Osteoimmunomodulatory effects of biomimetic porous carboxymethyl chitosan/alginate/hydroxyapatite composite hydrogel scaffolds

Recent advances in osteoimmunology have underscored the crucial role of the host immune response in bone regeneration. In this study, we developed a double-crosslinked carboxymethyl chitosan-alginate-hydroxyapatite composite hydrogel bone scaffold (CAHA) by directional freeze-casting. The content of HA (15 wt%, 25 wt%, 35 wt%, and 45 wt%) has significantly influenced the physical properties of the scaffold, including pore size, porosity, swelling behaviour, Ca 2+ ion release, surface stiffness, and compressive modulus. In vitro experiments indicated that the CAHA scaffold upregulated the expression of anti-inflammatory genes ( p <  0.0001) and downregulated the expression of proinflammatory genes ( p <  0.0001) in RAW 264.7 macrophages, promoting a shift from the M1 phenotype to the M2 phenotype. The M2 phenotype effectively enhanced osteogenic differentiation of MC3T3-E1 cells and angiogenesis in HUVECs. In vivo studies using an air pouch model revealed a greater number of M2 macrophages surrounding the CAHA scaffolds. Tibial defect experiments showed that 8 weeks after implantation, the group treated with the CAHA scaffold containing 45 wt% HA exhibited significant bone healing compared with the control group ( p <  0.05). Thus, the novel CAHA composite hydrogel scaffold shows promise as an immunomodulatory biomaterial for bone tissue engineering.