| Surface modification on metal implant for enhancing bone-like bonding (osseointegration) is a key issue in bone repair field. To establish surface micro-environment with micro-/nanostructure and bioactive composition, which is appropriate for cell/tissue growth, is an effective approach to improve growth of bone tissue on the interface between implant and host bone and enchance osseointegration. However, how to effectively combine the above two and make them exert biological effects simultaneously, is still a challenge. In this thesis, based on TiO2 nanostructure with excellent cell responses, surface-modificated layers (films) were established on tantalum via further optimizing micro-/nanostructure and loading bioactive elements (Mg or Zn). Moreover, cell adhesion, proliferation, differentiation and mineralization were adopted to evaluate the biological effects of the films. In addition, the mechanism of synergistic effect of micro-/nanostructures and bioactive elements on osteogenesis was discussed. The main results in 3 aspects are:1. Mg-containing TiO2 nanodot films.Mg was doped directly into TiO2 nanodots via sol-gel method. TiO2 nanodot films with various contents of Mg were obtained through controlling the addition of Mg into precursor sol (0～0.1Mg/Ti, molar ratio), keeping the nanodots with similar size and distribution. The nanodot was polycrystalline anatase, and Mg enriched in the superficial layer to some extent. The release amount of Mg were approximately proportional to the Mg contents in films. However, the accumulated amounts of Mg release were very low (0.017μg/mL for 0.1Mg-TiO2 after 14-day soaking). Mg doping improved the capacity of protein adsorption of the films. As the results of pre-osteoblasts culturing, Mg doping improved TiO2 nanodot films on 1-day cell adhesion quantity by 17.5% and 7-day cell proliferation quantity by 18.4%, indicating that Mg doping enhanced cell responses of TiO2 nanodot films.2. TiO2 nanorod films with Zn-loading bioactive glass.Zn-containing bioactive glass was filled into TiO2 nanorod films via sol-gel method. In the preparation process, the concentration of bioactive glass precursor sol was adjusted (concentration of Si from 0.160 mol/L to 0.320 mol/L). When the concentration of Si was in the range of 0.213～0.256 mol/L, the corresponding thickness of bioactive glass layer varied from 200 to 270 nm. Bioactive glass filling not only provided a carrier for Zn, but also further optimized the micro-/nanostructure of TiO2 nanorod films, which enhanced cell responses. Introduction of TiO2 into bioactive glass could effectively inhibit the overfast degradation of bioactive glass. When Ti was added with Ti/Si= 0.5 (molar ratio), bioactive glass layer had appropriate degradation behavior, which could meet the requirement of stimulating cells. A part of Ca was replaced by Zn, and results showed that it hardly impact the thickness and degradation of bioactive glass. The 14-day cumulative concentration of Zn release was lower than 0.1μg/mL.As the results of cell culturing, TiO2 nanorod films filled with Zn-containing bioactive glass (TiO2/Zn-BG) had significantly better cell responses than Zn-containing bioactive glass films (Zn-BG) and TiO2 nanorod films filled with Zn-free bioactive glass (TiO2/BG). Especially in the evaluations of osteogenic differentiation,7-day ALP activity of cells on TiO2/Zn-BG film was higher than that of Zn-BG film by 25.1% and that of TiO2/BG film by 43.9%,14-day OCN secretion by 130.9% and 1041.0% higher,14-day gene expression of Runx-2 by 309.8% and 505.6% higher, and 21-day extracellular matrix mineralization by 176.9% and 476.5% higher, respectively. In the condition of relatively low concentration of Zn release, cells on TiO2/Zn-BG films showed excellent activity of osteogenic differentiation in each stage, indicating that nanostructure and bioactive elements develop a synergistic effect, which could be described as:TiO2 nanorods structure improved initial cell adhesion and spreading; TiO2 nanorods, Zn-containing bioactive glass and cells adhered on their top established a semi-closed space together; this micro-environment facilitated the value effect of Zn on stimulating cells, and consequently qualified cells with high capacity of ostegenic differentiation.3. TiO2 nanorod films with Zn-loading calcium phosphate.Zn-containing calcium phosphate (Zn-CaP) was covered onto TiO2 nanorod films via spin-coating method. In the preparation process, various distributions of Zn-CaP were obtained through controlling modes and rotating speed of spin-coating process. Zn-CaP coating not only introduced bioactive Zn into the films, but also created microscaled structure on the films. For the films with dense distribution of Zn-CaP (TiO2/D-ZCP), the thickness of Zn-CaP was 314±20 nm, and the coverage percentage was 49.7%; for the films with sparse distribution of Zn-CaP (TiO2/S-ZCP), the thickness of Zn-CaP was 204±37 nm, and the coverage percentage was 37.6%. The length or width of uncovered TiO2 nanorods area was 50～150μm, which established a special topographic surface together with Zn-CaP coating in microscaled distribution. After heat treatment at 500℃, Zn-CaP coating firmly bonded with TiO2 nanorod films. Zn-CaP in the coating was HA with low crystallinity, and addition of Zn did not impact the crystalline phase of CaP. For TiO2/D-ZCP films and TiO2/S-ZCP films, the Zn-CaP coating showed fast degradation, while the accumulated concentrations of Zn release (0.104μg/mLand 0.087μg/mL, respectively) were much lower than fully covered Zn-CaP films (F-ZCP,0.200μg/mL).As the results of cell culturing, compared to those on TiO2/D-ZCP films and F-ZCP, MC3T3-E1 cells on TiO2/S-ZCP had more powerful ability of osteogenic differentiation. Particularly,14-day OCN secretion of cells on TiO2/Zn-BG film was higher than that of TiO2/D-ZCP film by 20.9% and that of F-ZCP film by 45.4%, 14-day gene expression of Runx-2 by 188.0% and 184.4% higher, and 21-day extracellular matrix mineralization by 109.2% and 108.2% higher, respectively. After the relatively short period of degradation and ions release, TiO2/S-ZCP films still showed excellent activity of osteogenesis in later stage, suggesting that structure and bioactive composition develop a combined effect, which could be depicted as: nanotopography improved cell adhesion and filopodia extension; microtopography improved cell proliferation; and the release of bioactive elements like Zn enhanced cell differentiation.In this thesis, TiO2 micro-/nanostructures and bioactive elements were combined to expand biological effect via synergistic mechanism, and it provides insight into design of a novel material surface on metal implant with a combination of micro-/nanotopography and bioactive elements, which consequently is of importance for efficiently enhancing osseointegration. |
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