14 We developed a novel skin regeneration therapy combining nevus tissue inactivated by high 15 hydrostatic pressure (HHP) in the reconstruction of the dermis with a cultured epidermal 16 autograft (CEA). The issue with this treatment is the unstable survival of CEA on the 17 inactivated dermis. In this study, we applied collagen/gelatin sponge (CGS), which can sustain 18 the release of basic fibroblast growth factor (bFGF), to the inactivated skin in order to 19 accelerate angiogenesis. Murine skin grafts from C57BL6J/Jcl mice (8 mm in diameter) were 20 prepared, inactivated by HHP and cryopreserved. One month later, the grafts were transplanted 21 subcutaneously onto the back of other mice and covered by CGS impregnated with saline or 22 bFGF. Grafts were taken after one, two and eight weeks, at which point the survival was 23 evaluated through the histology and angiogenesis-related gene expressions were determined by 24 real-time polymerase chain reaction. Histological sections showed that the dermal cellular 25 density and newly formed capillaries in the bFGF group were significantly higher than in the 26 control group. The relative expression of FGF-2, PDGF-A and VEGF-A genes in the bFGF 27 group was significantly higher than in the control group at Week 1. This study suggested that 28 the angiogenesis into grafts was accelerated, which might improve the survival in combination 29 with the sustained release of bFGF by CGSs.3 31 Introduction 32 Basic fibroblast growth factor (bFGF) is an essential mitogen that plays a crucial role in the 33 wound healing processes by not only stimulating cell growth and differentiation but also 34 inducing neovascularization and connective tissue synthesis [1][2][3]. As human recombinant 35 bFGF has been commercially available in Japan since 2001, its topical administration has been 36 shown to be effective for wound healing in clinical treatments and has recently been applied 37 more extensively [4][5][6]. However, the disadvantage of this medication is the need for its daily 38 administration on the wound due to its short half-life in vivo [2]. To overcome this issue, we 39 developed a novel collagen/gelatin scaffold (CGS) containing 10wt% acidic gelatin that is 40 capable of the sustained release of a charged growth factor, such as bFGF [7], platelet-derived 41 growth factor (PDGF) [8] or hepatocyte growth factor (HGF) [9] for more than 10 days. We 42 previously showed that CGS impregnated with bFGF at 7-14 μg/cm 2 achieved the most 43 efficient promotion of angiogenesis and dermis-like tissue formation, even in a delayed wound-44 healing model of diabetic mice [10][11][12].
45Recently, a number of different decellularization methods, including chemical, biological 46 and physical and miscellaneous agents, have been suggested to be useful for generating 47 superior bioengineering tissue [13]. Decellularized tissue retains its native structure or 48 mechanical properties and has been reported to show low or no immunogenicity [14, 15], 49 making it an ideal substitute or ...