Surface bioactivation through the nanostructured layer on titanium modified by facile HPT treatment

Facile fabrication of nanostructured surface is of great importance for the use of titanium (Ti) implants in biomedical field. In this study, a low-cost and easy-to-operate method called HPT (hydrothermal & pressure) here has been developed and used to fabricate the expected nanostructured surface on Ti substrates. The effects of experimental parameters on the morphology of Ti surface were investigated and characterized. The results indicated that by altering the hydrothermal pressure, NaOH concentration and treating time, surface nanostructure like nanopetals or nanoflakes could be formed on the surface of Ti substrates. The orthogonal experiments were conducted to demonstrate the optimized operation conditions. A formation mechanism of the nanostructured titanate layer was proposed, revealing that the nanostructured layer could be formed via a special upward and downward co-growth manner. In vitro cell culture showed that the HPT treated Ti substrates, especially the T-10 sample, could greatly enhance the cell-material interactions, i.e. the cell proliferation and differentiation, focal protein adhesion, and osteogenic factor expression. The HPT method paves a new way to modify the surface of Ti implants with better bioactivity and promising prospect for future biomedical applications.


SBF immersion test
The effects of surface nanostructure by HPT treatment on the bioactivity of Ti substrate were evaluated by immersing the treated Ti samples at 37°C in 24 mL of SBF with ion concentrations neatly equal to those of human blood plasma. The SBF recipe was prepared according to Kokubo's 1 formulation and buffered at pH 7.4 with Tris-hydroxymethyl amino methane and 1.0M HCl at 37°C . Samples were placed in an incubator at 37°C for 1, 4, 7 and 14 days, respectively. To keep the ion concentration constant, SBF solution was refreshed every 2 days. At each time point, the corresponding samples were taken out and gently rinsed with deionized water, then dried in a desiccator for further analysis.

Results
When the NaOH concentration is more than 15M, there are no special structure features present, and just a little round granules are dotted on the Ti surface   To evaluate the surface bioactivity of nanoflake-like titanate layer on T-10 sample prepared by HPT treatment in 10.0M NaOH solution for 4h, the SBF soaking experiments were carried out to investigate the formation of Ca-P precipitation on the treated Ti-10 sample. As shown in Fig. S2(a, c) after immersion in SBF for 1 and 3 day, plenty of nuclei have deposited on the surface of T-10 sample homogeneously. There are two Ca2p peaks at binding energies of 346.7 eV (Ca2p1/2) and 350.2 eV (Ca2p3/2) (Fig. S3e) and one P2p peak at 133.1eV (Fig.S3d) appeared in XPS spectra.
The Ti2p peaks at 458.5eV (Ti2p1/2) and 464.1 eV (Ti2p3/2) (Fig. S3a) are attributed to Ti−O bonds 2 , while Na1s peak at 1071.3 eV (Fig. S3c) is from the nanoflake-like titanate layer on Ti surface. The detected Ca and P peaks in XPS spectra demonstrate successful deposition of Ca-P aggregations in SBF which is generally regarded as the representative of surface bioactivity. In addition, it has been reported that the nucleation of calcium titanate readily occurred on sphere-like titanate layer. [2][3] The possible reasons is that the formation of Ti-OH is very easy via the exchange of Na + ions in sodium titanate with H3O + ions in body fluid. 5,[6][7][8] The Ti-OH groups are negatively charged 9 and, therefore, likely combine with the positively charged Ca 2+ ions in body fluid to form an amorphous calcium titanate. 10 Therefore, we can deduce that the deposited nuclei in SBF are mainly composed of Ca-P aggregations and may also contain amorphous calcium titanate.
With the progress of immersion from Day 3 to Day 7, the increase of Ca2p and P2p peak intensity and the decrease of Na1s and Ti2p peak intensity implied that calcium ions accumulated continuously, and the treated Ti surface became more positively charged, which attracted those negatively charged phosphate groups electrostatically to form plenty of amorphous Ca-P nuclei. 10 As shown in Fig. S2(e, f), the nuclei grew up and a large number of nanoflower-like Ca-P spheres with a diameter of 20~30 μm deposited on T-10 surface on Day 7.
After immersion for 14 days, the spherical Ca-P aggregations formed a precipitation layer covering the Ti surface entirely (Fig. S2g). In Fig. S3, the Ca2p and P2p peak intensity further increased markedly, and the Ti2p peaks almost disappeared completely, but small Na1s peak was still present. The EDS element mapping in Fig. S3f also testified that the precipitation layer was mainly Ca-P compound. According to the report of Kim et al, 10 the calcium phosphate precipitate could spontaneously transform into crystalline hydroxyapatite (HA), which is based on the fact that the solubility of HA is much lower than that of other calcium phosphates in body fluid.