Previous results have indicated that structured implants could improve osteoblastic function and bone formation. Therefore, we investigated the effect of nanoscaled and microscaled implants on osteogenesis. Our results indicate that nanotube surfaces can enhance cell proliferation and lead to a higher level of alkaline phosphatase activity compared with microstructured surfaces. Osteoblasts cultured on nanotube surfaces also exhibited upregulated levels of Cbfal, osteocalcin, osteoprotegerin, and collagen I. Both nanostructured and microstructured implants enhanced expression of vascular endothelial growth factor and increased the formation of capillaries in peri-implant tissues. The ultimate shear strength of the nanostructured implants was significantly boosted within four weeks of in vivo implantation.
It was shown that stimulation of cell growth was greater when the cells were cultivated on nanostructured titanium. Osteoblasts were reported to respond to nanoscale roughness, with a greater cell thickness11
due to a larger number of particle binding sites.12
It was demonstrated that nanostructured surfaces induced superhydrophilicity of samples.13
Recent reports indicated that hydrophilic surfaces could improve cell attachment and proliferation15
more than could hydrophobic surfaces. In addition to surface properties, such as contact area and wettability, Feng et al17
confirmed that chemical functional groups were strongly associated with the behavior of osteoblasts. Nanostructured surfaces seem to promote cellular growth and proliferation due to the higher number of hydroxyl groups.17
We also found that alkaline phosphatase activity and Cbfal, osteocalcin, osteoprotegerin, and collagen I were greater in cells cultivated on TiO2
nanotube layers than for those cultivated on microporous titanium and polished titanium plates. Some previous studies have demonstrated that cellular proliferation and differentiation showed the opposite trend, depending on surface roughness.20
However, our study revealed both increased proliferation and enhanced differentiation of osteoblasts on TiO2
nanotube layers. Implants modified with nanotubes may not only accelerate speed but may also promote quantity of bone formation, which is beneficial for enhancing osteointegration. This result may be due to the modulated cellular orientation, cell spreading,22
and increased apatite deposition24
induced by TiO2
nanotube layer surfaces. It is reasonable to hypothesize that the way in which osteoblasts sense nanostructures is quite different from how they sense microstructures. However, the mechanisms that underlie how nanoscaled surfaces affect osteoblast behavior remain to be determined.
Osteoprotegerin expressed in cells on TiO2
nanotube layers was 360% higher than that of cells on microporous titanium. Osteoprotegerin suppressed differentiation of osteoclasts and inhibited their activation.27
Increased osteoprotegerin levels on TiO2
nanotube layers may suggest that the inhibited activity of osteoclasts may be attributed to successful osteointegration at the bone-nanostructured implant interface.
Histological analysis indicated that more osteoblasts aggregated on the nanostructured titanium surfaces than on the surfaces of the other two groups. Osteoblast proliferation is a prerequisite for bone formation.22
An increased number of cells at the bone-nanostructured implant interface may contribute to the increase in cellular proliferation due to the topology mechanism. These hypotheses were supported by the MTT assay results. The nanostructured titanium surfaces may attract osteoblasts or mesenchymal stem cells due to high affinity and bioactivity9
of surfaces. Immunohistochemical analysis also indicated that osteocalcin and collagen proteins were highly expressed on the TiO2
nanotube layer specimens, which confirmed our in vitro study results. Interestingly, the high levels of vascular endothelial growth factor expressed both on the TiO2
nanotube layers and on the microporous titanium specimens were accompanied by a high degree of capillary formation.28
Capillaries can deliver sufficient nutrients, which may also contribute to bone regeneration.
Being a more direct and sensitive predictor of implant stability than histomorphometric analysis,29
biomechanical testing is utilized to assess implant anchorage in the surrounding bone in implant orthopedics research.30
nanotube layers showed a higher value of shear strength than microporous titanium and polished titanium plates four weeks after implantation, which indicates that the nanostructured titanium implants stimulated bone growth and accelerated formation of the bone-implant interface. During the early healing period, various environmental factors, such as occlusal forces, bacteria, and fibrous tissues, are capable of impairing interfacial formation and decreasing implant success due to the instability of the bone-implant interface.32
These results suggested that nanostructured implants may shorten the healing period and decrease the risk of implant failure. However, the shear strength of microporous titanium did not appear to be any different from that at 12 weeks post implantation. It is presumed that bone tissues can grow into the micropores of microporous titanium and form a micro-interlock,5
which greatly enhances bone-implant interfacial mechanical bonding. In summary, nanotopology is proven to have beneficial effects on the osteoblast response and bone growth.