Adipose-derived stem cell (ASC) is a potential source of abundant mesenchymal stem cells, which can be applied to promote tissue regeneration. We manipulated spheroid formation of human ASCs by culturing them on chitosan films and found enhanced expression of stemness markers Sox-2, Oct-4 and Nanog. ASCs within cell spheroids also exhibited enhanced neurogenic and hepatogenic transdifferentiation capabilities relative to monolayer ASCs, while adipogenic and osteogenic differentiation capacities of ASCs were still maintained. Moreover, spheroid-derived ASCs expressed significantly more angiogenic growth factors. We further aimed to develop chitosan-based biomaterials for ASC delivery, and composite films with different ratios of chitosan and gelatin were fabricated. While ASCs formed cell aggregates on pure chitosan films without obvious proliferation, ASC attachment and growth on pure gelatin films was similar to that of common culture plates. With an adequate ratio of chitosan and gelatin, ASCs were found to maintain their proliferative activity with enhanced motility, allowing them to gradually detach from the film for tissue repair.
We also developed a thermosensitive chitosan/gelatin hydrogel for ASC encapsulation and sustained release. By blending gelatin into the chitosan hydrogel, we significantly enhanced the viability of the encapsulated ASCs. During in vitro culturing, the gradual degradation of gelatin led to sustained release of ASCs from the chitosan/gelatin hydrogel. In vitro wound healing assays revealed significantly faster cell migration by co-culturing fibroblasts with ASCs encapsulated in chitosan/gelatin hydrogel compared to pure chitosan hydrogels. Additionally, co-culturing SVEC4-10 endothelial cells with ASC-encapsulated chitosan/gelatin hydrogels resulted in significantly more tube-like structures, indicating the hydrogel’s potential in promoting angiogenesis. Chick embryo chorioallantoic membrane assay and mice wound healing model showed significantly higher capillary density after applying ASC-encapsulated chitosan/gelatin hydrogel. Relative to ASC alone or ASC-encapsulated chitosan hydrogel, more ASCs were also found in the wound tissue on post-wounding day 5 after applying ASC-encapsulated chtosan/gelatin hydrogel. In summary, chitosan-based biomaterials are beneficial in the stemness enhancement and sustained delivery of ASCs for tissue regeneration.

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