Micro- and Nano-Structuring of Hydroxyapatite-MMT-Loaded Hydrogels for Bone Regeneration Applications.

Bone regeneration focuses on the creation of functional tissue to repair bone defects. Creating a biodegradable scaffold hydrogel that combines a hemostatic agent with bioactive ceramics can afford the biological and mechanical benefits of both components. In the present study, we developed an injectable gelatin-alginate dual-composite hydrogel, loaded with two functional fillers: hydroxyapatite (HA) and the hemostatic agent montmorillonite (MMT). HA (microparticles and nanoparticles) was incorporated at concentrations of 10-30 mg/mL, with and without MMT at 20 mg/mL. The effects of functional fillers and their concentration on the microstructure and resulting physical and mechanical properties were studied, and a qualitative model summarising these effects was developed. All formulations exhibited clinically appropriate gelation times (5-29 s). n-HA significantly prolonged gelation time, reaching 29 ± 3 s at 30 mg/mL, while MMT reduced gelation time at all concentrations. The tensile strength of the unloaded hydrogel reached 20 kPa and increased to 57 kPa with 30 mg/mL of n-HA. The tensile strength even increased further with the addition of MMT (77 kPa). The results indicate that the combination of HA and MMT produced dual micro-composite hydrogels with moderate reinforcement, whereas the combination of n-HA and MMT generated dual nano-micro composites with combined reinforcing effects. The latter exhibited the highest strength and sealing ability while maintaining clinically relevant gelation times and controlled swelling behaviour. In conclusion, the combination of MMT with n-HA or HA enables the creation of functional hydrogels with controlled properties, tailored to specific applications in bone regeneration.