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Abstract: The key to managing fracture is to achieve stable internal fixation, and currently, biologically and mechanically appropriate internal fixation devices are urgently needed. With excellent biocompatibility and corrosion resistance, titanium-niobium alloys have the potential to become a new generation of internal fixation materials for fractures. However, the role and mechanism of titanium-niobium alloys on promoting fracture healing are still undefined. Therefore, in this study, we systematically evaluated the bone-enabling properties of Ti45Nb via in vivo and in vitro experiments. In vitro, we found that Ti45Nb has an excellent ability to promote MC3T3-E1 cell adhesion and proliferation without obvious cytotoxicity. Alkaline phosphatase (ALP) activity and alizarin red staining and semiquantitative analysis showed that Ti45Nb enhanced the osteogenic differentiation of MC3T3-E1 cells compared to the Ti6Al4V control. In the polymerase chain reaction (PCR) experiment, the expression of osteogenic genes in the Ti45Nb group, such as ALP, OPN, OCN, Col-1 and Runx2, was significantly higher than that in the control group. Meanwhile, in the western blot (WB) experiment, the expression of osteogenic-related proteins in the Ti45Nb group was also significantly increased, and the expression of PI3K-Akt-related proteins was also higher, which indicated that Ti45Nb might promote fracture healing by activating the PI3k-Akt signaling pathway. In vivo, we found that Ti45Nb implants accelerated fracture healing compared to Ti6Al4V, and the biosafety of Ti45Nb was confirmed by histological evaluation. Furthermore, immunohistochemical staining confirmed that Ti45Nb may promote osteogenesis by upregulating the PI3K/Akt signaling pathway. Our study demonstrated that Ti45Nb exerts an excellent ability to promote fracture healing as well as enhance osteoblast differentiation by activating the PI3K/Akt signaling pathway, and its good biosafety has been confirmed, which indicates its clinical translation potential.