硫酸钙复合支架对大鼠骨髓间充质干细胞成骨分化的影响△

Effect of calcium sulfate composite scaffold on osteogenic differentiation of rat bone marrow mesenchymal stem cells

摘要:

[目的] 探讨阿司匹林/α-半水硫酸钙 （α-calcium sulfate hemihydrate, α-CSH） 复合支架对大鼠骨髓间充质干细胞 （bone marrow mesenchyml stem cells, BMSCs） 增殖及成骨分化的影响。[方法] 提取并培养 SD 大鼠 BMSCs，将其随机分为复合组 （阿司匹林/α-CSH 复合支架）、α-CSH 组 （α-CSH 支架） 与空白对照组 （不予以任何干预）。测定对比各组大鼠 BMSCs 增殖、成骨分化、迁移情况及相关指标。[结果]与培养第 3 d 比较，第 7 d 复合组和 α-CSH 组 CCK-8 检测 OD 值显著增加（P＜ 0.05），第 3、7 d 复合组和 α-CSH 组 CCK-8 检测 OD 值显著高于空白对照组 [OD 值, (1.4±0.3) vs (0.9±0.2) vs (0.2±0.1), P＜0.001； (2.0±0.4) vs (1.4±0.3) vs (0.2±0.1), P＜0.001]。与第 1 d 比较，第 3 d 复合组、α-CSH 组划痕试验愈合率显著增加（P＜0.05），第 1、3 d 复合组和 α-CSH 组划痕试验愈合率显著大于空白对照组 [%, (72.3±2.7) vs (60.2±2.5) vs (6.3±1.0), P＜0.001；(92.5±2.8) vs (83.2±3.0) vs (7.0±1.2), P＜0.001]。此外，复合组和 α-CSH 组 14 d 时钙化结节面积比显著大于空白对照组 [%, (43.2±4.3) vs (31.3±4.0) vs (20.4± 2.8), P＜0.001]。培养 7 d，复合组和 α-CSH 组培养液中 IL-1β、IL-6、TNF-α 含量均显著低于空白对照组（P＜0.05），复合组和 α-CSH 组成骨诱导 7 d 时 BMP-2、OPN、ALP、RUNX2 及 Wnt 3a、LRP5、β-catenin 蛋白相对表达量均显著高于空白对照组（P＜ 0.05），而复合组和 α-CSH 组 GSK-3β 蛋白显著低于空白对照组（P＜0.05）。[结论] 阿司匹林/α-CSH 复合支架可促进大鼠 BM- SCs 增殖及成骨分化，抑制炎症因子表达，其机制可能与调控 Wnt/β-catenin 信号通路相关。

Abstract:

[Objective] To investigate the effects of aspirin/α-calcium sulfate hemihydrate (α-CSH) composite scaffolds on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats. [Methods] BMSCs from SD rat were extracted and cultured, and randomly divided into the composite group (aspirin/α-CSH composite scaffold), the α-CSH group (α-CSH scaffold), and the blank control group (without any intervention). The proliferation, osteogenic differentiation, migration of BMSCs and related indicators were measured and compared among the 3 groups. [Results] The OD values of CCK-8 assay were significantly increased in the composite group and the α-CSH group 7 days compared with that 3 day of culture (P＜0.05), which in the composite group and the α-CSH group were significantly higher than those in the blank control group 3 days and 7 days [OD values, (1.4±0.3) vs (0.9±0.2) vs (0.2±0.1), P＜0.001; (2.0± 0.4) vs (1.4±0.3) vs (0.2±0.1), P＜0.001]. The scratch test healing rate were significantly increased at 3 days and 7 days in the composite group and the α-CSH group compared with that at 1 day (P＜0.05), which in the composite group and the α-CSH group was significantly higher than that in the blank control group at 1 day and 3 days [% (72.3±2.7) vs (60.2±2.5) vs (6.3±1.0), P＜0.001; (92.5±2.8) vs (83.2±3.0) vs (7.0±1.2), P＜0.001]. In addition, the area of calcified nodules in the composite group and the α-CSH group was significantly larger than that in the blank control group at 14 days [%, (43.2±4.3) vs (31.3±4.0) vs (20.4±2.8), P＜0.001]. After 7 days of culture, the IL-1β, IL-6, and TNF-α in the culture medium of the composite group and the α-CSH group were significantly lower than those of the blank control group (P＜ 0.05), while the relative expression levels of BMP-2, OPN, ALP, RUNX2, Wnt 3a, LRP5, and β-catenin proteins in the composite group and the α-CSH group at 7 days of osteoinduction culture were significantly higher than those in the blank control group (P＜0.05), whereas the GSK-3β protein in the composite group and the α-CSH group was significantly lower than that in the blank control group (P＜0.05). [Conclusion] Aspirin/α-CSH composite scaffolds does promote the proliferation and osteogenic differentiation of rat BMSCs, while inhibit the expression of inflammatory factors. The mechanism may be related to the regulation of the Wnt/β-catenin signaling pathway.