Demonstration of re-epithelialization in a bioprinted human skin equivalent wound model

Wound healing is life-dependent feature for animal survival. After a skin insult, keratinocytes, fibroblasts, and immune cells are recruited to restore the protective skin function. Immediately after injury, soluble fibrinogen is cleaved by thrombin and polymerize to insoluble fibrin clot. Immune cells infiltrate into the fibrin clot to activate the inflammatory response. Surrounding keratinocytes in contact with the fibrin clot migrate to the wounded area meanwhile distant keratinocyte proliferate. However, in vitro wound models have important limitations related to wound fibrinogen polymerization and immune cell recruitment. This study expands previous in vitro 3D skin equivalent co-culture platforms to include the installation of a physiologically relevant fibrin provisional matrix. Method: Here, we developed a fibrin provisional matrix installed into a wound facsimile of a bioprinted human skin equivalent (HSE). A mixture of plasma-derived fibrinogen-containing factor XIII, fibronectin, thrombin, and macrophages (an FPM “bioink”) was extruded into the wound site. The surrounding in vitro tissue culture became a source of keratinocytes to achieve wound closure by a re-epithelialization process signaled by the FPM. Results: An in vitro analog of wound closure and re-epithelialization by keratinocytes occurred over the FPM after a normal migration initiation at 3 days. Conclusion: This co-culture model was shown to temporally synchronize a re-epithelization process for initiation of keratinocyte migration from a surrounding tissue and the migration process over the top of an FPM. A future study of the analogous subepithelial healing pathway is envisioned using the same in vitro bioprinted tissue study platform for co-culture of keratinocytes, melanocytes, fibroblasts, endothelial cells, and macrophages using more specialized FPMs.