- Email: [email protected]
USE OF SKIN SUBSTITUTES IN DERMATOLOGY
Current Therapy
0733-8635/01 $15.00
+ .OO
Bruce H. Thiers, MD, Consulting Editor
USE OF SKIN SUBSTITUTES IN DERMATOLOGY Ysabel M. Bello, MD, and Anna F. Falabella, MD
The skin is the largest organ of the body, and its integrity is vital to survival. Significant skin loss, as seen in some thermal injuries, is associated with a high morbidity and mortality because of loss of fluids, proteins, and electrolytes, as well as overwhelming infections. Less significant skin loss, as seen with surgical skin excision (acute wounds) or leg ulcers (chronic wounds), may also result in significant morbidity and can negatively affect quality of life. A variety of skin substitutes are currently available for clinical use, and newer ones are being studied. Unlike autologous skin grafts, skin substitutes do not require the creation of a donor site, which can be painful and slow healing. Skin substitutes can act as a scaffold for tissue regeneration in vivo, or as tissue replacement, providing matrix material and cells when grown in c~1ture.l~ Skin substitutes can be categorized into three groups based on their structure: epidermal grafts, dermal replacements, or composite grafts with epidermal and dermal compon e n t ~ . They '~ can also be classified by other characteristics, including temporary vs. permanent; synthetic/biosynthetic vs. biological; autogeneic vs. allogeneiclxenogeneic; and acellular matrix vs. matrix with cells (Table 1).
EPIDERMAL GRAFTS
In 1975, Rheinwald and Green developed a method that made it possible to cultivate human keratinocytes serially in vitro. This technique permitted significant expansion of cultured keratinocytes, so that a l-2-cm' skin biopsy could be used to produce up to 1 m2 of keratinocyte cultured grafts in about 3 weeks.17 Cultured keratinocyte or epidermal autografts have been successfully used in the treatment of burns, chronic leg ulcers, scar revision, vitiligo, giant congenital nevi, chronic mastoiditis, and congenital hypospadias, and for corneal replacement. A study of 19 burn patients with an average of 71.9% of total body surface involved were treated with 31 treatments of cultured epithelial autografts and reported good adherence in patients with burns involving <50% of the total body surface.l8 After skin biopsy from the donor site, 2 to 3 weeks are required before the graft is available. The utility in using an epidermal autograft for a specific patient is therefore determined by the rapidity needed to treat a wound. The resultant product contains sheets of proliferative cultured autologous keratinocytes, two to eight cell layers thick, attached
From the Department of Dermatology and Cutaneous Surgery, University of Miami School of Medicine, Miami, Florida DERMATOLOGIC CLINICS VOLUME 19 NUMBER 3 JULY 2001
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Table 1. CATEGORIZATION OF SKIN SUBSTITUTES Skin Substitute
Cultured epidermal autograft Epicel (Genzyme Tissue Repair Co., Cambridge, MA) Vivoderm (ER Squibb & Sons Inc., Princeton, NT) Cultured epidermal allograft Biobrane (Winthrop Pharmaceuticals, New York, NY) E-ZDerm (Brennen Medical Inc., St Paul, MN) Oasis Wound Dressing (Cook Wound/Ostomy/Continence, Spencer, IN) Integra (Integra Life Sciences Co., Plainsboro, NJ) Alloderm (Life Cell Co., Woodlands, TX) Transcycle (Advanced Tissue Sciences Inc., La Joya, CA) Dermagraft (Advanced Tissue Sciences Inc., La Joya, CA) Human skin equivalent Apligraf (Organogenesis, Canton, MA/Novartis, East Hanover, NJ) Composite cultured skin (Ortec International Inc., New York, NY)
Characteristics
Permanent, biological, autogeneic cultured epidermal cells
Temporary, biological, allogeneic cultured epidermal cells Temporary, biosynthetic, xenogeneic, dermal matrix Temporary, biosynthetic, xenogeneic, acellular dermal matrix; shelf life up to 18 months Temporary, biological, xenogeneic, acellular dermal matrix; shelf life of 1-2 years Temporary, biosynthetic, xenogeneic, acellular dermal matrix Temporary, biological, allogeneic, acellular dermal matrix with intact basement membrane; cryopreserved, shelf life 2 years Temporary biosynthetic, allogeneic, nonviable cellular dermal matrix Temporary, biosynthetic, allogeneic, cellular dermal matrix Permanent, biological, allogeneic, cultured epidermal cells and cellular dermal matrix; shelf life 5 days Permanent, biological, allogeneic, cultured epidermal cells and cellular dermal matrix
to a petrolatum gauze backing. Each cultured epidermal autograft is approximately 30 cm2 in size. Once the graft is in place, the epidermal cells begin the process of attachment, replication, and differentiation. This is considered a permanent graft, requiring approximately 6 days to “take.” The dermoepidermal junction is reformed in 3 to 4 weeks, rete ridges regenerate 6 weeks to 1 year after grafting, and full maturation of anchoring fibrils takes more than 1 year.3 Epidermal autografts need to be stapled or sutured to prevent separation from the wound bed. A mesh gauze is placed over the graft and should be left undisturbed for 7 to 10 days. An outer dressing needs to be changed at least once per day depending on the amount of exudate produced. Adverse reactions from epidermal autografts have been reported. Cultured epidermal autografts are cultured in medium containing streptomycin/penicillin and/or gentamicin, and trace amounts of these antibiotics may adhere to the grafts. Increased nephrotoxicity has been reported in some patients, especially those with impaired renal function or those already receiving aminoglycosides and cephalosporins. Blisters some-
times occur and generally resolve spontane0us1y.~Grafted areas sometimes exhibit a contracted or scarred appearance, although it tends to be less than that caused by meshed grafts. At present, there are several cultured epidermal autografts commercially available, including Epicel (approved for use in the United States) and Vivoderm (available only in the United Kingdom, Germany, and Italy). Vivoderm is grown on a hyaluronic membrane. Laser-drilled micro-perforations allow keratinocytes to migrate through the membrane into the wound bed. It has been used for the treatment of burns and chronic wounds? For cultured epidermal allografts, the skin is obtained from allogeneic tissue, such as newborn foreskin, and can be grown in advance. Avoiding creation of a donor wound site, and ready availability, provide an advantage compared to a~t0grafts.l~ Cultured epidermal allografts have been used to treat donor sites, partial thickness burns, chronic leg ulcers, and epidermolysis bullosa. Cultured allografts do not survive permanently on the wound bedrg but they provide pain relief within hours after grafting and act as a bio-
USE OF SKIN SUBSTITUTES IN DERMATOLOGY
logical dressing, stimulating epithelialization from the wound edges and adnexa, probably through release of growth factor^.'^ Cultured epidermal allografts can be cryopreserved and stored in a - 70°C freezer and thawed at room temperature.' They are not commercially available in the United States, and further randomized placebo-controlled studies are needed to determine their effectiveness. DERMAL REPLACEMENTS
The dermis plays an important role in the healing process. The cellular component (fibroblasts) and the extracellular component (collagen, extracellular matrix proteins) influence epithelial migration and differentiation, dermoepidermal junction formation, wound contraction, and scar formation. Dermal cells also secrete growth factors and cytokines, which modulate cell proliferation and differentiation. Collagen synthesis by fibroblasts forms a scaffold for cellular migration. Biosynthetic dressings were developed to provide temporary coverage to optimize wound healing. They were first introduced in 1979 for the treatment of burns and donor sites. One product, Biobrane, is composed of a bilaminate biosynthetic synthetic material,
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constructed of silicone film with a nylon fabric partially embedded into the film. Type I porcine collagen peptides are present in both the silicone and nylon components as the biological component. Another dermal substitute, E-Z Derm (St. Paul, Minn), is a porcine-derived, biosynthetic, acellular dermal matrix, in which the porcine collagen has been chemically crosslinked using an aldehyde. It is available as a perforated or nonperforated dressing, attached to a gauze liner, which is discarded before grafting. E-Z Derm is indicated for patients requiring temporary coverage of partialthickness skin loss injuries, including burns and ulcers. Several advantages to the use of E-Z Derm are its long shelf life (18 months), immediate availability for serial grafting until autografts are available, and absence of human communicable disease (Fig. 1). Disadvantages include little clinical data to support its efficacy and safety, increase in the amount of exudates, and the possibility of disease transmission (xenograft). Oasis, a wound dressing derived from porcine small intestinal submucosa that has been processed to remove the serosa, smooth muscle, and mucosa layers results in an acellular collagen, extracellular matrix, which serves as a reservoir for cytokines and cell adhesion molecules, providing a scaffold for tissue
Figure 1. A, Venous ulcers before application of temporary skin substitute (E-Z Derm). B, Application of a temporary skin substitute. C, Healed ulcers several months after treatment with dermal skin substitute and compression therapy.
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Figure 2. Hydrated porcine small intestinal submucosa (Oasis, Spencer, Ind) applied over a leg ulcer using cotton applicators.
growth (Fig. 2). It gradually degrades so that the healed site consists entirely of host tissue. A small single-center pilot study performed on 15 patients with foot and leg ulcers using lyophilized versus hydrated small intestinal submucosa reported complete healing in lyophilized group (7/8) in 10 weeks.1° Although promising, further randomized controlled studies are warranted to evaluate the efficacy of this wound dressing. Integra, a temporary, biosynthetic, bilaminate skin substitute, has been approved by the FDA for postexcisional treatment of lifethreatening full-thickness or deep partialthickness thermal injury, when sufficient autograft is not available at the time of excision or not desirable because of the physiological condition of the patient. Developed by Burke et a1,2 it is composed of synthetic polysiloxane (silicone) and a three-dimensionalporous matrix of cross-linked bovine tendon collagen and shark-derived glycosaminoglycan (chondroitin-6-sulfate). A controlled multicenter, short-term trial compared the safety and efficacy of Integra versus conventional wound covers (autografts or temporary wound covers such as allografts, xenografts, or synthetics) in 149 patients with matched-pair thermal injuries (207 total wound sites). Results showed that healing was prolonged for all materials used, except autografts. No significant difference
was observed between total burn healingtime for wounds treated with Integra versus those treated with temporary covers. There appear to be some advantages to using Integra. Healing of donor sites was 14.3 days in those biopsied for conventional autografts versus 10.6 days in the Integra group from which ultra-thin epidermal autografts were obtained. Thinner autografts allow more rapid healing of donor sites, reharvesting can be accomplished with greater rapidity, and less hypertrophic scarring of the donor site is seen. At the treatment site, the silicone layer is more easily removed than allografts, and there is no known risk of rejection. One disadvantage to using Integra is the FDA requirement for clinicians to complete a company-sponsored training program. The application procedure is more complicated, and there is greater susceptibility to infection than with autografts. Cadaveric allograft skin has been used in burned patients to achieve temporary wound closure.’ Chemical treatment of human cadaver skin removes antigenic components (cellular components), resulting in an acellular collagen dermal matrix with an intact basement membrane complex that is immunologically inert. It can be used alone or in combination with cultured autografts. A human cryo-preserved, acellular cadaveric deepidermalized dermis, AlloDerm, has been used to treat burns since 1992, and for periodontal, plastic, and reconstructive surgery since 1994. Donor samples are screened for evidence of viral or bacterial infection. They are also tested for fast-growing microbes and fungi. At 10 days post-grafting, dermal allograft demonstrates repopulation with host fibroblasts and endothelial cells. Keratinocytes migrate from meshed autograft onto the surface of the dermal allograft, resulting in uniform distribution of epithelial cells across the dermoepidermal junction. Advantages of cadaveric grafts include the need for thinner autografts, less pain and faster healing at donor sites, no inflammatory response or clinical rejection at the treated site, and a small possibility of human disease transmission. Another skin substitute, TransCyte (formerly called Dermagraft-TC) is a temporary, biosynthetic, bi-laminate skin substitute that
USE OF SKIN SUBSTITUTES IN DERMATOLOGY
provides a temporary covering to help protect wounds from fluid loss and to reduce the risk of infection. It typically requires only one application. It has been approved by the FDA as a temporary covering for second- and third-degree burns. TransCyte is composed of a silicone polymer membrane and foreskinderived neonatal human fibroblasts grown in a three-dimensional nylon mesh. Fibroblasts are rendered nonviable after syntllesizing collagen, growth factors, and other extracellular matrix materials. A prospective, randomized, match-controlled trial performed in 14 patient with partial thickness burns reported decreased time to 90% of epithelialization of the wounds, 11.14 days in the TransCyte treated group, versus 18.14 days in the silver sulfadiazine treated group.12 A prospective, multicenter, randomized, controlled trial in 66 patients with 132 excised burn wounds compared the application of TransCyte versus human cadaveric allograft and reported equivalent adherence and graft take at day 14.16Investigators also reported that TransCyte was easier to remove and resulted in less bleeding than allograft. Dermagraft is a biosynthetic dermal matrix with cells that multiply and secrete collagen and growth factors. It is currently available in Canada, the United Kingdom, and Europe. Its manufacturer is currently seeking FDA approval for the treatment of diabetic foot ulcers, pressure ulcers, and venous ulcers. Dermagraft is produced by taking dermal fibroblasts from neonatal foreskin and seeding them onto a three-dimensional scaffold consisting of a bioabsorbable material (polyglactin mesh). The cells grow and divide, producing collagens, extracellular matrix proteins, and growth factors found in normal, healthy human dermis. Mature matrix is cryopreserved to -70°C and thawed before patient application. This skin substitute stimulates the formation of granulation tissue, reepithelialization, and angiogenesis. The fibroblasts produce collagens, fibronectin, and glycosaminoglycans. They also produce transforming growth factor Beta, which stimulates collagen formation. Fibroblasts produce keratinocyte growth factor and secrete
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vascular endothelial growth factor and hepatocyte growth factor. A prospective, multicenter, randomized, single-blind, controlled trail performed in 235 patients with diabetic foot ulcers, which were treated with once-per-week applications for 8 weeks, reported no significant statistical difference in healing in the treatment group (38.5%) compared with the in the control group (31.7?40).~~ COMPOSITE GRAFTS
Apligraf, a human skin equivalent with appearance similar to human skin, was the first biomedical device containing living human cells approved for use by the FDA (May 1998). It is a permanent, biosynthetic, bi-layered living construct of cultured human neonatal foreskin keratinocytes and fibroblasts cultured on a dermal matrix consisting of bovine type I collagen. It is approved to treat venous ulcers of at least l-month duration that have not adequately responded to conventional therapy (Fig. 3). In June 2000, the FDA approved Apligraf for the treatment of neuropathic diabetic ulcers. There is no evidence of host antibody or cell-mediated response or clinical rejection because there are no macrophages, lymphocytes, or Langerhans’ cells present in this skin substitute. A prospective, randomized, multicenter study in 293 patients with venous ulcers demonstrated that human skin equivalent plus compression therapy was superior to compression alone (63% vs. @YO),and patients healed more rapidly (61 days vs. 181 days).6 However, this study found no significant benefit between the human skin equivalent group and the control group in ulcers of less than 6 months duration. Healing was significantly faster than controls in ulcers greater than 6 months. A diabetic foot ulcer study reported that use of Apligraf was associated with a significantly better percentage of wounds with complete healing than the control group (56% vs. 38%), and average time to wound closure was significantly better (65 days vs. 90 days).20 A prospective, single-center, uncontrolled study performed in 15 patients with epidermolysis bullosa assessed the efficacy of Apli-
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Figure 3. A, Venous ulcer before treatment with human skin equivalent (Apligraf, Canton, MA). 8,Healed ulcer following treatment.
graf in 69 acute wounds and nine chronic wounds.5This study reported that 79% of the wounds were healed a week after treatment. Patients and their families felt that healing was faster and less painful when the tissueengineered skin was used and that quality of life was improved compared with conventional dressings. Further studies are needed to confirm these results. A multicenter, single-arm study evaluated the safety and efficacy of a single application of human skin equivalent in 107 patients with partial or full-thickness surgical Cosmetic results in 50% of the patients were reported to be the same or better than would be expected with split thickness autografts. No adverse effects were seen. Another composite skin substitute is composite cultured skin, which uses allogeneic cells derived from neonatal foreskins." Fibroblasts and keratinocytes are grown in vitro and seeded on opposite sides of a bi-layered matrix of bovine collagen. To provide a surface for the keratinocytes to create an epidermal layer, the porous collagen sponge is laminated with a nonporous collagen gel. The fibroblasts seeded on the underside of the porous sponge migrate and distribute inside the sponge. The process is complete within 10 to 14 days. This year, the FDA approved
this product for use in patients with recessive dystrophic epidermolysis bullosa. Clinical trials in the treatment of burns, epidermolysis bullosa, diabetic ulcers, and venous ulcers using composite cultured skin are currently underway.
SUMMARY
The use of skin substitutes to treat acute and chronic wounds should continue to increase as newer products are developed. The goals for the future are to eliminate the need for autografting, eliminate the risk for transmission of disease, improve the shelf life and simplify storage requirements, improve cosmetic outcomes, and reduce cost. A number of researchers are also looking to incorporate gene therapy into skin substitutes.
References 1. Bolivar-Flores J PE, Marsch-Moreno M, Montes de Oca G, Kuri-Harcuch W Use of cultured human epidermal keratinocytes for allografting bums and conditions for temporary banking of the cultured allografts. B u m s 163-8, 1990 2. Burke J, Yannas I, Quinby W, et al: Successful use of physiologically acceptable artificial skin in the treat-
USE OF SKIN SUBSTITUTES IN DERMATOLOGY ment of extensive burn injury. Ann Surg 194413428,1981 3. Compton CC GJ, Bradford DA, et a1 Skin regenerated from cultured epithelial autografts on full thickness bum wounds from 6 days to 5 years after grafting. Lab Invest 60600-612, 1989 4. Eaglstein WH, Alvarez OM, Auletta M, et al: Acute excisional wounds treated with a tissue-engineered skin (Apligraf). Dermatol Surg 25:195, 1999 5. Falabella AF, Valencia I, Eaglstein WH, Schachner LA: Tissue-engineered skin (Apligraf a) in the healing of patients with epidermolysis bullosa wounds. Arch Dermatol 136:1225-1230, 2000 6. Falanga V, Margolis D, Alvarez 0, et al: Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Human Skin Equivalent Investigators Group [see comments] Arch Dermatol 134:293-300,1998 7. Greenfield E, Jordan B Advances in bum wound care. Crit Care Nurs Clin North Am 8203-215, 1996 8. Harris PA DF, Barisoni D, et a1 Use of hyaluronic acid and cultured autologous keratinocytes and fibroblasts in extensive bums. Lancet 353:35-36, 1999 9. Leigh IM PP: Cultured grafted leg ulcers. Clin Exp Dermatol 11:65M52, 1986 10. Marie Brown-Etris MP, Shields D Final report on a pilot study to evaluate porcine small intestinal submucosa as a covering for partial thickness wounds (Abstract). Symposium on Advanced Wound Care. 2000 April 1 to 4, 2000, Dallas, Texas 11. Morgan JR, Yarmush M L Bioengineered skin substitutes. Sci Med July/August, 6-15,1997
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12. Noordenbos J, Dore C, Hansbrough JF: Safety and efficacy of TransCyte for the treatment of partialthickness burns. J Bum Care Rehabil20:275-278,1999 13. Phillips TJ, Kehinde 0, Green H, Gilchrest BA: Treatment of skin ulcers with cultured epidermal allografts. J Am Acad Dermatol21:191-199, 1989 14. Phillips TJ: New skin for old: Developments in biological skin substitutes (editorial). Arch Dermatol 134344-349, 1998 15. Pollak R, Edington H, Jensen J, et al: A human dermal replacement for the treatment of diabetic foot ulcers. Wounds 9:175-183, 1997 16. Purdue GF, Hunt JL, Still JM Jr, et al: A multicenter clinical trial of a biosynthetic skin replacement, Dermagraft-TC, compared with cryopreserved human cadaver skin for temporary coverage of excised bum wounds. J Bum Care Rehabil 1852-57,1997 17. Rheinwald J, Green H Serial cultivation of strains of human epidermal keratinocytes: Formation of keratinizing colonies from single cells. Cell 6331-344, 1975 18. Rue LW 111, Cioffi WG, McManus WF, et al: Wound closure and outcome in extensively bumed patients treated with cultured autologous keratinocytes. J Trauma 34:662-667,1993 19. Sefton MV, Woodhouse KA: Tissue engineering. J Cutan Med Surg 3(Suppl 1):S1-1%23 20. Veves A, Falanga V, Armstrong DG, Sabolinski M L Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic diabetic foot ulcers. Diabetes Care 24290-295,2001
Address reprint requests to Anna F. Falabella, MD Department of Dermatology and Cutaneous Surgery University of Miami School of Medicine PO Box 016250 Miami, FL 33101 e-mail: afalabelQmed.miami.edu
0733-8635/01 $15.00
+ .OO
Bruce H. Thiers, MD, Consulting Editor
USE OF SKIN SUBSTITUTES IN DERMATOLOGY Ysabel M. Bello, MD, and Anna F. Falabella, MD
The skin is the largest organ of the body, and its integrity is vital to survival. Significant skin loss, as seen in some thermal injuries, is associated with a high morbidity and mortality because of loss of fluids, proteins, and electrolytes, as well as overwhelming infections. Less significant skin loss, as seen with surgical skin excision (acute wounds) or leg ulcers (chronic wounds), may also result in significant morbidity and can negatively affect quality of life. A variety of skin substitutes are currently available for clinical use, and newer ones are being studied. Unlike autologous skin grafts, skin substitutes do not require the creation of a donor site, which can be painful and slow healing. Skin substitutes can act as a scaffold for tissue regeneration in vivo, or as tissue replacement, providing matrix material and cells when grown in c~1ture.l~ Skin substitutes can be categorized into three groups based on their structure: epidermal grafts, dermal replacements, or composite grafts with epidermal and dermal compon e n t ~ . They '~ can also be classified by other characteristics, including temporary vs. permanent; synthetic/biosynthetic vs. biological; autogeneic vs. allogeneiclxenogeneic; and acellular matrix vs. matrix with cells (Table 1).
EPIDERMAL GRAFTS
In 1975, Rheinwald and Green developed a method that made it possible to cultivate human keratinocytes serially in vitro. This technique permitted significant expansion of cultured keratinocytes, so that a l-2-cm' skin biopsy could be used to produce up to 1 m2 of keratinocyte cultured grafts in about 3 weeks.17 Cultured keratinocyte or epidermal autografts have been successfully used in the treatment of burns, chronic leg ulcers, scar revision, vitiligo, giant congenital nevi, chronic mastoiditis, and congenital hypospadias, and for corneal replacement. A study of 19 burn patients with an average of 71.9% of total body surface involved were treated with 31 treatments of cultured epithelial autografts and reported good adherence in patients with burns involving <50% of the total body surface.l8 After skin biopsy from the donor site, 2 to 3 weeks are required before the graft is available. The utility in using an epidermal autograft for a specific patient is therefore determined by the rapidity needed to treat a wound. The resultant product contains sheets of proliferative cultured autologous keratinocytes, two to eight cell layers thick, attached
From the Department of Dermatology and Cutaneous Surgery, University of Miami School of Medicine, Miami, Florida DERMATOLOGIC CLINICS VOLUME 19 NUMBER 3 JULY 2001
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Table 1. CATEGORIZATION OF SKIN SUBSTITUTES Skin Substitute
Cultured epidermal autograft Epicel (Genzyme Tissue Repair Co., Cambridge, MA) Vivoderm (ER Squibb & Sons Inc., Princeton, NT) Cultured epidermal allograft Biobrane (Winthrop Pharmaceuticals, New York, NY) E-ZDerm (Brennen Medical Inc., St Paul, MN) Oasis Wound Dressing (Cook Wound/Ostomy/Continence, Spencer, IN) Integra (Integra Life Sciences Co., Plainsboro, NJ) Alloderm (Life Cell Co., Woodlands, TX) Transcycle (Advanced Tissue Sciences Inc., La Joya, CA) Dermagraft (Advanced Tissue Sciences Inc., La Joya, CA) Human skin equivalent Apligraf (Organogenesis, Canton, MA/Novartis, East Hanover, NJ) Composite cultured skin (Ortec International Inc., New York, NY)
Characteristics
Permanent, biological, autogeneic cultured epidermal cells
Temporary, biological, allogeneic cultured epidermal cells Temporary, biosynthetic, xenogeneic, dermal matrix Temporary, biosynthetic, xenogeneic, acellular dermal matrix; shelf life up to 18 months Temporary, biological, xenogeneic, acellular dermal matrix; shelf life of 1-2 years Temporary, biosynthetic, xenogeneic, acellular dermal matrix Temporary, biological, allogeneic, acellular dermal matrix with intact basement membrane; cryopreserved, shelf life 2 years Temporary biosynthetic, allogeneic, nonviable cellular dermal matrix Temporary, biosynthetic, allogeneic, cellular dermal matrix Permanent, biological, allogeneic, cultured epidermal cells and cellular dermal matrix; shelf life 5 days Permanent, biological, allogeneic, cultured epidermal cells and cellular dermal matrix
to a petrolatum gauze backing. Each cultured epidermal autograft is approximately 30 cm2 in size. Once the graft is in place, the epidermal cells begin the process of attachment, replication, and differentiation. This is considered a permanent graft, requiring approximately 6 days to “take.” The dermoepidermal junction is reformed in 3 to 4 weeks, rete ridges regenerate 6 weeks to 1 year after grafting, and full maturation of anchoring fibrils takes more than 1 year.3 Epidermal autografts need to be stapled or sutured to prevent separation from the wound bed. A mesh gauze is placed over the graft and should be left undisturbed for 7 to 10 days. An outer dressing needs to be changed at least once per day depending on the amount of exudate produced. Adverse reactions from epidermal autografts have been reported. Cultured epidermal autografts are cultured in medium containing streptomycin/penicillin and/or gentamicin, and trace amounts of these antibiotics may adhere to the grafts. Increased nephrotoxicity has been reported in some patients, especially those with impaired renal function or those already receiving aminoglycosides and cephalosporins. Blisters some-
times occur and generally resolve spontane0us1y.~Grafted areas sometimes exhibit a contracted or scarred appearance, although it tends to be less than that caused by meshed grafts. At present, there are several cultured epidermal autografts commercially available, including Epicel (approved for use in the United States) and Vivoderm (available only in the United Kingdom, Germany, and Italy). Vivoderm is grown on a hyaluronic membrane. Laser-drilled micro-perforations allow keratinocytes to migrate through the membrane into the wound bed. It has been used for the treatment of burns and chronic wounds? For cultured epidermal allografts, the skin is obtained from allogeneic tissue, such as newborn foreskin, and can be grown in advance. Avoiding creation of a donor wound site, and ready availability, provide an advantage compared to a~t0grafts.l~ Cultured epidermal allografts have been used to treat donor sites, partial thickness burns, chronic leg ulcers, and epidermolysis bullosa. Cultured allografts do not survive permanently on the wound bedrg but they provide pain relief within hours after grafting and act as a bio-
USE OF SKIN SUBSTITUTES IN DERMATOLOGY
logical dressing, stimulating epithelialization from the wound edges and adnexa, probably through release of growth factor^.'^ Cultured epidermal allografts can be cryopreserved and stored in a - 70°C freezer and thawed at room temperature.' They are not commercially available in the United States, and further randomized placebo-controlled studies are needed to determine their effectiveness. DERMAL REPLACEMENTS
The dermis plays an important role in the healing process. The cellular component (fibroblasts) and the extracellular component (collagen, extracellular matrix proteins) influence epithelial migration and differentiation, dermoepidermal junction formation, wound contraction, and scar formation. Dermal cells also secrete growth factors and cytokines, which modulate cell proliferation and differentiation. Collagen synthesis by fibroblasts forms a scaffold for cellular migration. Biosynthetic dressings were developed to provide temporary coverage to optimize wound healing. They were first introduced in 1979 for the treatment of burns and donor sites. One product, Biobrane, is composed of a bilaminate biosynthetic synthetic material,
557
constructed of silicone film with a nylon fabric partially embedded into the film. Type I porcine collagen peptides are present in both the silicone and nylon components as the biological component. Another dermal substitute, E-Z Derm (St. Paul, Minn), is a porcine-derived, biosynthetic, acellular dermal matrix, in which the porcine collagen has been chemically crosslinked using an aldehyde. It is available as a perforated or nonperforated dressing, attached to a gauze liner, which is discarded before grafting. E-Z Derm is indicated for patients requiring temporary coverage of partialthickness skin loss injuries, including burns and ulcers. Several advantages to the use of E-Z Derm are its long shelf life (18 months), immediate availability for serial grafting until autografts are available, and absence of human communicable disease (Fig. 1). Disadvantages include little clinical data to support its efficacy and safety, increase in the amount of exudates, and the possibility of disease transmission (xenograft). Oasis, a wound dressing derived from porcine small intestinal submucosa that has been processed to remove the serosa, smooth muscle, and mucosa layers results in an acellular collagen, extracellular matrix, which serves as a reservoir for cytokines and cell adhesion molecules, providing a scaffold for tissue
Figure 1. A, Venous ulcers before application of temporary skin substitute (E-Z Derm). B, Application of a temporary skin substitute. C, Healed ulcers several months after treatment with dermal skin substitute and compression therapy.
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Figure 2. Hydrated porcine small intestinal submucosa (Oasis, Spencer, Ind) applied over a leg ulcer using cotton applicators.
growth (Fig. 2). It gradually degrades so that the healed site consists entirely of host tissue. A small single-center pilot study performed on 15 patients with foot and leg ulcers using lyophilized versus hydrated small intestinal submucosa reported complete healing in lyophilized group (7/8) in 10 weeks.1° Although promising, further randomized controlled studies are warranted to evaluate the efficacy of this wound dressing. Integra, a temporary, biosynthetic, bilaminate skin substitute, has been approved by the FDA for postexcisional treatment of lifethreatening full-thickness or deep partialthickness thermal injury, when sufficient autograft is not available at the time of excision or not desirable because of the physiological condition of the patient. Developed by Burke et a1,2 it is composed of synthetic polysiloxane (silicone) and a three-dimensionalporous matrix of cross-linked bovine tendon collagen and shark-derived glycosaminoglycan (chondroitin-6-sulfate). A controlled multicenter, short-term trial compared the safety and efficacy of Integra versus conventional wound covers (autografts or temporary wound covers such as allografts, xenografts, or synthetics) in 149 patients with matched-pair thermal injuries (207 total wound sites). Results showed that healing was prolonged for all materials used, except autografts. No significant difference
was observed between total burn healingtime for wounds treated with Integra versus those treated with temporary covers. There appear to be some advantages to using Integra. Healing of donor sites was 14.3 days in those biopsied for conventional autografts versus 10.6 days in the Integra group from which ultra-thin epidermal autografts were obtained. Thinner autografts allow more rapid healing of donor sites, reharvesting can be accomplished with greater rapidity, and less hypertrophic scarring of the donor site is seen. At the treatment site, the silicone layer is more easily removed than allografts, and there is no known risk of rejection. One disadvantage to using Integra is the FDA requirement for clinicians to complete a company-sponsored training program. The application procedure is more complicated, and there is greater susceptibility to infection than with autografts. Cadaveric allograft skin has been used in burned patients to achieve temporary wound closure.’ Chemical treatment of human cadaver skin removes antigenic components (cellular components), resulting in an acellular collagen dermal matrix with an intact basement membrane complex that is immunologically inert. It can be used alone or in combination with cultured autografts. A human cryo-preserved, acellular cadaveric deepidermalized dermis, AlloDerm, has been used to treat burns since 1992, and for periodontal, plastic, and reconstructive surgery since 1994. Donor samples are screened for evidence of viral or bacterial infection. They are also tested for fast-growing microbes and fungi. At 10 days post-grafting, dermal allograft demonstrates repopulation with host fibroblasts and endothelial cells. Keratinocytes migrate from meshed autograft onto the surface of the dermal allograft, resulting in uniform distribution of epithelial cells across the dermoepidermal junction. Advantages of cadaveric grafts include the need for thinner autografts, less pain and faster healing at donor sites, no inflammatory response or clinical rejection at the treated site, and a small possibility of human disease transmission. Another skin substitute, TransCyte (formerly called Dermagraft-TC) is a temporary, biosynthetic, bi-laminate skin substitute that
USE OF SKIN SUBSTITUTES IN DERMATOLOGY
provides a temporary covering to help protect wounds from fluid loss and to reduce the risk of infection. It typically requires only one application. It has been approved by the FDA as a temporary covering for second- and third-degree burns. TransCyte is composed of a silicone polymer membrane and foreskinderived neonatal human fibroblasts grown in a three-dimensional nylon mesh. Fibroblasts are rendered nonviable after syntllesizing collagen, growth factors, and other extracellular matrix materials. A prospective, randomized, match-controlled trial performed in 14 patient with partial thickness burns reported decreased time to 90% of epithelialization of the wounds, 11.14 days in the TransCyte treated group, versus 18.14 days in the silver sulfadiazine treated group.12 A prospective, multicenter, randomized, controlled trial in 66 patients with 132 excised burn wounds compared the application of TransCyte versus human cadaveric allograft and reported equivalent adherence and graft take at day 14.16Investigators also reported that TransCyte was easier to remove and resulted in less bleeding than allograft. Dermagraft is a biosynthetic dermal matrix with cells that multiply and secrete collagen and growth factors. It is currently available in Canada, the United Kingdom, and Europe. Its manufacturer is currently seeking FDA approval for the treatment of diabetic foot ulcers, pressure ulcers, and venous ulcers. Dermagraft is produced by taking dermal fibroblasts from neonatal foreskin and seeding them onto a three-dimensional scaffold consisting of a bioabsorbable material (polyglactin mesh). The cells grow and divide, producing collagens, extracellular matrix proteins, and growth factors found in normal, healthy human dermis. Mature matrix is cryopreserved to -70°C and thawed before patient application. This skin substitute stimulates the formation of granulation tissue, reepithelialization, and angiogenesis. The fibroblasts produce collagens, fibronectin, and glycosaminoglycans. They also produce transforming growth factor Beta, which stimulates collagen formation. Fibroblasts produce keratinocyte growth factor and secrete
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vascular endothelial growth factor and hepatocyte growth factor. A prospective, multicenter, randomized, single-blind, controlled trail performed in 235 patients with diabetic foot ulcers, which were treated with once-per-week applications for 8 weeks, reported no significant statistical difference in healing in the treatment group (38.5%) compared with the in the control group (31.7?40).~~ COMPOSITE GRAFTS
Apligraf, a human skin equivalent with appearance similar to human skin, was the first biomedical device containing living human cells approved for use by the FDA (May 1998). It is a permanent, biosynthetic, bi-layered living construct of cultured human neonatal foreskin keratinocytes and fibroblasts cultured on a dermal matrix consisting of bovine type I collagen. It is approved to treat venous ulcers of at least l-month duration that have not adequately responded to conventional therapy (Fig. 3). In June 2000, the FDA approved Apligraf for the treatment of neuropathic diabetic ulcers. There is no evidence of host antibody or cell-mediated response or clinical rejection because there are no macrophages, lymphocytes, or Langerhans’ cells present in this skin substitute. A prospective, randomized, multicenter study in 293 patients with venous ulcers demonstrated that human skin equivalent plus compression therapy was superior to compression alone (63% vs. @YO),and patients healed more rapidly (61 days vs. 181 days).6 However, this study found no significant benefit between the human skin equivalent group and the control group in ulcers of less than 6 months duration. Healing was significantly faster than controls in ulcers greater than 6 months. A diabetic foot ulcer study reported that use of Apligraf was associated with a significantly better percentage of wounds with complete healing than the control group (56% vs. 38%), and average time to wound closure was significantly better (65 days vs. 90 days).20 A prospective, single-center, uncontrolled study performed in 15 patients with epidermolysis bullosa assessed the efficacy of Apli-
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BELL0 & FALABELLA
Figure 3. A, Venous ulcer before treatment with human skin equivalent (Apligraf, Canton, MA). 8,Healed ulcer following treatment.
graf in 69 acute wounds and nine chronic wounds.5This study reported that 79% of the wounds were healed a week after treatment. Patients and their families felt that healing was faster and less painful when the tissueengineered skin was used and that quality of life was improved compared with conventional dressings. Further studies are needed to confirm these results. A multicenter, single-arm study evaluated the safety and efficacy of a single application of human skin equivalent in 107 patients with partial or full-thickness surgical Cosmetic results in 50% of the patients were reported to be the same or better than would be expected with split thickness autografts. No adverse effects were seen. Another composite skin substitute is composite cultured skin, which uses allogeneic cells derived from neonatal foreskins." Fibroblasts and keratinocytes are grown in vitro and seeded on opposite sides of a bi-layered matrix of bovine collagen. To provide a surface for the keratinocytes to create an epidermal layer, the porous collagen sponge is laminated with a nonporous collagen gel. The fibroblasts seeded on the underside of the porous sponge migrate and distribute inside the sponge. The process is complete within 10 to 14 days. This year, the FDA approved
this product for use in patients with recessive dystrophic epidermolysis bullosa. Clinical trials in the treatment of burns, epidermolysis bullosa, diabetic ulcers, and venous ulcers using composite cultured skin are currently underway.
SUMMARY
The use of skin substitutes to treat acute and chronic wounds should continue to increase as newer products are developed. The goals for the future are to eliminate the need for autografting, eliminate the risk for transmission of disease, improve the shelf life and simplify storage requirements, improve cosmetic outcomes, and reduce cost. A number of researchers are also looking to incorporate gene therapy into skin substitutes.
References 1. Bolivar-Flores J PE, Marsch-Moreno M, Montes de Oca G, Kuri-Harcuch W Use of cultured human epidermal keratinocytes for allografting bums and conditions for temporary banking of the cultured allografts. B u m s 163-8, 1990 2. Burke J, Yannas I, Quinby W, et al: Successful use of physiologically acceptable artificial skin in the treat-
USE OF SKIN SUBSTITUTES IN DERMATOLOGY ment of extensive burn injury. Ann Surg 194413428,1981 3. Compton CC GJ, Bradford DA, et a1 Skin regenerated from cultured epithelial autografts on full thickness bum wounds from 6 days to 5 years after grafting. Lab Invest 60600-612, 1989 4. Eaglstein WH, Alvarez OM, Auletta M, et al: Acute excisional wounds treated with a tissue-engineered skin (Apligraf). Dermatol Surg 25:195, 1999 5. Falabella AF, Valencia I, Eaglstein WH, Schachner LA: Tissue-engineered skin (Apligraf a) in the healing of patients with epidermolysis bullosa wounds. Arch Dermatol 136:1225-1230, 2000 6. Falanga V, Margolis D, Alvarez 0, et al: Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Human Skin Equivalent Investigators Group [see comments] Arch Dermatol 134:293-300,1998 7. Greenfield E, Jordan B Advances in bum wound care. Crit Care Nurs Clin North Am 8203-215, 1996 8. Harris PA DF, Barisoni D, et a1 Use of hyaluronic acid and cultured autologous keratinocytes and fibroblasts in extensive bums. Lancet 353:35-36, 1999 9. Leigh IM PP: Cultured grafted leg ulcers. Clin Exp Dermatol 11:65M52, 1986 10. Marie Brown-Etris MP, Shields D Final report on a pilot study to evaluate porcine small intestinal submucosa as a covering for partial thickness wounds (Abstract). Symposium on Advanced Wound Care. 2000 April 1 to 4, 2000, Dallas, Texas 11. Morgan JR, Yarmush M L Bioengineered skin substitutes. Sci Med July/August, 6-15,1997
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12. Noordenbos J, Dore C, Hansbrough JF: Safety and efficacy of TransCyte for the treatment of partialthickness burns. J Bum Care Rehabil20:275-278,1999 13. Phillips TJ, Kehinde 0, Green H, Gilchrest BA: Treatment of skin ulcers with cultured epidermal allografts. J Am Acad Dermatol21:191-199, 1989 14. Phillips TJ: New skin for old: Developments in biological skin substitutes (editorial). Arch Dermatol 134344-349, 1998 15. Pollak R, Edington H, Jensen J, et al: A human dermal replacement for the treatment of diabetic foot ulcers. Wounds 9:175-183, 1997 16. Purdue GF, Hunt JL, Still JM Jr, et al: A multicenter clinical trial of a biosynthetic skin replacement, Dermagraft-TC, compared with cryopreserved human cadaver skin for temporary coverage of excised bum wounds. J Bum Care Rehabil 1852-57,1997 17. Rheinwald J, Green H Serial cultivation of strains of human epidermal keratinocytes: Formation of keratinizing colonies from single cells. Cell 6331-344, 1975 18. Rue LW 111, Cioffi WG, McManus WF, et al: Wound closure and outcome in extensively bumed patients treated with cultured autologous keratinocytes. J Trauma 34:662-667,1993 19. Sefton MV, Woodhouse KA: Tissue engineering. J Cutan Med Surg 3(Suppl 1):S1-1%23 20. Veves A, Falanga V, Armstrong DG, Sabolinski M L Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic diabetic foot ulcers. Diabetes Care 24290-295,2001
Address reprint requests to Anna F. Falabella, MD Department of Dermatology and Cutaneous Surgery University of Miami School of Medicine PO Box 016250 Miami, FL 33101 e-mail: afalabelQmed.miami.edu