Titanium dioxide nanotubes increase purinergic receptor P2Y6 expression and activate its downstream PKCα-ERK1/2...

Titanium dioxide (TiO₂) nanotubes can improve the osseointegration of pure titanium implants, but this exact mechanism has not been fully elucidated. The purinergic receptor P2Y6 is expressed in bone marrow mesenchymal stem cells (BMSCs) and participates in the regulation of bone metabolism. However, it is unclear as to whether P2Y6 is involved in the osteogenic differentiation of BMSCs induced by TiO₂ nanotubes. TiO₂ nanotubes were prepared on the surface of titanium specimens using the anodizing method and characterized their features. Quantitative reverse transcriptase polymerase chain reaction and western blotting were used to detect the expression of P2Y6, markers of osteogenic differentiation, and PKCα-ERK1/2. A rat femoral defect model was established to evaluate the osseointegration effect of TiO₂ nanotubes combined with P2Y6 agonists. The results showed that the average inner diameter of the TiO₂ nanotubes increased with an increase in voltage (voltage range of 30-90V), and the expression of P2Y6 in BMSCs could be upregulated by TiO₂ nanotubes in osteogenic culture. Inhibition of P2Y6 expression partially inhibited the osteogenic effect of TiO₂ nanotubes and downregulated the activity of the PKCα-ERK1/2 pathway. When using in vitro and in vivo experiments, the osteogenic effect of TiO₂ nanotubes when combined with P2Y6 agonists was more pronounced. TiO₂ nanotubes promoted the P2Y6 expression of BMSCs during osteogenic differentiation and promoted osteogenesis by activating the PKCα-ERK1/2 pathway. The combined application of TiO₂ nanotubes and P2Y6 agonists may be an effective new strategy to improve the osseointegration of titanium implants. STATEMENT OF SIGNIFICANCE: Titanium dioxide (TiO₂) nanotubes can improve the osseointegration of pure titanium implants, but this exact mechanism has not been fully elucidated. The purinergic receptor P2Y6 is expressed in bone marrow mesenchymal stem cells (BMSCs) and participates in the regulation of bone metabolism. However, it is unclear as to whether P2Y6 is involved in the osteogenic differentiation of BMSCs induced by TiO₂ nanotubes. For the first time, this study revealed the relationship between TiO₂ nanotubes and purine receptor P2Y6, and further explored its mode of action, which may provide clues as to the regulatory role of TiO₂ nanotubes on osteogenic differentiation of BMSCs. These findings will help to develop novel methods for guiding material design and biosafety evaluation of nano implants.