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Nanomechanical properties and molecular structures of in vitro mineralized tissues on anodically-oxidized titanium surfaces
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 629 – 637
Original Article
nanomedjournal.com
Nanomechanical properties and molecular structures of in vitro mineralized tissues on anodically-oxidized titanium surfaces In-Kee Jang, BDS a , Reina Tanaka, PhD b , Wurihan, BDS a , Dai Suzuki, PhD c , Yo Shibata, PhD a,⁎, Naoki Fujisawa, PhD d , Yasuhiro Tanimoto, PhD e , Kayoko Ogura, PhD b , Ryutaro Kamijo, PhD c , Takashi Miyazaki, PhD a a
Department of Conservative Dentistry, Division of Biomaterials and Engineering, Showa University School of Dentistry, Tokyo, Japan Department of Conservative Dentistry, Division of Aesthetic Dentistry and Clinical Cariology, Showa University School of Dentistry, Tokyo, Japan c Department of Biochemistry, Showa University School of Dentistry, Tokyo, Japan d Hysitron, Inc., Minneapolis, MN, USA e Department of Dental Biomaterials, Nihon University School of Dentistry at Matsudo, Chiba, Japan Received 16 May 2013; accepted 26 September 2013
b
Abstract The biomechanical stability of mineralized tissues at the interface between implant surface and bone tissue is of critical importance. Anodically oxidized titanium prepared in a chloride solution results in enhanced mineralization of adherent osteoblasts and has antimicrobial activity against oral microorganisms. We evaluated the nanomechanical properties and molecular structures of the in vitro mineralized tissues developing around anodically oxidized titanium surfaces with and without preparation in chloride solution. Anodically oxidized titanium surfaces showed superior osteogenic gene expressions than those of thermally oxidized and bare titanium surfaces. Preparation of anodically oxidized titanium in chloride enhanced the production of mineralized tissue around it. However, the mineralized tissue around anodically oxidized titanium prepared without chloride had increased mineral:matrix and cross-linking ratios, resulting in higher hardness and lower elasticity. From the Clinical Editor: In this study anodically oxidized titanium was used to enhance the biomechanical stability of mineralized tissues at the implant surface – bone tissue interface. The mineralized tissue around anodically oxidized titanium prepared without chloride had increased mineral:matrix and cross-linking ratios, resulting in higher hardness and lower elasticity. © 2014 Elsevier Inc. All rights reserved. Key words: Titanium; Implant; Reactive oxygen species; Osteoblast; Nanoindentaion; Raman spectroscopy
Osseo-integrated titanium implants must be capable of forming rigid osseous anchors. Therefore, various surface modification techniques have been explored to improve the biological responses of the bone to titanium implants. 1-4 Implant surfaces should have not only wear and corrosion resistance, but also biological compatibility achieved by various surface related parameters such as surface Author contributions: Y.S. and I-K.J. performed project planning, experimental work, data interpretation and preparation of the manuscript. D.S., Wurihan, Y.T. and K.O. performed experimental work. R.T. and N.F. participated in data analysis. R.K. and T.M. supervised research and participated in project planning. Funding sources: This work was supported by a Grant-in-Aid for Scientific Research (B) and a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. None of the authors have any conflicts of interest to declare. ⁎Corresponding author: Department of Conservative Dentistry, Division of Biomaterials and Engineering, Showa University School of Dentistry, Tokyo 142-8555, Japan. E-mail address: [email protected] (Y. Shibata).
topography or surface chemistry. The anatase phase TiO2 in amorphous matrix accompanied with micro-submicron surface texture on anodically oxidized titanium surfaces results in enhanced expression of osteogenic genes and increased mineralization of adherent primary osteoblasts on its surface. These effects might be due to the superhydrophilicity of COO − and OH − oxidation products of reactive oxygen species (ROS) on these surfaces. 5 Anodically oxidized titanium incorporates the saturated ions of the electrolytes in the outer parts of the oxide layer at the interface between the oxide and electrolyte. 6-8 We have recently described a variety of anodically oxidized titanium surfaces prepared in electrolyte solutions with or without the addition of chloride. 5,9 Preparation in a solution containing chloride (Ti-Cl) generates surface hypo-chloride (HClO), whereas preparation in Na2HPO4 solution generates surface hydroxyl radicals (•OH). Both surfaces were found to enhance the expression of osteogenic gene and the mineralization of adherent osteoblasts. In addition, the surface hypochloride on the Ti-Cl surface destroyed the cell wall membrane of adherent microorganisms, thus demonstrating strong antimicrobial activity.
1549-9634/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2013.09.007 Please cite this article as: Jang I.-K., et al., Nanomechanical properties and molecular structures of in vitro mineralized tissues on anodically-oxidized titanium surfaces. Nanomedicine: NBM 2014;10:629-637, http://dx.doi.org/10.1016/j.nano.2013.09.007
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Figure 1. Surface topography of anodically oxidized titanium. The surface morphology of the titanium samples was examined using (A) a scanning electron microscope and (B) a laser microscope.
The biomechanical stability of titanium implants is of particular importance, especially if this property can be enhanced through structural manipulation of mineralized tissues between the implant surface and bone tissue. 10-14 Mineralized tissues such as bone and dentin are natural nano-biocomposites with a calcium phosphate and collagen matrix. 15-17 Densification and consolidation of mineralized tissue have been shown to increase its hardness and inelasticity. 14,18 Previous studies have reported that extracellular ROS might affect collagen cross-linking in human skin and cartilage, 19,20 but whether extracellular ROS has similar effects in mineralized tissues remains unclear. However, we hypothesize that the amount of surface ROS on anodically oxidized surfaces may affect the nanomechanical properties of the mineralized tissues interfacing with that surface. This study evaluated the expression of bone matrix proteinrelated genes in adherent osteoblasts on anodically oxidized titanium surfaces. We also investigated the nanomechanical and structural properties of mineralized tissues developing in vitro around anodically oxidized titanium surfaces, using nanoindentation tests with depth-dependent loading/partial unloading tests and Raman spectroscopy. 2,14,21-23
Methods Sample preparation JIS grade 2 titanium (KS-50; Kobe Steel, Tokyo, Japan) was prepared with dimensions of 10 × 10 × 1.0 mm. The surface of each specimen was mechanically polished under running water using waterproof polishing papers (up to #1200 grit) before finishing with 0.3 μm diameter alumina particles. Prepared specimens were
ultrasonically cleaned in acetone/detergent solution (7X; ICN Biomedicals, Aurora, OH, USA) and pure distilled water for 15 min, then dried and stored in a sealed desiccator for 1 week at a humidity of 50% and a temperature of 23 °C. For anodic oxidation, Ti specimens were connected to the anode of a device developed in our research group and immersed in electrolytes. A 50 × 100 × 0.1 mm stainless steel plate was used as the counter-electrode. Discharge (416 mA/cm 2) was generated between the electrolyte and the working electrode through a gas layer on the surface of the electrode for 60 s. Specimens were anodically oxidized in 100 mL of 1 M Na2HPO4 (Ao–Ti) or NaCl (Ti–Cl), then washed in pure distilled water. Untreated Ti was oxidized by heating at 600 °C for 30 min in a dental furnace (Ho-Ti). All titanium samples were stored in the dark at an ambient laboratory atmosphere for 24 h. Surface characterization Surface topography The surface morphology of the titanium samples was examined using a scanning electron microscope (S-2360 N; Hitachi) and a laser micro scope (OLS-4000; Shimadzu) (Figure 1). The average roughness (Sa), root mean square deviation (Sq), maximum peak height (Sp), maximum valley depth (Sv) peak to valley depth (Sz), degree of symmetry of the surface height about the mean plane (Ssk), and presence of inordinately high peaks/ deep valleys (Sku) were quantified using the proprietary software of these instruments with a scanning range of 643 × 644 μm. X-ray photoelectron spectroscopy (XPS) Sample surfaces were analyzed by XPS (ESCA-3400; Shimadzu, Kyoto, Japan). High-resolution spectra of Ti2p,
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O1s, C1s, P2p, and Cl2p were analyzed using a monochromatic X-ray source (Mg Kα radiation). A 20 mA emission current and 8 kV of accelerated voltage were applied in the analysis (UHV conditions at b 1 × 10 − 7 mbar). The binding energies for each spectrum were calibrated based on the C1s spectra of 285.0 eV.
Table 1 Primer sequences used in this study for qPCR analysis of osteoblast gene expression.
ROS detection on titanium surfaces by XPS The amount of ROS present on the titanium samples was determined by previously described methods. 24 Briefly, ammonium chloride solution (4 M, 500 ml) was mixed with zinc chloride solution (0.4 M, 250 ml) and the pH of the mixture adjusted to 6.9 with 30% ammonium hydroxide. The volume of the mixture was then adjusted to 1000 ml with deionized water. Titanium samples were immersed individually in 150 ml of this solution at ambient temperature for 300 s. During immersion, active radicals were mostly exchanged for zinc ions, and zinc chelates were formed. The disks were then immersed three times in 150 ml of deionized water to remove non-adsorbed chemical species. Sample surfaces were then analyzed by XPS. The relative concentration of zinc on the sample surfaces was also evaluated as a proxy measurement of the amount of surface ROS, to which it should be proportional.
Alkaline phosphatase (Alp) Type I collagen
Osteoblastic cell culture Primary osteoblasts were obtained from the calvariae of neonatal ddY mice (Sankyo Co. Inc., Tokyo, Japan) using 0.1% collagenase and 0.2% dispase. 25 Primary osteoblasts were cultured in α-MEM (Wako) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) containing penicillin-streptomycin and incubated at 37 °C in a CO2 incubator (5% CO2, 95% air). Before reaching confluence, cells were detached and seeded onto the titanium disks in polystyrene culture plates at a density of 4 × 10 4 cells/cm 2 in culture medium supplemented with 50 μg/mL ascorbic acid, 10 mM Na-β-glycerophosphate and 10 nM dexamethasone. Culture medium was replaced every 3 days. Quantitative real-time PCR (qPCR) Gene expression was analyzed and quantified by qPCR. Total RNA was extracted with TRIzol reagent (Invitrogen), then reverse-transcribed using SuperScript III enzyme preparation (Invitrogen). The qPCR assays were performed using a Fast SYBR Green PCR system (Applied Biosystems, CA, USA), using primer sequences as shown in Table 1. The mean relative fold changes in mRNA expression after normalization to Gapdh are shown with the standard deviation for all data points. The gene expressions on the samples were defined as relative to the Ti surface (defined as a relative expression level of 1). Mineralization assay Alizarin red staining was performed after 2 weeks of culture. Following three washes with PBS and two with pure distilled water, the titanium samples were dried in air, and stained for 5 min using 1% alizarin red (Sigma, St Louis, MO, USA) at pH 6.3-6.4. The mineralization capability of the cultured osteoblasts was quantified by colorimetric detection of calcium deposition. Samples stained with alizarin red were washed with PBS and incubated overnight in 1 ml of 0.5 M HCl solution with
Gene target
Primer sequence
Gapdh
5′-AAATGGTGAAGGTCGGTGTG-3′ and 5′-TGAAGGGGTCGTTGATGG -3′ 5′-TTCCCACGTTTTCACATTCG-3′ and 5′-GCCAGACCAAAGATGGAGTTG-3′ 5′-GCCTTGGAGGAAACTTTGCTT-3′ and 5′-GCACGGAAACTCCAGCTGAT-3′ 5′-CCTCTCCTGCTACCGCACAA-3′ and 5′-CTAGAGCCGCCAAATTTGCT-3′ 5′-CTGACAAAGCCTTCATGTCCAA-3′ and 5′-GGTAGCGCCGGAGTCTGTT-3′ 5′-GCTTTTGCCTGTTTGGCATT-3′ and 5′-AGCTGCCAGAATCAGTCACTTTC-3′ 5′-GAGTTAGCGGCACTCCAACTG-3′ and 5′-CACTTTTGGAGCCCTGCTTT-3′
Cyclin D1 Osteocalcin (Ocn) Osteopontin (Opn) Bone sialoprotein 2 (Bsp2)
gentle shaking. The solution was mixed with o-cresolphthalein complexone in alkaline medium (Calcium Binding and Buffer Reagent; Sigma) to produce a red calcium-cresolphthalein complexone complex. Color intensity was measured with an ELISA reader (Synergy HT; BioTek Instruments, Winooski, VT) at an absorbance wavelength of 575 nm. Nanoindentation test Indentation experiments were performed using a quantitative nanomechanical test system (TS-70 Triboscope; Hysitron, Inc., MN, USA) with a scanning probe microscope (SPM-9700; Shimadzu, Kyoto, Japan). A Berkovich indenter (Hysitron) was chosen for this study. Fused quartz acted as the standard material to calibrate the Berkovich nanoindenter tip area and instrument compliance. 26 Indentation tests were performed perpendicular to the surface of osteoblast-incubated samples after 2 weeks of cell culture. To minimize errors arising from surface roughness, appropriately smooth mineralized regions were chosen with a scanning range of 30 × 30 μm and then 3 × 3 μm. Depth-dependent loading/partial unloading tests 14 were conducted at a maximum load of 1 mN with a total of 66 loading and unloading cycles, with a 1 s holding time at each point. The distance between indents was more than 10 μm to avoid any influence of residual stresses from adjacent indentations. Hardness and elastic modulus were calculated from the force–displacement curves using the TS-70 proprietary software. The Berkovich tip area function used in this study produced an effective range of indenter contact depths of 20200 nm from the sample surface. For the standard nanoindentation analysis, it is ideal to have a smooth surface at the indenter penetration depth. 26,27 The loading/partial unloading test overcomes issues associated with biological structures because the evaluation of depth-dependent mechanical behaviors permits the normalization of data for any variation in contact between biological structures and the indenter tip. 28 Micro-Raman spectroscopy Raman spectra of randomly selected mineralized nodules on titanium samples were acquired using a confocal Raman
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microscope (RXN1; Kaiser Optical Systems, Inc., MI, USA) with a 785 nm laser diode source. Light was focused by a 50 × objective onto a 3 μm spot on the mineralized nodules. The Raman scatter (5 s exposure time) was collected by a spectrograph with a spectral resolution of 1 cm − 1. Peak intensities were recorded for Amide I (1660 cm − 1), Amide III (1250 cm − 1 ), B-type carbonate (1070 cm − 1 ), proline (856 cm − 1), and the υ1 phosphate (960 cm − 1). The mineralto-matrix ratios of mineralized nodules were calculated based on the intensity ratio of the υ1 phosphate peak to Amide III. 23 The second-derivative spectra obtained from original Amide I were used to calculate the cross-linking ratio of mineralized tissues on the titanium samples. 22 Two subbands were shown at 1690 and 1660 cm − 1 and may be indicative of secondary structure changes associated with collagen cross-linking. The intensity of the 1660/1690 cm − 1 sub-bands was used to calculate non-reducible/reducible crosslink ratios. 29 Statistical analysis Three samples were evaluated in the surface characterization and qPCR assays. Five samples were evaluated in the cell culture studies with alizarin red staining. In nanoindentation and Raman analysis, we expressed the random selection of five representative regions as the mean. Results are expressed as mean ± SD. The normal distribution of each value was confirmed using the Kolmogorov–Smirnov test. The appropriateness of the hypothesis of homogeneous variances was verified using Bartlett’s test. Data were analyzed by ANOVA followed by a post hoc Tukey test, with P b 0.01 considered statistically significant.
Results
Table 2 Mean surface roughness parameters of the titanium samples used in this study. Sp (μm) Sv (μm) Sz (μm) Sa (μm) Sq (μm) Ssk Ti 3.560 Ho-Ti 3.046 Ao-Ti 25.704 Ti-Cl 47.038
2.936 2.789 19.691 19.282
6.496 5.835 45.395 66.320
0.304 0.343 4.357 4.742
0.390 0.442 5.493 6.218
0.142 0.290 0.053 1.179
Sku 3.772 3.640 2.944 7.373
The average roughness (Sa), root mean square deviation (Sq), maximum peak height (Sp), maximum valley depth (Sv) peak to valley depth (Sz), degree of symmetry of the surface height about the mean plane (Ssk), and presence of inordinately high peaks/deep valleys (Sku) were quantified using the proprietary software of these instruments with a scanning range of 643 × 644 μm.
Gene expression in adherent osteoblasts After 1 week of osteoblast culture, the expression of Alp, Ocn, Opn and Bsp2 genes on the Ao-Ti and Ti-Cl surfaces were significantly higher than that on the Ti and Ho-Ti surfaces (Figure 2, A). On Ti-Cl, the expression of type I collagen was significantly higher (P b 0.01) than on the other samples. There were no obvious differences in gene expression profiles between cells grown on polystyrene cell culture plates and those seeded on Ti (Supplemental Figure 1). Mineralized nodules on titanium samples After 2 weeks of osteoblast cell culture, the areas of mineralized nodules on both Ao-Ti and Ti-Cl were found to be larger than those on Ti and Ho-Ti (Figure 2, B). As quantified in Figure 2, B, the areas of mineralization on anodically oxidized Ao-Ti and Ti-Cl were much greater (P b 0.01) than on Ti and Ho-Ti. However, the area of mineralization on Ti-Cl was significantly higher (P b 0.01) than on Ao-Ti.
Surface characterization Surface topography Pores with diameters in the micron or submicron scale were counted on Ao-Ti, whereas granular deposits forming on the TiCl surface were quantitated. The mean surface roughness parameters are summarized in Table 2. The height parameters of both anodically oxidized Ao-Ti and Ti-Cl were higher than those of Ti and Ho-Ti. XPS The relative atomic concentration of each element on the titanium samples is shown in Table 3. The energy position of Cl2p on Ti–Cl was detected at 201.4 eV (not shown). Therefore, it appeared that a portion of titanium tetrachloride had changed and combined with oxygen, resulting in the generation of ROS hypochloride. 5,30,31 After immersion in a solution containing ammonium chloride and zinc chloride, the relative concentration of zinc on both anodically oxidized Ao-Ti and Ti-Cl was significantly higher than that on Ti and Ho-Ti; hence the increased amount of ROS generated on the Ao-Ti and Ti-Cl surfaces.
Nanoindentation test After 2 weeks of cell culture, we calculated the representative hardness and elastic modulus of the mineralized tissues developing on the titanium samples using depth-dependent partial unloading curves, as shown in Figure 3. The hardness and elastic modulus of the bare Ti surface were 3.1 ± 0.3 GPa and 178.8 ± 4.5 GPa, respectively (data not shown). The hardness and elastic modulus (20-200 nm contact depth) of mineralized tissues on Ti were 0.25 ± 0.06 and 4.8 ± 0.05 GPa, respectively, while those on Ho-Ti were 0.24 ± 0.04 and 5.5 ± 0.04 GPa, respectively. In addition, the hardness and elastic modulus of the mineralized tissue on Ti-Cl were 0.25 ± 0.06 and 4.8 ± 0.05 GPa, respectively. There were no significant differences (P N 0.01) in the nanomechanical properties of any of the mineralized tissues grown on Ti, Ho-Ti and Ti-Cl surfaces. The hardness and elastic modulus of the mineralized tissue on Ao-Ti were significantly higher (P b 0.01) than those of Ti, Ho-Ti and Ti-Cl, being estimated at 0.78 ± 0.04 and 13.5 ± 0.08 GPa, respectively. This elastic modulus value is comparable to that reported for human cancellous bone and dentin. 10,14,32
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Table 3 Relative atomic concentrations of each element (%) on the titanium samples, with (+) or without immersion in a solution containing ammonium chloride and zinc chloride. Ti Ti Ho-Ti Ao-Ti Ti-Cl Ti (+) Ho-Ti (+) Ao-Ti (+) Ti-Cl (+)
13.9 23.6 6.2 20.7 10.2 9.8 2.7 14.3
O ± ± ± ± ± ± ± ±
0.4 1.3 0.8 1.8 1.6 0.9 0.6 2.3
54.5 61.3 59.2 54.2 43.6 40.7 40.4 51.6
C ± ± ± ± ± ± ± ±
2.8 3.1 3.8 2.2 4.2 2.2 3.1 4.4
31.6 15.1 15.5 15.7 44.5 47.1 40.3 16.4
± ± ± ± ± ± ± ±
2.1 1.9 2.2 1.2 5.4 3.2 4.1 2.2
P
Cl
Zn
15.6 ± 3.1 11.3 ± 1.2 -
9.4 ± 1.4 10.3 ± 2.4
1.6 2.2 5.2 6.1
± ± ± ±
0.4 0.7 1.4 2.4
Results are expressed as the mean ± SD of three independent repeats. Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test.
Figure 2. (A) Relative gene expression in samples after 1 week of cell culture. (B) Representative images of alizarin red staining and quantification of mineralized areas on sample surfaces after 2 weeks of cell culture. Results are expressed as the mean ± SD of three independent repeats. Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. P b 0.01 (*) was considered significant.
Molecular structure of mineralized nodules There were no distinctive Raman spectra observed on the Ti and Ho-Ti specimens, even after 2 weeks of culture (not shown). Representative Raman spectra (between 200 and 2000 cm − 1) of mineralized nodules on Ao-Ti and Ti-Cl are shown in Figures 4 and 5. Amide I (1660 cm − 1), Amide III (1250 cm − 1), B-type
carbonate (1070 cm − 1 ), proline (856 cm − 1 ), and the υ1 phosphate (960 cm − 1) were detectable on Ao-Ti and Ti-Cl. The ratio of υ1 phosphate peak to Amide III on Ao-Ti was 0.31 ± 0.02, while that of Amide III on Ti-Cl was 0.06 ± 0.01. The intensity of the second-derivative 1660/1690 cm − 1 , obtained from the original Amide I peak on Ao-Ti was 1.78 ± 0.02, while that of Ti-Cl was 1.04 ± 0.01. Both the ratio of υ1
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Figure 3. Representative hardness and elastic modulus vs. contact depth of mineralized tissues on samples after 2 weeks of cell culture, obtained from depthdependent partial unloading curves. Only values of indenter contact depth between 20 and 200 nm from the sample surface were considered, as this range corresponds to the effective range of the Berkovich tip area function used in this study.
phosphate peak to Amide III and the intensity of the secondderivative 1660/1690 cm − 1 on Ao-Ti were significantly higher (P b 0.01) than those on the Ti-Cl. Specifically, the value of the second-derivative 1660/1690 cm − 1 on Ao-Ti was similar to that described previously for bone or sound dentin. 22
Discussion Both surface chemistry and topography have been advocated as critical indicators on anodically oxidized titanium. 33,34 The present study elucidates biological activities such as gene
expression and mineralization of primary osteoblasts enhanced presumably by respective surface parameters on anodically oxidized titanium prepared with or without chloride solution. We have demonstrated the developing thickness of a titanium oxide layer observed on thermally oxidized Ho-Ti and anodically oxidized Ao-Ti or Ti-Cl. 5 The thick oxide film on the oxidized titanium surfaces would be responsible for high corrosion resistance and stable state at physiological pH condition. 35 Apart from the biological stability associated with thick titanium oxide layers, cells grown on anodically oxidized Ao-Ti and Ti-Cl showed higher expression of osteogenic genes than cells grown on Ti and Ho-Ti. Superhydrophilicity, caused by hydrophilic
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Figure 4. (A) Representative microscopic image of a mineralized nodule on Ao-Ti. (B) Raman spectra of mineralized nodules in the range of 200-2000 cm −1. The mineral-to-matrix ratios of mineralized nodules were calculated based on the intensity ratio of the υ1 phosphate peak to Amide III. (C) Second-derivative spectra obtained from original Amide I were used to calculate the cross-linking ratio of mineralized tissues on the titanium samples. Two sub-bands are shown (at 1690 and 1660 cm −1) that may be indicative of secondary structure changes associated with collagen cross-linking.
functional groups forming as the oxidation products of ROS, was observed only on the anodically oxidized titanium. 5 Besides the surface hydrophilicity, the results of surface topography showed large differences in height parameters, which has been identified as an important determinant of biological activity on titanium surfaces. 35,36 As a consequence of both surface chemistry and topography, Ao-Ti and Ti-Cl enabled adsorption of extracellular matrix proteins and cell adhesion, thereby increasing the rate of osteogenic gene expression. The areas of mineralized nodules on anodically oxidized AoTi and Ti-Cl were larger than those on Ti and Ho-Ti after 2 weeks of cell culture, perhaps because of the increased expression of bone matrix proteins such as Bsp2, Ocn, and Opn on Ao-Ti and Ti-Cl. However, the area of mineralization on TiCl was larger than that on Ao-Ti. Because both anodically oxidized Ao-Ti and Ti-Cl enhanced osteogenic gene expressions in adherent osteoblasts at equivalent level, gene expression alone cannot explain the elevated mineralization of Ti-Cl. The ROS generation of anatase TiO2 on anodically oxidized Ao-Ti and TiCl is higher than on rutile TiO2 on thermally oxidized Ho-Ti. Disregarding the production of hydroxy radicals, the Ti-Cl surface generated a larger amount of ROS, such as HClO, compared with the Ao-Ti surface. Mineralized tissues such as
bone and dentine are natural biocomposites comprising mainly of apatite biominerals and collagenous matrix proteins. 18 Apatite minerals occupy the collagen scaffold of bone and dentin during their development and mineralization. The presence of ROS, as well as the expression of bone matrix proteins, on anodically oxidized Ao-Ti and Ti-Cl appears to be necessary for the assembly of collagenous matrix proteins. 14 Therefore, the amount of ROS on Ti-Cl appears to be responsible for the higher mineralization potential of the surface. The Raman spectra of the mineralized nodules on anodically oxidized Ao-Ti and Ti-Cl were characteristic of the molecular structures in natural bone and dentine. Although Ti-Cl showed a higher extent of mineralization than Ao-Ti, the mineralized tissue on Ao-Ti was harder and more inelastic than that on Ti-Cl. With metallic or solid elastic-plastic material, elastic properties can be related to densification or increased hardness. 18 However, the hardness and elastic moduli of mineralized biocomposites such as bone and dentin do not increase linearly with increasing densification or mineralization. The elastic modulus is associated with fluid displacement and inelastic deformation of the collagen/apatite structure. The higher hardness and lower elasticity of mineralized tissue on Ao-Ti suggest that there is a reduction in permeability and consolidation brought about by
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Figure 5. (A) Representative microscopic image of a mineralized nodule on Ti-Cl. (B) Raman spectra of mineralized nodules in the range of 200-2000 cm −1. The mineral-to-matrix ratios of mineralized nodules were calculated based on the intensity ratio of the υ1 phosphate peak to Amide III. (C) Second-derivative spectra obtained from original Amide I were used to calculate the cross-linking ratio of mineralized tissues on the titanium samples. Two sub-bands are shown (at 1690 and 1660 cm −1) that may be indicative of secondary structure changes associated with collagen cross-linking.
cross-linking of this structure. As shown in the Raman spectra of the mineralized nodules on Ao-Ti, both the mineral-to-matrix and cross-linking ratios were increased on Ao-Ti. Generation of hydroxyl radicals on the anatase TiO2 surface of anodically oxidized Ao-Ti (in contrast to HClO on Ti-Cl) has been advocated in the present study as well as in previous studies. 5 A stronger oxidation effect of hydroxyl radicals against organic molecules other than HClO has been reported. 37 The oxidation of lysine residues in immature bone matrix proteins (e.g. collagen) is essential for cross-linking between molecules in vivo. 38 Because enhanced nanomechanical properties of in vitro mineralized tissue were observed only on Ao-Ti, it can be assumed that surface hydroxyl radicals, not HClO, enabled the oxidation of lysine residues in immature collagen and of lysine residues produced by natural oxidation enzymes such as lysyl oxidase. Meanwhile, one of the most notable features on Ao-Ti was surface containing phosphorous whereas a chloride compound was formed on Ti-Cl surface. As well as ROS, the surface phosphorous might be capable of harder and stiffer mineralized tissues on Ao-Ti. The efficacy of ROS or phosphorous on Ao-Ti should be explained in our next study by isolation of each elements onto the surface.
Anodically oxidized titanium surfaces enhanced the expression of osteogenic genes in adherent osteoblasts, regardless of whether chloride solution was used. The type and amount of ROS and the expression of bone matrix proteins on anodically oxidized titanium surfaces influenced the amount of mineralized tissue after cell culture. The ROS generated from hydroxyl radicals or phosphorous on anodically oxidized titanium without a chloride solution (rather than the HClO generated in chloride solutions) was responsible for the higher hardness and inelastic properties of the resultant mineralized tissue.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2013.09.007.
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I.-K. Jang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 629–637 2. Kim HM, Miyaji F, Kokubo T, Nakamura T. Preparation of bioactive Ti and its alloys via simple chemical surface treatment. J Biomed Mater Res 1996;32:409-17. 3. Nanci A, Wuest JD, Peru L, et al. Chemical modification of titanium surfaces for covalent attachment of biological molecules. J Biomed Mater Res 1998;40:324-35. 4. Puleo DA, Nanci A. Understanding and controlling the bone-implant interface. Biomaterials 1999;20:2311-21. 5. Shibata Y, Suzuki D, Omori S, et al. The characteristics of in vitro biological activity of titanium surfaces anodically oxidized in chloride solutions. Biomaterials 2010;3:8546-55. 6. Beutner R, Michael J, Förster A, Schwenzer B, Scharnweber D. Immobilization of oligonucleotides on titanium based materials by partial incorporation in anodic oxide layers. Biomaterials 2009;30:2774-81. 7. Cai Q, Paulose M, Varghese OK, Grimes CA. The effect of electrolyte composition on the fabrication of self-organized titanium oxide nanotube arrays by anodic oxidation. J Mater Res 2005;20:230-6. 8. Zwilling V, Aucouturier M, Darque-Ceretti E. Anodic oxidation of titanium and TA6V alloy in chromic media. An electrochemical approach. Electrochim Acta 1999;45:921-9. 9. Omori S, Shibata Y, Arimoto T, et al. Micro-organism and cell viability on antimicrobially modified titanium. J Dent Res 2009;88:957-62. 10. Butz F, Aita H, Wang CJ, Ogawa T. Harder and stiffer bone osseointegrated to roughened titanium. J Dent Res 2006;85:560-5. 11. Chen J, Birch MA, Bull SJ. Nanomechanical characterization of tissue engineered bone grown on titanium alloy in vitro. J Mater Sci Mater Med 2010;21(1):277-82. 12. Clark PA, Clark AM, Rodriguez A, Hussain MA, Mao JJ. Nanoscale characterization of bone-implant interface and biomechanical modulation of bone ingrowth. Mater Sci Eng C Mater Biol Appl 2007;27:382-93. 13. Nakamura H, Shim J, Butz F, et al. Glycosaminoglycan degradation reduces mineralized tissue-titanium interfacial strength. J Biomed Mater A 2006;77:478-86. 14. Shibata Y, He LH, Toda Y, et al. Micromechanical evaluation of mineralized multilayers. J Biomech 2008;41:3414-8. 15. Kikuchi M, Matsumoto HN, Yamada T, et al. Glutaraldehyde crosslinked hydroxyapatite/collagen self-organized nanocomposites. Biomaterials 2004;25:63-9. 16. Paschalis EP, Recker R, Dicarlo E, et al. Distribution of Collagen Cross-Links in Normal Human Trabecular Bone. J Bone Miner Res 2003;18:1942-6. 17. Shibata Y, Yamamoto H, Miyazaki T. Colloidal β-tricalcium phosphate prepared by discharge in a modified body fluid facilitates synthesis of collagen composites. J Dent Res 2005;84:827-31. 18. Shibata Y, He LH, Kataoka Y, Miyazaki T, Swain MV. Micromechanical property recovery of human carious dentin achieved with colloidal nano-β-tricalcium phosphate. J Dent Res 2008;87:233-7. 19. Daumer KM, Khan AU, Steinbeck MJ. Chlorination of pyridinium compounds. Possible role of hypochlorite, N-chloramines, and chlorine in the oxidation of pyridinoline cross-links of articular cartilage collagen type II during acute inflammation. J Biol Chem 2000;275:34681-92. 20. Ryu A, Naru E, Arakane K, et al. Cross-linking of collagen by singlet oxygen generated with UV-A. Chem Pharm Bull (Tokyo) 1997;45:1243-7. 21. Balooch G, Balooch M, Nalla RK, et al. TGF-β regulates the mechanical properties and composition of bone matrix. Proc Natl Acad Sci U S A 2005;102:18813-8.
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22. Xu C, Karan K, Yao X, Wang Y. Molecular structural analysis of noncarious cervical sclerotic dentin using Raman spectroscopy. J Raman Spectrosc 2009;40:1780-5. 23. Bi X, Patil CA, Lynch CC, et al. Raman and mechanical properties correlate at whole bone- and tissue-levels in a genetic mouse model. J Biomech 2011;44:297-303. 24. Hanawa T, Kon M, Doi H, et al. Amount of hydroxyl radical on calcium-ion-implanted titanium and point of zero charge of constituent oxide of the surface-modified layer. J Mater Sci Mater Med 1998;9: 89-92. 25. Suda T, Jimi E, Nakamura I, Takahashi N. Role of 1α,25-dihydroxyvitamin D3 in osteoclast differentiation and function. Methods Enzymol 1997;282:223-35. 26. Oliver WC, Pharr GM. Improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J Mater Res 1992;7:1564-80. 27. Oliver WC, Pharr GM. Measurement of hardness and elastic modulus by instrumented indentation: Advances in understanding and refinements to methodology. J Mater Res 2004;19:3-20. 28. Shibata Y, Suzuki D, Wurihan, et al. Lysyl oxidase like-2 reinforces unsatisfactory ossification induced by bone morphogenetic protein-2: Relating nanomechanical properties and molecular changes. Nanomedicine http://dx.doi.org/10.1016/j.nano.2013.04.008 29. Paschalis EP, Verdelis K, Doty SB, et al. Spectroscopic characterization of collagen cross-links in bone. J Bone Miner Res 2001;16:1821-8. 30. Deng JY, Arimoto T, Shibata Y, et al. Role of chloride formed on anodized titanium surfaces against an oral microorganism. J Biomater Appl 2010;25:179-89. 31. Shibata Y, Kawai H, Yamamoto H, Igarashi T, Miyazaki T. Antibacterial titanium plate anodized by being discharged in NaCl solution exhibits cell compatibility. J Dent Res 2004;83:115-9. 32. Angker L, Swain MV, Kilpatrick N. Characterising the micromechanical behaviour of the carious dentine of primary teeth using nano-indentation. J Biomech 2005;38:1535-42. 33. Omar O, Svensson S, Zoric N, et al. In vivo gene expression in response to anodically oxidized versus machined titanium implants. J Biomed Mater A 2010;92:1552-66. 34. Omar OM, Granéli C, Ekström K, et al. The stimulation of an osteogenic response by classical monocyte activation. Biomaterials 2011;32: 8190-204. 35. Sul YT, Johansson CB, Petronis S, et al. Characteristics of the surface oxides on turned and electrochemically oxidized pure titanium implants up to dielectric breakdown: the oxide thickness, micropore configurations, surface roughness, crystal structure and chemical composition. Biomaterials 2002;23:491-501. 36. Albrektsson T, Wennerberg A. Oral implant surfaces: part 1-review focusing on topographic and chemical properties of different surfaces and in vivo responses to them. Int J Prosthodont 2004;17:536-43. 37. Boudreau J, Bejan D, Li S, Bunce NJ. Competition between electrochemical advanced oxidation and electrochemical hypochlorination of sulfamethoxazole at a boron-doped diamond anode. Ind Eng Chem Res 2010;49:2537-42. 38. Vora SR, Palamakumbura AH, Mitsi M, et al. Lysyl oxidase propeptide inhibits FGF-2-induced signaling and proliferation of osteoblasts. J Biol Chem 2010;285:7384-93.
Original Article
nanomedjournal.com
Nanomechanical properties and molecular structures of in vitro mineralized tissues on anodically-oxidized titanium surfaces In-Kee Jang, BDS a , Reina Tanaka, PhD b , Wurihan, BDS a , Dai Suzuki, PhD c , Yo Shibata, PhD a,⁎, Naoki Fujisawa, PhD d , Yasuhiro Tanimoto, PhD e , Kayoko Ogura, PhD b , Ryutaro Kamijo, PhD c , Takashi Miyazaki, PhD a a
Department of Conservative Dentistry, Division of Biomaterials and Engineering, Showa University School of Dentistry, Tokyo, Japan Department of Conservative Dentistry, Division of Aesthetic Dentistry and Clinical Cariology, Showa University School of Dentistry, Tokyo, Japan c Department of Biochemistry, Showa University School of Dentistry, Tokyo, Japan d Hysitron, Inc., Minneapolis, MN, USA e Department of Dental Biomaterials, Nihon University School of Dentistry at Matsudo, Chiba, Japan Received 16 May 2013; accepted 26 September 2013
b
Abstract The biomechanical stability of mineralized tissues at the interface between implant surface and bone tissue is of critical importance. Anodically oxidized titanium prepared in a chloride solution results in enhanced mineralization of adherent osteoblasts and has antimicrobial activity against oral microorganisms. We evaluated the nanomechanical properties and molecular structures of the in vitro mineralized tissues developing around anodically oxidized titanium surfaces with and without preparation in chloride solution. Anodically oxidized titanium surfaces showed superior osteogenic gene expressions than those of thermally oxidized and bare titanium surfaces. Preparation of anodically oxidized titanium in chloride enhanced the production of mineralized tissue around it. However, the mineralized tissue around anodically oxidized titanium prepared without chloride had increased mineral:matrix and cross-linking ratios, resulting in higher hardness and lower elasticity. From the Clinical Editor: In this study anodically oxidized titanium was used to enhance the biomechanical stability of mineralized tissues at the implant surface – bone tissue interface. The mineralized tissue around anodically oxidized titanium prepared without chloride had increased mineral:matrix and cross-linking ratios, resulting in higher hardness and lower elasticity. © 2014 Elsevier Inc. All rights reserved. Key words: Titanium; Implant; Reactive oxygen species; Osteoblast; Nanoindentaion; Raman spectroscopy
Osseo-integrated titanium implants must be capable of forming rigid osseous anchors. Therefore, various surface modification techniques have been explored to improve the biological responses of the bone to titanium implants. 1-4 Implant surfaces should have not only wear and corrosion resistance, but also biological compatibility achieved by various surface related parameters such as surface Author contributions: Y.S. and I-K.J. performed project planning, experimental work, data interpretation and preparation of the manuscript. D.S., Wurihan, Y.T. and K.O. performed experimental work. R.T. and N.F. participated in data analysis. R.K. and T.M. supervised research and participated in project planning. Funding sources: This work was supported by a Grant-in-Aid for Scientific Research (B) and a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. None of the authors have any conflicts of interest to declare. ⁎Corresponding author: Department of Conservative Dentistry, Division of Biomaterials and Engineering, Showa University School of Dentistry, Tokyo 142-8555, Japan. E-mail address: [email protected] (Y. Shibata).
topography or surface chemistry. The anatase phase TiO2 in amorphous matrix accompanied with micro-submicron surface texture on anodically oxidized titanium surfaces results in enhanced expression of osteogenic genes and increased mineralization of adherent primary osteoblasts on its surface. These effects might be due to the superhydrophilicity of COO − and OH − oxidation products of reactive oxygen species (ROS) on these surfaces. 5 Anodically oxidized titanium incorporates the saturated ions of the electrolytes in the outer parts of the oxide layer at the interface between the oxide and electrolyte. 6-8 We have recently described a variety of anodically oxidized titanium surfaces prepared in electrolyte solutions with or without the addition of chloride. 5,9 Preparation in a solution containing chloride (Ti-Cl) generates surface hypo-chloride (HClO), whereas preparation in Na2HPO4 solution generates surface hydroxyl radicals (•OH). Both surfaces were found to enhance the expression of osteogenic gene and the mineralization of adherent osteoblasts. In addition, the surface hypochloride on the Ti-Cl surface destroyed the cell wall membrane of adherent microorganisms, thus demonstrating strong antimicrobial activity.
1549-9634/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2013.09.007 Please cite this article as: Jang I.-K., et al., Nanomechanical properties and molecular structures of in vitro mineralized tissues on anodically-oxidized titanium surfaces. Nanomedicine: NBM 2014;10:629-637, http://dx.doi.org/10.1016/j.nano.2013.09.007
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Figure 1. Surface topography of anodically oxidized titanium. The surface morphology of the titanium samples was examined using (A) a scanning electron microscope and (B) a laser microscope.
The biomechanical stability of titanium implants is of particular importance, especially if this property can be enhanced through structural manipulation of mineralized tissues between the implant surface and bone tissue. 10-14 Mineralized tissues such as bone and dentin are natural nano-biocomposites with a calcium phosphate and collagen matrix. 15-17 Densification and consolidation of mineralized tissue have been shown to increase its hardness and inelasticity. 14,18 Previous studies have reported that extracellular ROS might affect collagen cross-linking in human skin and cartilage, 19,20 but whether extracellular ROS has similar effects in mineralized tissues remains unclear. However, we hypothesize that the amount of surface ROS on anodically oxidized surfaces may affect the nanomechanical properties of the mineralized tissues interfacing with that surface. This study evaluated the expression of bone matrix proteinrelated genes in adherent osteoblasts on anodically oxidized titanium surfaces. We also investigated the nanomechanical and structural properties of mineralized tissues developing in vitro around anodically oxidized titanium surfaces, using nanoindentation tests with depth-dependent loading/partial unloading tests and Raman spectroscopy. 2,14,21-23
Methods Sample preparation JIS grade 2 titanium (KS-50; Kobe Steel, Tokyo, Japan) was prepared with dimensions of 10 × 10 × 1.0 mm. The surface of each specimen was mechanically polished under running water using waterproof polishing papers (up to #1200 grit) before finishing with 0.3 μm diameter alumina particles. Prepared specimens were
ultrasonically cleaned in acetone/detergent solution (7X; ICN Biomedicals, Aurora, OH, USA) and pure distilled water for 15 min, then dried and stored in a sealed desiccator for 1 week at a humidity of 50% and a temperature of 23 °C. For anodic oxidation, Ti specimens were connected to the anode of a device developed in our research group and immersed in electrolytes. A 50 × 100 × 0.1 mm stainless steel plate was used as the counter-electrode. Discharge (416 mA/cm 2) was generated between the electrolyte and the working electrode through a gas layer on the surface of the electrode for 60 s. Specimens were anodically oxidized in 100 mL of 1 M Na2HPO4 (Ao–Ti) or NaCl (Ti–Cl), then washed in pure distilled water. Untreated Ti was oxidized by heating at 600 °C for 30 min in a dental furnace (Ho-Ti). All titanium samples were stored in the dark at an ambient laboratory atmosphere for 24 h. Surface characterization Surface topography The surface morphology of the titanium samples was examined using a scanning electron microscope (S-2360 N; Hitachi) and a laser micro scope (OLS-4000; Shimadzu) (Figure 1). The average roughness (Sa), root mean square deviation (Sq), maximum peak height (Sp), maximum valley depth (Sv) peak to valley depth (Sz), degree of symmetry of the surface height about the mean plane (Ssk), and presence of inordinately high peaks/ deep valleys (Sku) were quantified using the proprietary software of these instruments with a scanning range of 643 × 644 μm. X-ray photoelectron spectroscopy (XPS) Sample surfaces were analyzed by XPS (ESCA-3400; Shimadzu, Kyoto, Japan). High-resolution spectra of Ti2p,
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O1s, C1s, P2p, and Cl2p were analyzed using a monochromatic X-ray source (Mg Kα radiation). A 20 mA emission current and 8 kV of accelerated voltage were applied in the analysis (UHV conditions at b 1 × 10 − 7 mbar). The binding energies for each spectrum were calibrated based on the C1s spectra of 285.0 eV.
Table 1 Primer sequences used in this study for qPCR analysis of osteoblast gene expression.
ROS detection on titanium surfaces by XPS The amount of ROS present on the titanium samples was determined by previously described methods. 24 Briefly, ammonium chloride solution (4 M, 500 ml) was mixed with zinc chloride solution (0.4 M, 250 ml) and the pH of the mixture adjusted to 6.9 with 30% ammonium hydroxide. The volume of the mixture was then adjusted to 1000 ml with deionized water. Titanium samples were immersed individually in 150 ml of this solution at ambient temperature for 300 s. During immersion, active radicals were mostly exchanged for zinc ions, and zinc chelates were formed. The disks were then immersed three times in 150 ml of deionized water to remove non-adsorbed chemical species. Sample surfaces were then analyzed by XPS. The relative concentration of zinc on the sample surfaces was also evaluated as a proxy measurement of the amount of surface ROS, to which it should be proportional.
Alkaline phosphatase (Alp) Type I collagen
Osteoblastic cell culture Primary osteoblasts were obtained from the calvariae of neonatal ddY mice (Sankyo Co. Inc., Tokyo, Japan) using 0.1% collagenase and 0.2% dispase. 25 Primary osteoblasts were cultured in α-MEM (Wako) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) containing penicillin-streptomycin and incubated at 37 °C in a CO2 incubator (5% CO2, 95% air). Before reaching confluence, cells were detached and seeded onto the titanium disks in polystyrene culture plates at a density of 4 × 10 4 cells/cm 2 in culture medium supplemented with 50 μg/mL ascorbic acid, 10 mM Na-β-glycerophosphate and 10 nM dexamethasone. Culture medium was replaced every 3 days. Quantitative real-time PCR (qPCR) Gene expression was analyzed and quantified by qPCR. Total RNA was extracted with TRIzol reagent (Invitrogen), then reverse-transcribed using SuperScript III enzyme preparation (Invitrogen). The qPCR assays were performed using a Fast SYBR Green PCR system (Applied Biosystems, CA, USA), using primer sequences as shown in Table 1. The mean relative fold changes in mRNA expression after normalization to Gapdh are shown with the standard deviation for all data points. The gene expressions on the samples were defined as relative to the Ti surface (defined as a relative expression level of 1). Mineralization assay Alizarin red staining was performed after 2 weeks of culture. Following three washes with PBS and two with pure distilled water, the titanium samples were dried in air, and stained for 5 min using 1% alizarin red (Sigma, St Louis, MO, USA) at pH 6.3-6.4. The mineralization capability of the cultured osteoblasts was quantified by colorimetric detection of calcium deposition. Samples stained with alizarin red were washed with PBS and incubated overnight in 1 ml of 0.5 M HCl solution with
Gene target
Primer sequence
Gapdh
5′-AAATGGTGAAGGTCGGTGTG-3′ and 5′-TGAAGGGGTCGTTGATGG -3′ 5′-TTCCCACGTTTTCACATTCG-3′ and 5′-GCCAGACCAAAGATGGAGTTG-3′ 5′-GCCTTGGAGGAAACTTTGCTT-3′ and 5′-GCACGGAAACTCCAGCTGAT-3′ 5′-CCTCTCCTGCTACCGCACAA-3′ and 5′-CTAGAGCCGCCAAATTTGCT-3′ 5′-CTGACAAAGCCTTCATGTCCAA-3′ and 5′-GGTAGCGCCGGAGTCTGTT-3′ 5′-GCTTTTGCCTGTTTGGCATT-3′ and 5′-AGCTGCCAGAATCAGTCACTTTC-3′ 5′-GAGTTAGCGGCACTCCAACTG-3′ and 5′-CACTTTTGGAGCCCTGCTTT-3′
Cyclin D1 Osteocalcin (Ocn) Osteopontin (Opn) Bone sialoprotein 2 (Bsp2)
gentle shaking. The solution was mixed with o-cresolphthalein complexone in alkaline medium (Calcium Binding and Buffer Reagent; Sigma) to produce a red calcium-cresolphthalein complexone complex. Color intensity was measured with an ELISA reader (Synergy HT; BioTek Instruments, Winooski, VT) at an absorbance wavelength of 575 nm. Nanoindentation test Indentation experiments were performed using a quantitative nanomechanical test system (TS-70 Triboscope; Hysitron, Inc., MN, USA) with a scanning probe microscope (SPM-9700; Shimadzu, Kyoto, Japan). A Berkovich indenter (Hysitron) was chosen for this study. Fused quartz acted as the standard material to calibrate the Berkovich nanoindenter tip area and instrument compliance. 26 Indentation tests were performed perpendicular to the surface of osteoblast-incubated samples after 2 weeks of cell culture. To minimize errors arising from surface roughness, appropriately smooth mineralized regions were chosen with a scanning range of 30 × 30 μm and then 3 × 3 μm. Depth-dependent loading/partial unloading tests 14 were conducted at a maximum load of 1 mN with a total of 66 loading and unloading cycles, with a 1 s holding time at each point. The distance between indents was more than 10 μm to avoid any influence of residual stresses from adjacent indentations. Hardness and elastic modulus were calculated from the force–displacement curves using the TS-70 proprietary software. The Berkovich tip area function used in this study produced an effective range of indenter contact depths of 20200 nm from the sample surface. For the standard nanoindentation analysis, it is ideal to have a smooth surface at the indenter penetration depth. 26,27 The loading/partial unloading test overcomes issues associated with biological structures because the evaluation of depth-dependent mechanical behaviors permits the normalization of data for any variation in contact between biological structures and the indenter tip. 28 Micro-Raman spectroscopy Raman spectra of randomly selected mineralized nodules on titanium samples were acquired using a confocal Raman
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microscope (RXN1; Kaiser Optical Systems, Inc., MI, USA) with a 785 nm laser diode source. Light was focused by a 50 × objective onto a 3 μm spot on the mineralized nodules. The Raman scatter (5 s exposure time) was collected by a spectrograph with a spectral resolution of 1 cm − 1. Peak intensities were recorded for Amide I (1660 cm − 1), Amide III (1250 cm − 1 ), B-type carbonate (1070 cm − 1 ), proline (856 cm − 1), and the υ1 phosphate (960 cm − 1). The mineralto-matrix ratios of mineralized nodules were calculated based on the intensity ratio of the υ1 phosphate peak to Amide III. 23 The second-derivative spectra obtained from original Amide I were used to calculate the cross-linking ratio of mineralized tissues on the titanium samples. 22 Two subbands were shown at 1690 and 1660 cm − 1 and may be indicative of secondary structure changes associated with collagen cross-linking. The intensity of the 1660/1690 cm − 1 sub-bands was used to calculate non-reducible/reducible crosslink ratios. 29 Statistical analysis Three samples were evaluated in the surface characterization and qPCR assays. Five samples were evaluated in the cell culture studies with alizarin red staining. In nanoindentation and Raman analysis, we expressed the random selection of five representative regions as the mean. Results are expressed as mean ± SD. The normal distribution of each value was confirmed using the Kolmogorov–Smirnov test. The appropriateness of the hypothesis of homogeneous variances was verified using Bartlett’s test. Data were analyzed by ANOVA followed by a post hoc Tukey test, with P b 0.01 considered statistically significant.
Results
Table 2 Mean surface roughness parameters of the titanium samples used in this study. Sp (μm) Sv (μm) Sz (μm) Sa (μm) Sq (μm) Ssk Ti 3.560 Ho-Ti 3.046 Ao-Ti 25.704 Ti-Cl 47.038
2.936 2.789 19.691 19.282
6.496 5.835 45.395 66.320
0.304 0.343 4.357 4.742
0.390 0.442 5.493 6.218
0.142 0.290 0.053 1.179
Sku 3.772 3.640 2.944 7.373
The average roughness (Sa), root mean square deviation (Sq), maximum peak height (Sp), maximum valley depth (Sv) peak to valley depth (Sz), degree of symmetry of the surface height about the mean plane (Ssk), and presence of inordinately high peaks/deep valleys (Sku) were quantified using the proprietary software of these instruments with a scanning range of 643 × 644 μm.
Gene expression in adherent osteoblasts After 1 week of osteoblast culture, the expression of Alp, Ocn, Opn and Bsp2 genes on the Ao-Ti and Ti-Cl surfaces were significantly higher than that on the Ti and Ho-Ti surfaces (Figure 2, A). On Ti-Cl, the expression of type I collagen was significantly higher (P b 0.01) than on the other samples. There were no obvious differences in gene expression profiles between cells grown on polystyrene cell culture plates and those seeded on Ti (Supplemental Figure 1). Mineralized nodules on titanium samples After 2 weeks of osteoblast cell culture, the areas of mineralized nodules on both Ao-Ti and Ti-Cl were found to be larger than those on Ti and Ho-Ti (Figure 2, B). As quantified in Figure 2, B, the areas of mineralization on anodically oxidized Ao-Ti and Ti-Cl were much greater (P b 0.01) than on Ti and Ho-Ti. However, the area of mineralization on Ti-Cl was significantly higher (P b 0.01) than on Ao-Ti.
Surface characterization Surface topography Pores with diameters in the micron or submicron scale were counted on Ao-Ti, whereas granular deposits forming on the TiCl surface were quantitated. The mean surface roughness parameters are summarized in Table 2. The height parameters of both anodically oxidized Ao-Ti and Ti-Cl were higher than those of Ti and Ho-Ti. XPS The relative atomic concentration of each element on the titanium samples is shown in Table 3. The energy position of Cl2p on Ti–Cl was detected at 201.4 eV (not shown). Therefore, it appeared that a portion of titanium tetrachloride had changed and combined with oxygen, resulting in the generation of ROS hypochloride. 5,30,31 After immersion in a solution containing ammonium chloride and zinc chloride, the relative concentration of zinc on both anodically oxidized Ao-Ti and Ti-Cl was significantly higher than that on Ti and Ho-Ti; hence the increased amount of ROS generated on the Ao-Ti and Ti-Cl surfaces.
Nanoindentation test After 2 weeks of cell culture, we calculated the representative hardness and elastic modulus of the mineralized tissues developing on the titanium samples using depth-dependent partial unloading curves, as shown in Figure 3. The hardness and elastic modulus of the bare Ti surface were 3.1 ± 0.3 GPa and 178.8 ± 4.5 GPa, respectively (data not shown). The hardness and elastic modulus (20-200 nm contact depth) of mineralized tissues on Ti were 0.25 ± 0.06 and 4.8 ± 0.05 GPa, respectively, while those on Ho-Ti were 0.24 ± 0.04 and 5.5 ± 0.04 GPa, respectively. In addition, the hardness and elastic modulus of the mineralized tissue on Ti-Cl were 0.25 ± 0.06 and 4.8 ± 0.05 GPa, respectively. There were no significant differences (P N 0.01) in the nanomechanical properties of any of the mineralized tissues grown on Ti, Ho-Ti and Ti-Cl surfaces. The hardness and elastic modulus of the mineralized tissue on Ao-Ti were significantly higher (P b 0.01) than those of Ti, Ho-Ti and Ti-Cl, being estimated at 0.78 ± 0.04 and 13.5 ± 0.08 GPa, respectively. This elastic modulus value is comparable to that reported for human cancellous bone and dentin. 10,14,32
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Table 3 Relative atomic concentrations of each element (%) on the titanium samples, with (+) or without immersion in a solution containing ammonium chloride and zinc chloride. Ti Ti Ho-Ti Ao-Ti Ti-Cl Ti (+) Ho-Ti (+) Ao-Ti (+) Ti-Cl (+)
13.9 23.6 6.2 20.7 10.2 9.8 2.7 14.3
O ± ± ± ± ± ± ± ±
0.4 1.3 0.8 1.8 1.6 0.9 0.6 2.3
54.5 61.3 59.2 54.2 43.6 40.7 40.4 51.6
C ± ± ± ± ± ± ± ±
2.8 3.1 3.8 2.2 4.2 2.2 3.1 4.4
31.6 15.1 15.5 15.7 44.5 47.1 40.3 16.4
± ± ± ± ± ± ± ±
2.1 1.9 2.2 1.2 5.4 3.2 4.1 2.2
P
Cl
Zn
15.6 ± 3.1 11.3 ± 1.2 -
9.4 ± 1.4 10.3 ± 2.4
1.6 2.2 5.2 6.1
± ± ± ±
0.4 0.7 1.4 2.4
Results are expressed as the mean ± SD of three independent repeats. Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test.
Figure 2. (A) Relative gene expression in samples after 1 week of cell culture. (B) Representative images of alizarin red staining and quantification of mineralized areas on sample surfaces after 2 weeks of cell culture. Results are expressed as the mean ± SD of three independent repeats. Data were statistically analyzed by ANOVA followed by a post-hoc Tukey test. P b 0.01 (*) was considered significant.
Molecular structure of mineralized nodules There were no distinctive Raman spectra observed on the Ti and Ho-Ti specimens, even after 2 weeks of culture (not shown). Representative Raman spectra (between 200 and 2000 cm − 1) of mineralized nodules on Ao-Ti and Ti-Cl are shown in Figures 4 and 5. Amide I (1660 cm − 1), Amide III (1250 cm − 1), B-type
carbonate (1070 cm − 1 ), proline (856 cm − 1 ), and the υ1 phosphate (960 cm − 1) were detectable on Ao-Ti and Ti-Cl. The ratio of υ1 phosphate peak to Amide III on Ao-Ti was 0.31 ± 0.02, while that of Amide III on Ti-Cl was 0.06 ± 0.01. The intensity of the second-derivative 1660/1690 cm − 1 , obtained from the original Amide I peak on Ao-Ti was 1.78 ± 0.02, while that of Ti-Cl was 1.04 ± 0.01. Both the ratio of υ1
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Figure 3. Representative hardness and elastic modulus vs. contact depth of mineralized tissues on samples after 2 weeks of cell culture, obtained from depthdependent partial unloading curves. Only values of indenter contact depth between 20 and 200 nm from the sample surface were considered, as this range corresponds to the effective range of the Berkovich tip area function used in this study.
phosphate peak to Amide III and the intensity of the secondderivative 1660/1690 cm − 1 on Ao-Ti were significantly higher (P b 0.01) than those on the Ti-Cl. Specifically, the value of the second-derivative 1660/1690 cm − 1 on Ao-Ti was similar to that described previously for bone or sound dentin. 22
Discussion Both surface chemistry and topography have been advocated as critical indicators on anodically oxidized titanium. 33,34 The present study elucidates biological activities such as gene
expression and mineralization of primary osteoblasts enhanced presumably by respective surface parameters on anodically oxidized titanium prepared with or without chloride solution. We have demonstrated the developing thickness of a titanium oxide layer observed on thermally oxidized Ho-Ti and anodically oxidized Ao-Ti or Ti-Cl. 5 The thick oxide film on the oxidized titanium surfaces would be responsible for high corrosion resistance and stable state at physiological pH condition. 35 Apart from the biological stability associated with thick titanium oxide layers, cells grown on anodically oxidized Ao-Ti and Ti-Cl showed higher expression of osteogenic genes than cells grown on Ti and Ho-Ti. Superhydrophilicity, caused by hydrophilic
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Figure 4. (A) Representative microscopic image of a mineralized nodule on Ao-Ti. (B) Raman spectra of mineralized nodules in the range of 200-2000 cm −1. The mineral-to-matrix ratios of mineralized nodules were calculated based on the intensity ratio of the υ1 phosphate peak to Amide III. (C) Second-derivative spectra obtained from original Amide I were used to calculate the cross-linking ratio of mineralized tissues on the titanium samples. Two sub-bands are shown (at 1690 and 1660 cm −1) that may be indicative of secondary structure changes associated with collagen cross-linking.
functional groups forming as the oxidation products of ROS, was observed only on the anodically oxidized titanium. 5 Besides the surface hydrophilicity, the results of surface topography showed large differences in height parameters, which has been identified as an important determinant of biological activity on titanium surfaces. 35,36 As a consequence of both surface chemistry and topography, Ao-Ti and Ti-Cl enabled adsorption of extracellular matrix proteins and cell adhesion, thereby increasing the rate of osteogenic gene expression. The areas of mineralized nodules on anodically oxidized AoTi and Ti-Cl were larger than those on Ti and Ho-Ti after 2 weeks of cell culture, perhaps because of the increased expression of bone matrix proteins such as Bsp2, Ocn, and Opn on Ao-Ti and Ti-Cl. However, the area of mineralization on TiCl was larger than that on Ao-Ti. Because both anodically oxidized Ao-Ti and Ti-Cl enhanced osteogenic gene expressions in adherent osteoblasts at equivalent level, gene expression alone cannot explain the elevated mineralization of Ti-Cl. The ROS generation of anatase TiO2 on anodically oxidized Ao-Ti and TiCl is higher than on rutile TiO2 on thermally oxidized Ho-Ti. Disregarding the production of hydroxy radicals, the Ti-Cl surface generated a larger amount of ROS, such as HClO, compared with the Ao-Ti surface. Mineralized tissues such as
bone and dentine are natural biocomposites comprising mainly of apatite biominerals and collagenous matrix proteins. 18 Apatite minerals occupy the collagen scaffold of bone and dentin during their development and mineralization. The presence of ROS, as well as the expression of bone matrix proteins, on anodically oxidized Ao-Ti and Ti-Cl appears to be necessary for the assembly of collagenous matrix proteins. 14 Therefore, the amount of ROS on Ti-Cl appears to be responsible for the higher mineralization potential of the surface. The Raman spectra of the mineralized nodules on anodically oxidized Ao-Ti and Ti-Cl were characteristic of the molecular structures in natural bone and dentine. Although Ti-Cl showed a higher extent of mineralization than Ao-Ti, the mineralized tissue on Ao-Ti was harder and more inelastic than that on Ti-Cl. With metallic or solid elastic-plastic material, elastic properties can be related to densification or increased hardness. 18 However, the hardness and elastic moduli of mineralized biocomposites such as bone and dentin do not increase linearly with increasing densification or mineralization. The elastic modulus is associated with fluid displacement and inelastic deformation of the collagen/apatite structure. The higher hardness and lower elasticity of mineralized tissue on Ao-Ti suggest that there is a reduction in permeability and consolidation brought about by
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Figure 5. (A) Representative microscopic image of a mineralized nodule on Ti-Cl. (B) Raman spectra of mineralized nodules in the range of 200-2000 cm −1. The mineral-to-matrix ratios of mineralized nodules were calculated based on the intensity ratio of the υ1 phosphate peak to Amide III. (C) Second-derivative spectra obtained from original Amide I were used to calculate the cross-linking ratio of mineralized tissues on the titanium samples. Two sub-bands are shown (at 1690 and 1660 cm −1) that may be indicative of secondary structure changes associated with collagen cross-linking.
cross-linking of this structure. As shown in the Raman spectra of the mineralized nodules on Ao-Ti, both the mineral-to-matrix and cross-linking ratios were increased on Ao-Ti. Generation of hydroxyl radicals on the anatase TiO2 surface of anodically oxidized Ao-Ti (in contrast to HClO on Ti-Cl) has been advocated in the present study as well as in previous studies. 5 A stronger oxidation effect of hydroxyl radicals against organic molecules other than HClO has been reported. 37 The oxidation of lysine residues in immature bone matrix proteins (e.g. collagen) is essential for cross-linking between molecules in vivo. 38 Because enhanced nanomechanical properties of in vitro mineralized tissue were observed only on Ao-Ti, it can be assumed that surface hydroxyl radicals, not HClO, enabled the oxidation of lysine residues in immature collagen and of lysine residues produced by natural oxidation enzymes such as lysyl oxidase. Meanwhile, one of the most notable features on Ao-Ti was surface containing phosphorous whereas a chloride compound was formed on Ti-Cl surface. As well as ROS, the surface phosphorous might be capable of harder and stiffer mineralized tissues on Ao-Ti. The efficacy of ROS or phosphorous on Ao-Ti should be explained in our next study by isolation of each elements onto the surface.
Anodically oxidized titanium surfaces enhanced the expression of osteogenic genes in adherent osteoblasts, regardless of whether chloride solution was used. The type and amount of ROS and the expression of bone matrix proteins on anodically oxidized titanium surfaces influenced the amount of mineralized tissue after cell culture. The ROS generated from hydroxyl radicals or phosphorous on anodically oxidized titanium without a chloride solution (rather than the HClO generated in chloride solutions) was responsible for the higher hardness and inelastic properties of the resultant mineralized tissue.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2013.09.007.
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