Developing a Skin Construct for Wound Healing Associated with Endothelial Cell Network

Researchers have recently engineered skin constructs incorporating epidermal and dermal layers from the collagen matrix. A significant disadvantage in designing this organotypic culture for wound healing applications is the lack of a vascular network that promotes anastomosis of the graft-host vasculature. Meanwhile, relying on the additive growth factor to promote vascularization and angiogenesis is not sustainable and could be costly. Mesenchymal cells like fibroblasts and dental pulp stem cells are known to secrete several pro-angiogenic growth factors. Hence, our objective in this research is to (a) establish a microvascular network by co-culturing human umbilical vein endothelial cells (HUVECs) with mesenchymal cells in vitro without additional growth factors, (b) develop a series of altered mediums where both the endothelial and the epithelial cells can be co-cultured, (c) determine the role of an endothelial cell network in predicting vascularization and anastomosis response in vivo and (d) evaluating the impact of these constructs on wound healing.<br/><br/>Living skin equivalents were prepared for 17 days according to the procedures from our previous work [1], modified with the HUVECs-EGFP and mesenchymal cells co-culture layer on the bottom of skin constructs, cultured in the altered mediums to enhance both vascularization and epithelial differentiations. The non-vascularized skin equivalents with the same bottom layer without HUVECs were prepared similarly for comparison. Three SCID Hairless Outbred mice were used as hosts for wound healing experiments. 8mm diameter wound beds were created on the back of the mice. The harvested skin equivalents were then placed on the wound beds and bandaged with silicone film to prevent dehydration during healing.<br/><br/>Following a 7-day period of postoperative recovery, skin tissue samples were collected for histological analysis. Hematoxylin and eosin (H&E) staining revealed a seamless integration between the graft and the surrounding epithelium and dermis. Additionally, lumens were scattered throughout the grafted tissue, identified as human using anti-EGFP staining, and filled with blood cells. The luminal diameters of the human blood vessels ranged from approximately 10um to 40um without observable leakage, confirming successful anastomosis. The joining of blood vessels between graft and host is a necessary milestone for successful tissue regeneration and long-term engraftment.<br/><br/>In conclusion, we have designed a highly effective scaffold that enables the simultaneous culture of HUVEC networks with skin organotypics, promoting anastomosis and accelerating wound healing.<br/><br/><i>We acknowledge the Morin Charitable Trust for funding.</i><br/><br/>[1] Li, Juyi, et al. "Engineering functional skin constructs: A quantitative comparison of three dimensional bioprinting with traditional methods." Experimental Dermatology 31.4 (2022): 516-527./