Abstract: BACKGROUND: Calcium sulfate has been widely used as a bone graft for the treatment of alveolar bone loss, endodontic lesions, and periodontal disease. Copper plays an important role in various biological processes, including angiogenesis and cell migration.
OBJECTIVE: To prepare copper-containing calcium sulfate bone cement and characterize its physicochemical properties and angiogenesis.
METHODS: (1) Calcium sulfate hemihydrate was used as the solid phase and copper sulfate pentahydrate solutions of varying concentrations were used as the liquid phase. The solid and liquid phases were mixed at a ratio of 1.7 g/1 mL. The mass ratios of copper sulfate pentahydrate to calcium sulfate hemihydrate were 0.1%, 0.5%, 1%, and 2.5%, respectively. The prepared copper-containing calcium sulfate bone cements were designated 0.1%Cu-CS, 0.5%Cu-CS, 1%Cu-CS, and 2.5%Cu-CS, respectively. Pure calcium sulfate bone cement was also prepared. The micromorphology, compressive strength, and copper and calcium ion release in the in vitro degradation solution of the five cements were characterized. (2) Thirty SD rats selected and a single cortical bone defect model with a diameter of 3 mm and a length of 5 mm was established on the left tibia. These models were randomly divided into three groups: a blank group (n=10) received no intervention; a control group (n=10) received calcium sulfate bone cement implantation; and an experimental group (n=10) received 0.5% Cu-CS bone cement implantation. Six weeks after surgery, angiogenesis at the tibial defect site was observed using micro-CT scans and CD31 immunohistochemical staining after vascular microfil perfusion.
RESULTS AND CONCLUSION: (1) Scanning electron microscopy revealed rod-shaped calcium sulfate crystals on the surface of calcium sulfate cement and plate-like gypsum crystals on the surface of copper-containing calcium sulfate cement. The number of plate-like gypsum crystals increased with increasing copper sulfate pentahydrate mass. The compressive strengths of 0.5% Cu-CS and 1% Cu-CS bone cements were higher than those of calcium sulfate and 0.1% Cu-CS bone cements (P < 0.05), and the compressive strength of 2.5% Cu-CS bone cement was higher than that of 0.5% Cu-CS and 1% Cu-CS bone cements (P < 0.05). After immersion in simulated body fluid for 6 weeks, calcium sulfate bone cement released the highest calcium ion concentration, while 2.5% Cu-CS bone cement released the lowest. Calcium sulfate bone cements did not release copper ions. Among copper-containing calcium sulfate bone cements, 2.5% Cu-CS bone cement released the highest copper ion concentration, while 0.1% Cu-CS bone cement released the lowest. (2) Micro-CT scanning showed that the blank group had the least neovascularization within the bone defect, while the experimental group had the most neovascularization within the bone defect. CD31 immunohistochemical staining revealed that only punctate or linear neovascular structures were observed in the bone defect of the blank and control groups, while the experimental group had abundant and dense blood vessel formation. (3) The results confirm that copper-containing calcium sulfate bone cement has excellent mechanical properties and sustained copper ion release, promoting angiogenesis.

Key words: copper, calcium sulfate, copper-containing calcium sulfate, bone cement, vascularization, tibial defect, tissue engineering