Abstract: BACKGROUND: Calcium sulfate bone graft materials have good biocompatibility but lack antibacterial properties, potentially leading to infections. Magnesium oxide has antibacterial effects and can promote bone regeneration and angiogenesis. 
OBJECTIVE: To develop novel calcium sulfate-magnesium oxide bone graft materials with antibacterial properties and the ability to promote bone regeneration, and to systematically evaluate its antibacterial capabilities, cytocompatibility, and osteogenic and angiogenic potential.
METHODS: (1) α-Calcium sulfate hemihydrate was synthesized by a hydrothermal method. α-Calcium sulfate hemihydrate was mixed with magnesium oxide at mass ratios of 2.5%, 7.5%, 15%, and 25%, respectively. Distilled water was added to the mixture and solidified to form calcium sulfate-magnesium oxide composites, designated CS-2.5MgO, CS-7.5MgO, CS-15MgO, and CS-25MgO, respectively. The surface morphology, compressive strength, in vitro degradation, and H2O2 production in PBS of α-calcium sulfate hemihydrate, CS-2.5MgO, CS-7.5MgO, CS-15MgO, and CS-25MgO were characterized. (2) Escherichia coli (or Staphylococcus aureus) cultures were co-cultured with the five composites. The antibacterial properties of the composites were evaluated by agar plate spread assay and inhibition zone assay. (3) MC3T3 cells were co-cultured with extracts from the five composites. The cytocompatibility of the composites was evaluated by CCK-8 assay and live-dead staining. MC3T3 cells were co-cultured with extracts from the five groups of materials. After osteogenic induction, the osteogenic mineralization-inducing ability of the materials was assessed by alkaline phosphatase and alizarin red staining. RUNX2 and WNT3a protein expression in the cells was detected by western blot assay. (4) Human umbilical vein endothelial cells were co-cultured with extracts from the five groups of materials. The angiogenesis-promoting ability of the materials was assessed by Matrigel tubule formation assay. Endothelial nitric oxide synthase protein expression in the cells was detected by western blot assay. (5) α-Calcium sulfate hemihydrate, CS-2.5MgO, CS-7.5MgO, CS-15MgO, and CS-25MgO containing Staphylococcus aureus were implanted into the muscle incision of SD rats. 1, 3, and 7 days after surgery, the materials and adjacent muscle tissue were rinsed, and the rinsate was collected. Colony formation was assessed by agar plate spread assay. Hematoxylin-eosin staining was performed to determine inflammatory cell infiltration in the muscle tissue surrounding the materials.
RESULTS AND CONCLUSION: (1) Scanning electron microscopy revealed that α-calcium sulfate hemihydrate was mostly short rod-shaped crystals, with a small number of elongated crystals and smooth surfaces. In the calcium sulfate-magnesium oxide composite, magnesium oxide particle aggregates were distributed on the crystal surfaces and between crystals, and the particle density increased with increasing magnesium oxide content. Compared with α-calcium sulfate hemihydrate, the compressive strength and degradation rate of the calcium sulfate-magnesium oxide composite decreased, while H₂O₂ production increased when immersed in PBS. Agar plate spread assays and inhibition zone assays demonstrated that the calcium sulfate-magnesium oxide composite exhibited excellent antibacterial properties, which increased with increasing magnesium oxide content. CCK-8 assay and live-dead staining demonstrated that α-calcium sulfate hemihydrate, CS-2.5MgO, and CS-7.5MgO exhibited good cytocompatibility. Alkaline phosphatase staining, Alizarin red staining, and western blot assay revealed that the CS-2.5MgO composite exhibited enhanced bone mineralization induction. Matrigel tubule formation assay and western blot assay showed that the CS-7.5MgO group exhibited the strongest angiogenesis ability. (2) Fluid agar plate spread assays revealed that the calcium sulfate-magnesium oxide composite material exhibited superior in vivo antibacterial properties compared with α-calcium sulfate hemihydrate, with this antibacterial activity increasing with increasing magnesium oxide content. Hematoxylin-eosin staining revealed significantly reduced inflammatory cell infiltration and exudation in muscle tissue in all calcium sulfate-magnesium oxide composite groups compared with the α-calcium sulfate hemihydrate group. (3) These results demonstrate that the calcium sulfate-magnesium oxide composite material exhibits excellent cytocompatibility and antibacterial properties, effectively promoting osteogenesis and angiogenesis. 

Key words: calcium sulfate, magnesium oxide, composite material, antibacterial material, bone, antibacterial property, osteomineralization, biomaterial