a guide to biological skin substitutes

British Journal of Plastic Surgery (2002), 55, 185-193 �9 2002 The British Association of Plastic Surgeons doi: 10.1054/hips.2002.3800

B R I T I S H J O U R N A L P L A S T I C S U R G E R Y

REVIEW

A guide to biological skin substitutes

I. Jones, L. Currie and R. Martin

Blond Mclndoe Centre, Queen Victoria Hospital, East Grinstead, West Sussex, UK

SUMMARY. The role of artificial skin substitutes in burn surgery and the treatment of chronic wounds is constantly evolving. New products are regularly being produced and approved for clinical use. Studies on existing products clarify their efficacy and effectiveness in different clinical scenarios. This review is aimed at busy clinicians in order to bring them up to date with the latest developments in the field of artificial skin substitutes. It examines the components, structure, performance and comparative costs of the main commercial skin substitutes, and reviews briefly technologies under development that have not yet become widely available. �9 2002 The British Association of Plastic Surgeons

Keywords: skin replacements, skin substitutes, bums, wounds.

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 bums, 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 mobilisa- tion, there remains a belief that a suitable replacement skin material could not only substantially improve the chances of bum 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 mor- tality 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 article reviews 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 gener- ated a measure of confusion amongst potential users of these products. Here, we examine the range of different products that are available, the current evidence for their effectiveness and their relative costs.

The function of normal skin

Anatomically and functionally, skin has two layers. The superficial epidermal layer provides a barrier against

infection and moisture loss. The deeper dermal layer is responsible for the elasticity and mechanical integrity of the skin, and contains the blood vessels that are responsible for the nutrition of the epidermal layer. Appendages, such as hair follicles or sweat glands, breach the epidermal and dermal layers. Cutaneous sensory nerves pass through the dermal tissue into the epidermal tissue. Regeneration of the epidermis relies on residues of epidermal cells that lie deep within dermal structures; ingrowth from the edges of a wound will be insufficient when the wound is more than a few cm across.

Wound closure requires a material to restore the epidermal barrier function and become incorporated into the healing wound, 1 whereas materials used for wound cover rely on the ingrowth of granulation tissue for adhe- sion. Materials for wound cover are most suited to superficial bums, where they create an improved environment for epidermal regeneration by providing a barrier against infection and controlling water losses. 2 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 infec- tive 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. 3'4 However, skin substitutes that have been manufactured

185

186 British Journal of Plastic Surgery

from intact skin are considered. Permanent wound closure relies on a subsequent epidermal graft to the inte- grated 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 covered in this review 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. In this review, the presence of any biological component qualifies the material for inclusion. Totally non-biological commercially available dressings, such as Opsite (Smith & Nephew Inc), Duoderm (ConvaTech Inc) and many more, are discussed in detail elsewhere. 5 These products are generally occlusive, providing a moist environment con- ducive to faster healing, although potentially allowing for increased proliferation of microorganisms beneath their surfaces.

Skin substitutes for wound cover

Biological wound cover encompasses both biosynthetic materials and unprocessed skin products, such as xenograft and human cadaveric and living allografts, but these whole-skin materials are not the subjects of this review. 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 unre- lated 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.

Biobrane (Dow Hickam/Bertek Pharmaceuticals, Sugar Land, TX, USA)

Biobrane is a bilaminate membrane consisting of nylon mesh fabric bonded to a thin layer of silicone. 6 The nylon mesh is coated with peptides derived from porcine type I collagen, in order to aid adherence to the wound bed and fibrovascular ingrowth. The silicone is semi-permeable. As the wound heals, Biobrane separates, and can readily be peeled away from the surface. It is recommended for use on donor sites and superficial partial-thickness burns within the first 6h of injury, as it is best reserved for 'clean' wounds. When used in this manner on burns expected to heal within 10-14 days, the length of inpa- tient treatment can be reduced by 46%. 7 Biobrane has been used as temporary cover for freshly excised full-thickness wounds. Adherence, fluid collection and subsequent autograft take are similar to results obtained with cryopreserved allograft, s providing that bacterial counts are less than 105 per gram tissue.

Transcyte -formerly Dermagraft- TC (Advanced Tissue Sciences Inc, La Jolla, CA, USA)

In Transcyte, the collagen-coated nylon mesh in Biobrane is seeded with neonatal fibroblasts in order to improve its

healing properties. As nylon is not biodegradable, this material cannot act as a dermal substitute. Formerly, this fibroblast-seeded Biobrane was known as Dermagraft- TC, where TC stood for temporary cover. The only com- fort in this confusion is that TC can now stand for Transcyte. The neonatal fibroblasts within the Transcyte inner biological layer are allowed to grow for 17 days, during which they produce fibronectin, type I collagen, proteoglycan and growth factors. The material is frozen to - 7 0 ~ for storage, a process that does not maintain the metabolic activity of the fibroblasts. The material is then stored at --20 ~ in specially designed cartridges.

Hansbrough et al performed a clinical trial comparing the use of cryopreserved allograft with Transcyte as a temporary dermal analogue in 10 patients. 9 The wounds were closed using meshed split-skin autograft. The results showed that autograft adherence and take were at least as good with Transcyte as with allograft. In a larger multicentre trial with 66 patients, it was noted that, as well as showing good adherence, Transcyte was easier to remove than allograft, resulting in less bleeding, l~ Histologically, a comparison of Transcyte with allograft in bum wounds showed the only significant difference to be increased granulation tissue in the allograft-treated wounds. 11 Paediatric burns of greater than 7% total body surface area that underwent wound closure using Transcyte have been shown to subsequently require a lower percentage of split-skin autograft than wounds treated using a standard therapy consisting of application of antimicrobial ointments and hydrodebridement, t2

The role for Transcyte is currently evolving to target partial-thickness bums; it adheres rapidly to a viable wound surface, stimulating epithelialisation. 13 In a recent study, a prospective randomised comparison of silver sulfadiazine and Transcyte was performed using paired wound sites on 14 patients. TM The results indicated that wounds treated with Transcyte healed with less hypertrophic scarring than wounds treated with silver sulfadiazine. Transcyte has also been shown to improve significantly the management and healing rate of partial- thickness facial bums, compared with the standard open topical-ointment technique. 15 Given that Transcyte costs 16 times more than ordinary Biobrane (Fig. 1) and requires frozen storage, a clinical trial of Transcyte versus Biobrane would seem highly desirable, but this has not yet been reported.

Cultured allogeneic keratinocytes

In an attempt to overcome the problem posed by the time delay in growing confluent autologous keratinocytes for wound closure, the use of pre-grown allogeneic keratinocytes has been examined. After 7 days of culture, keratinocytes fail to express MHC class II antigens. They fail to simulate allogeneic lymphocytes, although MHC class II can still be induced by cytokines such as ~/-interferon. 16,17 Expression of HLA class I antigens is also attenuated by serial cultivation and cryopreservation.18

In the clinical situation, there is no acute rejection reaction following the application of allogeneic sheets of keratinocytes; however, Y-chromosome and DNA probes have shown that the allogeneic cells survive less than

A guide to biological skin subst i tu tes 187

Trade Name Cost

Biobrsne' " ' (Dow Hickam/Bertek Pharmaceuticals, Sugar Land, TX).

Trauscyta | (Advanced Tissue Sciences, Inc. La Jolla, California, USA)

Apligraf ~ (Organogenesis Inc, Canton, MA and Novartis Pharmaceuticals Corporation, East Hanover, N J)

Dermagraft | (Advanced T;~sue Sciences, Inc. La Jolla, Califomia, USA)

Intogra | (Integra Life Science Corporation, Plainsboro, New Jersey)

AIIoderm | (LifeCell,Woodlands, Texas.)

Epicel TM

(Genzyme tissue repair corporation, Cambridge, MA.)

Laserskin" (Fidia Advanced Biopolymers, Italy) also marketed as Vivoderm TM by ER Squibb & sons Inc,

Cadaveric allograft (from not for profit skin banks)

Schematic Representation

�9 ~x~a~ ~ , ~ : ~ ~ < ~ e ~ i ~ ~"

Layers

1. Silicone

2. Nylon Mesh

3. Collagen

1, Silicone

2. Nylon Mesh

3. Collagen seeded with neonatal flbroblsets

1. Neonatal keratinocyl~s

2. Collagen seeded with neonatal fibroblasts

1. Polyglycolic acid (Dexon") or polygla_c_tin-910 (Vicryi TM) seeded with neonatal flbrobiasta

1. Silicone

2. Collagen and glycosaminoglycan

1. AceUular de- epithelialised cadaver dermis

1. Cultured autologous keratinocytes

1. Cultured autologous keraflnocytas

2. Hyaluronic acid with laser perforations

�9 cryopreserved in order to retain viability

�9 lyophilised �9 glycerolised

5xScm

s

13x9cm

s

7.5 cm diam disc

s

5xT.Scm

s

10x25cm

s

4x12cm

s

Not yet licensed in UK

Not yet licensed in UK

similar price for all three

various sizes

Cost p e r C m2

s

s

s

s

s

s

s

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 at the time of writing; firstly in a standard supplied size, and secondly as the cost in UK pounds per cm :. Costs should be treated as a guide, and have been converted into UK currency where necessary.


1 week when grafted onto tattoo-excision wounds or ulcers. 19,2~ This increases to over 6 weeks when they are applied to a split-skin-graft donor site. 21-23 Donor keratinocytes would appear to be replaced progressively rather than en masse. 24 Survival appears to be related to the wound bed, with the presence of dermis prolonging survival. The enhanced healing noted following the application of allogeneic keratinocytes is attributed to the secretion of growth factors and cytokines by the cells. 25 Cultured allogeneic keratinocytes are, therefore, regarded as materials for wound cover, since they will not in them- selves achieve wound closure. Nevertheless, there has been evidence of some allogeneic keratinocyte survival up to 30 months after application in patients undergoing tattoo excision, with predominant rapid replacement by host cell influx. This survival is assisted by the absence of detectable Langerhans cells, lymphocytes, melanocytes and endothelial cells (which all express HLA class II antigens) at the time of transplantation. 26 The main use for allogeneic keratinocytes remains as a dressing in chronic open wounds, such as leg ulcersY or to speed the healing of donor sites. 2s

Experimental studies have been performed to assess the value of chimeric grafts, which contain a combination of allogeneic and autologous keratinocytes, and take rates of greater than 80% have been achieved in immunocom- petent animal models. Cell-staining techniques show that by 30 days only autologous ceils remain. 29

Apligraf ( Graftskin) - living skin equivalent, human skin equivalent ( Organogenesis Ine, Canton, MA, USA and Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA)

Apligraf is derived by combining a gel of type I bovine collagen with living neonatal allogeneic fibroblasts with an overlying cornified epidermal layer of neonatal allogeneic keratinocytes. Consisting of two different cell types, this 'composite' is currently the most sophisticated commercially available tissue-engineered product, and also the most expensive (Fig. 1). Much of the above discussion on the survival of cultured allogeneic keratinocytes can be assumed for Apligraf. Apligraf (also known as Graftskin) is available in a ready-to-use form with a 5 day shelf life. Its primary role is as a treatment for chronic ulcers. It appears to hasten healing, particularly in deeper and more chronic wounds. A prospective randomised study showed significantly more patients achieved complete wound closure in the Apligraf-treated group than in the control group) ~ There were no clinical signs of rejection, although evidence of some healing by secondary intention was observed in over half of the group. These findings were confirmed by Sabolinski et a131 and Eaglstein et al.32 The efficacy of Apligraf in healing venous ulcers was evaluated in a prospective randomised study of 240 patients at 14 centres over a 6 month period. 33 Apligraf was three times more effective than compression therapy alone in achieving complete wound closure at 8 weeks. There are no reports, as yet, on how it compares clinically to cultured epidermal autografts, allogeneic keratinocytes or Dermagraft. The potential of Apligraf to improve cosmetic and functional outcomes when applied over meshed split-thickness

autografts was evaluated in a multicentre randomised within patient controlled clinical trial. 34 There was no difference in the percentage take of the autograft in the presence or absence of Apligraf, but the pigmentation, pliability and vascularity of the skin graft in the Apligraf- treated group were significantly better than in the control group.

Dermagraft (Advanced Tissue Sciences Inc, La Jolla, CA, USA)

Dermagraft is a cryopreserved living dermal structure, manufactured by cultivating neonatal allogeneic fibroblasts on a polymer scaffold (polyglycolic acid or polyglactin-910, marketed as Dexon or Vicryl, respec- tively). 35 The fibroblasts become confluent within the polymer mesh, secreting growth factors and dermal matrix proteins (collagens, tenascin, vitronectin and glycosaminoglycans), thus creating a living dermal structure. 36 This remains viable and metabolically active after implantation into the wound, despite cryopreservation. 37 The aim was to develop a living human material that can be permanently implanted to replace the patient's dam- aged dermis, although it is unclear exactly to what extent this aim has been fulfilled in clinical use. Again, much of the discussion above relating to the survival of allogeneic cells applies to Dermagraft, although it is believed that fibroblasts have a greater longevity in an immunocompe- tent host than epithelial cells, the donor fibroblasts, but not their newly produced matrix, will eventually be lost. Hence, Dermagraft facilitates healing by stimulating the ingrowth of fibrovascular tissue from the wound bed and re-epithelialisation from the wound edges. It does not close the wound and, as such, is marketed for stimulating the healing of chronic lesions, such as diabetic foot ulcers, rather than for closing burn wounds. 38'39 Although Dermagraft has not been used extensively for bums, it has been used beneath meshed split-skin grafts on full- thickness wounds. 4~ This could represent a useful means of replacing lost dermal tissue where thin split-skin graft would be desirable. The take rate of the split-skin graft was comparable to when it was grafted onto the wound bed alone. Wound histology showed extrusion of vicryl fibres from the wound, although this was not clinically apparent. Further studies will be needed to determine any long-term benefits following the application of Dermagraft under meshed split-skin grafts.

Skin substitutes for wound closure

Alloderm (LifeCell, Woodlands, TX, USA)

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 cryopreservation in order to avoid a specific immune response. 41 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. 1). Following application to a wound bed, it is repopulated by host

A guide to biological skin substitutes 189

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. In a multicentre study of 67 patients with full-thickness or deep partial-thickness bums, the wounds were excised and closed with either AlloDerm plus sprit- skin graft or split-skin graft alone. 41 In the later cases, the AlloDerm was meshed but not expanded, with the idea that cellular elements of the epidermis could be nour- ished by serum through the interstices. Wound assess- ment over time showed that thin split-thickness autografts with AlloDerm were equivalent to thicker split-thickness autografts. Lattari et al report three cases of AlloDerm used in conjunction with ultra-thin meshed split-skin grafts (0.2-0.15 mm grafts, meshed 1.5: 1) over the dorsum of three hands and one ankle. 42 They report an early recovery with no residual functional deficit. This approach is similar to the studies described above using Dermagraft. 4~

Integra (Integra Life Science Corporation, Plainsboro, N J, USA)

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. 43'44 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 Ixm 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 first clinical study of 10 patients highlighted problems with haematoma and seroma formation and identification, and premature silicone separation. 45 This was followed by a multicentre trial of 149 cases in 106 patients. 46 The median 'take' was 85%, compared with take rates of 95% in the controls treated with split-skin grafts. These results were comparable to using allograft. The trial also reported a subjective improvement in the cosmetic result, particularly of the donor sites, because the grafts were harvested at 0.15 mm (the average split- thickness skin graft is 0.33 mm thick) and the donor sites generally healed 4 days sooner with less hypertrophic scarring.

The longest reported Integra series spans 10 years, and successful engraftment rates were generally o v e r 80~ 47

There was no severe hypertrophic-scar formation, and 93% of patients had absent or minimal hypertrophic scarring. All patients achieved excellent function of involved joints. Children demonstrated that the areas grafted with the artificial skin were able to grow with the child. 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. Stem et al report no evidence of an immune reaction or scarring. 4s

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 autografts, 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, Orgill et al showed successful take of cultured epithelial autografts onto a pre-grafted Integra-like material in a porcine model. 49 The wounds showed nearly complete confluence of the cultured epidermal autografts at 7 days, whereas in the controls, when cultured epidermal autografts were applied 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. 5~

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 inher- ent with allografts. It is available immediately, and does not necessitate a narrow window of time in which to per- form 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 51 and antibiotics. 52 Cultured autologous keratinocytes have also been shown to produce a surface epithelium when seeded as a suspension into Integra and grafted onto athymic mice. 53 The seeded material exhibited good wound adherence, complete healing and minor wound contraction, and had the potential to recon- stitute an elastic functional and durable human skin. It remains to be seen whether this technique will work in patients.

Cultured autologous keratinocytes

The clonal growth of keratinocytes has been possible for over 20 years. 54'55 Confluent sheets of keratinocytes can


be grown in vitro and then applied to wounds such as burns, 56,57 chronic leg ulcers, 27 giant pigmented naevi, 58 epidermolysis bullosa 59 and neonatal scalp necrosis. 6~ 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. 28 Pre-grafting the wound with allograft 61 will encourage take, as will the presence of a non-granulated dermal bed of allogenic 62'63 or autologous dermis. 64-66

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 labora- tories. However, they are expensive as they require a high input of skilled labour and quality control, and it takes 3-5 weeks to produce 1.8m 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 coordi- nation 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%. 28

Cultured epithelial autografts are fragile sheets. They require separation from the tissue culture substrate using a proteolytic enzyme before they are applied to the wound bed. This process causes contraction and subsequent restricted proliferation. 67 The resulting epithelium is unstable, giving rise to spontaneous blistering many months after grafting, 68 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. 67 Histologically, the fragility of grafted cultured epithelial autograft sheets may be related to the immaturity of the dermo-epidermal junction, giving rise to inadequate anchoring. 69 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 present and the vascular organisation is normal. Elastin expression may take 4-5 years. 66'v~

Keratinocyte delivery systems

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. 71

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. 72 Subsequently, good take rates were achieved using a fibrin-glue suspension on a pre-grafted dermal bed in a rat model, 67 and beneath an allograft overlay in a clinical model. 73 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 bums. The fibrin was found to provide a satis- factory barrier for 10 days, while the keratinocytes grew to confluence. 74'75 Cultured autologous keratinocytes have also been applied to a wound surface in an autologous fibrin spray. 76

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 inno- vative is Laserskin (Fidia Advanced Biopolymers, Aban Terme, Italy), a membrane delivery system created from a laser-perforated derivative of esterified hyaluronic acid. 77 (The same material has also been marketed as Vivoderm (ER Squibb and Sons Inc, Princeton, NJ, USA).) Keratinocytes are seeded in vitro onto the membrane, 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 subsequent 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, 78 as well as to resurface Integra. 79

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. 8~ 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.

Composite epidermal-dermal skin substitutes for wound closure

The results of the alternative delivery systems are encour- aging, but where cultured keratinocytes are used alone, the quality of healing is poor. Efficacy is improved by


combining keratinocytes with some form of dermal matrix, but the fundamental truth is that detached sheets or subconfluent cells take time to re-establish a new basement membrane. It is this structure that is the key to a robust and adherent epidermal barrier. The obvious solu- tion is to allow the epithelium to form a basement membrane during the culture process and to keep this intact during cultured epidermal autograft delivery. This can be achieved by growing the cells on some kind of biomaterial. If the epidermal layer can receive adequate nutrition and the biomaterial can bond to the wound, then the vital epidermal barrier can be replaced. This approach is often referred to as a 'composite' in skin-replacement technol- ogy. Synthesising a skin composite with dermal and epidermal components may not only improve techniques for keratinocyte transfer, but also influence epidermal attachment, growth and differentiation. Collagen VII, the major structural protein of anchoring fibrils, is markedly increased when keratinocytes and fibroblasts are cultured together, st Composites developed to date encompass sim- ple collagen gels (similar in form to Apligraf covered above), processed dermis and collagen-glycosaminoglycan-based composites.

Allograft- and xenograft-based composites. Ghosh et al compared methodologies for the preparation of composites based on allogeneic dermis, investigating the sterilisation, de-epithelialisation and introduction of fibroblasts and keratinocytes to the composite, with the aim of producing a sterile dermis rendered acellular with the intact basement membrane necessary for good epidermal adhe- sion. 82 A standardised glycerolisation technique gave the optimum sterilisation results while maintaining a pliable dermis with good preservation of the normal histological pattern. Irradiation rendered the dermis rigid and histologically abnormal, and ethylene-oxide treatment had unpredictable results. If ethylene oxide had given consis- tent results it would have been preferable, because it is a time-efficient standard sterilisation procedure. De- epidermisation with 1 M sodium chloride preserved the basement membrane better than Dispase and was quicker than using phosphate-buffered saline. Fibroblast entry into the dermis appeared to occur only on the reticular dermis, unless co-seeded with keratinocytes. The authors of this study recommend a composite formed from standardised glycerolised or improved ethylene-oxide treated allograft, pre-seeded with fibroblasts, then seeded with keratinocytes. Preliminary promising reports of an ethylene- oxide sterilised composite have been presented. 83 Currently, this is only available in sizes of less than 6 cm in diameter, while further work into producing a large- scale composite is underway.

Collagen-glycosaminoglycan matrix derived composite. In vitro studies placing sub-confluent human keratinocytes on the surface of a collagen-glycosaminoglycan matrix, similar to that used in Integra, showed attachment and growth to confluence, s4 Subsequent mod- ifications of the collagen-glycosaminoglycan membrane to contain autologous fibrobtasts have allowed the growth of established stratified layers of cultured autologous keratinocytes on the modified surface. Clinical use of this

material on excised full-thickness skin wounds showed acceptable take, with basement membrane formation within 9 days. s5 Further work by this group using keratinocytes together with fibroblasts has shown that inclusion of these two cell types results in improved basement membrane formation, increased production of laminin and type IV collagen, and thicker epithelial layers in vitro. 37 Cultured skin substitutes consisting of human keratinocytes and fibroblasts attached to collagen- glycosaminoglycan substrates have now been tested in athymic mice, and show rapid wound closure, s6 More recently, in burn patients, cultured skin substitutes have been successfully applied to vascularised Integra for the closure of excised full-thickness wounds, s7

Conclusions

In summary, the current state of play in the development of skin substitutes is as follows. Products formed from allogeneic cultured cells are commercially available in combination with biomaterials as treatments to provide wound cover and stimulate wound closure. Autologous materials that might provide immediate wound closure have not reached the same level of sophistication, and are not yet commercially available. As clinicians, our aim is to provide wounded patients with the best quality of skin in the shortest possible time. As scientists, we have not yet achieved the ultimate goal of a universal 'off the shelf' skin product. At present, this elusive target is some way off, but, by constantly reviewing the current options and using them appropriately, we can at least fulfil our role as clinicians in the knowledge that we are using the correct material in the correct situation. Limited resources have made the need for comparisons of clinical effectiveness and cost ever more important. In the future, skin-substitute materials may be able to stimulate regeneration rather than repair, and tissue-engineered skin may match the quality of split-skin autografts, our present gold standard.

Acknowledgements

This work was funded by the Blond Mclndoe Centre for Medical Research (registered charity no. 258154) in conjunction with the Royal College of Surgeons of England and the John Byrne Fellowship (I.J.), and the Mark Hanna Research Fellowship (L.C.). We thank Mr Ian Grant for his advice on an early draft of this manuscript. I.J. and L.C. contributed equally to this review.

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T h e A u t h o r s

Isabel Jones MBBS, FRCS Lachlan Currie MBBS, BSc, FRCS Robin Martin BSc, PhD

Blond Mclndoe Centre, Queen Victoria Hospital, Holtye Road, East Grinstead, West Sussex RH 19 3DZ, UK.

Correspondence to Dr Robin Martin.

Paper received 2 November 2001. Accepted 7 January 2002.

a guide to biological skin substitutes

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