CL283
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CL283
Wound closure with human keratinocytes cultured on a polyurethane dressing overlaid on a cultured human dermal replacement Hans O. Rennekampff, MD,John F. Hansbrough, MD, VereDa Kiessig, BS, SalwanAbiezzi, and Vlrgll Woods Jr., MD, San Diego,Calif Background. Bum excisionlollowed by immediate lvound coveragehas becomethe clinical standard lor managing extensivebum injuries in much 01 the world. VVhensufficient autogralt skin to achieve permanent lvound closureis unavailable, cell culture technologyhas made the use 01 cultured human keratinocyte(HK) sheetsclinically leasible. VVhereas previous techniqueshavelocused on devetopment01 multilayered, differentiated HK sheets,OUTattention has beendrawn to using HK in a highly proliferative, lessdifferentiated state. Time requirementslor preparation 01 multistratijied cultured HK are high, and preparatory steps may destroyimportant integrin adhesion molecules. Methods. We describethe use01 HK cultured to single layer confluente on a polyurethane membrane(HD), lvith serumjree medium. HK-HD gralts lvere transplanted tolull-thickness wounds on athymic mice (n = 31). A secondgroup DImite (DG-HK-HD, n = 28) receiveda living human dermal replacementcontaining cultured fibroblasts beforeplacement 01 HK-HD. Control mice receivedHD alone (n = 4). Basementmembraneproteins on healed wounds and surfaceintegrins on cultured HK were identijied by means01 immunostaining and direct microscopicvisualization. Results. HK cultured just to the confluent state on polyurethane membranewerepositive lor integrins (X5and (X6,major integrins on proliferating HK Histologic analysis showedepithelialized wounds in all groups alter 21 days. Using an anti-human involucrin antibody we demonstratedthe presente01 HK in 64.5 % 01 the HK-HD group, 61 % 01 the DG-HK-HD group, and 0% in the HD group. Mice that receivedthe living human dennal replacementcontaining cultured fibroblasts in combination with HK-HD gralts devetopeda thick, lvell-vascularized neodermis.Strong laminin and collagen N staining lvas observedin wound aTeascoveredwith HK. Conclusions. Thesedata show that lull-thickness wounds can be closedby application 01 a single layer 01proliferating HK cultured on a biocompatiblepolyurethane membrane.1'his technique is an alternative to the use01 multilayered, differentiated HK sheets.Preparation timeslor HK-HD gralts should be signijicantly shorter than requiredlor multilayered HK sheets,technical efforts should be less, and more extensivel/Jound aTeascould be covered. (Surgery 1996,'120:16-22.)~ From theDepartmentso/ Surgery,Medicine,and Orthopedics, Universityo/ California,Sanniego Medical Genter,SanDiego,Calif WHENTHERi\W.INJURIES to the skin are extensive, the availability of donar sites for autografting is limited. Although temporary skin substitutes have been useful for short-term wound coverage,eventuallypermanentgrafting with autologous skin grafts is necessary. An ap,.H. O. RennekampITissupported byagrdntfrom Deutsche Forschungsgemeinschaft. Accepted for publication Oct. 11, 1995. Reprint requests: Oliver RennekampIT, MD, Department of Surgery 8896, Universii). ofCalifomia, San Diego Medical Cerner, 200 W. Arbor Dr., San Diego, CA 92103. Copyright @ 1996 by Mosby-Year Book, Inc. 003%060/96/S5.00 16 SURGERY + O 11/56/71954 proach to accelerate fue availability of graftable tissue is fue expansion of autologous skin by culturing human keratinocytes (HK) in fue laboratory. Advances in serial subculture techniques of human epiderrnal celIs result in high proliferation rates ofHK and fue ability to generate large amounts of epithelium suitable for grafting.l The time period required for fue culture of suitable epithelium is at least 3 to 4 weeks because fue celI sheets must be physicalIy removed from fue culture surface and transferred to the wound; fue sheetsmust be at least four to six layers in thickness to withstand fue trauma. In addition, the culture and preparation processesmar reduce adhesion properties of fue cultured celIs. We report the transplantation of single layers of ke- Surgery Volume120, ¡Vumber1 RennekamPJJel al. Skin Sample HK Suspension 17 HK Culture Epldennls Dennis Panniculus camosus , , , , ;V~~ ~-.~1!!fI ,..Jj ~ I~~~~~~~~~~ ~-H K Hydroderm@ ,, If'Jj ,.jJ .-=7 Fig. l. Sequenceofsteps involved in preparation ofHydroderm-HKgrafts and their application to excised wounds. ratinocytes, cultured on a synthetic polyurethane membrane (Hydroderm; Wilshire Medical Inc., DalIas, Texas), anta fulI-thickness excised wounds in athymic mice. In some of the animals we placed the grafts overlaid on a dermal replacement composed of human fibroblasts cultured on a biodegradable mesh. We algo examined the expression of specific integrins during the culture process and structural characteristics of the healed skin in this animal model of wound closure. MATERIAL AND METHODS Cell culture. Fig. 1 shows fue overall scheme for setting up primary cultures from human skin, establishing secondary cultures on fue polyurethane membrane, and then transfer of grafts to excised wounds on animals. Human keratinocytes (HK) were isolated from cadaveric donor skin obtained from fue University of California SanDiego Regional Tissue Bank. Donor skin was placed overnight in 0.25% trypsin/0.02% ethylenediamine tetraacetic acid (Gibco BRL Life Technologies, Grand Island, N.Y.) at 40C. Mter isolation into a single cell suspension, epidermal cells were resuspended and expanded in number in 75 cm2 polystyrene tissue culture flasksby using Keratinocyte-SFM medium (Gibco). HK were released from the plastic surface with 0.25% trypsin/0.02% ethylenediamine tetraacetic acid. Preparation of fue polyw:ethane-HK grafts. HK were seeded onto a sypthetic hydrophilic dressing, Hydro- derm. Hydroderm has high but variable water vapor permeability and has a biocompatible adhesive coating applied in a diamond-shaped pattem to one sirle of the membrane. Hydroderm has the unique characteristic of markedly but reversibly increasing its moisture-vapor transmission characteristics (MVfR) when it becomes moist, facilitating the local control of wound exudates and wound evaporation. A frame was designed to keep the membrane flat while it was submerged in culture medium. A 7 x 8 cm sterile piece ofHydroderm wasstretched flat in a sterile polycarbonate frame that was constructed in our biomedical engineering shop. HK were seeded at a density of 5 x 104 cells/40 cm2 of Hydroderm and red every 3 days with Keratinocyte-SFM medium. Mter 3 to 5 days when HK had just begun to attain confluent growth (Fig. 2), the membranewas removed from fue culture medium and cut into 2 x 2 cm sections, and sections were transplanted to athymic mice. Application of Hydroderm/HK grafts to animal wounds. Animal experiments were performed with arproval oftheUniversityofCalifomia, San DiegoAnimal ResearchCommittee and in accordance with guidelines prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources,National ResearchCouncil. Athymic mice, 4 to 8 weeks old (Balb/c-nu/nu; Simonsen Laboratories, Gilroy, Calif.) ,were kept in isolation rooms. Surgery and 18 Rennekampff el al. Surgery fuiy 1996 Fig. 2. Photomicrograph of cultured HK attached to Hydroderm 4 days after seeding of cell anta membrane. HK havejust reached the stageof confluent growth. Staining with hematoxylin-eosin;original magnification x400. dressing changes were perfonned in laminar-flow hoods under anesthesia with 10 mg intraperitoneal Avertin (Aldrich Chemical Inc., Milwaukee, Wis.) in 0.4 mI normal saline salution. The dorsolateral surface of the mouse was washed with povidone-iodine and 70% isopropanol. Full-thickness skin defects (2 x 2 cm) were created to a depth sparing the panniculus camosis; grafts were sutured ayer fue defect with 6-0 silk. In fue first group (n = 4) the defect wascovered with Hydrodenn without keratinocytes, which was kept in Keratinocyte-SFM medium for at least 24 hours before fue operation. The second group (n= 11) received Hydrodenn seeded with HK (Hydrodenn-HK). In the third group (n = 14) a 2 x 2 cm piece ofDennagraft, a living dennal replacement composed of human neonatal fibroblasts cultured on polyglactin mesh (Advanced Tissue Sciences Inc., Lajolla, Calif.), was sutured into the defect and covered with a 2 x 2 cm piece ofHydrodenn-HK Grafts were covered with a bulky gauze dressing (Band-aid; johnson & johnson, New Brunswick, NJ.), and animals were wrapped circurnferentially. Mter operation animals were given daily injections of 3 mg ceftazidime (Glaxo Inc., ResearchTriangle Park, N.C.) intraperitoneally for 7 daysafter operation. Sulfamethoxazole and trimethoprim (Biocraft Laboratories, Elmwood Park, N J.) were added to autoclaved drinking water. Animals were examined for the integrity of dressings each dar and were killed on dar 21. Wound areas were excised through the dorsal muscle bed for histologic evaluation. Histologic analysis of ceIls and tissues. For light microscopy, tissue samples were flXed in 10% fonnalin, embedded in paraffin, sectioned at 5 pm thickness, mounted anta slides, and stained with hematoxylineosin. HK-seeded Hydrodenn (1 x 1 cm) wasmounted on slides and stained with hematoxylin-eosin. Immunohistochemical staining was used to identify specific proteins.ldentification ofhuman involucrin in healed wounds on fue athymic mice wasobtained after following fue instructions described in fue Histostain-SP Kit (Zymed Laboratories Inc., San Francisco, Calif.). Frozen sections were fixed, rehydrated, and treated with 10% nonimmune goat serum to block nonspecific binding. Slides were incubated with primary antibody (rabbit anti-human involucrin; Biomedical Technologies Inc., Stoughton, Mass.) for 60 minutes, rinsed, and incubated for 10 minutes with biotinylated secondary goat anti-rabbit antibody. Mter rinsing, enzyme conjugate was applied for 10 minutes and rinsed, and slides were incubated 10 minutes in Substrate-Chromogen Mixture (3-amino-9-ethylcarbazole,AEC). Positive staining revealed a red precipitate in fue superficial epidermal layer. Staining for human collagen type IV and laminin was performed on 7 11mthick, frozen sections, which were treated as above. Specimens were incubated with fue collagen IV primary antibody (rabbit anti-human collagen IV; Chemicon, Temecula, Calif.) and laminin primary antibody, respectively (rabbit anti-human laminin 1;Chemicon). Mter washing,fue biotinylated secondary antibody \vas applied, followed by an additional rinsing and then application afilie enzyme conjugate and SuDstrate-Chromogen Mixture (AEC). Staining for fue integrin Q5and integrin Q6was performed on fue HK~eeded Hydroderm according to fue instructions of fue Vectastain ABC kit (Vector Laboratories Inc., Burlingame, Calif.). Specimenswere blocked with horse serum, followed by human AB serum. Sections were incubated ovemight with fue primary antibody to fue integrins Q5 (1VF4,2.5 l1g/ml) and Qij (S3-41, 2.5 l1g/ml) , respectively. Mterwashing, a biotinylated horse anti-mouse secondary antibody wasapplied. Additional washing was followed by 60 minutes of incubation with biotinylated alkaline phosphate H and by 25 minutes of incubation with fue substrate alkaline phosphatase suDstrate kit 1 (Vector red; Vector Laboratories Inc.). RennekamPff el al. 19 Surgery Volume 120, Number 1 / Fig. 3. A. Photomicrograph ofhealed wound 21 daysafter application ofHydroderm-HK to excisedwound. Stratified layer of epidermal cells restson thin well-vascularizedlayer of connective tissuecovering skeletal muscle. Hematoxylin-eosin stain; original magnification x200. B, Healed wound 21 daysafter application of Hydroderm-HK overlaid on Dermagraft on excisedwound. In contrast to A a very thick layerof well-vascularlzed connective tissue can be seen,containing large numbers of fibroblasts. Hematoxylin-eosinstain; original magnification x200. RESULTS Keratinocyte culture on Hydrodenn membranes. Initial attempts to culture cells on Hydrodenn were perfonned by immersing fue membrane in plastic tissue culture dishes. This resulted in wrinkIing of fue Hydroderrn and floatingofthe membrane to fue surface ofthe culture medium. To avoid wrinkIing of fue Hydroderrn membrane during the culture process an adjustable frame wasdeveloped, asdescribed above. In this wayfue celJscould be uniforrnly seeded onto fue stretched and flat Hydroderrn, and wrinkIing of fue membrane was prevented. The HK became adherent to fue Hydrodenn by 24 hou:rs after inoculation of cells to fue membrane surface. Under light microscopy a sample ofthe cultured graft (Hydroderrn-HK) 4 days after inoculation just before transplantation is shown in Fig. 2. Multiple colonies of HK were seen, resulting in almost confluent cell coverage ofthe membrane. No multilayered epiderrnal afeas were seen at this time point. Cells seemed to attach and proliferate on fue polyurethane membrane and on fue bioadhesive that coated a portion of fue membrane surface in a crisscross pattem. During operation fue Hydroderrn-HK was convenient to handle and could be moved easilyfrom fue culture apparatus to the wounds. No shrinkage or tearing occurred \'(hile culturing celJSon fue membrane or while transferring or suturing fue Hydroderrn to fue wound. However, fue markedly convex con tour of fue dorsum of fue animals, coupled with their constant activity, created partial wrink1ing ofthe Hydrodenn membrane within severa! days of its application to fue wounds. Wrink1ing mar have resulted in some loss of adherence of cultured HK to fue wound surface after transplantation. Gross appearance of healed wounds. Seeded and nonseeded Hydroderm were adherent to fue wound from postoperative dar 2. Peeling of fue Hydrodenn occurred from dar 17 after grafting. Wounds were closed in all groups after17 to 21 days,and a silvery pink appearance of fue woundsurface was observed, which preceded spontaneous separation of fue Hydrodenn from fue healing epithelium. Histologic examination of healed wounds. Mice that received Hydroderm coverage without HK exhibited wound closure by epithelialization and marked wound contraction after 21 days,with onlya small reepithelialized area. Typical photomicrographs of healed wounds 21 days after application of Hydrodenn-HK to an' excised wound are shown in Fig. 3. Mter application of Hydrodenn-HK a stratified layer of epidennal cells can be seen, resting on a thin layer of connective tissue covering skeletal muscle (Fig. 3, A). In Fig. 3, B, a healed wound is shown 21 days after application of Hydrodenn-HK overlaid on Dermagraft on fue excised wound. A thick layer of well-vascularized neodennal tissue can 20 RennekamPff et al. Surgery fUI] 1996 Fig. 4. junction of healed,grafted wound and normal mouse skin is shown. Red staining of suprabasalepitheliallayers on right sideof section (belowcornified layer) with anti-human involucrin antibody identifies human epidermal cells,proving persistenceof transplantedHK. Nonstaining epidermis on leftis murine origin. Hematoxylin-eosinstain; original magnification x200. be seen, containing large numbers of fibroblasts and many blood vessels. Immunohistochemical analysis was used to prove persistence of HK on fue murine wounds. Animals that were treated with Keratinocyte-seeded Hydroderm (HDHK) exhibited a "take" of HK in 20 (64.5%) of 31 animals in this group, determined by positive staining for human involucrin (Fig. 4). Up to 90% of fue length of fue wound section stained with involucrin. The remainder of fue wound showed reepithelialization with murine keratinocytes, which had migrated from the wound margins. In wounds that were closed with Hydroderm atolle, no human skin wasidentified by means of staining for involucrin. Analysis of fue 28 animals that received Dermagraft before HK-HD revealed that 17 (61 %) of fue animals showed positive take of HK, as proved by staining for human involucrin. The areas of human skin were characterized by a well-defined multilayered epidermis at dar 21 and a thick, well-vascularized "neodermis" as described above. Human collagen type IV and laminin were identified at fue dermal-epidermaljunction by means ofimmunohistochemical staining. No collagen type IV and laminin were seen in wounds covered with non-HK-seeded Hydroderm. Collagen type IV and laminin expression was seen in fue animals closed with Hydroderm-HK and with Hydroderm-HK overlaid on Dermagraft, fue living dermal replacement. It was our impression thatstronger, more continual expression ofbasementmembrane antigens was seen in animals that received Dermagraft folIowed by Hydroderm-HK However, no definitive conclusions could be drawn because fue staining tech:' nology does not yield quantifiable data. Analysis of keratinocyte expression of integrins. Integrin expression on HK wasexamined by means of direct staining of cells attached to fue Hydroderm mem- brane. HK that were cultured on Hydrodenn exhibited anti-«6 integrin irnrnunoreactivity at fue cell borders, as exhibited by others in cells cultured in plastic flasks.2 Staining wasalso seen on areas of fue Hydrodenn where cells had detached during specirnen preparation. Distinct Q5 integrin expression by attached cells was also noticed. DISCUSSION Numerous reports on fue use of cultured HK sheets have appeared with variable results. Long-term take is frequently less than 50%,1.-' and late graft loss and friability of healed skin are reported.4 Early take of cultured epithelium is affected by storage and handling of fue cultured sheet grafts.5 Optimal take of cultured HK sheets mar relate to their adhesion to fue wound. Keratinocyte adhesion to the wound bed is mediated by integrins, a family of cell surface receptors.6.7The use of the enzyme dispase,a neutral protease derived from BaciUuspolymyxa,to separate epithelial sheets from fue surface of fue culture flask was an advance in fue development of cultured epithelium. Dispase, a fibronectifiase and type IV collagenase, avoids traumatizing fue thin, fragile epithelial sheets as they separate from fue culture flask surface.8 However, dispase-detached HK sheets mar lose adhesion properties.9 Furthermore, cultured HK, when forming a multilayered epithelium, change their characteristics from a proliferating state to .' a more differentiated epithelium with modified expression of adhesion molecules,10 including loss of fue a5~1 integrinll that promotes cell adherence to fue woundl2. 13and basement membrane. A specialized polyurethane membrane, Hydroderm, wasused as a support for culturing HK in fuese experiments. Hydroderm has unique characteristics that make it a satisfactory dressing for moist wounds, and it also supports fue growth of human censoThe MVfR of Surgery Volume 120, Number 1 Hydroderm is a function of fue degree of wetnessof the membrane. Normal skin has a range of MVTR of 200 to 2000gm-2. day-l , whereas damaged or burned skin mar have water losses as high as 3000 to 5000 gm-2.day-l.14 Conventional hydrophobic polyether urethane films with equilibrium water contents of <1 % have MVTRs of 1500 to 2000 gm-2.day-l. However, when coated with a conventional medical adhesive, fue MVfRfalls to 500 to 1000 gm-2.day-l because fue adhesive blocks water vapor transmission. Conventional films with working MVTRs of <1000 gm-2.day-l are unsatisfactory for use on moist wounds because exudates rapidly accumulate. The high MVTR of Hydroderm results in avoidance of exudate collections. Hydroderm, maintained taut in a frame and submerged in culture medium, promoted attachment and proliferation of HK. Hydroderm with attached HK was easily removed from fue culture chamber and transferred to fue wound without further manipulation. Ease of achieving cell transfer to the wound at an early stage in fue culture process is important. Instead ofwaiting 3 to 4 weeks until multilayered HK sheetsare achieved, cultured cells can be transferred much earlier to the wound bed. In addition, enzymatic release of cultured epithelium from fue culture flask surface and tedious transfer of fragile cell sheets to a support dressing are unnecessary. Finally, cultured HK sheetscontract markedly after their enzymatic release, thereby limiting the wound area that can be closed. Use ofHydroderm as a support for cultured HK not only permits application of grafts to fue wound surface far earlier after injury, but shrinkage of fue cultured graft is avoided during the transplantation process. Calculations suggestthat at fue seeding density we used, a smafi section of skin could yield 500 cm2 ofHydroderm-HKin 17 to 18 days on average, and 14 days later 1 m2could be furnished for fue patient. The development and use of effective dermal replacements appear necessary to achieve functional skin replacements. 1he dermis is now known to play an important role in development and stability of the epidermis. Reconstitution of the dermal-epidermal junction and in particular fue basement membrane is mandatory for optimal skin strength and durability .15Full-thickness wounds that are closed by cultured epithelium frequently exhibit subsequent fragility and breakdown.l, 4 Ultrastructural studies show that after application of cultured HK sheets healed wounds lack critical anatomic structures such as anchoring fibrils,16 perhaps contributing to clinical instability of the 'I'ounds. We have demonstrated that composite grafts, which contain both demial and epidermal elements, achieve accelerated formation of basement membrane stn¡ctures.l For thest; reasons we used a living dermal replacement, Dermagraft, in some afilie experimental groups RennekamPJJ el al. 21 to achieve both epidermal and dermal replacement in fue full-thickness wounds. Dermagraít is a living human dermal replacement tissue that consists of neonatal fibroblasts that are cultured on a biodegradable polyglacrin mesh. During fue culture process fue cells secrete multiple matrix proteins and multiple growth factors.17,18 Dermagraft supports fue adherence and growth of overlaid meshed skin grafts on both animals and human beings and supports growth of cultured keratinocytes in vitro. Because fibroblasts arrear to be nonantigenic in fue allogeneic situation, Dermagraít can be clinically used as a permanent dermal replacemento OUT experiments show that grafting of proliferating keratinocytes attached to Hydroderm, in conjunction with their placement on the living dermal replacement Dermagraft, results in wound closure with a multilayered, differentiated epidermis growing on a well-vascularized tissue thatresembles dermis. Strong and continual staining for laminin and typeIV collagen, important structural proteins at fue dermal-epidermal junction, suggeststhat these graíts replace important anatomic structures in the full-thickness wound. Closure of fue wounds, or take of the cultured grafts, was equally successful in experimental groups with and without fue inclusion of Dermagraít. Subjectively, staining for specific basement membrane proteins appeared more intense and continual in fue wounds that received Dermagraít before application ofHydroderm-HD, although no firm conclusions could be drawn in this regard. Hydroderm is an effective dressing on wounds, controlling exudates because of its high moisture vapor transmission. As HK proliferation progresses on fue wounds and fue cells stratify and differentiate, fue Hydroderm separates from fue epithelialized wound. If this technique can be used in full-thickness wounds on bumed human beings, it mar be useful for replacing both dermal and epidermal components by using a simpler approach than fue use of delicate cultured HK sheets. In addition, coverage of extensive bum wounds with a layer of viable autologous keratinocytes, in conjunction with a living humandermal substrate,could be achieved 1 to 2 weeks earlier compared with cultured epithelial autograít sheets. The loss of integrins during enzymatic detachment of HK sheets mar relate to suboptimal early adherence of cultured cells to fue wound bed. These results mar relate to the relatively low and inconsistent clinical success' in grafting dispase-<letached epithelial cell sheets to wounds. Becauseculture of HK on Hydroderm and direct transfer of fue membrane containing attached cells to fue wound do not require fue use of enzytnatic treatment to detach fue cells before transplantation, thus preserving integrin expression, this potencial obstacle to cultured skin transplantation success might be over- ~ Surgery 22 Rennekampffetal. july 1996 come by this new process. Clinical trials to test this methodology in bumed human beings are planned. 9. Poumay Y, Leclercq-Smekens M, Grailly S, et al. Specific internalization ofbasal membrane domains containing the integrin «6~4 in Dispase-detached cultured human keratinocytes. Eur J These studies were perfonned with fue assistance of fue University of California, San Diego, Institute for Biomedical Engineering Adhesion Receptor Core Facility. Cell BioI1993;60:12-20. 10. Hotchin NA, Watt FM. Transcriptional and post-translational regulation of ~1 integrin expression during keratinocyte termi- REFERENCFS l. Hansbrough]F. Wound coverage with biologic dressings and cultured skin substitUtes. Austin: RG Landes lnc, 1992. 2. Marchisio PC. BondanzaS, CremaDa O. Cancedda R, DeLuca M. Polarized expression ofintegrin receptors (a6r.4,a2r.¡, a3r.¡, and avr.5) and their relationship with the cytoskeleton and basement membrane matrix in cultUred human keratinocytes. J Cell Biol 1991;112:761-73. 3. Rue LW lII, Cioffi WG, McManus WF, Pruitt BAJr. Wound closure and outcome in extensively bumed patients treated with cultured autologous keratinocytes. J Trauma 1993;34:662-8. 4. Desai MH, Mlakar JM, McCauley RL, et al. Lack oflong-term durability of cultured keratinocyte bum-wound coverage: a case report.J Bum Care Rehabill991;12:540-5. 5. Poumay Y, Boucher F, Degen A. Leloup R lnhibition ofbasal cell proliferation during storage of detached epidermal keratinocyte sheets. Acta Derm Venereoll991;71:195-8. . 6. Hynes RO. lntegrins: a family of cell surface receptors. Cell 1987;48:549-54. 7. Marchisio PC. Bondanza S, Cremona O, et al. Polarized expression of integrin receptors and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes. J Cell Bioll991;112:761-73. 8. Stenn K. Link R, Moellmann G. et al. Dispase. a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase. J Invest Dermatol 1989;93:287-90. nal difIerentiation.J Biol Chem 1992;267:14852-8. 11. Adarns JC, Watt FM. Changes in keratinocyte adhesion during terminal difIerentiation: reduction in fibronectin binding precedes a5~1 integrin loss from the cell surface. Cell 1990;63:42535. 12. GailitJ, Clark RAF. Wound repair in the context of extracellular matrix. Curr Opin Cell BioI1994;6:717-25. 13. Takashima A, Grinnel F. Fibronectin-mediated keratinocyte migration and initiation of fibronectin receptor function in vitTO.J Invest DermatoI1985;85:304-8. 14. Larnke LO, Liljedahl SO. Evaporative water loss from bums, grarts and donar sites. ScandJ Plast Surg 1971;5:17-22. 15. Briggarnan RA, Wheeler CEo Epidermal-dermal interactions in adult human skin: role of dermis in epidermal maintenance. J Invest DermatoI1968;51:454-65. 16. Woodley DT, Peterson HD, Herzog SR, et al. Burn wounds resurfaced by cultured epidermal autograrts show abnormal reconstitution of anchoring fibrils. JAMA 1988;259:2566-71. 17. HansbroughJF, MorganJL, GreenleafGE, Bartel R Composite grarts of human keratinocytes grown on a polyglactin mesh-<ultured fibroblast dermal substitute function as a bilayer skin replacement in full-thickness wounds on athymic mice. J Bum Care RehabiI1993;I4:485-94. 18. HansbroughJF, Doré C, Hansbrough WB. Clinical trials ora living dermal tissue replacement placed beneath meshed, splitthickness skin grarts on excised burn wounds. J Burn Care RehabiI1992;13:519-29. ~