘s remarkable efforts to repair tissues at all costs,
abnormal invasion by conjunctival cells provides the damaged cornea with a protective surface
layer . The consequences are dire, resulting in vascularization of the cornea, chronic
inflammation, stromal scarring and, ultimately, corneal opacity and loss of sight2.Allogeneic
corneal transplantations, which involve transplanting cornea from a genetically non-identical
donor, have to some extent been successful in restoring patients‘ vision.
Eventually, however, conjunctiva cells invade and replace the transplanted cornea. What‘s
more, two other factors make treating patients with ocular burns by corneal transplantation
problematic: the number of available donors is insufficient to meet demand, and the increasingly
popular corrective laser eye surgery often makes the cornea unsuitable for transplantation.
Pellegrini, De Luca and colleagues1 now report that limbal stem cells maintained in culture can be
a viable alternative source of cells for transplantation to treat burned human corneas.Stem-cell
transplantation is not a new concept.More than half a century ago, E. Donnall Thomas showed
that intravenous infusion of donor bone-marrow cells can repopulate the bone marrow and
produce new blood cells4;he later won a Nobel prize for this first demonstration of the use of stem
cells for regeneration of damaged or diseased tissues and organs. By the 1970s, physicians were
successfully performing bone-marrow transplants, which are now used to treat blood disorders
ranging from severe combined immunodeficiency to sicklecell anaemia to leukaemias, as well as
other cancers of the human immune system.
By the early 1980s, human skin stem cells were being cultured to make epidermal sheets to
repair the skin of badly burned patients. Pellegrini, De Luca and colleagues have been culturing
corneal stem cells from small biopsies of human limbal tissue for the past decade. The appreciable
similarities between limbal and epidermal cells allowed the researchers to adapt methods
developed for human epidermal stem-cell cultures. Cultured epidermalcell colonies can be
classified according to cell number and capacity for growth. The smallersized colonies generate
epidermal cells that stop growing over time. By contrast, the largersized colonies — referred to as
holoclones —display quintessential features of stem cells, namely long-term self-renewal and the
ability to regenerate tissue. This makes them suitablefor burn therapy.Pellegrini, De Luca and co-
workers7 discovered that human limbal cells cultured using a similar protocol also form small and
large colonies.Interestingly, only the limbal holoclones and not the smaller colonies expressed
p63,a transcription factor that is essential for theproliferative potential of epidermal stem cells8.In
the impressive accompanying clinical studies9,the researchers obtained limbal stem cells from the
healthy eye of 112 patients with ocular burns, cultivated them and then transplanted the cultured
cells onto the patients‘ damaged eye. After an extensive 10-year monitoring period, the authors
now report1 permanent restoration of a transparent, self-renewing corneal epithelium in three-
quarters of the study patients (Fig. 1c).
Notably, 78% of the successful transplantations involved cultures in which p63-expressing
cells constituted more than 3% of the cells capable of forming colonies.These observations unveil
a direct correlation between the percentage of p63-positive corneal stem cells in a culture and their
transplantability.The correlation presents a powerful diagnostic tool for predicting whether any
given limbal culture is likely to be suitable for long-term transplantation.This work1 also offers
hope for exploring alternative sources of limbal stem cells to treatpatients who have suffered
severe injuries to both eyes, and who therefore lack limbal stem cells. Indeed, in the future it
might be possible to create corneal stem cells by culturing other cells from the patient — for
instance, skin stem cells — and then either directly inducing their transdifferentiation to limbal
cells, or transforming them first to an embryonic-stemcell-like state (induced pluripotent stem
cells,or iPS cells) before inducing their differentiation along the limbal lineage.
engineering.An overview about the most common scaffold types applied in cardiovascular tissue
engineering is given in table 1. After seeding, the constructs are cultured in nutrient media
supplemented with ascorbic acid and 10% foetal calf serum in a pulse duplicator in vitro system
(bioreactor) mimicking the in vivo environment. In order to improve cell migration, proliferation
and extracellular matrix production mechanical load by means of shear stress have been applied to
the seeded valves in pulsatile flow bioreactors .
There, the tissue formation takes place and after several days the constructs are ready for
implantation. This concept for in vitro heart valve tissue engineering was earlier applied in an
animal model using completely autologous tissue engineered heart valves based on polyglycolic
acid coated with poly-4-hydroxybutyrate (PGA/P4HB) (figure 2) starter matrices . In this ―proof
of principle‖ study trileaflet heart valve scaffolds were fabricated from PGA/P4HB bioabsorbable
polymers and sequentially seeded with autologous ovine myofibroblasts and endothelial cells. The
constructs were grown for 14 days in a pulse duplicator in vitro system under gradually increasing
flow and pressure conditions and implanted into a growing sheep model (n = 6 lambs; mean
weight at cell harvest 9 ± 2.8 kg). Echocardiography demonstrated mobile, functioning leaflets
without stenosis, thrombus or aneurysm. These autologous tissue engineered valves functioned up
to 5 months in vivo and resembled normal heart valves as to microstructure, mechanical properties
and extracellular matrix formation.
With regard to future routine clinical realization of the tissue engineering concept, human marrow
stromal cells are a promising cell source. In contrast to vascular cells, these cells can be obtained
without surgical interventions representing an easy-to-access cell source in a possible routine
clinical scenario. The usage of marrow stromal cells may offer several advantages in i) easy
collection by a simple bone marrow puncture avoiding the sacrifice of intact vascular structures,
ii) showing the potential to differentiate into multiple cell lineages, and iii) demonstrating unique
immunological characteristics allowing persistence in allogenic settings. Recently, human marrow
stromal cells have been used for the fabrication of trileaflet heart valves (figure 3) . Histology of
the tissue engineered valve leaflets revealed viable tissue organized in a layered fashion with
extracellular matrix proteins characteristic of heart valve tissue such as collagen I and III, and
glycosaminoglycans. However, the typical three layered structural composition of native valve
leaflets comprising a ventricularis, spongiosa and fibrosa layer was not achieved. The ultra-
structural analysis of the tissue engineered heart valves supported this observation demonstrating
cell elements typical of viable, secretionally active myofibroblasts such as actin/myosin filaments
as well as collagen fibrils and elastin. The quantitative extracellular matrix protein analysis
revealed values significantly lower compared to human native valve tissue.
Epithelial stem cells can be harvested from the palate while mesenchymal stem cells can
be obtained from either bone marrow or the pulp tissue of an extracted third molar
Stem cells are isolated and multiplied in vitro by using appropriate supportive culture
media
At the late cap or bell stage the tooth germ is transplanted into the jaw
Development:-
The treatment of damaged cartilage has long been one of the aims of orthopedic
surgery. Professor Mitsuo Ochi of Hiroshima University has taken small amounts of cartilage
from patients with cartilage damage and produced cultured cartilage, which is then implanted into
the defective area. The therapeutic technique he established is known as autologous cultured
cartilage transplantation. J-TEC was quick to take notice of this method and obtained the guidance
of Professor Ochi concerning his culture technique, in order to develop Japan's first ever cultured
cartilage.
Culture:-
By creating these new offices for regulation, the FDA has begun to evolve into a more
nimble approval agency capable of handling the advances of the 21st century .To complement
these new offices, the FDA has also begun collaborating with various NIH Institutes and
participating in the Multi-Agency Tissue engineering Sciences Working Group to raise awareness
of FDA guidelines and procedures. Another innovative initiative the FDA has begun in
conjunction with the National Cancer Institute allows cancer researchers to link their
investigational new drug (IND) applications to the FDA. This program has the goal of reducing
process and submission times for researchers. If successful, this program will provide a model for
IND applications in other fields, including regenerative medicine. While the FDA has made great
strides towards embracing new technologies, the private sector still views the regulatory process
as somewhat difficult. FIRM will provide a cohesive framework whereby public and private
funding organizations will partner with the FDA very early in the development of regenerative
medicine products to facilitate transparency in the regulatory oversight process for these new
products. By partnering in this manner, FIRM will set a new standard for industry and FDA
cooperation that will lead to faster product approvals without sacrificing safety or efficacy.
Despite 10 years of study in regenerative medicine, there has been a lack of directed
research. Although much has been learned on these subjects the field is still in an embryonic
stage. Without this fundamental research, the potential of fully engineered complex tissues will
never be realized. If regenerative medicine researchers and clinicians are able to gain a detailed
understanding of how cells interact with each other and how to mass-produce, preserve, catalogue,
and build these cells, they can then apply this knowledge towards developing tissue and organ
based therapies. One example of a complex regenerative medicine issue that scientists must solve
is the growth of vasculature in tissues and organs. These vascular tissues are blood vessels
responsible for transporting nutrients and waste through tissue. Due to a lack of understanding of
cellular interaction, scientists have not been successful in creating vasculature in tissues and
organs, limiting regenerative medicine products to ―two dimensional‖ materials, such as skin and
bone, which do not require vascular tissue upport. To achieve the promise of regenerative
medicine ,growing vascularized tissues is a necessary next step. To do this, scientists must gain a
better understanding of tissue interactions and scaffold technology. Once scientists understand
these concepts, they will be able to apply this knowledge and create more advanced tissue
systems. Ultimately, the application of knowledge of cellular interactions and tissue growth will
culminate in two branches of regenerative medicine research, in vivo cell based therapy and in
vitro grown tissues and organs. Cell-based therapy focuses on cellular treatments that lead to
regeneration by having the body ―gather‖ the necessary reparative cells and bring them to the
damaged site. The second branch is the in vitro growth of tissues and organs that are then
implanted within the body, either to prompt regeneration or to replace damaged tissue.30 Each
branch of research offers substantial advances over current medicine, and discoveries from one
branch may be directly applicable to the other. Both of these research branches are vital for fully
realizing all of the potential therapies of regenerative medicine. For example, preliminary research
has shown that spinal cord regeneration through implantation of seeded scaffolds is feasible.31
Seed-driven regeneration is a form of cell-driven therapy that will not require transplantation of a
new tissue or organ. On the other hand, it is believed that treatment of a cancerous lung would
require removing the lung and replacing it with a laboratory grown healthy lung. In the case of
cancer, seeding the lung for regeneration would not work, as the cancer would still be present.
These two different applications of regenerative medicine demonstrate why it is essential to
research both cellular therapies and full organ growth techniques to maximize the potential of
regenerative medicine. It may be possible to use regenerative medicine to cure a diseased lung
without removing it, or to cure spinal injuries through neural cell transplants. What is most
important is that these two branches be investigated fully: findings from both will further our
knowledge of regenerative medicine .Fulfilling these goals will create a foundation for future
regenerative medicine products and work .A cohesive effort focused on advancing the science and
the field as a whole is essential.
With a cohesive Government initiative and appropriate funding, within 20 years
regenerative medicine will be the standard of care for replacing all tissue/organ systems in the
body in addition to extensive industrial use for pharmaceutical testing. The ultimate goal at the
end of 20 years is to have real time mass customization of tissues on demand, in vivo. During
those 20 years, as our knowledge of tissues grows, it is reasonable to expect to see treatments
discovered along the way, roughly at the 5, 10 and 20 year marks. In 5 years the following
milestones are hoped for:
• Develop multiple applications for skin, cartilage, bone, blood vessel, and some
urological products
• Develop insurance reimbursable regenerative therapies • Establish standards for FDA
regenerative medicine therapy product approvals
• Solve cell sourcing issues, giving researchers access to the materials they need to
design new therapies
• Establish cost-effective means of production, paving the way for future products •
Establish specialized cell banks for tissue storage, allowing storage of viable ―off the
shelf‖ products
In 10 years, effective regenerative medicine therapies will be available for patient care and
industrial research and development purposes. At this time, the following may be achieved:
• Further understand stem cell and progenitor cell biology
• Engineer smart degradable biocompatible scaffolding
• Develop microfabrication and nanofabrication technologies to produce tissues with
their own complete vascular circulation
• Develop complex organ patches, that could repair damaged pieces of the heart or other
organs
Ultimately, within 20 years the full benefits of regenerative medicine therapies will be
reached. Some of the applications of regenerative medicine could be:
• Harness regenerative medicine materials to produce in situ regeneration of diseased and
damaged structures in many areas of the body
• Regenerate most damaged tissues and organs either in vivo or through implanted
regeneration therapies
• Produce in vitro sophisticated 3-D tissues and organs that cannot be regenerated through in
vivo techniques, such as an entire heart or lung Without a Federal initiative supporting
this research, this timeline could extend over the next 40 to 50 years. Considering the
many economic and health advances this technology may bring, it is absolutely vital that
regenerative medicine advance as quickly as possible.
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