Influence Of Anodization On The Biological Behaviors Of Osteoblasts Cultured On Ti-5Zr-3Sn-5Mo-15Nb

The aim of this research is to evaluate the effects of anodization on the biological behaviors of osteoblasts cultured on Ti-5Zr-3Sn-5Mo-15Nb (TLM). TLM surfaces were modified using an anodization technique, with different anodizing voltage 5V and 20V. Field emission Scanning electron microscope (FE-SEM) was used to observe the surface topography of the prepared samples. The crystallization of samples was evaluated by X-ray diffraction (XRD). Contact angle measurements were performed by the sessile-drop method with an contact angle measuring system. The protein absorption of samples was determined by commercial Protein Assay Kit. Primary rat calvarial osteoblasts were obtained by digestion of calvarial bone of one day old Sprague–Dawley rats. Cell adhesion were assessed by the method of staining and counting with ?uorescence microscope. Cell proliferation was assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Total intracellular protein synthesis and alkaline phosphatase (ALP) activity were assessed using commercial kits. The cell morphology was observed using FE-SEM. The expression of several osteogenesis-related genes was assessed using Realtime-PCR. The results showed that:1. After treatment of anodization, a nanonet-like topography was fabricated at 5V and the average diameter of the pores was about 15 nm. A nanotubular topography was fabricated at 20V and the average diameter of the hollow nanotubes was about 80 nm.2. XRD pattern shows that the crystallization of samples was changed to anatase nature.3. The contact angle results obtained with ultrapure water and diiodomethane show differences among polished, 5V anodized and 20V anodized surfaces, and all differences are found to be statistically significant(p < 0.05). After anodization especially at 20V voltage, contact angles of liquid on TLM surface became smaller and surface energy of samples increased.4. The 20V anodized surfaces absorb significantly more protein than 5V anodized and polished surfaces(p < 0.05).5. It was found that 20V anodized surfaces presented a significantly enhanced cell adhesion compared with 5V anodized and polished surfaces(p < 0.05). Not only a better cell proliferation was observed on the anodized surfaces than that on polished surfaces, but also an inhibition was observed on the 5V anodized TLM surfaces(p < 0.05).6. Remarkable improved ALP activity and total intracellular protein synthesis of osteoblasts were observed on 20V anodized surfaces compared with 5V anodized and polished surfaces(p < 0.05).7. After 4 days culture, the osteoblasts on two kinds of anodized surfaces spread well and present flat shapes, whereas those on polished surfaces spread along the grooves formed by polish. In higher magnification images, it can be seen that the osteoblasts on 20V anodized surfaces spread out with a large amount of lamellopodia and fillopodia. The protrusions of osteoblasts on 5V anodized and polished surfaces were less than those on 20V anodized surfaces. 8. Ther was no significant difference in the expressions of Integrin-β1 and RUNX2 observed on three different TLM surfaces. But strikingly higher expressions of ALP, OCN and BMP-2 were observed on the 20V anodized surfaces than that on 5V anodized and polished surface after 3 days of culture. And osteoblasts on 5V anodized surfaces anodized showed up-regulated expression of OPN contrasted with the other surfaces after 10 days culture.Conclusion:Nanoscale topography on TLM surface can be fabricated by anodization, of which the characteristics are closely related to the anodized voltage. Compared with polished surfaces and nanonet like surfaces anodized at 5V, the nanotubular TLM surfaces fabricated at 20V present significantly enhanced osteoblast adhesion, spread, ALP activity, total intracellular protein synthesis and the initial expressions of osteogenesis-related genes such as ALP, OCN and BMP-2. But the 5V anodized surfaces almost have little effect on the osteoblasts cultured on them except for inhibition of proliferation and up-regulated expression of OPN.