Guanidinylated/PEGylated chitosan in the bioink promotes the formation of multi-layered keratinocytes in a human skin equivalent

The biological differences of skin between rodent and human beings and the strong appeal to replace the experimental animals have led to the development of alternative models with structures similar to the real human skin. Keratinocytes cultured in vitro on conventional dermal scaffolds tend to form monolayer rather than multi-layer epithelial tissue architectures. How to construct human skin or epidermal equivalents with multi-layered keratinocytes similar to real human epidermis remains one of the greatest challenges. Herein, a human skin equivalent with multi-layered keratinocytes was constructed by 3D bioprinting fibroblasts and subsequent culturing epidermal keratinocytes. Biocompatible guanidinylated/PEGylated chitosan (GPCS) was used as the main component of bioink to 3D bioprint tissue-engineered dermis. The function of GPCS to promote HaCat cell proliferation and connection was confirmed at the genetic, cellular, and histological levels. Compared with the skin tissues with mono-layered keratinocytes engineered with collagen and gelatin, adding GPCS in the bioink generated tissue-engineered human skin equivalents with multi-layered keratinocytes. Such human skin equivalents could be alternative models for biomedical, toxicological, and pharmaceutical research.