摘要：

目的: 探究钛表面纳米形貌介导的巨噬细胞极化对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的影响。方法: 选用机械加工的纯钛片作为对照组(control-Ti组);将180 ℃下水热反应12 h后的纯钛片作为纳米组(nm-Ti组)。通过扫描电镜(scanning electron microscope,SEM)和水接触角测试其表面性能,细胞学实验检测其生物相容性,实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测RAW264.7巨噬细胞极化的相关基因和BMSCs成骨相关基因的表达,酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测巨噬细胞炎症相关因子的分泌水平。结果: 经过水热处理后,钛片表面形成纳米形貌,且具有良好的亲水性。相比于control-Ti组,nm-Ti组表面细胞增殖和黏附更为活跃。ELISA结果显示,随时间增加,nm-Ti组分泌白细胞介素-10(interleukin-10,IL-10)的水平显著升高(P<0.01)。M2极化标志物Arg-1和M1极化标志物iNOS均随时间递增而增多,以Arg-1增多为主,经白细胞介素-4(interleukin-4,IL-4)刺激后,nm-Ti组的Arg-1表达显著增多(P<0.01),iNOS表达降低(P<0.05)。巨噬细胞条件培养液培养BMSCs后,nm-Ti组成骨相关基因骨形态发生蛋白-2(bone morphogenetic protein 2,BMP-2)和碱性磷酸酶(alkaline phosphatase,ALP)表达量比正常培养时减少,IL-4刺激培养后,BMP-2和ALP表达均有所升高。结论: 钛表面纳米形貌能够影响巨噬细胞极化方向,进而影响BMSCs的成骨分化能力。

关键词: 钛种植体, 纳米形貌, 巨噬细胞极化, 骨整合

Abstract:

Objective: The study aimed to explore the effect of macrophage polarization mediated by nano-topography of titanium (Ti) surface on bone marrow mesenchymal stem cells (BMSCs). Methods: Mechanically treated pure Ti discs were chosen as control group(control-Ti group). The pure Ti discs that were hydrothermally treated in NaOH solution at 180 ℃ for 12 h were used as nano-topography group(nm-Ti group). Surface characteristics were analyzed by scanning electron microscope(SEM) and water contact angle measurement. Biocompatibility was measured by cellular assays. The mRNA expression of polarization related genes in RAW264.7 macrophage and osteogenesis related genes in BMSCs was measured by real-time quantitative polymerase chain reaction (RT-qPCR). Secretion levels of macrophage related inflammatory factors were tested by enzyme-linked immunosorbent assay(ELISA). Results: After hydrothermal treatment, nano-structure like topography was formed on the surface of nm-Ti with good hydrophilicity. And compared with control-Ti, the surface of nm-Ti showed better cellular adhesion and proliferation. ELISA results showed that the secretion level of interleukin-10 (IL-10) in nm-Ti group was much higher than that of control-Ti group. M2 macrophage polarization marker Arg-1 and M1 macrophage polarization marker iNOS increased with time, the increase in Arg-1 is dominant. And after IL-4 stimulation, in nm-Ti group, the expression of M2 macrophage polarization marker Arg-1 increased (P<0.01), while the expression of M1 macrophage polarization marker iNOS decreased(P<0.05). After culturing the BMSCs by the conditional medium of macrophage, the expression levels of osteogenesis related genes such as bone morphogenetic protein 2(BMP-2) and alkaline phosphatase(ALP) were reduced than that of normal culture in nm-Ti group. After IL-4 stimulation, the expression levels of both BMP-2 and ALP increased. Conclusion: The nano-topography of titanium surface can induce the polarization of macrophages in the direction of M1, which in turn will affect the osteogenic differentiation ability of BMSCs.

Key words: titanium implant, nano-structure, macrophage polarization, osseointegration

中图分类号: 

• R782