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Biomaterials & Bioengineering |
Characterization of Apatite Formed on Alkaline-heat-treated Ti
N. Chosa1,*, ,
M. Taira2,,
S. Saitoh2,
N. Sato1 and
Y. Araki2
1 Departments of Biochemistry and
2 Dental Materials Science and Technology, Iwate Medical University School of Dentistry, Morioka, Iwate, 020-8505 Japan;
Correspondence: * corresponding author, nchosa{at}iwate-med.ac.jp
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ABSTRACT
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Alkaline-heat-treated titanium self-forms an apatite surface layer in vivo. The aim of the present study was to materialistically characterize the surface of alkaline-heat-treated titanium immersed in simulated body fluid (AHS-TI) and to examine the differentiation behavior of osteoblasts on AHS-TI. SEM, thin-film XRD, FTIR, and XPS analyses revealed that AHS-TI contained a 1.0-µm-thick, low-crystalline, and [002] direction-oriented carbonate apatite surface. Human osteoblast-like SaOS-2 cells were cultured on polystyrene, titanium, and AHS-TI, and RT-PCR analyses of osteogenic differentiation-related mRNAs were conducted. On AHS-TI, the expression of bone sialoprotein mRNA was up-regulated as compared with that on polystyrene and titanium (p < 0.05). On AHS-TI, the expression of osteopontin and osteocalcin mRNAs was up-regulated as compared with that on polystyrene (p<0.05). The results indicate that the apatite was bone-like and accelerated the osteogenic differentiation of SaOS-2, suggesting that alkaline-heat treatment might facilitate better integration of titanium implants with bone.
Key Words: apatite • titanium • alkaline-heat treatment • osteoblasts • RT-PCR
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INTRODUCTION
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Titanium has been widely used in endosseous dental implants. Alkaline-heat treatment is a simple and cost-effective method of coating titanium with apatite (Kokubo et al., 1996). It has been reported that NaOH treatment of pure titanium causes a sodium titanate hydrogel surface layer to form. Subsequent heat treatment at 600°C results in an amorphous sodium titanate surface layer. When immersed in simulated body fluid or placed in vivo, alkali-heat-treated titanium forms an apatite surface layer (coded as AHS-Ti) (Kokubo et al., 1996). There have been few reports, however, which scrutinized this apatite coating (Takadama et al., 2001). The apatite layer was reported to be both osteo-conductive and -inductive, which is advantageous for the clinical usage of titanium implants (Yan et al., 1997). Furthermore, there have been few attempts to examine, systematically, the osteogenic differentiation of human osteoblasts cultured on apatite itself (Massas et al., 1993; Nishio et al., 2000).
We hypothesized that an apatite layer did exist on AHS-TI in a form analogous to natural bone and thus could accelerate the osteogenic differentiation of osteoblasts. In this study, therefore, we first characterized the surface structure of alkali-heat-treated titanium analytically before and after soaking it in SBF. Then, on the apatite layer (AHS-TI) as well as polystyrene culture dishes and titanium, we cultured human osteoblast-like cells, SaOS-2, and examined the expression of 6 osteogenic differentiation-related marker mRNAs by RT-PCR up to 4 wks after confluence. The osteogenic differentiation proceeds sequentially with the appearance of specific osteogenic marker mRNAs. Usually, alkaline phosphatase (ALP), type I collagen (COL), and osteonectin (OSN) mRNAs are expressed first, followed by osteopontin (OPN) and bone sialoprotein (BSP) mRNAs, while osteocalcin (OSC) mRNA emerges last (Beck et al., 2000). The expression of these mRNAs on AHS-TI needed to be clarified.
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MATERIALS & METHODS
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Alkali-heat Treatment of Ti
Commercially pure (c.p.) Ti plates (10 x 10 x 1 mm for surface analyses and 25 x 25 x 1 mm for cell culture) (Ti > 99.8%, Kobe Steel, Kobe, Japan) were mechanically polished with 100-, 180-, and 320-grit sand paper, successively, and ultrasonically washed in acetone and distilled water. The polished titanium plates (TI) were then soaked in a 5M NaOH solution at 60°C for 24 hrs, followed by gentle washing in distilled water and drying in an oven at 37°C for 24 hrs (A-TI). A-TI plates were subsequently heated to 600°C at a rate of 5°C/min in an electrical furnace, kept at 600°C for 1 hr, and allowed to cool in the furnace (AH-TI).
Soaking in Simulated Body Fluid
The AH-TI plates were dipped at 37°C in 50 mL of simulated body fluid (SBF) with a pH of 7.4 and ionic concentrations (142.0 mM Na+, 5.0 mM K+, 1.5 mM Mg2+, 2.5 mM Ca2+, 147.8 mM Cl–, 4.2 mM HCO3–, 1.0 mM HPO42–, and 0.5 mM SO42–) resembling those of human blood plasma (Kokubo et al., 1996). We prepared the SBF by dissolving reagent-grade NaCl, NaHCO3, KCl, K2HPO4•3H2O, MgCl2•6H2O, CaCl2, and Na2SO4 in distilled water, and buffered it at pH 7.4 with tris-hydroxymethyl aminomethane [(CH2OH)3CNH2] and hydrochloric acid at 37°C. The SBF solution was replaced every two days. After soaking for 8 days, the plates were washed with distilled water, and dried on a clean bench (AHS-TI).
Surface Analyses
The surfaces of TI, A-TI, AH-TI, and AHS-TI were examined by scanning electron microscopy (SEM) (S-700, Hitachi, Tokyo, Japan), thin-film x-ray diffraction (XRD) (JDX-3500, JEOL, Tokyo, Japan), Fourier transform infrared spectroscopy (FTIR) (Spectrum One, Perkin Elmer Japan, Kanagawa, Japan), and x-ray photoelectron spectroscopy (XPS) (AXIS-HSi, Kratos, Manchester, UK). In the thin-film XRD measurements, Cu-K radiation was used as an x-ray source. The glancing angle of the specimen ( angle) was fixed at 1° against the incident beam. The characteristic XRD peaks were labeled with reference to standard powder diffraction data (JCPDS, 1991). In the XPS measurements, Al-K radiation was used as an x-ray source. The XPS take-off angle was set at 90°. Following XPS wide scans, XPS narrow scans of O 1s, Ti 2p, Ca 2p, 2p, and Na 1s regions were conducted for the quantification of atomic compositions. The depth profile was taken on AHS-TI.
Cell Culture
Human osteoblast SaOS-2 cells were cultured in Eagle’s -modified minimum essential medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco BRL, Rockville, MD, USA). The cells (5 x 105) were plated in polystyrene culture dishes (PS) (#150288, Nalge Nunc International, Tokyo, Japan), or on TI plates in PS and on AHS-TI plates in PS and cultured. Every 3 days, the medium was exchanged, and TI and AHS-TI plates were transferred to new PS so that the spreading of the cells from the plates to PS could be minimized. After confluence (dated at 0 wk), the cells were further cultured for 1, 2, 3, or 4 wks.
RT-PCR
Total RNA of the cells cultured on PS, TI, or AHS-TI was isolated with ISOGEN reagent (Nippongene, Tokyo, Japan), and the cDNA was synthesized by TrueScript II (Sawady Technology, Tokyo, Japan) according to the manufacturer’s instructions. PCR was performed with the following primers: alkaline phosphatase (ALP, 5'-CTCGTTGACACCTGGAAGAGC-3' and 5'-ACAGGATGG CAGTGAAGGGCT-3'); type-I collagen (COL, 5'-ACTGGGG AAACCTGTATCCGG-3' and 5'-AAGGGCAGGCGTGAT GGCTTA-3'); osteonectin (OSN, 5'-CCGAAGAGGAGGTGG TGGCGG-3' and 5'-ACGGGGTGGTCTCCTGCCTCC-3'); osteopontin (OPN, 5'-CCTAGCCCCACAGACCCTTCC-3' and 5'-CTGTCCTTCCCACGGCTGTCC-3'); bone sialoprotein (BSP, 5'-CAACACTGGGCTATGGAGAGGACGC-3' and 5'-GTAATTGTCCCCACGAGGTTCCCCG-3'); osteocalcin (OSC, 5'-CAGCAAAGGTGCAGCCTTTGT-3' and 5'-TCCTGAAA GCCGATGTGGTC-3'); glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 5'-TGGTATCGTGGAAGGACTCATG-3' and 5'-TCTCTTCCTCTTGAGCTCTTGC-3'). PCR cycle conditions were 95°C for 30 sec, 60°C for 60 sec, and 72°C for 90 sec for 20, 22, or 24 cycles. The relative expression levels of each mRNA were determined by their densitometric value divided by that of the corresponding GAPDH control, with the use of NIH Image (National Institutes of Health, Bethesda, MD, USA).
Statistics
Data were presented as the mean ± standard deviation (SD). Statistical analysis was performed with the Student t test, and p-values < 0.05 were considered ‘significant’.
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RESULTS
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Surface Characterization
It became evident from SEM photo-micrographs (Fig. 1 ), thin-film XRD (Fig. 2A), FTIR (Fig. 2B), XPS wide-scan spectra (Figs. 3A–D), and the depth profile of AHS-TI (Fig. 3E) that AHS-TI had (a) a domed carbonate-apatite surface layer which was about 1.0 µm thick, low-crystalline, and preferentially oriented in the [002] direction with (b) a first graded zone, (c) a Ti-O layer, (d) a second graded zone, and (e) a titanium base.
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Figure 1. Surface image of AHS-TI. SEM photo-micrographs at low (left) and high (right) magnification of (A,B) TI (polished titanium), (C,D) A-TI (alkali-treated titanium), (E,F) AH-TI (alkali-heat-treated titanium), and (G,H) AHS-TI (alkali-heat-treated titanium soaked in SBF) (n = 1). Note: TI had a rough scratched surface, both A-TI and AH-TI had porous honeycomb structures, and AHS-TI had a domed structure.
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Figure 2. AHS produced carbonate-apatite on TI. (A) Thin-film XRD profiles of TI (polished titanium), A-TI (alkali-treated titanium), AH-TI (alkali-heat-treated titanium), and AHS-TI (alkali-heat-treated titanium soaked in SBF) (n = 1). Note: TI had 5 metallic titanium peaks; A-TI had peaks of amorphous sodium titanate and titanium; AH-TI had peaks of rutile, sodium titanate, and titanium; and AHS-TI had peaks of hydroxyapatite along with peaks of trace titanium. (B) FTIR charts of AH-TI, AHS-TI, and hydroxyapatite (n = 1). Note: Arrow indicates existence of CO32– in AHS-TI.
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Figure 3. AHS-TI consisted of 1.0 µm carbonate-apatite and underlying layered structures. XPS wide-scan spectra of the surfaces of (A) TI (polished titanium), (B) A-TI (alkali-treated titanium), (C) AH-TI (alkali-heat-treated titanium), and (D) AHS-TI (alkali-heat-treated titanium soaked in SBF) (representative case). Note: The chemical composition (at%) of each element is also depicted (mean ± SD, n = 5). (E) The depth profile of AHS-TI (n = 1). Note: AHS-TI consisted of 5 distinctive layers. From the surface, the apatite layer continued with a thickness of about 1.0 µm. Below the first graded zone, there is a Ti-O layer presumably consisting of titanium and TiO2 oxide about 1.0 µm thick. Below the second graded zone, there is base metallic titanium.
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Osteogenic Differentiation
Expression levels of ALP and COL mRNAs on TI and AHS-TI declined as the culture period increased to more than 3 wks, while that on PS remained quasi-constant. Expression levels of OSN mRNA were kept constant on the 3 materials examined (Fig. 4A). Expression levels of OPN mRNA were significantly higher on AHS-TI than on PS and TI at 3 and 4 wks (p < 0.05), although the former levels were not different from those on TI at 2 wks. The expression of BSP mRNA on AHS-TI was induced at 1 wk, and overwhelmed that on PS and TI in all 4 culture periods (p < 0.05). The expression of OSC mRNA on AHS-TI appeared after 2 wks, but its magnitude gradually declined at 3 and 4 wks, being greater than that of PS after 2 wks (p < 0.05) (Fig. 4B). [Supplemental material can be found in the APPENDIX.]
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Figure 4. On AHS-TI, the expression of early-stage differentiation-related mRNAs was down-regulated and that of middle- to late-stage differentiation-related mRNAs was up-regulated. (A) The changes in the levels of expression of 6 osteogenic differentiation marker mRNAs and GAPDH of SaOS-2 cultured for 1, 2, 3, or 4 wks on PS (polystyrene dish), TI (polished titanium), and AHS-TI (alkali-heat-treated titanium soaked in SBF) (representative case). (B) The relative expression levels of OPN, BSP, and OSC on PS, TI, and AHS-TI (mean ± SD, n = 5). *p < 0.05. Note: AHS-TI accelerated the osteogenic differentiation of SaOS-2 cells with ALP and COL mRNAs down-regulated and OPN, BSP, and OSC mRNAs up-regulated.
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DISCUSSION
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Our structural findings of AHS-TI were basically consistent with those reported previously (Takadama et al., 2001). Our new findings were that the apatite was oriented in the [002] direction, contained CO32–, and possessed complicated layered structures beneath the surface. The domed structure of the apatite appeared to consist of many needle-shaped crystals, along the length of which columnar Ca might line up. The carbonate apatite identified here was analogous to the inorganic phase of human bones (Bigi et al., 1997). Its composition and structure are regarded as more suitable for osseous implants than those of hydroxyapatite (Hasegawa et al., 2003). The apatite structure of AHS-TI may differ from that precipitated with other methods (Massaro et al., 2001).
Our results clarified that AHS-TI accelerated osteogenic differentiation in that the expression of early-stage differentiation-related mRNAs was down-regulated and that of middle- to late-stage differentiation-related mRNAs was up-regulated, compared with the results for PS and TI. This might be attributed to the early settling of SaOS-2 on apatite. On apatite, the cells started proliferating earlier, but their motility ceased earlier, leading to earlier osteogenic differentiation (Okamura et al., 2001). Another reason might stem from the existence of phosphate on the apatite surface, which might up-regulate the production of OPN (Beck et al., 2000). Other factors, such as up-regulation of osteoblast-specific transcription factor Cbfa1, might be involved, but this is beyond the scope of this study. The expression of BSP mRNAs best clarified the change in osteogenic differentiation, and is considered a more reliable indicator of osteogenic differentiation of osteoblasts than often-unstable OSC mRNAs (Cooper et al., 2001). TI might contain traces of calcium-phosphate precipitates after immersion in SBF (Hanawa and Ota, 1991), and thus, could slightly accelerate the osteogenic differentiation. It appeared that PS alone little accelerated the osteogenic differentiation but had an important role in improving cell adhesion. The use of SaOS-2 seemed reasonable to examine the material’s influence on osteogenic differentiation.
Referring to c.p. endosseous titanium implants, alkaline-heat treatment would be beneficial because it can improve the osteo-integration of implants with bone through the apatite layer.
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ACKNOWLEDGMENTS
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The study was supported by (1) a Grant-in-Aid for High-performance Biomedical Materials Research, (2) a Grant-in-Aid for the Open Research Project from the Ministry of Education Culture, Sports, Science and Technology, Japan, and (3) CREST of JST (Japan Science and Technology Corporation).
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FOOTNOTES
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 authors contributing equally to this work; 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.
Received for publication April 30, 2003.
Revision received March 29, 2004.
Accepted for publication March 30, 2004.
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REFERENCES
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Journal of Dental Research, Vol. 83, No. 6,
465-469 (2004)
DOI: 10.1177/154405910408300606
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ABSTRACT
INTRODUCTION