Effect of osteon-like concentric microgroove structures of different sizes on the osteoclastic differentiation of macrophages

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Objective: To 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. Methods: The 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 200 μ m, the minimum diameter of 20 μ m, the spacing of concentric circles of 10 or 30 μm, the width of microgrooves of 10 or 30 μm, and the depth of microgrooves of 5 or 10 μm) specimens (the total sample size in each group was 27). The width of microgrooves of C10-5 and C10-10 groups was 10 μm, the depth was 5 and 10 μm, and the width of microgrooves of C30-5 and C30-10 groups was 30 μ m, the depth was 5 and 10 μ m, 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. Results: Macrophages 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). 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 (P<0.05). 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) and CtsK (0.445±0.037) in the C10-10 group were lower compared to the smooth group and other concentric groups, with statistical significance (P<0.05), the expression levels of osteoclast differentiation-related proteins TRAP (0.648±0.041), matrix metalloproteinase 9 (MMP-9) (0.688±0.026), and CtsK (0.491±0.016) in the C10-10 group were also lower compared to the smooth group and other concentric groups, with statistical significance (P<0.05). Conclusions: The osteon-like concentric microgroove structures inhibit the osteoclastic differentiation of macrophage-like cell line RAW264.7, with the microgrooves 10 μm wide and 10 μm deep showing the most significant inhibitory effect on the osteoclastic differentiation.