BIOLOGY ASSIGNMENT WOUND HEALING SPRAY

GROUP : C4 Beta 1

GROUPS MEMBERS : (1) Tan Jing Peng (1300433) (2) Koh Wei See (1300723) (3) Sangeeta A/P Rajindran (1200225) (4) Meera A/P Kalaiselvan (1300607) (5) Thiviya Gunalan (1300633) (6) Velaraasi A/P Mathiyalagan (1300608)

DATE OF SUBMISSION : 24 January 2014

CONTENT
NO. 1. Introduction 1.1 Definition of wound 1.2 Type and Causes of wound 1.3 Complications 1.4 Risk factors which causes developing of wounds 1.5 Motivation 1.6 Justification 1.7 Stages of wound healing 1.8 Barriers that slows down the healing of the wound 2. 3. 4. 5. 6. 7. 8. 9. Objective Ingredient Method Literature Review Expected Results Results Discussion Reference 2 3-6 7-9 10 11 12-19 20 21 22-24 25 26 27-32 33 34-38 39-62 63-64 Title Page

1.1 Definition Of Wound

A wound is the breakage of normal alignment of body cells with or without destroying them, which may impair or stop their function. Wound causes can be internal or external in origin. Wounds of internal origin are mainly due to impaired circulation, neuropathy or medical illness. Wounds of external origin are due to an outside force or trauma that causes open or closed wounds.

1.2

Type and Causes of wound

Internal Wounds
Disturbance of the different regulating systems of the human body can lead to wound formation, and may include the following:

Impaired circulation: This can be from either ischemia or stasis. Ischemia is the result of reduced blood supply caused by the narrowing or blockage of blood vessels, which leads to poor circulation. Stasis is caused by immobilization (or difficulty moving) for long periods or failure of the regulating valves in the veins, which leads to blood pooling and failing to flow normally to the heart.

Neuropathy: This is seen mostly in cases of prolonged uncontrolled diabetes mellitus, where high blood sugars, derivative proteins and metabolites accumulate and damage the nervous system. The patients are usually unaware of any trauma or wounds, mainly due to loss of sensation in the affected area.

Medical illness: When chronic and uncontrolled for long periods (such as hypertension, hyperlipidemia, arthrosclerosis, diabetes mellitus, AIDS, malignancy, morbid obesity, hepatitis C virus, etc.), medical illnesses can lead to impairment of the immune system functions, diminishing the circulation and damaging other organs and systems.

External Wounds
External wounds can either be open or closed. In cases of closed wounds, the skin is intact and the underlying tissue is affected but not directly exposed to the outside environment. The following are the most common types of closed wounds:

Contusions: These are a common type of sports injury, where a direct blunt trauma can damage the small blood vessels and capillaries, muscles and underlying tissue, as well the internal organs or bone. Contusions present as a painful bruise with reddish to bluish discoloration that spreads over the injured area of skin.

Hematomas: These include any injury that damages the small blood vessels and capillaries resulting in blood collecting and pooling in a limited space. Hematomas typically present as a painful, spongy rubbery lump-like lesion. Depending on the severity and site of the trauama, hematomas can be small or large, deep inside the body or just under the skin.

Crush injuries: These are usually caused by an external high-pressure force that squeezes part of the body between two surfaces. The degree of injury can range from a minor bruise to a complete destruction of the crushed area of the body, depending on the site, size, duration and power of the trauma.

In cases of open wounds, the skin is cracked open, leaving the underlying tissue exposed to the outside environment. The following are the major types of open wounds:

Abrasions: These are shallow irregular wounds of the upper skin layers, due to brushing against either a rough surface or a smooth surface at high speed.

Abrasions usually present with minor to no bleeding, and some pain that subsides shortly after the initial injury.

Lacerations: These wounds are tear-like wounds with irregularly torn edges that are usually deeper than abrasions and cause more pain and bleeding. Lacerations are generally caused by trauma or contact with an object, as might result from hard blows, collusions or accidents.

Incisions: These are most likely the result of a surgical procedure or skin cut with a sharp object such as a scalpel or knife. Incisions are mostly linear in shape with smooth, even edges. Depending on the depth and site of the wound, an incision can be life threatening and cause serious damage, especially if it involves vital organs, major blood vessels or nerves.

Punctures: These are small rounded wounds that result from objects with thin pointed tips, such as needles, nails or teeth (in cases of human or animal bites). The wound size, depth, bleeding and pain are directly related to the size and force of the causative object.

Penetrating: This type of wound can be caused by any object or force that breaks through the skin to the underlying organs or tissue. A penetrating wound has variable sizes, shapes and presentations depending on the cause. Penetrating

wounds can be life threatening, causing serious injury, especially if involving vital organs, major blood vessels or nerves.

Gunshot wounds: These are considered to be penetrating wounds that are exclusively caused by bullets from firearms. The wounds at the entrance site of a bullet are regular, rounded, and smaller than the bullet size. Entrance wounds may have burn marks or soot on the edges and surrounding tissue, depending on the distance from which the bullet was fired. If the bullet goes all the way through the body, the exit wound will have an irregular shape that is larger than the entrance wound and usually bleeds more. Aside from the risk of hitting vital organs or major blood vessels, the fast, spinning movement of the bullet may cause serious damage to the surrounding tissue it passes through.

1.3 Complications
Internal wound complications:

Impaired circulation: In cases of both ischemia and stasis, the supply of blood, oxygen and nutrients, as well as flushing of extra waste products, is impaired. This results in tissue death and wound formation that may develop later to venous and arterial ulcers, or gangrene.

Neuropathy may give rise to:

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Diabetic foot ulcers: The part of nervous system responsible for sweating and moisturizing the skin is impaired, leaving the skin on the feet dry and vulnerable to cracks and injuries. Due to the reduced sensation of that area, many skin cuts, cracks or injuries can go unnoticed and progress to ulcers.

Charcot or neuropathic joints: The part of nervous system that controls the muscle of the leg and foot is damaged, leading to improper distribution of force and pressure while performing routine activities. This can eventually cause joint dislocation, deformity, fracture and injury.

Medical illness: A weakened immune system and other malfunctioning or diseased systems decrease the ability of the human body to defend itself against infections, inflammations, ulcers or wounds. A weakened immune system can also delay or prevent wound healing.

Externally caused closed wounds can be complicated by:

Severe bleeding Large bruises Nerve damage Bone fractures Internal organ damage Compartment syndrome: This syndrome involves the lower and/or upper limbs (especially the legs and forearms), where the damage causes swelling and increased pressure in the fascia that surrounds the muscles, nerves and blood vessels in that area. The increased pressure can block the blood supply to the affected limbs, causing severe damage to the muscles and nerves. The damage can be permanent, leading to loss of function, and may necessitate amputation.

Externally caused open wounds can be complicated by:

Infections: Except for surgical incisions, most open wounds are caused by dirty, contaminated objects that carry different types of bacteria and organisms. An infected wound may present with a foul odor, pus or yellowish drainage, fever and pain.

Inflammation: This can result from the body's immune response to a foreign material that caused a wound. Inflammation can make the wound area red, hot, swollen and painful.

Loss of function: Whether because of the pain or the trauma itself, loss of function can be temporary or permanent, depending on the extent of the wound and the damage to the affected limb or area.

Scarring: Many open wounds will leave a scar after healing, and some may even cause a deformity of the affected area, especially with penetrating, gunshot or deep laceration wounds.

1.4 Risk Factors Which Causes Developing Of Wounds

Having one or more of the following can increase the risk of developing wounds:

Heavy smoking, alcohol consumption and increased age: These factors can reduce the elasticity of blood vessels and increase the probability of blood clots, leading to vascular related disease and improper healing.

Immobility: Whether standing, sitting or lying down for long periods, as in cases of severe burns, multiple surgeries or car accidents, immobility can cause stasis and increase the risk of developing bed ulcers, venous ulcers, deep vein thrombosis and varicose veins.

Unhealthy lifestyle: These risk factors include limited exercise, poor diet, obesity and poor hygiene.

Weakened immune system: Patients taking corticosteroids, chemotherapy, radiotherapy or receiving a transplantation

History of chronic medical illnesses or vascular disease: These illnesses include cancers, diabetes, high cholesterol, AIDS, heart disease, hypertension, atherosclerosis, anemia, varicose veins or deep venous thrombosis.

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1.5 Motivation

The motivation of this proposal is to help humans to heal their wounds at a faster rate without going through operation and suffering from any side effects. This product is convenient because it is small and easy to carry. It can be applied to any wounds no matter how serious the wound is. It can prevent excessive bleeding by just applying this spray to the wound because the product contains blood clotting factor. As a result, this can reduce the risk of fatality caused by excessive bleeding.

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1.6 Justification

Wound healing is an intricate process whereby the skin (or another organ-tissue) repairs itself after injury. So, we need to increase the rate of mitosis of skins. Mitosis is the process in which a eukaryotic cell nucleus splits in two, followed by division of the parent cell into two daughter cells. The word "mitosis" means "threads," and it refers to the threadlike appearance of chromosomes as the cell prepares to divide. Early microscopists were the first to observe these structures, and they also noted the appearance of a specialized network of microtubules during mitosis. These tubules, collectively known as the spindle, extend from structures called centrosomes with one centrosome located at each of the opposite ends, or poles, of a cell. As mitosis progresses, the microtubules attach to the chromosomes, which have already duplicated their DNA and aligned across the center of the cell. The spindle tubules then shorten and move toward the poles of the cell. As they move, they pull the one copy of each chromosome with them to opposite poles of the cell. This process ensures that each daughter cell will contain one exact copy of the parent cell DNA. Mitosis consists of five morphologically distinct phases: prophase, prometaphase, metaphase, anaphase, and telophase. Each phase involves characteristic steps in the process of chromosome alignment and separation. Once mitosis is complete, the entire cell divides in two by way of the process

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called cytokinesis. Prophase is the first stage in mitosis, occurring after the conclusion of the G2 portion of interphase. During prophase, the parent cell chromosomes which were duplicated during S phase condense and become thousands of times more compact than they were during interphase. Because each duplicated chromosome consists of two

identical sister chromatids joined at a point called the centromere, these structures now appear as X-shaped bodies when viewed under a microscope. Several DNA binding proteins catalyze the condensation process,including cohesin and condensin. Cohesin forms rings that hold the sister chromatids together, whereas condensin forms rings that coil the chromosomes into highly compact forms. The mitotic spindle also begins to develop during prophase. As the cell's two centrosomes move toward opposite poles, microtubules gradually assemble between them, forming the network that will later pull the duplicated chromosomes apart. When prophase is complete, the cell enters prometaphase the second stage of mitosis . During prometaphase, phosphorylation of nuclear lamins by M-CDK causes the nuclear membrane to break down into numerous small vesicles. As a result, the spindle microtubules now have direct access to the genetic material of the cell.Each microtubule is highly dynamic, growing outward from the centrosome and collapsing backward as it tries to locate a chromosome. Eventually, the microtubules find their targets and connect to each chromosome at its kinetochore, a complex of proteins positioned at the centromere. The actual number of microtubules that attach to a kinetochore varies between species, but at least one microtubule from each pole attaches to the kinetochore

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of each chromosome. A tug-of-war then ensues as the chromosomes move back and forth toward the two poles. As prometaphase ends and metaphase begins, the chromosomes align along the cell equator. Every chromosome has at least two microtubules extending from its kinetochore with at least one microtubule connected to each pole. At this point, the tension within the cell becomes balanced, and the chromosomes no longer move back and forth. In addition, the spindle is now complete, and three groups of spindle microtubules are apparent. Kinetochore microtubules attach the chromosomes to the spindle pole;interpolar microtubules extend from the spindle pole across the equator, almost to the opposite spindle pole; and astral microtubules extend from the spindle pole to the cell membrane.Metaphase leads to anaphase, during which each chromosome's sister chromatids separate and move to opposite poles of the cell. Enzymatic breakdown of cohesin which linked the sister chromatids together during prophase causes this separation to occur. Upon separation, every chromatid becomes an independent chromosome. Meanwhile, changes in microtubule length provide the mechanism for chromosome movement. More specifically, in the first part of anaphase sometimes called anaphase A the kinetochore microtubules shorten and draw the chromosomes toward the spindle poles. Then, in the second part of anaphase sometimes called anaphase B the astral microtubules that are anchored to the cell membrane pull the poles further apart and the interpolar microtubules slide past each other, exerting additional pull on the chromosomes .

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During telophase, the chromosomes arrive at the cell poles, the mitotic spindle disassembles, and the vesicles that contain fragments of the original nuclear membrane assemble around the two sets of chromosomes. Phosphatases then dephosphorylate the lamins at each end of the cell. This dephosphorylation results in the formation of a new nuclear membrane around each group of chromosomes. Cytokinesis is the physical process that finally splits the parent cell into two identical daughter cells. During cytokinesis, the cell membrane pinches in at the cell equator, forming a cleft called the cleavage furrow. The position of the furrow depends on the position of the astral and interpolar microtubules during anaphase.The cleavage furrow forms because of the action of a contractile ring of overlapping actin and myosin filaments. As the actin and myosin filaments move past each other, the contractile ring becomes smaller, akin to pulling a drawstring at the top of a purse. When the ring reaches its smallest point, the cleavage furrow completely bisects the cell at its center, resulting in two separate daughter cells of equal size . To increase the rate of mitosis, the number of chromosomes and the amount of DNA which needed to be replicated and separated have to increase. Therefore, we can produce different types of healing sprays of different organs such as skin by inserting different types of chromosome. For example, a skin healing spray only consists of chromosomes and DNA that codes for skin cells.

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Advantages of The Instant Spray For its Wound Healing Spray forming offers certain advantages over both conventional ingotmetallurgy and more specialized techniques such as powder metallurgy. Firstly, it is a flexible process and can be used to manufacture a wide range of materials, some of which are difficult to produce by other methods, e.g. Al-5wt% Li alloys or Al-SiC, Al-Al2O3metal matrix composites (MMCs). The atomisation of the melt stream into droplets of 10-500 m diameter, some of which, depending on diameter, cool quickly to the solid and semi-solid state provide a large number of nucleants for the residual liquid fraction of the spray formed material on the billet top surface. The combination of rapid cooling in the spray and the generation of a large population of solid nucleants in the impacting spray leads to a fine equiaxed microstructure, typically in the range 10100 m, with low levels and short length scales of internal solute partitioning. These microstructural aspects offer advantages in material strength because of fine grain size, refined distribution of dispersion and/or secondary precipitate phases, as well as tolerance to impurity tramp elements. This fine structure in the as sprayed condition means homogenising heat treatments can often be avoided. Because of the complex solidification path (i.e. the rapid transition from superheated melt to solid, liquid or semi-solid droplet to temperature equilibration at semi-solid billet top and final slow cooling to fully solid) of the spray formed material, extended solubility of alloying elements and the formation of metastable and quasi-crystalline phases has also been reported. One of the major attractions of spray forming is the potential economic benefit to be gained from reducing the number of process steps between melt and finished product. Spray forming can be used to produce strip, tube, ring, clad bar / roll and cylindrical extrusion feed stock products, in each case with a relatively fine-scale microstructure even in large cross-sections. The benefits of GASF over powder metallurgy accrue from the reduced number of process steps where powder sieving, pressing, de-gassing and handling steps and their attendant safety and contamination issues may be removed. Generally speaking, this spray is also very effective as it is only needed to apply once in order to heal any types and degree of wounds, burns and bruises. Clinically speaking, the whole healing process can be done in one office visit. This spray can also be used to treat larger areas and only small quantity is needed to so

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Disadvantages of the Instant Spray for its Wound Healing There are two major disadvantages to the gas atomisation spray forming process. The most significant disadvantage is the relatively low process yield with typical losses of ~30%. Losses occur because of overspray (droplets missing the emerging billet), splashing of material from the billet surface, and material bouncing off the semi -solid top surface. Many operators of the spray forming process now use a particle injector system to re-inject the overspray powder, and thus recycle material that would otherwise be lost, or sell the overspray powder as a product in its own right. The second major disadvantage is one of process control. As it is essentially a free-forming process with many interdependent variables, it has proved difficult to predict the shape, porosity or deposition rate for a given alloy. Much of the control is based on operator experience and empirical relationships. It is partly the process complexity and lack of robust process control that has prevented the widespread commercialisation of this process. Some developments using feed-back control have proved successful in improving the variations in billet diameter and improving yield in specific systems but these have yet to find widespread implementation. Porosity resulting from gas entrapment and solidification shrinkage is a significant problem in spray formed materials. A typical spray formed billet will contain 1-2% porosity with a pore size dependent on alloy freezing range and various process parameters. Hot isostatic pressing (HIPing) or thermo-mechanical processing can heal these pores if they are small (less than 30 m). Despite these disadvantages, spray forming remains an economic process for the production of difficult to manufacture, niche alloys. Large-scale porosity is more difficult to heal effectively and must be minimised by careful process control. In some cases, porosity is controlled by alloy additions which react with dissolved and entrapped gas to form a solid phase e.g. titanium added to copper billets to form titanium nitride with dissolved and entrapped nitrogen gas. Porosity, even after consolidation, can limit the applications of spray formed material, for example rotating gas turbine components must have zero porosity because of the detrimental effect on high-cycle fatigue (HCF).Besides, and the spray is also not effective for all the patients as the ingredients may cause allergic reaction on patients affected regions. This can also be due to different types of skin .Apart from that,using this spray on a normal and healthy skin is dangerous as it can cause formation of new layer of skin developing on the healthy skin. This also causes this newly formed layer of cells look like bruise. Development of this new layer of skin due to excessive mitosis has high chances of developing cancer. Furthermore, the amount of usage of this spray on infected areas should be given extra attention as it too has its pros and cons. Excessive usage of this spray can cause further degeneration of the cells and this will not lead to any sorts of improvement as it would most likely worsen the bruise. As a result, the healing process takes a longer time and worst case scenario the spray might not even work.

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Disadvantages of Excessive Use of Ingredients Antibacteria Due to immunosuppressants this will cause kidney failure and can bring negative effects to the immune system. Vitamin C Excessive vitamin can cause DNA due to oxidation of guanine to oxoguanine . Centellaasiatica Excessive use of this ingredient in the spray can give negative effect as it will cause inflammation of blood vessel . Glucosamine Excessive use of glucosamine can affect the measure of insulin in diabetes patients hence this is a drawback Bromelain Contains chemicals that might interfere with the growth of tumour cells and slow blood clotting. L-arginie Excessive use of this substances can cause itchiness and even rash. Glutamine Too much of glutamine can cause skin darkening hence it should be used moderately CALENDULA Calendula is not advisable to be used on pregnant women as it can bring about negative effects when breastfeeding

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Ways to Overcome the Disadvantages This is especially for those with allergic skin. An effective method done to help those with this dilemma is firstly, an experienced doctor usually takes a sample of the individuals skin sample. The skin sample is then processed and various research as well as laboratory experiments are to done to identify the type of skin ranging from the type of tissues and cells present in the specific sample of the skin. Proceeding this, scientist then add the necessary remedies as additional components to the spray or even extract any allergic causing remedies to the patient concerned. To overcome excessive usage of the spray to avoid any unwanted scenario is by precisely aiming the nozzle to the affected areas to make sure it usage is appropriate .

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1.7 Stages Of Wound Healing

Healing process involves 3 main stages: inflammatory stage which is the constriction of blood vessels to prevent excess blood loss and platelets form clot. After the clot is formed, blood vessels expand back to allow maximum blood flows. This is why the newly healed wound looked warm and red. To reduce this problems, the spray must be dissolve in ammonia because ammonia has the highest specific heat capacity, 6.74 kJ kg-1

C-1 in Lithium container which has the specific heat capacity of

3.58 J kg-1 C-1 .

High heat capacity can absorb more heat and reduce the temperature. The second stage is fibroblastic stage, is the growth of the protein fiber collagen to strengthen the wound or the skin. Since peroxide anion and oxidants are the culprit of destroying collagen in the body. The spray can add in liquid Vitamin C because Vitamin C can directly absorbed by the skin and it boosts the production of collagen. The final stage of wound healing is maturation stage. In this stage, collagen is constantly added to the wounded area and this process maybe taking months or years.

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1.8 Barriers That Slow The Healing of Wound

There are some barriers that slow the healing of wound. For example, dryness of the wounded area, infection of pathogen and haemorrhage, which is the persistent bleeding that will keep the wound margins apart. To avoid these problems, the spray can add in salt water because when water and ice are mixed together some molecules of ice are melting and some of the water molecules are freezing. The salt water works when the temperature reaches 0 C the two processes balance each other out in what is known as dynamic equilibrium. However, salt upsets this balance. This is because salt dissolves in water but can't interact with ice. The salt molecules start to replace the water molecules so there are fewer water molecules freezing compared to the number of ice molecules melting. The water cools down below 0 C so more molecules will freeze to achieve balance once again. The more salt you add, the colder the icy water gets. On the other hand, some supplements such as zinc, calcium and vitamins can be added into the spray because these supplements can effectively increase the rate of healing. The other end of the spray can have a small tube having filled with distilled water, this can be used to clean the wound and clear off the debris from the wounded area.

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Objective
1. Can prevent excessive bleeding caused by injury .This is because once apply this spray on any wound ,the wound will heal immediately and no more bleeding . 2. No need rush to go to hospital to treat the wound but we just need to apply this spray on the wound . 3. To make human being life easier and reduced the risk of death caused by excessive bleeding or infection because no need to take time to go to hospital . 4. No need to undergo surgery but the wound can heal in a short time .

5. Can boost the economy of a country .This is because this product has a lot of benefits for all the people and this may attract people to buy it .

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Benefits and side effects

The average healing time for patients with second degree burns takes weeks, which the skin cell gun reduces to days. Traditional skin grafting can be risky, in that chances for infection are relatively high. The skin cell gun alleviates this concern because the increased speed in which the wound heals directly correlates to the decreased time the wound can be vulnerable to infection. Although it takes several months for the skin to regain the exact texture and color before the incident, harmful side effects that can result from an open wound are not a factor. Applying the skin cells is quick and doesn't harm the patient because only a thin layer of the patients healthy skin is extracted from the body into the aqueous spray. The electronic spray distributes the skin cells uniformly without damaging the skin cells, and patients feel as if they are sprayed with salt water. Because the skin cells are actually the patients own cells, the skin that is regenerated looks more natural than skin grown from traditional methods. During recovery, the skin cells grow into fully functional layers of the skin, including the dermis, epidermis, and blood vessels. The regenerated skin leaves little scarring. The basic idea of optimizing regenerative healing techniques to damaged biological structures demonstrated by the skin cell gun in the future may also be applied to engineering reconstruction of vital organs, such as the heart and kidneys.

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There are major limitations: the method will not work on deep burns that go through bone and muscle, specifically below the dermis. As of 2011, only several dozen patients have been treated; it remains an experimental, not a proven, method. As of 2011, the skin cell gun was still in its prototyping stage, since it has only treated a dozen patients in Germany and the US, compared to over 50,000 treated with Dermagraft bioengineered skin substitute. There is thus a lack of published peer reviewed clinical evidence, and no knowledge of long-term stability of the newly generated skin.

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INGREDIENT:
750 000 IU of Vitamin A 60 g of Vitamin C 0.45 g of Vitamin E 0.9 g of Zinc 45 g of Glucosamine 1.44 g of Adequate protein 270 g of Arginine 120 g of Glutamine 360 ml of pure Centella asiatica Extract 450 ml of pure Aloe vera Juice 300 ml of pure Calendula succus Extract 3 mg of pure Symphytum officinale Extract 100 ml of tea tree oil 500 ml of Isopropyl alcohol 20 ml of Blood clotting agent 1000 ml of Distilled water 30 g of Lechitin

APPARATUS:
Saucepan Bowls Stirrer Stove

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METHODS
1. Add 750 000 IU of Vitamin A and 0.45 g of Vitamin E into 100 ml of Tea tree oil. The solution named solution A. 2. Heat and stir the solution A. 3. Add 60 g of Vitamin C, 0.9 g of Zinc, 45 g of Glucosamine, 1.44 g of Adequate protein, 270 g of Arginine, 120 g of Glutamine, 360 ml of pure Centella asiatica Extract, 450 ml of pure Aloe vera Juice, 300 ml of pure Calendula succus Extract, 3 mg of pure Symphytum officinale Extract, 500 ml of Isopropyl alcohol, and 20 ml of Blood clotting agent into 1000 ml of the distilled water. The solution named solution B. 4. Heat and stir the solution B. 5. Pour the solution A, solution B and 30 g of the Lechitin into the saucepan. 6. Boil and stir the mixture simultaneously. 7. Let the mixture cool down until 43 C. 8. After cooling down, insert the mixture into specially made 20 spray bottles and each of the bottles contains 50ml of the mixture . 9. Before applying on the human beings skin, take 80 white mices as experimental apparatus to apply the spray on them .

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Literature Review( Previous Research)

(A)Skin cell gun
The skin cell gun is an experimental device for the treatment of second degree burns developed by Jrg C. Gerlach and colleagues at Stem Cell Systems GmbH in Berlin. With this technique, individual adult stem cells from the patient's uninjured skin are applied to the wound site, where they differentiate into normal skin. (The conventional methods use mesh grafting) The hope is that with the skin cell gun, damaged skin tissue can be regenerated more quickly than with traditional methods.

Operation
Stem cells from a biopsy of the patient's healthy skin are isolated, placed into a sterile syringe with a fitted nozzle, and sprayed directly through the nozzle into the wound. Using computer precision, the gun distributes cells at a uniform velocity throughout the wound. Then, a temporary artificial wound capillary system is applied. A tube is attached to each end of the dressings, one doing the work of an artery and the other of a vein. A bioreactor is attached to this artificial vascular system to provide nutrition such as glucose, sugar, amino acids, antibiotics and electrolytes; and support the fragile skin stem cells until they start to grow and generate new skin. The newly introduced stem cells are able to regenerate and differentiate into their respective parts in a matter of days. The first phase of gathering the patients stem cells,

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creating a solution, and applying the stem cells takes approximately 1.52 hours. Within a week, the wound dressing procedure allows the stem skin cells to fully generate normal skin, and after a couple of months the skin regains its color and texture.

Past approaches
The skin cell gun is not the first ground-breaking method used to treat burns. The genesis of tissue engineered products as skin substitutes began more than 20 years ago with improved culture methods for the growth of keratinocytes, and the production of natural and synthetic matrices used as delivery systems for cells or cell products. Keratinocytes are epidermal cells that synthesize keratin and other proteins. They are formed from undifferentiated (basal) cells and comprise 95% of the epidermis. Access to populations of keratinocytes that can be grown and expanded in vitro and still serve as stem cells subsequent to transplant is a requirement for producing epidermal skin substitutes. The overall goal of tissue engineering is the recapitulation of normal skin and normal skin function. As such, any epidermal replacement must restrict trans-epidermal water loss, limit bodily damage induced by environmental chemical and physical insults, and minimize microbial load. Any dermal replacement must provide epidermal support, neovascularization, and functional pliability. Some of the most popular methods of skin regeneration are as follows:

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Rheinwald and Green experimented with treating whole skin with trypsin to separate the epidermis from the dermis and to disaggregate the epidermal cells, which were then grown on a feeder layer of lethally irradiated mouse cells in a complex culture medium. Three to four weeks later, confluent stratified sheets of epidermal keratinocytes were available for grafting. Cultured epithelial autografts (CEA) began to move into mainstream use for deep partialthickness injury, full-thickness burn injury, and congenital nevi in 1988. When allowed to grow in a lab setting, individual keratinocytes produce colonies that fuse and form a multilayered epithelium.[8] When these keratinocyte cultures are incubated with Dipase II, the epithelial tissue detaches itself from the surface of the culture vessel and is readily available as a skin substitute. Skin glue has been used to successfully treat deep partial and full-thickness burn injuries. Histologically normal epidermis has been shown to develop in situ. The direct application of keratinocyte cell suspensions has also shown promise. When delivered within a fibrin spray, keratinocytes promote the closure of deep partial and fullthickness burns, and of chronic wounds. The fibrin helps secure even placement of the keratinocytes. The keratinocyte spray technology is being commercialized for human use pending clinical trials and regulatory approvals.

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2 Acellular dermal substitutes have been evaluated for their ability to jumpstart neodermis formation and to provide a matrix compatible with reepithelialization and neovascularization. De-epidermized dermis is structurally identical to dermis and can be purchased as Alloderm . It can be used in conjunction with either STSG or CEA. De-epidermized dermis offers the advantage of being able to support engraftment of thin rather than thick STSG. Dermal matrix molecules require extensive remodeling to achieve normal dermal structure. Sold as Integra , the pore size of the matrix is designed to be sufficient to allow fibroblast and endothelial cell migration to occur, and for the product to be remodeled by invading host cells. Neodermis is formed in about 3 weeks, at which time the silastic layer readily detaches. As with de-epidermized dermis, to achieve permanent wound closure, the treated area must be grafted with either a thin STSG or with CEA.

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Comparison to traditional methods

Whereas it takes mere hours to prepare and administer stem cells with the stem cell gun, it takes 23 weeks to produce a skin sheet and harvest it from an external lab. Once the skin sheet has been attached to the wound, blisters form under the newly attached skin, pushing the sheet up, damaging the wound and increasing the risk of infection. The skin cell gun applies its stem cells directly to the patient's cells, which alleviates the concern of further tissue damage. The artificial vascular system network also provides a reliable
source of protection to the skin stem cells. After the wound has been treated, it takes months for the skin sheet to heal over, yet only days for the skin cell gun to fulfill its function. Reducing the fragility of the cells and time frame of the operation by cleverly employing differentiated stem skin cells in such a way that offers a renewable source of replacement is an essential component of the skin cell guns capability.

Applications
The current method is applicable to different burn damaged areas. Patients who have incurred second-degree burns, patients with infected wounds or patients with delay in wound healing are suitable for cell grafting treatment. Third-degree burns, however, completely deprive victims of both their epidermis and dermis skin levels, which exposes the tissue surrounding the muscles. The skin cell gun has not progressed to the point where it can be used for such advanced wounds, and these patients must seek more traditional treatment methods. The skin cell gun is generally not used for burn victims with anything less than a second-degree burn either. First degree-burns still maintain portions of the epidermis and can readily heal on their own, thus they do not need this expensive technology.

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Currently, the skin cell gun's applications have not been extended to include the regeneration of skin lost due to other injuries or skin diseases. It is also limited in that it is only effective immediately following the burn incident.

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EXPECTED RESULTS
Healing is very rapid. The amount of spray on the wound depends on the seriousness of the wound. The deeper skin layer will cure first followed by the next layer of skin .The deeper the wound the more time taken (few minutes). Scratches will only take few seconds .The nozzle of the spray must be sharp so that the aiming toward the wound will be accurate. Deeper the wound more times it is sprayed .The result is more accurate if the specificity is increased .For sensitive skin individual are advised to order more specific spray for your skin.

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Results
Since 2008 the skin cell gun has been under development for the treatment of second degree burns. It is not yet approved by the FDA. Stem cell damage during the spraying procedure is a current research hurdle. This treatment is only for recently burned victims; it will not yet work for those who suffered the injury a few months prior. A few days are required for the wounds to internally heal with this new process; but after those few days, the wound still looks damaged. The skin will not start to look as it did before the injury until months afterward. Because pigment cells are so much deeper in the part of the skin than keratinocytes are, pigment cells need much more time to develop.

(B)"Spray-on skin" product may treat flesh wounds, promote healing

UA researchers are studying the effectiveness of a spray-on skin intended to treat flesh wounds such as chronic leg ulcers that develop in elderly people. Currently screening patients for their study, a team of researchers from the Southern Arizona Limb Salvage Alliance is one of the few groups in the country working with the wound treatment tool. Im excited because, for a lot of this stuff, it sounds and it seems like Star Trek, where youre spraying [skin] on, said Dr. David G. Armstrong, a UA professor of surgery and director of SALSA, but the future is now. The spray-on skin product, developed by the Texas-based HealthPoint Biotherapeutics, contains living skin cells that work with the patients cells to promote healing. The

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substance could prove especially useful in treating venous leg ulcers, which are currently dealt with either by using compression bandages or through skin graft surgery. Occurring most often in the elderly, venous skin ulcers result from the deterioration of the valves in the veins of the leg. As blood is pumped up through the legs and back to the heart, it has to overcome the force of gravity pulling it down, Armstrong said. To do this, tiny valves open and close with each pump of the heart, keeping the blood cells from sliding down and pooling in the legs. For some people, those valves have deteriorated to the point where the blood does pool, causing pressure that can kill the surrounding cells and creating a wound from the inside out, Armstrong said. Its really hard to treat these things, he said, adding that the medical treatment of such ulcers has progressed considerably over the past few hundred years despite the challenges involved. The study uses HP 802-247, a liquid cocktail of two different types of dermal cells suspended in various proteins that can be sprayed as an aerosol. Cultured from the skin of a donor, the cells are applied on the wound and interact with the existing cells in a symphony of healing, promoting the development of growth hormones and other growth factors, Armstrong said. As opposed to a skin graft, which is a flat layer of skin plastered onto the area, the liquid treatment can slip into the cracks and undulations of the wound, which could potentially

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make it a more effective option, said Dr. Nicholas Giovinco, assistant professor in the Department of Surgery and director of education for SALSA. Manish Bharara, assistant professor in the Department of Surgery and director of operations for SALSA, emphasized that the spray-on treatment is being developed in concert with a number of other regenerative techniques. Its not the kind of thing that alone is going to revolutionize the [wound treatment] industry, Bharara said. It is one of the tools that will work in complement with other standard techniques that are out there. If the technology proves effective, it represents a sort of Holy Grail in regenerative medicine, Giovinco said. [The spray-on skin] is pretty fantastic in how much easier it is to apply and the results that we are likely to get without exposing patients to unnecessary risks, Giovinco said. Im pretty excited about that.

(C)The 'spray-on skin' that heals wounds

A new sprayable solution of recycled skin and blood-clotting proteins could revolutionize how we treat hard-to-cure wounds Healthpoint Biotherapeutics, a company out of Fort Worth, has successfully tested a spray-on treatment that promises to help wounds heal faster and without the need of skin grafts. Dr. Robert Kirsner at the University of Miami and his colleagues tested the "spray-on skin" on 228 patients with leg ulcers, or open wounds that are notoriously hard to heal. The result was "superior healing and a faster time frame than anything else we've

36

seen in the treatment of venous leg ulcers," Kirsner tells MedNews Today. The study, published in the prestigious British medical journal The Lancet, is preliminary, but outside experts are impressed. Here's a look at the possible future of wound therapy: What's the skin spray made of? Healthpoint's spray, currently sporting the decidedly un-mellifluous name HP802-247, is made of donated skin cells (keratinocytes) and an anti-clotting agent (fibroblasts). The two substances are believed to work together to speed up healing and stimulate the body's production of new skin to cover the wound. The standard treatment for leg ulcers is a compression bandage, which heals up to 75 percent of the lesions within six months. Does this study prove that the skin-in-a-bottle works? No. But the point was to make sure the spray was safe and determine the optimal dose, which turned out to be the lowest one: Spraying patients every 14 days. The results exceeded expectations: After three months, 70 percent of the patients receiving both the optimal dose of the spray-on skin treatment and traditional compression had completely healed versus 46 percent of the control group, which was using a placebo spray and compression bandages. Patients sprayed with the solution every 14 days also saw their wounds close 21 days before the control group, in 50 days versus 71 days. The size of the wound started shrinking almost immediately after spraying started with the skin solution. What's next for spray-on skin? Kirsner is already enrolling people in the U.S. and Europe for a Phase III clinical trial, to see if the treatment is feasible. Part of the issue is cost it's expensive to turn the donated neonatal foreskin (circumcision trimmings) and blood-clotting proteins into a usable spray. In an accompanying commentary, Matthias Augustin at the University
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Medical Center in Hamburg, Germany, said the price tag may make more sense over the long term, especially for hard-to-treat wounds like leg ulcers: "The temporary higher costs for additional cell and tissue-engineered therapy can be justified as an investment in improved healing." Is there a downside? Besides cost, there's the risk of false hope for those suffering from painful leg ulcers, says leg ulcer theorist Irene Anderson at Britain's University of Hertfordshire. "Leg ulcers are becoming increasingly complex, and when using the range of treatments available there needs to be clear evidence that there will be a beneficial effect to ensure cost effectiveness and to make sure that patients are not given false expectations of a cure."

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Discussion
1. What is the benefits of using vitamin A, C ,E, Zinc, Bromelain and Glucosamine to do the wound spray ?

Vitamin A
Vitamin A is required for epithelial and bone tissue development, cellular differentiation, and immune system function. Substantial evidence supports the use of vitamin A as a perioperative nutritional supplement. In addition to facilitating normal physiological wound repair, Ehrlich and Hunt have shown vitamin A reverses the corticosteroid induced inhibition of cutaneous and fascial wound healing.Vitamin A has also corrected non-steroid induced, post-operative immune depression and improved survival in surgically induced abdominal sepsis. Levensonetal suggest vitamin A benefits the wound by enhancing the early inflammatory phase, including increasing the number of monocytes and macrophages at the wound site, modulating collagenase activity, sup- porting epithelial cell differentiation, and improving localization and stimulation of the immune response. Animal studies show vitamin A may increase both collagen cross-linkage and wound- breaking strength. Greenwald et al inflicted surgical flexor profundus damage and immediate repair on adult chickens. They found chickens that ate a diet supplemented with vitamin A (150,000 IU/kg chicken chow) demonstrated wound breaking strength more than double that of controls fed standard chicken chow.In addition, rats with dorsal skin incisions and concurrent comminuted femoral fractures exhibited delayed cutaneous healing. Supplemental vitamin A enhanced wound healing in these animals, demonstrated by

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increased breaking strength of the dorsal skin incisions in rats fed supplemental vitamin A compared to the non-supplemented group. The authors believe the improved wound healing is a result of an increased rate of collagen cross-linkage. Levenson and Demetrio recommend vitamin A supplementation of 25,000 IU daily be- fore and after elective surgery.Research supports perioperative vitamin A supplementation in patients known to be immune depleted or steroid treated. Surgical patients with sepsis and those with fractures, tendon damage, or vitamin A deficiency may also benefit from perioperative vitamin A supplementation. Additional research is necessary to establish the effectiveness of universal perioperative vitamin A supplementation in healthy individuals. Concern among some practitioners regarding the potential toxicity of higher doses of vitamin A has led to uneasiness about using it perioperatively. The vast majority of toxicity cases have occurred at daily vitamin A dosages of 50,000-100,000 IU in adults over a period of weeks to years.Short-term supplementation of 25,000 IU daily appears to be safe for most non- pregnant adults. Caution must be exercised in supplementing vitamin A in patients for whom the antiinflammatory effect of steroids is essential, such as in rheumatoid arthritis or organ transplants, as well as in pregnant women and women of childbearing age.

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Vitamin C
Ascorbic acid is an essential cofactor for the synthesis of collagen, proteoglycans, and other organic components of the intracellular matrix of tissues such as bones, skin, capillary walls, and other connective tissues. Ascorbic acid deficiency causes abnormal collagen fibers and alterations of the intracellular matrix that manifests as cutaneous lesions, poor adhesion of endothelium cells, and decreased tensile strength of fibrous tissue. Clinical manifestations of ascorbic acid deficiency include bleeding gums, poor immunity, easy bruising and bleeding, and slow healing of wounds and fractures. Ascorbic acid is necessary for the hydroxylation of proline and lysine residues in procollagen, which is necessary for its release and subsequent conversion to collagen. Hydroxyproline also stabilizes the collagen triple-helix structure. In addition to collagen production, ascorbic acid enhances neutrophil function, increases angiogenesis, and functions as a powerful antioxidant. Although ascorbic acid is required for reparation of damaged tissue, researchers have demonstrated the benefit of vitamin C only in vitamin C-deficient individuals using low doses of ascorbic acid.In a study by Hodgesetal, four subjects (ages 33-44) were depleted of vitamin C for 99 days to induce scurvy. On day 100, a 5-cm incision was made in the left thigh of each subject and they began the oral administration of 4, 8, 16, or 32 mg ascorbic acid daily. Healing was measured by histological and electron microscope technique. It was shown that 4 mg daily of vitamin C was just as effective as 32 mg daily for wound healing in these vitamin C-deficient sub- jects.The efficacy of using vitamin C to improve wound healing in non-deficient individuals remains uncertain. It should be noted, however, that even the highest dose in this study (32 mg) is below the RDA for vitamin C. Higher doses and larger differences

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between doses might have yielded more significant differences. Humans lack the ability to store vitamin C, and certain populations are more likely to be deficient in ascorbic acid, including the elderly, alcoholics, drug abusers, and under-nourished individuals. Subclinical vitamin C deficiency is being recognized increasingly in the general population. Published cases show that restricted eating patterns, prolonged hospitalization, severe illnesses, and poor dietary intake in both children and adults cause deficiency with significant clinical consequences. In one study 12 patients with postsurgical diffuse hemorrhage, each exhibiting normal coagulation parameters, were found to have low plasma ascorbic acid levels. Each patient received 250-1,000 mg oral vitamin C daily. Within 24 hours of vitamin C administration there was no further evidence of bleeding or need for subsequent blood transfusions in any patient. The authors concluded vitamin C deficiency should be included in the differential diagnosis for nonspecific bleeding in surgical patients.4 In mammals, ascorbic acid is necessary for a normal response to physiological stressors, with the need for ascorbic acid increasing during times of injury or stress.Studies have shown the physiological stress of intense exercise generates excess reactive oxygen species (ROS), increasing the demand on the antioxidant defense system. A similar elevation of ROS has been noted within wounds; therefore, substances that increase tissue antioxidants are thought to benefit healing .Events leading to wounds, including trauma and surgery, are perceived as physiological stressors that have also been correlated with a decrease in plasma ascorbic acid. Thus, the acute stress experienced by trauma or surgery patients may unmask marginal vitamin C deficiencies, leading to deficiency symptoms. Cutaneous healing wounds have been found to have lower ascorbic acid content than intact tissue. Levels of vitamin C were

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compared to normal skin in two, four, seven, and 14-day- old wounds in animals. Vitamin C levels decreased approximately 60 percent post-wound and had not exhibited full recovery by day 14.36 In addition, low levels of antioxidants, including ascorbic acid, accompanied by elevated levels of markers of free radical damage have been detected in elderly rat cutaneous wounds exhibiting delayed healing. Eighteen-month-old wounded male rats were compared to 3-4 month-old rats pre-wound and seven days postwound. Normal skin of aged and young rats showed no difference in ascorbic acid content; however, a 59-percent decrease in ascorbic acid content was observed in wound tissues of aged animals compared to its content in young adult wounds.Rasik and Shukla propose the delay in wound healing of older rats is at least partially a result of increased free radical damage.The programmed sequences of the cellular and molecular processes occurring during wound repair are also dependent on immune function. Infection resulting from impaired immunity is one of the most commonly encountered and clinically significant impediments to wound healing.In addition, cellular immunity and dysregulation of cytokines can impair wound healing. Ascorbic acid has been shown to improve immune function in humans. Human volunteers who ingested 2-3 g ascorbate daily for several weeks exhibited enhanced neutrophil motility to chemotactic stimulus and stimulation of lymphocyte transformation. Neutrophil motility and lymphocyte transformation were also stimulated by 1 g intravenous ascorbic acid in six healthy volunteers. Alterations in these activities were related to serum ascorbic acid levels.The combined effect of ascorbic acid on collagen synthesis, antioxidant status, and immunomodulation make it an appropriate supplement for wound repair protocols. Research provides evidence for the use of low doses of vitamin C in vitamin C deficient

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individuals, but many practitioners believe larger doses of ascorbic acid in non-deficient individuals are indicated for optimal wound repair. Levenson and Demetriou recommend supplementing 1-2 g ascorbic acid daily from wound onset until healing is complete. Such doses may be justified due to the lack of adverse effects at these levels 44 combined with the potential for deficiency in certain individuals. In addition, the transient increase in metabolic requirements for vitamin C resulting from the physiologic stress of trauma or surgery and the metabolic requirement of vitamin C for collagen synthesis are indications for higher doses of vitamin C in non-deficient individuals.

Zinc
Approximately 300 enzymes require zinc for their activities. Zinc is an essential trace min- eral for DNA synthesis, cell division, and protein synthesis, all necessary processes for tissue regeneration and repair. Zinc deficiency has been associated with poor wound healing and decreased breaking strength of animal wounds, which can result from decreased protein and collagen synthesis during healing found in zinc deficient animals. Senapati and Thompson found zinc levels were 50-percent higher in muscle and skin from abdominal wounds of rats during wound healing, but mild deficiency reduced this accumulation. Zinc demands are thought to be the highest from time of wounding throughout the early inflammatory phase. Sequential changes in zinc concentrations were studied in the incisional wound model in the rat. Zinc levels increased from wounding and peaked on the fifth day at a time of high inflammation, granulation tissue formation, and epidermal cell proliferation. Zinc concentrations returned to normal by the seventh day, when inflammation had regressed. It has been suggested that increased local demand
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for zinc resulting from surgery and wounding exposes otherwise marginal zinc deficiencies in humans.Perioperative zinc supplementation is recommended for zinc depleted patients. Data is lacking to show zinc supplementation improves healing in nondeficient individuals; however, zinc deficiency in humans is widespread, and injured and stressed individuals are more prone to developing deficiencies. Ehrlichetal suggest zinc is lost in significant amounts after surgery because of fistulas, stress, and diarrhea.Zinc deficiencies have also been identified in individuals with deep partial- or full-thickness burns and chronic venous leg ulceration. Further research is needed on the efficacy of zinc supplements for wound healing. Justification for perioperative zinc supplementation includes the absence of adverse effects at moderate doses (15-30 mg daily) and evidence that zinc deficiency impairs wound healing. Zinc supplementation of 15-30 mg daily is recommended perioperatively to prevent unmasking of marginal deficiencies. Higher levels of zinc supplementation may be necessary in patients with malnutrition, malabsorption, chronic diarrhea, or other risk factors of zinc deficiency .

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Vitamin E
Vitamin E is popular among consumers for skin care and to prevent scar formation. It functions as the major lipophilic antioxidant, preventing peroxidation of lipids and resulting in more stable cell membranes. The antioxidant-membrane stabilizing effect of vitamin E also includes stabilization of the lysomal membrane, a function shared by glucocorticoids. Systemic vitamin E and glucocorticoids inhibit the inflammatory response and collagen synthesis, thereby possibly impeding the healing process. The effect of vitamin E on wound healing is complex; it may have alternate effects in different types of wounds and in the presence of other nutrients, as well as different functions for water soluble versus lipid soluble preparations of vitamin E. Animal studies of vitamin E supplementation on surgical wounds show conflicting results. Greenwaldetal showed flexor tendon repair in chickens treated with vitamin E had breaking strength less than half that of controls measured after days 7 and 45 from surgical repair.Another animal study showed impaired collagen synthesis in rats treated with vitamin E after wounding.The researchers cite the glucocorticoid like effect of vitamin E as the cause of the negative results. However, these effects are mitigated by vitamin A, as vitamin A is a lysomal destabilizer that reverses several of the deleterious effects of glucocorticoids.Paradoxical results found by Galeanoetal showed a hydrophilic vitamin E preparation positively impacted delayed wound healing in diabetic mice. Increased breaking strength and collagen content of the wound was found in treated animals. These authors speculate inhibition of lipid peroxidation accounted for the positive results.In addition, prophylactic administration of vitamin E has been shown to increase breaking strength and normalize healing of wounds exposed to preoperative

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irradiation and to decrease the development of intraperitoneal adhesions in animals. Since the discovery of vitamin E as the major lipid-soluble antioxidant in skin, it has been used topically for a wide variety of skin lesions. Anecdotal reports claim topical vitamin E is valuable for speeding wound healing and improving cosmetic outcome of burns and other wounds, including surgical scars. Such claims are disputed by two human clinical trials. In a double blind study of 15 patients with surgically-induced wounds, emollient lotion and emollient lotion mixed with vitamin E were applied to healing wounds. The wounds were randomly divided into two parts and the different topical applications were applied to the same half of each wound twice daily. Physicians and patients independently evaluated the scars for cosmetic appearance on weeks 1, 4, and 12. In 90 percent of cases, topical vitamin E either had no effect, or actually worsened the cosmetic appearance of scars. In addition, 33 percent of the patients studied developed contact dermatitis to topical vitamin E. A response to this study, published in Dermatologic Surgery, pointed out that d-alpha tocopherol is an extremely unstable compound, rendering details of its source, formulation, storage condition, and stability over time critical to interpretation of this study. It was also noted that breakdown products and contami- nants could account for the inflammatory response encountered.In a second, larger blinded study, the effects of topical steroids, vitamin E, or the base cream carrier for these substances on scar outcome of 159 post-operative patients were evaluated. Both topical steroids and topical vitamin E failed to impact scar thickness, range of motion, or ultimate cosmetic appearance.The available data on vitamin E and wound healing could lead to several possible conclusions: (1) systemic vitamin E may have a negative impact on surgical wounds due to its lysosomal stabilizing properties; (2) vitamin A may mitigate these

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negative effects; and (3) hydrophilic and hydrophobic preparations of vitamin E may have different actions related to wounds. The benefit of topical vitamin E on surgical wound healing and scar formation remains inconclusive and, although anecdotal reports support topical use of vitamin E for scar therapy, research shows it may have a negative effect on scarring and wound outcome.

Bromelain
Bromelain is a general name given to a family of proteolytic enzymes derived from Ananas comosus, the pineapple plant. Through- out the 1960s and 1970s a series of studies found the effects of orally administered bromelain in- clude the reduction of edema, bruising, pain, and healing time following trauma and surgical procedures.More recently, researchers from the Czech Republic found that patients with long bone fractures administered a proteolytic enzyme combination containing 90 mg bromelain per tablet had less post-operative swelling compared to patients given placebo. Fractures were treated by surgically inserting rods through the long axis of the fractured bone (intramedullary fixation) or by constructing an external framework of pins and rods going through the skin and muscle to connect to the fractured bone (external fixators). The treatment group was given three 90-mg tablets three times daily for three days after surgery and, subsequently, two tablets three times daily for two weeks. On the fourteenth post-operative day the limb volume of the treatment group was reduced by 17 percent compared with nine percent in the control group. The total number of analgesics consumed by the treatment group was also significantly reduced in comparison to the

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control group.Studies by Tassman et al show bromelain reduced swelling, bruising, pain, and healing time in patients following dental surgeries. In a double-blind study of dental surgery patients, bromelain was found to decrease swelling to 3.8 days, compared with seven days in patients given placebo. In addition, duration of pain was reduced to five days in the treatment group, compared to eight days in the placebo group. In an uncontrolled trial, bromelain was reported to positively influence swelling, pain at rest and during movement, and tenderness in patients with blunt injuries to the musculoskeletal system. Although bromelain has been shown to reduce post-operative and trauma-related pain, this is probably related to its anti-inflammatory action rather than a direct analgesic effect. Aside from its documented anti-inflammatory activity, bromelain is of interest to surgeons because of its ability to increase resorption rate of hematomas. Bromelains influence on hematoma resorption was demonstrated using artificially induced hematomas in humans. Hematomas in the treatment group resolved significantly faster than controls when oral bromelain was given at the time of hematoma induction and for seven days there after.Seltzer investigated two different doses of bromelain in patients undergoing rhinoplasty. Fifty-three patients were randomized to receive either one of two doses of bromelain or placebo. In patients receiving placebo, swelling and ecchymosis persisted for seven days, compared to two days in both bromelain groups. However, a randomized trial of 154 facial plastic surgery patients receiving either 400 mg bromelain daily or placebo for one day before and four days after surgery found no statistically significant differences inedema between the two groups. subsequently, two tablets three times daily for two weeks. On the fourteenth postoperative day the limb volume of the treatment group was reduced by 17 percent

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compared with nine percent in the control group. The total number of analgesics consumed by the treatment group was also signifi- cantly reduced in comparison to the control group. Studies by Tassman et al show bromelain reduced swelling, bruising, pain, and healing time in patients following dental surgeries. In a double-blind study of dental surgery patients, bromelain was found to decrease swelling to 3.8 days, compared with seven days in patients given placebo. In addition, duration of pain was reduced to five days in the treatment group, compared to eight days in the placebo group. In an uncontrolled trial, bromelain was reported to positively influence swelling, pain at rest and during movement, and tenderness in patients with blunt injuries to the musculoskeletal system. Although bromelain has been shown to reduce post-operative and trauma-related pain, this is probably related to its anti-inflammatory action rather than a direct analgesic effect. Aside from its documented anti-inflammatory activity, bromelain is of interest to surgeons because of its ability to increase resorption rate of hematomas. Bromelains influence on hematoma resorption was demonstrated using artificially induced hematomas in humans. Hematomas in the treatment group resolved significantly faster than controls when oral bromelain was given at the time of hematoma induction and for seven days there after.69 Seltzer investigated two different doses of bromelain in patients undergoing rhinoplasty. Fifty-three patients were randomized to receive either one of two doses of bromelain or placebo. In patients receiving placebo, swelling and ecchy- mosis persisted for seven days, compared to two days in both bromelain groups. However, a ran domized trial of 154 facial plastic surgery patients receiving either 400 mg bromelain daily or placebo for one day before and four days after surgery found no statistically significant differences in edema between the two groups.

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Tassmanetal noted that, while post-surgical oral bromelain administration was effective in reducing pain, swelling, and healing time, a protocol using pre- and post-surgical bromelain is recommended. Studies have shown bromelain prevents aggregation of blood platelets in patients with high platelet aggregation values, which has led to recommendations by physicians and surgeons to avoid oral bromelain prior to any surgical procedure. In one human trial, bromelain was administered orally to 20 volunteers with a history of heart attack or stroke, or with high platelet aggregation values. Bromelain decreased platelet aggregation in 17 of the subjects and normalized values in eight of the nine subjects who previously had high aggregation values.Contrary to this, other human studies have shown oral bromelain to be free of any significant effects on clotting parameters.In one study, 47 patients with various disorders leading to edema and inflammation found no significant effects of oral bromelain (40 mg four times daily for one week) on bleeding, coagulation, and prothrombin time. It is noteworthy that the studies pertaining to bromelain and platelet aggregation are over 30 years old. The potential benefit of pre- and post- surgical oral bromelain on hematoma resorption, pain, inflammation, and healing time justifies the need for concise, well-designed clinical trials evaluating different doses of bromelain on clotting parameters. Until further data is available regarding bromelains action on platelets, oral bromelain administration should be withheld or used with caution before surgery.

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Glucosamine
Hyaluronic acid is an important part of the extracellular matrix and one of the main glycosaminoglycans secreted during tissue repair. Production of hyaluronic acid by fibroblasts during the proliferative stage of wound healing stimulates the migration and mitosis of fibroblasts and epithelial cells. Glucosamine appears to be the rate limiting substrate for hyaluronic acid synthesis.In vitro studies suggest the mechanism of glucosamine on repair processes involves stimulation of the synthesis of glycosaminoglycans and collagen. Animal studies have shown the content of glycosaminoglycans within the site of partially ruptured muscles increased maximally five days after trauma and decreased thereafter. This suggests the timing of glucosamine supplementation may determine its therapeutic impact on wounds. Clinical trials using glucosamine for perioperative support are lacking. However, the administration of oral glucosamine both before as well as the first few days after surgery or trauma might enhance hyaluronic acid production in the wound, promoting swifter healing and possibly fewer complications related to scarring.

2.

Any other ingredients are good for wounds healing other than those mentioned in the

1st question?

Adequate protein intake is essential for proper wound healing. Protein

depletion appears to delay wound healing by prolonging the inflammatory phase; by inhibiting fibroplasia, collagen and proteoglycan synthesis, and neoangiogenesis (proliferation phase); and by inhibiting wound remodeling.Experimental protein

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depletion in animals caused a decrease in the tensile strength of wounds. Rats fed a diet deficient in protein exhibited decreased wound integrity and strength versus control animals.In a study of 108 human patients with experimental wounds, individuals with either low serum protein or serum albumin had significantly weaker wounds than those with normal protein values. Protein calorie malnutrition increases morbidity and mortality in the surgical/trauma patient. Many studies have found hospitalized patients in a state of malnutrition at admission. Thus, it is important to increase protein intake to optimize healing and immune function, and to prevent post-surgical complications in these individuals.Protein supplementation of elderly patients with liquid protein formulas significantly enhanced healing of pressure ulcers. The change in ulcer area was significantly correlated with the amount of protein in the diet. The surgical or trauma patient exists in a state of metabolic stress, with the severity of the stress depending on the severity of the wounded state. An injured patient requires more protein than a noninjured patient because of the increased metabolic activity of wound healing, acute-phase protein production in response to stress, and amino acid mobilization from muscle used for hepatic gluconeogenesis. In a non-injured state, adults require approximately 0.8 g dietary protein/kg body wt/day. Elderly patients have a higher protein requirement (1-1.2 g/kg body wt/ day) due to a decreased ability to synthesize proteins. The surgical/trauma patient can require significantly more protein. Minor surgery may not significantly increase the protein requirement; however, if the patient is already protein malnourished, wound healing will be adversely affected unless dietary protein intake is increased. Major surgery can increase protein requirements 10 percent, while a patient with multiple traumas may need 75-percent more protein. Burn wounds cause tremendous metabolic

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stress and have the greatest impact on protein requirements, increasing protein need 75100 percent.

Amino Acids in wound healing It is well accepted that sufficient protein is

necessary for wound healing. This appears to be due to the increased overall protein need for tissue regeneration and repair. Researchers have investigated the effects of specific amino acids on the healing process and determined that arginine and glutamine appear to be necessary for proper wound healing.

Arginine is a non-essential amino acid that plays a key role in protein and amino
acid synthesis. It is acquired from the diet and derived en-dogenously from citrulline in a reaction catalyzed by the enzyme arginine synthetase. Adequate tissue arginine appears to be essential for efficient wound repair and immune function. Arginine (17 g/day) was given to 30 elderly patients (>65 years of age) who sustained an experimental surgical injury. Supplemented patients demonstrated significantly greater hydroxyproline (a sign of collagen deposition) and protein accumulation at the wound site, compared to nonsupplemented controls. Lymphocyte response, signifying greater immune activity, was elevated in the supplemented group, as was insulin like growth factor 1, which is a control molecule for wound repair.

Glutamine is used by inflammatory cells within the wound for proliferation and
as a source of energy. Fibroblasts use glutamine for these same purposes, as well as for protein and nucleic acid synthesis. Because optimal functioning of these cells is

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paramount to the healing process, glutamine is a necessary component of the process of tissue repair. Glutamine is a non-essential amino acid that can become a conditionally essential amino acid in certain circumstances, including tissue injury.Glutamine is released from skeletal muscle following injury or surgery, which can cause a relative deficiency of glutamine in skeletal muscle and the gut, as intestinal uptake is frequently diminished as well. Studies utilizing oral glutamine pre- and post-surgery, and in burn patients, have shown mixed results. Oral feeding of glutamine in surgery patients did not affect plasma glutamine or nitrogen turnover. Intravenous glutamine in surgery patients as an alanine-glutamine dipeptide showed consistently better post-operative results, as seen by significantly decreased length of hospital stays (average of four days or less).92 A significantly smaller incidence of pneumonia, bacteremia, and sepsis was noted in patients with multiple trauma given enteral glutamine feedings.94 Whether glutamine supplementation will enhance wound healing in less severely injured individuals is not known. A mixture of arginine (14 g/day), glutamine (14 g/day), and beta-hydroxy-betamethylbutyrate (HMB) (3 g/day) was given to 18 elderly (>70 years) individuals who then under- went experimental implantation of sterile polytetrafluoroethylene tubes that could later be excised and studied for fibroblastic migration and collagen deposition. Supplementation with this mixture resulted in significantly greater wound collagen deposition than in 17 controls not supplemented.

Aloe vera Centella asiatica and Aloe vera have been used for
decades as folk remedies for burns, wounds, and scars. Improved wound healing has been reported from topical or internal application of these two botanical medicines. Continued

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use of these plants as healing agents has led to scientific investigation of their efficacy as wound healing agents. Centella asiatica (gotu kola) has been documented to aid wound healing in several scientific studies. One of the primary mechanisms of action of Centella appears to be the stimulation of type-1 collagen production. Animal studies have consistently shown topical application of Centella asiatica to a sutured wound significantly increased the breaking strength of the wound. Asiaticoside, a saponin extracted from Centella asiatica, is thought to be one of its active constituents. Shukla et al showed a 0.2-percent asiaticoside solution applied topically twice daily for seven days to punch wounds in guinea pigs resulted in 56-percent increase in hydroxyproline, 57percent increase in tensile strength, increased collagen content, and better epithelialization compared to controls. Using the same punch wound model the researchers demonstrated an oral dose of 1 mg/kg for seven days produced a 28 percent reduction in wound area and a significant increase of tensile strength and hydroxyproline content of the wound. Topical treatment with Aloe vera has been shown to improve healing in frostbite and electrical injury in animals.In addition, Aloe vera has improved the healing of wounds in both normal and diabetic rats.Topical application and oral administration of Aloe vera to rats with healing dermal wounds increased the collagen con- tent of the granulation tissue as well as the degree of cross linkage. Collagen increased 93 percent with topical treatment and 67 percent with oral treatment compared to controls. The increase was attributed to increased stimulation by Aloe vera of collagen synthesis or increased proliferation of fibroblast synthesis of collagen, or both.In a similar study, the effects of oral and topical Aloe vera on full thickness dermal wounds in rats exhibited an increase in glycosaminoglycan components of the extracellular matrix and,

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in particular, hyaluronic acid and dermatan sulphate levels. Aloe vera and Centella asiatica have been widely used for a host of curative purposes including facilitating wound repair. In spite of their wide use as folk remedies the biochemical basis for their action or influence on tissue repair is just beginning to be understood. Human clinical trials are needed to determine safety and benefits of perioperative oral administration of these botanicals. Topical application of both Aloe vera and Centella asiatica extracts to healing wounds or surgical scars appears to be safe and facilitates improved wound repair.

3. What are the nutrient impacts on the phases of wound healing ?

Wounding
Calendula succus topical antimicrobial

Hemostasis
Drugs, herbs, vitamins, amino acids, or minerals that effect blood-clotting mechanisms should be avoided prior to surgery.

Inflammatory Phase
Vitamin A enhances early inflammatory phase Bromelain and adequate protein intake prevent prolonging inflammatory phase Vitamin C enhances neutrophil migration and lymphocyte transformation

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Proliferative Phase
Vitamin C necessary for collagen synthesis Centella asiatica promotes type-1 collagen synthesis Glucosamine enhances hyaluronic acid production Vitamin A promotes epithelial cell differentiation Zinc required for DNA synthesis, cell division, and protein synthesis Calendula succus and Aloe vera support formation of granulation tissue

Remodeling
Protein deficiency inhibits wound remodelling

4.

Why the product is made in spray form but not in ointment form or in tablet form ?

The product is made in spray form but not in tablet form because some of the ingredients used is not eatable. For example, Isopropyl alcohol and blood clotting agent. This product is prefer to made in spray form instead of ointment because in the case of big and serious wound, we are not practically to apply ointment on it and we dont know how much do we need to apply and if apply using hand, we will face a big problem that we are unable to sterilise our hand fully and this may caused infection on the wound. Furthermore, we want instant effect after apply the product on the wound. Therefore by using spray, its contents are able to penetrate into the skin in a very short time because spray has very

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high momentum compared to ointment. Its contents can move out from the spray and travel in the air with a speed of 300 m per seconds .

5.

Explain why we cannot simply apply that spray product on normal without injuries

skin?

This is because some of the ingredients to make this product will cause irritation to the normal skin. It may caused scars on the normal skin because one of the ingredients using is fibrinogen which has ability to make the wound to heal and if the skin without any wounds ,it will forming scar .

6.

Explain why not everyone can use this spray product ?

Some of the people will have allergic on some of the contents of this product .

7.

Necessity to have Mitosis process in wound healing and importances of it .

Mitosis is the process of cell division. There are three reasons why cells divide mitotically:

1. Cell Replacement 2. Growth 3. Asexual Reproduction

1) Cell Replacement

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Cells divide in order to produce new cells that replace damaged, dying or lost cells. Your skin, for example, consists of millions of cells that protect your body against damage and pathogens. The cells on the surface slough off after a while, for example, when you scratch yourself or because of other forms of abrasion. The cells lost need to be replaced by new cells. These cells are produced in deep layers of your skin through mitotic cell division.

2) Growth

We all started out as a single cell. When your mother's egg cell and your father's sperm cell fused, this single cell was created. Then the cell divided mitotically often enough to produce the millions of cells in your body. Some cells, for example, will become part of your heart, while others will develop into bone cells. All the cells of your body work together in tissues and organs in very complex ways so that you can speak, learn, eat, play and perform all the other activities that are part of your daily life.

3) Asexual Reproduction

Sometimes, mitosis even functions to produce a whole new organism. To the left, you see a microscopic picture of an organism that consists of a single cell (author of the picture: The Alpha Wolf). This organism, called a cilliate, reproduces by mitotic cell division: it simply divides in half. Thus, the new cell has a single parent cell and is genetically identical with its parent. Asexual reproduction occurs in single-celled organisms, but also in plants and more complex animals. If you clip part of a plant off, for example, it can

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develop into a whole new plant. Sea stars can also reproduce asexually. If a sea star loses an arm, for example, this arm can regenerate into a whole new sea star.

8.

Why we are unable to actually getting the result same as the expected result ?

Theoretically we are able to get the result same as the expected result but in practically we cannot get exactly the effect same as in expected result. This may because

(1) The ingredients is not mix properly.

(2) The amount of ingredients in the product is not enough to presenting out so perfect effect.

(3) The boiling temperature is not high enough.

9.

What are the natural wound healing process ?

The natural skin healing process is a grouping of the mechanisms that allows the skin to repair itself after a tear, burn or other injury. For healthy skin, the epidermis and dermis layers, together form a protective barrier that shields the inner body. Once that barrier is broken, the normal healing process is set in motion. Wound healing generally has three different stages: the inflammatory stage, the proliferative stage and the remodeling stage. Once the skin is damaged, a series of interrelated events take place in close succession in order to repair the skin. Within minutes after an injury occurs, blood platelets collect at the site of injury to form a clot. This clot limits bleeding at the injury site. The inflammatory phase causes bacteria and debris to be removed from the wound, and signals are released that result in the division of cells for repair. The proliferative phase is

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shown by the formation of new tissue at the injury site, the replacement of new skin at the site and the general shrinking and eventual disappearance of the wound. New blood vessels are also established during the healing process. The wound is made smaller by myofibroblasts, which hold on to the edges of the wound and slowly get smaller by a system similar to the contraction of muscle cells. When cellular repair is complete unneeded cells are disposed of through apoptosis.

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References
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22. Levenson SM, Demetrio AA. Metabolic factors. In: Cohen IK, Diegelmann RF, Linblad WJ, eds. Wound Healing: Biochemical and Clinical Aspects. Philadelphia, PA: WB Saunders Co; 1992:264. 23. Levenson SM, Gruber CA, Rettura G, et al. Supplemental vitamin A prevents the acute radiation-induced defect in wound healing. Ann Surg 1984;200:494-512. 24. Petry JJ. Surgically significant nutritional supplements. Plast Reconstr Surg 1996;97:233-240. 25. Seifter E, Crowley LV, Rettura G, et al. Influence of vitamin A on wound healing in rats with femoral fracture. Ann Surg 1975;181:836-841. 26. Stadelmann WK, Digenis AG, Tobin GR. Impediments to wound healing. Am J Surg 1998;176:39S-47S. 27. Stadelmann WK, Digenis AG, Tobin GR. Physiology and healing dynamics of chronic cutaneous wounds. Am J Surg 1998;176:26S- 38S.

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