Functional Group Chemistry Modulates Cellular Responses to Soluble Gelatin Derivatives Independent of Crosslinking

Gelatin functionalized with methacrylate (GelMA), norbornene (GelNB), and phenol (GelPH) has been studied as a precursor for crosslinkable biomaterials in tissue engineering. Cytocompatibility is usually assessed post-hydrogel formation, but cells briefly encounter soluble precursors during preparation and residual functional groups that may remain post-gelation. However, the biological effects of these uncrosslinked derivatives remain poorly understood. This study systematically investigated the impact of functional group chemistry on cellular responses under uncrosslinking conditions. Four cell lines, including fibroblasts (BALB/3T3), cervical cancer cells (HeLa), mesenchymal stem cells (UE7T-13), and neuronal cells (PC-12), were exposed to soluble GelMA, GelNB, and GelPH at matched polymer concentrations and degrees of functionalization (~50%). The results showed no overt cytotoxicity. The detectable differences were subtle and strongly cell type-dependent. HeLa and PC-12 cells showed no clear differences from untreated controls in most parameters. BALB/3T3 fibroblasts showed mainly GelMA-associated differences in mitochondrial activity, proliferation, and morphology. In the UE7T-13 cells, GelMA-treated cells showed the largest deviations from controls across the readouts examined, including mitochondrial activity, cell area, phenotype-related gene expression, and some osteogenic markers, while GelPH was intermediate and GelNB was comparable to controls. These findings underscore the importance of considering residual precursor effects in gelatin-based biomaterial design.