Abstract: BACKGROUND: The micro/nanostructured gradient biomimetic surface of implant materials can simulate the structure of the extracellular environment in human bone tissue, thereby achieving perfect bone integration function. However, further research is needed on the mechanisms by which the surface microstructure of bone implant materials regulates cell function and promotes osteogenesis.
OBJECTIVE: To analyze the effect of titanium sheet microstructure surface on osteogenic differentiation of MC3T3-E1 osteoblasts. 
METHODS: (1) At a constant voltage of 5 V or 20 V, nanotube arrays of different diameters were prepared on the surface of titanium sheets by acid etching and anodic oxidation techniques, and were recorded as group R5 and group R20, respectively. The surface morphology, roughness, and hydrophilicity of pure titanium sheet (without acid etching or anodizing treatment) were measured in group R5 and group R20. (2) MC3T3-E1 osteoblasts of logarithmic growth stage were inoculated on the surface of pure titanium sheets, R5 group and R20 group respectively. After 24 hours of osteogenic induction culture, the expression of mechanical sensitive channel protein 1 was analyzed by RT-PCR and immunofluorescence staining. Osteoblast inducible base with or without the mechanosensitive channel protein 1 activator Yada1 was added, and alkaline phosphatase staining was performed after 7 days of culture. Alizarin red staining was performed after 14 days of culture.
RESULTS AND CONCLUSION: (1) The surface of pure titanium sheets was smooth under scanning electron microscope. Relatively uniform and orderly nanotube arrays with average diameters of about 30 nm and 100 nm were observed on the surface of titanium sheets of groups R5 and R20, respectively. The results of scanning electron microscope were further verified by atomic force microscopy. The surface roughness of titanium sheet of group R5 was higher than that of pure titanium (P < 0.05), and the water contact angle was lower than that of pure titanium (P < 0.05). The surface roughness of titanium sheet in group R20 was higher than that in group R5 (P < 0.05), and the water contact angle was lower than that in group R5 (P < 0.05). (2) RT-PCR and immunofluorescence staining showed that the expression of mechanosensitive channel protein 1 in group R5 was higher than that in pure titanium group (P < 0.05), and the expression of mechanosensitive channel protein 1 in group R20 was higher than that in group R5 (P < 0.05). Under the osteogenic induction, compared with the condition without Yada1, there were no significant changes in the activity of alkaline phosphatase and the deposition of calcified nodules in pure titanium group after Yada1 addition, while the activity of alkaline phosphatase and the deposition of calcified nodules in groups R5 and R20 after Yada1 addition were significantly increased (P < 0.05). With or without Yada1, the alkaline phosphatase activity and calcified nodule deposition in group R5 were higher than those in pure titanium group (P < 0.05), and the alkaline phosphatase activity and calcified nodule deposition in group R20 were higher than those in group R5 (P < 0.05). (3) The results show that the surface microstructure of titanium sheet can promote the osteogenic differentiation of osteoblast MC3T3-E1 by activating mechanosensitive channel protein 1.

Key words: bone implant material">, titanium implant">, material microstructure">, mechanosensitive channel protein">, osteogenic differentiation">, PIEZO1