[Preparation and in vitro biological performance evaluation of tannic acid-Brushite in situ composite bone cement material]

To investigate the effects of tannic acid (TA) doping on the physicochemical properties, biocompatibility, in vitro osteogenic performance, and antibacterial activity of Brushite bone cement, and to evaluate its feasibility for bone defect repair.

TA was incorporated into Brushite bone cement at concentrations of 0, 1.0, 3.0, 5.0, and 10.0 mg/g of solid powder, designated as Brushite, Brushite/TA-1, Brushite/TA-2, Brushite/TA-3, and Brushite/TA-4, respectively. The compressive strength and microstructure of each group were evaluated. The extracts of the bone cements were prepared and co-cultured with MC3T3-E1 cells. Cell proliferation was assessed using the cell counting kit 8 (CCK-8) assay. The cytotoxicity was observed by Calcein/propidium iodide live/dead cell staining. Cell adhesion was observed via scanning electron microscopy. After osteogenic induction, alkaline phosphatase (ALP) activity was measured and ALP staining was performed. The expression levels of osteogenic-related genes, including runt-related transcription factor 2 (Runx2), osteocalcin (OCN), osteopontin (OPN), collagen type Ⅰ (Col-Ⅰ), and integrin-binding sialoprotein (IBSP), were detected by real-time fluorescent quantitative PCR (qRT-PCR). The antibacterial activity of the bone cement against Escherichia coli was assessed using the inhibition zone method.

Compared with the Brushite group, the Brushite/TA-3 and Brushite/TA-4 groups exhibited significantly increased compressive strength ( P<0.05). TA doping resulted in a higher crystal content and a more regular and dense crystal arrangement. Regarding cytocompatibility, the Brushite/TA-3 group demonstrated the most pronounced enhancement of cell proliferation ( P<0.05), whereas the Brushite/TA-4 group showed relatively lower cell proliferative activity ( P<0.05). All groups exhibited low cytotoxicity with good cell viability. Cell adhesion density and pseudopodia extension were superior in all TA-doped groups compared with the Brushite group. Regarding osteogenic activity, after 14 days of osteogenic induction, ALP activity was higher in all TA-doped groups than in the Brushite group ( P<0.05) in a dose-dependent manner. The relative expression of Runx2, OCN, OPN, Col-Ⅰ, and IBSP mRNA also increased to varying degrees in a dose-dependent manner compared with the Brushite group. Regarding antibacterial performance, only the Brushite/TA-4 group exhibited inhibitory effects against Escherichia coli, with an inhibition zone diameter of approximately 7 mm.

Doping with an appropriate concentration of TA (3.0-5.0 mg/g) improves the mechanical properties, cytocompatibility, and osteogenic activity of Brushite bone cement. A higher concentration (10.0 mg/g) confers antibacterial properties but may partially inhibit cell proliferation. TA-doped Brushite bone cement demonstrates good application potential in the field of bone defect repair.