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Bonelike apatite formation on carbon microspheres
Materials Letters 61 (2007) 2502 – 2505 www.elsevier.com/locate/matlet
Bonelike apatite formation on carbon microspheres Chengtie Wu a,b,⁎, Jiang Chang a a
b
Biomaterials and Tissue Engineering Research Center, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, People's Republic of China Biomaterials and Tissue Engineering Research Unite, The School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, Sydney, NSW 2006, Australia Received 20 July 2006; accepted 22 September 2006 Available online 12 October 2006
Abstract Carbon microspheres with a diameter of 2 μm were prepared by hydrothermal process. The apatite-formation ability of the carbon microspheres was evaluated by soaking them in a simulated body fluid (SBF) for 5 and 10 d and apatite-formation mechanism was also analyzed. The result showed that bonelike apatite was formed on the surface of carbon microspheres. Our study indicates that the carbon microspheres synthesized by this method possess apatite-formation ability and may be used as a bioactive injectable filler for bone tissue regeneration. © 2006 Elsevier B.V. All rights reserved. Keywords: Carbon microspheres; SBF; Apatite-formation ability
1. Introduction Recently, significant attention has been drawn to the use of microparticles as injectable scaffolds for tissue regeneration [1]. The main advantage of this approach, compared with the traditional block scaffolds, is that small particles can be combined with a vehicle and be administered by injection, thus giving the possibility of filling defects of different shapes and sizes through minimally invasive surgery. Upon implantation, the microspheres–vehicle system is expected to easily conform to the irregular implant site, whereas the interstices between the particles may provide a space for both tissue and vascular ingrowth, as required for effect healing [2]. As for a injectable filler for bone tissue regeneration, not only the regular size and morphology of the filler are needed, which can improve the injectability, but also the bioactivity is important. Many researchers consider that a bonelike carbonate-containing apatite layer, which precipitates on the surface of a bioactive material in human body, plays an essential role in forming the chemical ⁎ Corresponding author. Biomaterials and Tissue Engineering Research Unite, The school of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, Sydney, NSW 2006, Australia. Tel.: +61 2 93515527; fax: +61 2 93517060. E-mail address: [email protected] (C. Wu). 0167-577X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2006.09.045
bond of the bioactive material to the living bone. This carbonatecontaining apatite layer can be reproduced in vitro in SBF [3,4]. Previously, Sun and Li synthesized a novel carbon microsphere and found that abundant hydroxyl groups (OH−) remained on the surface of the carbon microspheres [5]. OH− groups are important for the formation of apatite when soaking materials in SBF, which provides favorable sites for calcium phosphate nucleation [6]. Therefore, it is reasonable to assume that the carbon microspheres may possess apatite-formation ability in SBF because of the existing OH− groups on the surface. Therefore, in this study, we prepared carbon microspheres using hydrothermal method and evaluated the apatite-formation ability of carbon microspheres by soaking them in SBF. 2. Experimental The carbon microspheres were prepared by the hydrothermal process described in the literature [5]. Briefly, 70 ml glucose Table 1 Ion concentrations of SBF and human blood plasma (mM)
SBF Blood plasma
Na+
K+
Mg2+
Ca2+
Cl−
HCO−3
HPO2− 4
142.0 142.0
5.0 5.0
1.5 1.5
2.5 1.5
148.8 103.0
4.2 27.0
1.0 1.0
C. Wu, J. Chang / Materials Letters 61 (2007) 2502–2505
Fig. 1. XRD patterns of carbon microspheres before and after soaking in SBF for 5 d. (a) Before soaking; (b) after soaking.
water solution with a concentration of 1 M was transferred to a Teflon-lined autoclave, sealed and maintained at 180 °C for 20 h. The dark product was collected by centrifugation at 8000 rpm for 10 min, then washed with distilled water and ethanol three times,
2503
respectively, and dried at 60 °C in vacuum for 12 h. The dried powders were analyzed by X-ray diffraction (XRD, Geigerflex, Rigaku, Japan) with a monochromated CuKα radiation, and the microstructure of the powders was observed by scanning electron microscopy (SEM, Jsm-6700F, JEOL, Tokyo, Japan). For the evaluation of apatite formation on the carbon microspheres, the obtained carbon microspheres were soaked in SBF solution at 37 °C for 5 and 10 d at a solid/liquid ratio of 1.5 mg/ml. The SBF solution was prepared according to the method described by Kokubo [7]. Briefly, reagent-grade CaCl2, K2HPO4·3H2O, NaCl, KCl, MgCl2·6H2O, NaHCO3, and Na2SO4 were dissolved in distilled water and pH was adjusted with Tris and HCl. The ion concentrations were similar to those in human blood plasma as shown in Table 1. After soaking, the products were dried at 100 °C for 1 d and characterized by XRD, and SEM. In addition, the carbon microspheres after soaking in SBF was calcined in air at 500 °C for 2 h to burn carbon and the obtained apatite powder products were characterized by Fourier transitioned-infrared spectroscopy (FTIR; Nicolet Co., USA). 3. Results and discussion The XRD patterns of carbon microspheres before and after soaking in the SBF solution for 5 d are shown in Fig. 1. It is obvious that the
Fig. 2. SEM microstructures of carbon microspheres before and after soaking in SBF for 5 and 10 d. (a) and (b) before soaking; (c) and (d) after soaking for 5 d; (e) and (f) after soaking for 10 d.
2504
C. Wu, J. Chang / Materials Letters 61 (2007) 2502–2505
Fig. 3. FTIR spectrum of the SBF-soaked carbon microspheres after being calcined at 500 °C.
formed carbon microspheres prepared at this condition are amorphous (Fig. 1a). On the contrary, after soaking for 5 d, the crystalline peaks of apatite at 31.7° and 25.8° 2θ corresponding to the 211 and 002 reflections of apatite (JCPD 250166) are evident in the XRD patterns (Fig. 1b), which suggests that carbon microspheres induce the apatite formation when soaked in SBF. Fig. 2 shows the SEM micrographs of carbon microspheres before and after soaking in SBF solution for 5 and 10 d. The prepared carbon microspheres present regular morphology and the size is about 2 μm (Fig. 2a). Previously, carbon microspheres with different size were prepared by Sun and Li through the variation of
reaction temperature, time, and concentration of starting material [5]. In this study, we increased the reaction time to 20 h and prepared carbon microspheres with a larger size as compared with those of Sun and Li. As for a bioactive injectable filler for bone tissue regeneration, the microspheres with larger size can provide larger interstices between two cumulate microspheres, which benefits the tissue ingrowth and blood and nutrients supply after implanted. In addition, the surface of the prepared carbon microspheres is smooth (Fig. 2b), however, after 5 d of soaking in SBF, numerous apatite clusters with cotton morphology are formed on the surface of carbon microspheres (Fig. 2c). The higher magnification SEM micrograph shows that numerous uniform and lathlike apatite crystallites are aggregated on the surface of carbon microspheres and the size of the crystallites is about 200– 300 nm in length (Fig. 2d), which shows a typical apatite morphology [8,9]. After 10 d of soaking, more bonelike apatite forms on the surface of microspheres as compared with that after soaking for 5 d. Furthermore, the apatite almost packs the surface of carbon microspheres and forms a layer (Fig. 2e and f). FTIR spectrum of carbon microspheres soaked in SBF for 5 d is shown in Fig. 3. The broad bands from 900 to 1300 cm− 1 are mainly attributed to the P–O stretching vibration of the PO3− 4 unit in the apatite and the P–O bending vibration in PO3− occurs around 598 cm− 1 [10]. Furthermore, the C–O 4 −1 stretching of CO2− is observed, which 3 groups at 1490 and 872 cm indicates that the formed apatite on the surface of carbon microspheres is bonelike carbonate-containing apatite. Previous studies have shown that apatite formation on the materials in SBF is much sensitive to the
Fig. 4. Schematic mechanism for the apatite formation on the surface of carbon microspheres in SBF.
C. Wu, J. Chang / Materials Letters 61 (2007) 2502–2505
OH− group on the surface of the materials according to Hench and Kokubo [4,6]. In addition, according to the literature [5], the surface of the carbon spheres possesses a distribution of OH− groups. Therefore, the potential mechanism of apatite formation on the surface is presented in Fig. 4. The surface of carbon microspheres is negative because of OH− groups, which provide favorable sites for calcium 2− phosphate nucleation. The Ca2+, PO3− 4 and CO3 ions will be assembled on the surface of carbon microspheres to form amorphous carbonate apatite. With the increase of soaking time, the amorphous carbonate apatite will grow into crystalline carbonate apatite. It is considered that a bonelike carbonate-containing apatite layer, which precipitates on the surface of a bioactive material in the human body, plays an essential role in forming the chemical bond of the bioactive material to the living bone. In this study, we prepared bioactive carbon microspheres and found that they possessed bonelike apatite-formation ability. Therefore, the bioactive carbon microspheres can be combined with a vehicle and be administered by injection, thus giving the possibility of filling defects of different shapes and sizes through minimal invasive surgery. Further study will be conducted by in vitro cell culture and in vivo implantation using the carbon microspheres.
4. Conclusions Carbon microspheres were prepared by hydrothermal reaction 20 h at 180 °C using glucose solution, and the diameter of the microspheres was about 2 μm. The in vitro study showed that carbon microspheres could induce bonelike apatite formation on their surface after 5 and 10 d of soaking in SBF. The potential mechanism of apatite formation is directly relative to the existing
2505
OH− groups on the surface of microspheres. Our study suggests that the carbon microspheres synthesized by this method possess apatite-formation ability and may be a potentially bioactive injectable filler for bone tissue regeneration. Acknowledgements This work is supported by the National Basic Science Research Program of China (973 Program) (Grant No: 2005CB522700) and the Science and Technology Commission of Shanghai Municipality (Grant No: 05JD14005). References [1] T.J. Wu, H.H. Huang, C.W. Lan, F.Y. Hsu, Y.J. Wang, Biomaterials 25 (2004) 651. [2] C.C. Barrias, C.C. Ribeiro, M. Lamghari, C.S. Miranda, M.A. Barbosa, J. Biomed. Mater. Res. 72 (2005) 57. [3] E. Ghaith, T. Hayakawa, T. Kasuga, M. Nogami, Mater. Lett. 60 (2006) 194. [4] L.L. Hench, J. Am. Ceram. Soc. 7 (1991) 1487. [5] X. Sun, Y. Li, Angew. Chem., Int. Ed. Engl. 43 (2004) 597. [6] T. Kokubo, H. Kushitani, S. Saka, T. Kitsgi, T. Kitsugi, T. Yamamuro, J. Biomed. Mater. Res. 24 (1990) 721. [7] T. Kokubo, J. Non-Cryst. Solids 120 (1990) 138. [8] K. Zhang, Y. Ma, F.F. Lorraine, J. Biomed. Mater. Res. 61 (2002) 52. [9] A.J. Salinas, M. Vallet-Regi, I. Izquierdo-Barba, J. Sol-Gel Sci. Technol. 21 (2001) 13. [10] B.O. Fowler, Inorg. Chem. 13 (1974) 194.
Bonelike apatite formation on carbon microspheres Chengtie Wu a,b,⁎, Jiang Chang a a
b
Biomaterials and Tissue Engineering Research Center, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, People's Republic of China Biomaterials and Tissue Engineering Research Unite, The School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, Sydney, NSW 2006, Australia Received 20 July 2006; accepted 22 September 2006 Available online 12 October 2006
Abstract Carbon microspheres with a diameter of 2 μm were prepared by hydrothermal process. The apatite-formation ability of the carbon microspheres was evaluated by soaking them in a simulated body fluid (SBF) for 5 and 10 d and apatite-formation mechanism was also analyzed. The result showed that bonelike apatite was formed on the surface of carbon microspheres. Our study indicates that the carbon microspheres synthesized by this method possess apatite-formation ability and may be used as a bioactive injectable filler for bone tissue regeneration. © 2006 Elsevier B.V. All rights reserved. Keywords: Carbon microspheres; SBF; Apatite-formation ability
1. Introduction Recently, significant attention has been drawn to the use of microparticles as injectable scaffolds for tissue regeneration [1]. The main advantage of this approach, compared with the traditional block scaffolds, is that small particles can be combined with a vehicle and be administered by injection, thus giving the possibility of filling defects of different shapes and sizes through minimally invasive surgery. Upon implantation, the microspheres–vehicle system is expected to easily conform to the irregular implant site, whereas the interstices between the particles may provide a space for both tissue and vascular ingrowth, as required for effect healing [2]. As for a injectable filler for bone tissue regeneration, not only the regular size and morphology of the filler are needed, which can improve the injectability, but also the bioactivity is important. Many researchers consider that a bonelike carbonate-containing apatite layer, which precipitates on the surface of a bioactive material in human body, plays an essential role in forming the chemical ⁎ Corresponding author. Biomaterials and Tissue Engineering Research Unite, The school of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, Sydney, NSW 2006, Australia. Tel.: +61 2 93515527; fax: +61 2 93517060. E-mail address: [email protected] (C. Wu). 0167-577X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2006.09.045
bond of the bioactive material to the living bone. This carbonatecontaining apatite layer can be reproduced in vitro in SBF [3,4]. Previously, Sun and Li synthesized a novel carbon microsphere and found that abundant hydroxyl groups (OH−) remained on the surface of the carbon microspheres [5]. OH− groups are important for the formation of apatite when soaking materials in SBF, which provides favorable sites for calcium phosphate nucleation [6]. Therefore, it is reasonable to assume that the carbon microspheres may possess apatite-formation ability in SBF because of the existing OH− groups on the surface. Therefore, in this study, we prepared carbon microspheres using hydrothermal method and evaluated the apatite-formation ability of carbon microspheres by soaking them in SBF. 2. Experimental The carbon microspheres were prepared by the hydrothermal process described in the literature [5]. Briefly, 70 ml glucose Table 1 Ion concentrations of SBF and human blood plasma (mM)
SBF Blood plasma
Na+
K+
Mg2+
Ca2+
Cl−
HCO−3
HPO2− 4
142.0 142.0
5.0 5.0
1.5 1.5
2.5 1.5
148.8 103.0
4.2 27.0
1.0 1.0
C. Wu, J. Chang / Materials Letters 61 (2007) 2502–2505
Fig. 1. XRD patterns of carbon microspheres before and after soaking in SBF for 5 d. (a) Before soaking; (b) after soaking.
water solution with a concentration of 1 M was transferred to a Teflon-lined autoclave, sealed and maintained at 180 °C for 20 h. The dark product was collected by centrifugation at 8000 rpm for 10 min, then washed with distilled water and ethanol three times,
2503
respectively, and dried at 60 °C in vacuum for 12 h. The dried powders were analyzed by X-ray diffraction (XRD, Geigerflex, Rigaku, Japan) with a monochromated CuKα radiation, and the microstructure of the powders was observed by scanning electron microscopy (SEM, Jsm-6700F, JEOL, Tokyo, Japan). For the evaluation of apatite formation on the carbon microspheres, the obtained carbon microspheres were soaked in SBF solution at 37 °C for 5 and 10 d at a solid/liquid ratio of 1.5 mg/ml. The SBF solution was prepared according to the method described by Kokubo [7]. Briefly, reagent-grade CaCl2, K2HPO4·3H2O, NaCl, KCl, MgCl2·6H2O, NaHCO3, and Na2SO4 were dissolved in distilled water and pH was adjusted with Tris and HCl. The ion concentrations were similar to those in human blood plasma as shown in Table 1. After soaking, the products were dried at 100 °C for 1 d and characterized by XRD, and SEM. In addition, the carbon microspheres after soaking in SBF was calcined in air at 500 °C for 2 h to burn carbon and the obtained apatite powder products were characterized by Fourier transitioned-infrared spectroscopy (FTIR; Nicolet Co., USA). 3. Results and discussion The XRD patterns of carbon microspheres before and after soaking in the SBF solution for 5 d are shown in Fig. 1. It is obvious that the
Fig. 2. SEM microstructures of carbon microspheres before and after soaking in SBF for 5 and 10 d. (a) and (b) before soaking; (c) and (d) after soaking for 5 d; (e) and (f) after soaking for 10 d.
2504
C. Wu, J. Chang / Materials Letters 61 (2007) 2502–2505
Fig. 3. FTIR spectrum of the SBF-soaked carbon microspheres after being calcined at 500 °C.
formed carbon microspheres prepared at this condition are amorphous (Fig. 1a). On the contrary, after soaking for 5 d, the crystalline peaks of apatite at 31.7° and 25.8° 2θ corresponding to the 211 and 002 reflections of apatite (JCPD 250166) are evident in the XRD patterns (Fig. 1b), which suggests that carbon microspheres induce the apatite formation when soaked in SBF. Fig. 2 shows the SEM micrographs of carbon microspheres before and after soaking in SBF solution for 5 and 10 d. The prepared carbon microspheres present regular morphology and the size is about 2 μm (Fig. 2a). Previously, carbon microspheres with different size were prepared by Sun and Li through the variation of
reaction temperature, time, and concentration of starting material [5]. In this study, we increased the reaction time to 20 h and prepared carbon microspheres with a larger size as compared with those of Sun and Li. As for a bioactive injectable filler for bone tissue regeneration, the microspheres with larger size can provide larger interstices between two cumulate microspheres, which benefits the tissue ingrowth and blood and nutrients supply after implanted. In addition, the surface of the prepared carbon microspheres is smooth (Fig. 2b), however, after 5 d of soaking in SBF, numerous apatite clusters with cotton morphology are formed on the surface of carbon microspheres (Fig. 2c). The higher magnification SEM micrograph shows that numerous uniform and lathlike apatite crystallites are aggregated on the surface of carbon microspheres and the size of the crystallites is about 200– 300 nm in length (Fig. 2d), which shows a typical apatite morphology [8,9]. After 10 d of soaking, more bonelike apatite forms on the surface of microspheres as compared with that after soaking for 5 d. Furthermore, the apatite almost packs the surface of carbon microspheres and forms a layer (Fig. 2e and f). FTIR spectrum of carbon microspheres soaked in SBF for 5 d is shown in Fig. 3. The broad bands from 900 to 1300 cm− 1 are mainly attributed to the P–O stretching vibration of the PO3− 4 unit in the apatite and the P–O bending vibration in PO3− occurs around 598 cm− 1 [10]. Furthermore, the C–O 4 −1 stretching of CO2− is observed, which 3 groups at 1490 and 872 cm indicates that the formed apatite on the surface of carbon microspheres is bonelike carbonate-containing apatite. Previous studies have shown that apatite formation on the materials in SBF is much sensitive to the
Fig. 4. Schematic mechanism for the apatite formation on the surface of carbon microspheres in SBF.
C. Wu, J. Chang / Materials Letters 61 (2007) 2502–2505
OH− group on the surface of the materials according to Hench and Kokubo [4,6]. In addition, according to the literature [5], the surface of the carbon spheres possesses a distribution of OH− groups. Therefore, the potential mechanism of apatite formation on the surface is presented in Fig. 4. The surface of carbon microspheres is negative because of OH− groups, which provide favorable sites for calcium 2− phosphate nucleation. The Ca2+, PO3− 4 and CO3 ions will be assembled on the surface of carbon microspheres to form amorphous carbonate apatite. With the increase of soaking time, the amorphous carbonate apatite will grow into crystalline carbonate apatite. It is considered that a bonelike carbonate-containing apatite layer, which precipitates on the surface of a bioactive material in the human body, plays an essential role in forming the chemical bond of the bioactive material to the living bone. In this study, we prepared bioactive carbon microspheres and found that they possessed bonelike apatite-formation ability. Therefore, the bioactive carbon microspheres can be combined with a vehicle and be administered by injection, thus giving the possibility of filling defects of different shapes and sizes through minimal invasive surgery. Further study will be conducted by in vitro cell culture and in vivo implantation using the carbon microspheres.
4. Conclusions Carbon microspheres were prepared by hydrothermal reaction 20 h at 180 °C using glucose solution, and the diameter of the microspheres was about 2 μm. The in vitro study showed that carbon microspheres could induce bonelike apatite formation on their surface after 5 and 10 d of soaking in SBF. The potential mechanism of apatite formation is directly relative to the existing
2505
OH− groups on the surface of microspheres. Our study suggests that the carbon microspheres synthesized by this method possess apatite-formation ability and may be a potentially bioactive injectable filler for bone tissue regeneration. Acknowledgements This work is supported by the National Basic Science Research Program of China (973 Program) (Grant No: 2005CB522700) and the Science and Technology Commission of Shanghai Municipality (Grant No: 05JD14005). References [1] T.J. Wu, H.H. Huang, C.W. Lan, F.Y. Hsu, Y.J. Wang, Biomaterials 25 (2004) 651. [2] C.C. Barrias, C.C. Ribeiro, M. Lamghari, C.S. Miranda, M.A. Barbosa, J. Biomed. Mater. Res. 72 (2005) 57. [3] E. Ghaith, T. Hayakawa, T. Kasuga, M. Nogami, Mater. Lett. 60 (2006) 194. [4] L.L. Hench, J. Am. Ceram. Soc. 7 (1991) 1487. [5] X. Sun, Y. Li, Angew. Chem., Int. Ed. Engl. 43 (2004) 597. [6] T. Kokubo, H. Kushitani, S. Saka, T. Kitsgi, T. Kitsugi, T. Yamamuro, J. Biomed. Mater. Res. 24 (1990) 721. [7] T. Kokubo, J. Non-Cryst. Solids 120 (1990) 138. [8] K. Zhang, Y. Ma, F.F. Lorraine, J. Biomed. Mater. Res. 61 (2002) 52. [9] A.J. Salinas, M. Vallet-Regi, I. Izquierdo-Barba, J. Sol-Gel Sci. Technol. 21 (2001) 13. [10] B.O. Fowler, Inorg. Chem. 13 (1974) 194.