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Effect of Mg ion on formation of bone-like apatite on the plasma modified titanium surface
Surface & Coatings Technology 228 (2013) S404–S407
Contents lists available at SciVerse ScienceDirect
Surface & Coatings Technology journal homepage: www.elsevier.com/locate/surfcoat
Effect of Mg ion on formation of bone-like apatite on the plasma modified titanium surface Yeong-Mu Ko, Kang Lee, Byung-Hoon Kim ⁎ Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, 309 Pilmun-daero, Dong-gu, Gwangju, 501‐759 South Korea
a r t i c l e
i n f o
Available online 24 May 2012 Keywords: Plasma surface modification Titanium Bone-like apatite Magnesium
a b s t r a c t Magnesium (Mg) is naturally found in bone tissue and is essential to human metabolism. It is well known that Mg ion improved bone-like apatite nucleation and growth. In this study, Mg ions were ion exchanged onto poly acrylic acid (PAA) thin film containing the carboxyl groups deposited on the commercially pure titanium (CP-Ti) surface using a plasma polymerization at discharge power 50 W for 5 min. Surface morphology and chemical composition of all samples were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Bioactivity of the Mg ion exchanged CP-Ti samples was evaluated by immersing in simulated body fluid (SBF) and MC3T3-E1 cell proliferation. The bone-like apatite forming ability was significantly influenced by Mg ion concentrations. Mg ion promotes bone-like apatite nucleation and growth on CP-Ti surface in SBF solution and improves MC3T3-E1 cell proliferation. It is therefore expected that CP-Ti and Ti alloys having a high biocompatibility can be obtained by ion exchanging the Mg ion after PAA plasma modified Ti surface. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Commercially pure titanium (CP-Ti) and Ti alloys have been intensively studied for the applications of orthopedic and dental implants because of their excellent mechanical properties, corrosion resistance, and outstanding biocompatibility. Hydroxyapatite (HAp) is a bioceramic that has excellent bioactivity and thus has received much attention for its applications as an implant material. In order to improve the biocompatibility of the Ti and Ti alloys, a variety of approaches has been adopted to coat calcium-phosphate (Ca-P) on Ti and Ti alloys. Plasma spray [1], electrophoretic deposition [2], sol–gel [3], sputtering [4], ion beam deposition [5], and biomimetic growth in SBF [6] have been reported. Among the methods, biomimetic process has been noticed owing to its low temperature. Recently, as new approach to enhancing capability of inducing Ca-P coating on Ti, ion implantation method has suggested. Different kinds of metals have been ion-implanted and enhanced Ca-P growth has been reported [7]. Magnesium (Mg) is one of the essential elements for all living organisms and the seventh most abundant element in the earth's crust by mass and eighth by molarity [8]. Mg deficiency affects all skeletal metabolism stages causing cessation of bone growth, decrease of osteoblastic and osteoclastic activities, and generation of osteopenia and bone fragility [9]. Mg ions are the most often described dopants for calcium in the HAp structure. Kuwahara et al. and Li et al. noted that an apatite was precipitated on the surface of
⁎ Corresponding author. Tel.: + 82 62 230 6447; fax: + 82 62 226 6876. E-mail address: [email protected] (B-H. Kim). 0257-8972/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2012.05.058
commercial grade pure Mg from SBF [10]. Howlett et al. reported that the attachment and spreading of human bone derived cells onto the Mg-coated Al2O3 were significantly enhanced as compared to the uncoated Al2O3 [11]. Plasma surface modification techniques are used in biomedical engineering to modify the surface of biomaterial in order to improve adhesion, spreading and proliferation of cells [12]. Hydrophilic coatings with tunable surface density of COOH groups, such as plasma polymerization acrylic acid, have been investigated as cell-adhesive and functional layers for the immobilization of biomolecules [13]. In this study, Mg ions were exchanged to COOH groups of PAA thin films by modifying the Ti surface with acrylic acid (AA) plasma polymerization in order to improve the bone-like apatite growth and MC3T3-E1 cell proliferation. Bioactivity of the Mg ions exchanged CP-Ti samples was evaluated by immersing in SBF and MTT assay.
2. Experimental procedure 2.1. Materials CP-Ti disks (NSC, grade 2, Japan) with diameter 20 mm and thickness 2 mm were used substrates and mechanically polished by utilizing 100 grit emery paper down to 1200 grit emery paper. In brief, CP-Ti disks were cleaned by immersing in ethanol-acetone mixture (1:1) to remove organic and inorganic residues. The cleaning was assisted by ultrasonic cleaner for 15 min. Acrylic acid and magnesium nitrate hexahydrate (Mg(NO3)2) were purchased from Sigma-Aldrich.
Y-M. Ko et al. / Surface & Coatings Technology 228 (2013) S404–S407
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exchange the Mg ions to COOH groups on the CP-Ti surface. These samples are referred to as “Mg/COOH/Ti.” 2.4. Immersing in SBF To examine the bioactivity, the Mg ions exchanged and untreated CP-Ti samples (control) were immersed in SBF for 4 and 7 days. The SBF was prepared by dissolving the ion concentrations such as Na + 142.0, K + 5.0, Ca2+ 2.5, Mg2+ 1.5, CI− 147.8, HCO3− 4.2, HPO42− 1.0, and SO42− 0.5 mM nearly equal to those in human blood plasma. The pH and temperature of SBF were adjusted at 7.4 and 36.5 °C with tris-hydroxymethyl-aminomethane ((CH2OH)3CNH3) and hydrochloric acid. Fig. 2 shows that experimental procedure involves two processes, (a) preparation of Mg ion exchanged CP-Ti surfaces, (b) process of HAp formation in SBF. 2.5. Surface characterization
Fig. 1. Schematic diagram for plasma device.
2.2. Plasma polymerization CP-Ti surface modification was carried out by depositing an PAA thin films containing carboxyl groups through plasma polymerization of hydrophilic monomer, AA using radio frequency (RF) discharge plasma device (Mini Plasma Station, Korea) and the plasma schematic diagram is shown in Fig. 1. Plasma polymerization process carried out at a discharge power of 50 W for 5 min and 200 mTorr working pressure. These samples are referred to as “COOH/Ti.”
2.3. Mg ions exchange to COOH groups on CP-Ti surface To activate the AA plasma polymerized CP-Ti surface, COOH/Ti samples were pretreated by immersing in 25% NH4OH solution for 1 h. Pretreated COOH/Ti samples were immersed in 0.01 M, 0.1 M and 1 M Mg(NO3)2 solutions for 1 h at room temperature in order to
The HAp films on the CP-Ti surfaces were characterized by a thinfilm X-ray diffractometer (TF-XRD, X'pert Philips, Netherlands). The surfaces of the SBF-immersed CP-Ti samples were observed by a field emission scanning electron microscopy (FE-SEM, S-4800 Hitachi, Japan). X-ray photoelectron spectroscopy (XPS, MultiLab 2000 System, SSK, USA) was used to analyze the Mg 1s and calcium and phosphorus (in terms of Ca 2p and P 2p) spectra after process of Mg ions exchanged CP-Ti surfaces and process of HAp formation in the SBF for 7 days. 2.6. Cell culture and MTT assay The bioactivities of the CP-Ti and Mg ions exchanged CP-Ti were examined by evaluating the cytocompatibility of the MC3T3-E1 cells (ATCC CRL-2593). MC3T3-E1, a clonal pre-osteoblastic cell line derived from newborn mouse calvaria, was cultured in an α-modified minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) and a penicillin–streptomycin solution (100 units/ ml penicillin and 100 units/ml streptomycin) at 37 °C in an CO2 incubator (MCO-15AC, Sanyo Electric Co. Ltd.) with 5% CO2 mixed gas. A MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
Fig. 2. The experiment process (a) preparation of Mg ion exchanged CP-Ti surfaces and (b) process of HAp formation in SBF.
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Fig. 3. FE-SEM images of the surfaces of (a) CP-Ti, (b) COOH/Ti, (c) 0.01 M Mg/COOH/Ti and (d) 0.1 M Mg/COOH/Ti after immersing in SBF for 4 days.
to a purple formazan product, Sigma-Aldrich Co.) was used to examine the level of cell proliferation, as described in previous study [14]. 3. Results and discussion
the surfaces of specimens, which, in turn, results in elevated calcium concentration [16], namely improved supersaturation level. The enhancement of supersaturation level leads the improvement of Ca‐P precipitation on the surfaces of Mg ions exchanged CP-Ti. This mechanism has also been reported by Pham et al. [17].
3.1. FE-SEM observations 3.2. Surface characterization Fig. 3 shows the FE-SEM micrographs of the precipitates on the various CP-Ti samples after immersing in SBF for 4 days. Fig. 3a shows that deposits are rarely present on the CP-Ti surface. More precipitates are clearly observed on the AA plasma treated CP-Ti sample (Fig. 3b). This globular morphology of the Ca-P precipitates has been widely reported [15]. For the Mg ions exchanged CP-Ti sample, Mg ions play a role in formation and growth of bone-like apatite, so thick film like precipitates are observed compared to CP-Ti and AA plasma treated CP-Ti samples (Fig. 3c and d). In addition, presence of Mg ions is thought to increase the concentration of Mg ions in the SBF solution adjacent to
TF-XRD analysis demonstrated the presence of HAp in the samples immersed in SBF solution. Fig. 4 shows the TF-XRD patterns of CP-Ti surface and HAp formed on CP-Ti surface after 4 and 7 days of immersion in the SBF. TF-XRD patterns of CP-Ti revealed characteristic peaks of Ti, and no HAp peaks were detected (Fig. 4a). Diffraction peaks in Fig. 4b and c show similar patterns; only relative peak strength differs due to the difference in HAp coating thickness. It is probably because two samples were different in the immersing period in SBF. The characteristic peaks of HAp were observed at 25.9°, 31.7°, and 32.2° 2θ
Fig. 4. TF-XRD patterns of (a) CP-Ti, (b) 0.1 M Mg/COOH/Ti in SBF for 4 days and (c) 0.1 M Mg/COOH/Ti in SBF for 7 days.
Fig. 5. XPS spectra of (a) COOH/Ti, (b) 0.1 M Mg/COOH/Ti and (c) 0.1 M Mg/COOH/Ti in SBF for 7 days.
Y-M. Ko et al. / Surface & Coatings Technology 228 (2013) S404–S407
Fig. 6. MTT assay from the MC3T3-E1 cell seeded on control (tissue culture plate), CP-Ti, COOH/Ti, 0.01 M Mg/COOH/Ti, 0.1 M Mg/COOH/Ti and 1 M Mg/COOH/Ti surface for 1 day and 4 days (*P b 0.05).
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0.01 M Mg/COOH/Ti, 0.1 M Mg/COOH/Ti and 1 M Mg/COOH/Ti surfaces with the tissue culture plate as the control. The cell viability was assessed using a MTT assay, which is a direct mitochondrial activity assay. The cell viability is expressed as a percentage of the control value. The initial cell viability (1 day after cell seeding) of the all samples showed similar results except in the case of control. The 0.1 M Mg/ COOH/Ti sample showed a significant increase in cell viability after 4 days compared to other samples. However, high concentration of Mg ions could lead to decreasing of cell viability. This means that appropriate Mg ions can have some positive effects on MC3T3-E1 cell viability. Fig. 7 shows the MC3T3-E1 cell spread out images on the CP-Ti and 0.1 M Mg ions exchanged Ti surfaces after 3 days of cell incubation. The cell morphologies showed that the attached cells exhibited a good spreading appearance on the Mg ions exchanged Ti surface as compared to that on the CP-Ti surface. When cell adhesion progresses on the Mg ions exchanged Ti, a thin membrane is extended, following the spreading of filopodia on the cell (Fig. 7b). 4. Conclusion
values that correspond closely to those observed in the ICDD file no. 09‐0432 for HAp. The characteristic peaks of HAp crystals suggested that HAp crystals are formed on the Mg-implanted titanium samples [16]. XPS was used to investigate each process step because it can provide information on chemical bonds and atomic concentrations. In order to confirm the Mg ion which substitutes to hydrogen of carboxyl group and HAp formation immersed in SBF for 7 days, XPS analysis was performed (Fig. 5). The O 1s and C 1s peaks strongly appeared on the COOH/Ti surface (Fig. 5a). As seen in Fig. 5b, the Mg ions exchanged CP-Ti surface shows an Mg 1s (1305 eV) peak. Fig. 5c showed that spectra corresponding to Ca 2p (347.5 eV) and P 2p (133.2 eV) are clearly present, and correspond closely to those reported for HAp in the literature [18]. 3.3. Cell proliferation and morphology seeded on the Mg ions exchanged Ti surfaces
Plasma polymerization of AA was used to provide functional groups for CP-Ti surface. The enhancement of the HAp-forming ability arises from Mg/COOH/Ti surface, which has formed the reduction of the Mg ions. The HAp growth and MC3T3-E1 cell viability were significantly influenced by Mg ion concentration. The in vitro cell biological assay showed that introducing Mg ions to the COOH/Ti surfaces had a beneficial effect on MC3T3-E1 cell spreading and proliferation. The current experimental findings confirmed that Mg ions improve the bioactivity of Ti as biomaterials substrate. Acknowledgment This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. R13-2008-010-00000‐0). References
Fig. 6 shows the viability of MC3T3-E1 seeded on the prepared samples at 1 and 4 days. The samples used were CP-Ti, COOH/Ti,
Fig. 7. Morphology of MC3T3-E1 cell seeded on (a) CP-Ti surface and (b) 0.1 M Mg ions exchanged CP-Ti surface for 3 days.
[1] T. Kitsugi, T. Nakamura, M. Oka, Y. Senaha, T. Goto, T. Shibuya, J. Biomed. Mater. Res. 30 (1996) 261. [2] M. Weir, A.J. Rays, B.K. Misthropies, C.C. Sorrell, J.H. Evans, J. Sol-Gel Sci. Technol. 21 (2001) 39. [3] E. Tkalcec, M. Sauer, R. Nonninger, H. Schmidt, J. Mater. Sci. 36 (2001) 5253. [4] W. Kowalski, J. Mater. Sci. 18 (1983) 2531. [5] P. Sioshansi, E.J. Tobin, Surf. Coat.Technol. 83 (1996) 175. [6] J. Ma, W. Huifen, L.B. Kong, K.W. Peng, Nanotechnology 14 (2003) 619. [7] X. Liu, R.W.Y. Poon, S.C.H. Kwok, P.K. Chu, C. Ding, Surf. Coat.Technol. 191 (2005) 43. [8] R.J. Elin, Clin. Chem. 33 (1987) 1965. [9] C.M. Serre, M. Papillard, P. Chavassieux, J.C. Voegel, J. Biomed. Mater. Res. 42 (1998) 626. [10] L. Li, J. Gao, Y. Wang, Surf. Coat.Technol. 185 (2004) 92. [11] C.R. Howlett, H. Zreiqat, R. O'Dell, J. Noorman, P. Evans, B.A. Dalton, C. McFarland, J.G. Steele, J. Mater. Sci. Mater. Med. 5 (1994) 715. [12] Y. Wei-En, H. Ming-Lun, L. Mau-Chin, C. Zhi-Hwa, C. Li-Kai, H. Her-Hsiung, J. Alloys Compd. 479 (2009) 642. [13] I. Bisson, J. Hilborn, F. Wurm, B. Meyrat, P. Frey, Biomaterials 60 (2002) 176. [14] H.S. Seo, Y.M. Ko, B.H. Kim, Prog. Org. Coat. 69 (2010) 38. [15] X. Lu, Y. Leng, Biomaterials 25 (2004) 1779. [16] K.S. TenHuisen, P.W. Brown, J. Biomed. Mater. Res. 36 (1997) 306. [17] M.T. Pham, M.F. Maitz, W. Matz, H. Reuther, E. Richter, G. Steiner, Thin Solid Films 379 (2000) 50. [18] S. Koutsopoulos, J. Biomed. Mater. Res. 62 (2002) 600.
Contents lists available at SciVerse ScienceDirect
Surface & Coatings Technology journal homepage: www.elsevier.com/locate/surfcoat
Effect of Mg ion on formation of bone-like apatite on the plasma modified titanium surface Yeong-Mu Ko, Kang Lee, Byung-Hoon Kim ⁎ Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, 309 Pilmun-daero, Dong-gu, Gwangju, 501‐759 South Korea
a r t i c l e
i n f o
Available online 24 May 2012 Keywords: Plasma surface modification Titanium Bone-like apatite Magnesium
a b s t r a c t Magnesium (Mg) is naturally found in bone tissue and is essential to human metabolism. It is well known that Mg ion improved bone-like apatite nucleation and growth. In this study, Mg ions were ion exchanged onto poly acrylic acid (PAA) thin film containing the carboxyl groups deposited on the commercially pure titanium (CP-Ti) surface using a plasma polymerization at discharge power 50 W for 5 min. Surface morphology and chemical composition of all samples were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Bioactivity of the Mg ion exchanged CP-Ti samples was evaluated by immersing in simulated body fluid (SBF) and MC3T3-E1 cell proliferation. The bone-like apatite forming ability was significantly influenced by Mg ion concentrations. Mg ion promotes bone-like apatite nucleation and growth on CP-Ti surface in SBF solution and improves MC3T3-E1 cell proliferation. It is therefore expected that CP-Ti and Ti alloys having a high biocompatibility can be obtained by ion exchanging the Mg ion after PAA plasma modified Ti surface. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Commercially pure titanium (CP-Ti) and Ti alloys have been intensively studied for the applications of orthopedic and dental implants because of their excellent mechanical properties, corrosion resistance, and outstanding biocompatibility. Hydroxyapatite (HAp) is a bioceramic that has excellent bioactivity and thus has received much attention for its applications as an implant material. In order to improve the biocompatibility of the Ti and Ti alloys, a variety of approaches has been adopted to coat calcium-phosphate (Ca-P) on Ti and Ti alloys. Plasma spray [1], electrophoretic deposition [2], sol–gel [3], sputtering [4], ion beam deposition [5], and biomimetic growth in SBF [6] have been reported. Among the methods, biomimetic process has been noticed owing to its low temperature. Recently, as new approach to enhancing capability of inducing Ca-P coating on Ti, ion implantation method has suggested. Different kinds of metals have been ion-implanted and enhanced Ca-P growth has been reported [7]. Magnesium (Mg) is one of the essential elements for all living organisms and the seventh most abundant element in the earth's crust by mass and eighth by molarity [8]. Mg deficiency affects all skeletal metabolism stages causing cessation of bone growth, decrease of osteoblastic and osteoclastic activities, and generation of osteopenia and bone fragility [9]. Mg ions are the most often described dopants for calcium in the HAp structure. Kuwahara et al. and Li et al. noted that an apatite was precipitated on the surface of
⁎ Corresponding author. Tel.: + 82 62 230 6447; fax: + 82 62 226 6876. E-mail address: [email protected] (B-H. Kim). 0257-8972/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2012.05.058
commercial grade pure Mg from SBF [10]. Howlett et al. reported that the attachment and spreading of human bone derived cells onto the Mg-coated Al2O3 were significantly enhanced as compared to the uncoated Al2O3 [11]. Plasma surface modification techniques are used in biomedical engineering to modify the surface of biomaterial in order to improve adhesion, spreading and proliferation of cells [12]. Hydrophilic coatings with tunable surface density of COOH groups, such as plasma polymerization acrylic acid, have been investigated as cell-adhesive and functional layers for the immobilization of biomolecules [13]. In this study, Mg ions were exchanged to COOH groups of PAA thin films by modifying the Ti surface with acrylic acid (AA) plasma polymerization in order to improve the bone-like apatite growth and MC3T3-E1 cell proliferation. Bioactivity of the Mg ions exchanged CP-Ti samples was evaluated by immersing in SBF and MTT assay.
2. Experimental procedure 2.1. Materials CP-Ti disks (NSC, grade 2, Japan) with diameter 20 mm and thickness 2 mm were used substrates and mechanically polished by utilizing 100 grit emery paper down to 1200 grit emery paper. In brief, CP-Ti disks were cleaned by immersing in ethanol-acetone mixture (1:1) to remove organic and inorganic residues. The cleaning was assisted by ultrasonic cleaner for 15 min. Acrylic acid and magnesium nitrate hexahydrate (Mg(NO3)2) were purchased from Sigma-Aldrich.
Y-M. Ko et al. / Surface & Coatings Technology 228 (2013) S404–S407
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exchange the Mg ions to COOH groups on the CP-Ti surface. These samples are referred to as “Mg/COOH/Ti.” 2.4. Immersing in SBF To examine the bioactivity, the Mg ions exchanged and untreated CP-Ti samples (control) were immersed in SBF for 4 and 7 days. The SBF was prepared by dissolving the ion concentrations such as Na + 142.0, K + 5.0, Ca2+ 2.5, Mg2+ 1.5, CI− 147.8, HCO3− 4.2, HPO42− 1.0, and SO42− 0.5 mM nearly equal to those in human blood plasma. The pH and temperature of SBF were adjusted at 7.4 and 36.5 °C with tris-hydroxymethyl-aminomethane ((CH2OH)3CNH3) and hydrochloric acid. Fig. 2 shows that experimental procedure involves two processes, (a) preparation of Mg ion exchanged CP-Ti surfaces, (b) process of HAp formation in SBF. 2.5. Surface characterization
Fig. 1. Schematic diagram for plasma device.
2.2. Plasma polymerization CP-Ti surface modification was carried out by depositing an PAA thin films containing carboxyl groups through plasma polymerization of hydrophilic monomer, AA using radio frequency (RF) discharge plasma device (Mini Plasma Station, Korea) and the plasma schematic diagram is shown in Fig. 1. Plasma polymerization process carried out at a discharge power of 50 W for 5 min and 200 mTorr working pressure. These samples are referred to as “COOH/Ti.”
2.3. Mg ions exchange to COOH groups on CP-Ti surface To activate the AA plasma polymerized CP-Ti surface, COOH/Ti samples were pretreated by immersing in 25% NH4OH solution for 1 h. Pretreated COOH/Ti samples were immersed in 0.01 M, 0.1 M and 1 M Mg(NO3)2 solutions for 1 h at room temperature in order to
The HAp films on the CP-Ti surfaces were characterized by a thinfilm X-ray diffractometer (TF-XRD, X'pert Philips, Netherlands). The surfaces of the SBF-immersed CP-Ti samples were observed by a field emission scanning electron microscopy (FE-SEM, S-4800 Hitachi, Japan). X-ray photoelectron spectroscopy (XPS, MultiLab 2000 System, SSK, USA) was used to analyze the Mg 1s and calcium and phosphorus (in terms of Ca 2p and P 2p) spectra after process of Mg ions exchanged CP-Ti surfaces and process of HAp formation in the SBF for 7 days. 2.6. Cell culture and MTT assay The bioactivities of the CP-Ti and Mg ions exchanged CP-Ti were examined by evaluating the cytocompatibility of the MC3T3-E1 cells (ATCC CRL-2593). MC3T3-E1, a clonal pre-osteoblastic cell line derived from newborn mouse calvaria, was cultured in an α-modified minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) and a penicillin–streptomycin solution (100 units/ ml penicillin and 100 units/ml streptomycin) at 37 °C in an CO2 incubator (MCO-15AC, Sanyo Electric Co. Ltd.) with 5% CO2 mixed gas. A MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
Fig. 2. The experiment process (a) preparation of Mg ion exchanged CP-Ti surfaces and (b) process of HAp formation in SBF.
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Fig. 3. FE-SEM images of the surfaces of (a) CP-Ti, (b) COOH/Ti, (c) 0.01 M Mg/COOH/Ti and (d) 0.1 M Mg/COOH/Ti after immersing in SBF for 4 days.
to a purple formazan product, Sigma-Aldrich Co.) was used to examine the level of cell proliferation, as described in previous study [14]. 3. Results and discussion
the surfaces of specimens, which, in turn, results in elevated calcium concentration [16], namely improved supersaturation level. The enhancement of supersaturation level leads the improvement of Ca‐P precipitation on the surfaces of Mg ions exchanged CP-Ti. This mechanism has also been reported by Pham et al. [17].
3.1. FE-SEM observations 3.2. Surface characterization Fig. 3 shows the FE-SEM micrographs of the precipitates on the various CP-Ti samples after immersing in SBF for 4 days. Fig. 3a shows that deposits are rarely present on the CP-Ti surface. More precipitates are clearly observed on the AA plasma treated CP-Ti sample (Fig. 3b). This globular morphology of the Ca-P precipitates has been widely reported [15]. For the Mg ions exchanged CP-Ti sample, Mg ions play a role in formation and growth of bone-like apatite, so thick film like precipitates are observed compared to CP-Ti and AA plasma treated CP-Ti samples (Fig. 3c and d). In addition, presence of Mg ions is thought to increase the concentration of Mg ions in the SBF solution adjacent to
TF-XRD analysis demonstrated the presence of HAp in the samples immersed in SBF solution. Fig. 4 shows the TF-XRD patterns of CP-Ti surface and HAp formed on CP-Ti surface after 4 and 7 days of immersion in the SBF. TF-XRD patterns of CP-Ti revealed characteristic peaks of Ti, and no HAp peaks were detected (Fig. 4a). Diffraction peaks in Fig. 4b and c show similar patterns; only relative peak strength differs due to the difference in HAp coating thickness. It is probably because two samples were different in the immersing period in SBF. The characteristic peaks of HAp were observed at 25.9°, 31.7°, and 32.2° 2θ
Fig. 4. TF-XRD patterns of (a) CP-Ti, (b) 0.1 M Mg/COOH/Ti in SBF for 4 days and (c) 0.1 M Mg/COOH/Ti in SBF for 7 days.
Fig. 5. XPS spectra of (a) COOH/Ti, (b) 0.1 M Mg/COOH/Ti and (c) 0.1 M Mg/COOH/Ti in SBF for 7 days.
Y-M. Ko et al. / Surface & Coatings Technology 228 (2013) S404–S407
Fig. 6. MTT assay from the MC3T3-E1 cell seeded on control (tissue culture plate), CP-Ti, COOH/Ti, 0.01 M Mg/COOH/Ti, 0.1 M Mg/COOH/Ti and 1 M Mg/COOH/Ti surface for 1 day and 4 days (*P b 0.05).
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0.01 M Mg/COOH/Ti, 0.1 M Mg/COOH/Ti and 1 M Mg/COOH/Ti surfaces with the tissue culture plate as the control. The cell viability was assessed using a MTT assay, which is a direct mitochondrial activity assay. The cell viability is expressed as a percentage of the control value. The initial cell viability (1 day after cell seeding) of the all samples showed similar results except in the case of control. The 0.1 M Mg/ COOH/Ti sample showed a significant increase in cell viability after 4 days compared to other samples. However, high concentration of Mg ions could lead to decreasing of cell viability. This means that appropriate Mg ions can have some positive effects on MC3T3-E1 cell viability. Fig. 7 shows the MC3T3-E1 cell spread out images on the CP-Ti and 0.1 M Mg ions exchanged Ti surfaces after 3 days of cell incubation. The cell morphologies showed that the attached cells exhibited a good spreading appearance on the Mg ions exchanged Ti surface as compared to that on the CP-Ti surface. When cell adhesion progresses on the Mg ions exchanged Ti, a thin membrane is extended, following the spreading of filopodia on the cell (Fig. 7b). 4. Conclusion
values that correspond closely to those observed in the ICDD file no. 09‐0432 for HAp. The characteristic peaks of HAp crystals suggested that HAp crystals are formed on the Mg-implanted titanium samples [16]. XPS was used to investigate each process step because it can provide information on chemical bonds and atomic concentrations. In order to confirm the Mg ion which substitutes to hydrogen of carboxyl group and HAp formation immersed in SBF for 7 days, XPS analysis was performed (Fig. 5). The O 1s and C 1s peaks strongly appeared on the COOH/Ti surface (Fig. 5a). As seen in Fig. 5b, the Mg ions exchanged CP-Ti surface shows an Mg 1s (1305 eV) peak. Fig. 5c showed that spectra corresponding to Ca 2p (347.5 eV) and P 2p (133.2 eV) are clearly present, and correspond closely to those reported for HAp in the literature [18]. 3.3. Cell proliferation and morphology seeded on the Mg ions exchanged Ti surfaces
Plasma polymerization of AA was used to provide functional groups for CP-Ti surface. The enhancement of the HAp-forming ability arises from Mg/COOH/Ti surface, which has formed the reduction of the Mg ions. The HAp growth and MC3T3-E1 cell viability were significantly influenced by Mg ion concentration. The in vitro cell biological assay showed that introducing Mg ions to the COOH/Ti surfaces had a beneficial effect on MC3T3-E1 cell spreading and proliferation. The current experimental findings confirmed that Mg ions improve the bioactivity of Ti as biomaterials substrate. Acknowledgment This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. R13-2008-010-00000‐0). References
Fig. 6 shows the viability of MC3T3-E1 seeded on the prepared samples at 1 and 4 days. The samples used were CP-Ti, COOH/Ti,
Fig. 7. Morphology of MC3T3-E1 cell seeded on (a) CP-Ti surface and (b) 0.1 M Mg ions exchanged CP-Ti surface for 3 days.
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