Crosslinking of genipin and autoclaving in chitosan-based nanofibrous scaffolds: structural and physiochemical properties

Chitosan-based electrospun nanofibrous scaffolds have been selected as wound healing/tissue scaffolds because of their extracellular matrix nature and biocompatible properties. However, crosslinking of scaffolds is necessary to avoid lysozyme degradation in an aqueous environment, as a stable scaffold is crucial for the activities of fibroblasts, including adhesion and proliferation during wound healing. Autoclaving (physical) and genipin crosslinking (chemical) methods have been employed to stabilize chitosan-based scaffolds individually. However, the differences in scaffold microstructure induced by the individual or combined crosslinking methods have yet to be investigated systematically. In this study, autoclaving crosslinking improved mainly the structural properties (tensile strength and crystallinity), but it also expanded the chitosan and PEO network by hydrolysis, which enlarged the fiber diameter and caused chitosan chain degradation. Meanwhile, genipin crosslinking improved the physiochemical properties, primarily hydrophilicity. On the other hand, the combined crosslinking significantly improved both the structural and physiochemical properties through the unique reorganization of the polymeric network. The confined geometry of the nanofiber as well as the genipin crosslinks resulted in maximal crystallization of chitosan and amorphization of PEO chains. Unfortunately, the combined crosslinking resulted in the lowest antibacterial activity because of the consumption of amino and protonated amino groups in the crosslinking process. Despite this, the combined crosslinking scaffold achieved the best stability under lysozyme degradation and therefore it is preferred over autoclaving or genipin crosslinking alone. In conclusion, the results show that chemical and physical crosslinking methods induce different changes in crystallinity and hydrophilicity that affect the physicochemical properties. Therefore, crystallinity and hydrophilicity are significant considerations when designing a tissue scaffold.