Dual crosslinking strategy to generate mechanically viable cell-laden printable constructs using methacrylated collagen bioinks.

Photopolymerization of methacrylated collagen (CMA) allows for 3D bioprinting of tissue scaffolds with high resolution and print fidelity. However, photochemically crosslinked CMA constructs are mechanically weak and susceptible to expedited enzymatic degradation in vivo. The goal of the current study was to develop a dual crosslinking scheme for the generation of mechanically viable cell-laden printable constructs for tissue engineering applications. Dual crosslinking was performed by first photochemical crosslinking of CMA hydrogels using VA-086 photoinitiator and UV exposure followed by chemical crosslinking with two different concentrations of genipin (i.e., 0.5 mM (low dual) or 1 mM (high dual)). The effect of dual crosslinking conditions on gel morphology, compressive modulus, stability and print fidelity was evaluated. Additionally, human MSCs were encapsulated within CMA hydrogels and the effect of dual crosslinking conditions on viability and metabolic activity was assessed. Uncrosslinked, photochemically crosslinked, and genipin crosslinked CMA hydrogels were used as controls. SEM results showed that gel morphology was maintained upon dual crosslinking. Further, dual crosslinking significantly improved the compressive modulus and degradation time of cell-laden and acellular CMA hydrogels. Cell viability results showed that high cell viability (i.e., >80%) and metabolic activity in low dual crosslinked CMA hydrogels. On the other hand, cell viability and metabolic activity decreased significantly (p < 0.05) in high dual crosslinked CMA hydrogels. Quantitative fidelity measurements showed the measured parameters (i.e., line widths, pore size) were comparable between photochemically crosslinked and dual crosslinked constructs, suggesting that print fidelity is maintained upon dual crosslinking. In conclusion, application of low dual crosslinking is a viable strategy to yield mechanically superior, cell compatible and printable CMA hydrogels.