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1 Journal of the Ceramic Society of Japan 114 [8] (2006) Paper eóçêçñó~é~íáíécçêãáåö ^Äáäáíó ~åç jéåü~åáå~ä mêçééêíáéë çñ lêö~åáå fåçêö~åáå eóäêáçë oéáåñçêåéç Äó `~äåáìã müçëéü~íéë Tomohiro UCHINO, Chikara OHTSUKI, Masanobu KAMITAKAHARA, Masao TANIHARA and Toshiki MIYAZAKI Graduate School of Materials Science, Nara Institute of Science and Technology, , Takayama-cho, Ikoma-shi, Nara Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2 4, Hibikino, Wakamatsu-ku, Kitakyushu-shi, Fukuoka é ÙÛ { û œ ú Å Û ø Û ÉÊ 2 4 The bone-bonding ability, known as bioactivity, of ceramic biomaterials is usually evaluated by the potential for hydroxyapatite HAp formation on their surfaces after exposure to a simulated body fluid SBF proposed by Kokubo et al. We previously reported that an organic inorganic hybrid synthesized from 2-hydroxyethylmethacrylate and 3-methacryloxypropyltrimethoxysilane showed formation of HAp on its surface in SBF, when calcium ions were incorporated into the hybrid. In the present study, the hybrid was combined with a-tricalcium phosphate porous body or calcium phosphates powder CPP consisting of dicalcium phosphate anhydrous and tetracalcium phosphate as the Ca 2 sources, to improve the mechanical strength of the hybrid. These composites formed HAp on their surfaces in SBF. The mechanical strength of the hybrid was improved by the reinforcement with calcium phosphates. When CPP was used, the compressive strength of the composite increased after soaking in SBF for one day. The combination of the hybrid and calcium phosphates offers a novel design for bioactive materials. Received March 30, 2006; Accepted June 15, 2006 Key-words : Organic inorganic hybrid, Composite, Mechanical strength, Bioactivity, Hydroxyapatite, Calcium phosphate 1. Introduction Bioactive ceramics, such as Bioglass, 1 glass-ceramic A W 2 and sintered hydroxyapatite 3 are attractive as bone substitutes because they spontaneously bond to living bone after implantation in bony defects. 4 However, their applications are still limited because of their brittleness. An essential requirement for artificial materials to show bone-bonding ability, i.e., bioactivity, is to form a bone-like hydroxyapatite HAp layer on their surfaces in the body environment. 5,6 The potential to form the bone-like HAp layer is evaluated by in vitro examination using a simulated body fluid SBF proposed by Kokubo et al. 7,8 SBF is an acellular solution that has ion concentrations similar to those of human blood plasma. Previous studies on the formation of bone-like HAp layer in SBF have already revealed that silanol groups Si OH on the materials provide effective sites for heterogeneous nucleation of HAp in the body environment, 9 and the release of calcium ions Ca 2 from the materials enhances HAp formation by increasing the degree of supersaturation with respect to HAp in surrounding fluids. 10 It is expected that if calcium ions are incorporated into an organically modified silicate structure, the hybrid obtained could show both HAp-forming ability on its surface in the body environment and flexibility. Tsuru et al. 11 reported that some polydimethylsiloxane PDMS CaO SiO 2 hybrids prepared by the sol gel method formed HAp in SBF. This indicates that these hybrids can be bioactive. Since this report, many researchers have reported such bioactive hybrids Recently, we reported synthesis of an organic inorganic 692 hybrid starting from 2-hydroxyethylmethacrylate HEMA and 3-methacryloxypropyltrimethoxysilane MPS, in combination with calcium chloride CaCl Such an organic inorganic hybrid has the potential to show not only bioactivity but also flexibility. However, hybrids that contain calcium ions may show a significant decrease in their mechanical strength in the body environment when the calcium ions are added as highly water-soluble calcium salts. 19 Therefore, we focused on calcium phosphates with adequate solubility as a source of calcium ions, because they could also provide reinforcement to the hybrid. In the present study, we fabricated a composite of HEMA MPS hybrid and a-tricalcium phosphate a-tcp porous body. 20,21 Two types of composites with different porosities were prepared in order to reveal the effect of the porosity of the composites on their mechanical properties and HAp-forming ability. One was prepared by impregnating an ethanol solution containing HEMA MPS polymer into the a-tcp porous body to result in the higher porosity, and the other was HEMA MPS monomer solution polymerized by bulk polymerization in the a-tcp porous body to result in the lower porosity. We also prepared a composite of HEMA MPS hybrid and self-setting calcium phosphates powder CPP, 22,23 which we selected because we expected the self-setting reaction to occur in SBF and increase the mechanical strength of the composite. The HAp-forming ability in SBF and the mechanical strength of the composites before and after exposure to SBF were examined.

2 Tomohiro UCHINO et al. Journal of the Ceramic Society of Japan 114 [ 8 ] Experimental 2.1 Preparation of HEMA MPS hybrid Reagent-grade 2-hydroxyethylmethacrylate HEMA: CH 2 C CH 3 COO CH 2 2 OH, Wako Pure Chemical Industries, Ltd. and 3-methacryloxypropyltrimethoxysilane MPS: CH 2 C CH 3 COO CH 2 3 Si OCH 3 3, Chisso Corporation, Japan were used as starting reagents without further purification. The HEMA and MPS in the molar ratio of 9 : 1 were dissolved in ethanol, and 100 cm 3 of the solution at a total concentration of 1 mol dm 3 was prepared. The solution was heated at 75?C for3hwith0.001molbenzoylperoxide BPO as an initiator for the polymerization of HEMA and MPS. 16 The resultant solutions were cast in polypropylene containers and dried under ambient condition until the weight loss became less than 2 in 24 h. 2.2 Preparation of composites of a-tcp porous body and HEMA MPS hybrid a-tcp porous body was prepared according to the previous research. 20 The slurry consisting of b-tcp Nacalai Tesque Inc., potato starch Nacalai Tesque Inc. and water was prepared at a mass ratio of b-tcp : potato starch : water 4 : 4 : 7. This was impregnated in a polyurethane sponge. After drying at 60?C for 1 h, it was calcined at 1000?C for 3 h, followed by sintering at 1400?C for 12 h. The porous body obtained was designated TCP PB. Two types of composites were prepared from the a-tcp porous body and a hybrid consisting of HEMA and MPS. One was prepared by impregnation of an ethanol solution containing HEMA MPS polymer into pores in the a-tcp porous body. Monomers of HEMA and MPS at a molar ratio of 9 : 1 were dissolved in ethanol to make 100 cm 3 of solution at a total concentration of 1 mol dm 3. The solution was heated at 75?C for 3 h with BPO. The a-tcp porous body was impregnated in the HEMA MPS polymer solution, followed by drying under ambient condition. The composite obtained was designated TCP SP. The other composite was prepared by bulk polymerization of HEMA and MPS in the a-tcp porous body. The a-tcp porous body was immersed in the mixture of the HEMA and MPS monomers at a molar ratio of 9 : 1 with BPO under vacuum. Then the a-tcp porous body with monomer solution was heated at 60?C for2h,followed by drying under ambient condition. This was designated TCP BP. 2.3 Preparation of composite of CPP and HEMA MPS hybrid A composite consisting of HEMA MPS hybrids and CPP was prepared as follows. Dicalcium phosphate anhydrous DCPA: CaHPO 4 was prepared by heating commercial dicalcium phosphate dihydrate DCPD: CaHPO 4 2H 2 O, Nacalai Tesque Inc. at 250?C for 10 h. Tetracalcium phosphate TTCP: Ca 4 P 2 O 9 was synthesized from commercial b- TCP and calcium carbonate CaCO 3 : Nacalai Tesque Inc.. b-tcp and CaCO 3 were mixed at a molar ratio of 1 : 1. The mixture was calcined at 1500?C for 10 h twice to obtain TTCP. CPP was prepared by mixing DCPA and TTCP at a molar ratio of 1 : 1. The average particle sizes of DCPA and TTCP were 1.9 mm and1.4mm, respectively. Monomers of HEMA and MPS at a molar ratio of 9 : 1 were dissolved in ethanol to make 50 cm 3 of solution at a total concentration of 2 mol dm 3. The solution was heated at 75?C for3hwithbpo.the obtained HEMA MPS polymer solution was mixed with the CPP powder at a mass ratio of the HEMA MPS polymer : powder 3 : 7, followed by drying under ambient condition. This sample was designated CPP SP. 2.4 Calculation of porosity The density of the HEMA MPS hybrid was calculated from its weight and volume, assuming that the HEMA MPS hybrid did not contain pores. The porosity of TCP PB was calculated from its weight and volume. The density of a-tcp is 2.86 g cm The porosities of TCP SP and TCP BP were calculated from their weight and volume by using the density of the HEMA MPS hybrid and the porosity of TCP PB. The porosity of CPP SP was calculated from its weight and volume by assuming that the mass ratio of HEMA MPS hybrid : powder is 3 : 7. The densities of DCPA and TTCP are 2.89 g cm 325 and 3.05 g cm 3, 26 respectively. 2.5 Examination of bioactivity To estimate the bioactivity of the samples, the HAp-forming ability of the composites was evaluated by soaking in SBF Na 142.0, K 5.0, Mg 2 1.5, Ca 2 2.5, Cl 147.8, HCO3 4.2, HPO and SO mmol dm 3 at ph 7.25 at 36.5?C. 7,8 A rectangular specimen, mm 3 in size, was cut from the composites and soaked in 35 cm 3 of SBF. After it was kept at 36.5?C for seven days, the surfaces of the samples before and after soaking in SBF were characterized by thin-film X-ray diffraction TF XRD, RINT2200VPC LR, Rigaku Co., and scanning electron microscopy SEM, S 4800, Hitachi, Ltd Examination of mechanical properties The mechanical properties of the HEMA MPS hybrid on its own were examined using a tensile test with a universal testing machine Model 5566, Instron, Co., USA. Dumbbelltype specimens in conformity to the JIS K 7113 Type No. 1, small size were stamped out using a cutting die. The gauge length was 12.5 mm and the crosshead speed was 5 mmmin. The mechanical properties of the TCP PB, TCP SP, TCP BP and CPP SP specimens were evaluated by a compressive test. The maximum stress was defined as the compressive strength when a mm 3 rectangular sample was loaded using a constant crosshead speed of 20 mmmin. 3. Results Figure 1 shows the SEM images of the cross sections of TCP PB, TCP SP, TCP BP and CPP SP. TCP PB had continuous pores ranging from 10 to 50 mm, which were filled with the HEMA MPS hybrid in samples TCP SP and TCP BP. The porosities of samples TCP PB, TCP SP, TCP BP and CPP SP were 80, 71, 30 and 28, respectively. In sample CPP SP, homogeneous distribution of calcium phosphate powders was observed. Figure 2 shows TF XRD patterns of the HEMA MPS hybrid, TCP PB, TCP SP, TCP BP and CPP SP before and after soaking in SBF for seven days. The HEMA MPS hybrid was identified as the amorphous phase. All the peaks in samples TCP PB, TCP SP and TCP BP were assigned to an a-tcp phase JCPDS before soaking in SBF, while peaks in sample CPP SP were assigned to DCPA and TTCP phases JCPDS , , respectively. After soaking in SBF for seven days, no change was observed in the HEMA MPS hybrid, TCP PB or TCP SP. Broad additional peaks at about 26?and 32?were detected in samples TCP BP and CPP SP and assigned to the HAp phase JCPDS The peak at 2u 26?was assigned to the 002 diffraction line of HAp, whereas the one at about 2u 32?was an envelope of the 211, 112, and 300 diffraction lines of HAp. This indicates that bone-like HAp was formed on the surface of those samples after soaking in SBF for less than seven days, because those peaks corresponded to conventional bone-like HAp formation. Figure 3 shows SEM images of

3 694 Hydroxyapatite-Forming Ability and Mechanical Properties of Organic Inorganic Hybrids Reinforced by Calcium Phosphates the surfaces of the samples before and after soaking in SBF for seven days. TCP SP, TCP BP and CPP SP formed precipitates on their surfaces after soaking in SBF within seven days, while HEMA MPS hybrid and TCP PB did not form them. These precipitates were assigned to HAp deposited on the specimens after exposure to SBF. Although the precipitates were observed on TCP SP, XRD could not detect HAp. This indicated that the amount of HAp formed was too small to be detected. Figure 4 shows representative stress-strain curves of TCP PB, TCP SP, TCP BP and CPP SP through compressive Fig. 1. SEM images of cross sections of TCP PB, TCP SP, TCP BP and CPP SP. measurement before and after soaking in SBF for one day, as well as that of HEMA MPS hybrid through tensile measurement. Table 1 summarizes the strength and Young's modulus of those samples. The samples TCP SP and TCP BP showed higher mechanical strength than TCP PB. TCP BP's lower porosity gave it higher mechanical strength and Young's modulus than TCP SP before soaking in SBF. Although sample CPP SP showed lower porosity than sample TCP SP, it had lower mechanical strength and Young's modulus. The porous body of the ceramic was effective in reinforcing the hybrid. After soaking in SBF, the tensile strength and Young's modulus of HEMA MPS hybrid decreased, and the compressive strength and Young's modulus of TCP PB, TCP SP and TCP BP also decreased after soaking in SBF for one day. In contrast, the compressive strength and Young's modulus of CPP SP increased after soaking in SBF for one day. 4. Discussion It is clear that the potential for HAp formation of HEMA MPS hybrid free from calcium ions is quite low in SBF. Moreover, a-tcp porous body on its own did not show formation of HAp in SBF within seven days. In contrast, the ability for HAp formation in SBF was induced by combining calcium phosphates, such as a-tcp porous body and CPP powders, with the organic inorganic hybrids synthesized from HEMA and MPS through sol gel processing. This suggests that the release of calcium and phosphate ions from the a- TCP porous body did not allow heterogenous nucleation on its surface, while the existence of silanol Si OH groups formed on the surface of the HEMA MPS hybrid by hydrolysis of MPS may also not be sufficient to allow heterogenous nucleation of HAp on its surface. The coexistence of released calcium and phosphate ions and silanol groups is important for inducing the heterogeneous nucleation of HAp in a body environment. Such cooperative effects from both materials are quite interesting for the design of novel bioactive compo- Fig. 2. TF XRD patterns of the HEMA MPS hybrid, TCP PB, TCP SP, TCP BP and CPP SP before and after soaking in SBF for 7 d. 0d: Before soaking.

4 Tomohiro UCHINO et al. Journal of the Ceramic Society of Japan 114 [ 8 ] Fig. 3. SEM images of the surfaces of HEMA MPS hybrid, TCP PB, TCP SP, TCP BP and CPP SP before and after soaking in SBF for 7 d. 0d: Before soaking. Fig. 4. Representative stress-strain curves of HEMA MPS hybrid, TCP PB, TCP SP, TCP BP and CPP SP before and after soaking in SBF for 1. 0d: Before soaking Tensile test, the others: compressive test. sites that show bone-bonding properties. As the surface area of TCP SP is larger than TCP BP due to its higher porosity, TCP SP would be expected to show higher release rate of calcium ions than that of TCP BP. However, the present results showed that the HAp-forming ability of TCP SP was lower than that of TCP BP. For the HAp formation, silanol groups which induce the heterogeneous nucleation of HAp and release of calcium ions which accelerate the nucleation are important. These indicate that silanol groups in the HEMA MPS hybrid govern the HAp formationinthepresentsystem.thatis,thehema MPS hybrid prepared by bulk polymerization had higher potential to induce heterogeneous nucleation of HAp than that prepared by solution polymerization in ethanol. Polymerization condition of HEMA MPS in TCP BP is quite different from that in TCP SP. This might cause differences in molecular structure, such as concentration and arrangement of silanol groups, as well as the porosity in the dried hybrid. Cho et al. showed that the HAp-forming ability of silica gels rich in silanol groups was dependent on their structures. 27 Therefore, the polymerized HEMA MPS through bulk polymerization might produce a preferable structure to induce heterogeneous nucleation of HAp in SBF. Further investigations are needed to examine the structure in detail. Incorporation of calcium phosphates with HEMA MPS hybrid resulted in reinforcement of mechanical strength and Young's modulus before soaking in SBF. The continuous ceramic framework of the a-tcp porous body was more effective in increasing the mechanical strength and Young's modulus than CPP. The tensile strength of the HEMA MPS hybrid and the compressive strength of TCP PB, TCP SP and TCP BP decreased after soaking in SBF for one day. The decrease in compressive strength of the composites TCP SP and TCP BP is caused by the decrease in mechanical strength

5 696 Hydroxyapatite-Forming Ability and Mechanical Properties of Organic Inorganic Hybrids Reinforced by Calcium Phosphates Table 1. Mechanical Strength and Young's Modulus of the HEMA MPS Hybrid, TCP PB, TCP SP, TCP BP and CPP SP Before and After Soaking in SBF for 1 d. n 5 of both parts of the HEMA MPS hybrid and TCP PB. In the case of TCP BP, the decrease seemed larger than that for TCP SP. The swelling of the HEMA MPS hybrid component in SBF might induce many cracks in the composite because it shows low porosity, and there is little space to relax the stress because of the swelling. In contrast, CPP SP increased its compressive strength after soaking in SBF for one day. It has been reported that the mixture of DCPA and TTCP sets in a physiological aqueous solution with transformation into HAp. 22,23 Transformation of DCPA and TTCP into HAp was confirmed by XRD. This suggests that a continuous HAp framework was formed in the composites in SBF and that this increased the compressive strength. 5. Conclusion HEMA MPS hybrid was reinforced by a-tcp porous body or calcium phosphates powder. The resulting composites formed HAp on their surfaces in SBF within seven days. Cooperative effects of organic inorganic hybrids with calcium phosphates will provide a potential to increase not only mechanical strength but also bioactivity suitable for bone repairing. Acknowledgment This work was partially supported by Nippon Sheet Glass Foundation for Materials Science and Engineering. References 1 Hench, L. L., Splinter, R. J., Allen, W. C. and Greenlee, T. K., J. Biomed. Mater. Res. Symp., Vol. 2, pp Kokubo, T., Shigematsu, M., Nagashima, Y., Tashiro, M., Nakamura, T., Yamamuro, T. and Higashi, S., Bull. Inst. Chem. Res., Kyoto Univ., Vol. 60, pp Jarcho, M., Kay, J. L., Gumaer, R. H. and Drobeck, H. P., J. Bioeng., Vol. 1, pp Hench, L. L., J. Am. Ceram. Soc., Vol. 81, pp Kokubo, T., J. Ceram. Soc. Japan Seramikkusu Ronbunnshi, Vol. 99, pp Kim, H.-M., J. Ceram. Soc. Japan, Vol. 109, pp. S49 S Kokubo, T., Kushitani, H., Sakka, S., Kitsugi, T. and Yamamuro, T., J. Biomed. Mater. Res., Vol. 24, pp Cho, S. B., Nakanishi, K., Kokubo, T., Soga, N., Ohtsuki, C., Nakamura, T., Kitsugi, T. and Yamamuro, T., J. Am. Ceram. Soc., Vol. 78, pp Li, P., Ohtsuki, C., Kokubo, T., Nakanishi, K., Soga, N., Nakamura, T. and Yamamuro, T., J. Am. Ceram. Soc., Vol. 75, pp Ohtsuki, C., Kokubo, T. and Yamamuro, T., J. Non-Cryst. Solids, Vol. 143, pp Tsuru, K., Ohtsuki, C., Osaka, A., Iwamoto, T. and Mackenzie, J. D., J. Mater. Sci.: Mater. Med., Vol. 8, pp Chen, Q., Miyaji, F., Kokubo, T. and Nakamura, T., Biomaterials, Vol. 20, pp Kamitakahara, M., Kawashita, M., Miyata, N., Kokubo, T. and Nakamura, T., J. Sol Gel Sci. Tech., Vol. 21, pp Kamitakahara, M., Kawashita, M., Miyata, N., Kokubo, T. and Nakamura, T., J. Mater. Sci.: Mater. Med., Vol. 13, pp Rhee, S.-H., Choi, J.-Y. and Kim, H.-M., Biomaterials, Vol. 23, pp Ohtsuki, C., Miyazaki, T. and Tanihara, M., Mater. Sci. Eng. C, Vol.22, pp Miyazaki, T., Ohtsuki, C. and Tanihara, M., J. Nanosci. Nanotech., Vol. 3, pp Ohtsuki, C., Kamitakahara, M. and Miyazaki, T., Ann. Chim. Sci. Mat., Vol. 29, pp Kamitakahara, M., Kawashita, M., Miyata, N., Kokubo, T. and Nakamura, T., J. Am. Ceram. Soc., Vol. 87, pp Kitamura, M., Ohtsuki, C., Iwasaki, H., Ogata, S., Tanihara, M. and Miyazaki, T., J. Mater. Sci.: Mater Med., Vol. 15, pp Kitamura, M., Ohtsuki, C., Ogata, S., Kamitakahara, M. and Tanihara, M., Materials Transactions, Vol.45, pp Brown, P. and Chow, L.C., Cement Research Progress, Am. Ceram. Soc., Westfield 1986 pp Ishikawa, K., Takagi, S. Chow, L. C. and Suzuki, K., J. Biomed. Mater. Res., Vol. 46, pp Mathew, M., Schroeder, L. W., Dickens, B. and Brown, W. E., Acta Cryst., Vol. B33, pp Smith, J. P., Lehr, J. R. and Brown, W. E., Am. Miner., Vol. 40, pp Dickens, B., Brown, W. E., Kriger, G. J. and Stewart, J. M., Acta Cryst., Vol. B29, pp Cho, S. B., Nakanishi, K., Kokubo, T., Soga, N., Ohtsuki, C. and Nakamura, T., J. Biomed. Mater. Res., Vol. 33, pp