Development of injectable microgel-based scaffolds via enzymatic cross-linking of hyaluronic acid-tyramine/gelatin-tyramine for potential bone tissue engineering.

Currently, the healing of large bone defects relies on invasive surgeries and the transplantation of autologous bone. As a less invasive treatment option, the provision of microenvironments that promote the regeneration of defective bones holds great promise. Here, we developed hyaluronic acid (HA)/gelatin (Ge) microgel-based scaffolds to guide bone regeneration. To enable the formation of microgels by enzymatic cross-linking in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂), we modified the polymers with tyramine (TA). Spectrophotometry and proton nuclear magnetic resonance (¹H NMR) spectroscopy analysis confirmed successful tyramine substitution on polymer backbones. To enable the formation of microgels by a water-in-oil emulsion approach, the HRP and H₂O₂ concentrations were tuned to achieve the gelation in a few seconds. By varying the stirring speed from 600 to 1000 rpm, spherical microgels were produced with an average size of 116 ± 8.7 and 68 ± 4.7 μm, respectively. The results showed that microgels were injectable through needles and showed good biocompatibility with the cultured human osteosarcoma cell line (MG-63). HA/Ge-TA microgels served as a promising substrate for MG-63 cells since they improved the alkaline phosphatase activity and level of calcium deposition. In summary, the developed HA/Ge-TA microgels are promising injectable microgel-based scaffolds in bone tissue engineering.