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 .