BIOMATERIALS GROUP

The skin is a complex organ, which serves as a protective barrier against several hazards of the environment, at least as long as the skin stays intact. However, when the skin gets wounded by trouma, injury or skin disease, this may lead to the loss of several functions and hence to illness and even to death.

Major injuries resulting in extensive damage to the skin necessitate immediate coverage to aid repair and regeneration to restore normal skin function. However skin injuries are traumatic events, which are seldom accompanied by complete structural and functional restoration of the original tissue. There is a substantial expense by way of health care and loss of personal freedom, quality of life and productivity. Research efforts to deal with skin loss, have focused on three principal directions. Firstly, towards improvement of wound healing by factors which speed up the process and reduce scarring. Secondly, the design and development of skin substitutes as functional equivalents of autograft skin. Thirdly, to identify cues that induce the skin to heal by regeneration rather than repair (scarring).

Management of wounds due to injury, burns, surgery, other types of trauma and non-healing ulcers poses a great challenge to the surgeons, plastic surgeons and all others associated in this mission. The type of wounds (acute or chronic), extent and depth of lesion and factors like nutritional status, age, systemic disease and medication that is specific to the individual, com-plicate the healing process.

The potential for a patient to benefit from the attention of a plastic surgeon frequently depends on the availability of cutaneous tissue for use as an autologous graft or flap. This is significantly so in burns, but it is also an issue in surgical approaches to chronic wounds and scar revision. Despite the continued development of skin-expansion techniques and the large variety of flaps available to achieve mobilisation, there remains a belief that a suitable replacement skin material could not only substantially improve the chances of burn survival but also lead to a better recovery of function and appearance for other surgical procedures.

Many skin substitutes are in development or have recently been introduced onto the market. Some of these are designed as suitable materials with which to facilitate early burn wound excision regimes. Others are targeted at chronic wounds. The hard commercial reality is that materials aimed at burn wound closure are unlikely to be as economically profitable as materials that could be used for chronic wounds, such as venous and diabetic ulcers, which are substantially more prevalent. Nevertheless, as the mortality from burns continues to fall in developed countries, the desire to develop protocols that reduce morbidity is furnishing a continued interest in the improvement of burn wound healing, and, consequently, new skin substitute products are being introduced for these applications.

This term paper investigates and reviews the functions of skin, the large number of new approaches and products that are emerging in the quest to develop biologically based skin substitutes for clinical use. The variety of materials has understandably generated a measure of confusion amongst potential users of these products. Here, the range of different products that are available, the current evidence for their effectiveness and their relative costs are examined. Lastly, some future prospects and some expectations about the new coming technologies will be described.

The skin is the largest human organ. It covers between 1.5 and 2 m² , comprising about one sixth of total body weight. It 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.

growth factors and contact direction for epidermal replication and dermal repair

2.1. 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 are 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.

Figure 2. (1)Epidermis, (2)Dermis, (3)Subcutis, (4) Hair Follicle, (5) Sebaceus Gland, (6) Sweat Gland

Replacement of the epidermal layer by this regenerative process takes 2-3 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.

ability to regenerate every 2-3 weeks resulting from biologic cues and contact direction provided by dermis, basement membrane

inner layer: epidermal cells which are proliferating and migratory to surface and will become keratinocytes

innermost layer: basal epidermal cells anchored to basement membrane by adhesion molecules

Langerhans’ cells, contain granules, fix antigens (felt to be responsible for antigen-antibody and allergy functions)

2.2. 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 a scaffolding for cell migration.

factory for all the components required for replication and repair of epidermis and dermis

papillary dermis: upper dermis containing anchoring rete pegs and also is the most biologically active part of the dermis

reticular dermis: the thicker deeper portion responsible for durability and anchoring of skin appendages

matrix proteins
- collagen is the predominant protein, mainly collagen Type 1. (besides structure; collagen type 1 provides a contract orientation for dividing and migrating epithelial cells)
- fibronectin is the primary adhesive protein playing a major role in healing
- other adhesive proteins

ground substance (glycosaminoglycan)
- carbohydrates protein complexes
- hyaluronic acid

2.3. THE SUBCUTIS (HYPODERMIS): The subcutis (sub = under; cutis = skin/Lat.) refers to the fat tissue below the skin. It consists of spongy connective tissue interspersed with energy-storing adipocytes (fat cells).

Fat cell clusters: Fat cells are grouped together in large cushion-like clusters held in place by collagen fibres called connective tissue septa or sheaths.

Nourishment, insulation and padding: The subcutis is heavily interlaced with blood vessels, ensuring a quick delivery of stored nutrients as needed. The functions carried out by the subcutaneous fatty tissue, beside the storage of nutrients in the form of liquid fats, include the insulation of the body from cold and shock absorption. On the palms of the hand, the soles of the feet and the buttocks, fat padding serves almost exclusively for shock absorption. (Note: Fats, also triglycerides or acylglycerins, are the most plentiful and simplest fatty acid-containing lipids. They are esters of the triol alcohol, glycerine with three saturated and/or unsaturated fatty acids. Fats make up the main component of the fat depots.)

Fat distribution in men and women: The fat content of the subcutis is not the same in all body regions. Also men and women differ in the distribution of subcutaneous fat. An example is cellulite - it is characterized by a special arrangement of the subcutaneous fat tissue septa and predisposes to fat deposition on the hips, thighs and buttocks - which occurs mostly in women. Men on the other hand tend to store fat on the torso.

EPITHELIAL CELLS: These cells make up the majority of the epidermis. Immature cells are programmed to divide, migrate and mature to keratin producing cells called keratinocytes. The signals to activate this process come from messenger proteins called growth factors as well as through contact direction from key dermal adhesive proteins, especially collagen.

FIBROBLASTS: The cells of mesenchymal original are normal present in the dermis and produce normal dermal replacement components. After injury these cells migrate into the wound and proliferate; in order to produce increased quantities of these dermal proteins and matrix.

conduit for living fibrin, fibronectin and growth factors in contact with the wound surface

scaffold for surface deposition of fibrin and fibronectin i.e., cell guidance proteins

ENDOTHELIAL CELLS: These cells make up the lining of micro and macro vessels and also make up the lining of new capillaries produced after injury. These cells like fibroblasts do differentiate from local mesenchymal cells and are also attracted into the wound by local signals.

MACROPHAGES: These cells of mesenchymal origin are normally present in tissue but increase in number after injury, attracted by chemical messages released by the activation of inflammation. The long lived cells release the protein chemical messages, growth factors and growth stimulants which orchestrate healing in an organized fashion.

PLATELETS: The factor-rich particles release a host of growth factors and adherence proteins during the initial post burn period.

As can be seen, skin is a very biologically active organ. Duplication of skin properties typically requires a Bilayer structure, the outer layer having protection properties and the inner layer properties stimulating tissue growth. Temporary skin substitutes are not concerned with dermal replacement as would be the case with a permanent skin substitute.

Whenever the skin gets wounded, tissue is destroyed and substance lost. If the wound is superficial (only the epidermis destroyed), regeneration takes place and the original is exactly restored. Whenever the wound is deeper, regeneration is not possible and a repair process is started. Scar tissue that differs from the originalhealthy tissue closes the gap of the wound. In some cases wound healing is not complete or delayed because of underlying disease in the patient. Some of the most obvious examples are diabetes and vascular insufficiency.

The wound healing is a complex process that can be divided into several phases. The first phase is a vascular response to the injury.

Each major injury causes the disruption of blood vessels and extravasation of blood. This process has a cleaning effect and lasts until the blood coagulation reestablishes hemostasis. Platelets are very important in the formation of a hemostatic plug, but also secrete several mediator of wound healing, such as growth factors, cytokines, etc. The different pathways activated in the case of coagulation generate another number of vasoactive mediators and chmotacti factors and hence induce inflammation.

During this inflammatory process, several types of cells are recruited to the site of injury.

Infiltrating neutrophils cleanse the wounded area of foreign elements and bacteria. Monocytes become activated macrophages, which are important mediators in the initiation and propagation of the formation of new tissue in the wounds.

Reepithelialization with epidermal cells from the skin appendages and the wound edges begins quite early in the wound haling process.

Phenotypic alterations of the cells during the first hours after injury, including the dissolution of intercellular desmosomes and hemidesmoomal links and the formation of peripheral cytoplasmic actin filaments, allow some cells movement. The migrating cells dissect the wound, separating the eschar from the underlying viable tissue. This process of dissection seems to be regulated by the interaction between integrins and integrin receptors on the epidermal cells on the one hand, and the production of collagenase, which facilitates degradation of collagen and extracellular-matrix proteins, on the other han.

After one or two days the migration process is accelerated by the proliferation o epidermal cells. Prolifration is probably induced by th loss of cell contact and by the production of growth factors such as EGF, TGF, KGF.

Granulation tissue (the new stroma) begins to invade the wound after three to four days. The macrophages in this new stroma provide a source of growth factors necessary to stimulate the fibroblasts to produce the new extracellular matrix and stimulate the blood vessels to grow into the wound. A number of growth factors, especially PDGF and transforming growth factor 1 stimulate the fibroblast to form a matrix with fibrin, fibronectin and hyaluronic acid. Once the fibroblast have moved into this provisional matrix, thy synthesize collagen and replace the provisional matrix with a collagenous matrix. After the deposition of a sufficient amount of collagen amtrix the fibroblasts stop further production and die off by an apoptotic process. If this apoptosis does not take place in a correct way, fibrotic disorders such as keloid formation and hypertropic scarring can be induced.

New vessles are necessary to sustain the newly formed granulation tissue with oxygen and nutrients. Growth factors such as acidic and basic fibroblast growth factor,vscular endothelial growth factor,transforming growth factor and a number of others induce. Once the wound is filled with new granulation tissue, a process of apoptosis will stop the angigenesis process.

Wound contraction and wound modelling is the last step in the wound healing. Due to wound contraction, the healed wounds gain strength. However the maximal strength of scar is only approximately 70% of this normal skin.

tissue desiccation: occurs with open wound, impedes epithelial migration, risk of wound conversion

wound infection: due to impaired local defense, exposure to microbes in the environment, increases inflammation induced injury

wound trauma: environmental insult, use of toxic chemicals, traumatic dressing changes

Although the wound healing process is very complex,most wounds do get healed with conventional wound treatment.

A caregiver, confronted with a wound,will clean th wound using water and/or anticeptics. The necrotic tisue is removed during the wound debriment and an appropriate wound dressin will be chosen, taking into account the principles of moist wound healing.

But when applying conventional treatent modalities in an appropriate way, wounds will have difficulties to heal. Conventional treatment is not fesible in wounds that are too large such as extensive burns and wounds in patients with epidermolysis bullosa, an inherited blistering disease. In those wounds in which conventional treatment is not feasible or ineffective, there is need for more than conventional treatment. A temporary or permanent closure should be established with some type of biological and/or synthetic material.

This artificial skin should mimic some of the most important features of normal skin. The artificial device should protect the wound by providing a barrier to the outside. It should also control water evaporation and protein and electrolyte loss. It is not necessary that an artificial skin product keeps the wound strile, but a dynamic procees between the skin substitute and the host should prevent local and sytemic infection. Other essential properties include limiting excessive heat los, decreasing pain, allowing early mobilizaiton, providing an environment for accelerated wound healing and improving the cosmetic appearence of the scar.

Also important are the physical characteristics of the actual product. Is it easy to manuplate the product? Is it feasible to place and dress the skin substitute effectively?

Another consideration is the availibilty of the product. Is the product readily available (off the shell) or is it custom-made?

In skin substitutes containing biological material, the risk of infection shoul be taken into account. Careful screening, using the most performant laboratory techniques, should be carried out.

A last consideration concerning the the use of artificial skin product is the cost. The high price of a skin substitute will limit reimbursement and might preclude the use of the device.

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.

People have tried to improve healing in wounds where conventional treatment fails.The most important milestone in this aspect is the possibility to cultivate human keratinocytes, a technique described for the first time by Rheinwald and green in 1975 (1).

But long before this date, since the beginning of recorded time, humans have struggled with the treatment of wounds. The ancient greeks were among the first to recognize the benefits of proteckting a wound with derssings. In ancient India the oldest descriptions of autologous soft tissue flaps were found (2).

During the Renaissance the work of Amboise-Paré and others gave a foundation for the modern day understanding of wound healing and wound coverage.

In the second half of the nineteen century Reverdin and Thiersch were the first to use autologous skin grafts (both epidermal and dermo-epidermal) for traumatic skin loss (3).

The first person to cultivate human kerotinocytes in vitro was Kreiblich in 1914 (4). He separated the upper part of the skin mechanically from the underlying layers and cultivated the resulting epidermis on solid agar prepared with human plasma.

A fallowing important step was the autotransplantation of keratinocytes, in which Medawar in 1948 suceeded. Together with Billingham, he was able to separate epidermis from dermis with trypsin. Later on they were able to get a cell suspension which could be retransplanted to the wound surface (5.6).

Karasek was the first to transplant cultivated human keratinocytes. These cells originated from explants of skin. The number of cells obtained was to be able subcultivate the cells (7).

Only in 1975 Rheinwald and Green were able to obtain passeges of proliferating keratinocytes. The big difference with the former culture techniques was the introduction of a feeder layer of fibroblasts, which had been treated with a sublethal dose of radiation before the keratinocytes were seeded upon. A culture medium containing 10% serum and several kinds of growth factors were needed to promote the proliferation of the keratinocytes. Using this technique Rheinwald and Green were able to induce a augmentation of cells, resulting in a 10.000 fold augmentation of surface of the initial biopsy within 3 to 4 weeks .

In order to prevent the use of fetal calf serum, Boyce and Ham introduced, in 1985, an alternative culturing method using a completely defined medium (8). From 1989 onwards it was possible to cryo-preserve keratinocyte sheets (9).

O’Connor and his collegues from Boston were the first to use a large amounts of cultured keratinocytes in a patient in a clinical setting. They did an autootransplantation of cultured keratinocytes in a patient with major burns (10).

In 1983, Hefton and his collegues used cultured allografts in burns (11.12). A few years later cultured keratinocytes were also introduced in the treatment of other skin defects. There have been many papers on the use of cultured autografts and allografts in leg ulcers (13). Another application field was the use in defects after tatoo removal and in patients with epidermolysis bullosa(14).

Meanwhile Yannas and Burke have described the use of bilaminate collagen-glycosaminoglycan matrix covered with a silicon surface. After take of the matrix, the silicon surface could be removed and be replaced by autologous cultured epidermal cells (15). In 1981, Bell and be coworkers constructed the first living skin equivalent consisting of collagen fibroblast gel with keratinocytes cultured on top of the contracted gel (16).

For the majority of skin substitutes, the most extensive clinical experience has been in burns. Since the need for an artificial skin is mostly comes from the burn patients,which suffer from moderate to extensive burns, the researches are concentrated mostly in this area. 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 were 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.

APPROACHES TO BURN CARE

ancient Greece
-cleanse, apply animal fat and wrap

Roman Empire
-cleanse, apply ashes and oil, herbs, then wrap

Middle Ages
-wax plus herbs or boiling oil

1800’s - heat or ice

early 1900's
-expose wound, apply tannic acid or variety of pigments to dry wound

1950’s to present
-use of topical antibiotics using the exposure or the closed dressing method

1900’s to present
-introduction of the use of skin substitutes

1960’s to present
-rapid wound closure with surgery, skin, skin substitutes:

The evolution and current technology of these temporary and permenant skin substitutes are very different and the new technology will be discussed separetely.

Rheinwald J.G., Green H., Serial cultivation of strains of human epidermal keratinocytes: the formation of colonies from single cells. Cell 1975; 6: 331-44

Cairns B.A., de Serres S., Peterson H.D., Meyer A.A., Skin replacements: the biotechnological quest for optimal wound closure. Arch Surg 1993; 128: 1246-52

Kreiblc K., Kultur erwachsener Haut auf festem Naehrboden. Arch Dermatol Syph 1914; 120:168-77

Medawar P. The cultivation of adult mammalian skin epithelium in vitro. Q J Micro Sci Ser; 1948:187-96

Billingham R.E., Reynolds J. Transplantation studies on sheets of pure epidermal epithelium and epidermal cell suspensions. Br J Plast Surg 1953; 5:25-36

Karasek M. In vitro culture of human epithelial cells. J Invest Dermatol 1966; 47:533-40

Boyce S.T., Ham R.G. Cultivation, frozen storage and clonal growth of normal human epidermal keratinocytes in serur free medium. J Tissue Culture Meth 1985; 9: 83-93.

Teepe R.G.C., Koegbrugge E.J., Ponec M, Vermeer B.J., Fresh versus cryopreserved cultured allografts for the treatment of chronic skin ulcers. Br J Dermatol 1990; 122:81-9

O’Connor N.E., Mulliken M.R., Finkelstein J.L., Shires G.T., Grafting of burn Patients with allografts of cultured epidermal cell. Lancet 1983; 2:428-30

Hefton J.M., Caldwell D., Biozes D.G., Balin A.K., Carter D.M. Grafting of skin ulcers with cultured autologous epidermal cells. J Am Acad Dermatol 1986; 14:399-405

Leigh I.M., Pukis P.E., Navsaria H.A., Phillips T.J., Treatment of chronicVenous ulcers with sheets of cultured allogenic keratinocytes. Br J Dermatol 1987; 117:591-7

Hill J., Grimwood R.E., Parsons D.S., Treatment of chronic erosions of junctional epidermylosis bullosa with humar epidermal allografts. J Dermatol Surg Oncol 1992; 18:396-400

Yannas I.V., Burke J.F., Design of an artificial skin. !. Basic Design principles. J Biomed Mater Res 1980; 67: 386-92

Bell E., Ehrlich H.P., Sher S., et al. Development and use of a living skin equivalent. Plast Reconstr Surg 1981; 67: 386-92

As we make a literature survey, we see that there are different types of classifications of skin substitutes. Conceptually, skin substitutes are temporary or permanent; epidermal, dermal or composite; and biologic or synthetic. Biologic components are xenogeneic, allogenic or autogenic. There is a research effort centered on many of the possible permutations of these traits. Moreover, skins substitutes are divided into two groups for wound cover and for wound closure. Another method available for the classification is wether the artificial skin is tissue engineered or non tissue engineered. In these classifications, most of the artificial skin products overlap to one another, that is, we can see the same skin substitute under many different headlines.

For example, we can see Appligraf in literature classified as: 1)Biological skin substitute-allogeneic (allograft) or 2) Engineered cell containing biological substitute or 3) Composite skin substitute or 4) permanent skin substitute or 5) Skin substitutes for wound cover.

Lack of coherent classification aggreement in literature, in this term paper, I suggest two steps. First, the types of classifications will be described information about the artificial skin substitutes

Second, the skin substitutes will be investigated deeply 1) as being Xenografts, Allografts and Autografts and 2)under the head of their commercial names (except the tissue engineereds) 3)as Composites.

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 a 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 physiologic 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. There are a large number of such membranes in common use, classes of which are:

A useful permanent skin substitute remains the `holy grail' of burn research. Although no ideal substitute exists at present, there are a number of devices currently available that contribute to permanent coverage of burn wounds:

Having established the design principles for cultured skin substitutes and a directory defining many of those currently available, the next stage is to review some of the animal and clinical studies utilising these materials. The aim is not to provide a comprehensive list of all in vivo studies published, but rather, at this early stage, to tease out certain characteristics of each category of graft materials, on a range of cutaneous defects. Aside from the clinical data that has accumulated through the years on the use of cultured epidermal autografts (CEA), there are few large-scale multi-centre prospective trials for the newer tissue engineered skin substitutes. Nevertheless, even at this early stage there are preliminary indications of where the materials may show promise clinically, and where further research and development can be guided by both positive and negative in vivo performance.

As with approaches to the engineering of other tissue grafts, strategies for the fabrication of skin substitutes can be allocated to alternative categories, outlined in Table 1. Although these categories are artificial in that there is a good deal of overlap, nevertheless they are a useful starting point for analysing the clinical data. There are also differences in approach to the clinical use of skin substitutes and the role(s) that they are expected to perform in vivo. This spectrum is outlined in Table 2.

There have been two major approaches to the provision of a cell-free matrix for replacement of the dermis component of skin. The first has been to fabricate a matrix with the required physical and chemical structure. One example of this approach is the material developed in the 1980s by Burke and Yannas and which has recently been released for clinical use as Integra ® artificial skin (Integra LifeSciences Corpora-tion, Plainsboro NJ). The second approach has been to utilise allogeneic human dermis rendered cell-free and preserved. An example of this is Alloderm ® produced by LifeCell Corporation (The Woodlands, TX).

As with the cell-free matrices, there have been ap-proaches based on an initially synthetic matrix comprising of, for example, polyglycolic acid or polyglactin, and alternative approaches using natural biological substrates such as collagen and glycosaminoglycans. An example of the former is Dermgraft and of the latter approach, Apligraf.

Wound closure requires a material to restore the epidermal barrier function and become incorporated into the healing wound, whereas materials used for wound cover rely on the ingrowth of granulation tissue for adhesion. Materials for wound cover are most suited to superficial burns, where they create an improved environment for epidermal regeneration by providing a barrier against infection and controlling water losses. For the purposes of this review, we will define biological skin substitutes in terms of provision of either wound cover or wound closure. Temporary wound closure can be achieved with cadaveric allograft. The pathological immunosuppression present in the early stages of a severe burn injury protects allografts from rejection during this period. Cadaveric material is supplied by skin banks, where it is frequently cryopreserved. Careful screening of prospective donor material aims to reduce the risk of transmission of infective agents, but this risk may not be eliminated. The potential for disease transmission is a significant factor in the development of skin substitutes. All biological materials have the potential for disease transmission, and careful manufacturing and sourcing of raw materials is essential and tightly regulated in Europe, North America and many other countries. The use of intact human allograft and animal-skin xenograft have been reviewed in detail elsewhere, and will not be covered in depth here. However, skin substitutes that have been manufactured from intact skin are considered. Permanent wound closure relies on a subsequent epidermal graft to the integrated allodermis. This may be from an autologous skin graft or, potentially, from a skin substitute of some description involving the delivery of epidermal cells. The structure and composition of the main substitutes are shown diagrammatically in Figure 1 along with a cost per cm 2 . Many skin substitutes contain a mixture of biologically derived components and non-biological substances, such as synthetic polymers. Totally non-biological commercially available dressings, such as Opsite (Smith & Nephew Inc), Duoderm (ConvaTech Inc) and many more, are available either. These products are generally occlusive, providing a moist environment conducive to faster healing, although potentially allowing for increased proliferation of microorganisms beneath their surfaces.

Biological wound cover encompasses both biosynthetic materials and unprocessed skin products, such as xenograft and human cadaveric and living allografts. Two of the most readily available biological-based wound covering materials are Dermagraft and Transcyte. Transcyte was very confusingly once called Dermagraft-TC, but it is structurally completely unrelated to the product currently known as Dermagraft. Both products were developed by Advanced Tissue Sciences Inc, La Jolla, San Diego, CA, USA, and are now supplied in a joint venture with Smith & Nephew Ltd, York, UK.

Figure 1—A guide to biological skin substitutes. The first column gives the product name and its manufacturer. The second column is a schematic representation of the components of the product, with the contents of each layer described in the third column. The final two columns give the cost of the product in 2001

Alloderm is processed human cadaveric skin from which the epidermis has been removed and the cellular components of the dermis have been extracted prior to cryo-preservation in order to avoid a specific immune response. Alloderm functions as a dermal graft but, because it has little barrier function, it is questionable whether it can be classed as a skin substitute for wound closure and, in many respects, it could be considered equivalent to Dermagraft, which has a similar cost (Fig.). Following application to a wound bed, it is repopulated by host cells, revascularised and incorporated into the tissue. Its role is as a template for dermal regeneration. It is reported to have good take rates and to reduce subsequent scarring of full-thickness wounds, while allowing grafting of an ultra-thin split-skin graft as a one-stage procedure.

Integra artificial skin is currently the most widely accepted synthetic skin substitute to be developed for use in burns patients, and was described originally by Yannas et al. Integra has a bilaminar structure, consisting of cross-linked bovine collagen and glycosaminoglycan, coated on one side with a silicone membrane that provides epidermal function. The pore size has been designed at 70–200 _m in order to allow migration of the patient’s own endothelial cells and fibroblasts. Smaller pores delay, or even prevent, biointegration, whereas larger pores provide an insufficient attachment area for invading host cells. Following application to a freshly excised wound, the collagen layer is biointegrated with the wound to form a vascular ‘neodermis’, a process that takes approximately 3–6 weeks. Once this stage has been reached, the silastic layer can be removed and an ultra-thin split-skin graft applied. The general opinion of the patients was that the areas grafted with artificial skin were cosmetically superior to those where autograft alone was used, although in no instances was it felt to be identical to normal skin. Histological follow-ups at 2 years reported that the dermal component was gradually remodelled over the first month, resembling papillary and reticular dermis, but failing to show rete ridges. The use of Integra requires a two-stage procedure, with a minimum interval of 3 weeks between the application of the Integra and the split-skin grafting, to allow neodermis formation. In some instances, this can increase the time taken to achieve wound healing. If Integra could be used in combination with cultured epidermal auto-grafts, then a 3 week delay would be advantageous as it allows for expansion of a skin biopsy into cultured epidermal sheets. In a recent study, successful take of cultured epithelial autografts onto a pre-grafted Integra-like material in a porcine model was shown. The wounds showed nearly complete confluence of the cultured epidermal autografts at 7 days, whereas in the controls, when cultured epidermal autografts wereapplied to freshly excised full-thickness wounds, they did not take very well at all. Histological analysis suggested that anchoring fibrils were present by 7 days. However, there are very few reports of the successful combination of cultured epidermal autograft sheets with Integra in clinical practice, for reasons that are as yet unclear. There are disadvantages to the use of this product. It is relatively expensive when compared with cadaveric allograft skin from skin banks, and the learning curve is reported to be steep, with high failure rates initially. The advantages are that it provides improved elasticity and cosmesis compared with an ultra-thin split-skin graft, with reduced donor-site morbidity compared with a standard-thickness split-skin graft, as it heals faster with less scarring. It avoids the risks of cross infection inherent with allografts. It is available immediately, and does not necessitate a narrow window of time in which to perform the second stage. Integra has an important role in providing immediate wound cover following early excision in patients with insufficient autograft. Donor sites can be recropped more often. Whether it improves the elasticity and cosmesis obtained from meshed autograft is not yet proven. Current research is focusing on modifying the collagen–glycosaminoglycan matrix through the incorporation of peptides and antibiotics. Cultured autologous keratinocytes have also been shown to produce a surface epithelium when seeded as a suspension into Integra and grafted onto athymic mice. The seeded material exhibited good wound adherence, complete healing and minor wound contraction, and had the potential to reconstitute an elastic functional and durable human skin. It remains to be seen whether this technique will work in patients.

The clonal growth of keratinocytes has been possible for over 20 years. Confluent sheets of keratinocytes can be grown in vitro and then applied to wounds such as burns, chronic leg ulcers, giant pigmented naevi, epidermolysis bullosa and neonatal scalp necrosis. As with split-skin grafts, the take of cultured epithelial autografts depends on the wound bed, which should be early granulating tissue or muscle fascia, rather than chronic granulation tissue. Pre-grafting the wound with allograft will encourage take, as will the presence of a non-granulated dermal bed of allogenic or autologous dermis. Cultured keratinocyte sheets are available commercially from a number of companies, e.g. Epicel (Genzyme Tissue Repair Corporation, Cambridge, MA,USA), and are also a relatively straightforward under-taking for suitably equipped university or hospital laboratories. However, they are expensive as they require a high input of skilled labour and quality control, and it takes 3–5 weeks to produce 1.8 m 2 confluent sheets of cells from a 2 cm 2 biopsy. To use cultured epithelial autograft sheets at their optimum requires a high degree of coordination between burn theatre and laboratory. To address this limitation, cultured keratinocytes have been stored frozen, both as suspensions and as cultured epithelial autografts, and remained viable, although the colony-forming efficiency is decreased by about 40%–50%. Cultured epithelial autografts are fragile sheets. They require separation from the tissue culture substrate using proteolytic enzyme before they are applied to the wound bed. This process causes contraction and subsequent restricted proliferation. The resulting epithelium unstable, giving rise to spontaneous blistering many months after grafting, increased susceptibility to infection, and contractures. Around the edges of a graft that has taken well, there is a zone of disturbed wound healing, and a ‘bricklaying’ pattern of scar formation has been noted. Histologically, the fragility of grafted cultured epithelial autograft sheets may be related to the immaturity of the dermo-epidermal junction, giving rise inadequate anchoring. At the time of transplant, the keratinocytes are undifferentiated and lack cornified and granular layers. By 6 days they have differentiated into all the normal strata, but lack rete ridges. At 3 weeks there is a basal lamina with hemidesmosomes. Anchoring fibrils (collagen VII) are sparse and immature for the first 6–12 months, although at this stage rete ridges are pre-sent and the vascular organisation is normal. Elastin expression may take 4–5 years.

Many groups are working on the development of alternative systems for the delivery of cultured autologous keratinocytes, in the hope that this may reduce the costs and improve the take and quality of the resulting epidermis. Many of these developments have not been fully proven in a clinical setting, and controlled trials with defined end points are lacking. Currently, it is doubtful exactly how great a contribution alternative delivery systems make to wound closure. A demonstration that cultured allogenic keratinocytes achieve significantly worse results than cultured autologous cells would be significant. In pigs the incorporation of cultured autologous cells into wound-derived re-epithelialisation has been demonstrated using retroviral gene transfer.

Fibrin-glue suspension. Some success has been achieved by applying cells together with fibrin glue, in a suspension of growth medium or using a membrane for delivery. Keratinocytes have been fixed to wounds by fibrin, and complete healing was achieved within 14–21 days. Subsequently, good take rates were achieved using a fibrin-glue suspension on a pre-grafted dermal bed in a rat model, and beneath an allograft overlay in a clinical model. A stable skin was formed with good mechanical qualities. This is an easier technique, which allows grafting at an earlier stage, when the cells are in an actively proliferative state.

Fibrin-glue sheets. Subconfluent cultured keratinocytes have been grown on fibrin glue, and then transferred as a sheet onto the wounds in three patients with excised full-thickness burns. The fibrin was found to provide a satis-factory barrier for 10 days, while the keratinocytes grew to confluence. Cultured autologous keratinocytes have also been applied to a wound surface in an autologous fibrin spray.

Upside-down membrane delivery systems. Several membranes have been used to deliver keratinocytes to the wound in an upside-down manner. One of the most innovative is Laserskin (Fidia Advanced Biopolymers, Aban Terme, Italy), a membrane delivery system created from a laser-perforated derivative of esterified hyaluronic acid. (The same material has also been marketed as Vivoderm (ER Squibb and Sons Inc, Princeton, NJ, USA).) Keratinocytes are seeded in vitro onto the mem-brane, and populate the laser-drilled pores. The cell colonies then grow above and below the membrane, which can be peeled off the Petri dish without enzymatic digestion. It is proposed that there is a potential advan-tage in this membrane not requiring inversion and subse-quent cell reorientation for application to the wound bed, and that the presence of hyaluronic acid may promote cell migration, proliferation and angiogenesis. It has been used for the treatment of vitiligo, as well as to resurface Integra.

Sprayed cell suspensions. Sprayed cultured keratinocytes have been applied to wounds with autologous split-skin grafts meshed 3 : 1 in pigs. In these cases, the cells have been suspended in their growth medium and sprayed directly onto the wound without the use of fibrin. The wound is reported to heal faster and to be of superior quality where cells were sprayed. There is, as yet, no analogous clinical controlled trial to substantiate this view in patients.