Comparison of Crosslinking Reagents and Their Impacts on Bone Derived ECM Hydrogels

Extracellular matrix (ECM) derived biomaterials are becoming more prevalent for regenerative medicine applications as they can provide bio-instructive cues to damaged tissues, facilitating constructive remodeling of the injured site. Existing clinical products based on ECM can be found in sheet or powder formats, however recent clinical trials have utilized ECM hydrogels. Further exploration of ECM hydrogels and modulation of their mechanics, degradation and swelling behaviors could provide increased utility of these materials. Mechanical modulation through crosslinking can be achieved through application of chemical reagents such as glutaraldehyde (GA). However long-term effects of GA warrant increased research of alternatives such as the plant-based derivatives genipin (GP) and proanthocyanidins (PA). In this work, crosslinking was confirmed through alterations to the mechanical moduli of bECM hydrogels with both GP and PA conferring significantly increased moduli compared to non-crosslinked controls. In vitro examination determined that cytotoxic effects were observed for PA to SH-SY5Y cells at the concentration used, however no cytotoxicity was observed from PA when tested on L929 cells and primary tenocytes. There was no cytotoxicity observed from GP treatment. Therefore, use of these reagents could provide a further toolkit to manipulate physicochemical properties and increase utility of ECM hydrogels.