In Vitro Cell.Dev.Biol.-Animal 31:432-439, June1995 © 1995 Society for In Vitro Biology 1071-2690195 $05.00 + 0.00 SKIN EQUIVALENT PRODUCED WITH HUMAN COLLAGEN FRANCOIS A. AUGER, 1 CARLOS A. LOPEZ VALLE, RINA GUIGNARD, NATHALIETREMBLAY,BERNARD NOEL, FRANCINE GOULET,and LUCIE GERMAIN Laboratoire de Recherchedes Grands Bral~s/LOEX, Saint-SacrementHospital, Quebec, QC, Canada, GIS 4L8; and Department of Surgery and Department of Microbiology, Laval UniversityFaculty of Medicine, Sainte-Foy, QC, Canada, G1K 7P4. (Received 6 July 1994; accepted 10 February 1995) SUMMARY Several studies have recently been conducted on cultured skin equivalent (SE), prepared using human keratinocytes seeded on various types of dermal equivalents (DE). We previously showed the advantages of our anchorage method in preventing the severe surface reduction of DE due to fibroblast contractile properties in vitro. A new anchored human SE was established in our laboratory in order to obtain a bioengineered tissue that would possess the appropriate histological and biological properties. In order to compare the effects of different collagen origins on the evolution of SE in vitro, human keratinocytes were seeded on three types of anchored DE. A comparative study was carried out between bovine SE (bSE), human SE (hSE), and human skin equivalent containing additional dermal matrix components (hSE + ). Immunohistological analysis showed that hSE and hSE + presented good structural organization, including the deposition of several basement membrane constituents. Higher amounts of transglutaminase, ceramides, and keratin 1 were detected in the epidermal layers of all SE when cultured at the air-liquid interface. However, a 92 kDa gelatinase activity was higher in bovine skin equivalent (bSE) compared to hSE cultures. The use of human collagens comparatively to bovine collagen as SE matricial component delayed the degradation of the dermal layer in culture. Key words: skin substitute; human collagen. INTRODUCTION Since 1983, the method of autologous epithelial sheet grafting has been used for permanent burn wound coverage and had significant impact on the therapeutical options of burn victims (10,17,29). Al- though full terminal epithelial differentiation is obtained in vivo post- grafting (1,2), some drawbacks have been revealed over time. This treatment does not allow a complete skin regeneration before 2 to 5 yr in young patients because the dermal component undergoes a slow reorganization (10). Moreover, elastic fibers are absent during the 3 yr that follow the transplantation. The basement membrane and an- choring structure formation postgrafting only appear when some mes- enchyme-epithelium interactions occur in situ (13). Therefore, the addition of a dermal counterpart to these epithelial sheets would be highly desirable to accelerate wound healing and skin regeneration posttransplantation. In this regard, the use of collagen gels as sup- portive matrix to grow cells was first described by Ehrmann and Gey (12). Three-dimensional dermis-like structure obtained after the con- traction of collagen lattices seeded with human fibroblasts in vitro was introduced by Bell et al. (5). A first floating SE was then reported in 1981 (4). The main limitation of this skin model remains its severe contraction that occurs very early in culture and greatly reduces its use for wound coverage in vivo. During the last few years, various skin models have been proposed ~To whom correspondence should be addressed at Laboratoire de Re- cherche des Grands Brfilds, Hrpital du Saint-Sacrement, 1050 Chemin Sainte-Foy, Qudbec, QC, Canada, G1S 4L8. 432 by different laboratories for pharmacological, toxicological, or bio- logical studies or for human wound coverage. These models are ob- tained by human keratinocytes that will grow into a differentiated epidermis when plated on different DE such as de-epidermized der- mis (26), biodegradable sponges (8,18,30), fibroblasts grown on nylon mesh (11), or within collagen gels (7,22). However, most of these skin models are produced using dermal collagens from animal source or allogeneic human material and thus may not always be suitable for transplantation onto patients because the physiological and im- munological effects of such components have not been yet clearly defined in vivo. Indeed, some allogeneic collagen epitopes can be recognized by the graft recipient in vivo (9,20) and may lead to im- munological reactions following the transplantation of human SE containing animal collagen matrix. A new skin substitute was produced in our laboratory using cells and collagens from human source in order to obtain an improved graftable cutaneous equivalent for the treatment of extensively burned patients. In the present study, various characteristics of our SE were evaluated in order to compare bovine and human collagen matrix as to their effects on proliferation and differentiation of kerati- nocytes in vitro. MATERIALS AND METHODS Human Cutaneous Cells Human keratinocytes and dermal fibroblasts were isolated from skin biop- sies removed during mammaryreductive breast surgery of healthy subjects.