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Plast Reconstr Surg Skin Anatomy and Antigen Expression after Burn Wound Closure with Composite Grafts of Cultured Skin Cells and ~io~bl~mers Steven T. Boyce, Ph.D., David G. Greenhalgh, M.D., Richard J. Kagan, M.D., Terry Housinger, M.D., J. Michael Sorrell, Ph.D., Charles P. Childress, M.A.T., Mary Rieman, R.N., and Glenn D. Warden, M.D. Ciitriilitnti nil(/ Clnl~lnilrl,Ohio Closure of large skin wounds (i.e., burns, congenital dures. Cultured epidermal auto graft^^.^'^ are ad- giant nevus, reconstruction of traumatic injury) with ministered over fascia, granulation tissue, or a110 split-thickness skin grafts requires extensive harvesting dermis, but are known to blister and ulcerate of autologous skin. Composite grafts consisting of col- lagen-glycosaminoglycan (GAG) substrates populated from slow development of dermal-epidermal with cultured dermal fibroblasts and epidermal kerati- junction (DEJ) for several months after graft- nocytes were tested in a pilot study on full-thickness burn ing.7,"-13 By contrast, skin analogues with mes- wounds of three patients as an alternative to split-thick- enchymal and epithelial components that are ap- ness skin. Light microscopy and transmission electron microscopy showed regeneration of epidermal and der- plied in one procedure are reported not to blister mal tissue by 2 weeks, with degradation of the collagen- after regeneration of epidermal tis~ue~~'~in GAG implant associated with low numbers of leukocytes, greater similarity to split-thickness skin au- and deposition of new collagen by fibroblasts. Complete tograft. basement membrane, including anchoring fibrils and an- To provide maximum availability of skin sub- choring plaques, is formed by 2 weeks, is mature by 3 stitutes for permanent closure of full-thickness months, and accounts for the absence of blistering of healed epidermis. All skin antigens tested (involucrin, burns, composite grafts consisting of cultured filaggrin, laminin, collagens IV and VII, fibronectin, and autologous keratinocytes and fibroblasts at- chondroitin-sulfate) were expressed by 16 days after tached to collagen-glycosaminoglycan (GAG) grafting. This cultured skin analogue provides an exper- substrates can be prepared consistently and imental alternative to split-thickness skin graft that de- velops histiotypic markers of skin anatomy and antigen stored conveniently in large q~antities.~.'~-" expression after wound closure. (Plast. Reconstr. Surg. 9 1 : This pilot study describes initial evidence that 632, 1993.) normal markers of epidermal differentiation (in- vol~crin,~~filaggrinlg), DEJ (collagens IV and VII,20 laminin21), and dermis (fibrone~tin,~~ Closure of full-thickness skin wounds is accom- chondroitin-sulfate23) are expressed by 16 days plished safely and effectively by grafting of split after grafting of this cultured skin analogue onto thickness autologous skin as sheets or full-thickness excised burns in pediatric patients. However, inadequate availability of skin au- Microscopic analyses of patient biopsies begin- tograft in large total body surface area burns has ning 2 weeks after grafting confirm develop- led to development of alternative materials from ment of skin anatomy and ultrastructure corre- ex vivo and in vitro sources4-" for repair of sponding to antigenic markers of normal human epidermal and/or dermal skin. These materials skin. Together, these qualitative data demon- are applied in either one or two surgical proce- strate that a cultured cell-biopolymer implant From the Shriners Burns Institute, the Department of Surgery at the University of Cincinnati, and Case Western Reserve University. Received for publication June 19, 1991; revised March 25, 1992. Presented 04 Apr 91 at the 23rd Annual Meeting of the American Burn Association. Supported by the Shriners of North America, grants #I5893 and #15837. 632 Vol. 91, No. 4 / ANATOMY AND ANTIGENS OF cu 'LTURED CELLS ON BURNS 633 can be applied in a single procedure, and can type to very large numbers, dermal fibroblasts rapidly regenerate epithelial and mesenchymal were inoculated onto the porous surface of col- tissues for skin wound repair. Early formation of lagen-GAG substrate^.^^'^^" One day later, sub- DEJ eliminates complications of skin blistering strates were inverted to orient the nonporous and ulceration associated with application of cul- lamination on top, and epidermal keratinocytes tured epidermal autografts after enzymatic re- were inoculated to cover substrate surfaces. Af- lease from tissue culture vessels. Increased avail- ter inoculation, cultured cell-collagen-GAG ability of cultured skin compared to skin composites were cultured from 4 to 14 days in autograft for large skin loss injuries may de- serum-free medium MCDB 153 either sub- crease requirements for donor skin for comple- merged or lifted to the air-liquid inter- tion of wound closure. fa~e.~~,~~,~~For 24 hours before surgery, grafts were incubated in medium in which 1 to 10 METHODS AND MATERIALS ng/ml epidermal growth factor was replaced Patient Information with 10 ng/ml basic fibroblast growth factor. Three patients admitted to the Shriners Burns Surgical Application, Dressings, and Wound Care Institute in Cincinnati were enrolled by in- Sites receiving cultured grafts included the ab- formed consent into a clinical study of cultured domen, flank, back, and knee. Wounds were ex- skin grafts (CSG) for burn treatment that was cised to viable tissue (fat or deep dermis) and approved by the University of Cincinnati Insti- soaked overnight in wet dressings with 0.5% Sul- tutional Review Board (IRB). Patient informa- famylon (mafenide acetate) solution (adminis- tion is summarized in Table I. Two patients were tered under separate IRB approval). The follow- 3 years old and one was 14 years old at the time ing day, wounds were irrigated well with saline, of grafting. Full-thickness burn size ranged from and composite cultured grafts were placed onto 65 to 80 percent, and the size of the area grafted wounds with a backing of N-Terface and stapled with CSG ranges from 120 to 2 16 cm2 (two or to the wound bed. Grafts were then covered ei- three grafts, 8.5 X 8.5 cm each). ther with dry dressings consisting of Xeroform Graft Preparation and Composition gauze, cotton gauze bolster, and spandex, or with wet dressings consisting of fine mesh gauze, Biopsies ranging in size from 10 to 20 cm2 cotton gauze, perforated red rubber catheters were collected at the first scheduled skin graft (0.5 cm diameter), additional cotton gauze, and procedure after acquisition of informed consent. spandex stapled to surrounding skin. Wet dress- Epidermal keratinocytes and dermal fibroblasts ings were irrigated with antimicrobial solutions were isolated by enzymatic and mechanical dis- administered through catheters at 2-hour inter- aggregation and cultured in selective nutrient vals on a protocol of 0.5% Sulfamylon solution media for each cell type as previously de- followed by two additions of double antibiotic s~ribed.~~'~-"~~~After propagation of each cell TABLE I Patient Information Patient No. 1 2 3 Age 14 3 3 Sex M F M % TBSA 65 64 80 PBD donor skin 6 6 5 PBD CSG applied 34 3 1 33,40 PBD CSG biopsy 48, 118 45, 58, 108 56 CSG site Abdomen Left flank Right knee, back CSG area (cm2) 144 120 72, 144 CSG % engraft- FIG. 1. Histology of cultured skin graft. Composite ment* 15 65 15 grafts consist of cultured human epidermal keratinocytes Microscopy LM, TEM LM, TEM IFM (HK) and fibroblasts (HF) attached to a thin sponge of col- * Engraftment as CSG was estimated at day 10 after grafting and is ex- lagen-glycosaminoglycan (COL-GAG) that has a nonporous pressed as '%" of area with dry, keratinized epithelium on the wound. Ab- film as an epithelial culture surface. The total thickness of breviations: TBSA, total body surface area; PBD, post-burn day; CSG, cultured skin graft: LM, light microscopy; TEM, transmission electron microscopy; the skin substitute is no greater than 0.5 mm (0.020 inches) IFM, immunofluorescence microscopy. to minimize time of vascularization. Scale bar = 0.1 mm. 634 PLASTIC AND RECONSTRUCTIVE SURGERY,April 1993 (DAB; 40 pg/ml neomycin sulfate and 200 ble I. Biopsies were rinsed in saline, bisected, and units/ml polymyxin B sulfate). Wet or dry dress- fixed in 2% glutaraldehyde and 2% paraformal- ings were changed on day 2 or 3 and on day 5, dehyde in 0.1 M Na cacodylate buffer, pH 7.4, at which time wet dressings were discontinued. and subsequently processed by standard TEM Thereafter, all dressings were changed twice methods. daily with Adaptic and Aquaphor containing bacitracin-zinc ointment (500 pg/gm), or 3 parts ImmunoJuorescence Microscopy and Skin Antigens bacitracin ointment plus part 1% silver sulfadi- Comparison was made of skin tissue regener- azine cream. Grafted wounds were observed at ated after application of cultured skin grafts to each dressing change until day 5 and once daily ungrafted cadaver skin (control) by indirect im- until day 10. After reepithelialization was com- munofluorescence with a limited panel of mono- plete, .pressure garments were applied to the clonal antibodies to skin antigens. The panel in- grafted sites. cluded: (1) involucrin of stratum corneum; (2) Light and Transmission Electron Microscopy (LM filaggrin of stratum granulosum; (3) collagen and TEM) type IV of basement membrane lamina densa; (4) laminin of lamina lucida; (5) collagen type VII of Biopsies (3 mm) for LM and TEM were col- anchoring fibrils; (6) fibronectin; and (7) chon- lected under general or local anesthesia from droitin-sulfate of dermis. All primary antibodies patients after 14 days, approximately 1 month, were reacted with unfixed cryostat sections (3-6 and approximately 3 months, as indicated in Ta- pm) of skin samples on glass slides, followed by reaction with biotinylat&d secondary antibodies, FIG. 2. Clinical result. Toppanel: Ten days after grafting keratinized epithelium covers greater than 50 percent of the FIG. 3. Histology of healed skin after 2 weeks.
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