摘要
目的探讨钛表面仿骨单位同心圆形微沟槽结构尺寸对巨噬细胞破骨分化的影响, 为种植体表面改性提供参考。方法选择表面溅射钛的硅片制作对照组(表面光滑的试件)与4个同心圆组表面制作最大直径为200 μm, 最小直径为20 μm, 同心圆间隔为10或30 μm, 微沟槽宽度为10或30 μm, 微沟槽深度为5或10 μm的同心圆试件(每组样本量为27), C10-5组、C10-10组微沟槽宽度为10 μm, 深度分别为5和10 μm, C30-5组、C30-10组微沟槽宽度为30 μm, 深度分别为5和10 μm。使用扫描电镜、接触角测量仪对各组试件表面理化性能进行表征, 通过细胞计数试剂盒(CCK-8)、免疫荧光染色和激光扫描共聚焦显微镜观察各组试件表面巨噬细胞样细胞株RAW264.7的增殖、黏附以及破骨细胞肌动蛋白环的形成, 通过抗酒石酸酸性磷酸酶(TRAP)定量检测、荧光实时定量PCR和蛋白质印迹法探讨试件表面仿骨单位同心圆形微沟槽结构对巨噬细胞破骨分化的调控作用。结果细胞在对照组试件表面无序成片聚集生长, 在同心圆组试件表面沿微沟槽结构呈同心圆状排列。培养5 d后C30组(C30-5组、C30-10组A值分别为1.335±0.018、1.340±0.033)细胞增殖均显著大于C10组(C10-5组、C10-10组A值分别为0.967±0.015、1.182±0.020), 4个同心圆组细胞增殖均显著大于对照组(A值为0.796±0.012)(P<0.05)。C10-5组和C10-10组诱导的破骨细胞肌动蛋白环较小且数量较少。各同心圆组TRAP活性均显著小于对照组(P<0.05), C10-10组破骨分化相关基因TRAP(0.610±0.022)、组织蛋白酶K(CtsK)(0.445±0.037)的基因表达量最小, 与对照组和其余各同心圆组相比差异均有统计学意义(P<0.05), C10-10组破骨分化相关蛋白TRAP(0.648±0.041)、基质金属蛋白酶9(MMP-9)(0.688±0.026)、CtsK(0.491±0.016)的蛋白表达量最小, 与对照组和其余各同心圆组差异均有统计学意义(P<0.05)。结论钛表面仿骨单位同心圆形微沟槽结构可抑制巨噬细胞的破骨分化, 10 μm宽10 μm深的微沟槽抑制细胞破骨分化的效果最明显。
ObjectiveTo investigate the effect of the sizes of osteon-like concentric microgroove structures on the osteoclastic differentiation of macrophages on titanium surfaces,and to provide reference for the surface modification of implants.MethodsThe silicon wafers sputtered with titanium were selected as the control group(smooth surface specimens)and four concentric groups(concentric circles with the maximum diameter of 200μm,the minimum diameter of 20μm,the spacing of concentric circles of 10 or 30μm,the width of microgrooves of 10 or 30μm,and the depth of microgrooves of 5 or 10μm)specimens(the total sample size in each group was 27).The width of microgrooves of C10-5 and C10-10 groups was 10μm,the depth was 5 and 10μm,and the width of microgrooves of C30-5 and C30-10 groups was 30μm,the depth was 5 and 10μm,respectively.The physicochemical properties of the material surfaces were characterized using scanning electron microscopy and contact-angle measurement.The proliferation,adhesion of macrophage-like cell line RAW264.7 and the formation of osteoclast actin-rings on the specimen surfaces were observed by cell counting kit-8(CCK-8),immunofluorescence staining and laser confocal microscopy.Tartrate resistant acid phosphatase(TRAP)quantitative detection,real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting were used to investigate the regulation of osteon-like concentric microgroove structures on the specimen surfaces on the osteoclastic differentiation of macrophages.ResultsMacrophages aggregated and grew disorderly on the surface of the smooth group,and arranged in concentric circles along the microgroove structures on the surfaces of the concentric groups.After 5 days of culture,the cell proliferation of C30 groups(the A values of C30-5 group and C30-10 group were 1.335±0.018 and 1.340±0.033,respectively)was significantly higher than that of C10 groups(the A values of C10-5 group and C10-10 group were 0.967±0.015 and 1.182±0.020,respectively)(all P<0.05).The cell proliferation of the four concentric groups was significantly higher than that of the control group(the A value was 0.796±0.012),with statistical significance(all P<0.05).After osteoclastic induction for 5 days the osteoclasts induced in the C10-5 and C10-10 groups exhibited smaller actin rings and fewer numbers.The TRAP activity in each concentric group was significantly lower than that in the control group(P<0.05).The expression levels of osteoclast differentiation-related genes TRAP(0.610±0.022)in the C10-10 group was lowest,and CtsK(0.489±0.136,0.445±0.037)in the C10-5 and C10-10 groups were lower compared to the smooth group and other concentric groups,with statistical significance(all P<0.05),the expression levels of osteoclast differentiation-related proteins TRAP(0.648±0.041),MMP-9(0.688±0.026)in the C10-10 group were lowest,and CtsK(0.491±0.016,0.453±0.010)in the C10-10 and C30-10 groups were also lower compared to the smooth group and other concentric groups,with statistical significance(all P<0.05).ConclusionsThe osteon-like concentric microgroove structures inhibit the osteoclastic differentiation of macrophage-like cell line RAW264.7,with the microgrooves 10μm wide and 10μm deep showing the most significant inhibitory effect on the osteoclastic differentiation.
作者
翁鑫泽
黄臣
赖颖真
尹戈
雷奕宣
许志强
Weng Xinze;Huang Chen;Lai Yingzhen;Yin Ge;Lei Yixuan;Xu Zhiqiang(Department of Stomatology,Xiamen Medical College,Engineering Research Center of Stomatological Biomaterials,Fujian Province University,Xiamen 361023,China;Department of Stomatology,Affiliated Hospital of Putian University,Putian 351106,China)
出处
《中华口腔医学杂志》
北大核心
2025年第2期160-168,共9页
Chinese Journal of Stomatology
基金
福建省自然科学基金(2022J011408)
福建省大学生创新创业训练计划(202412631003)
厦门市医疗卫生指导性项目(3502Z20244ZD1134)。
关键词
钛
牙种植
巨噬细胞
破骨分化
仿骨单位
同心圆形微沟槽
Titanium
Dental implantation
Macrophages
Osteoclastic differentiation
Osteon-like
Concentric microgroove
