Evaluation of Sodium Titanate Coating on Titanium by Sol-Gel Method in vitro

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1 Key Engineering Materials Online: ISSN: , Vols , pp doi: / Trans Tech Publications, Switzerland Evaluation of Sodium Titanate Coating on Titanium by Sol-Gel Method in vitro Fangfang Wang 1a, Yunmao Liao 2, Min Wang 1, Ping Gong 1,2,b, Xiaoyu Li 2, Hua Tang 1, Yi Man 1, Quan Yuan 1, Na Wei 1, Zhen Tan 1, Yu Ban 1 1 West China College of Stomatology, Sichuan University, Chengdu, China P.R. 2 Key Lab for Oral Biomedical Engineering, Minister of Health, West China College of Stomatology, Sichuan University, Chengdu, China P.R. a fangfangwang613@gmail.com b corresponding anthor,gp602002@163.com Keywords: sol-gel, sodium titanate, bioactivity Abstract. In this study, an exclusive sodium titanate (Na 2 Ti 6 O 13 ) coating on titanium was fabricated by sol-gel method and evaluated in vitro. The coating was characterized by SEM and XRD. The bioactivity of the Na 2 Ti 6 O 13 coating was evaluated by the biomimetic growth of apatite on its surface after soaked in an acellular simulated body fluid (SBF) for a period of time. In vitro osteoblasts culture was carried out to determine cytocompatibility by the measurement of the proliferation and alkaline phosphatase (ALP) activity of the cells. XRD patterns showed that Na 2 Ti 6 O 13 was well crystallized when the coating was heated at 800. SEM observation exhibited that the Na 2 Ti 6 O 13 coated titanium had a homogeneous surface without any cracks. After immersion in SBF, the apatite layer can be formed on the coating. The cells culture showed that the osteoblasts grew well on the Na 2 Ti 6 O 13 coated titanium. It can be concluded that Na 2 Ti 6 O 13 coating on titanium obtained by sol-gel method is bioactive. Introduction It is already known that the essential requirement for an artificial material to bond to living bone is the formation of a bone-like apatite layer on its surface in the body environment. The bone-like apatite formation on bioactive materials can be reproduced even in an acellular simulated body fluid (SBF). Generally, titanium is covered with a passive oxide layer and is bioinert. But titanium can be modified to be bioactive when previously treated in NaOH. In NaOH solution, the passive oxide layer dissolves and sodium titanate is formed on the surface. When exposed to SBF, the Na + ions are released from the sodium titanate and hydronium ions enter into the surface layer, resulting in the formation of Ti-OH groups in the surface. And Ti-OH groups can induce apatite nucleation on the titanium surface [1-3]. However, since the composition and thickness of the passive oxide layer on titanium differ and there are several kinds of sodium titanate, the sodium titanate formed by NaOH treatment is hard to be unique. Whether all kinds of sodium titanate are bioactive is still unknown. Among numerous methods used to improve the bioactivity of titanium, sol-gel processing represents an alternative approach for the coating preparation with potential advantages, such as higher purity and chemical homogeneity, finer grain structure, lower processing temperature, easier procedure and controllable thickness. It is promising to use sol-gel method to provide an exclusive and uniform sodium titanate by controlling the proportion of the components. Thus we can use this method to study the properties of sodium titanate more deeply. In this study, an exclusive sodium titanate (Na 2 Ti 6 O 13 ) was fabricated and coated on commercially pure titanium (c.p Ti) discs by sol-gel method. The characterization and evaluation of the coating in vitro were carried out. All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, (ID: , Pennsylvania State University, University Park, USA-09/05/16,08:26:59)

2 778 Bioceramics 19 Materials and methods Tetraisopropyl titanate was dissolved into ethanol to form TiPT solution. Nitric acid, distilled water and sodium nitrate were added to the TiPT solution by molar ratio of TiPT :HNO 3 :H 2 O:NaNO 3 = 3:1.5:12:1 and mixed with magnetic stirrer at room temperature for 1.5 h. Commercially pure titanium(c.p Ti) discs with a diameter of 12mm were abraded, polished and pickled. After washed and dried, the titanium discs were dipped into the sol solution described above for 15 s, withdrawn at a rate of 10 mm/min, dried at room temperature for 5 min, and then heated at 300 for 30 min in a furnace. Thus obtained specimens were heated at different temperatures (500, 600, 700, and 800 ) for 20 min respectively and then allowed to cool with the furnace. Phase characterization of the coatings treated at different temperatures was identified by X-ray diffraction (XRD) (Philips X Pert pro MPD). The surface morphology of the coating heat-treated at 800 was observed under a scanning electron microscopy (SEM, JEOL JEM-5900LV, Japan) The bioactivity of the coated samples heated at 800 was evaluated by the biomimetic growth of apatite on its surface after soaked in SBF with ion concentrations nearly equal to those of human blood plasma at 36.5 for a period of time. The SBF was prepared by dissolving reagent-grade chemicals of NaCl, NaHCO 3, KCl, K 2 HPO. 4 3H2O, MgCl. 6H 2 O, CaCl 2 and Na 2 SO 4 into distilled water, and buffered at ph 7.40 with tris-hydroxymethylamminomethane ((CH 2 OH) 3 CNH 3 ) and HCl at After soaked for 7days, 14 days and 4weeks the specimens were taken out from the SBF, washed with distilled water and dried in air. The culture and harvest of MG63 osteoblast-like cell line followed the description of literature strictly [5]. The cells were plated at a density of cells/ml on all the specimens for 3-(4, 5-dimethilthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and cells/ml for alkaline phosphatase (ALP) activity assay. The MTT assay was performed on day 1, 3 and 5 to determine cell proliferation and ALP assay was performed on day 5, 8 and 11 to determine cell differentiation. The uncoated titanium discs were taken as control. The cell morphology was observed with SEM. The data were processed with SPSS11.5 software. Results and discussion Characterization of the coatings. Figure 1A shows the XRD diagrams of the coated titanium heat-treated at 500, 600, 700 and 800. When heated at 800, the coating gives peaks ascribed to Na 2 Ti 6 O 13, whereas under other temperatures it does not give the Na 2 Ti 6 O 13 peaks. Fig1.XRD diagrams of the coating, (A) heat-treated at 500, 600, 700 and 800 ; (B) heat-treated at 800 and soaked in SBF for 4 weeks. Since we are interested in the performance of Na 2 Ti 6 O 13 coating, we only evaluated the coating heated at 800. Figure 2A shows the SEM image of the surface morphology of the coating

3 Key Engineering Materials Vols heat-treated at 800. The coating is homogeneous, dense tightly adhered to the substrate without any cracks or disintegration. Sound bonding strength is thus suggested from the SEM photograph. Immersion of the Na 2 Ti 6 O 13 coated titanium in SBF. Figure 1B shows the XRD pattern of the coated titanium heat-treated at 800 and soaked in SBF for 4 weeks. It gives a peak ascribed to an apatite. Figure 2B and 2C show the SEM photographs of the apatite induced on the coating after soaked in SBF for 7 days and 14 days respectively. The apatite takes a nano-scale, which is much similar to the structure of natural hydroxyapatite. The fine grain structure of the coating may be the reason. It is reported that a material able to have apatite formed on its surface in SBF has apatite produced on its surface in the living body, and bonds to living bone through this apatite layer. This method can be used for screening bone bioactive materials before animal testing [4]. The mechanism of the formation of apatite may be similar to NaOH-treated titanium, which exists in the release of the Na + ions and the formation of Ti-OH groups in the surface. Fig2. SEM photographs of the coating heat-treated at 800. (A): surface of the coating, 1000; (B): and soaked in SBF for 7 days, 20,000; (C): and soaked in SBF for 14 days, 20,000. Cell proliferation and differentiation. Figure 3(left) shows the MTT value of Na 2 Ti 6 O 13 coating group and c.p Ti group. On day 3 and 5, Na 2 Ti 6 O 13 group has higher MTT value than c.p Ti group. And on day 3, there is statistic difference between two groups. It is clear that the Na 2 Ti 6 O 13 coating has better performance than c.p Ti group at the early stage of cell proliferation, which can be explained with the characteristics of the chemical composition of the coating, such as Ti-OH groups and wettability. Figure 3(right) shows the ALP value of two groups. Its peak is found early in culture (day 5) for two groups. On day 5 and 8, the ALP value of Na 2 Ti 6 O 13 coating group is lower than c.p Ti group. But on day 11, Na 2 Ti 6 O 13 coating group has higher value than c.p Ti. The results maybe suggest the Na 2 Ti 6 O 13 coating has better performance than c.p Ti in terms of differentiation of the osteoblasts at later stage of cell culture. Fig3. MTT value (left) and ALP activity (right) p<0.05. Figure 4 shows the SEM images of the osteoblasts on the surface of Na 2 Ti 6 O 13 coating after seeded for 6hours (A, B, C) and 1day (D, E, F). After seeded for 6 hours, the osteoblasts have already spread and interconnected with each other. They stretch many pseudopods onto the coating. Many of them

4 780 Bioceramics 19 begin to become spindle shape. After 1 day, all osteoblasts become spindle form. And they grow actively and rapidly, the surface of the coating is almost coved by mature osteoblasts. The evaluation of cytocompatibility suggests that the Na2Ti6O13 coating is suitable for the growth of osteoblasts and superior to c.p Ti in some aspects. Fig 4 SEM images of osteoblasts on the surface of Na2Ti6O13 coating (A), (B), (C): 6h after seeding, 200, 500, 1500; D, E, F: 1 day after seeding, 200, 500, Summary It can be concluded that Na2Ti6O13 coating on titanium can be obtained by sol-gel method and is bioactive. The method used in this paper provided another option to modify titanium. Moreover, in further studies, the bioactivity of different kinds of sodium titanate can be investigated through this method thus to make it clear the effect of the content of sodium in sodium titanate on its bioactivity. Acknowledgements The authors would like to thank Xiaoguang Liu for his support of partial materials used in this research. References [1] H.M. Kim, F. Miyaji and T. Kokubo: J. Biomed. Mater. Res. Vol.32 (1996), p.409 [2] H.M. Kim, F. Miyaji, and T. Kokubo: J. Biomed. Mater. Res. Vol.45 (1999), p.100 [3] H. Takadama, H.M. Kim and T. Kokubo: J. Biomed. Mater. Res. Vol.55 (2001), p.185 [4] T. Kokubo, H. Takadama: Biomaterials Vol.27 (2006), p.2907 [5] H.W.Kim, Y.M.Kong and C.J.Bae: Biomaterials Vol.25 (2004), p.2919

5 Bioceramics / Evaluation of Sodium Titanate Coating on Titanium by Sol-Gel Method In Vitro /