磷酸钙骨水泥/聚乳酸羟基乙酸降解产物促进小鼠单核细胞破骨向分化

Calcium phosphate cement/poly(lactic-co-glycolic acid)degradation products promote osteoclast differentiation of mouse monocytes

背景:前期研究发现磷酸钙骨水泥/聚乳酸羟基乙酸复合材料可加速骨改建过程,其降解产物对单核细胞破骨向分化的影响有待进一步研究。目的:观察磷酸钙骨水泥/聚乳酸羟基乙酸降解产物对小鼠RAW264.7单核细胞破骨向分化的影响。方法:实验分为空白对照组、羟基乙酸组、磷酸钙骨水泥/聚乳酸羟基乙酸组,按照实验分组,将配制好的溶液和50μg/L核因子κB受体活化因子配体添加到完全培养基中制备成不同条件的培养基,培养小鼠RAW264.7细胞5 d,诱导其分化。应用CCK-8法分析各组培养液对细胞增殖作用的影响;RT-PCR和Western blot检测小鼠RAW264.7细胞破骨向分化相关因子活化T细胞核因子c1、基质金属蛋白酶9、核因子κB受体活化因子、抗酒石酸酸性磷酸酶基因和蛋白表达水平的变化;使用抗酒石酸酸性磷酸酶染色法鉴定破骨样细胞。结果与结论:①CCK-8结果显示,细胞在前72 h处于快速生长期,96 h后开始下降;②RT-PCR、Western blot结果显示磷酸钙骨水泥/聚乳酸羟基乙酸组的活化T细胞核因子c1、核因子κB受体活化因子、抗酒石酸酸性磷酸酶mRNA的表达水平及活化T细胞核因子c1、核因子κB受体活化因子的蛋白表达水平显著高于羟基乙酸组、对照组(P<0.05);③羟基乙酸组和磷酸钙骨水泥/聚乳酸羟基乙酸组基质金属蛋白酶9mRNA和蛋白的表达水平差异无显著性意义,但高于对照组(P<0.01);④抗酒石酸酸性磷酸酶染色可见磷酸钙骨水泥/聚乳酸羟基乙酸组破骨样细胞数高于对照组和羟基乙酸组,差异有显著性意义(P<0.05);⑤结果表明磷酸钙骨水泥/聚乳酸羟基乙酸组降解产物形成的微环境可促进小鼠RAW264.7细胞破骨向分化。

BACKGROUND:Preliminary studies found that calcium phosphate cement/poly(lactic-co-glycolic acid)composite can accelerate the process of bone remodeling,and the effect of its degradation products on the differentiation of mononuclear osteoclasts needs further study.OBJECTIVE:To observe the effect of calcium phosphate cement/poly(lactic-co-glycolic acid)degradation products on the differentiation of mouse RAW264.7monocytes and osteoclasts.METHODS:The experiment was divided into blank control group,glycolic acid group,and calcium phosphate cement/poly(lactic-co-glycolic acid)group.According to the experimental group assignment,the prepared solution and 50μg/L ligand of nuclear factorκB receptor activator were added to complete medium to prepare media with different conditions.Mouse RAW264.7 cells were cultured for 5 days to induce their differentiation.Cell counting kit-8 assay was used to analyze the effect of different culture media on cell proliferation.RT-PCR and western blot assay were used to detect the changes of gene and protein expression levels of nuclear factor of activated T-cells cytoplasmic 1,matrix metalloproteinase-9,nuclear factorκB receptor activator and tartrate-resistant acid phosphatase activated by osteoclast differentiation-related factors in mouse RAW264.7 monocytes.The osteoclast-like cells were identified by tartrate-resistant acid phosphatase staining.RESULTS AND CONCLUSION:Cell counting kit-8 assay results showed that the cells were in a rapid growth period within 72 hours,and then began to decline after 96 hours.The results of RT-PCR and western blot assay showed that the expression levels of nuclear factor of activated T-cells cytoplasmic 1,nuclear factor-κB receptor activator and tartrate-resistant acid phosphatase mRNA and protein expression levels of nuclear factor-1 and nuclear factor-κB receptor activator in activated T cells in calcium phosphate cement/poly(lactic-co-glycolic acid)group were significantly higher than those in glycolic acid group and control group(P<0.05).There was no significant difference in gene and protein expression levels of matrix metalloproteinase-9 between glycolic acid group and calcium phosphate cement/poly(lactic-co-glycolic acid)group,but it was higher than that of the control group(P<0.01).Tartrate-resistant acid phosphatase staining results showed that the number of osteoclast-like cells in the calcium phosphate cement/poly(lactic-co-glycolic acid)group was significantly higher than that in the control group and the glycolic acid group(P<0.05).Overall,these results indicate that the microenvironment formed by the degradation products of calcium phosphate cement/poly(lactic-co-glycolic acid)can promote the osteoclast differentiation of mouse RAW264.7 monocytes.