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Bioactive nano-titania ceramics with biomechanical compatibility prepared by doping with piezoelectric BaTiO3
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Acta Biomaterialia 5 (2009) 2189–2195 www.elsevier.com/locate/actabiomat
Bioactive nano-titania ceramics with biomechanical compatibility prepared by doping with piezoelectric BaTiO3 Zhensheng Li, Yang Qu, Xingdong Zhang, Bangcheng Yang * Engineering Research Center in Biomaterials, Sichuan University, Wangjiang Road, No. 29, Chengdu 610064, China National Engineering Research Center for Biomaterials, Chengdu 610064, China Received 25 June 2008; received in revised form 6 February 2009; accepted 9 February 2009 Available online 14 February 2009
Abstract Piezoelectric BaTiO3 was employed as a crystal growth inhibitor additive for the preparation of bioactive nano-titania ceramics in this study. It is found that the additive could significantly inhibit nano-titania ceramic crystal growth during the pressureless sintering process. This inhibitory ability has great effects on the mechanical properties and bioactivities of the nano-titania ceramics, making it possible to obtain bioactive nano-titania ceramics with mechanical properties analogous to human bone. In this study, the crystal grain sizes of the nano-titania ceramics ranged from 18 to 68 nm and the particle sizes ranged from 187 to 580 nm by changing the additive content from 1% to 20%. The elastic modulus of the nano-titania ceramics ranged from 6.2 to 10.6 GPa, which is analogous to that of human bone, by adjusting the additive content. The piezoelectric properties of the additive also showed the enhancing effects on the bioactivity of the nano-titania ceramics, which made the osteoblasts proliferate faster on the nano-titania ceramics in cell culture experiments. It might be a potential way to prepare bioactive nano-titania ceramics with biomechanical compatibility by using BaTiO3 as a crystal growth inhibitor. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Nano-titania ceramics; BaTiO3; Bioactivity; Grain growth inhibitor; Biomechanical compatibility
1. Introduction Nano-titania ceramics has been proved to be a potential bioactive material for bony tissue applications. It induced apatite formation in simulated body fluid and enhanced osteoblast differentiation [1–3], which implies that it could form bioactive bonding with bony tissue in the biological environment. It could even inhibit the activity of germs in the biological environment, which would make clinical applications less infectious [4–6]. However, it is very difficult to produce nanophase titania ceramics, because of the aggregation of crystals during the sintering process for ceramics preparation. A number * Corresponding author. Address: Engineering Research Center in Biomaterials, Sichuan University, Wangjiang Road, No. 29, Chengdu 610064, China. Tel.: +86 28 85416391. E-mail address: [email protected] (B. Yang).
of additives have been employed to inhibit crystal growth during this process, such as MgO [7]. In our previous studies [8,9], hydroxyapatite (HA) was successfully used as an additive to inhibit the crystal growth of titania for biomedical applications. The HA additive not only inhibits the crystal growth of titania, thereby making the ceramics bioactive and biomechanically compatible, but also improves the bioactivity of the ceramic by the bioactivity of the additive itself. Barium titanate (BaTiO3) has also been reported to be a potential bioactive material. It could induce apatite formation in simulated body fluid [10,11], and it could enhance bone formation in the biological environment because of its piezoelectric properties [12,13]. In order to obtain bioactive nono-titania ceramics with higher bioactivity and better piezoelectric properties, barium titania was employed in this study to act as a grain growth inhibitor in the preparation of a new type of nano-titania ceramic. The effects of
1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.02.013
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2. Materials and methods
The compressive test was carried out at 1 mm min 1 on a multi-mechanical testing machine (SLP-5 Biomechanical Testing Machine, Chaoyang Instrument, Changchun, China). The elastic moduli of the ceramics were calculated from the compressive test curves.
2.1. Materials preparation
2.3. Fast calcification solution soaking
The nano-titania powder used in this study was synthesized by the sulfate process in Zhejiang Mingri Nano-powder Co, and had an average grain size of 10 nm. For details of the method, see Ref. [14]. Its X-ray diffraction (XRD) pattern showed the powder to be in anatase phase. Nano-BaTiO3 powder supplied by Hebei Kingway Chemical Industry, with an average grain size less than 100 nm, was used as a grain growth inhibitor. The nano-titania powders were doped with different amounts of BaTiO3 (Table 1). The mixed powders were then milled in ethanol by an ultrasonic generator for 10– 20 min. After the powders were dried at room temperature, the mixtures were subjected to cold isostatic pressing at 180 MPa for 30 min for casting and then sintered at 1000 °C for 2 h to obtain nanocomposite ceramics in a normal muffle furnace. The nanocomposite ceramics were cut into / 10 2 mm for scanning electron microscopy (SEM; JSW-5900LV-JEOL) and thin-film XRD (TF-XRD; X’pert Pro MPD, Philips) analysis. Moreover, pure TiO2 ceramic was prepared as control. All of the TiO2/BaTiO3 composite ceramics (TB01, TB05, TB10, TB15 and TB20) were polarized at a voltage of 2500 V for 30 min to produce polarized TiO2/BaTiO3 composite ceramics (PTB01, PTB05, PTB10, PTB15 and PTB20).
The TiO2, TB01, TB20 and PTB20 ceramics were cut into plates of / 10 2 mm to study the formation of apatite. The four ceramics were soaked in 40 ml of fast calcification solution (FCS) for 5 or 10 days at 36.5 °C. FCS was prepared according to Ref. [15], with the following ionic concentrations: Na+ (137 mM), K+ (3.71 mM), Ca2+ (3.10 mM), Cl (145 mM) and HPO42 (1.86 mM). After the plates were taken out from the FCS, they were analyzed by SEM and TF-XRD.
the BaTiO3 additive content on the particle size, the bioactivity and the biomechanical compatibility of bioactive nano-titania ceramics are reported in this paper.
2.4. Cell culture
2.2. Mechanical tests
TB and PTB series ceramic samples, of size / 10 2 mm, were put into 24 multi-well plates for cell culture to test the biocompatibility of the materials according to ISO10993-5:1999. During the cell culture, pressure was exerted on the PTB series ceramic specimens to generate piezoelectric. Cells from the rat osteoblast-like cell line Ros17/28 were suspended, at a density of 5 103 cells ml 1, in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Then 1 ml of the suspension was added to each well containing the substrates, and then placed under a standard cell culture conditions (i.e. 37 °C in a 5% CO2 environment) for 2, 4 and 6 days. The cell medium was changed every 2 days. In this study, pure TiO2 ceramic plates, / 10 2 mm in size, were used as the control.
In accordance with the GB/T 4740-1999 standard compressive resistance test, each ceramic was cut into / 10 20 mm samples. Before the mechanical tests, all the samples were ground with a No. 400 whetstone and cleaned in distilled water, then dried in an oven at 60 °C for 24 h.
2.4.1. MTT assay After 2, 4 and 6 days, 0.2 ml of 3-(4,5-dimethylthiazol-2yl)-2,5-dipheyltetrazolium bromide (MTT) solution (5 mg ml 1 in DMEM) was added to each well. After the cells were cultured for a further 4 h, the plates were washed
Table 1 Nano-titania ceramics doped with different BaTiO3 additive contents. Specimen
TiO2 content (vol.%) BaTiO3 content (vol.%) Average grain size (nm) Average particle size (nm) Average compressive strength (MPa) Average elastic modulus (GPa)
Pure TiO2
TB01
TB05
TB10
TB15
TB20
Human bonea
Ti alloyb
100 0 103 ± 9.4 319.2 ± 29.7 126.3 ± 11.1
99 1 62.3 ± 6.5 187.0 ± 22.4 178.1 ± 12.4
95 5 68.2 ± 5.8 222.1 ± 26.8 157.6 ± 10.2
90 10 18.1 ± 2.6 240.0 ± 22.9 132.2 ± 11.5
85 15 41.9 ± 5.1 399.4 ± 41.4 124.4 ± 9.8
80 20 45.3 ± 4.4 579.2 ± 55.7 89.0 ± 6.7
– – – – 110–170
– – – – –
6.2 ± 0.5
10.6 ± 0.6
9.6 ± 0.4
9.1 ± 0.7
8.9 ± 0.5
7.1 ± 0.4
0.09–18.6
110
The grain size of TB10 is the smallest of all the ceramics, and with increasing BaTiO3, the crystal particle size of titania composite ceramics also increased. However, the average compressive strength and average elastic modulus decreased with increasing BaTiO3 additive contents. a Data from Ref. [16]. b Data from Ref. [17].
Z. Li et al. / Acta Biomaterialia 5 (2009) 2189–2195
twice with D-Hanks solution, then 1 ml of dimethyl sulfoxide was added to each well. The wells were then shaken for 10 min, after which their optical densities (ODs) were read at 490 nm. 2.4.2. SEM analysis Osteoblast morphology on the ceramics films were observed by SEM. At 4 days, the substrates were washed twice with phosphate-buffered solution. The cells were fixed with 2.5% glutaraldehyde buffer for 24 h at 4 °C, then dehydrated in sequentially graded ethanol and subsequently dealcoholized in sequentially graded isoamyl acetate. The samples were next subjected to critical point desiccation, followed by gold coating for SEM observation.
TiO2 BaTiO3 BaO(TiO2)2
Intensity
1500
f
1000
e d 500
c b a
0 20
30
40
50
60
2θ(degree) Fig. 1. XRD patterns of ceramics. (a) TiO2, (b) TB01, (c) TB05, (d) TB10, (e) TB15 and (f) TB20.
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3. Results 3.1. Materials analysis The XRD patterns of the titania ceramics are shown in Fig. 1. When the additive content is 5 vol.%, there is a little new material (BaO(TiO2)2) in the composite ceramics (PCPDF Card, No. 850476). In TB10 the BaO(TiO2)2 signal was higher than that in TB15 and in TB20, as was the BaTiO3 signal (PCPDF Card, No. 340129). The reason for this phenomenon is not clear, but it might be caused by the interaction between TiO2 and BaTiO3 at the higher concentrations, especially the formation of a solid solution of BaTiO3 in TiO2 resulting in a change in the interplanar spacing or crystal face orientation, so the signal of BaTiO3 was not detected. The aggregation of BaTiO3 with itself at the higher concentration might make it less likely to form BaO(TiO2)2 in the ceramics. From the XRD patterns, it could be calculated using the Scherrer equation (crystal plate 110) that the average grain size of the titania in all of the composite ceramics was less than 70 nm but that of the pure TiO2 ceramic was more than 100 nm, as shown in Table 1. It is interesting that the grain size of titania composite ceramics decreased from 62.25 to 18.10 nm when the BaTiO3 additive was increased from 1% to 10%, but it increased from 18.10 to 45.27 nm when the BaTiO3 additive was increased from 10% to 20%. The SEM photographs of titania composite ceramics with different BaTiO3 additive contents are shown in Fig. 2, together with those of the pure TiO2 ceramic. The results showed that the crystal particle sizes of the ceramics were influenced by their BaTiO3 content. From the SEM photographs, it can be seen that the average crystal particle sizes of the composite ceramics TB01, TB05 and TB10 were
Fig. 2. SEM photographs of ceramics added with different BaTiO3 additive contents. (A) TB01, (B) TB05, (C) TB10, (D) TB15, (E) TB20 and (F) TiO2.
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smaller than that of the pure TiO2 (Table 1), but those of TB15 and TB20 were larger. Using the Smile View Software on the SEM, it could be calculated from the SEM photograph that the particle crystal sizes of the TB ceramics ranged from 187.0 to 579.2 nm when the BaTiO3 additive was increased from 1% to 20%.
3.2. Mechanical test 3.2.1. Compressive test The mechanical test results of the compressive strength of the titania composite ceramics were shown in Table 1. The compressive strength of composite ceramics decreased
Fig. 3. SEM micrograph of four titania ceramics soaked in FCS for 5 days (left) and 10 days (right). Titania (A1 and A2), TB01 (B1 and B2), TB20 (C1 and C2), polarized TB20 (D1 and D2).
from 178.1 to 89.01 MPa with increasing BaTiO3 additive content. The pure TiO2 ceramic’s compressive strength was 126.3 MPa. The composite ceramics have compressive strengths analogous to that of human cortical bone, which ranges from 110 to 170 MPa [16]. 3.2.2. Elastic modulus As shown in Table 1, the elastic moduli of the composite ceramics with increasing BaTiO3 additive content ranged from 10.57 to 7.05 GPa. Compared to the elastic modulus of titanium alloy, which is 110 GPa [17], the elastic modulus of the composite ceramics corresponded more closely with that of human bone, which ranges from 0.09 to 18.6 GPa [16]. The elastic modulus of the TiO2 ceramic was 6.21 GPa. 3.3. FCS soaking After the pure titania ceramic was soaked in FCS for 5 days, there is few granular mineral on its surface. There is more mineral on the surface of TB01 after 5 days, as shown in Fig. 3. The surfaces of TB20 and polarized TB20 were fully covered by the mineral. When the four kinds of titania ceramics were soaked in FCS for 10 days, the surfaces of all the ceramics were fully covered by the mineral. XRD (Fig. 4) showed that the mineral on all the ceramics was apatite. This meant that the four titania ceramics could induce apatite formation in FCS. 3.4. Cell culture 3.4.1. MTT assay Fig. 5 shows the MTT assay results after osteoblasts were cultured on the pure titania ceramic and the polarized and unpolarized titania composite ceramics for 2, 4 and 6 days. The OD values of all the titania ceramics increased with time. During the cell culture, the OD value of the pure TiO2 ceramic was lower than those of TB01 and TB20, and was also lower than those of all the polarized nano-titania
Intensity
Fig. 5. OD value of osteoblasts cultured on polarized and unpolarized titania composite ceramics and pure titania ceramic for 2, 4 and 6 days.
composite ceramics (p < 0.05, Student’s t-test). In addition, the OD values of all the PTB series ceramics were significantly higher than those of all TB series ceramics (p < 0.05, Student’s t-test). Notably, the OD value of PTB01 was almost similar to that of PTB20, while both were significantly higher than that of PTB10 (p < 0.05, Student’s t-test). This indicates that the activity of the cells on the PTB01 and PTB20 ceramics were almost the same, while both were higher than that on PTB10. Also, the OD value of TB01 was similar to that of TB20, while both were all significantly higher than that of TB10 (p < 0.05). The activities of the cells on TB01 and TB20 were almost the same, while both were higher than that on TB10. 3.4.2. SEM analysis After the rat osteoblast-like cell line Ros17/28 was cultured on the pure titania ceramic and the polarized titania and unpolarized composite ceramics for 4 days, the osteoblast morphology on the ceramics films was observed with SEM, as shown in Fig. 6. The osteoblasts on all the materials had spread widely. There were numerous microvilli and pseudopodia present on all of the ceramics. Moreover, on all materials, where the cells were connected with microvilli and pseudopodia, coverage was complete. 4. Discussion
1000
Rutile Apatite
500
0 20
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OD value
Z. Li et al. / Acta Biomaterialia 5 (2009) 2189–2195
d c b a 30
40
50
60
2θ(degree) Fig. 4. XRD patterns of four titania ceramics soaked in FCS for 10 days. The mineral apatite was found on all the ceramics. (a) Titania, (b) TB01, (c) TB20 and (d) polarized TB20.
From the XRD and SEM patterns, the titania ceramics with different barium titanate additive contents were seen to have different micro- and nanometer structures. The average grain sizes of all the titania composite ceramics is shown in Fig. 7. The titania composite ceramics’ grain size ranged from 62.25 nm (TB01) to 18.10 nm (TB10), then increased back to 45.27 nm (TB20). The grain size of TB10 was the smallest of all the nano-titania composite ceramics. This might be caused by the generation of the new substance BaO(TiO2)2, which restricted further grain boundary slippage. In TB15 and TB20, the BaO(TiO2)2 content decreased, which weakened its ability to inhibit titania grain growth. This implies that the ability of the
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Fig. 6. SEM micrograph showing osteoblasts cultured on the titania composite ceramics and TiO2 ceramics for 4 days.
new substance BaO(TiO2)2 to inhibit titania grain growth surpasses that of BaTiO3. In our previous study, with increasing HA additive content, the compressive strength and the elastic modulus of tita-
nia composite ceramics also increased. These facts implied that the HA additive enhances the strength of the nanoceramics. However, in this study, with the increasing of BaTiO3 additive content, the mechanical properties, compres-
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Acknowledgments
80
Grain size(nm)
70
The SEM photographs were courtesy of the Analytical & Testing Center, Sichuan University, PR China. This work was supported by the National Natural Science Foundation of China (Nos. 50672062 and 30870615) and Key Programs for Science and Technology Development of Sichuan Province, China (No. 2008SZ0104).
60 50 40 30 20 10 0 TB01
TB05
TB10
TB15
TB20
References
Titania composite ceramics
Fig. 7. Average grain size of all the titania composite ceramics.
sive strength and elastic modulus of titania composite ceramics decreased. This indicates that, with increasing additive content in nano-titania ceramics, the ability of BaTiO3 to enhance the strength of the ceramics is less than that of the HA. In nano-ceramics, a smaller particle size could increase the specific surface area and wettability, which would benefit the adhesion and proliferation of cells [18]. The particle size of TB01 was smaller than that of TiO2, so the osteoblast proliferation and differentiation on TB01 was faster than that on TiO2. Moreover, BaTiO3 particles, of which TB20 had the largest content of all the titania composite ceramics, might congregate together and sinter themselves into bigger BaTiO3 ceramics particles during the sintering process. It is reported that BaTiO3 itself is a bioactive material [19]. So the OD value (Fig. 5) of TB20, which was higher than that of TiO2, might be the effect of the bioactivity of BaTiO3. In addition, for TB10, the new substance BaO(TiO2)2, the content of which was higher than in the other titania ceramics, might weaken the bioactivity. So the OD value of TB10 was smallest among the nano-titania ceramics. In this experiment, we utilized the negatively charged PTB series ceramic surfaces for cell culture [10]. The results of the MTT assay (Fig. 5) provided that the bioactivity of polarized nano-TiO2 ceramics was higher than that of nonpolarized nano-TiO2 ceramics. The bioactivity of the samples was enhanced by negatively polarizing the film surface. This implies that the ferroelectric and piezoelectric effects of TiO2 composite ceramics caused by BaTiO3 play an important role in cell culture. 5. Conclusion The use of BaTiO3 as a grain growth inhibitor could adjust the grain/particle size and the mechanical properties of nano-titania ceramics to make them analogous to that of human bone. The results of this experiment show that the piezoelectric effect caused by the piezoelectric material BaTiO3 in titania composite ceramics promotes the bioactivity of those ceramics. Titania ceramics doped with BaTiO3 are potential bony tissue replacement materials under load-bearing conditions.
[1] Kasuga T, Kondo H, Nogami M. Apatite formation on TiO2 in simulated body fluid. J Cryst Growth 2002;23:5235–40. [2] Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R. Enhanced functions of osteoblasts on nanophase ceramics. Biomaterials 2000;21:1803–10. [3] Liu XY, Zhao XB, Ricky KY, Joan PY, Ding CX, Chu PK. Plasmatreated nanostructured TiO2 surface supporting biomimetic growth of apatite. Biomaterials 2005;26:6143–50. [4] Colon G, Ward BC, Webster TJ. Increased osteoblast and decreased Staphylococcus epidermidis functions on nanophase ZnO and TiO2. J Biomed Mater Res 2006;78A:595–604. [5] Cueto LF, Sanchez E, Torres-Martınez LM, Hirata GA. On the optical, structural, and morphological properties of ZrO2 and TiO2 dip-coated thin films supported on glass substrates. Mater Charact 2005;55:263–71. [6] Yoshinari M, Oda Y, Kato T, Okuda K. Influence of surface modifications to titanium on antibacterial activity in vitro. Biomaterials 2001;22:2043–8. [7] Yu QF, Yang BC, Wu Y, Zhang XD. Preparation of bioactive nanophase titania ceramics by alkali-heat treatment. Key Eng Mater 2005;288–289:215–8. [8] Yang BC, Yu QF, Li ZS, Qu Y, Huang Y, Chen JY, et al. Preparation of bioactive nano-titania ceramics with biomechanical compatibility. J Biomed Mater Res 2006;79A:210–5. [9] Li ZS, Qu Y, Yang BC, Zhang B, Kim HM, Zhao HB, et al. Effects of hydroxyapatite additive content on the bioactivity and biomechanical compatibility of bioactive nano-titania ceramics. J Biomed Mater Res 2008;86A:333–8. [10] Park YJ, Hwang KS, Song JE, Ong JL, Rawls HR. Growth of calcium phosphate on poling treated ferroelectric BaTiO3 ceramics. Biomaterials 2002;23:3859–64. [11] Hwang KS, Song JE, Jo JW, Yang HS, Park YJ, Ong JL, et al. Effect of poling conditions on growth of calcium phosphate crystal in ferroelectric BaTiO3 ceramics. J Mater Sci Mater Med 2002;13:133–8. [12] Park YJ, Jeong YH, Lee YR, Noh SR, Song HJ. Effect of negatively polarized barium titanate thin film, formed on Ti, on osteoblast cell activity. J Dent Res 2003;82:B212. [13] Feng JQ, Yuan HP, Zhang XD. Promotion of osteogenesis by a piezoelectric biological ceramic. Biomaterials 1997;18:1531–4. [14] Jensen H, Soloviev A, Li ZS, Sogaard EG. XPS and FTIR investigation of the surface properties of different prepared titania nano-powders. Appl Surf Sci 2005;246:239–49. [15] Wen HB, van den Brink J, de Wijn JR, Cui FZ, de Groot K. Crystal growth of calcium phosphate on chemically treated titanium. J Cryst Growth 1998;186:616–23. [16] Park JB, Lakes RS. Structure–property relationships of biological materials. In: Park JB, Lakes RS, editors. Biomaterials, an introduction. New York: Plenum Press; 1992. p. 185–222. [17] Ratner BD. A perspective on titanium biocompatibility. In: Brunette DM, Tengvall P, Textor M, Thomsen P, editors. Titanium in medicine. Berlin: Springer; 2001. p. 1–12. [18] Webster TJ, Ejiofor JU. Increased osteoblast adhesion on nanophase metals: Ti, Ti6Al4V, and CoCrMo. Biomaterials 2004;25:4731–9. [19] Song HJ, Park YJ. Fabrication of BaTiO3 films on titanium by microarc oxidation method and improvement of bioactivity by electric poling treatment. Mater Lett 2007;61:3473–6.
Acta Biomaterialia 5 (2009) 2189–2195 www.elsevier.com/locate/actabiomat
Bioactive nano-titania ceramics with biomechanical compatibility prepared by doping with piezoelectric BaTiO3 Zhensheng Li, Yang Qu, Xingdong Zhang, Bangcheng Yang * Engineering Research Center in Biomaterials, Sichuan University, Wangjiang Road, No. 29, Chengdu 610064, China National Engineering Research Center for Biomaterials, Chengdu 610064, China Received 25 June 2008; received in revised form 6 February 2009; accepted 9 February 2009 Available online 14 February 2009
Abstract Piezoelectric BaTiO3 was employed as a crystal growth inhibitor additive for the preparation of bioactive nano-titania ceramics in this study. It is found that the additive could significantly inhibit nano-titania ceramic crystal growth during the pressureless sintering process. This inhibitory ability has great effects on the mechanical properties and bioactivities of the nano-titania ceramics, making it possible to obtain bioactive nano-titania ceramics with mechanical properties analogous to human bone. In this study, the crystal grain sizes of the nano-titania ceramics ranged from 18 to 68 nm and the particle sizes ranged from 187 to 580 nm by changing the additive content from 1% to 20%. The elastic modulus of the nano-titania ceramics ranged from 6.2 to 10.6 GPa, which is analogous to that of human bone, by adjusting the additive content. The piezoelectric properties of the additive also showed the enhancing effects on the bioactivity of the nano-titania ceramics, which made the osteoblasts proliferate faster on the nano-titania ceramics in cell culture experiments. It might be a potential way to prepare bioactive nano-titania ceramics with biomechanical compatibility by using BaTiO3 as a crystal growth inhibitor. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Nano-titania ceramics; BaTiO3; Bioactivity; Grain growth inhibitor; Biomechanical compatibility
1. Introduction Nano-titania ceramics has been proved to be a potential bioactive material for bony tissue applications. It induced apatite formation in simulated body fluid and enhanced osteoblast differentiation [1–3], which implies that it could form bioactive bonding with bony tissue in the biological environment. It could even inhibit the activity of germs in the biological environment, which would make clinical applications less infectious [4–6]. However, it is very difficult to produce nanophase titania ceramics, because of the aggregation of crystals during the sintering process for ceramics preparation. A number * Corresponding author. Address: Engineering Research Center in Biomaterials, Sichuan University, Wangjiang Road, No. 29, Chengdu 610064, China. Tel.: +86 28 85416391. E-mail address: [email protected] (B. Yang).
of additives have been employed to inhibit crystal growth during this process, such as MgO [7]. In our previous studies [8,9], hydroxyapatite (HA) was successfully used as an additive to inhibit the crystal growth of titania for biomedical applications. The HA additive not only inhibits the crystal growth of titania, thereby making the ceramics bioactive and biomechanically compatible, but also improves the bioactivity of the ceramic by the bioactivity of the additive itself. Barium titanate (BaTiO3) has also been reported to be a potential bioactive material. It could induce apatite formation in simulated body fluid [10,11], and it could enhance bone formation in the biological environment because of its piezoelectric properties [12,13]. In order to obtain bioactive nono-titania ceramics with higher bioactivity and better piezoelectric properties, barium titania was employed in this study to act as a grain growth inhibitor in the preparation of a new type of nano-titania ceramic. The effects of
1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.02.013
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2. Materials and methods
The compressive test was carried out at 1 mm min 1 on a multi-mechanical testing machine (SLP-5 Biomechanical Testing Machine, Chaoyang Instrument, Changchun, China). The elastic moduli of the ceramics were calculated from the compressive test curves.
2.1. Materials preparation
2.3. Fast calcification solution soaking
The nano-titania powder used in this study was synthesized by the sulfate process in Zhejiang Mingri Nano-powder Co, and had an average grain size of 10 nm. For details of the method, see Ref. [14]. Its X-ray diffraction (XRD) pattern showed the powder to be in anatase phase. Nano-BaTiO3 powder supplied by Hebei Kingway Chemical Industry, with an average grain size less than 100 nm, was used as a grain growth inhibitor. The nano-titania powders were doped with different amounts of BaTiO3 (Table 1). The mixed powders were then milled in ethanol by an ultrasonic generator for 10– 20 min. After the powders were dried at room temperature, the mixtures were subjected to cold isostatic pressing at 180 MPa for 30 min for casting and then sintered at 1000 °C for 2 h to obtain nanocomposite ceramics in a normal muffle furnace. The nanocomposite ceramics were cut into / 10 2 mm for scanning electron microscopy (SEM; JSW-5900LV-JEOL) and thin-film XRD (TF-XRD; X’pert Pro MPD, Philips) analysis. Moreover, pure TiO2 ceramic was prepared as control. All of the TiO2/BaTiO3 composite ceramics (TB01, TB05, TB10, TB15 and TB20) were polarized at a voltage of 2500 V for 30 min to produce polarized TiO2/BaTiO3 composite ceramics (PTB01, PTB05, PTB10, PTB15 and PTB20).
The TiO2, TB01, TB20 and PTB20 ceramics were cut into plates of / 10 2 mm to study the formation of apatite. The four ceramics were soaked in 40 ml of fast calcification solution (FCS) for 5 or 10 days at 36.5 °C. FCS was prepared according to Ref. [15], with the following ionic concentrations: Na+ (137 mM), K+ (3.71 mM), Ca2+ (3.10 mM), Cl (145 mM) and HPO42 (1.86 mM). After the plates were taken out from the FCS, they were analyzed by SEM and TF-XRD.
the BaTiO3 additive content on the particle size, the bioactivity and the biomechanical compatibility of bioactive nano-titania ceramics are reported in this paper.
2.4. Cell culture
2.2. Mechanical tests
TB and PTB series ceramic samples, of size / 10 2 mm, were put into 24 multi-well plates for cell culture to test the biocompatibility of the materials according to ISO10993-5:1999. During the cell culture, pressure was exerted on the PTB series ceramic specimens to generate piezoelectric. Cells from the rat osteoblast-like cell line Ros17/28 were suspended, at a density of 5 103 cells ml 1, in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Then 1 ml of the suspension was added to each well containing the substrates, and then placed under a standard cell culture conditions (i.e. 37 °C in a 5% CO2 environment) for 2, 4 and 6 days. The cell medium was changed every 2 days. In this study, pure TiO2 ceramic plates, / 10 2 mm in size, were used as the control.
In accordance with the GB/T 4740-1999 standard compressive resistance test, each ceramic was cut into / 10 20 mm samples. Before the mechanical tests, all the samples were ground with a No. 400 whetstone and cleaned in distilled water, then dried in an oven at 60 °C for 24 h.
2.4.1. MTT assay After 2, 4 and 6 days, 0.2 ml of 3-(4,5-dimethylthiazol-2yl)-2,5-dipheyltetrazolium bromide (MTT) solution (5 mg ml 1 in DMEM) was added to each well. After the cells were cultured for a further 4 h, the plates were washed
Table 1 Nano-titania ceramics doped with different BaTiO3 additive contents. Specimen
TiO2 content (vol.%) BaTiO3 content (vol.%) Average grain size (nm) Average particle size (nm) Average compressive strength (MPa) Average elastic modulus (GPa)
Pure TiO2
TB01
TB05
TB10
TB15
TB20
Human bonea
Ti alloyb
100 0 103 ± 9.4 319.2 ± 29.7 126.3 ± 11.1
99 1 62.3 ± 6.5 187.0 ± 22.4 178.1 ± 12.4
95 5 68.2 ± 5.8 222.1 ± 26.8 157.6 ± 10.2
90 10 18.1 ± 2.6 240.0 ± 22.9 132.2 ± 11.5
85 15 41.9 ± 5.1 399.4 ± 41.4 124.4 ± 9.8
80 20 45.3 ± 4.4 579.2 ± 55.7 89.0 ± 6.7
– – – – 110–170
– – – – –
6.2 ± 0.5
10.6 ± 0.6
9.6 ± 0.4
9.1 ± 0.7
8.9 ± 0.5
7.1 ± 0.4
0.09–18.6
110
The grain size of TB10 is the smallest of all the ceramics, and with increasing BaTiO3, the crystal particle size of titania composite ceramics also increased. However, the average compressive strength and average elastic modulus decreased with increasing BaTiO3 additive contents. a Data from Ref. [16]. b Data from Ref. [17].
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twice with D-Hanks solution, then 1 ml of dimethyl sulfoxide was added to each well. The wells were then shaken for 10 min, after which their optical densities (ODs) were read at 490 nm. 2.4.2. SEM analysis Osteoblast morphology on the ceramics films were observed by SEM. At 4 days, the substrates were washed twice with phosphate-buffered solution. The cells were fixed with 2.5% glutaraldehyde buffer for 24 h at 4 °C, then dehydrated in sequentially graded ethanol and subsequently dealcoholized in sequentially graded isoamyl acetate. The samples were next subjected to critical point desiccation, followed by gold coating for SEM observation.
TiO2 BaTiO3 BaO(TiO2)2
Intensity
1500
f
1000
e d 500
c b a
0 20
30
40
50
60
2θ(degree) Fig. 1. XRD patterns of ceramics. (a) TiO2, (b) TB01, (c) TB05, (d) TB10, (e) TB15 and (f) TB20.
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3. Results 3.1. Materials analysis The XRD patterns of the titania ceramics are shown in Fig. 1. When the additive content is 5 vol.%, there is a little new material (BaO(TiO2)2) in the composite ceramics (PCPDF Card, No. 850476). In TB10 the BaO(TiO2)2 signal was higher than that in TB15 and in TB20, as was the BaTiO3 signal (PCPDF Card, No. 340129). The reason for this phenomenon is not clear, but it might be caused by the interaction between TiO2 and BaTiO3 at the higher concentrations, especially the formation of a solid solution of BaTiO3 in TiO2 resulting in a change in the interplanar spacing or crystal face orientation, so the signal of BaTiO3 was not detected. The aggregation of BaTiO3 with itself at the higher concentration might make it less likely to form BaO(TiO2)2 in the ceramics. From the XRD patterns, it could be calculated using the Scherrer equation (crystal plate 110) that the average grain size of the titania in all of the composite ceramics was less than 70 nm but that of the pure TiO2 ceramic was more than 100 nm, as shown in Table 1. It is interesting that the grain size of titania composite ceramics decreased from 62.25 to 18.10 nm when the BaTiO3 additive was increased from 1% to 10%, but it increased from 18.10 to 45.27 nm when the BaTiO3 additive was increased from 10% to 20%. The SEM photographs of titania composite ceramics with different BaTiO3 additive contents are shown in Fig. 2, together with those of the pure TiO2 ceramic. The results showed that the crystal particle sizes of the ceramics were influenced by their BaTiO3 content. From the SEM photographs, it can be seen that the average crystal particle sizes of the composite ceramics TB01, TB05 and TB10 were
Fig. 2. SEM photographs of ceramics added with different BaTiO3 additive contents. (A) TB01, (B) TB05, (C) TB10, (D) TB15, (E) TB20 and (F) TiO2.
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smaller than that of the pure TiO2 (Table 1), but those of TB15 and TB20 were larger. Using the Smile View Software on the SEM, it could be calculated from the SEM photograph that the particle crystal sizes of the TB ceramics ranged from 187.0 to 579.2 nm when the BaTiO3 additive was increased from 1% to 20%.
3.2. Mechanical test 3.2.1. Compressive test The mechanical test results of the compressive strength of the titania composite ceramics were shown in Table 1. The compressive strength of composite ceramics decreased
Fig. 3. SEM micrograph of four titania ceramics soaked in FCS for 5 days (left) and 10 days (right). Titania (A1 and A2), TB01 (B1 and B2), TB20 (C1 and C2), polarized TB20 (D1 and D2).
from 178.1 to 89.01 MPa with increasing BaTiO3 additive content. The pure TiO2 ceramic’s compressive strength was 126.3 MPa. The composite ceramics have compressive strengths analogous to that of human cortical bone, which ranges from 110 to 170 MPa [16]. 3.2.2. Elastic modulus As shown in Table 1, the elastic moduli of the composite ceramics with increasing BaTiO3 additive content ranged from 10.57 to 7.05 GPa. Compared to the elastic modulus of titanium alloy, which is 110 GPa [17], the elastic modulus of the composite ceramics corresponded more closely with that of human bone, which ranges from 0.09 to 18.6 GPa [16]. The elastic modulus of the TiO2 ceramic was 6.21 GPa. 3.3. FCS soaking After the pure titania ceramic was soaked in FCS for 5 days, there is few granular mineral on its surface. There is more mineral on the surface of TB01 after 5 days, as shown in Fig. 3. The surfaces of TB20 and polarized TB20 were fully covered by the mineral. When the four kinds of titania ceramics were soaked in FCS for 10 days, the surfaces of all the ceramics were fully covered by the mineral. XRD (Fig. 4) showed that the mineral on all the ceramics was apatite. This meant that the four titania ceramics could induce apatite formation in FCS. 3.4. Cell culture 3.4.1. MTT assay Fig. 5 shows the MTT assay results after osteoblasts were cultured on the pure titania ceramic and the polarized and unpolarized titania composite ceramics for 2, 4 and 6 days. The OD values of all the titania ceramics increased with time. During the cell culture, the OD value of the pure TiO2 ceramic was lower than those of TB01 and TB20, and was also lower than those of all the polarized nano-titania
Intensity
Fig. 5. OD value of osteoblasts cultured on polarized and unpolarized titania composite ceramics and pure titania ceramic for 2, 4 and 6 days.
composite ceramics (p < 0.05, Student’s t-test). In addition, the OD values of all the PTB series ceramics were significantly higher than those of all TB series ceramics (p < 0.05, Student’s t-test). Notably, the OD value of PTB01 was almost similar to that of PTB20, while both were significantly higher than that of PTB10 (p < 0.05, Student’s t-test). This indicates that the activity of the cells on the PTB01 and PTB20 ceramics were almost the same, while both were higher than that on PTB10. Also, the OD value of TB01 was similar to that of TB20, while both were all significantly higher than that of TB10 (p < 0.05). The activities of the cells on TB01 and TB20 were almost the same, while both were higher than that on TB10. 3.4.2. SEM analysis After the rat osteoblast-like cell line Ros17/28 was cultured on the pure titania ceramic and the polarized titania and unpolarized composite ceramics for 4 days, the osteoblast morphology on the ceramics films was observed with SEM, as shown in Fig. 6. The osteoblasts on all the materials had spread widely. There were numerous microvilli and pseudopodia present on all of the ceramics. Moreover, on all materials, where the cells were connected with microvilli and pseudopodia, coverage was complete. 4. Discussion
1000
Rutile Apatite
500
0 20
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OD value
Z. Li et al. / Acta Biomaterialia 5 (2009) 2189–2195
d c b a 30
40
50
60
2θ(degree) Fig. 4. XRD patterns of four titania ceramics soaked in FCS for 10 days. The mineral apatite was found on all the ceramics. (a) Titania, (b) TB01, (c) TB20 and (d) polarized TB20.
From the XRD and SEM patterns, the titania ceramics with different barium titanate additive contents were seen to have different micro- and nanometer structures. The average grain sizes of all the titania composite ceramics is shown in Fig. 7. The titania composite ceramics’ grain size ranged from 62.25 nm (TB01) to 18.10 nm (TB10), then increased back to 45.27 nm (TB20). The grain size of TB10 was the smallest of all the nano-titania composite ceramics. This might be caused by the generation of the new substance BaO(TiO2)2, which restricted further grain boundary slippage. In TB15 and TB20, the BaO(TiO2)2 content decreased, which weakened its ability to inhibit titania grain growth. This implies that the ability of the
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Fig. 6. SEM micrograph showing osteoblasts cultured on the titania composite ceramics and TiO2 ceramics for 4 days.
new substance BaO(TiO2)2 to inhibit titania grain growth surpasses that of BaTiO3. In our previous study, with increasing HA additive content, the compressive strength and the elastic modulus of tita-
nia composite ceramics also increased. These facts implied that the HA additive enhances the strength of the nanoceramics. However, in this study, with the increasing of BaTiO3 additive content, the mechanical properties, compres-
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Acknowledgments
80
Grain size(nm)
70
The SEM photographs were courtesy of the Analytical & Testing Center, Sichuan University, PR China. This work was supported by the National Natural Science Foundation of China (Nos. 50672062 and 30870615) and Key Programs for Science and Technology Development of Sichuan Province, China (No. 2008SZ0104).
60 50 40 30 20 10 0 TB01
TB05
TB10
TB15
TB20
References
Titania composite ceramics
Fig. 7. Average grain size of all the titania composite ceramics.
sive strength and elastic modulus of titania composite ceramics decreased. This indicates that, with increasing additive content in nano-titania ceramics, the ability of BaTiO3 to enhance the strength of the ceramics is less than that of the HA. In nano-ceramics, a smaller particle size could increase the specific surface area and wettability, which would benefit the adhesion and proliferation of cells [18]. The particle size of TB01 was smaller than that of TiO2, so the osteoblast proliferation and differentiation on TB01 was faster than that on TiO2. Moreover, BaTiO3 particles, of which TB20 had the largest content of all the titania composite ceramics, might congregate together and sinter themselves into bigger BaTiO3 ceramics particles during the sintering process. It is reported that BaTiO3 itself is a bioactive material [19]. So the OD value (Fig. 5) of TB20, which was higher than that of TiO2, might be the effect of the bioactivity of BaTiO3. In addition, for TB10, the new substance BaO(TiO2)2, the content of which was higher than in the other titania ceramics, might weaken the bioactivity. So the OD value of TB10 was smallest among the nano-titania ceramics. In this experiment, we utilized the negatively charged PTB series ceramic surfaces for cell culture [10]. The results of the MTT assay (Fig. 5) provided that the bioactivity of polarized nano-TiO2 ceramics was higher than that of nonpolarized nano-TiO2 ceramics. The bioactivity of the samples was enhanced by negatively polarizing the film surface. This implies that the ferroelectric and piezoelectric effects of TiO2 composite ceramics caused by BaTiO3 play an important role in cell culture. 5. Conclusion The use of BaTiO3 as a grain growth inhibitor could adjust the grain/particle size and the mechanical properties of nano-titania ceramics to make them analogous to that of human bone. The results of this experiment show that the piezoelectric effect caused by the piezoelectric material BaTiO3 in titania composite ceramics promotes the bioactivity of those ceramics. Titania ceramics doped with BaTiO3 are potential bony tissue replacement materials under load-bearing conditions.
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