Dual-crosslinked chitosan nanofibres - Balancing mechanical enhancement with cytotoxic risk

This study evaluated the effects of dual-crosslinking strategies on the mechanical performance and biocompatibility of chitosan-norbornene (Chi-NB) nanofibres for tissue reconstruction. Chi-NB, bearing 28.6 ± 1.4 mol% alkene groups, was processed into nanofibres via solution blow spinning. Dual crosslinking was conducted by photo-initiated thiol-ene crosslinking with dithiothreitol (DTT), achieving >95% norbornene conversion with minimal thiol oxidation, followed by exposure to tannic acid (TA). Structural and chemical validation was performed using SEM, NMR, XPS, FT-IR, and tensile testing. In vitro, unmodified fibres supported robust cell viability, however, TA/DTT-modified fibres caused up to 15-fold higher LDH release at low seeding-densities, mirrored in indirect conditioned medium exposure, implicating cytotoxic leachates. Confocal imaging confirmed cell adhesion and cytoskeletal organisation whereas transcript analysis revealed altered expression of genes related to stress response, metabolism, growth, and differentiation. Overall, TA/DTT crosslinking improved nanofibre mechanical properties but introduced bioactive residues, highlighting the need for post-processing and seeding density optimisation. These findings support the translational potential of Chi-NB scaffolds in skin and neural tissue engineering, but future refinements are required for these applications.