Multivalent ion-based in situ gelling polysaccharide hydrogel as an injectable bone graft

•In situ gelling ALG/HA hydrogel with a controllable gelation rate is prepared.•BMP-2 immobilized in the ALG/HA hydrogel is continuously released over 5 weeks.•Osteogenic differentiation of hBMSCs is improved with increasing HA composition.•The hBMSCs/BMP-2-immobilized ALG/HA hydrogel greatly enhances bone regeneration. We prepared in situ gelling alginate (ALG)/hyaluronic acid (HA) hydrogels with a controllable gelation rate using CaSO4 as a crosslinking agent and Na2HPO4 as a crosslinking retardation agent. The ALG/HA hydrogels provided sustained release of bone morphogenetic protein-2 (BMP-2) immobilized in the hydrogels over 5 weeks. The BMP-2-immobilized ALG/HA hydrogels with different ALG/HA ratios were investigated for their in vitro osteogenic differentiation behavior of human bone marrow stem cells (hBMSCs) and in vivo bone regeneration behavior using an animal model (mandibular defect model of miniature pigs). Our findings from cell culture and animal study demonstrated that the osteogenic differentiation of hBMSCs was improved with increasing HA composition in the hydrogel. The hBMSCs/BMP-2-immobilized ALG/HA hydrogel allowed greatly enhanced osteogenic differentiation of hBMSCs (in vitro) and bone regeneration (in vivo) compared with the ALG/HA hydrogel itself and single hBMSCs- or BMP-2-immobilized hydrogel groups.