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Nanoporous Biocompatible Layer on Ti–6Al–4V Alloys Enhanced Osteoblast-like Cell Response
J Exp Clin Med 2013;5(3):92e96
Contents lists available at SciVerse ScienceDirect
Journal of Experimental and Clinical Medicine journal homepage: http://www.jecm-online.com
ORIGINAL ARTICLE
Nanoporous Biocompatible Layer on Tie6Ale4V Alloys Enhanced Osteoblast-like Cell Response Wei-Fang Lee 1, 2, Tzu-Sen Yang 2, a, Yi-Chieh Wu 2, Pei-Wen Peng 1, 2 * 1 2
Research Center for Biomedical Devices and Prototyping Production, Taipei Medical University, Taipei, Taiwan School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan
a r t i c l e i n f o Article history: Received: Jun 1, 2012 Revised: Jun 22, 2012 Accepted: Mar 15, 2013 KEY WORDS: MG-63 cells; osteoblast; titanium alloys; X-ray photoelectron spectroscopy
Background: The surface properties of Tie6Ale4V (Ti64) alloys extensively affect the biological responses in a physical environment. To enhance the surface biocompatibility of Ti64 specimens, in the present study, electrical discharge machining (EDM) was performed to produce the modified layer on the surface of the Ti64 specimen. Methods: The EDM-functionalized surfaces were obtained at three different pulse durations, which varied from 10 ms to 60 ms. The surface properties of the EDM-functionalized specimen were characterized with scanning electron microscopy and X-ray photoelectron spectroscopy. The properties of adhesion and proliferation of MG-63 cells were evaluated for the interactions between the EDMfunctionalized layer and cells. Results: The incorporation of oxygen roughened the EDM-functionalized surface on a microscale, where the nanoscale pores were superimposed. The EDM-functionalized layer, which can generate the thick anatase TiO2 on the Ti64 surface, afforded a cytocompatible environment. In cell culture, alkaline phosphatase activity could be enhanced on the EDM-functionalized surfaces as compared to the untreated surface. In addition, the increase in pulse durations to the EDM functionalization led to the enhancement of multiple osteoblast functions. Conclusion: The present study revealed that the chemistry and crystallinity of the EDM-functionalized layer played important roles in affecting osteoblastic responses to the specimens, thereby providing insight into the development of new biomedical implant surfaces. Copyright Ó 2013, Taipei Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
1. Introduction Titanium (Ti) and its alloys have been widely used in dental and orthopedic applications owing to their favorable mechanical properties, corrosion resistance, and biocompatibility.1,2 Numerous studies have demonstrated that surface properties of Ti-based alloys, such as surface chemistry and morphology, extensively affect osteoblast proliferation, extracellular matrix production, local factor production, and stimulation of an osteogenic microenvironment.3,4 Among these alloys, the Tie6Ale4V alloy (Ti64) is the most common (aþb)-phases alloy and is well established as a metallic biomaterial for orthopedic and dental implants.5,6 The excellent biocompatibility of Ti64 is attributed to the spontaneous formation of a passive titanium dioxide (TiO2) film,
* Corresponding author. Pei-Wen Peng, School of Dental Technology, College of Oral Medicine, Taipei Medical University, Taipei 11031, Taiwan. E-mail: P.-W. Peng a Wei-Fang Lee and Tzu-Sen Yang contributed equally.
which has the affinity to form a biologically active carbonatecontaining hydroxyapatite layer on Ti surfaces within a physiological environment. The TiO2 film can provide a favorable implante bone interface, which improves the apatite precipitation, osteoblast adhesion, proliferation, osteoblast proliferation, and direct chemical bonding.7,8 Nevertheless, osseointegration via such spontaneously formed TiO2 layer takes about 6 months to complete, which cannot meet the increasing needs of an aging society (e.g., compromised bone density and decreased healing time).9 With the increasing clinical use of Ti64 and need for rapid healing, there have been persistent efforts to search for optimized surfaces for better in vitro response and in vivo osseointegration, and such studies reveal that modification of the physical structure of Ti64 may be beneficial in the long term in the design of artificial implants.10e12 Different surface properties provoke specific protein expressions of mineralized tissue and determine the biological capability.13 Despite significant advances in surface modifications for improving cellular responses, there are still controversial statements on which surface morphologies determine the biological capability of Ti64.
1878-3317/$ e see front matter Copyright Ó 2013, Taipei Medical University. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.jecm.2013.04.002
Nanoporous layers enhanced MG-63 cell response
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Electrical discharge machining (EDM) is a relatively modern machining process based on the conversion of electrical energy into thermal energy.14,15 There is indirect contact between the electrode and the workpiece, both of which are immersed in a dielectric fluid, where the generated discrete spark erosion effectively eliminates mechanical stresses, chatter, and vibration problems.16 Hence, EDM was used in dentistry for fabricating titanium implant-retained restorations17 and for correcting the marginal fit18 of Ti64 crowns. Recently, surfaces subjected to EDM have demonstrated major changes in morphology and composition of the Ti64 substrate.19e21 The bond strength of titaniumeporcelain interface using EDM was compatible with that using sandblasting.22 After the EDM process, a rough texture with craters and pores at the nanoscale level was formed on the Ti-based alloy. We speculated that this modification of physical structure may be beneficial in the long-term survival rate of the Ti64 implants. However, reliable information on cellular responses to EDM-treated surfaces with a defined topography is rather lacking. Thus, we used EDM to functionalize Ti64. We have assessed the surface characteristics and cellular responses of MG-63 osteoblast-like cells on EDMfunctionalized Ti64 surfaces to validate this hypothesis. 2. Materials and methods 2.1. Specimen preparation Commercially available disk-type Ti64 specimen, 10 mm in diameter and 1 mm in thickness (ASTM F136-92; Hung Chun Bio-S Co. Ltd., Kaohsiung, Taiwan), was used as a substrate. The chemical composition of Ti64 was C (0.08 wt.%), Fe (0.25 wt.%), O (0.13 wt.%), Al (6.75 wt.%), V (4.5 wt.%), and Ti (tracer ). After cleaning in distilled water and drying in air, the specimens were subjected to EDM using the die-sinking EDM (Aristech 250, Lien Sheng Mechanical and Electrical Co., Ltd., Taichung, Taiwan), as described previously.21 The positive electrode was made of grade II pure titanium, whereas the negative electrode was a pretreated-Ti64 specimen. Distilled water was used as the dielectric solvent, and the experiments were repeated at three different pulse duration, which varied from 10 ms to 60 ms. A summary of the experimental parameters is given in Table 1. After EDM functionalization, specimens were rinsed with deionized water and air dried. The surface topographies of each specimen were qualitatively characterized using scanning electron microscopy (SEM; JEOL6500F, Joel Ltd, Tokyo, Japan) and quantitatively measured using atomic force microscopy [Dip Pen Nanolithography (DPN) 5000, NanoInk Inc, Skokie, IL, USA]. X-ray photoelectron spectroscopy [XPS; Thermo Electron (VG Scientific Microlab), East Grinstead, UK] was used to analyze the depth profiles of the elements. The contact angle was assessed by dropping 0.4 mL distilled water on specimens Table 1 Experimental conditions for EDM functionalization on Ti64 specimens* Work condition
Description
Work piece Electrode Dielectric fluid Power supply voltage (V) Gap discharge voltage (V) Working time (min) Peak current (A) Off time (ms)
Tie6Ale4V Ti Distilled water 150 30 10 10 30
* Pulse duration (untreated, 10 ms, 30 ms, 60 ms); denotation (EDM-10, EDM-30, EDM-60). EDM ¼ electrical discharge machining.
and using a contact angle goniometer (Jeteazy EA-01, JetEazy system Co., Ltd., Hsinchu City, Taiwan) equipped with a digital camera. 2.2. Cell culture model and evaluation MG-63 cells (60279; Bioresource Collection and Research Center, Hsinchu City, Taiwan) were cultured in Dulbecco’s modified Eagle’s medium (D5648; Sigma-Aldrich, Saint Louis, MO, USA) containing 10% fetal bovine serum (100-106; Gemini Bio-Products, West Sacramento, USA), and 100 units/mL penicillinestreptomycin (P4333; Sigma-Aldrich), in an atmosphere of 95% humidity, 5% CO2, and at a temperature of 37 C. At 80% confluence, the cells were seeded onto specimens of three Ti surface types at a density of 1 105 cells/cm2. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays were performed after the MG-63 cell was cultured on specimens for 1 day, 3 days, and 7 days. At the determined time point, culture medium was removed, and the MTT reagent (50 mL/ well, M2128; Thiazolyl Blue Tetrazolium Bromide, Sigma-Aldrich) was added to the culture plate and incubated at 37 C for 4 hours. Then, the MTT reagent was removed and dimethyl sulfoxide (50 mL) was added to each well to dissolve the formazan crystals. Results of the MTT assay were expressed as the optical density, which was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader (ELx800; Biotek, Winooski, VT, USA). The morphology of the adherent cells on the SLAffinity-Ti surface was observed by SEM after 3 days of incubation. The specimens were fixed with 2.5% (v/v) glutaraldehyde for 30 minutes and washed in 0.1M cacodylate solution. Finally, the specimens were dehydrated using a graded series of ethanol, sputter-coated with Pt/Au, and observed using SEM. After being cultured on specimens for 1 day, 3 days, and 7 days, the alkaline phosphatase (ALP) activity in the MG-63 cell layer was analyzed. The ALP activity (Metra Biosystems, Santa Clara, CA, USA) was evaluated with ELISAs. The optical density was measured at 405 nm using an ELISA reader. The results of the assays were determined using an ELISA reader at the wavelength of 405 nm. Activity values were normalized to the total protein concentration, which was detected in biuret reaction (BCA Protein Assay Kit; Pierce Biotechnology Inc., Rockford, IL, USA) at 570 nm (Microplate reader, Bio-Rad Laboratories Inc., Hercules, CA, USA). 3. Results and discussion The SEM images of the Ti64 specimens with or without EDM functionalization are presented in Figure 1. The untreated specimen revealed relatively smooth surfaces with regular polishing grooves (data not shown). The EDM-10 surface had volcanic-like configuration including globules of debris and a low density of nanoscale pits (Figure 1A). The larger and deeper pores were observed at higher pulse durations, which resulted in a greater surface roughness (Figure 1B). However, these pores were still retained in the nanoscale level. Furthermore, the mean roughness (Ra) and the root mean square (Rq) at different pulse durations were determined quantitatively by atomic force microscopy, as shown in Table 1. The measured Ra and Rq of the untreated surface were 20.56 nm and 25.60 nm, respectively. Compared with the untreated specimens, the Ra and Rq of EDM-60 specimens increased significantly (p < 0.01), which was in agreement with the SEM observations. Figure 2 presents Ti 2p high-resolution XPS spectra of EDM-60 specimens, showing the peaks attributed to TiO2 and TiO.23 The peak belonging to metal Ti was not observed, inferring that a thick oxide layer was formed on the EDM-60 specimen. XRD patterns of the EDM-functionalized layer confirmed that EDM-60 specimens
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Figure 1 SEM textured images of (A) EDM-10 and (B) EDM-60 specimens. EDM ¼ electrical discharge machining; SEM ¼ scanning electron microscopy.
had the diffraction patterns attributed to the anatase TiO2 (not shown here). Table 1 also shows the relationship between the contact angle values and the pulse durations. EDM-60 specimens presented the
Figure 3 MG-63 cells attachment and proliferation on the (A) untreated specimen, (B) EDM-10 specimen, and (C) EDM-60 specimen at day 7. EDM ¼ electrical discharge machining.
Figure 2 Ti 2p high-resolution XPS spectra of the EDM-60 specimen. EDM ¼ electrical discharge machining; XPS ¼ X-ray photoelectron spectroscopy. The black line represents the sum of the peaks; the blue line represents the peak belonging to TiO; the red represents the peak belonging to TiO2.
most hydrophilic surface property, whereas the untreated specimens presented an intermediate hydrophobic property. The results of the contact angle test revealed an increase in the hydrophilicity of the EDM-functionalized layers at high pulse durations owing to the presence of anatase TiO2. The biological properties of EDM-functionalized specimens are assessed by measuring the in vitro cellular responses, as shown in
Nanoporous layers enhanced MG-63 cell response
95 Table 2 Surface roughness parameter (Ra), and contact angle ( ) for the Ti64 with and without EDM functionalization
Roughness (nm) Ra Rq Contact angle ( )
Untreated
EDM-10
EDM-30
EDM-60
20.6 8.2 25.6 5.9 95.1 6.1
124 10.5 254 19.2 66.3 4.3
152 9.4 268 13.3 49.8 5.5
182 12.6 299 12.8 45.3 5.2
Data are presented as mean SD (n ¼ 6). EDM ¼ electrical discharge machining; Ra ¼ mean roughness; Rq ¼ root mean square.
Figure 4 MTT assay of MG-63 cells on specimens after 1 day, 3 days, and 7 days in culture. MTT ¼ 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.
Figures 3, 4, and 5. A high attachment of MG-63 cells onto the untreated surfaces was observed, as shown in Figure 3A. By contrast, Figure 3B and 3C show that the cells were widely spread and completely covered the surfaces of the EDM-10 and EDM-60 specimens. The clear extensions of filopodia penetrating into the pores were observed after EDM functionalization, and this upregulation was significant at the higher pulse duration. Based on the MTT assay (Figure 4), MG-63 cells grown on untreated specimens showed an increase in their proliferation as a function of incubation period. The same behaviors were also observed for those grown on the EDM-functionalized specimens. As compared to untreated specimens, cells seeded on EDM-60 specimens showed a significant increase in their proliferation after 3 days of culture (p < 0.05). The differentiation of MG-63 cells is examined using ALP activity at 1 day, 3 days, and 7 days as shown in Figure 5. A significantly higher level of ALP (p < 0.01) activity is shown by cells grown on EDM-treated specimens compared to untreated specimens after 7 days of growth. Lee and Yur24 reported that pulse current and pulse duration were the two most efficient parameters to affect the surface integrity of the material among the other EDM parameters. Hence, in this study, we selected pulse duration as the experimental variation, and EDM functionalization resulted in the topographic modification and a TiO2 layer formation on Ti64 specimens. EDM modified not only the surface chemistry but also the wettability of Ti64 specimens. During the EDM process, the dielectric, pure water was decomposed into oxygen and hydrogen. The fieldassisted migration of oxygen ions and species diffused into the Ti64 substrate to form a thick oxide layer under the high pulse duration.
Figure 5 ALP activity of MG-63 cells on specimens after 1 day, 3 days, and 7 days in culture. ALP ¼ alkaline phosphatase.
The anatase-TiO2 phase accompanying the nanopore size and density on the Ti64 specimens exhibits hydrophilicity (Table 2). Osteoblast adhesion and proliferation were associated with the functions of the surface topography, chemistry, and crystallinity of the biomaterial.2,7 The present study determined the effect of the principle EDM parameter on the responses of MG63 cells. Our results indicated that the chemistry and nanoscale topography of the EDM treatment played important roles in affecting osteoblastic responses to the Ti64 specimens. The porous anatase-TiO2 layers stimulated the recruitment of osteoblasts, which is consistent with results reported in previous studies.25,26 In conclusion, this research demonstrated that EDM functionalization not only produced the microscale surface roughness but also created the nanoporous TiO2 layers. This structure had a significant increase in ALP activity, which was associated with osteoblastic differential expression during the early osseointegration period without any side effects. An increase in the adherence, proliferation, and differentiation of MG-63 cells was observed on the EDM-functionalized layer, suggesting that this treatment could be a useful process for further enhancing osseointegration and clinical outcome. References 1. Cheng HC, Lee SY, Chen CC, Shyng YC, Ou KL. Influence of hydrogen charging on the formation of nanostructural titania by anodizing with cathodic pretreatment. J Electrochem Soc 2007;154:E13e8. 2. Ou KL, Shih YH, Huang CF, Chen CC, Liu CM. Preparation of bioactive amorphous-like titanium oxide layer on titanium by plasma oxidation treatment. Appl Surf Sci 2008;255:2046e51. 3. Park JH, Olivares-Navarrete R, Baier RE, Meyer AE, Tannenbaum R, Boyan BD, Schwartz Z. Effect of cleaning and sterilization on titanium implant surface properties and cellular response. Acta Biomater 2012;8:1966e75. 4. Le Guéhennec L, Soueidan A, Layrolle P, Amouriq Y. Surface treatments of titanium dental implants for rapid osseointegration. Dent Mater 2007;23: 844e54. 5. Park JW, Park KB, Suh JY. Effects of calcium ion incorporation on bone healing of Ti6Al4V alloy implants in rabbit tibiae. Biomaterials 2007;28:3306e13. 6. Zreiqat H, Valenzuela SM, Nissan BB, Roest R, Knabe C, Radlanski RJ, Renz H, et al. The effect of surface chemistry modification of titanium alloy on signalling pathways in human osteoblasts. Biomaterials 2005;26:7579e86. 7. Ou KL, Lin CT, Chen SL, Huang CF, Cheng HC, Yeh YM, Lin KH. Effect of multinano-titania film on proliferation and differentiation of mouse fibroblast cell on titanium. J Electrochem Soc 2008;155:E79e84. 8. Das K, Bose S, Bandyopadhyay A. Surface modifications and cellematerials interactions with anodized Ti. Acta Biomater 2007;3:573e85. 9. Huang CF, Cheng HC, Liu CM, Chen CC, Ou KL. Microstructure and phase transition of biocompatible titanium oxide film on titanium by plasma discharging. J Alloys Compd 2009;476:683e8. 10. Dey T, Roy P, Fabry B, Schmuki P. Anodic mesoporous TiO2 layer on Ti for enhanced formation of biomimetic hydroxyapatite. Acta Biomater 2011;7: 1873e9. 11. Kim H, Choi SH, Chung SM, Li LH, Lee IS. Enhanced bone forming ability of SLAtreated Ti coated with a calcium phosphate thin film formed by e-beam evaporation. Biomed Mater 2010;5. 044106. 12. Ou KL, Wu J, Lai WF, Yang CB, Lo WC, Chiu LH, Bowley J. Effects of the nanostructure and nanoporosity on bioactive nanohydroxyapatite/reconstituted collagen by electrodeposition. J Biomed Mater Res A 2010;92A:906e12. 13. Sugita Y, Ishizaki K, Iwasa F, Ueno T, Minamikawa H, Yamada M, Suzuki T, et al. Effects of pico-to-nanometer-thin TiO2 coating on the biological properties of microroughened titanium. Biomaterials 2011;32:8374e84. 14. Huertas Talón JL, Cisneros Ortega JC, López Gómez C, Ros Sancho E, Faci Olmos E. Manufacture of a spur tooth gear in Tie6Ale4V alloy by electrical discharge. Comput Aided Design 2010;42:221e30.
96 15. Hasçalık A, Çaydas¸ U. Electrical discharge machining of titanium alloy (Tie6Ale 4V). Appl Surf Sci 2007;253:9007e16. 16. Beri N, Maheshwari S, Sharma C, Kumar A. Technological advancement in electrical discharge machining with powder metallurgy processed electrodes: a review. Mater Manuf Process 2010;25:1186e97. 17. Walter M, Böning K, Reppel PD. Clinical performance of machined titanium restorations. J Dent 1994;22:346e8. 18. Contreras EFR, Henriques GEP, Giolo SR, Nobilo MAA. Fit of cast commercially pure titanium and Tie6Ale4V alloy crowns before and after marginal refinement by electrical discharge machining. J Prosthet Dent 2002;88:467e72. 19. Jane cek M, Nový F, Stráský J, Harcuba P, Wagner L. Fatigue endurance of Tie 6Ale4V alloy with electro-eroded surface for improved bone in-growth. J Mech Behav Biomed 2011;4:417e22. 20. Harcuba P, Ba cáková L, Stráský J, Ba cáková M, Novotná K, Jane cek M. Surface treatment by electric discharge machining of Tie6Ale4V alloy for potential application in orthopaedics. J Mech Behav Biomed 2012;7:96e105.
W.-F. Lee et al. 21. Peng PW, Ou KL, Lin HC, Pan YN, Wang CH. Effect of electrical-discharging on formation of nanoporous biocompatible layer on titanium. J Alloys Compd 2010;492:625e30. _ 22. Inan Ö Acar A, Halkacı S. Effects of sandblasting and electrical discharge machining on porcelain adherence to cast and machined commercially pure titanium. J Biomed Mater Res B 2006;78B:393e400. c M, Strehblow HH. Passive film on orthopaedic 23. Milosev I, Metikos-Hukovi TiAlV alloy formed in physiological solution investigated by X-ray photoelectron spectroscopy. Biomaterials 2000;21:2103e13. 24. Lee HT, Yur JP. Characteristic analysis of EDMed surfaces using the Taguchi approach. Mater Manuf Process 2000;15:781e806. 25. Guo J, Padilla RJ, Ambrose W, De Kok IJ, Cooper LF. The effect of hydrofluoric acid treatment of TiO2 grit blasted titanium implants on adherent osteoblast gene expression in vitro and in vivo. Biomaterials 2007;28:5418e25. 26. Zhao G, Zinger O, Schwartz Z, Wieland M, Landolt D, Boyan BD. Osteoblast-like cells are sensitive to submicron-scale surface structure. Clin Oral Implants Res 2006;17:258e64.
Contents lists available at SciVerse ScienceDirect
Journal of Experimental and Clinical Medicine journal homepage: http://www.jecm-online.com
ORIGINAL ARTICLE
Nanoporous Biocompatible Layer on Tie6Ale4V Alloys Enhanced Osteoblast-like Cell Response Wei-Fang Lee 1, 2, Tzu-Sen Yang 2, a, Yi-Chieh Wu 2, Pei-Wen Peng 1, 2 * 1 2
Research Center for Biomedical Devices and Prototyping Production, Taipei Medical University, Taipei, Taiwan School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan
a r t i c l e i n f o Article history: Received: Jun 1, 2012 Revised: Jun 22, 2012 Accepted: Mar 15, 2013 KEY WORDS: MG-63 cells; osteoblast; titanium alloys; X-ray photoelectron spectroscopy
Background: The surface properties of Tie6Ale4V (Ti64) alloys extensively affect the biological responses in a physical environment. To enhance the surface biocompatibility of Ti64 specimens, in the present study, electrical discharge machining (EDM) was performed to produce the modified layer on the surface of the Ti64 specimen. Methods: The EDM-functionalized surfaces were obtained at three different pulse durations, which varied from 10 ms to 60 ms. The surface properties of the EDM-functionalized specimen were characterized with scanning electron microscopy and X-ray photoelectron spectroscopy. The properties of adhesion and proliferation of MG-63 cells were evaluated for the interactions between the EDMfunctionalized layer and cells. Results: The incorporation of oxygen roughened the EDM-functionalized surface on a microscale, where the nanoscale pores were superimposed. The EDM-functionalized layer, which can generate the thick anatase TiO2 on the Ti64 surface, afforded a cytocompatible environment. In cell culture, alkaline phosphatase activity could be enhanced on the EDM-functionalized surfaces as compared to the untreated surface. In addition, the increase in pulse durations to the EDM functionalization led to the enhancement of multiple osteoblast functions. Conclusion: The present study revealed that the chemistry and crystallinity of the EDM-functionalized layer played important roles in affecting osteoblastic responses to the specimens, thereby providing insight into the development of new biomedical implant surfaces. Copyright Ó 2013, Taipei Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
1. Introduction Titanium (Ti) and its alloys have been widely used in dental and orthopedic applications owing to their favorable mechanical properties, corrosion resistance, and biocompatibility.1,2 Numerous studies have demonstrated that surface properties of Ti-based alloys, such as surface chemistry and morphology, extensively affect osteoblast proliferation, extracellular matrix production, local factor production, and stimulation of an osteogenic microenvironment.3,4 Among these alloys, the Tie6Ale4V alloy (Ti64) is the most common (aþb)-phases alloy and is well established as a metallic biomaterial for orthopedic and dental implants.5,6 The excellent biocompatibility of Ti64 is attributed to the spontaneous formation of a passive titanium dioxide (TiO2) film,
* Corresponding author. Pei-Wen Peng, School of Dental Technology, College of Oral Medicine, Taipei Medical University, Taipei 11031, Taiwan. E-mail: P.-W. Peng
which has the affinity to form a biologically active carbonatecontaining hydroxyapatite layer on Ti surfaces within a physiological environment. The TiO2 film can provide a favorable implante bone interface, which improves the apatite precipitation, osteoblast adhesion, proliferation, osteoblast proliferation, and direct chemical bonding.7,8 Nevertheless, osseointegration via such spontaneously formed TiO2 layer takes about 6 months to complete, which cannot meet the increasing needs of an aging society (e.g., compromised bone density and decreased healing time).9 With the increasing clinical use of Ti64 and need for rapid healing, there have been persistent efforts to search for optimized surfaces for better in vitro response and in vivo osseointegration, and such studies reveal that modification of the physical structure of Ti64 may be beneficial in the long term in the design of artificial implants.10e12 Different surface properties provoke specific protein expressions of mineralized tissue and determine the biological capability.13 Despite significant advances in surface modifications for improving cellular responses, there are still controversial statements on which surface morphologies determine the biological capability of Ti64.
1878-3317/$ e see front matter Copyright Ó 2013, Taipei Medical University. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.jecm.2013.04.002
Nanoporous layers enhanced MG-63 cell response
93
Electrical discharge machining (EDM) is a relatively modern machining process based on the conversion of electrical energy into thermal energy.14,15 There is indirect contact between the electrode and the workpiece, both of which are immersed in a dielectric fluid, where the generated discrete spark erosion effectively eliminates mechanical stresses, chatter, and vibration problems.16 Hence, EDM was used in dentistry for fabricating titanium implant-retained restorations17 and for correcting the marginal fit18 of Ti64 crowns. Recently, surfaces subjected to EDM have demonstrated major changes in morphology and composition of the Ti64 substrate.19e21 The bond strength of titaniumeporcelain interface using EDM was compatible with that using sandblasting.22 After the EDM process, a rough texture with craters and pores at the nanoscale level was formed on the Ti-based alloy. We speculated that this modification of physical structure may be beneficial in the long-term survival rate of the Ti64 implants. However, reliable information on cellular responses to EDM-treated surfaces with a defined topography is rather lacking. Thus, we used EDM to functionalize Ti64. We have assessed the surface characteristics and cellular responses of MG-63 osteoblast-like cells on EDMfunctionalized Ti64 surfaces to validate this hypothesis. 2. Materials and methods 2.1. Specimen preparation Commercially available disk-type Ti64 specimen, 10 mm in diameter and 1 mm in thickness (ASTM F136-92; Hung Chun Bio-S Co. Ltd., Kaohsiung, Taiwan), was used as a substrate. The chemical composition of Ti64 was C (0.08 wt.%), Fe (0.25 wt.%), O (0.13 wt.%), Al (6.75 wt.%), V (4.5 wt.%), and Ti (tracer ). After cleaning in distilled water and drying in air, the specimens were subjected to EDM using the die-sinking EDM (Aristech 250, Lien Sheng Mechanical and Electrical Co., Ltd., Taichung, Taiwan), as described previously.21 The positive electrode was made of grade II pure titanium, whereas the negative electrode was a pretreated-Ti64 specimen. Distilled water was used as the dielectric solvent, and the experiments were repeated at three different pulse duration, which varied from 10 ms to 60 ms. A summary of the experimental parameters is given in Table 1. After EDM functionalization, specimens were rinsed with deionized water and air dried. The surface topographies of each specimen were qualitatively characterized using scanning electron microscopy (SEM; JEOL6500F, Joel Ltd, Tokyo, Japan) and quantitatively measured using atomic force microscopy [Dip Pen Nanolithography (DPN) 5000, NanoInk Inc, Skokie, IL, USA]. X-ray photoelectron spectroscopy [XPS; Thermo Electron (VG Scientific Microlab), East Grinstead, UK] was used to analyze the depth profiles of the elements. The contact angle was assessed by dropping 0.4 mL distilled water on specimens Table 1 Experimental conditions for EDM functionalization on Ti64 specimens* Work condition
Description
Work piece Electrode Dielectric fluid Power supply voltage (V) Gap discharge voltage (V) Working time (min) Peak current (A) Off time (ms)
Tie6Ale4V Ti Distilled water 150 30 10 10 30
* Pulse duration (untreated, 10 ms, 30 ms, 60 ms); denotation (EDM-10, EDM-30, EDM-60). EDM ¼ electrical discharge machining.
and using a contact angle goniometer (Jeteazy EA-01, JetEazy system Co., Ltd., Hsinchu City, Taiwan) equipped with a digital camera. 2.2. Cell culture model and evaluation MG-63 cells (60279; Bioresource Collection and Research Center, Hsinchu City, Taiwan) were cultured in Dulbecco’s modified Eagle’s medium (D5648; Sigma-Aldrich, Saint Louis, MO, USA) containing 10% fetal bovine serum (100-106; Gemini Bio-Products, West Sacramento, USA), and 100 units/mL penicillinestreptomycin (P4333; Sigma-Aldrich), in an atmosphere of 95% humidity, 5% CO2, and at a temperature of 37 C. At 80% confluence, the cells were seeded onto specimens of three Ti surface types at a density of 1 105 cells/cm2. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays were performed after the MG-63 cell was cultured on specimens for 1 day, 3 days, and 7 days. At the determined time point, culture medium was removed, and the MTT reagent (50 mL/ well, M2128; Thiazolyl Blue Tetrazolium Bromide, Sigma-Aldrich) was added to the culture plate and incubated at 37 C for 4 hours. Then, the MTT reagent was removed and dimethyl sulfoxide (50 mL) was added to each well to dissolve the formazan crystals. Results of the MTT assay were expressed as the optical density, which was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader (ELx800; Biotek, Winooski, VT, USA). The morphology of the adherent cells on the SLAffinity-Ti surface was observed by SEM after 3 days of incubation. The specimens were fixed with 2.5% (v/v) glutaraldehyde for 30 minutes and washed in 0.1M cacodylate solution. Finally, the specimens were dehydrated using a graded series of ethanol, sputter-coated with Pt/Au, and observed using SEM. After being cultured on specimens for 1 day, 3 days, and 7 days, the alkaline phosphatase (ALP) activity in the MG-63 cell layer was analyzed. The ALP activity (Metra Biosystems, Santa Clara, CA, USA) was evaluated with ELISAs. The optical density was measured at 405 nm using an ELISA reader. The results of the assays were determined using an ELISA reader at the wavelength of 405 nm. Activity values were normalized to the total protein concentration, which was detected in biuret reaction (BCA Protein Assay Kit; Pierce Biotechnology Inc., Rockford, IL, USA) at 570 nm (Microplate reader, Bio-Rad Laboratories Inc., Hercules, CA, USA). 3. Results and discussion The SEM images of the Ti64 specimens with or without EDM functionalization are presented in Figure 1. The untreated specimen revealed relatively smooth surfaces with regular polishing grooves (data not shown). The EDM-10 surface had volcanic-like configuration including globules of debris and a low density of nanoscale pits (Figure 1A). The larger and deeper pores were observed at higher pulse durations, which resulted in a greater surface roughness (Figure 1B). However, these pores were still retained in the nanoscale level. Furthermore, the mean roughness (Ra) and the root mean square (Rq) at different pulse durations were determined quantitatively by atomic force microscopy, as shown in Table 1. The measured Ra and Rq of the untreated surface were 20.56 nm and 25.60 nm, respectively. Compared with the untreated specimens, the Ra and Rq of EDM-60 specimens increased significantly (p < 0.01), which was in agreement with the SEM observations. Figure 2 presents Ti 2p high-resolution XPS spectra of EDM-60 specimens, showing the peaks attributed to TiO2 and TiO.23 The peak belonging to metal Ti was not observed, inferring that a thick oxide layer was formed on the EDM-60 specimen. XRD patterns of the EDM-functionalized layer confirmed that EDM-60 specimens
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Figure 1 SEM textured images of (A) EDM-10 and (B) EDM-60 specimens. EDM ¼ electrical discharge machining; SEM ¼ scanning electron microscopy.
had the diffraction patterns attributed to the anatase TiO2 (not shown here). Table 1 also shows the relationship between the contact angle values and the pulse durations. EDM-60 specimens presented the
Figure 3 MG-63 cells attachment and proliferation on the (A) untreated specimen, (B) EDM-10 specimen, and (C) EDM-60 specimen at day 7. EDM ¼ electrical discharge machining.
Figure 2 Ti 2p high-resolution XPS spectra of the EDM-60 specimen. EDM ¼ electrical discharge machining; XPS ¼ X-ray photoelectron spectroscopy. The black line represents the sum of the peaks; the blue line represents the peak belonging to TiO; the red represents the peak belonging to TiO2.
most hydrophilic surface property, whereas the untreated specimens presented an intermediate hydrophobic property. The results of the contact angle test revealed an increase in the hydrophilicity of the EDM-functionalized layers at high pulse durations owing to the presence of anatase TiO2. The biological properties of EDM-functionalized specimens are assessed by measuring the in vitro cellular responses, as shown in
Nanoporous layers enhanced MG-63 cell response
95 Table 2 Surface roughness parameter (Ra), and contact angle ( ) for the Ti64 with and without EDM functionalization
Roughness (nm) Ra Rq Contact angle ( )
Untreated
EDM-10
EDM-30
EDM-60
20.6 8.2 25.6 5.9 95.1 6.1
124 10.5 254 19.2 66.3 4.3
152 9.4 268 13.3 49.8 5.5
182 12.6 299 12.8 45.3 5.2
Data are presented as mean SD (n ¼ 6). EDM ¼ electrical discharge machining; Ra ¼ mean roughness; Rq ¼ root mean square.
Figure 4 MTT assay of MG-63 cells on specimens after 1 day, 3 days, and 7 days in culture. MTT ¼ 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.
Figures 3, 4, and 5. A high attachment of MG-63 cells onto the untreated surfaces was observed, as shown in Figure 3A. By contrast, Figure 3B and 3C show that the cells were widely spread and completely covered the surfaces of the EDM-10 and EDM-60 specimens. The clear extensions of filopodia penetrating into the pores were observed after EDM functionalization, and this upregulation was significant at the higher pulse duration. Based on the MTT assay (Figure 4), MG-63 cells grown on untreated specimens showed an increase in their proliferation as a function of incubation period. The same behaviors were also observed for those grown on the EDM-functionalized specimens. As compared to untreated specimens, cells seeded on EDM-60 specimens showed a significant increase in their proliferation after 3 days of culture (p < 0.05). The differentiation of MG-63 cells is examined using ALP activity at 1 day, 3 days, and 7 days as shown in Figure 5. A significantly higher level of ALP (p < 0.01) activity is shown by cells grown on EDM-treated specimens compared to untreated specimens after 7 days of growth. Lee and Yur24 reported that pulse current and pulse duration were the two most efficient parameters to affect the surface integrity of the material among the other EDM parameters. Hence, in this study, we selected pulse duration as the experimental variation, and EDM functionalization resulted in the topographic modification and a TiO2 layer formation on Ti64 specimens. EDM modified not only the surface chemistry but also the wettability of Ti64 specimens. During the EDM process, the dielectric, pure water was decomposed into oxygen and hydrogen. The fieldassisted migration of oxygen ions and species diffused into the Ti64 substrate to form a thick oxide layer under the high pulse duration.
Figure 5 ALP activity of MG-63 cells on specimens after 1 day, 3 days, and 7 days in culture. ALP ¼ alkaline phosphatase.
The anatase-TiO2 phase accompanying the nanopore size and density on the Ti64 specimens exhibits hydrophilicity (Table 2). Osteoblast adhesion and proliferation were associated with the functions of the surface topography, chemistry, and crystallinity of the biomaterial.2,7 The present study determined the effect of the principle EDM parameter on the responses of MG63 cells. Our results indicated that the chemistry and nanoscale topography of the EDM treatment played important roles in affecting osteoblastic responses to the Ti64 specimens. The porous anatase-TiO2 layers stimulated the recruitment of osteoblasts, which is consistent with results reported in previous studies.25,26 In conclusion, this research demonstrated that EDM functionalization not only produced the microscale surface roughness but also created the nanoporous TiO2 layers. This structure had a significant increase in ALP activity, which was associated with osteoblastic differential expression during the early osseointegration period without any side effects. An increase in the adherence, proliferation, and differentiation of MG-63 cells was observed on the EDM-functionalized layer, suggesting that this treatment could be a useful process for further enhancing osseointegration and clinical outcome. References 1. Cheng HC, Lee SY, Chen CC, Shyng YC, Ou KL. Influence of hydrogen charging on the formation of nanostructural titania by anodizing with cathodic pretreatment. J Electrochem Soc 2007;154:E13e8. 2. Ou KL, Shih YH, Huang CF, Chen CC, Liu CM. Preparation of bioactive amorphous-like titanium oxide layer on titanium by plasma oxidation treatment. Appl Surf Sci 2008;255:2046e51. 3. Park JH, Olivares-Navarrete R, Baier RE, Meyer AE, Tannenbaum R, Boyan BD, Schwartz Z. Effect of cleaning and sterilization on titanium implant surface properties and cellular response. Acta Biomater 2012;8:1966e75. 4. Le Guéhennec L, Soueidan A, Layrolle P, Amouriq Y. Surface treatments of titanium dental implants for rapid osseointegration. Dent Mater 2007;23: 844e54. 5. Park JW, Park KB, Suh JY. Effects of calcium ion incorporation on bone healing of Ti6Al4V alloy implants in rabbit tibiae. Biomaterials 2007;28:3306e13. 6. Zreiqat H, Valenzuela SM, Nissan BB, Roest R, Knabe C, Radlanski RJ, Renz H, et al. The effect of surface chemistry modification of titanium alloy on signalling pathways in human osteoblasts. Biomaterials 2005;26:7579e86. 7. Ou KL, Lin CT, Chen SL, Huang CF, Cheng HC, Yeh YM, Lin KH. Effect of multinano-titania film on proliferation and differentiation of mouse fibroblast cell on titanium. J Electrochem Soc 2008;155:E79e84. 8. Das K, Bose S, Bandyopadhyay A. Surface modifications and cellematerials interactions with anodized Ti. Acta Biomater 2007;3:573e85. 9. Huang CF, Cheng HC, Liu CM, Chen CC, Ou KL. Microstructure and phase transition of biocompatible titanium oxide film on titanium by plasma discharging. J Alloys Compd 2009;476:683e8. 10. Dey T, Roy P, Fabry B, Schmuki P. Anodic mesoporous TiO2 layer on Ti for enhanced formation of biomimetic hydroxyapatite. Acta Biomater 2011;7: 1873e9. 11. Kim H, Choi SH, Chung SM, Li LH, Lee IS. Enhanced bone forming ability of SLAtreated Ti coated with a calcium phosphate thin film formed by e-beam evaporation. Biomed Mater 2010;5. 044106. 12. Ou KL, Wu J, Lai WF, Yang CB, Lo WC, Chiu LH, Bowley J. Effects of the nanostructure and nanoporosity on bioactive nanohydroxyapatite/reconstituted collagen by electrodeposition. J Biomed Mater Res A 2010;92A:906e12. 13. Sugita Y, Ishizaki K, Iwasa F, Ueno T, Minamikawa H, Yamada M, Suzuki T, et al. Effects of pico-to-nanometer-thin TiO2 coating on the biological properties of microroughened titanium. Biomaterials 2011;32:8374e84. 14. Huertas Talón JL, Cisneros Ortega JC, López Gómez C, Ros Sancho E, Faci Olmos E. Manufacture of a spur tooth gear in Tie6Ale4V alloy by electrical discharge. Comput Aided Design 2010;42:221e30.
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