Objectives: This study aimed to investigate the effect of new biomimetic micro/nano surfaces on the osteoclastic differentiation of RAW264.7 macrophages by simulating natural osteons for the design of concentric circular structures and modifying graphene oxide (GO).
Methods: The groups were divided into smooth titanium surface group (SS), concentric microgrooved titanium surface group (CMS), and microgroove modified with GO group (GO-CMS). The physicochemical properties of the material surfaces were studied using scanning electron microscopy (SEM), contact-angle measurement, atomic force microscopy, X-ray photoelectron spectroscopy analysis, and Raman spectroscopy. The effect of the modified material surface on the cell biological behavior of RAW264.7 was investigated by cell-activity assay, SEM, and laser confocal microscopy. The effect on the osteoclastic differentiation of macrophages was investiga-ted by tartrate-resistant acid phosphatase (TRAP) immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) experiments.
Results: Macrophages were arranged in concentric circles along the microgrooves, and after modification with GO, the oxygen-containing groups on the surface of the material increased and hydrophilicity increased. Osteoclasts in the GO-CMS group were small in size and number and had the lowest TRAP expression. Although it promoted the proliferation of macrophages in the GO-CMS group, the expression of osteoclastic differentiation-related genes was lower than that in the SS group, and the difference was statistically significant (P<0.05).
Conclusions: Concentric circular microgrooves restricted the fusion of osteoclasts and the formation of sealing zones. Osteomimetic concentric microgrooves modified with GO inhibited the osteoclastic differentiation of RAW 264.7 macrophages.
目的: 本研究通过模拟天然骨单位进行同心圆结构的设计，并修饰氧化石墨烯（GO），探究新的仿生微纳米结构表面对巨噬细胞RAW264.7破骨分化的影响。方法: 实验分为光滑钛片组（SS）、微沟槽组（CMS）和微沟槽表面修饰GO组（GO-CMS），利用扫描电子显微镜（SEM）、接触角测量仪、原子力显微镜、X射线光电子能谱分析仪和拉曼光谱仪研究材料表面的理化性能，通过细胞活性检测、SEM和激光共聚焦显微镜研究修饰后的材料表面对RAW264.7的细胞生物学行为的影响，通过抗酒石酸酸性磷酸酶（TRAP）免疫荧光染色、TRAP定量检测和荧光实时定量聚合酶链反应（qRT-PCR）研究其对巨噬细胞破骨分化的影响。结果: 巨噬细胞沿着微沟槽排列成同心圆状，修饰GO后，材料表面含氧基团增多，亲水性增加。GO-CMS组诱导形成的破骨细胞体积小，数量少，TRAP表达量最少，TRAP单位酶活性也最低。GO-CMS组虽然促进巨噬细胞的增殖，但破骨分化相关基因的表达低于SS组，差异具有统计学意义（P<0.05）。结论: 同心圆微沟槽限制了破骨细胞的融合及封闭区的形成，GO修饰类骨单位同心圆微沟槽抑制了巨噬细胞RAW264.7的破骨分化。.
Keywords: concentric microgroove; graphene oxide; macrophages; osteoclastic differentiation; osteomimetic.