Key Takeaways & Executive Findings
- •• Calcium sulfate-magnesium oxide composites exhibit enhanced antibacterial activity against both Escherichia coli and Staphylococcus aureus, with efficacy increasing proportionally to magnesium oxide content. • The composites maintain good cytocompatibility, particularly at lower magnesium oxide ratios (2.5% and 7.5%), supporting MC3T3 cell viability and proliferation. • CS-2.5MgO significantly enhances osteogenic differentiation and mineralization, as evidenced by increased alkaline phosphatase activity, alizarin red staining, and RUNX2/WNT3a expression. • CS-7.5MgO demonstrates the strongest pro-angiogenic effect, promoting tube formation in HUVECs and upregulating endothelial nitric oxide synthase expression.
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%, and distilled water was added to form calcium sulfate-magnesium oxide composites, denoted as CS-2.5MgO, CS-7.5MgO, CS-15MgO, and CS-25MgO. The surface morphology, compressive strength, in vitro degradation, and H2O2 production in PBS were characterized. (2) Escherichia coli (or Staphylococcus aureus) suspensions were co-cultured with the five groups of materials, and antibacterial properties were evaluated by agar plate coating and inhibition zone tests. (3) MC3T3 cells were co-cultured with material extracts, and cytocompatibility was assessed by CCK-8 and live/dead staining. After osteogenic induction, alkaline phosphatase staining and alizarin red staining were used to evaluate osteogenic mineralization, and Western blot detected RUNX2 and WNT3a protein expression. (4) Human umbilical vein endothelial cells were co-cultured with material extracts, and angiogenic potential was evaluated by Matrigel tube formation assay, and Western blot detected endothelial nitric oxide synthase protein expression. (5) α-Calcium sulfate hemihydrate, CS-2.5MgO, CS-7.5MgO, CS-15MgO, and CS-25MgO loaded with Staphylococcus aureus were implanted into muscle incisions of SD rats. At 1, 3, and 7 days postoperatively, materials and adjacent muscle tissues were rinsed, and the rinse fluid was collected for colony counting by agar plate coating. Hematoxylin-eosin staining was used to observe inflammatory cell infiltration in surrounding muscle tissues. RESULTS AND CONCLUSION: (1) Scanning electron microscopy showed that α-calcium sulfate hemihydrate mostly exhibited short rod-like crystals with a few long strip crystals and smooth surfaces; in the composites, magnesium oxide particle aggregates were distributed on crystal surfaces and between crystals, with density increasing with magnesium oxide ratio. Compared with α-calcium sulfate hemihydrate, the compressive strength and degradation rate of the composites decreased, while H2O2 production in PBS increased. Agar plate coating and inhibition zone tests showed that the composites had excellent antibacterial properties, which increased with magnesium oxide ratio. CCK-8 and live/dead staining showed that α-calcium sulfate hemihydrate, CS-2.5MgO, and CS-7.5MgO had good cytocompatibility. Alkaline phosphatase staining, alizarin red staining, and Western blot showed that CS-2.5MgO enhanced osteogenic mineralization. Matrigel tube formation and Western blot showed that CS-7.5MgO had the strongest angiogenic ability. (2) Rinse fluid agar plate coating showed that the composites had good in vivo antibacterial properties compared with α-calcium sulfate hemihydrate, increasing with magnesium oxide ratio. Hematoxylin-eosin staining showed that inflammatory cell infiltration and exudation in muscle tissues were significantly reduced in all composite groups compared with α-calcium sulfate hemihydrate group. (3) These results indicate that calcium sulfate-magnesium oxide composites have good cytocompatibility and antibacterial properties, and can effectively promote osteogenesis and angiogenesis.
1. Introduction
Orthopedic surgeries (such as trauma repair, tumor resection, and joint revision) often require bone grafting [1]. Currently, various clinical grafts (e.g., autograft, bioglass, bioceramics, metals) have advantages and disadvantages. For example, autograft has osteoinductivity but limited supply and requires a second surgery [2]; bioceramic bone repair materials (e.g., calcium phosphate, calcium sulfate) have good biocompatibility [3] but insufficient mechanical properties for load-bearing; metals have high strength but are difficult to shape intraoperatively and are mostly non-degradable [4]. An ideal bone graft material should possess multiple favorable properties to meet clinical needs.
Calcium sulfate (CaSO4) is an implantable material that is moldable, degradable, biocompatible, and supports bone regeneration [5], and has been used clinically since the 1950s [6-7]. However, calcium sulfate itself lacks antibacterial properties; for example, ZIRAN et al. [8] used a calcium sulfate-deminaralized bone matrix composite to treat 44 cases of fracture nonunion, with a postoperative infection rate of 34%. Many studies have combined calcium sulfate with antibiotics, copper ions, silver ions, antimicrobial peptides, etc. [9-10], but antibiotics can induce drug resistance, copper and silver heavy metals have significant cytotoxicity [11], and antimicrobial peptides are not tolerant to sterilization. Furthermore, calcium sulfate alone has limited osteogenic and angiogenic properties.
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HU Liqun, XIAO Dongqin, MA Chenxi, LI Zhuohan, YAN Jiyuan, LI Zhong, HE Kui, DUAN Ke (2026). Performance of calcium sulfate-magnesium oxide composites as anti-infective bone graft materials. Chinese Journal of Tissue Engineering Research. https://doi.org/10.12307/2026.21539
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Frequently Asked Questions
What are the key properties of calcium sulfate-magnesium oxide composites?
The composites exhibit enhanced antibacterial activity, good cytocompatibility, and promote osteogenesis and angiogenesis, making them promising anti-infective bone graft materials.
How does the magnesium oxide content affect the composite properties?
Increasing magnesium oxide content enhances antibacterial activity but reduces compressive strength and degradation rate. Lower ratios (2.5% and 7.5%) maintain good cytocompatibility, while 2.5% optimally promotes osteogenesis and 7.5% optimally promotes angiogenesis.
What methods were used to evaluate the antibacterial performance?
Antibacterial performance was evaluated using agar plate coating and inhibition zone tests against Escherichia coli and Staphylococcus aureus, as well as in vivo colony counting in a rat muscle infection model.
What are the potential clinical applications of this composite?
The composite can be used as an anti-infective bone graft material in orthopedic surgeries, such as trauma repair, tumor resection, and joint revision, to reduce postoperative infections and enhance bone healing.
What is the significance of the H2O2 production in the composites?
H2O2 production is associated with the antibacterial mechanism of magnesium oxide, as it can generate reactive oxygen species that kill bacteria, contributing to the composite's antimicrobial effect.
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