More than millions of bone graft procedures are expected to be performed yearly to fill bone defects or improve fracture healing and repair. This number is expected to continue to increase with the rapid growth of the elderly population. Therefore, developing a suitable material for repairing and regenerating bones that have been fractured due to disease, trauma and aging is a significant clinical challenge1. Titanium (Ti) and Ti-based alloys are widely used as biomaterials for orthopedic and dental implant applications due to their suitable mechanical properties, exceptional corrosion resistance and the highest biocompatibility with bone among all metallic biomaterials2,3. Although Ti and Ti-based alloys have specific and excellent properties for biomedical applications, some problems must be addressed for the long-term implantation of these materials. One of the most important issues is biomechanical incompatibility, especially in the elastic modulus of the commercially used Ti and Ti-based alloys, with human bone because both bone overload and excessive stress protection can result in bone resorption4,5.
Compared with conventional metallic materials, such as stainless steel and Co-Cr-Mo alloys, Ti and Ti-based alloys show a relatively lower elastic modulus than those of stainless steel (200 GPa) and Co-Cr-Mo alloys (200-230 GPa). However, the elastic modulus of most of the commonly used commercial Ti and Ti-based alloys, including commercially pure Ti (CP-Ti), Ti-6Al-4V and Ti-6Al-7Nb (100-110 GPa)6, is still an order of magnitude higher than that of human cortical bone (10-30 GPa)7,8. This mismatch between the elastic modulus of bone and that of implants will cause an insufficient load transfer from the implant to the adjacent bone and thus induce a stress-shielding effect during long-term implantation at the sites of load-bearing bones. This type of stress-shielding effect will lead to bone resorption at the bone-implant interface and eventually result in implant failure9,10.
β-type Ti alloys have been the most attractive materials, for overcoming the incompatibility between the elastic modulus of Ti-based implants and that of bone, for orthopedic applications due to their non-toxic components, high mechanical strength and low elastic modulus3,11,12. Of all β-type Ti alloys, Ti-24Nb-4Zr-8Sn (wt%, hereafter designated Ti2448) is a recently developed β-type Ti alloy for biomedical applications.
This novel alloy consists of non-toxic and non-allergic elements and possesses a low elastic modulus of approximately 45 GPa13,14, close to that of human cortical bone.
Such a low elastic modulus may prevent the stress-shielding effect caused by the inhomogeneous stress transfer between metal implants and the adjacent bone.
Furthermore, this alloy not only has a low elastic modulus that is close to that of human bone, but it also has high mechanical strength - an ideal combination/balance of properties that is not available in the other β-type Ti alloys that have been developed so
far.
For long-term clinical use, the corrosion resistance of metallic materials is a great concern, especially when a metallic substitute is implanted in the virulent electrolytic environment of the human body, because corrosion is a gradually degradation of materials by electrochemical attack. Therefore, the corrosion performance of Ti2448 is extremely important for orthopedic and dental implant applications. Cheng et al.
reported that for dental implant applications, the Ti2448 alloy showed a corrosion resistance similar to that of CP-Ti and the Ti6Al4V alloy in simulated oral environments, such as modified Fusayama artificial saliva and a lactic acid solution15. Moreover, Bai et al. showed that the corrosion resistance of the Ti2448 alloy was better than that of Ti6Al4V but comparable to that of CP-Ti in different simulated physiological solutions16,17. It has been commonly accepted that Ti and Ti-based alloys show exceptional corrosion resistance due to the natural formation of a passive oxide film.
However, this protective passive film, which inhibits the release of metal ions, may become unstable in the human body due to deterioration during long-term implantation.
Once the passive surface film is disrupted, corrosion proceeds, and metal ions are released continuously, which leads to ion accumulation and thus results in biological side effects and eventually implant failure18,19. Although the corrosion behavior of the Ti2448 alloy is similar to that of Ti, there is still a potential risk of metal ion release from the Ti2448 alloy due to the corrosion process. Therefore, determining how to further improve the bio-corrosion resistance of the Ti2448 alloy for future long-term implantation is one of the important purposes of this study.
Moreover, the passive oxide film on metallic materials plays an influential role not only in corrosion resistance but also in the biocompatibility of materials. It is well known that surface properties, including surface topography, roughness, chemical composition and wettability, are important factors in biocompatibility because they affect the biological responses at the bone-implant interface20,22. Surface topography is a key surface property because it is able to regulate the responses of cells that are located on material surfaces. Some studies have reported the design and creation of surface geometries with a suitable nanoscale topography that were able to improve cell responses, such as cell adhesion23, migration24, proliferation25 and differentiation26. There are various surface modification treatments that can create a nanoscale topography on a metallic material surface to provide the desired biological responses.
In our previous studies, a fast and simple electrochemical anodization treatment was used to produce a nano-networked oxidelayer on the surfaces of CP-Ti and Ti-based alloys to improve the hemocompatibility and cell responses. Moreover, the layer was also able to enhance the corrosion resistance of biomedical Ti alloys27-29.
Our hypothesis is that a Ca/P-containing oxide layer with a nanoscale porous topography could be produced and thickened on the surface of the Ti2448 alloy using a simple and fast electrochemical anodization treatment in Ca/P-containing solution to improve the bio-corrosion resistance and biocompatibility of this new β-type Ti alloy with low elastic modulus. To analyze this hypothesis, the polished Ti2448 specimens were created, as well as polished Ti control specimens, and Ti2448 specimens treated with electrochemical anodization treatment in Ca/P-containing solution to produce a nanoscale porous topography containing Ca/P composition. Within this research, the surface characteristics, including the surface morphology, oxide layer thickness, crystal structure and surface wettability, were investigated after the anodization treatment.
Furthermore, the corrosion behaviors of the anodized Ti2448 alloys in simulated body environments were evaluated. To provide helpful indicators for further in vivo studies and clinical applications, the protein adsorption of the anodized Ti2448 alloy was studied as well.
2. Materials and Methods
2.1 Specimen preparation
Discs (diameter of 15 mm; thickness of 1 mm) of Ti2448, the new β-type of Ti alloys, which were polished with SiC papers from #120 up to #1200 and then cleaned in ethyl alcohol were used as substrates. Pure commercial Ti disks (diameter of 15 mm;
thickness of 1 mm) that were polished and cleaned according to the above-mentioned procedure were used as a reference control.
A potentiostat was used for the electrochemical anodization treatment to apply two different anodic currents, A1 and A2 (A1< A2 < 0.5 ampere), to the polished Ti2448 substrate for a few tens of min in Ca/P-containing alkaline solution (Ca+2 < 0.5M; PO4 -3 < 0.5M). The polished Ti and Ti2448 specimens that were not subjected to the electrochemical anodization treatment were termed Ti-M and Ti2448-M, respectively.
The polished Ti2448 specimen treated by electrochemical anodization with the applied currents A1 and A2 were termed Ti2448-A1 and Ti2448-A2, respectively.
2.2 Surface characteristics analysis
The surface morphology and average roughness of the test specimens were evaluated using field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM), respectively. The crystallographic structure and thickness of the outermost surface layer of the anodized Ti2448 specimens were evaluated using transmission electron microscopy (TEM). Prior to the TEM evaluation, the cross-sectional test specimens were prepared using a focused ion beam milling process. The
chemical composition of the outer surface layer of the specimens was analyzed by X-ray photoelectron spectroscopy (XPS). The surface wettability of the test specimens was analyzed using a contact angle goniometer, and the surface free energy of the specimens was calculated with two different solutions: polar double-distilled H2O and non-polar diiodomethane.
2.3 Bio-corrosion resistance analysis
The surface corrosion resistance of the polished Ti-M and Ti2448-M and the electrochemical anodization-treated Ti2448-A1 and Ti2448-A2 was evaluated using a potentiostat. A saturated calomel electrode (SCE) and a platinum sheet were used as the reference and counter electrode, respectively. The test specimens with and without the electrochemical anodization treatment were used as the working electrodes. Neutral simulated blood plasma (SBP; pH 7.4)30 and modified Fusayama artificial saliva (AS;
pH 5.2)31 were used as the corrosion test electrolyte and were maintained at 37°C during the experiment. The SBP was used to simulate the environment inside human body, and the AS was used to simulate that of the human mouth. All test specimens were placed into two different electrolytes, and the polarization curves of all specimens were measured from -1 V to + 1.5 V (with respect to the SCE).
2.4 Protein adsorption analysis
Two different types of protein were used as model proteins to evaluate the difference in protein adsorption on the test specimens. One was bovine serum albumin (BSA, Sigma), the most abundant protein in human body plasma32; the other was fibronectin (Sigma), an important protein involved in cell adhesion, migration, proliferation and differentiation33. The test specimens were immersed in two different phosphate buffered saline (PBS) solutions, one containing 5 mg/ml BSA and the other containing 50 μg/ml fibronectin. After 5 min of incubation at 37°C, the test specimens were washed with double-distilled H2O and then dried at room temperature. XPS was used to analyze the nitrogen spectra (in terms of N1s) of the test specimens to estimate the ability for proteins to adsorb on the various treated test specimens.
2.5 Statistical analysis
The number of samples was 3 for all measurements, and the results were expressed as the means ± standard deviation (SD). Student’s two-tailed t-test was used to determine the level of the significance, and p<0.05 was considered statistically significant.