Development and characterization of crosslinked collagen biomaterial inks for 3D bioprinting applications

Limited organ availability and transplantation risks have driven the development of tissue engineering approaches. This study developed and characterized crosslinked collagen biomaterial inks extracted from calf skin for three-dimensional bioprinting applications. Collagen was extracted using pepsin digestion and purified through dialysis. Biomaterial inks were prepared at 3%, 4%, and 5% (w/v) concentrations and crosslinked using genipin (1, 3, 5 mM) and riboflavin (1 mM) with UV-A activation. Optimal printing parameters were determined as 5% (w/v) collagen concentration with 0.26 mm nozzle diameter. Synchrotron FTIR spectroscopy confirmed successful crosslinking through characteristic peak shifts in amide regions. Mechanical testing revealed enhanced compressive strength: riboflavin-crosslinked scaffolds (1.5 ± 0.08 MPa) > genipin-crosslinked scaffolds (1.19 ± 0.12 MPa) > uncrosslinked scaffolds (0.66 ± 0.03 MPa). Cell viability assessments demonstrated that genipin crosslinking at 1 mM concentration significantly enhanced fibroblast viability (181.2 ± 29.32% compared to uncrosslinked controls), while higher concentrations exhibited cytotoxic effects. Riboflavin biocompatibility assessment was limited by methodological constraints due to spectral interference, preventing reliable comparative evaluation. These results demonstrate that genipin crosslinking successfully enhances both mechanical properties and biocompatibility at appropriate concentrations, while riboflavin crosslinking provides superior mechanical reinforcement but requires alternative biocompatibility assessment methods for comprehensive characterization.