Exploration of the importance of viable cells in skin micrograft for wound healing

Abstract

Abstract Background: Skin micrograft (MG) has been reported to promote tissue regeneration and is clinically used for intractable wounds. MG contains diverse cells and an extracellular matrix (ECM); however, the importance of the proliferative potential of MG cells for wound healing is not clear. Therefore, we investigated the wound healing-promoting effect of MG, in which the cells were inactivated using several methods. Methods: Skin MG was prepared from the skin of C57BL mice using Rigenera protocol, and then four types of MGs were prepared: Fresh-MG, HHP-MG (treated with high hydrostatic pressure of 200MPa for 10 min), LN-MG (treated with liquid nitrogen for 20 min), and Heat-MG (treated heat with 80℃ for 30 min). The outgrowth potential, growth factor content (basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF)), and in vitro cell proliferation-promoting potential were examined for each type of MG. For in vivo studies, MG was used to treat murine skin defects. The wound area, neoepithelial length, granulation tissue formation, and newly formed capillaries were evaluated on day 7. Results: All types of MGs, except for Heat-MG, contained bFGF, EGF and VEGF, promoted cell proliferation, and accelerated wound closure. In addition, the neoepithelial lengths in the HHP-MG and LN-MG groups were significantly longer than those in the Heat-MG and control groups. The area of granulation tissue, and the number and area of capillaries in the Fresh-MG, HHP-MG, and LN-MG groups were significantly larger than those in the control groups. Conclusions: The results confirmed that proliferative cells are not essential for the promotion of wound healing by MG. This effect was attenuated in the Heat-MG group, suggesting that soluble factors, such as growth factors, which are inactivated by heat, play an important role.