The optimal temperature for the alkaline treatment and subsequent heat treatment is determined to optimize the nanoporous structures formed on Ti6Al4V titanium alloy plates. Surface characterization of the alkali-heat treated samples was performed by scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The effects of heating temperatures on albumin adhesion, rat bone marrow mesenchymal stem cells (BMMSCs) adhesion, alkaline phosphatase activity, osteocalcin production, calcium deposition, and Runx2 mRNA expression were evaluated. The nanotopography, surface chemistry, and surface roughness were unchanged even after heat treatments at 200, 400, and 600°C. Only the amorphous sodium titanate phase changed, increasing with the temperature of the heat treatments, which played a crucial role in promoting superior cell adhesion on the nanoporous surface compared with the sodium hydrogen titanate obtained by a single alkali treatment. The heat treatment at 800°C did not enhance cell attachment on the surface because the nanostructure was dramatically destroyed with the reappearance of Al and V. This study reveals that nanoporous structures with amorphous sodium titanate were fabricated on Ti6Al4V surface through an amended alkali-heat treatment process to improve BMMSCs adhesion.
Titanium alloys are the most frequently used metallic for medical implants, due in part to the spontaneous protective oxide coating that forms on their surface, although titanium-6-aluminium-4-vanadium (Ti6Al4V) alloy has been used most frequently as a biomaterial. The implants must have surface treatments in advance for a successful osseointegration. The surface characteristics of the implant material affect the rate and extent of osseointegration. Vandrovcová and Bačáková [
The structures used in this study were nanostructures similar to the titanium dioxide (titania, TiO2) nanotubes, created by titanium deposition using the process of TiO2 sputtering, and were named titanium nanosheets (TNS) [
In recent study, we found TNS-modified titanium alloy surfaces induce RBM cell bone differentiation [
Recently, methods for inducing bioactivity for direct bonding between the bone and implant by means of chemical surface modification of the biomaterial have been widely studied. Kim et al. introduced combined alkali- with heat-treatment as a method of surface modification of titanium alloys to improve bioactivity [
The aims of the present study were to investigate the combination of alkali treatment with various heat treatments of the Ti6Al4V alloy and evaluate the ability of the modified surface to affect the morphology, osteogenic differentiation, and biocompatibility of RBM cells to increase the success rate of titanium implants. The null hypothesis was that there would be no difference in cellular behavior on titanium surfaces as a result of different heat treatment at several temperatures after NaOH treatment.
Ti6Al4V disks (15 mm in diameter and 1 mm in thickness) of Ti grade 5 (chemical composition in wt.% N: 0.02; C: 0.03; H: 0.011; Fe: 0.22; O: 0.16; Al: 6.12; V: 3.93; and Ti: balance) were prepared as the substrate material (Daido Steel, Osaka, Japan). Nanoporous Ti6Al4V alloy was produced by treatment with concentrated alkali solution at room temperature, as published earlier. In short, the alloy disks were successively polished with SiC abrasive paper (600, 800, 1000, and 1500 grits), ultrasonically rinsed in acetone, ethanol, and distilled water for 10 min each, and dried in air. The disks were soaked in 10 M NaOH solution at 30°C for 24 h, washed with distilled water, and dried at room temperature overnight to obtain the nanostructured alloy samples. Following the alkaline treatment at 30°C, the samples were heated to 200, 400, 600, and 800°C at a rate of 5°C/min in an electrical furnace, maintained at the desired temperature for 1 h, and cooled naturally to room temperature. An alloy subjected to only NaOH treatment was used as a control sample.
The surface characterization of the alkali-heat treated samples was observed by scanning electron microscopy (SEM) (S-4800; Hitachi, Tokyo, Japan) with an acceleration voltage of 10 kV.
Atomic force microscopy (AFM) (SPM-9600; Shimadzu Tokyo, Japan) was also performed to obtain the mean average surface roughness (
Bovine serum albumin (BSA), fraction V (Pierce Biotechnology), was used as a model protein. Protein solution (300
Rat BMMSCs were isolated and cultured according to our previously published procedures [
Rat BMMSCs were seeded on the specimens at an initial density of 4 × 104 cells/cm2, and cell attachment was analyzed after 72 h and 7 d. The nonadherent cells were removed by washing with phosphate-buffered saline (PBS) after the appropriate incubation time. CellTiter-Blue Reagent (50
After 7 and 14 days of culture, cells were washed with PBS, lysed with 200
The sandwich enzyme immunoassay used in this study was specific for rat osteocalcin (OCN) and can measure its levels directly in cell culture supernatant after 21 and 28 days of culture. The OCN levels in cell culture supernatant were measured using a commercially available ELISA Kit (Rat Osteocalcin ELISA Kit DS, DS Pharma Biomedical Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions.
Calcium deposited in the extracellular matrix was measured after dissolution with 10% formic acid. The amount of calcium was quantified using a Calcium E-Test Kit (Wako Pure Chemical Industrials Ltd.). After 21 and 28 days of culture, 1 mL Calcium E-Test reagent and 2 mL kit buffer were added to 50
After 3 days of culture, the total RNA was extracted from the cells and cDNA was synthesized from 1
Statistical analysis was performed by one-way analysis of variance followed by Turkey’s test. All results were based on a mean ± standard deviation from five random fields of each sample. Differences were considered statistical at
The alkali treatment with 10 M NaOH at 30°C created a nanoporous network structure on Ti6Al4V alloy surfaces. Observation of the porous network structure at the nanoscale showed that the pores were well interconnected with an average diameter of about 50 to 100 nm at 30°C. Figure
SEM and AFM photographs of the specimens subjected to alkali treatment (A30) with 10 M NaOH solution and alkali-heat treatment (AH) performed various heating temperatures at 200°C, 400°C, 600°C, and 800°C.
The maintenance of the nanostructure on the Ti6Al4V alloy after heat treatment over the 200–600°C range was also verified by AFM (Figure
Surface roughness of the specimens subjected to alkali treatment (A30) and alkali-heat (AH) treatments under various subsequent heating temperatures.
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|
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---|---|---|
A30 | 26.581 | 291.280 |
AH200 | 23.412 | 288.217 |
AH400 | 25.635 | 340.186 |
AH600 | 25.444 | 272.379 |
AH800 | 165.384 | 372.140 |
The broad-range XPS surface chemical analysis of the alkali-heat treated Ti6Al4V alloys is shown in Figure
Surface chemical compositions of the specimens subjected to alkali treatment (A30) and alkali-heat (AH) treatments under various subsequent heating temperatures.
Treatment | Chemical composition (at%) | ||||
---|---|---|---|---|---|
O | Na | Al | Ti | V | |
A30 | 66.39 | 5.15 | 0 | 28.46 | 0 |
AH200 | 66.68 | 5.08 | 0 | 28.24 | 0 |
AH400 | 67.03 | 4.89 | 0 | 27.93 | 0 |
AH600 | 67.18 | 4.97 | 0 | 27.65 | 0 |
AH800 | 70.23 | 0.12 | 14.62 | 10.20 | 4.83 |
XPS survey spectra of Ti-6Al-4V alloy specimens subjected to alkali treatment (A30) and alkali-heat (AH) treatments with the subsequent heating temperatures at 200°C, 400°C, 600°C, and 800°C.
The crystallographic structures (Figure
TF-XRD pattern of the surfaces subjected to alkali treatment (A30) and alkali-heat (AH) treatments with the subsequent heating temperatures at 200°C, 400°C, 600°C, and 800°C.
The amount of protein adsorbed from bovine serum albumin on the surface after 1, 3, 6, and 24 h incubation was assayed (Figure
Initial number of albumin on the surfaces of Ti6Al4V specimens subjected to alkali treatment (A30) and alkali-heat (AH) treatments with the subsequent heating temperatures at 200°C, 400°C, 600°C, and 800°C.
Figure
Number of adherent BMMSCs on the surfaces of Ti6Al4V specimens subjected to alkali treatment (A30) and alkali-heat (AH) treatments with the subsequent heating temperatures at 200°C, 400°C, 600°C, and 800°C.
ALP activity on the surface after 7 and 14 days of incubation was assayed (Figure
ALP activity on the surfaces of Ti6Al4V specimens subjected to alkali treatment (A30) and alkali-heat (AH) treatments with the subsequent heating temperatures at 200°C, 400°C, 600°C, and 800°C.
The presence of osteocalcin in the supernatant of the specimens after 3 and 4 weeks of culture is shown in Figure
OCN production on the surfaces of Ti6Al4V specimens subjected to alkali treatment (A30) and alkali-heat (AH) treatments with the subsequent heating temperatures at 200°C, 400°C, 600°C, and 800°C.
Calcium deposition in the supernatant of the specimens after 3 and 4 weeks of culture is shown in Figure
Calcium deposition on the surfaces of Ti6Al4V specimens subjected to alkali treatment (A30) and alkali-heat (AH) treatments with the subsequent heating temperatures at 200°C, 400°C, 600°C, and 800°C.
Runx2 is a transcription factor involved in the early stages of osteogenic differentiation. As shown in Figure
Runx2 mRNA on the surfaces of Ti6Al4V specimens subjected to alkali treatment (A30) and alkali-heat (AH) treatments with the subsequent heating temperatures at 200°C, 400°C, 600°C, and 800°C.
This study investigated whether RBM cells respond differently to titanium alloy implants with chemical and heat treatment surface modifications compared with those that were untreated. The results of this study showed that, after the proliferation of cells, expressions of Runx2 transcription factor and RBM cell differentiation markers, such as ALP and OCN, were elevated in alkali- and heat-treated alloy disks compared with alkali modified titanium alloy disks. This study found that calcium deposition in the extracellular matrix of the RBM cells was increased in the presence of surface-treated titanium alloy disks compared with controls disks. Our results suggest that titanium alloy disks modified by alkali and heat treatment at 600°C promote RBM cell proliferation, differentiation, and activation, which augment calcium deposition.
Alkali- and heat-treated titanium alloy was reported to bond to bone so it is thought to be clinically applicable as an implant and orthopedic material. In theory, if alkali treatment without heat treatment could induce the bone-bonding ability with titanium alloy, it would be possible to prepare bioactive titanium alloy implants using NaOH solution. Kim et al. [
From the surface characterizations results, alkali treatments with 10 M NaOH solution at 30°C provided a fine nanoporous network. This study proposed that the pore size of the porous network was sensitive to the concentration of alkali solution. The mechanism of the formation of a porous network on Ti metal or its alloys during alkali treatment is the corrosive attack of the hydroxyl groups [
In XPS analysis, the results confirm that the surface chemistry of the alkali-treated alloy did not include Al and V before and after heat treatments until 600°C. It has been demonstrated that the alloy species of Al and V selectively dissolve in the alkaline solution, and Al and V oxides are formed in the TiO2 layer after heat treatment, which contribute to eliminating the toxicity of the Ti6Al4V alloy [
The XRD findings were in agreement with the results of conventional alkali-heat treatment and showed that the sodium hydrogen titanate was gradually transformed into amorphous sodium titanate and/or crystalline sodium titanate after the heat treatments. When the NaOH-treated Ti metal is subjected to a heat treatment, its surface sodium titanite hydrogel layer is dehydrated and transformed into an amorphous sodium titanate at 400 to 500°C, fairly deified at 600°C, and then converted into crystalline sodium titanate and rutile above 800°C [
In this study, albumin and cell proliferation of BMMSCs, Runx2 expression, ALP activity, OCN production, and Ca deposition were performed to demonstrate the biocompatibility of the modified Ti6Al4V alloy surface, which is considered as a critical prerequisite for cell proliferation and differentiation. The adsorption of ECM adhesive proteins onto implanted materials is the first essential step in bone tissue response and affects cell adhesion and proliferation [
Surface characterization studies revealed that the nanotopography, surface chemistry, and surface roughness of the modified layer obtained by alkali treatment were maintained even after heat treatments at 200, 400, and 600°C. Only the phase structure was altered, particularly the amorphous sodium titanate phase, which might play a crucial role in promoting cell adhesion on the nanoporous surface with the increase in heating temperatures. The heat treatment of 800°C transformed the nanoporous structure into a crystalline structure at the macroscale level with the reappearance of Al and V, which induced a decline in cell attachment. Consequently, these results suggest that nanoporous surface features and BMMSCs cell proliferation and bone differentiation on Ti6Al4V surface may be controlled and improved by varying the temperature of the initial alkali immersion and subsequent heat treatment. The materials should be further studied as a novel bioactive material for dental implants. Further development of advanced implant materials using nanotechnology should further improve their osseointegration.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by Grant-in Aid for Scientific Research (26861664) from the Japan Society for the Promotion of Science. The authors would like to thank Mr. H. Hori, Central Institute of Dental Research, Osaka Dental University, Japan, for his help with experimental techniques.