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Effect of sterilization and water rinsing on cell adhesion to titanium surfaces
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APSUSC-27914; No. of Pages 5
Applied Surface Science xxx (2014) xxx–xxx
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Effect of sterilization and water rinsing on cell adhesion to titanium surfaces Mitsuhiro Hirano a , Taro Kozuka a , Yuta Asano a , Yuko Kakuchi a , Hirofumi Arai b , Naofumi Ohtsu a,∗ a b
Instrument Analysis Center, Kitami Institute of Technology, 165 Koen-cho, Kitami, Hokkaido 090-8507, Japan Department of Biotechnology and Environmental Chemistry, Kitami Institute of Technology, 165 Koen-cho, Kitami, Hokkaido 090-8507, Japan
a r t i c l e
i n f o
Article history: Received 2 October 2013 Received in revised form 7 May 2014 Accepted 14 May 2014 Available online xxx Keywords: Titanium Sterilization Water rinsing MC3T3-E1 cell Hydroxide groups
a b s t r a c t In this study, the effects of sterilization and water rinsing on cell adhesion to titanium (Ti) surfaces were investigated. Ti substrates were treated using autoclave, dry-heating, and 70% ethanol. Thereafter, some of the substrates were rinsed with sterilized ultrapure water. Osteoblast-like MC3T3-E1 cells were seeded on the Ti surfaces and the numbers of adhered cells were counted after cultivation for 24 h. The number of cells adhered to ethanol-treated plates was lower than that on autoclave- and dry-heat-sterilized Ti substrates. However, interestingly, the cell adhesion performance on the ethanol-treated substrates was superior compared to that of the other substrates, after rinsing with ultrapure water. To investigate the origin of these differences, the chemical state of the treated surfaces was analyzed by X-ray photoelectron spectroscopy. We found a clear correlation between the number of adhered cells and the concentration of hydroxide groups (OH− ) on the surface, thus indicating that a change in OH− concentration affects the cell adhesion performance on Ti substrates. Since the sterilization and subsequent water rinsing affect the cell adhesion on Ti substrates, we suggest that the sterilization methods should be unified to correctly evaluate the cytocompatibility of metallic materials. © 2014 Published by Elsevier B.V.
1. Introduction Metallic materials are used as orthopedic and dental devices because of their excellent mechanical strength and ductility [1–4]. However, metallic ions released from the metallic devices are often toxic to biological tissues [5,6]. Therefore, cytocompatibility tests for newly developed metallic material are indispensable to check for their biological safety. The cytocompatibility of metallic materials is generally tested using the following protocol: animal cells are seeded on a metallic surface, and biological safety is evaluated by viability tests at its surface. Prior to seeding, test materials must be sterilized to avoid any contamination by microorganisms such as bacteria and fungi. Among the sterilization methods available, autoclave sterilization using steam at high temperature and pressure, dry-heat sterilization in air, and ethanol disinfection by soaking in 70% ethanol are commonly used. However, during these
∗ Corresponding author. Tel.: +81 157 26 9563; fax: +81 157269563. E-mail address: [email protected] (N. Ohtsu).
treatments, the chemical state of the materials surface often change owing to adsorption of organic compounds and/or a reaction with water existing in the atmosphere [7,8]. It is well known that cell adhesion on a metallic surface is achieved by the adsorption of cell adhesion proteins such as fibronectin. If the chemical state of a metallic surface is altered, it can be expected that the adsorption behavior of the adhesion proteins will change, thereby affecting the cell adhesion performance [9]. Serro et al. [10] carefully analyzed the chemical state of a titanium (Ti) surface sterilized by various sterilization methods and revealed that the hydrophilicity varied depending on the sterilization method used. Park et al. [11] investigated the relationship between the adsorption of carbon (C) and cell adhesion on Ti surfaces sterilized and rinsed using various methods, and indicated that a difference in C adsorption affected cell adhesion on their surfaces. Although the effect of C adsorption on cell adhesion has already been reported, studies focusing on the effect of hydroxide groups (OH− ) on cell adhesion are rare. In this study, we analyzed Ti surfaces (treated using three types of sterilization or disinfection methods and rinsed with water), using X-ray photoelectron spectroscopy (XPS). We then evaluated
http://dx.doi.org/10.1016/j.apsusc.2014.05.096 0169-4332/© 2014 Published by Elsevier B.V.
Please cite this article in press as: M. Hirano, et al., Effect of sterilization and water rinsing on cell adhesion to titanium surfaces, Appl. Surf. Sci. (2014), http://dx.doi.org/10.1016/j.apsusc.2014.05.096
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Fig. 1. SEM image of the adhered cells on the treated Ti surfaces; (a) autoclave, (b) dry-heat, (c) 70% ethanol, (d) autoclave with water rinsing, (e) dry-heat with water rinsing, (f) 70% ethanol with water rinsing.
the cell adhesion performance on the differently treated surfaces. By comparing these results, we also attempted to discuss the correlation between the cell adhesion performance and the amount of OH− groups on their surface. 2. Experimental procedures Ti substrates with sizes of 15 mm × 1 mm and 10 mm × 10 mm × 1 mm were used for cell cytocompatibility tests and surface analyses, respectively. They were polished using colloidal silica suspensions and then sequentially washed with ethanol and distilled water using an ultrasonicator. The substrates were then treated under the following conditions: (1) autoclave sterilization at 121 ◦ C and 0.12 MPa for 30 min (KTS-2322, ALP, Japan) [7], (2) dry-heat sterilization in air at 180 ◦ C for 60 min (FT-300, Yamato Scientific, Japan) [12], and (3) ethanol disinfection using 70% ethanol for 30 min [13]. After the treatment, the substrates were dried at 60 ◦ C for 60 min in the incubator or were cooled up to room temperature. The seeding of the cells and XPS analysis was carried out within 10 min immediately after the treatment. Osteoblast-like MC3T3-E1 cells (RIKEN BioResource Center, Japan), derived from mouse calvaria, were employed for cell adhesion assays. MC3T3-E1 cells were cultivated in ␣modified minimum essential medium (GIBCO BRL, USA) containing 10% fetal bovine serum (JR Science, USA) and 1% antibioticantimycotic (100 U mL−1 penicillin, 100 g mL−1 streptomycin, and 0.25 g mL−1 amphotericin B; GIBCO BRL, USA). Prior to seeding of the cells on the specimens, the cells were cultivated until they reached 100% confluence in a polystyrene dish at 37 ◦ C in a fully humidified atmosphere containing 5% CO2 . The treated substrates were placed on 24-well cell-culture polystyrene plates, and thereafter some of the substrates were rinsed using sterilized ultrapure water (Milli-Q, Millipore, USA). MC3T3-E1 cells were seeded at 5 × 103 cells cm−2 on the substrates. After incubating for 24 h, morphology of the cells was observed using scanning electron microscopy (SEM; JCM-5000 NeoScope, JEOL, Japan). Before observation, the cells were fixed with 4% glutaraldehyde solution for 1 h, and thereafter were dehydrate by sequential soaking in 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol
for 15 min. Furthermore, the number of living cells adhered on the Ti surface was measured using a Cell Counting Kit-8 assay (DOJINDO LABORATORIES, Japan), based to the standard protocol. The data were statistically analyzed using analysis of variance (ANOVA) and Student’s t-test. The chemical state of the treated Ti substrates was analyzed using XPS (PHI 5000 VersaProbe, ULVAC-PHI, Japan). Al K␣ radiation (h = 1486.8 eV) was adopted as the X-ray source, and the photoelectron take-off angle was set at 45◦ . The binding energy of the spectra was corrected by the C 1s peak corresponding to naturally adsorbed hydrocarbon (284.8 eV) [14]. The background of the spectra was subtracted by Shirley’s methods, and overlapping peaks were deconvoluted using a Gaussian–Lorentzian function. 3. Results and discussions 3.1. Effect of sterilization on cell adhesion Fig. 1(a)–(c) shows the morphological image of cells on the Ti surfaces treated by autoclave, dry-heat, and 70% ethanol, without water rinsing, respectively. Difference in the morphology depending on the treatment was not found in the images. Fig. 2 shows the number of cells adhered to the treated Ti surfaces after cultivation for 24 h, normalized to the number of seeded cells. Results of a statistical analysis using ANOVA revealed that the number of cells adhered to the surface treated by 70% ethanol was lower than that on the surface treated by autoclave. On the other hand, there was no significant difference between the cell numbers of the autoclave- and the dry-heat-sterilized substrate. These results indicated that the cell adhesion performance on an autoclave- and dry-heat-sterilized Ti surface was superior to the case of a Ti surface treated by 70% ethanol. To elucidate the reason for this difference, we analyzed the chemical state of oxygen at the topmost surface layer using XPS. Fig. 3 shows O 1s XPS spectra for the Ti surfaces treated using each method. The spectral intensities were normalized to the highest intensity. When focusing on the spectra around 533 eV, the spectral intensity for the ethanol-treated surface was remarkably lower than those for the other surfaces. Fig. 4 shows the deconvolution
Please cite this article in press as: M. Hirano, et al., Effect of sterilization and water rinsing on cell adhesion to titanium surfaces, Appl. Surf. Sci. (2014), http://dx.doi.org/10.1016/j.apsusc.2014.05.096
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Fig. 2. Cell adhesiveness on treated Ti surfaces after cultivation for 24 h. Here, cell adhesiveness means the number of adhered cells normalized to the number of seeded cells. The data were statistically analyzed using ANOVA (*p < 0.05).
analysis of the O 1s spectra. The spectra comprise three peaks corresponding to the chemical states of O2− (530.1 eV), OH− (531.8 eV) and H2 O (533.2 eV) [15–17]. These results indicate that in the case of ethanol treatment, the concentration of OH− on the metal surface was remarkably lower than that on Ti surfaces sterilized using either the autoclave or the dry-heat method, and was the cause for the difference in cell adhesion performance. Based on these results, we insist that, in order to accurately assess the cytotoxicity of metallic materials, the sterilization methods used should be unified among metallic materials tested in each experiment. Otherwise, the test results are unreliable because they provide an erroneous numbers of adhered cells owing to differences in the surface chemical state. 3.2. Effect of water rinsing on cytocompatibility In cell adhesion test, metallic surfaces are often rinsed with sterilized ultrapure water to remove adhered organic compounds before seeding cells. This treatment may induce adsorption and/or a reaction of the water with the metallic surface, which may change the surface chemical state. Therefore, the effect of water rinsing on the cell adhesion to Ti surfaces was also investigated. Fig. 5 shows the number of cells adhered to the Ti surfaces with and without water rinsing, after cultivation for 24 h. In this case too, the number of adhered cells was normalized to the number of seeded cells. Statistical analysis using Student’s t-test revealed that there was no significant difference in the number of adhered
Fig. 3. O 1s XPS spectra for the differently treated Ti surfaces.
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Fig. 4. Typical peak deconvolution analysis of the O 1s XPS spectrum.
cells between the water-rinsed and non-rinsed Ti surface in the case of autoclave and dry-heat sterilization. On the other hand, in case of ethanol treatment, the number of adhered cells drastically increased after water rinsing. It should be noted that difference of the cell morphology between the water-rinsed and non-rinsed surfaces was not found in the SEM images (Fig. 1). Fig. 6 shows changes in the O 1s XPS spectra of the treated Ti surface on water rinsing. We could not find any significant change in the spectra when employing autoclave or dry-heat sterilization methods. By contrast, in the case of ethanol treatment, the spectral intensity corresponding to the OH− was larger after the water rinsing, compared to the spectral intensity for the non-rinsed surface. These results suggested that, in case of autoclave or dry-heat sterilizations methods, water rinsing did not affect the surface chemical state and, hence, the cell adhesion performance was not altered. In contrast, when employing ethanol treatment method, the concentration of OH− increased on water rinsing. As a result, the cell adhesion performance was remarkably improved. 3.3. Effect of sterilization and water rinsing on the topmost surface layer Comparing the OH− concentration on the topmost surface layer immediately after the treatment, we found the concentration on
Fig. 5. Cell adhesiveness on the treated Ti surface with and without water rinsing after cultivation for 24 h. Here, cell adhesiveness means the number of adhered cells normalized to the number of seeded cells. The data were statistically analyzed using Student’s t-test (*p < 0.05).
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Fig. 7. Atomic concentration ratio of C to Ti ([C]/[Ti]) on Ti surfaces treated using different methods and evaluated by XPS.
known that ethanol can decompose organic compounds on a metallic surface. Accordingly, we focused on the amount of adsorbed C. Fig. 7 shows comparison of the atomic ratios of C to Ti ([C]/[Ti]) on Ti surfaces treated using the different sterilization methods. As evident the ratio of [C]/[Ti] on the ethanol-treated Ti surface is notably lower than in case of autoclave and dry-heat sterilization. This means that on the ethanol-treated surface, water present during rinsing can react with the topmost surface layer easily. Consequently, the OH− concentration on the ethanol-treated surface increased drastically after water rinsing. 3.4. Correlation between the cell adhesion performance and the OH− concentration on Ti surfaces Fig. 8 shows number of cells adhered to the treated and/or water-rinsed Ti surfaces after cultivation for 24 h, plotted against the atomic ratio of OH− to O2− ([OH− ]/[O2− ]). A pronounced correlation between the cell adhesiveness and [OH− ]/[O2− ] is found. This result suggests that the OH− groups on the outermost surface layer of the Ti substrate may be one of the important factors that determine the cell adhesion performance. For seeding cells contained in a culture medium, the biological reaction that occurs firstly is the adsorption of proteins on Ti surfaces. It is generally accepted that the selective adsorption of cell adhesion proteins dominates the cell adhesion onto a surface. It is conjectured that OH− existing at the topmost surface layer are beneficial to the adsorption of cell adhesion proteins. However, further study is required to elucidate this phenomenon. Fig. 6. Changes in the O 1s XPS spectra of the treated Ti surface on water rinsing (a) autoclave, (b) dry-heat, and (c) ethanol treatment.
the ethanol-treated surface to be notably lower. We consider that this difference originated from the temperature and atmosphere conditions during the treatment. In the cases of autoclave and dry-heat treatment, the Ti surface is exposed to an atmosphere containing water vapor during heating. This situation facilitates the reaction between the topmost surface and water, resulting in an increase of the OH− concentration. In contrast, it is well known that ethanol has a dehydration effect. Accordingly, in the case of ethanol treatment, the surface could not react with water even under the condition that it is present. Consequently, the OH− concentration on the ethanol-treated Ti surface was comparatively low. Conversely, the OH− concentration on the ethanol-treated surface was comparatively high following water rinsing. It is well
Fig. 8. Correlation between cell adhesiveness (after cultivating for 24 h) and [OH− ]/[O2− ] ratio of Ti surfaces.
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In contrast to the number of adhered cells, change in the morphology depending on the surface treatment was not observed, notwithstanding the different concentrations of the OH− groups (Fig. 1). Duske et al. [18] reported that although the difference in cell spreading depending on a surface roughness was observed at 30 and 60 min after the seeding, the difference was hardly found after cultivating for 24 h because the cells attained stable state. In the present study, SEM observation was carried out after cultivation for 24 h, which corresponds to the periods reaching the stable state. We therefore speculated that the difference in the cell morphology might be found when observing the cells cultivated for more short periods. We plan to perform such test and to report in near future. 4. Conclusion Ti surfaces were treated by autoclave, dry heat, or 70% ethanol and subsequently rinsed with sterilized ultrapure water. The chemical state of oxygen at the topmost surface layer was carefully analyzed by XPS, and cell adhesion performance on the treated Ti surfaces was evaluated. The atomic fraction of OH− groups on the surface varied depending on the combination of the sterilization and water rinsing treatments. The cell adhesiveness on ethanol-disinfected substrates was lower than on autoclaveand dry-heat-sterilized substrates. Conversely, after rinsing with ultrapure water, the cell adhesion performance on the ethanoldisinfected substrates was superior compared to that for the other substrates. There was a good correlation between the cell adhesiveness and the fraction of OH− groups, indicating that a change in the OH− fraction affects the adhesion of cells to a Ti surface. In conclusion, we would like to emphasize that the sterilization methods should be unified in each experiment to evaluate cytotoxicity of metallic materials in terms of cell-adhesion behavior. Acknowledgements The authors gratefully acknowledge Mr. M. Yamane and Mr. W. Saito from Kitami Institute of Technology for their help in XPS analysis. This work was supported by Grants-in-aid for Scientific Research (C) (No. 24560841) from the Ministry of Education, Science, Sports, and Culture (MEXT) of Japan.
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References [1] A.F. Mavrogenis, R. Dimitriou, J. Rarvizi, G.C. Babis, Biology of implant osseointegration, J. Musculoskelet. Neuronal Interact. 9 (2009) 61–71. [2] R. Van Noort, Titanium: the implant material of today, J. Mater. Sci. 22 (1987) 3801–3811. [3] M. Long, H.J. Rack, Titanium alloys in total joint replacement – a materials science perspective, Biomaterials 19 (1998) 1621–1639. [4] T. Akahori, M. Niinomi, Fracture characteristics of fatigued Ti-6Al-4V ELI as an implant material, Mater. Sci. Eng. A 243 (1998) 237–243. [5] R.E. McMahon, J. Ma, S.V. Verkhoturov, D. Munoz-Pinto, I. Karaman, F. Rubitschek, H.J. Maier, M.S. Hahn, A comparative study of the cytotoxicity and corrosion resistance of nickel–titanium and titanium–niobium shape memory alloys, Acta Biomater. 8 (2012) 2863–2870. [6] G. Schmalz, U. Schuster, H. Schweikl, Influence of metals on IL-6 release in vitro, Biomaterials 19 (1998) 1689–1694. [7] M. Pegueroless, F.J. Gil, J.A. Planell, C. Aparicio, The influence of blasting and sterilization on static and time-related wettability and surface-energy properties of titanium surfaces, Surf. Coat. Technol. 202 (2008) 3470–3479. [8] B. Thierry, M. Tabrizian, O. Savadogo, L’H. Yahia, Effects of sterilization processes on NiTi alloy: Surface characterization, J. Biomed. Mater. Res. 49 (2000) 88–98. [9] H. Aita, N. Hori, M. Takeuchi, Y. Suzuki, M. Yamada, M. Anpo, T. Ogawa, The effect of ultraviolet functionalization of titanium on integration with bone, Biomaterials 30 (2009) 1015–1025. [10] A.P. Serro, B. Saramago, Influence of sterilization on the mineralization of titanium implants induced byincubation in various biological model fluids, Biomaterials 24 (2003) 4749–4760. [11] J.H. Park, R. Olivars-Navarrete, R.E. Baier, A.E. Meyer, R. Tannenbaum, B.D. Boyan, Z. Schwartz, Effect of cleaning and sterilization on titanium implant surface properties and cellular response, Acta Biomater. 8 (2012) 1966–1975. [12] X.L. Liu, W.R. Zhou, Y.H. Wu, Y. Cheng, Y.F. Zheng, Effect of sterilization process on surface characteristics and biocompatibility of pure Mg and MgCa alloys, Mater. Sci. Eng. C 33 (2013) 4144–4154. [13] K.C. Popat, M. Eltgroth, T.J. Latempa, C.A. Grimes, T.A. Desai, Decreased Staphylococcus epidermis adhesion and increased osteoblast functionality on antibiotic-loaded titania nanotubes, Biomaterials 28 (2007) 4880–4888. [14] N. Ohtsu, T. Ashino, M. Ishihara, F. Sakamoto, T. Hanawa, Calcium-phosphate formation on titanium modified with newly developed calcium-hydroxideslurry treatment, Mater. Trans. 48 (2007) 105–110. [15] N. Ohtsu, N. Masahashi, Y. Mizukoshi, K. Wagatsuma, Hydrocarbon decomposition on a hydrophilic TiO2 surface by UV irradiation: spectral and quantitative analysis using in-situ XPS technique, Langmuir 25 (2009) 11586–11591. [16] N. Ohtsu, K. Sato, K. Saito, K. Asami, T. Hanawa, Calcium phosphates formation on CaTiO3 coated titanium, J. Mater. Sci. Mater. Med. 18 (2007) 1009–1016. [17] O. Linnik, I. Petri, N. Smirnova, V. Kandyba, O. Korduban, A. Eremenko, G. Socol, N. Stefan, C. Ristoscu, I.N. Mihailescu, C. Sutan, V. Malinovschi, V. Djokic, D. Janakovic, TiO2 /ZrO2 thin films synthesized by PLD in low pressure N-, Cand/or O-containing gases: structural, optical and photocatalytic properties, Dig. J. Nanomater. Biostruct. 7 (2012) 1343–1352. [18] K. Duske, I. Koban, E. Kindel, K. Schroder, B. Nebe, B. Holtfreter, L. Jablonowski, K.D. Weltmann, T. Kocher, Atmospheric plasma enhances wettability and cell spreading on dental implant metals, J. Clin. Periodontol. 39 (2012) 400–407.
Please cite this article in press as: M. Hirano, et al., Effect of sterilization and water rinsing on cell adhesion to titanium surfaces, Appl. Surf. Sci. (2014), http://dx.doi.org/10.1016/j.apsusc.2014.05.096
ARTICLE IN PRESS
APSUSC-27914; No. of Pages 5
Applied Surface Science xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Effect of sterilization and water rinsing on cell adhesion to titanium surfaces Mitsuhiro Hirano a , Taro Kozuka a , Yuta Asano a , Yuko Kakuchi a , Hirofumi Arai b , Naofumi Ohtsu a,∗ a b
Instrument Analysis Center, Kitami Institute of Technology, 165 Koen-cho, Kitami, Hokkaido 090-8507, Japan Department of Biotechnology and Environmental Chemistry, Kitami Institute of Technology, 165 Koen-cho, Kitami, Hokkaido 090-8507, Japan
a r t i c l e
i n f o
Article history: Received 2 October 2013 Received in revised form 7 May 2014 Accepted 14 May 2014 Available online xxx Keywords: Titanium Sterilization Water rinsing MC3T3-E1 cell Hydroxide groups
a b s t r a c t In this study, the effects of sterilization and water rinsing on cell adhesion to titanium (Ti) surfaces were investigated. Ti substrates were treated using autoclave, dry-heating, and 70% ethanol. Thereafter, some of the substrates were rinsed with sterilized ultrapure water. Osteoblast-like MC3T3-E1 cells were seeded on the Ti surfaces and the numbers of adhered cells were counted after cultivation for 24 h. The number of cells adhered to ethanol-treated plates was lower than that on autoclave- and dry-heat-sterilized Ti substrates. However, interestingly, the cell adhesion performance on the ethanol-treated substrates was superior compared to that of the other substrates, after rinsing with ultrapure water. To investigate the origin of these differences, the chemical state of the treated surfaces was analyzed by X-ray photoelectron spectroscopy. We found a clear correlation between the number of adhered cells and the concentration of hydroxide groups (OH− ) on the surface, thus indicating that a change in OH− concentration affects the cell adhesion performance on Ti substrates. Since the sterilization and subsequent water rinsing affect the cell adhesion on Ti substrates, we suggest that the sterilization methods should be unified to correctly evaluate the cytocompatibility of metallic materials. © 2014 Published by Elsevier B.V.
1. Introduction Metallic materials are used as orthopedic and dental devices because of their excellent mechanical strength and ductility [1–4]. However, metallic ions released from the metallic devices are often toxic to biological tissues [5,6]. Therefore, cytocompatibility tests for newly developed metallic material are indispensable to check for their biological safety. The cytocompatibility of metallic materials is generally tested using the following protocol: animal cells are seeded on a metallic surface, and biological safety is evaluated by viability tests at its surface. Prior to seeding, test materials must be sterilized to avoid any contamination by microorganisms such as bacteria and fungi. Among the sterilization methods available, autoclave sterilization using steam at high temperature and pressure, dry-heat sterilization in air, and ethanol disinfection by soaking in 70% ethanol are commonly used. However, during these
∗ Corresponding author. Tel.: +81 157 26 9563; fax: +81 157269563. E-mail address: [email protected] (N. Ohtsu).
treatments, the chemical state of the materials surface often change owing to adsorption of organic compounds and/or a reaction with water existing in the atmosphere [7,8]. It is well known that cell adhesion on a metallic surface is achieved by the adsorption of cell adhesion proteins such as fibronectin. If the chemical state of a metallic surface is altered, it can be expected that the adsorption behavior of the adhesion proteins will change, thereby affecting the cell adhesion performance [9]. Serro et al. [10] carefully analyzed the chemical state of a titanium (Ti) surface sterilized by various sterilization methods and revealed that the hydrophilicity varied depending on the sterilization method used. Park et al. [11] investigated the relationship between the adsorption of carbon (C) and cell adhesion on Ti surfaces sterilized and rinsed using various methods, and indicated that a difference in C adsorption affected cell adhesion on their surfaces. Although the effect of C adsorption on cell adhesion has already been reported, studies focusing on the effect of hydroxide groups (OH− ) on cell adhesion are rare. In this study, we analyzed Ti surfaces (treated using three types of sterilization or disinfection methods and rinsed with water), using X-ray photoelectron spectroscopy (XPS). We then evaluated
http://dx.doi.org/10.1016/j.apsusc.2014.05.096 0169-4332/© 2014 Published by Elsevier B.V.
Please cite this article in press as: M. Hirano, et al., Effect of sterilization and water rinsing on cell adhesion to titanium surfaces, Appl. Surf. Sci. (2014), http://dx.doi.org/10.1016/j.apsusc.2014.05.096
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Fig. 1. SEM image of the adhered cells on the treated Ti surfaces; (a) autoclave, (b) dry-heat, (c) 70% ethanol, (d) autoclave with water rinsing, (e) dry-heat with water rinsing, (f) 70% ethanol with water rinsing.
the cell adhesion performance on the differently treated surfaces. By comparing these results, we also attempted to discuss the correlation between the cell adhesion performance and the amount of OH− groups on their surface. 2. Experimental procedures Ti substrates with sizes of 15 mm × 1 mm and 10 mm × 10 mm × 1 mm were used for cell cytocompatibility tests and surface analyses, respectively. They were polished using colloidal silica suspensions and then sequentially washed with ethanol and distilled water using an ultrasonicator. The substrates were then treated under the following conditions: (1) autoclave sterilization at 121 ◦ C and 0.12 MPa for 30 min (KTS-2322, ALP, Japan) [7], (2) dry-heat sterilization in air at 180 ◦ C for 60 min (FT-300, Yamato Scientific, Japan) [12], and (3) ethanol disinfection using 70% ethanol for 30 min [13]. After the treatment, the substrates were dried at 60 ◦ C for 60 min in the incubator or were cooled up to room temperature. The seeding of the cells and XPS analysis was carried out within 10 min immediately after the treatment. Osteoblast-like MC3T3-E1 cells (RIKEN BioResource Center, Japan), derived from mouse calvaria, were employed for cell adhesion assays. MC3T3-E1 cells were cultivated in ␣modified minimum essential medium (GIBCO BRL, USA) containing 10% fetal bovine serum (JR Science, USA) and 1% antibioticantimycotic (100 U mL−1 penicillin, 100 g mL−1 streptomycin, and 0.25 g mL−1 amphotericin B; GIBCO BRL, USA). Prior to seeding of the cells on the specimens, the cells were cultivated until they reached 100% confluence in a polystyrene dish at 37 ◦ C in a fully humidified atmosphere containing 5% CO2 . The treated substrates were placed on 24-well cell-culture polystyrene plates, and thereafter some of the substrates were rinsed using sterilized ultrapure water (Milli-Q, Millipore, USA). MC3T3-E1 cells were seeded at 5 × 103 cells cm−2 on the substrates. After incubating for 24 h, morphology of the cells was observed using scanning electron microscopy (SEM; JCM-5000 NeoScope, JEOL, Japan). Before observation, the cells were fixed with 4% glutaraldehyde solution for 1 h, and thereafter were dehydrate by sequential soaking in 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol
for 15 min. Furthermore, the number of living cells adhered on the Ti surface was measured using a Cell Counting Kit-8 assay (DOJINDO LABORATORIES, Japan), based to the standard protocol. The data were statistically analyzed using analysis of variance (ANOVA) and Student’s t-test. The chemical state of the treated Ti substrates was analyzed using XPS (PHI 5000 VersaProbe, ULVAC-PHI, Japan). Al K␣ radiation (h = 1486.8 eV) was adopted as the X-ray source, and the photoelectron take-off angle was set at 45◦ . The binding energy of the spectra was corrected by the C 1s peak corresponding to naturally adsorbed hydrocarbon (284.8 eV) [14]. The background of the spectra was subtracted by Shirley’s methods, and overlapping peaks were deconvoluted using a Gaussian–Lorentzian function. 3. Results and discussions 3.1. Effect of sterilization on cell adhesion Fig. 1(a)–(c) shows the morphological image of cells on the Ti surfaces treated by autoclave, dry-heat, and 70% ethanol, without water rinsing, respectively. Difference in the morphology depending on the treatment was not found in the images. Fig. 2 shows the number of cells adhered to the treated Ti surfaces after cultivation for 24 h, normalized to the number of seeded cells. Results of a statistical analysis using ANOVA revealed that the number of cells adhered to the surface treated by 70% ethanol was lower than that on the surface treated by autoclave. On the other hand, there was no significant difference between the cell numbers of the autoclave- and the dry-heat-sterilized substrate. These results indicated that the cell adhesion performance on an autoclave- and dry-heat-sterilized Ti surface was superior to the case of a Ti surface treated by 70% ethanol. To elucidate the reason for this difference, we analyzed the chemical state of oxygen at the topmost surface layer using XPS. Fig. 3 shows O 1s XPS spectra for the Ti surfaces treated using each method. The spectral intensities were normalized to the highest intensity. When focusing on the spectra around 533 eV, the spectral intensity for the ethanol-treated surface was remarkably lower than those for the other surfaces. Fig. 4 shows the deconvolution
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Fig. 2. Cell adhesiveness on treated Ti surfaces after cultivation for 24 h. Here, cell adhesiveness means the number of adhered cells normalized to the number of seeded cells. The data were statistically analyzed using ANOVA (*p < 0.05).
analysis of the O 1s spectra. The spectra comprise three peaks corresponding to the chemical states of O2− (530.1 eV), OH− (531.8 eV) and H2 O (533.2 eV) [15–17]. These results indicate that in the case of ethanol treatment, the concentration of OH− on the metal surface was remarkably lower than that on Ti surfaces sterilized using either the autoclave or the dry-heat method, and was the cause for the difference in cell adhesion performance. Based on these results, we insist that, in order to accurately assess the cytotoxicity of metallic materials, the sterilization methods used should be unified among metallic materials tested in each experiment. Otherwise, the test results are unreliable because they provide an erroneous numbers of adhered cells owing to differences in the surface chemical state. 3.2. Effect of water rinsing on cytocompatibility In cell adhesion test, metallic surfaces are often rinsed with sterilized ultrapure water to remove adhered organic compounds before seeding cells. This treatment may induce adsorption and/or a reaction of the water with the metallic surface, which may change the surface chemical state. Therefore, the effect of water rinsing on the cell adhesion to Ti surfaces was also investigated. Fig. 5 shows the number of cells adhered to the Ti surfaces with and without water rinsing, after cultivation for 24 h. In this case too, the number of adhered cells was normalized to the number of seeded cells. Statistical analysis using Student’s t-test revealed that there was no significant difference in the number of adhered
Fig. 3. O 1s XPS spectra for the differently treated Ti surfaces.
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Fig. 4. Typical peak deconvolution analysis of the O 1s XPS spectrum.
cells between the water-rinsed and non-rinsed Ti surface in the case of autoclave and dry-heat sterilization. On the other hand, in case of ethanol treatment, the number of adhered cells drastically increased after water rinsing. It should be noted that difference of the cell morphology between the water-rinsed and non-rinsed surfaces was not found in the SEM images (Fig. 1). Fig. 6 shows changes in the O 1s XPS spectra of the treated Ti surface on water rinsing. We could not find any significant change in the spectra when employing autoclave or dry-heat sterilization methods. By contrast, in the case of ethanol treatment, the spectral intensity corresponding to the OH− was larger after the water rinsing, compared to the spectral intensity for the non-rinsed surface. These results suggested that, in case of autoclave or dry-heat sterilizations methods, water rinsing did not affect the surface chemical state and, hence, the cell adhesion performance was not altered. In contrast, when employing ethanol treatment method, the concentration of OH− increased on water rinsing. As a result, the cell adhesion performance was remarkably improved. 3.3. Effect of sterilization and water rinsing on the topmost surface layer Comparing the OH− concentration on the topmost surface layer immediately after the treatment, we found the concentration on
Fig. 5. Cell adhesiveness on the treated Ti surface with and without water rinsing after cultivation for 24 h. Here, cell adhesiveness means the number of adhered cells normalized to the number of seeded cells. The data were statistically analyzed using Student’s t-test (*p < 0.05).
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Fig. 7. Atomic concentration ratio of C to Ti ([C]/[Ti]) on Ti surfaces treated using different methods and evaluated by XPS.
known that ethanol can decompose organic compounds on a metallic surface. Accordingly, we focused on the amount of adsorbed C. Fig. 7 shows comparison of the atomic ratios of C to Ti ([C]/[Ti]) on Ti surfaces treated using the different sterilization methods. As evident the ratio of [C]/[Ti] on the ethanol-treated Ti surface is notably lower than in case of autoclave and dry-heat sterilization. This means that on the ethanol-treated surface, water present during rinsing can react with the topmost surface layer easily. Consequently, the OH− concentration on the ethanol-treated surface increased drastically after water rinsing. 3.4. Correlation between the cell adhesion performance and the OH− concentration on Ti surfaces Fig. 8 shows number of cells adhered to the treated and/or water-rinsed Ti surfaces after cultivation for 24 h, plotted against the atomic ratio of OH− to O2− ([OH− ]/[O2− ]). A pronounced correlation between the cell adhesiveness and [OH− ]/[O2− ] is found. This result suggests that the OH− groups on the outermost surface layer of the Ti substrate may be one of the important factors that determine the cell adhesion performance. For seeding cells contained in a culture medium, the biological reaction that occurs firstly is the adsorption of proteins on Ti surfaces. It is generally accepted that the selective adsorption of cell adhesion proteins dominates the cell adhesion onto a surface. It is conjectured that OH− existing at the topmost surface layer are beneficial to the adsorption of cell adhesion proteins. However, further study is required to elucidate this phenomenon. Fig. 6. Changes in the O 1s XPS spectra of the treated Ti surface on water rinsing (a) autoclave, (b) dry-heat, and (c) ethanol treatment.
the ethanol-treated surface to be notably lower. We consider that this difference originated from the temperature and atmosphere conditions during the treatment. In the cases of autoclave and dry-heat treatment, the Ti surface is exposed to an atmosphere containing water vapor during heating. This situation facilitates the reaction between the topmost surface and water, resulting in an increase of the OH− concentration. In contrast, it is well known that ethanol has a dehydration effect. Accordingly, in the case of ethanol treatment, the surface could not react with water even under the condition that it is present. Consequently, the OH− concentration on the ethanol-treated Ti surface was comparatively low. Conversely, the OH− concentration on the ethanol-treated surface was comparatively high following water rinsing. It is well
Fig. 8. Correlation between cell adhesiveness (after cultivating for 24 h) and [OH− ]/[O2− ] ratio of Ti surfaces.
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In contrast to the number of adhered cells, change in the morphology depending on the surface treatment was not observed, notwithstanding the different concentrations of the OH− groups (Fig. 1). Duske et al. [18] reported that although the difference in cell spreading depending on a surface roughness was observed at 30 and 60 min after the seeding, the difference was hardly found after cultivating for 24 h because the cells attained stable state. In the present study, SEM observation was carried out after cultivation for 24 h, which corresponds to the periods reaching the stable state. We therefore speculated that the difference in the cell morphology might be found when observing the cells cultivated for more short periods. We plan to perform such test and to report in near future. 4. Conclusion Ti surfaces were treated by autoclave, dry heat, or 70% ethanol and subsequently rinsed with sterilized ultrapure water. The chemical state of oxygen at the topmost surface layer was carefully analyzed by XPS, and cell adhesion performance on the treated Ti surfaces was evaluated. The atomic fraction of OH− groups on the surface varied depending on the combination of the sterilization and water rinsing treatments. The cell adhesiveness on ethanol-disinfected substrates was lower than on autoclaveand dry-heat-sterilized substrates. Conversely, after rinsing with ultrapure water, the cell adhesion performance on the ethanoldisinfected substrates was superior compared to that for the other substrates. There was a good correlation between the cell adhesiveness and the fraction of OH− groups, indicating that a change in the OH− fraction affects the adhesion of cells to a Ti surface. In conclusion, we would like to emphasize that the sterilization methods should be unified in each experiment to evaluate cytotoxicity of metallic materials in terms of cell-adhesion behavior. Acknowledgements The authors gratefully acknowledge Mr. M. Yamane and Mr. W. Saito from Kitami Institute of Technology for their help in XPS analysis. This work was supported by Grants-in-aid for Scientific Research (C) (No. 24560841) from the Ministry of Education, Science, Sports, and Culture (MEXT) of Japan.
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Please cite this article in press as: M. Hirano, et al., Effect of sterilization and water rinsing on cell adhesion to titanium surfaces, Appl. Surf. Sci. (2014), http://dx.doi.org/10.1016/j.apsusc.2014.05.096