| A novel nearβtitanium alloy Ti-5Zr-3Sn-5Mo-15Nb (TLM) has been developed, which possesses the characteristics of avoidance of controversial alloying elements aluminum (Al) and vanadium (V) ions, lower elastic modulus (～69 GPa), higher strength and better plasticity and toughness matching than otherβTi alloys reported so far. Animal experiments showed that TLM exhibits good biocompatibility with no irritating response to oral mucous membrane. Our in vitro study proved that osteoblasts adhesion, proliferation on TLM is similar to those on Ti-6Al-4V, and osteoblasts differentiation is promoted on TLM. In this study we modified TLM by micro-arc oxidation (MAO) at two different final voltages in order to improve its biocompatibility. Surface morphology and composition of the specimens were analyzed by scanning electron microscope (SEM) and energy dispersive spectroscopy (EDS). Surface roughness and contact angle were measured using a surface profilometer and a contact angle measuring system, and surface energy was calculated based on Owens-Wendt-Rabel-Kaelble theory. Cell adhesion and proliferation were assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Total intracellular protein synthesis and alkaline phosphatase (ALP) activity were assessed using commercial kits. The gene expression of several osteogenesis-related genes was surveyed by semi-quantitative RT–PCR.The results indicated that:1. A porous bioactive oxide layer was grown on TLM, and the diameters of holes were larger when higher voltage applied. Both Ca and P were incorporated into the oxidized surfaces. Surface roughness was enhanced on the micro-arc oxidized surfaces. After MAO treatment, water contact angles became smaller and surface energies of the TLM surfaces were increased, especially the polar component.2. Cell culture experiments showed an enhanced osteoblasts adhesion and proliferation on the micro-arc oxidized surfaces, and better cell proliferation was found on the surface formed at 450 V than that at 300 V.3. After 3-d culture, on the polished surface the osteoblasts exhibited monolayer proliferation with large amount of fillopodia and lamellipodia, most of which showed an elongated shape and aligned along the direction of the grooves. While on the micro-arc oxidized surfaces, the osteoblasts formed a double layer net-like structure, with more lamellipodia coming out from the cells. It seemed that the osteoblasts spread better than those on the polished surface, especially on the surface formed at high voltage.4. After 7-d culture, Total intracellular protein synthesis was higher on the micro-arc oxidized surfaces. While ALP activity was lower on the micro-arc oxidized surfaces, and showed the lowest on the micro-arc oxidized surface formed at higher voltage.5. No obvious variations in gene expression of integrinβ1 (Itgβ1), core binding factor-α1 (Cbfa1), osteopontin (OPN), collagen type Iα2-chain (Coll-Iα2) and fibronection (FN) by osteoblasts were observed on different surfaces. However, the expression of osteocalcin (OCN) was strikingly increased on MAO surfaces after 72 h. Interestingly, obvious enhanced bone morphogenetic protein (BMP)-2/-4 expression was observed on MAO surfaces. Conclusions:MAO process can enhance surface roughness, wettability and surface energy of TLM, and incorporate Ca/P into the surface, which are of dependence on the MAO voltage. Initial cell adhesion, spread and proliferation are enhanced, while ALP activity is inhibited by MAO. The enhanced BMP-2/-4 expression may be a reason for the enhanced osteoblasts behaviours by MAO.
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