World J. Surg. 16, 47-52, 1992

World Journal of Surgery © 1992 by the Soei~t '~ lntcrnationale de Chirurgie

Burn Wound Closure Using Permanent Skin Replacement Materials Ronald G. T o m p k i n s , M . D . , Sc.D, and J o h n F. Burke, M.D. Surgical and Trauma Services, Massachusetts General Hospital, Department of Surgery, Harvard Medical School, and Shriners Burns Institute, Boston Unit, Boston, Massachusetts, U.S.A. Over the past decade, very significant advances in the development of clinically useful, permanent skin replacement materials have taken place. The most prominent and successful approaches to the physiological closure of an open wound have been either by creating a totally artificial dermal matrix material, by using culture techniques to expand cell populations for autologous transplantation, or by using a combination of these methods. As a result of substantial early progress in this field, permanent skin replacement materials as a treatment modality promise significant contributions to improved wound management and increased survival rates for patients with devastating soft tissue destruction such as massive burn injuries.

Destruction of the epidermal and dermal layers of skin produces two very serious short-term and life-threatening problems, the escape of essential body fluids and the invasion of bacteria. The epidermal layer of normal skin, consisting of a differentiating layer of keratinocytes attached to an underlying basement membrane, normally prevents water loss and microbial invasion. These short-term problems of life-threatening dehydration and invasive sepsis which are normally solved by the epidermal layer of skin must be addressed very soon after injury by physiologically-acceptable and permanent wound closure materials. In addition to these short-term issues, serious long-term Problems with cosmesis and function are also important. The dermal layer of skin supports the durable and pliable nature of skin which provides optimal cosmesis and useful functional activities of skin. The combination of short-term and long-term issues must be adequately resolved by a permanent skin replacement material before the material should be considered as a success. Because the development of skin replacement materials is relatively new, clinical applications of these materials have been generally restricted to use in massive burn injuries (~70% total body surface area) in which the available donor skin is severely limited and the patient's survival is threatened. Only the few permanent skin replacement methods that have been tried under these circumstances will be reviewed here in any detail. Permanent skin replacement materials should fulfill the foi-

Reprint requests: Ronatd G. Tompkins, M.D., Sc.D., Trauma SerVice, Bigelow 1302, Mass~.chusetts General Hospital, Boston, Massachusetts, 021 t4, U.S.A.

lowing desig n criteria to mimic normal dermis and epidermis: (1) adhere intimately to the wound bed and maintain normal water and heat transport rates; (2) provide an intact microbial barrier which is nontoxic, antiseptic, noninflammatory, and nonantigenic; (3) participate in normal host defense and wound repair mechanisms; (4) provide a permanent surface that will grow with the patient; (5) maintain elasticity and long-term durability; and (6) display long-term mechanical and cosmetic function with wound contracture properties comparable to split-thickness autografts. The best biological solution to these issues takes advantage of the host's own cellular and structural recycling processes available to all normal tissues. In order that these criteria can be met by nonbiological materials, the replacement materials must interact in a positive sense with the host's cellular systems of the wound and not simply remain inert and nonreactive, as seen with temporary skin replacements. Temporary skin materials rely upon incorporation into the wound coagulum and ingrowth of granulation tissues for adhesion; they do not biodegrade and therefore, they can only be temporary substitutes and must be surgically replaced with the patient's own skin. This review includes progress in permanent skin replacements and does not address these temporary wound coverage materials. General Strategies

Three general strategies to skin replacement have been followed. The first strategy uses a totally artificial device to mimic the 3 dimensional structure and character of dermis and the physical properties of epidermis; the second strategy uses culture methods to replace the keratinocytes of epidermis; and the third strategy uses a combination of these techniques to create a composite graft material, The totally artificial matrix approach successfully solves the problem of permanent dermal replacement whereas the cultured epidermal cell approach addresses the epidermal problem alone. In regard to construction of a dermal matrix device, the first and most successful approach was taken by Yannas and Burke [1] with the construction of a bilayer, polymeric membrane comprising an upper layer of medical-grade silastic (epidermal layer) and a lower layer of highly porous cross-linked collagen and chondroitin 6-sulfate (dermal layer) (Table 1). The most

48

World J. Surg. VoL 16, No. 1, Jam/Feb. 1992

Table 1. Artificial dermis and composite grafts for permanent skin replacement. Author (reference) Artificial dermis" Burke (6) Heimbach (8) Tompkins (7) Composite materials AIlograft skin and epidermal cells Heck (30) Clark (31) Cuono (32) Zhi-Ren (33) Langdon (34) Modified collagen-GAG c and cells Hansbrough (37)

Year

Number of patients

Cadaver allograft

Epidermal removal

Epidermal cell source

1981 1988 1989

10 149 21

N.A. N.A. N.A,

N.A. N.A. N.A.

0.004 in autograft 0.004 in autograft 0.004 in autograft

1985 1986 1986 1986 1988

8 3 I 4 6

YES YES, 6:1 YES YES YES

Stripped Abrasion Abrasion h Abrasion

"Suction blister" uncultured epidermal cells Cultured autologous epidermal cells Cultured autologous epidermal cells

1989

4

N.A.

N.A.

Cultured autologous epidermal cells and fibroblasts

b

Cultured autologous epidermal cells

N.A.: not applicable. "Cotlagen-chondroitin 6-sulfate dermal matrix with silastic epidermis. hUncultured, autologous epidermal cells injected into autograft. CGlycosaminoglycan. Table 2. Cultured epidermal cells alone for permanent skin replacement.

Author (Reference) Autologous epidermal cells O'Conner (11) Gallico (12) Gallico (13) Pittelkow (17) Eldad (14) Laterjet (15) Herzog (18) Kamagai (16) Woodley (19) Compton (20) Gallico (21) Munster (22) Peterson (23) Allogenic epidermal cells He(ton (26) Madden (27) Brain (28) Burt (29)

Year

Number of patients

Comments"

1981 1984 1985 1986 1987 1987 1988 1988 1988 1989 1989 1990 1990

1 2 5 I 25 2 8 7 4 21 8 7 3

Initial clinical report Early clinical studies with extensive coverage Additional follow-up study Use of 2 phase culture method Combination of allografts and autografts yielding "disappointing results" "'Disappointing take" No anchoring fibrils; 0-85% "take" Lack of rete ridge and elastin "Defective anchoring fibrils" Normal dermal-epidermal junction Fascial excision of giant hairy nevi; 68% "take" 75% "take" "Abnormal keratinocyte differentiation"

1983 1986 1989 1989

3 26 19 20

Partial-thickness wounds; accelerated healing Accelerated healing on partial-thickness; no "take" on full-thickness Partial-thickness excision of tatoos; last grafts -< 3 weeks Last grafts -< 1 week

"Descriptive comments or from the text of the article.

extensively utilized example of the second strategy involves the culture of the patient's own epidermal cell using methods described by Green and coworkers [2] or the transplantation of cultured allogenic keratinocytes for wound closure [3] (Table 2). The best example of a composite device is a system described by Boyce and Hansbrough [4] which is a modification o f the collagen-chondroitin 6-sulfate matrix of Yannas and Burke (Table 1). This composite is a nonporous surface of collagen and glycosaminoglycan which is laminated to the upper layer of the dermal membrane to provide a nonporous and planar surface. The nonporous surface is seeded with cultured, proliferating autologous epidermal cells and the porous dermal collagen-chondroitin 6-sulfate matrix is seeded with proliferating autologous (or potentially allogenic) fibroblasts. Multiple literature references illustrating all three strategies are included in Tables 1 and 2, however, these tables are

not intended to be an exhaustive list but are intended to be representative of the field. Dermal Matrix

The earliest and largest clinical experience with permanent skin replacement materials has been with the artificial dermis which was designed by Yannas and Burke [1] and is currently manufactured by Marion Merrell Dow as Integra ®. The design considerations, construction, and clinical results have recently been reviewed in the World Journal o f Surgery [5] and will be only briefly summarized here. Integra is a totally artificial device which is acellular and is composed of two layers, an upper layer of silastic with a porosity sufficient tO control water loss and to prevent invasion of microbes and a lower, highly porous layer which is a

R.G. Tompkins and J.F. Burke: Development of Artificial Skin

49

Fig. 1. Steps in the clinical use of artificial skin in the definitive skin replacement of a full-thickness flame burn of the right forearm. A. Artificial skin sutured in place following surgical excision of full-thickness burn eschar promptly postinjury. B. Removal of the temporary epidermis 2 weeks postinjury. C. The excellent vascularized "neodermis" immediately following silastic removal. D. The neodermis covered with an ultrathin epidermal autograft harvested immediately before silastic removal and autograft placement. Cross-linked coprecipitate of bovine collagen and shark chondroitin 6-sulfate. An extremely important design consideration of the material is the pore size of the lower dermal layer (20-125 t~rn) which has been optimized to allow the migration of the Patient's own endothelial cells and fibroblasts into the matrix. These host cells provide vascularization and the eventual replacement of the artificial matrix with a new dermal structure or the "neodermis". Once the burned tissue has been excised the acellular artificial skin is sutured in place using the same techniques as for skin autograft placement (Figure IA). Once the full-thickness of the lower layer of the Integra has been POpulated by host cells, the silastic layer can be easily removed (Figure IB), providing an excellent vascularized "neodermal"

base (Figure IC). The patient's own freshly harvested thin (0.002-0.004 in) epidermal autograft seeds the neodermis (Figure ID). A 7-year follow-up of this patient's artificial skin is shown in Figure 2. Integra is not currently available but is involved in the final stages of FDA trials. Clinical experience with Integra in burn injuries was reported in an initial 10 patients from the Boston Unit of the Shriners Burns Institute and the adult burn unit of the Massachusetts General Hospital in 1981 [6] and later, long-term results of a total of 21 adult patients at the Massachusetts General Hospital were reported in 1989 [7]. In this latter study, a survival benefit with the use of Integra was suggested by an adjusted odds of dying of 0.52 (half the odds of dying as those patients not

50

Fig. 2. The patient's forearm 7 years following burn injury. There is no evidence of hypertrophic scar or contracture formation. The skin has functioned well without abnormality or symptoms since graft healing and hospital discharge.

treated with Integra). As can be appreciated in this later study, the use of Integra has been limited to massive burn injuries (->70% total body surface area) in adults whose determinants for survival depend upon many parameters independent of the use of Integra. From this later study, it was estimated that 160 adult massively burned patients would be necessary to demonstrate any potential statistical significance of a survival benefit. In a recently published report of an 11 center prospectively randomized trial [8], Integra was used in 149 patients with final long-term evaluations in 82 patients. The " t a k e " of the Integra was a very acceptable 80% as compared to 95% for meshed autograft. In these patients, donor sites healed an average of 4 days sooner because they were harvested more superficially at an average of 0.006 in (0.15 mm) as compared to the usual depth of 0.013 in (0.33 mm) for standard split-thickness donor skin. At the completion of the study, there was less hypertrophic scarring of the Integra, and more patients preferred the areas covered with Integra to the control areas with meshed autograft. The conclusion of this multicenter trial was that Integra provided a permanent cover that was at least as satisfactory as currently available skin grafting techniques, and allowed donor grafts that were thinner and donor sites that healed more rapidly. Significant technical problems including wrinkles, hematomas, and infections however occurred, but it was suggested by the authors that these problems could be eliminated by meshing the Integra in a fashion similar to meshing of autografts. Additional recent investigations using this dermal matrix together with the centrifugation of a dermal-epidermal cell suspension into the junction between the silastic and dermal matrix has resulted in significant improvements in regeneration of the dermis in an animal model system [9]. Cultured Epidermal Cells

Technology to culture the cellular components of skin has been available for fibroblasts and endothelial cells for many years, however, methods to culture epidermal cells were improved by Rheinwald and Green [10] with more recent further modifica-

World J. Surg. VoL 16, No. 1, Jan./Feb. 1992

tions by Green and associates [2] which have been sufficient to allow their use in human applications (Table 2). In 1981, the first reported clinical experience using cultured autologous epidermal cells using these methods occurred at the Brigham and Women's Hospital in Boston in which two thermally injured patients received autologous epidermal cell transplantations to very limited body surface areas [11]. In 1984 at the Boston Unit of the Shriners Burn Institute, two extensively burned patients, who were also receiving multiple other therapeutic modalities, received transplants of cultured epidermal autografts to an extent which reportedly covered 50% of the body surface area [12]; further experience with this cultured cell approach rapidly ensued [13]. Additional experience with this technology has been reported from both Europe [14, 15], Japan [16], and the U.S.A. [17-23] (Table 2). Controversial issues have arisen using this cultured cell approach including the degree of " t a k e " of the cultured epidermal autografts (the efficiency in which the cultured cells adhere and persist in vivo), the long-term morphology of the dermalepidermal junction in the development of normal histological features of this junction, and the long-term cosmetic and functional results. The efficiency of " t a k e " has been reported to vary widely between 0 and 85% in burned patients (Table 2) and to be 68% when used in a purely elective circumstance in the excision of congenital hairy nevi [21]. A wider difference of opinion arises over the presence and temporal development of normal dermal-epidermal structures including hemidesmisomes and anchoring fibrils which are considered necessary for longterm adherence of the cultured cell grafts. In a recent report by Compton and colleagues [201 in follow-up of 21 pediatric patients with burn injuries, hemidesmisomes and anchoring fibrils were completely reconstituted within 3 to 4 weeks post-transplantation. In an animal study of human keratinocyte transplants in athymic mice by Regauer and coworkers [24] the Type VII collagen constituting the anchoring fibrils was shown to be keratinocyte in origin. In contrast, Woodley and associates [19] reported that in four patients the formation of these same structures remained abnormal and was associated with abnormal keratinocyte differentiation [23]. Investigators have recognized that formation of a proper dermal-epidermal junction is not only highly desirable but is likely necessary to develop a pliable and durable long-term result. Further analysis of studies using this skin replacement strategy will be necessary to resolve these issues. In addition to the application of cultured analogous epidermal cells, in a parallel effort, investigators have transplanted cultured allogenic keratinocytes using methods described by Eisinger and colleagues [25] to accelerate would healing by repopulating the wound with epidermal cells. In initial studies [26], acceleration of healing of partial-thickness wound was seen, however in later studies [27], essentially no " t a k e " of these transplanted epidermal cells was found in full-thickness wounds. In later studies in patients with partial-thickness excisions of tatoos [28] and full-thickness wounds in burned patients [29], the transplanted allogenic cells were not found to persist in the wound beyond the first few weeks post-transplantation by using gender chromosomal analysis of the cells remaining in the wound (Table 2). Therefore, the beneficial effects of allogenic epidermal cells on the acceleration of partial-thickness wound healing must be dependent upon effects

R.G. Tompkins and J.F. Burke: Development of Artificial Skin unrelated to the persistence of the transplanted cells within the Wound.

Composite Materials Multiple investigators have attempted to develop composite materials to include the following methods (Table 1): (1) transplantation of allograft skin combined with cultured autologous epidermal cells [30-34]; (2) transplantation of gelled collagen Seeded with cultured epidermal cells and fibroblasts [35]; and (3) construction of a modified collagen-GAG (glycosaminoglycan) matrix with cultured epidermal cells and fibroblasts [36-40]. One of the earliest reported efforts with this composite approach involved transplantation of allograft; after successful "take" of the allograft, the allogenic epidermal cells were removed and replaced by "suction blister" autologous keratinocytes with partial success [30]. This experience was rapidly followed by Clark [31] and Cuono and coworkers [32] using cultured autologous keratinocytes and allograft. One advantage of this approach is that the allograft provides wound closure in addition to sufficient time for proliferation of the autologous epidermal cells in vitro (approximately 3 weeks). In this system, presumably the host's own fibroblasts and endothelial cells m~grated into the dermal matrix and repopulated the allogenic dermis by host tissues. Another composite graft has been described by Bell and associates [35] which is a "living skin equivalent" consisting of a gelled collagen matrix that is seeded with cultured fibroblasts and keratinocytes. Although multiple in vitro and animal studies have investigated the living skin equivalent properties, very limited if any clinical results of this composite material have been reported. The composite material is currently available from Organogenesis (Cambridge, Massachusetts, U.S.A.) exclusively for in vitro testing purposes. As previously mentioned, in 1988 Boyce and Hansbrough [36] described a modification of the collagen-chondroitin 6-sulfate dermal matrix material described by Yannas and Burke in Which a nonporous surface was created on the superficial layer of the, dermal matrix with the silastic layer omitted. In this system, autologous fibroblasts were seeded into the dermal Portion of the graft and the patient's own epidermal cells were Placed upon the nonporous surface. In four patients, Hansbrough and colleagues [37] demonstrated partial success of this composite when applied to limited areas of full-thickness WOUnds; the "take" was acceptable with formation of a basement membrane within 9 days of transplantation. The basic C°llagen-chondroitin 6-sulfate matrix has been further modified to include peptide growth factors [38] and matrix peptides containing the arginine-glycine-aspartic acid sequence considered to be the cell attachment site for many extracellular matrix tnolecules [39]. More recently, this group has pursued an artificial dermal matrix composed of human fibroblasts which ~Vere grown on surgical mesh materials made from polyglycolic acid prior to transplantation [40]. Clinical results of this latter approach however, are currently limited but appear promising.

Summary In general, results of the relatively few and limited clinical investigations are promlsing but do not allow a definitive

51 resolution regarding the optimal permanent skin replacement material. However, one generality can be made from the available studies. The most successful short-term and long-term results have been achieved only if both dermal and epidermal replacement has been taken into account in a timely fashion. Only with the physiological replacement of both normal layers of skin can the success criteria previously outlined most likely be achieved. Of the permanent skin replacement efforts to date, the most extensively studied and the only approach evaluated by a prospectively randomized clinical trial has been by the creation of an artificial dermal matrix combined with a silastic epidermis such as Integra. Ultimately, the best candidates for permanent skin replacements will require such prospectively randomized clinical trials to determine the best approach under defined clinical conditions.

R~sum~ Dans la derni~re d6cennie, d'importants progr~s dans le remplacement tissulaire permanent ont 6t6 accomplis. Les approches les plus s6duisantes, qui r6ussissent le plus souvent, sprit celles qui spit cr6ent une matrice compl6tement artificielle, spit utilisent des techniques de culture cellulaire en vue de transplantations autologues ou encore qui combine plusieurs de ces m6thodes. Du fait de ces progr~s, le traitement par remplacement cutan6 permanent est une m6thode pleine de promesses pour l'avenir. Elle pourrait am61iorer le traitement des plaies et la survie des patients ayant eu des pertes tissulaires importantes, comme darts le cas de brOlures 6tendues.

Resumen En la dltima d6cada se han logrado avances de gran significaci6n en el desarrollo de materiales de reemplazo permanente de la piel para uso clfnico. Los aproches mds destacados y titiles para el cierre fisiol6gico de una quemadura abierta han sido la creaci6n de una matriz d6rmica totalmente artifcial mediante el uso de t6cnicas de cultivo para expander la poblaci6n celular para trasplante aut61ogo o mediante la combinaci6n de estos m6todos. Como resultado del sustancial progreso inicial en este campo, los materiales para el reemplazo permanente de piel como modalidad terap6utica aparecen promisorios en cuanto a contribuciones significativas al mejor manejo de la herida y a un incremento en la tasa de sobrevida de pacientes con desvastadoras destrucciones de los tejidos blandos, tal como ocurre en las quemaduras masivas.

Acknowledgment Supported in part by the General Medical Services of the National Institutes of Health grants GM 21700 and GM 07035 and the Shriners Hospitals for Crippled Children.

References 1. Yannas, l.V., Burke, J.F., Orgill, D.P., Skrabut, E.M.: Wound tissue can utilize a polymeric template to synthesize a functional extension of skin. Science 215:174, 1982 2. Green, H., Kehinde, O., Thomas, J.: Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc. Natl. Acad. Sci. 76:5665, 1979

52

3. Madden, M.R., Finkelstein, J.L., Staiano-Coico, L., Goodwin, C.W., Shires, G.T., Nolan, E.E., Hefton, J.M.: Grafting of cultured allogeneic epidermis on second- and third-degree burn wounds on 26 patients. J. Trauma 26:955, 1986 4. Boyce, S.T., Hansbrough, J.F.: Biologic attachment, growth, and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate. Surgery 103: 421, 1988 5. Tompkins, R.G., Burke, J.F.: Progress in burn treatment and the use of artificial skin. World J. Surg. 14:819, 1990 6. Burke, J.F., Yannas, I.V., Quinby, W.C., Bondoc, C.C., Jung, W.K.: Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Ann. Surg. 194:413, 1981 7. Tompkins, R.G., Hilton, J.F., Burke, J.F., Schoenfeld, D.A., Hegarty, M.T., Bondoc, C.C., Quinby, W.C., Behringer, G.E., Ackroyd, F.W.: Increased survival after massive thermal injuries in adults: Preliminary report using artificial skin. Crit. Care Med. •7:734, 1989 8. Heimbach, D., Luterman, A. Burke, J., Cram, A., Herndon, D., Hunt, J., Jordan, M., McManus, W., Solem, L., Warden, G., Zawacki, B.: Artificial dermis for major burns: A multicenter randomized clinical trial. Ann. Surg. 208:313, 1988 9. Yannas, I.V., Lee, E., Orgill, D.P., Skrabut, E.M., Murphy, G.F.: Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin. Proc. Natl. Acad. Sci. 86:933, 1989 10. Rheinwald, J.G., Green, H.: Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265:421, 1977 11. O'Conner, N.E., Mulliken, J.B., Banks-Schlegel, S., Kehinde, O., Green, H.: Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet 1:75, 1981 12. Gallico, G.G., O'Conner, N.E., Compton, C.C., Kehinde, O., Green, H.: Permanent coverage of large burn wounds with autologous cultured human epithelium. N. Engl. J. Med. 311:448, 1984 13. Gallico, G.G., O'Conner, N.E.: Cultured epithelium as a skin substitute. Clin. Plast. Surg. 12:149, 1985 14. Eldad, E., Burr, A., Clarke, J.A.: Cultured epithelium as a skin substitute. Burns 13:173, 1987 15. Latarjet, J., Gangolphe, M., Hezez, G., Masson, C., Chomel, P.Y., Cognet, J.B., Galoisy, J.P., Joly, R., Robert, A., Foyatier, J.L., Faure, M., Thivolet, J.: The grafting of burns with cultured epidermis as autografts in man. Scand. J. Plast. Reconstr. Surg. 21:241, 1987 16. Kumagai, N., Nishina, H., Tanabe, H., Hosaka, T., lshida, H., Ogino, Y.: Clinical application of autologous cultured epithelia for the treatment of burn wounds and burn scars. Plast. Reconstr. Surg. 82:99, 1988 17. Pittelkow, M.R., Scott, R.E.: New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns. Mayo Clin. Proc. 51:771, 1986 18. Herzog, S.R., Meyer, A., Woodley, D., Peterson, H.D.: Wound coverage with cultured autologous keratinocytes: Use after burn wound excision, including biopsy followup. J. Trauma 28:195, 1991 19. Woodley, D.T., Peterson, D.T., Herzog, S.R., Stricklin, G.P., Burgeson, R.E., Briggaman, R.A., Cronce, D.J., O'Keffe, E.J.: Burn wounds resurfaced by cultured epidermal autografts show abnormal reconstitution of anchoring fibrils. J.A.M.A. 259:2566, 1988 20. Compton; C.C., Gill, J.M., Bradford, D.A., Regauer, S., Gallico, G.G., O'Conner, N.E.: Skin regenerated from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. Lab. Invest. 60:600, 1989

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21. Gallico, G.G., O'Conner, N.E., Compton, C.C., Remensnyder, J.P., Kehinde, O., Green, H.: Cultured epithelial autografts for giant congenital nevi. Plast. Reconstr. Surg. 84:1, 1989 22. Munster, A.M., Weiner, S.H., Spence, R.J.: Cultured epidermis for the coverage of massive burn wounds. Ann. Surg. 211:676, 1990 23. Peterson, M.J., Lessane, B., Woodley, D.T.: Characterization of cellular elements in healed cultured keratinocyte autografts used to cover burn wounds. Arch. Dermatol. 126:175, 1990 24. Regauer, S., Seller, G.R., Barrandon, Y., Easley, K.W., Compton, C.C.: Epithelial origin of cutaneous anchoring fibrils. J. Cell. Biol. 111:2109, 1990 25. Eisenger, M., Lee, J.S., Hefton, J.M., Darzynkieqicz, Z., Chiao, J.W., de Harven, E.: Human epidermal cell cultures: Growth and differentiation in the absence of dermal components and medium supplements. Proc. Natl. Acad. Sci. 76:5340, 1979 26. Hefton, J.M., Finkelstein, J.L., Madden, M.R., Shires, G.T.: Grafting of burn patients with allografts of cultured epidermal cells. Lancet 2:428, 1983 27. Madden, M.R., Finkelstein, J.L., Staiano-Coico, L., Goodwin, C.W., Shires, G.T., Nolan, E.E., Hefton, J.M.: Grafting of cultured allogencic epidermis on second- and third-degree burn wounds on 26 patients. J. Trauma 26:955, 1986 28. Brain, A., Purkis, P., Coates, P., Hackett, M., Navsaria, H., Leigh, I.: Survival of cultured allogeneic keratinocytes transplanted to deep dermal bed assessed with probe specific for Y chromosome. BMJ 298:917, 1989. 29. Burt, A.M., Pallett, C.D., Sloane, J.P., O'Hare, M.J., Schafler, K.F., Yardeni, P., Eldad, A., Clarke, J.A., Gusterson, B.A.: Survival of cultured allografts in patients with burns assessed with probe specific for Y chromosome. Br. Med. J. 298:915, 1989 30. Heck, E.L., Bergstresser, P.R., Baxter, C.R.: Composite skin graft: Frozen dermal allografts support the engraftment and expansion of autologous epidermis. J. Trauma 25:106, 1985 3 I. Clarke, J.A.: Cultured skin for burn injury. Lancet 2:809, 1986 32. Cuono, C., Langdon, R., McGuire, J.: Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury. Lancet 1:1123, 1986 33. Zhi-Ren, G., Zhi-Qan, H., Lan-Jan, N., Gui-Fen, L.: Coverage of full skin thickness burns with allograft inoculated with autogenous epithelial cells. Burns 12:220, 1986 34. Langdon, R.C., Cuono, C.B., Birchall, N., Madri, J.A., Kuklinska, E., McGuire, J., Moellmann, G.E.: Reconstitution of structure and cell function in human skin grafts derived from cryopreserved allogeneic dermis and autologous cultured keratinocytes. J. Invest. Dermatol. 91:478, 1988 35. Bell, E., Ehrlich, H.P., Buttle, D.J., Nahatsuji, T.: Living tissue formed tn vitro and accepted as skin-equivalent tissue of fullthickness. Science 211:1052, 1981 36. Boyce, S.T., Hansbrough, J.F.: Biologic attachment, growth, and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate. Surgery 103: 421, 1988 37. Hansbrough, J.F., Boyce, S.T., Cooper, M.L., Foreman, T.J.: Burn wound closure with cultured autologous keratinocytes and fibroblasts attached to a collagen-glycosaminoglycan substrate. J.A.M.A. 262:2125, 1989 38. Stompro, B.E., Hansbrough, J.F., Boyce, S.T.: Attachment of peptide growth factors to implantable collagen. J. Surg. Res. 46:413, 1989 39. Cooper, M.L., Hansbrough, J.F., Foreman, T.J.: In vitro effects of matrix peptides on a cultured dermal-epidermal skin substitute. J. Surg. Res. 48:528, 1990 40. Hansbrough, J.F.: Current status of skin replacements for coverage of extensive burn wounds. J. Trauma 30:S155, 1990