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In order to improve the regeneration of full-layer skin defects, hydrogels were developed based on the combination of chitosan (Cs), Daba silk fibroin (DSF), and graphene oxide (GO): CS, DSF/Cs and DSF/Cs/GO. The biocompatibility of hydrogels with human dermis fibroblasts in vitro was evaluated using the MTS assay. To assess the regenerative potential of hydrogels, a model of a full-layer skin defect was reconstructed on the back of rats and closed the wound surface with CS, DSF/Cs and DSF/Cs/GO hydrogels. The morphological and morphometric characteristics of regenerate tissues were obtained by staining with hematoxylin-eosin, Heidengain azocarmine, and immunohistochemistry on days 7 and 14 of the experiment. It has been shown that the use of DSF/Cs and DSF/Cs/GO promotes enhanced healing and epithelization of a full-layer skin wound. The addition of GO to the hydrogel increased the synthetic activity of fibroblasts and improved the characteristics of the produced collagen fibers.