Inhibitory effects of tangeretin on osteoclastogenesis and titanium particle-induced osteolysis

Abstract

Objective To explore the effects of tangeretin on osteoclastogenesis and bone resorption. Methods Primary bone marrow mononuclear macrophages were isolated and purified from C57BL/6 mice, and different concentrations of tangeretin were added for co-culture. Cell count kit (CCK-8) method was used to detect the cytotoxicity of tangeretin. Macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) were used to induce the differentiation of primary bone marrow mononuclear macrophages into osteoclasts. No tangeretin was given in the control group, and different concentrations of tangeretin (3.125, 6.250 μmol/L) were added in the experimental groups. The number of osteoclasts was counted by tartrate resistant acid phosphatase (TRAP) staining. Cells were planted on bone slices and treated with different concentrations of tangeretin until osteoclasts formed, and bone slices were imaged and analyzed using field-emission scanning electron microscopy. Twenty mice were randomly assigned to four groups (n=5 for each): sham PBS control (sham), Ti particles with PBS (vehicle), and Ti particles with low [10 mg/(kg·day)] or high [20 mg/(kg·day)] concentrations of tangeretin. After 14 days, the effects of tangeretin on osteoclasts in vivo were studied using the titanium particle-induced calvarial osteolysis model. Results Tangeretin cytotoxicity was concentration-dependent. Cell viabilities at 6.250, 12.500 and 25.000 μmol/L of tangeretin were not significantly different from those in the control group (P>0.05). The maximum concentration used in our study was 25.000 μmol/L, which was proven to have no cytotoxic effects on primary bone marrow mononuclear macrophages. Compared to the control group, the number of TRAP positive multinucleated osteoclasts in tangeretin 3.125 μmol/L group (P<0.01) and 6.250 μmol/L group (P<0.01) was significantly decreased. Similarly, the bone resorption area in tangeretin 3.125 μmol/L group (P<0.01) and 6.250 μmol/L group (P<0.01) was also significantly decreased. The In vivo results showed significant osteolysis of the skull in the control group compared to the sham group (P<0.01). As compared with the control group, the osteolysis was significantly inhibited in both the low-concentration tangeretin group (P<0.01) and the high-concentration tangeretin group (P<0.01), and the degree of osteolysis in the high-concentration tangeretin group was significantly lower than that in the low-concentration tangeretin group (P<0.01). Conclusion Tangeretin could inhibit the osteoclastogenesis and osteolysis. Key words: Tangeretin; Osteoclasts; Bone resorption; Osteoporosis