Abstract: BACKGROUND: Injectable sodium alginate hydrogel can repair bone defects by non-invasive or minimally invasive methods, but the mechanical properties are poor and polyethylene glycol-based hydrogel has elasticity. The composite of materials is expected to improve the mechanical properties and cellular biocompatibility of hydrogel scaffolds.
OBJECTIVE: To investigate the physical and chemical properties of glucolactone-sodium alginate/β-tricalcium phosphate-polyethylene glycol hydrogel and its effect on the proliferation and differentiation of murine bone marrow mesenchymal stem cells. 
METHODS: Gluconolactone as cross-linking agent was set to prepare glucolactone-sodium alginate/β-tricalcium phosphate-polyethylene glycol hydrogel (concentrations of cross-linking agent were 5, 10, and 20 g/L, respectively, which were marked as groups A, B, and C) and glucolactone-sodium alginate/β-tricalcium phosphate hydrogel (concentration of cross-linking agent was 10 g/L, which was marked as group D). The morphology, mechanical properties, and gelation time of hydrogels in the four groups were characterized. Rat bone marrow mesenchymal stem cells were co-cultured with four groups of hydrogels. The proliferation of cells was detected by CCK-8 assay. Cell survival was observed by Live/Dead fluorescence staining. Osteogenic differentiation was detected by immunocytochemical staining of type I collagen. 
RESULTS AND CONCLUSION: (1) The filamentous structure similar to adhesion was observed by scanning electron microscope in the groups A, B and C. The pore size distribution of group A was uneven. The pore size of group B was uniform and the porosity was high. The group C had high porosity but different pore sizes. The group D had uneven pore size distribution and low porosity. (2) Physicochemical properties: The compressive stress in group B was higher than that in group D (P < 0.05). With the increase of gluconolactone concentration, the compressive stress of hydrogel in groups A, B and C increased (P < 0.05). The macroscopic performance of hydrogel after 50% compression showed that there were larger cracks in group D and only smaller cracks in group B after compression. The gelation time of groups A, B and C shortened with the increase of gluconolactone concentration, and the gelation time of group B was slightly longer than that of group D (P < 0.05). (3) CCK-8 assay showed that the cell proliferation rate of groups A and B was higher than that of group C (P < 0.05), and that of group B was higher than that of group D (P < 0.05). Live/Dead staining showed that the cell survival rate of each group was higher, but the number of dead cells was more in group C than that in the other three groups. (4) Immunocytochemical staining showed that the cells in groups A, B, and D were polygonal, and the cells in group C were partially round. The green fluorescence intensity in group B was higher than that in the other three groups, and the morphology of the cytoskeleton and microfilaments and the number of cells were also better than those in the other three groups. (5) The results show that the glucolactone-sodium alginate/β-tricalcium phosphate-polyethylene glycol hydrogel has perfect mechanical properties and stability; hydrogel prepared with 10 g/L gluconolactone can effectively promote the proliferation and differentiation of murine bone marrow mesenchymal stem cells.

Key words: tissue-engineered scaffold, injectable hydrogel, sodium alginate, β-tricalcium phosphate, polyethylene glycol, gluconolactone