Commercially available cultured epithelial keratinocyte sheets (KSs) have played an essential role in wound healing over the past four decades. Despite the initial uptake by the dermal elements, the survival rate of KS on the dermis-like tissue generated by conventional artificial dermis (AD) is low, making this method unsuitable for standard treatments. Therefore, an innovative AD such as collagen-gelatin sponge (CGS) that maintains the release of human recombinant basic fibroblast growth factor (bFGF) may promote wound healing. In this study, we examined whether combination therapy with KSs and CGS with bFGF (bFGF-CGS) could enhance KS survival by heterologous grafting by transplantation of human-derived KSs in an athymic nude rat wound model of staged skin reconstruction. The CGSs were implanted into skin defect wounds on athymic nude rats, which were then divided into two experimental groups: the bFGF group (CGSs containing bFGF, n = 8) and the control group (CGSs with saline, n = 8). Two weeks after implantation, human epithelial cell-derived KSs were grafted onto the dermis-like tissue, followed by assessment of the survival and morphology at 1 week later using digital imaging, histology (hematoxylin and eosin and Masson's trichrome staining), immunohistology (von Willebrand factor), immunohistochemistry (cytokeratin 1-5-6, Ki-67), and immunofluorescence (collagen IV, pan-cytokeratins) analyses. The bFGF group showed a significantly higher KS survival area (86 ± 58 mm2 vs. 32 ± 22 mm2; p < 0.05) and increased epidermal thickness (158 ± 66 μm vs. 86 ± 40 μm; p < 0.05) compared with the control group, along with higher dermis-like tissue regeneration, neovascularization, epidermal maturation, and basement membrane development. These results indicate that the survival rate of KSs in the dermis-like tissue formed by bFGF-CGS was significantly increased. Therefore, combination treatment of bFGF-CGS and KSs shows potential for full-thickness skin defect reconstruction in clinical situations. Impact statement This study highlights how using a combination of cultures, keratinocyte sheets, and collagen-gelatin sponge containing basic fibroblast growth factors can significantly improve cell survival in athymic nude rats with staged skin reconstruction. Our study makes a significant contribution to the literature because it highlights a novel and improved strategy for treating a very common condition such as skin wounds arising from many conditions. Clinical translation of this study may be useful for treating skin wounds.
Keywords: artificial dermis; basic fibroblast growth factor; collagen–gelatin sponge; epithelial keratinocyte sheet; keratinocyte; survival.