Characterization of pentenoate-functionalized hyaluronic acid and pentenoate-functionalized gelatin hydrogels for printing and future surgical placement in regenerative medicine applications

Injectable hydrogels with in situ crosslinking may be more suitable than pre-fabricated scaffolds for surgical delivery to clinical injuries. However, low viscosity hydrogel precursors may be challenging to surgically place into an injury if the precursor leaks or is washed out. The biomaterials field for extrusion bioprinting is a fertile ground for discovering biomaterials with injectable and paste-like precursor rheology with in situ gelation capabilities, which may promote better material retention in clinical injuries. We previously developed and evaluated one formulation of a pentenoate-functionalized hyaluronic acid (PHA) / pentenoate-functionalized gelatin (PGel) hydrogel with a paste-like, printable precursor and rapid photocrosslinking in a spinal cord injury application. Further characterization of the material and cell response to PHA/PGel hydrogel formulations is needed to expand the bioprinting and other regenerative medicine opportunities for PHA/PGel hydrogels. In the current study, we utilized 2D NMR methods (i.e., ¹H–¹H TOCSY) to confirm and quantify a high degree of pentenoate functionalization of PGel and PHA. We characterized the stiffness, swelling, and cell viability using varying formulations of PGel or PHA/PGel hydrogels. For compression testing, a straightforward application of the Ogden model enabled evaluation of the full stress-strain range for improved moduli comparisons. We identified two formulations that best supported cell viability (i.e., 3%/10% and 4%/5% PHA/PGel). Furthermore, one of the identified formulations (4%/5% PHA/PGel) had superior printability compared to the other. With better printability and potentially better clinical surgical placement, the new PHA/PGel hydrogel formulations may be more widely applied in the bioprinting and regenerative medicine fields.