ARTIFICIAL SKIN

By Sibel ÇINAR

[A] SKIN (BIOLOGIC PROPERTIES)

Skin is a Bilayer organ whose functions are essential for survival. Although the Bilayers works as a unit, each component has specific properties, which need to be recognized if one is to duplicate these properties with a skin substitute. [1] Skin is a complex organ. Functionally, it has two layers with a highly specialized and effective bonding mechanism. The epidermis, consisting of strata basale, spinosum, granulosum and corneum provides a vapor and bacterial barrier. The dermis provides strength and elasticity. Thin epidermal layer is constantly replacing itself from its basal layer, with new keratinocytes undergoing terminal differentiation over approximately 4 weeks to anuclear keratin filled cells that make up the stratum corneum, which provides much of the barrier function of epidermis The basal layer of epidermis is firmly attached to the dermis by a complex bonding mechanism containing collagen types 4-6.When this bond fails, serious morbidity results. [2] Skin is the largest organ in the body (1,5-2 m²) and the thickness is about 1-2 mm. in adult but it is thinner in children and elderly.

[B] FUNCTIONS OF THE SKIN

Skin Functions

Epidermis Protection from desiccation Protection from bacterial entry Protection from toxins Fluid balance: avoiding excess evaporative loss Neurosensory Social-interactive Dermis Protection from trauma due to elasticity, durability, properties Fluid balance thru regulation of skin blood flow Thermoregulation thru control of skin blood flow Growth factors and contact direction for epidermal replication and dermal repair

 [C] FUNCTIONAL COMPONENTS OF SKIN

EPIDERMIS: The outer thinner layer known as the epidermis is composed mainly of epithelial cells. The outermost cells contain the protein keratin and are known as keratinocytes. The basal or deepest epidermal cells are anchored to the basement membrane by adhesion molecules (or glue), namely fibronectin. These immature cells are continually dividing and migrating toward the surface to replace lost surface cells e.g. after an injury. The same type of regenerating epidermal cells is found in hair follicles and other skin appendages, which are anchored in the dermis. As the cells mature and migrate to the surface they form keratin which becomes an effective barrier to environmental hazards such as infection and to excess water evaporation. Replacement of the epidermal layer by this regenerative process takes 3-4 weeks. Cues and biologic stimuli at the wound surface are necessary to direct proper orientation and mitotic response of the epidermal cells. Many of the cues come from dermal elements, especially the matrix proteins and matrix glycosaminoglycan.

    DERMIS: The dermis is a very dynamic layer of thick connective tissue, also in constant turnover. The dermis is divided into a thin superficial layer known as the papillary dermis containing the anchoring epidermal rete pegs and the thicker deeper portion known as the reticular dermis. The papillary dermis is the major factory for the proteins providing direction for epidermal replication. The upper dermis also contains the highest blood flow. The primary cell type is the fibroblast, which produces the key structural extra cellular matrix proteins collagen and elastin as well as matrix or ground substance. In addition these cells produce the key adhesion proteins used to attach epidermal cells to the basement membrane and for used epidermal cell migration and replication. Fibronectin is a key fibroblast derived signal protein for orchestration of healing. The ground substance or matrix is made up of complex polysaccharide - protein complex known as glycosaminoglycan or the GAG component as well as hyaluronic acid. The matrix provides a semi fluid, which allows for cell and connective tissue orientation as well as nutrient diffusion to the cells and scaffolding for cell migration. 

[D] EVOLUTION OF BURN WOUND CARE

(THE CONCEPT OF EARLY WOUND CLOSURE WITH SKIN, SKIN SUBSTITUTES)

The predominant approach to the management of burns throughout history has been to alter the burn surface in an attempt to improve healing. Initial approach was based on the "theories of the time" and appears quite unusual to us now. However, it is important to recognize that our current understanding of wound healing has evolved only recently and is still evolving. Current concepts of wound healing principles are only just beginning to be recognized and implemented

SKIN SUBSTITUTES IN BURN MANAGEMENT (HISTORICAL PERSPECTIVE)

A)   Overview

It has been recognized for centuries (but not widely practiced) that wound care using a dressing with "skin like" properties increases healing. In order to better understand the principles used in the development of the "optimum skin substitutes" ; it is best to review the history of skin substitutes from antiquity to the present. The skin substitute restores the optimal biological environment to a clean wound surface and protects the wound from conversion. Thus, its use is relegated to the wound free of non-viable tissue and infection. The impetus for the use of temporary skin substitutes evolved centuries ago for the acute partial thickness wound described as: redness and blistering of the skin or withering without charring" Fabricius Hildanus in his book on burns, DeCombustionbus 1607.The use of skin substitutes for the burn with "eschar formation and charring" (DeCombustionbus 1607) awaited the recent advances in burn excision in which massive burn injuries commonly survive.

B) Xenograft Use{16^th-17^th century}

The importance of wound closure in healing is well documented beginning in Ancient Greece where burn wounds were occluded with dressings rather than left open. The concept of occlusion is the first step toward the concept of wound closure. As there were no synthetic materials with the properties of skin, animal or reptile skin (Xenograft, a tissue graft transferred from one species to another) was used to mimic human skin.

The use of animal and reptile skin as a "skin substitute" dates back several hundred years. Frog and lizard skin use was reported in the 16^th and 17^th century and frog skin is used today in Brazil. The skin of a variety of animal species were used beginning in the early 1900’s. Pigskin became popularized in the 1960’s and is currently the most common Xenograft used. The objective remains to close the burn wound using a bi layer tissue likes found with skin. The inner (dermal layer) having surface collagen, can bind to the wound surface if there is no non-viable tissue.

 C) Allograft Tissue Use {1950’s}

Human tissue use as a skin substitute was reported in the mid- 19^th century but really became popularized beginning about 1950. Cadaver skin and human amnion have and continue to be used. Allograft skin is used primarily to cover excised full thickness wound as opposed to just partial thickness injuries. Human amnion has been more commonly used on partial thickness wounds or excised wounds. Human amnion has fibronectin, a collagen, lattice as well as an epithelial cell layer which can act as a barrier similar to the epidermis while also adhering, although weak; to the burn. Amnion was introduced in 1910 as a biologic dressing and was popular as a burn-wound dressing until the 1960’s when alternatives like pigskin became more available.

D) First Cultivation of Human Epidermal Cells {1960’s}

Rheinwalk and Green at the Massachusetts Institute of Technology cultivated human epidermal cells in serial culture, allowing for a larger amount of these cells to be created. Many researchers utilized this method and produced autologous keratinocytes that could be used for grafting.  The epithelial sheets that were produced were successfully grafted to athymic mice in most cases.  This step led to clinical application of this method by O’Connor et al where they successfully treated 2 burn patients with cultured autologous expanded keratinocyte sheets.  This procedure, including some adaptations that had to be made, is used to treat burns, venous ulcers and other procedures that require grafts.  The advantages of this procedure are that it is able to cover a large surface area from one skin biopsy, leads to eventual regeneration of the dermis, and it is autologous.  Its major drawback is that there is a 3-4 week period between the actual biopsy and the graft. This graft has a 50% to 60% permanent take.  It also does not have a dermal component, possibly requiring years for the neodermis formation to occur, which is also another drawback [3]

E) First successful Application of Allogenic Keratinocyte Grafts {1983}

the first successful application of allogenic keratinocyte grafts were performed on burn patients.  There is a large disparity as to the percentage of these grafts that actually take, ranging from 30%-100%.  These grafts act as a wound covering and often promote healing in venous leg ulcers.  Leigh et al. reported on the efficiency of allogenic kerationocyte grafts for treating venous leg ulcers in 1987.  The crucial advantage of this procedure is that a biopsy does not have to be done, which cuts down on the time for the procedure. One disadvantage to the procedure is that epithelial sheets have no dermis.  This causes blistering of the graft and the appearance of the graft is not what many people are looking for. [3]

F) Alloderm and Integra Use To Treat Burn Patient {1980-present}

The next step, or one that paralleled that of autologous/allogenic composites was acellular matrices, which included Alloderm, and Integra, which are primarily used to treat, burn patients.  Burke et al. lead the development of a bilayered artificial skin composed of a temporary Silastic “epidermis”.  The matrix was successful in closing the wounds of the burn patients and many human skin characteristics were seen in the matrix.  When compared to epithelial sheets this product was more efficient at producing functional tissue at the graft site studies that were conducted.  [3] The brainchild of a trauma surgeon and a mechanical engineer, Integra® is a prime example of NIGMS' investment in collaborative research. In the early 1970s, the surgeon, Dr. John F. Burke, then director of the Burn Center at Massachusetts General Hospital and Shriners Burns Institute, came up with the idea that completely removing badly burned skin (as opposed to letting it slough off over time) might offer greater protection against wound infection and improve the very poor prognosis that severely burned patients faced. Dr. Burke recognized that a necessary follow-up to the removal of burned skin would be immediate and permanent skin replacement. Once developed, his idea ultimately became standard practice for treating major burn injuries.

At first, Dr. Burke pioneered the use of skin from related donors (such as family members with similar genetic markers). But doing so required that the burn patient be given powerful immunosuppressant drugs, to dampen the patient's immune system so that the graft would not be rejected. Unfortunately, crippling the immune system in this way posed many serious problems for the patient. Instead, Dr. Burke began using the patient's own unburned skin (often from the scalp, which is rarely burned) as a source of graft material.

However, since using these sorts of grafts (or even skin from cadavers) did not permanently solve the problem, Dr. Burke saw the need for some type of artificial means to recover skin. Using a synthetic product would also offer an advantage in that such a material is free of viruses and bacteria, which can transmit disease. Dr. Burke, who had a penchant for engineering, recruited a clever mechanical engineer at neighboring MIT, Dr. Ioannas Yannas, to cooperate in this effort. The collaboration, marrying biomedical engineering with clinical medicine, proved fruitful. After initial testing in animals, the artificial skin that Drs. Burke and Yannas developed proceeded to rigorous scientific testing in humans, in a multi-center clinical trial. [4] Another product resulting from NIGMS-sponsored research that is similar to Integra® is called AlloDerm™. Removing from cadaver skin all cell produces this product, which is sold and manufactured by LifeCell Corporation of The Woodlands, Texas

Components that cause a burn patient's immune system to reject a graft from any other person. The principle behind the product AlloDerm™ got its start in the mid-1980s in the laboratory of Dr. Charles Baxter of the University of Texas Southwestern Medical Center at Dallas, as well as in other laboratories working in this area of research. A key feature of the process is preserving to the greatest extent possible the "natural," three-dimensional structure of the dermis. Properly approximating this scaffold, whether from real dermis (as in AlloDerm™) or artificial dermis (as in Integra®), is crucial to the ability of the patient's remaining cells to regenerate themselves into a new, functioning skin. [4]

G) Dermagraft and Apligraf Use {present}

Refining the matrix technology, Advanced Tissue Technologies developed its product of Dermagraft.  “Dermagraft is a living, metabolically active immunologically inert dermal tissue.”  “This product through its trials has shown that it takes with athymic mice.”    “Clinical trials in patients with diabetic foot ulcers have demonstrated more complete healing with Dermagraft than with conventional treatment.”  There are clinical trials under way looking into the efficacy of Dermagraft.  “The advantages of this living dermis include avoidance of non-human tissue, immediate graft application, mesh absorption, mesh absorption in 60 to 90 days and less wound contracture and scarring.”  [3]

This new research led to the development of a product that had both dermal and epidermal components, with each housing living cells.  This was a major step that led to the development of Apligraf.  Apligraf uses a procedure that is a modification of Bell et al’s work.  “Apligraf is morphologically, biochemically, and metabolically similar to human skin.  “Hansbrough et al evaluated the properties of Graft skin in full-thickness wounds on athymic mice and found that it adhered quickly, with excellent take of all grafts.” [3]

The First Tissue Engineered Organ, Which Has Progressed From Lab Bench To The First Accepted Patient Care, Has Been Skin. [5]

[E] COMMERTIALLY AVAILABLE SKIN SUBSTITUTES

With advancing technology, a host of both permanent and temporary biologically active skin substitutes are available to replace allograft and Xenografts. [1]

Naturally occurring tissues
- Cutaneous allografts
- Cutaneous xenografts
- Amniotic membranes

Skin substitutes
- Synthetic bilaminate
- Collagen based composites
       Biobrane
       TransCyte
       Integra

Collagen based dermal analogs
- Deepithelized allograft
- Alloderm

Culture-derived tissue
- Bilayer human tissue (Apligraf)
- Cultured autologous keratinocytes
- Fibroblast seeded dermal analogs
- Collagen-glycosaminoglycan matrix
- Polyglycolic or acid mesh (Dermagraft)
- Epithelial seeded dermal analog

[F] IDEAL PROPERTIES OF BIOSYNTHETIC SKIN SUBSTITUTES [1]

Rapid and sustained adherence to wound surface*

Impermeable to exogenous bacteria

Water vapor transmission similar to normal skin

Inner surface structure that permits cell migration, proliferation and in growth of new tissue

Flexibility and pliability to permit conformation to irregular wound surface, elasticity to permit motion of underlying body tissue

Resistance to linear and shear stresses

Prevention of proliferation of wound surface flora

Tensile strength to resist fragmentation when removed

Biodegradability (important for "permanently" implanted membranes

Low cost

Indefinite shelf life

Minimal storage requirements

Absence of antigenicity

Tissue compatibility

Absence of local and systemic toxicity

*The most important criteria is Adherence

[G] DIFFERENCES IN APPLICATION [6]

1} Materials that are applied for short periods then removed or replaced at intervals.

Function: To stimulate autologous healing.

2} Cell free materials that encourage colonization by autologous cells and/or biodegradation and re-modeling.

Function: To stimulate neo-skin formation.

3} Cell containing skin substitutes

Function: To provide an immediate functional skin replacement.

[H] TYPES OF SKIN SUBSTITUTES

Skin substitutes can be classified into two according to their time of use; Temporary skin substitutes and Permanent skin substitutes.

TEMPORARY SKIN SUBSTITUTES

Temporary skin substitutes provide transient physiologic wound closure. Physiologic wound closure implies a degree of protection from mechanical trauma, vapor transmission characteristics similar to skin and a physical barrier to bacteria. These membranes therefore contribute to the creation of a moist wound environment with low bacterial density. There are four common uses for temporary skin substitutes:(1)as a dressing on donor sites to facilitate pain control and epithelialization  from skin appendages,(2) as a dressing on clean superficial wounds to a similar end,(3) to provide temporary physiological closure of deep dermal and full thickness wounds after excision while  awaiting autografting or healing of underlying widely meshed autografts and(4) as a test graft in questionable wound beds.[2]

PER MANENT SKIN SUBSTITUTES

This area can be arbitrarily divided into two approaches. The first approach is the use of a bilayer skin substitute with the inner layer being incorporated as a dermis rather than removed like a temporary product. The outer layer is either a synthetic to be replaced by autograft (epidermis) or both together. The outer layer of these products is usually not sufficiently developed to act as a barrier upon initial placement.

The second approach is the provision of either an epidermal or dermal components or simply a co-culture of cells containing elements of both. These products are technically not skin substitutes upon initial placement as there is no bilayer structure. [1]

[I] EXAMPLES TO VARIOUS SKIN SUBSTITUTES

1/Naturally Occurring Tissue

Porcine Xenograft

[1]

Xenografts have been used as intact split thickness grafts from variety of species from many years to provide cover of wounds. The most commonly utilized species employed in this Faison has been domestic swine (pig). Porcine xenograft is often used as a reconstituted product consisting of homogenized porcine dermis, which is fashioned into sheets and meshed. It is widely used for temporary coverage of clean wounds such as superfacial second-degree burns and donor sites. Its use has been favorably reported in patients with toxic epidermal necrolysis and it has been combined with silver to suppress wound colonization. Although it does not vascularize, it will adhere to a clean superfacial wound and can provide excellent pain control while the underlying wound epithelializes. It is applied to a cleansed wound and covered with a dry dressing, no sutures being required. [2].However after  these excellent properties ,xenograft has some disadvantages such as :It can not  obtain blood supply from wound and will slough, I t is the potential for serious disease transmission   and since it  belongs to  different species, tissue rejection occurs .[1]

Allograft (Amniotic Membrane)

Human amnion has fibronectin, a collagen, lattice as well as an epithelial cell layer which can act as a barrier similar to the epidermis while also adhering, although weak; to the burn. Amnion was introduced in 1910 as a biologic dressing and was popular as a burn-wound dressing until the 1960’s when alternatives like pigskin became more available. This material has also some advantages and disadvantages;

 Advantages include:

Acts like biologic barrier

Easy to apply, remove

Transparent

Disadvantages include:

 Difficult to obtain, prepare and store

Need to change every 2 days

Disintegrate easily

risk of disease transfer

Allograft (Human Cadaver Skin)

Human cadaver skin is currently the most commonly used biologic temporary covering for surgically excised thermal and chemical burns, but there are drawbacks. Foremost is insufficient supply: The American Red Cross estimates that only one-sixth of the skin supply needed for burn victims is available from the nation's tissue banks. Other drawbacks include epidermal sloughing (requiring painful and costly removal and reapplication) and disease transmission.

Biomedical engineers have been attacking the problem for a couple of decades. They do not get easy breaks, but they have developed a means of generating replacement tissue free from most of the complications of using borrowed human skin. [7]

BIOBRANE [1]

Biobrane is a bilayer synthetic skin substitute. Outer epidermal analog constructed of a thin silicone film with barrier functions comparable to skin Small pores present in silicone to allow for exudates removal, permeability to topical antibiotics. Inner dermal analog composed of a three dimensional irregular nylon filament weave upon which is bonded type I collagen peptides

Surface binding of inner membrane potentiated by collagen-fibrin bonds as well as fibrin deposition between nylon weave. Subsequently fibronectin, produced by migrated fibroblasts, enhances binding to the fibrin entrapped in mesh .New epithelial cells growing along mesh measures adherence. Thin water layer at surface maintained for epidermal cell migration. Removed after re-epithelialization (or prior to skin graft on excised wound).Silicone and nylon weave provides flexibility. Long shelf life: maintained at room temperature Bertek Pharmaceuticals Inc.

Biobrane Bilayer Structure is shown. Silicone outer layer acts like a protective epidermal barrier. The inner surface is composed of a three - dimensional interwoven nylon filament upon which collagen peptides are bonded. Initial wound adherence is the result of bonding of membrane collagen to surface fibrin. The second phase of adherence results from epidermal cells proliferating between the nylon matrixes

BIOBRANE STRUCTURE AND PROPERTIES

Trifilament threads bonded with collagen exposed to wound (Biobrane)

Adherence:

Biobrane contains a nylon fabric that is woven from tri-filament threads and covalently bonded with collagen peptides. The multiple filaments provide a high exposure to the wound surface resulting in an increasing adherence to the wound surface of partial thickness burn. Adherence is biphasic: the initial adherence phase is due to fibrin-collagen (nylon) bonding and the second is the ingrowth of epithelial cells and fibrin between the threads. Adherence initially is comparable to allograft but biobrane adherence exceeds allograft by 3-5 days

  Water Vapor Transport

A decrease in water vapor transport is essential to avoid wound desiccation. However, some water vapor permeability is beneficial in preventing excess fluid accumulation.

Barrier to Bacteria

Assessment performed by surface cultures of wounds excised and covered with biobrane, allograft or treated open.

Optimizing healing environment

Biobrane increases re-epithelialization rate compared to topical antibiotics or exeroform gauze

Decrease pain

 Biobrane decreases pain compared to topical antibiotics or xeroform gauze

Decrease wound exudate

 Biobrane decreases exudate compared to fine mesh gauze

Durability,flexibility

A minimal elongation of 350± 50% and a burst strength of 10.3± 2 lb/inch have been reported (Tavis, et al)

Permeability to antibiotics

Permeability to 1% silver Sulfadiazine comparing biobrane without pores to the standard with pores.

The standard biobrane is very permeable to topical antibiotics. (Tavis et.al.)

Safety

- The value for the primary skin irritation index falls within the "non-irritant" category

- Intracutaneous toxicity studies were reportedly negative

-Pyrogenicity studies were reported negative

PROCESS OF HEALING

Figure1:A superficial partial thickness burn is shown. The zone of necrosis is confined to the upper (papillary) dermis and is usually separated by a layer of edema from the viable wound surface 

Figure2: Superficial burn derided to viable wound bed. Note surface fibrin and collagen. Also note increased proteolytic activity which is not suppressed by immediate wound closure, will produce increased injury

Figure3: Bilayer biobrane ready to be placed on clean wound. Note outer silicone layer (epidermal analog) and inner nylon mesh coated with collagen to adhere to surface (dermal analog)

Figure4: Biobrane in place, adhered to surface by nylon-collagen mesh. Note preservation of thin water layer on surface to allow epithelial migration along inner layer.

Figure5: Biobrane peeled back from surface to demonstrate rapid migration of new epithelium along nylon-collagen mesh. As epithelialization increases the biobrane becomes more opaque. The minimal exudate produced, drains out the pores.

Figure6: Biobrane removed with re-epithelialization.

PROCESS OF APPLICATION

Identify appropriate wound

Remove the sterile biobrane sheet from package

Cut to fit and apply under a moderate stretch,

Attached product to surrounding unburned skin with steri-strips

BIOBRANE REMOVAL

 

When healed biobrane turns whitish and dry in appearance Gently peel off then that wound with moisturizer If small area still open, treat with bacitracin or Neosporin (triple mix).

TRANSCYTE

Trancyte is a bilayer skin substitute. Outer epidermal analog is a thin nonporous silicone film with barrier functions. Inner dermal analog is layered human fibroblast products mainly collagen type 1,fibronectin and Glycosaminoglycan. Subsequent cryo-preservation destroys fibroblasts but preserves activity of fibroblast-derived products. Thin water layer at surface is maintained for epidermal cell migration. It is removed after re-epithelialization (or prior to skin graft or excised wound). Silicone provides flexibility. It must be kept frozen until use.(Smith and Nephew Wound Management).[1] [ No published reports of the above properties for TransCyte use on a partial thickness wound.

 TransCyte is stored and sealed in a cassette with two pieces per cassette. Product is thawed just prior to use.

1]

TRANCYTE IN PLACE

The area of coagulation or eschar has been completely removed exposing a viable but injured wound surface (zone of injury), which can deepen. The surface itself has an increased content of fibrin produced by activation of the clotting cascade and fibronectin produced by the dermal cells. There is also evidence of the onset of inflammation with vasodilatation, increased neutrophils and macrophages. The wound surface also has increased proteolytic activity which if persistent can denature new tissue formation and growth factors. . The outer synthetic layer (knitted nylon) protects the wound surface from environmental insults. The inner bioactive membrane is composed mainly of human fibronectin and collagen Type I which will produce a contact adherence to the wound surface. These elements and the other components of the inner membrane are produced by human fibroblasts. 

ALLODERM (An Example Of A Natural cell Free Matrix)[8]

 

AlloDerm is donated human tissue that is processed using a patented technique to remove all epidermal and dermal cells while preserving the remaining biological dermal matrix. AlloDerm is used as you would an autograft; following transplantation AlloDerm begins to regenerate into the patient's own tissue.

ALLODER

Day 7-10: By day 7 to 10 host fibroblast cells and blood vessels respond to the transplantation of the AlloDerm matrix initiating the revascularization and normal tissue remodeling process.
Day 45: Replacement and revascularization of the transplant continues as normal connective tissue for Day 90: AlloDerm repopulated with the patient's own cells has become integrated as the patient's own natural soft tissue. Fibroblasts continue to lay down autologous collagen. Ms through host collagen deposition.
Day 180: AlloDerm is naturally remodeled into the patient's own tissue 6 to 8 months after the procedure. The healing process culminates in natural tissue remodeling, with no fibrosis

Alloderm is based on allogeneic human dermis from which all cellular material is removed using techniques claimed to optimize the residual matrix for subsequent implantation. The matrix is preserved by freezedrying. Although the skin is retrieved from screened donors and subjected to processing steps that are likely to inactivate many bacteria and viruses ,there appears to be normal sterilization method applied. The patented process is below.

Step 1: Dermal tissue consists of collagens (1) Fibroblasts (2), elastin (3) and blood vessels (4	Step 2: The epidermis (5) is removed without causing damage to the dermal

Step 3: The dermal cells are removed without damaging the structural components essential for repopulation	Step 4: The dermal matrix is freeze-dried .

Step 6: Revascularization begins through the blood vessels (4).

Step 7: AlloDerm is repopulated with cells and begins remodeling.

Step 8: AlloDerm is remodeled into the patient's own tissue

AlloDerm is donated, human dermal tissue that has been decellularized to remove the risk of rejection or inflammation. It is then freeze dried through a patented process that does not damage the crucial elements of the tissue structure (collagens, elastin and proteoglycans) and packaged with a shelf life up to two years. Once AlloDerm is used as a graft or implant, it quickly revascularizes and repopulates with cells naturally remodeling into the patient's own tissue. [8]

Alloderm is found to facilitate many of the key biological events in wound healing like re-epithelialization and angiogenesis. The use of Alloderm began in1992 for burn patients. Since all the cells are removed no component necessary for survival and transmission of viruses is present. Since it is human tissue no inflammatory response or allergic reaction is incited.[5]Clinical experience with this material in acute and re-constructive burn wounds is still early and limited, but appears favorable.[1]

INTEGRA (An Example Of A Synthetic Cell-free Matrix)

Product Description

INTEGRA® Dermal Regeneration Template is a bilayer membrane system for skin replacement. The dermal replacement layer is made of a porous matrix of fibers of cross-linked bovine tendon collagen and a glycosaminoglycan (chondroitin-6-sulfate) that is manufactured with a controlled porosity and defined degradation rate. The temporary epidermal substitute layer is made of synthetic polysiloxane polymer (silicone) and functions to control moisture loss from the wound. The collagen dermal replacement layer serves as a matrix for the infiltration of fibroblasts, macrophages, lymphocytes, and capillaries derived from the wound bed. As healing progresses an endogenous collagen matrix is deposited by fibroblasts; simultaneously, the dermal layer of

INTEGRA® Dermal Regeneration Template is degraded. Upon adequate vascularization of the dermal layer and availability of donor autograft tissue, the temporary silicone layer is removed and a thin, meshed layer of epidermal autograft is placed over the "neodermis." Cells from the epidermal autograft grow and form a confluent stratum corneum, thereby closing the wound reconstituting a functional dermis and epidermis.

INTEGRA® Dermal Regeneration Template Function

INTEGRA® Dermal Regeneration Template is a skin replacement system for the treatment of deep partial-thickness or full-thickness thermal injury to the skin. INTEGRA® Dermal Regeneration Template is applied following excision of the burn wound to viable tissue. It serves two critical functions:

It is available without delay and functionally closes the excised wounds immediately without the need to create donor site wounds. Following application, it functions as an "artificial skin" that provides immediate postexcisional wound homeostasis, facilitating patient recovery and relieving metabolic stress.

It serves as a template to generate "neodermis," a dermal-like tissue that readily accepts very thin epidermal autografts. Formation of the neodermis typically takes 14-21 days. After the neodermis is formed, the silicone layer is easily removed and a very thin meshed and widely spread epidermal autograft can be applied over the neodermis. These thin epidermal autografts result in less donor site morbidity than conventional split-thickness autografts. The cosmetic results are excellent, exceeding the results for conventional meshed autograft. Since the epidermal autograft can be applied immediately after the neodermis has formed (usually 14-21 days after application), the application of the epidermal autograft can also be scheduled at a time when sufficient donor sites are available and/or the patient's condition is suitable for the grafting procedure. In a multicenter clinical trial, INTEGRA® Dermal Regeneration Template remained in place successfully for up to 73 days prior to epidermal autografting.

The neodermis tissue formed by INTEGRA® Dermal Regeneration Template is distinct from granulation tissue, which is not a desirable bed for epidermal autografts. Granulation tissue, which may form at joints between INTEGRA® Dermal Regeneration Template sheets or in areas affected by infection or other problems (see below) will typically have a deep red, granular appearance and bleeds easily. In contrast, the neodermis that is visible under the silicone layer, or that is exposed after removal of the silicone layer, is typically yellow to orange in color with patches of light red. After final healing of the wound, the neodermis tissue histologically and functionally is similar to normal dermis. [9]. The preliminary  clinical results on the use of artificial skin as a permanent wound cover for excised burns were published by Burke and Yannas et al in 1981 and encouraged further clinical trials[2]

Researchers at MIT and Massachusetts General Hospital (MGH) are clinically testing an artificial skin that should provide a permanent replacement for irreparably damaged skin, according to Dr. Ioannis V. Yannas '59 of the Department of Mechanical Engineering. Yannas, Dr. Dennis Orgill '83 and Mr. Eugene Skrabut '69, all of MIT's Department of Mechanical Engineering, have collaborated with Dr. John F. Burke of MGH to develop Stage 2 artificial skin. Stage 2 artificial skin is an improvement over Stage 1, which Yannas and his research team have worked on for over a decade. He has already successfully tested Stage 1 skin on human burn victims. Whereas Stage 1 skin only promotes regrowth of the dermis -- the innermost layer of skin -- Stage 2 also promotes the growth of new epidermis. The first step in making artificial skin is identical for Stages 1 and 2. Yannas makes a template of collagen fibers taken from cowhide. The template provides a lattice around which the body's own cells can begin to grow. The collagen lattice slowly degrades, ideally at roughly

the same rate as new tissue forms in the wound. If a burn wound is smaller than a half-dollar, the skin can regenerate itself without too much damage by growing inward from the edge of the wound. But with a burn wound any larger, "the dermis is not spontaneously regenerable, and instead you get scar tissue .The purpose of the collagen template is to "channel the process of wound healing away from scar synthesis to the synthesis of dermis. To prevent water loss and infection, Yannas places a silicone layer over the collagen template, which acts as a temporary epidermis. After the dermis has regrown, which usually takes about 20days, Yannas replaces the silicone layer with patches of epidermis taken from unburned parts of the victim's body. These skin patches grow together, creating a new epidermis. The problem with Stage 1 skin was that when large portions of the patient's body are severely burned, it was difficult to find enough unburned skin to patch up the epidermis.

Stage 2 skin eliminates this problem by providing an epidermis as well as a dermis. Yannas takes basal cells, "the innermost, baby cells," from a quarter-sized patch of unburned skin. He then seeds them into the collagen template in a centrifuge. Basal cells in normal skin grow in a layer on top of the dermis and work their way up through the epidermis to replace old cells. In the seeded artificial skin they proliferate to form new epidermis complete with nerves and blood vessels. The actual laboratory procedure for seeding the skin takes about 100 minutes from start to finish. MGH surgeons take a quarter-sized layer of thin, unburned skin tissue from the patient and hand it over to the waiting Yannas. He puts the skin sample in a jar with the enzyme trypsin, which cuts the bonds between the dermis and epidermis. Back at the MIT lab, Yannas and his research team throw away the epidermis and spin the remaining dermis in a vortex. The vortex separates the basal cells from the dermis. Yannas then takes the basal cells and seeds them into the collagen template to create artificial skin custom-made for the particular burn victim. The new skin is capable of "temperature regulation and almost every neurological sensation’’. However, the new epidermis does not promote regrowth of hair follicles or sweat glands. The body uses sweat glands to regulate temperature. However, the body has "another method of moisture loss, which is diffusion of water through the epidermis." In addition to its ability to save lives and prevent disfigurement of burn victims, the Stage 2 artificial skin interests Yannas because the regrowth of skin is similar to embryonic development. Yannas has not yet studied the metabolism of the Stage 2 skin, which would show whether the skin could perform functions such as producing Vitamin D. He is now studying the immunology of the artificial skin. A stage 2 artificial skin has proven successful in tests with guinea pigs. Yannas expects the ongoing clinical tests to end in May, at which time he will report his findings in a scientific journal. [10].

Yannas also studies artificial skin (by the use of dermal regeneration template (DRT) ) by the use of island graft  to isolate organ regeneration from scar synthesis and other processes leading to skin wound closure A porous analog of the extra cellular matrix, composed of a graft copolymer of type 1 collagen and chondroitin 6-sulfate,was seeded with uncultured autologous keratinocytes and served to induce regeneration of the dermis and the epidermis. Histologic study of island grafts on day 14 showed that the copolymer grafts had largely degraded and that a new epidermis and dermis had been synthesized in its place The thickness of the new epidermis increased as the density of cells seeded into the graft increased. The island graft procedure clearly separates graft healing from extraneous processes of the wound site which encroach upon the regenerate, and complicate its identification.[11].Although Integra Artificial Skin is becoming widely used in burns and reconstructive surgery, poor take and loss due to infection remains a concern for some patients.[12] Disadvantages of Integra also includes its high price, complex and labour-intensive dressing requirements.[13].

DERMAGRAFT (An Example Of A Synthetic Matrix Combined With Allogenic Fibroblasts)

Dermal fibroblasts, a type of skin cell, are seeded onto the cadaver skin for transplants.biocompatible Vicryl scaffold to form a living tissue, a sterile alternative to human

ATS (Advanced Tissue Science) makes artificial skin, which it markets under the name Dermagraft, for treating severe wounds. The product is grown under laboratory conditions from human cells on a chemical base called a scaffold. ATS's ability to create this tissue involves many disciplines—not just cell biology, but also biochemistry and biomedical engineering. ATS takes stromal cells, like fibroblasts from neonatal tissue (which reproduces the fastest and is the most likely to be healthy), and expands and seeds them onto biocompatible scaffolds. The scaffolds are made of polyglycolic acids, the basis of many "resorbable" medical materials, such as surgical sutures and new surgical glues. Dermagraft is grown on Vicryl, a Johnson & Johnson trademarked blend of polylactic and polyglycolic acids.

Vicryl (Polyglactin 910) knitted mesh is prepared from a synthetic absorbable copolymer of glycolide and lactide, derived respectively from glycolic and lactic acids. This knitted mesh is prepared from uncoated, undyed fiber identical in composition to that used in Vicryl synthetic absorbable suture, which has been found to be inert, nonantigenic, nonpyrogenic, and to elicit only a mild tissue reaction during absorption. Vicryl knitted mesh is intended for use as buttress to provide temporary support during the healing process.[14,15]. When Dermagraft is applied to the body, the scaffold breaks down into glycolic acid and lactic acid, which are carried away by the bloodstream and metabolized to carbon dioxide, oxygen, and water. As they develop over a period of a few weeks, the cells on the scaffold are kept under optimal conditions in bioreactors. The bioreactors are challenging to build because they have to create perfect conditions to grow the implants. In them, one neonatal sample of a square inch or so yields cells sufficient for manufacturing 250,000 square feet of final product; each 3x4-inch lot produces 1,000 units of Dermagraft. The tissue must be cryopreserved at a sufficiently low temperature to maintain sterility. Dermagraft is stored at -70°C and shipped in dry ice to the clinical site.

Tough demands are put on ATS for quality control. The tissue must be free of all pathogens. The rinse for sterility requires that the scaffold be laser z-welded to the inside of the transport bioreactor for stability. The reactor must also be translucent to allow tracing of the wound and precise trimming of the implant with sterile scissors. Then, after cryopreservation and just before implantation, the material is tested to ensure proper metabolic activity—that is, that the cells can generate enough energy on their own for growth and reproduction. ATS found out the hard way in a long clinical trial that if tissue metabolic activity was not within a certain range, healing suffered.  Dermagraft is a total skin replacement for full thickness burns and chronic wounds like diabetic foot ulcers.

The evidence ATS provides is inspiring. Patients on whom Dermagraft is used have shown reduction of pain within 30 minutes, and can be released from the hospital within one or two days rather than the 10 to 12 typically needed for healing. [7]When both fibroblasts and keratinocytes are used within the dermagraft mesh the effectiveness of the procedure decreases. Keratinocyte outgrowth across the Dermagraft increased as the viability of the fibroblasts grown within the Vicryl mesh, decreased. The authors suggest that in this system the complex extracellular matrix secreted by the fibroblasts and the matrix bound growth factors, rather than   ongoing fibroblast-secreted factors, may be the important stimulatory factors and that viable fibroblasts would simply compete with keratinocytes for limiting nutrients.[6].It is also reported that the formation the fibroblast-aggregates was accelerated by the addition of insulin, dexamethasone, basic fibroblast growth factor,and ascorbic acid to basal medium and by the control of shaking time. In fibroblast-aggregates formed under a basal medium, the level of gene expression related to wound healing was higher than that of monolayer when one added the hormones and growth factors Therefore, tissue-engineered skin products with aggregates and scaffolds would become an artificial skin with potentiality of higher wound healing.[16]The general hypothesis is that wound healing is accelerated by the presence of more fibroblasts and ECM(Extra cellular matrix) in the dermal substitute. In the case of Demagraft the preculture of fibroblasts in the vicryl mesh caused Degradation and the replacement of the substitute material with fibroblast-synthesized ECM. This resulted in a reduction in the inflammatory reaction occurring in vivo with hydrolysis of the Vicryl material [17].It is also reported for the Collagen –GAG matrix .A cell population enriched with proliferating cells can be expected to generate an epidermis more efficiently than uncultured epithelial cells when seeded into a CG matrix graft. Cultivation of keratinocytes also makes massive epithelial expansion possible. Thus, combining the seeded CG matrix technique with the cellular expansion pf in vitrocultivation would allow for reconstruction of large surfaces from small skin donor sites.[18]

Dermagraft-TC is a temporary cover to promote faster healing in burn patients without the pain and complexity involved in current treatment programs. Working with ATS's polymer chemists and molecular biologists, IDEO worked on the fluid mechanics and the human factors of the bioreactor cassette, which serves as an aseptic growing chamber and shipping container and withstands temperature changes from the 37 degrees C of human body temperature to the -70 degrees C of cryogenicstorage.[19]

Dermagraft cassettes ,ready for patient use

APLIGRAF (An Example of A Largely Biological Matrix Combined With Skin Cells)

The recent advances in the field of tissue engineering have forever altered the course of human scientific history. With the advent of the necessary laboratory techniques to develop human tissues in vitro, researchers have gained the ability to manipulate nature in a remarkable manner. Tissue engineered skin, the first engineered organ to emerge from the lab to the marketplace, has opened the door to the variety of other tissue-engineered products. Living skin equivalents have proven successful in treating patients with FDA-approved indications and patients involved in controlled clinical trials examining future applications. One such living skin equivalent is Organogenesis and Novartis' Apligraf, which consists of a type-I collagen matrix seeded with fibroblasts and covered with a layer of cultured keratinocytes.  On May 26, 1998, Apligraf was approved by the FDA to treat patients exhibiting venous leg ulcers. In the United States alone, approximately 1,000,000 people annually seek treatment for these lesions. In July of 2000, Apligraf was also approved for diabetic foot ulcer treatment, potentially improving the lives of another 800,000 Americans. [3]

APLIGRAF is supplied as a living, bi-layered skin substitute. Like human skin, APLIGRAF consists of living cells and structural proteins. The lower dermal layer combines bovine type 1 collagen and human fibroblasts (dermal cells), which produce additional matrix proteins. The upper epidermal layer is formed by promoting human keratinocytes (epidermal cells) first to multiply and then to differentiate to replicate the architecture of the human epidermis. Unlike human skin, APLIGRAF does not contain melanocytes, Langerhans' cells, macrophages, and lymphocytes, or other structures such as blood vessels, hair follicles or sweat glands. [20,21]

Formation of Apligraf

1}Dermal layer formation

Removed from master cell bank, thawed, and expanded

• Placed on a semipermeable membrane along with bovine type I collagen and neutralizing medium^1,2

• During 6-day incubation period
- Fibroblasts divide and multiply
- Fibroblasts contract the collagen filaments^1,2
- Fibroblasts produce human matrix proteins
- Dermal matrix forms and condenses

2} Epidermal Layer Formation

Removed from master cell bank, thawed, and expanded

• Seeded onto the contracted dermal matrix^1,2

• During 4-day incubation period
- Keratinocytes attach to matrix¹
- Keratinocytes proliferate and differentiate
- Epidermal layer forms

3} Cornification

•Keratinocyte medium is replaced by a maintenance medium
•Cells continue to mature
•Distinct epidermal and dermal layers are present
•Developing bilayer is air-lifted (exposed to air)
•Incubated (air-liquid interface) for 10 days
- Enhances maturation of keratinocytes in epidermal layer
- Produces a stratum corneum (cornification)

4} Maintenance ,Harvesting, Packaging

•Resulting product is a bilayered, viable skin construct
•11-day harvesting window
•APLIGRAF^® is individually packaged for use
- 5-day shelf life from day of packaging
- Maintained in a nutrient medium
- Packaged and sealed in polybag
- Shipped via overnight courier

Apligraf vs. other skin substitutes:

Apligraf Advantages:

First multi-layer skin substitute available (neodermis and enoepidermis layers)

Not external – becomes a part of the patient

A long-term permanent solution

FDA approved

Economically viable

Readily available

Synthetic and Biologically active component

Faster wound therapy than other compression therapy    (complete healing 61 days vs. 181 days)

No donor site re-injury necessary

Disadvantages:

Cadaver tissue cells must be screened and sanitized

More expensive than compression bandages

Technical application more complicated

Multiple applications necessary

^{SOME CASE STUDIES FOR APLIGRAF APPLICATION}

Case 1:

71-year –old female with venous leg ulcer of approximately 3 months duration. Treated with Apligraf

Day 0: Wound 2.5 cm in length and 1.3 cm in width	Week 1: Wound 0.63 cm in length by 0.32 cm in width
Week 2: Revascularization and healing	Week 56: The wound remains completely healed

CASE STUDY2

 Healing acute excisional wounds from Mohs Micrographic Surgery:

Mohs micrographic surgery is now universally recognized as a precise method for treating skin cancers. It is especially effective in cancers of the face and other sensitive areas because it can eliminate virtually all the cancer cells while causing minimal damage to the surrounding normal skin. Mohs micrographic surgery is also ideal for the removal of recurrent skin cancers -- tumors that reappear after treatment and can plague a patient repeatedly. As a study by Eaglstein, Alvarez et. al. indicates, this surgery creates relatively deep skin wounds, which can be effectively treated with Apligraf     (Eaglstein, Alvarez et. al. Dermatol Surg. 25:3:1999) . One hundred and seven patients participated in this study. The tissue-engineered skin was applied once, immediately after excisional surgery and patients were followed for up to one year. One such patient who underwent Mohs Micrographic surgery and subsequent Apligraf treatment is presented below.

Excision site immediately after surgery	Day 7 after Apligraf application
3 months after Apligraf application

The Apligraf was applied once,immediately after excisional surgery (largely skin cancer)and patients followed up for 1 year.There were no indications of any immunological rejection[6]

[J] CONCLUSION

Following on from developments in the1980’student involving the use of cultured keratinocyte grafts in wound healing, the last decade has seen great progress in the fabrication of composite grafts. This has been the part of a general movement towards in vitro fabrication of bioactive or living tissue replacement materials that has been termed Tissue Engineering .The momentum in this new scientific field has built up rapidly during the latter half of the decade There is little doubt that the volume of research and development in tissue replacements for bone, cartilage, cardiovascular tissue and even nerve replacements together with fundamental studies on the isolation and exploitation of mesenchymal stem cells and other pluripotent (e.g. embryonic stem cells) or even totipotent stem cells; will cross-fertilise with skin research. It is therefore, perhaps not too optimistic to expect tissue engineered skin grafts with a clinical performance equivalent to the current gold standard skin autografts, in the not too distant future

REFERENCES;

1} www.burnsurgery.org

2} Burns 25(1999) 97-103 Skin Substitutes in burns

3} http://www.brown.edu

4} http://www.nigms.nih.gov/news/features/artificial_skin.html

5} Burns 27 (2001) 534-544 Skin substitutes: a review

6} Burns 27 (2001) 545-551 Clinical Evaluation of skin substitutes

7} http://www.memagazine.org/backissues/february99/features/skin/skin.html

8} www.lifecell.com

9} http://www.integra-Is.com

10} http://www-tech.mit.edu/V105/N18/skin.18n.html

11} J. Biomed. Mater Res, 39,531-535,1998 Design of an artificial skin4:use of island graft to isolate organ regeneration from scar synthesis and other processes leading to skin wound closure.

12} Burns27 (2001) 699-707 Strategies to improve the take of commercially available collagen/glycosaminoglycan wound repair material investigated in an animal model.

13} British Journal Of Plastic Surgery (2001), 54, 208-212 The use of a bilaminate artificial skin substitute (integra) in acute resurfacing of burns : an early experience.

14} http://www.ethiconinc.com/page/healthcare/ETHICON_products.html#

15} http://www.johnsonandjohnson.com

16} Materials Science and Engineering C17 (2001) 59-62 Effects of hormone and growth factor on formation of fibroblast-aggregates for tissue-engineered skin

17} Journal Of Pathology (2000), 190, 595-603 Higher numbers of autologous fibroblasts in an artificial dermal substitute improve tissue regeneration and modulate scar tissue formation.

18} British Journal Of Plastic Surgery (1999), 52, 127-132 Comparison of cultured and uncultured keratinocytes seeded into a collagen-GAG matrix for skin replacements.

19} http://ideo.com/studies/ats.htm

20} www.apligraf.com

21} www.novartis.com

22} Burns 27(2001) 523-533 Design principles for composition and performance of cultured skin substitutes

23} Burns23, supplement no1 (1997) 30-32 Evaluation of artificial skin (Integra) in a rodent model

24} www.advancedtissuescience.com

25} Burns 27 (2001) 517-522 Burn wound healing and skin substitutes