Osteoblast differentiation on the TiBP-SF-coated titanium surfaces.
The above data demonstrated that TiBP-SF and SF have distinct behaviors in terms of binding to the titanium surface. However, it was not clear whether the altered properties of TiBP-SF can improve the performance of titanium for medical devices. To address this question, we performed biological assessments using an osteoblastic cell line (MC3T3-E1), which has often been used to evaluate osteoblast differentiation. After coating titanium plates with either SF or TiBP-SF (as described in MATERIALS and METHODS), MC3T3-E1 was grown on the plates for 14 and 21 days in the differentiation-inducing medium (OIM). The morphologies of the cells on the plates were then observed by SEM. As shown in Figure 5 a, the enhanced differentiation of MC3T3-E1, which is characterized by an elongated sheet-like cell structures with multiple filopodia-like intercellular connections protruding from their leading edges, was evident after 21 days when the titanium plates were coated with TiBP-SF. Without coating (or SF-coating), these characteristic morphologies were not evident.
The formation of filopodia from MC3T3-E1 after 21 days of growth on the TiBP-SF coated Ti plate suggested the cells were differentiated into a hydroxyapatite-mineralizing state, which was further confirmed by the increased alkaline phosphatase (ALP) activity and osteocalcin (OC) production. As shown in Figure 5 b, both the ALP activity and OC production from the cells grown on the TiBP-SF coated Ti substrate were the highest among the 3 groups after 21 days of growth. Moreover, the ALP activity of the SF group was lower than that of the TiBP-SF group (p < 0.05), indicating that TiBP-SF was more beneficial than SF for inducing cell differentiation and promoting cell mineralization. The mineralization of MC3T3-E1 cells was also evaluated by alizarin red staining (Figure 5 c). Cells grown on TiBP-SF displayed higher mineralization capacities than those grown on SF (p < 0.01), as well as the control after culture for 21 days. The calcified deposits were uniform and clustered in all locations. To evaluate the cell differentiation state in more detail, the expression levels of osteoblast-related genes (including Runx2, Osterix, OC, OPN, ALP, and Col I) were evaluated by real-time PCR analyses after culture for 14 and 21 days (Figure 5 d). Runx2 and Osterix are master transcription factors involved in regulating the process of MC3T3-E1cell differentiation. After culture for 14 days, the expression levels of both the Runx2 and Osterix genes in cells cultured on TiBP-SF were up-regulated more than those cultured on SF or on the control. Although the expression level of the Runx2 gene decreased in all groups after culture for 21 days, the TiBP-SF group remained much higher than the SF group. A similar trend was also found for Osterix expression. The early markers of osteoblasts (such as the transcription of ALP and Col I) were also up-regulated after culture for 21 days on TiBP-SF compared with the other groups. Furthermore, culture on TiBP-SF up-regulated the mRNA expression levels of OC and OPN at most time intervals. Thus, TiBP-SF was shown to have an ability for enhancing the differentiation of an osteoblast model cell line in vitro .