Surface thermal oxidation on titanium implants to enhance osteogenic activity and in vivo osseointegration

Abstract

Thermal oxidation, which serves as a low-cost, effective and relatively simple/facile method, was used to modify a micro-structured titanium surface in ambient atmosphere at 450 °C for different time periods to improve in vitro and in vivo bioactivity. The surface morphology, crystallinity of the surface layers, chemical composition and chemical states were evaluated by field-emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Cell behaviours including cell adhesion, attachment, proliferation, and osteogenic differentiation were observed in vitro study. The ability of the titanium surface to promote osseointegration was evaluated in an in vivo animal model. Surface thermal oxidation on titanium implants maintained the microstructure and, thus, both slightly changed the nanoscale structure of titanium and enhanced the crystallinity of the titanium surface layer. Cells cultured on the three oxidized titanium surfaces grew well and exhibited better osteogenic activity than did the control samples. The in vivo bone-implant contact also showed enhanced osseointegration after several hours of oxidization. This heat-treated titanium enhanced the osteogenic differentiation activity of rBMMSCs and improved osseointegration in vivo, suggesting that surface thermal oxidation could potentially be used in clinical applications to improve bone-implant integration.

Figure 1

Surface morphology of four samples at different magnifications: (a,b) TO-0, (c,d) TO-2, (e,f) TO-4, (g,h) TO-6.

Figure 2. Surface chemical components and chemical states of different samples examined by XPS.

Figure 3

XRD pattern of the four specimens (a) and SEM images of cross-section of TO-2, TO-4 and TO-6 (b–d).

Figure 4. Surface biocompatibility.

(a) Optical images of water contact angles, (b) Adsorption of proteins on different titanium surfaces. (c) Statistical results for adhesive cell numbers. (d) MTT assay for cell metabolism on titanium substrates. (e) Cell nuclei stained with DAPI at 4 hours after seeding at a 100-foldmagnification. (f) Actin cytoskeletons were labelled to observe cell attachment at 24 hours after seeding at 400-foldmagnification. (g) Actin cytoskeletons were labelled to observe cell morphology 7 days after cell-seeding on these four titanium surfaces.

Figure 5. SEM observation of cell morphology on titanium surfaces after 2 days of seeding.

Figure 6. Osteogenic differentiation.

Alkaline phosphatase (a) and matrix mineralization (b) semi-quantitative assay; (c) Expression of osteogenic-related differentiation genes (ALP, OCN, OPN and BSP) were measured by real-time PCR. (d) Alizarin Red S staining at 10-fold magnification. (e) Expression of OCN was detected by immunofluorescent staining after 14 days culture.

Figure 7. Sequential fluorescent labelling observations.

(a) Red and green represent labelling by Alizarin Red S (AL) and Calcein (CA), respectively (bar = 300 um). (b) The blue rectangle region was selected to evaluate the new bone rate. (c) The area of the two fluorochromes stained bone.

Figure 8. Histological observations and histomorphometric measurements.

(a) Undecalcified sections are stained with Van Gieson’s picro fuchsin. The results of BV/TV (b) and BIC (c) from the histomorphometric measurements. (Notes: ^#P < 0.05, ^##P < 0.01 versus TO-0 group; *P < 0.05, **P < 0.01 versus TO-2 group; ^&P < 0.05, ^&&P < 0.01 versus TO-4 group).