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RESEARCH REPORT |
1 Departments of Biochemistry and
2 Dental Materials Science and Technology, Iwate Medical University School of Dentistry, Morioka, Iwate, 020-8505 Japan;
* corresponding author, nchosa{at}iwate-med.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: apatite • titanium • alkaline-heat treatment • osteoblasts • RT-PCR
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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), 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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). 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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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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 April 30, 2003; Last revision March 29, 2004; Accepted March 30, 2004
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REFERENCES |
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