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Nanoporous hydroxy-carbonate apatite scaffold made of natural bone
Materials Letters 60 (2006) 2844 – 2847 www.elsevier.com/locate/matlet
Nanoporous hydroxy-carbonate apatite scaffold made of natural bone R. Murugan a,⁎, S. Ramakrishna a , K. Panduranga Rao b a
NUS Nanoscience and Nanotechnology Initiative (NUSNNI), Division of Bioengineering, Faculty of Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore b Spark Biotech, 3720 Sunburst Lane, Naperville, IL 60564, USA Received 7 September 2005; accepted 31 January 2006 Available online 20 February 2006
Abstract A processing method of hydroxy-carbonate apatite (HCAp) from the natural bone and their phase purity, crystal structure, chemical functionality, thermal stability, morphology, and solubility are described in this investigation. The X-ray diffraction analysis suggested that the processed bone corresponds characteristically to carbonate (CO2− 3 ) containing hydroxyapatite. Fourier transform infrared spectroscopic study 3− − confirmed the presence of CO2− 3 in addition to hydroxyl (OH ) and phosphate (PO4 ) functional groups, indicating the formation of HCAp. Environmental scanning electron microscopy was employed to identify the surface morphology of HCAp, which obviously shows a nanoporous structure throughout the matrix. The in vitro solubility study performed under physiological conditions confirms the resorbable nature of HCAp. These findings indicate the feasibility of processing nanoporous HCAp scaffolds from the natural bone by a straightforward method. © 2006 Elsevier B.V. All rights reserved. Keywords: Hydroxy-carbonate apatite; Calcination; Bone scaffold; Nanoporous structure
1. Introduction Clinical treatment of bone defects arising from traumatic or non-traumatic events often requires artificial bone grafts. A variety of biomaterials so far have been investigated as bone graft substitutes. Among them, hydroxyapatite (HA) is frequently used in orthopedic, dental and maxillofacial applications owing to its biocompatibility, osteoconductivity and bioactivity, meaning that it supports bone growth and osteointegration [1–3]. Multiple techniques have been employed for the preparation of HA either from natural or from synthetic sources, which chiefly include coral exoskeleton, animal bone and chemical synthesis [4–9]. It is known that most of the synthetic HA are stoichiometric with a chemical composition of Ca10(PO4)6(OH)2. By contrast, human bones do not have a pure or a stoichiometric HA, which has other ions mainly of CO32− and trace of Na+, Mg2+, Fe2+, Cl−, F− [10]. Although
⁎ Corresponding author. Tel.: +65 6874 6593; fax: +65 6874 3346. E-mail address: [email protected] (R. Murugan). 0167-577X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2006.01.104
presence of these ions is significantly low, they play a vital role in the biochemical reactions associated with bone metabolism. As a result, CO32− containing HA has gained much attention, not only due to its ionic composition but also due to its functional properties favorable for bone growth. Further, it can be easily resorbed by the living cells compared to stoichiometric HA, which might lead to faster bone regeneration. There are several reports that describe the substitution of CO32− into apatite phase [11–13]. It can be substituted for either OH− (A-type) or PO43− (B-type) or it can exist on the crystal surface (labile). Sometimes, both A- and B-type substitutions can also occur [14]. The problem of substitutional site of CO32− has not yet been solved by a direct measurement and work is under process in this direction as well. Further, such ionic substitution can affect the crystal structure, crystallinity, surface charge, solubility and other vital properties, leading to major changes in the biological performance upon implantation. As an alternative, HCAp can be processed from the natural bone without any extraneous ionic substitution but leaving only the natural trace elements, which is reported in this investigation in a detailed fashion.
R. Murugan et al. / Materials Letters 60 (2006) 2844–2847
2. Experimental 2.1. Processing of HCAp The HCAp was derived from chemically and thermally processed bovine bone. Briefly, the cortical portion of bovine bone samples were sliced into required size under running water. The macroscopic impurities of soft tissues and bone marrow associated with the bone were removed by cleaning with a 2% NaCl solution followed by degreasing in acetone– ether mixture (3 : 2) for 24 h. The degreased bone was then treated with 4% NaOH aqueous solution under hydrothermal condition. The chemically treated bone samples were calcined overnight at 500 °C in a muffle furnace under atmospheric pressure and ambient humidity. The resultant product was washed with deionized water and dried in a vacuum oven. 2.2. Characterization The phase purity and crystallographic structure of HCAp was identified with a powder X-ray diffractometer (XRD) (Seifert XRD 3000, Germany). The measurement was carried out with a Cu Kα1 radiation at the wavelength of 1.5406 Å. The chemical functionality was determined by spectroscopic method using a Fourier transform infrared (FTIR) spectrophotometer (ThermoNicolet Avatar 360, USA) over the range between 4000 and 400 cm− 1 at 2 cm− 1 resolution averaging 100 scans. The CO32− content was qualitatively determined from the infrared spectrum of HCAp by comparing the extinction coefficient (E) of the 1450 cm− 1 (carbonate) and 569 cm− 1 (phosphate) peaks using the formula: % CO32− =13.5(E1450/E569)− 0.2, where (E1450/ E569)=Ei =log(T2,i/T1,i). Here, i is 1450 or 569 and T2,i and T1,i are the transmission intensities at the peak maximum and local baseline, respectively. The CO32− content was also quantitatively determined as carbon using a CHN analyzer (Medac, UK). Thermal stability was studied using a thermogravimetric analyzer (TGA) (PerkinElmer, USA). The thermogram was recorded from 50 to about 800 °C at 10 °C/min heating rate under nitrogen atmosphere in order to avoid oxidation and other unwanted side reactions with atmospheric air. The characteristic surface morphology was studied by an environmental scanning electron microscopy (ESEM) (Quanta 200F, FEI) with an accelerating voltage of 20 kV. The in vitro solubility test of HCAp was performed in phosphate buffer with pH 7.2. The following protocol was used in the experiment: 50 mg of powder sample was added to 50 ml of buffer and kept at 37 °C in a thermostatic water bath. The change in pH of the buffer was quantitatively measured at pre-determined time intervals. The dissolution of HCAp was also determined on a palletized sample, prepared by a hot-pressing method, in the same buffer used for the solubility study at room temperature. The weight loss of the pellet was measured at pre-determined time intervals and plotted in a graph against immersion time.
human calcified tissue concerning crystallographical, chemical, thermal, porous morphological and resorbable properties, and, at the same time, to use as a scaffold for bone tissue engineering. In this regard, cortical portion of bovine bone was thoroughly analyzed at various calcination temperatures, ranging from 200 to 900 °C, and the temperature at 500 °C was found to be suitable for the removal of organic impurities associated with the native bone. Therefore, this temperature was chosen for further studies intended. The XRD technique was employed to evaluate the phase purity and the crystallographic structural properties of HCAp (Fig. 1). The result indicates all the characteristic Bragg peaks pertaining to HCAp without any structural deformation or extraneous phase substitution. The Bragg peaks appearing approximately at 26°, 30–34°, 40° and 46° (2θ) corresponds to the characteristic peaks of HA with reference to ICDD file #9-432 [15]. The intense peak around 12° (2θ) corresponds to the background peak of silicon that was used as an adhesive for the powder grains. The crystallographical geometry of HCAp processed in this study has a resemblance to that of human bone apatite [16] and shows a poor crystallinity compared to pure HA [17,18] owing to the CO2− 3 substitution into apatite, suggesting the formation of HCAp phase. The chemical functionality of HCAp was determined from its FTIR spectrum (Fig. 2). It can be seen from the result that all the absorptional peaks corresponding to apatite phase are detectable. Briefly, a band at 3450 cm− 1 is due to the presence of surface hydroxyl groups. The 1043 cm− 1 band can be assigned to a −1 stretching vibration of PO3− are 4 ions and bands at 590–610 cm 3− due to deformation vibration of PO4 ions. In addition, it can be easily seen that the bands pertain to the CO2− 3 functional group at 1500 and 870 cm− 1, indicating the substitution of CO2− 3 ions into the apatite and thus confirms the formation of HCAp. The CO2− 3 peaks at 1550 and 1480 cm− 1 are assigned to the A- and B-types and at 870 cm− 1 it is associated with the labile surface CO2− 3 . The low intense peaks noticed at about 2900 cm− 1 may be attributed to H–C–O functional group; thereby it is obvious that the processed HCAp has a close chemical structure with that of biominerals of human calcified tissue. The amount of CO2− 3 content was estimated by a spectroscopic method using an equation given in the Experimental and it was found to be 2 wt.%. As human calcified tissue also contains CO2− 3 to about 3– 6 wt.% depending upon age factor, its presence in the HCAp is highly beneficial. Further, it is reported that the lower concentration of CO2− 3 ions substantially improves the mechanical strength [19]. The CO2− 3 content was also determined by CHN analyzer and was found to be 1.8 wt.%, which corroborates well with the results of spectroscopic
3. Results and discussion A major intention of this investigation is to process a bone graft substitute from bovine bone with quite similar biominerals of the
2845
Fig. 1. X-ray powder diffraction pattern of HCAp.
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R. Murugan et al. / Materials Letters 60 (2006) 2844–2847
Fig. 2. FTIR spectrum of HCAp, arrow denotes substitution of carbonate ions (A) A-type, (B) B-type.
method. Accordingly, qualitative and quantitative analyses proved the presence of CO2− 3 into the apatite phase and noticed that the total amount of the CO2− 3 content falls near to the preferred range. Fig. 3 shows a representative TGA trace of HCAp in terms of weight loss as a function of temperature. There is no significant weight loss pertaining to endothermic or exothermic reactions observed from the thermogram, indicating its thermal stability upon calcination. The initial weight loss from 50 to 200 °C could be responsible for the evaporation of surface adsorbed water molecules and the weight loss that occurred between 580 and 620 °C could be due to the 2− decomposition of trace CO2− 3 . The endothermic dissociation of CO3 is reported to occur at the temperatures between 400 and 600 °C in air and between 500 and 890 °C in nitrogen atmosphere [20], which concur well with our results. Therefore, TGA analysis also confirms the formation of HCAp and supports further FTIR analysis. The surface morphological structure of HCAp is shown in Fig. 4. The result indicates that the HCAp has a porous architecture throughout the matrix. As it can be seen from the gross morphology, almost all the pores are in the order of nanometer size range. The average pore size was estimated as 12 nm. Here, the average pore size denotes the number of pores within a single micrograph of processed bone. These pores were created due to the removal of organic constituents associated with the native bone; thereby its native structure was disturbed, but, however, quite analogous to the morphological
Fig. 3. TGA trace of HCAp, which shows the weight loss of carbonate due to its decomposition.
Fig. 4. ESEM image of HCAp, which shows nanoporous architecture throughout the matrix.
structure of human trabecula. It has been reported that nanoporous structure of the biomaterials tremendously enhance the cells adhesion, proliferation and differentiation required for tissue functions [21–23]. Therefore, it can be expected that HCAp would have the capability to support the bone tissue in-growth upon implantation. However, the mechanical integrity of the processed material might be lost due to the removal of organic matters; thereby this kind of HCAp can only be used for low-weight bearing bone grafting and coating on bioimplants. Further, the porous architecture with nanopores could enhance the efficacy of HCAp if we use them for bone tissue engineering applications. It is also worth mentioning that the cells live in a complex mixture of pores, ridges, and fibers of extracellular matrix in a nano-featured environment; thereby the processed nanoporous HCAp may be highly beneficial for bone tissue engineering as a scaffold. The dissolution rate was determined by using two different approaches: (i) pH method and (ii) weight loss method. First method is a direct method, in which pH was determined using an electrode. In second method, the weight loss was determined by calculating weight of the pellet before and after immersion into the physiological medium. This is a common method in which the surface observed water was carefully removed and dried to a weight. All the experiments were carried out for one dissolution run and the results were compared with a pure HA. The in vitro solubility of HCAp performed in phosphate buffer (a common buffering agent used in cell/tissue cultures) under
Fig. 5. In vitro solubility of HCAp under physiological conditions.
R. Murugan et al. / Materials Letters 60 (2006) 2844–2847
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scaffold from the natural bone. The overall results indicated that the HCAp has characteristic resemblance with the biominerals of human calcified tissues. The retention of nanoporous architecture, chemical constituents, and the resorbable nature of HCAp would be beneficial if we use them for bone tissue engineering. Acknowledgements The financial support of the National University of Singapore and the Singapore Millennium Foundation are gratefully acknowledged. Fig. 6. Dissociation weight loss of HCAp in phosphate buffer.
physiological conditions is depicted in Fig. 5, which obviously shows the pH change on soaking of HCAp. The solubility test was also conducted for a pure HA, without carbonate substitution, and included in the same figure for comparison. The pH of the buffer medium without sample was found to be stable throughout the experimental period, which act as a control, but the addition of apatite has more or less alkaline effect depending on its solubility. The pH of the medium containing HA was found to have an almost unvarying trend as it did not undergo a considerable resorption, indicating its stability during the period of study. By contrast, HCAp shows a decreasing trend in the pH as it has a significant amount of CO2− 3 ions, indicating its resorbable nature. Moreover, a poorly crystalline phase of the HCAp is also responsible for its higher solubility than pure HA. The initial rapid increase in the pH may be attributed to the thermodynamical changes of apatite [24]. These results are consistent with previous findings [25,26] in which they reported that the solubility mainly depends on the preparation methodology, ionic substitution, in particular, carbonate, and crystallinity of the apatite. As solubility is highly sensitive to structural and chemical compositions of the apatite, the functional composition and crystallinity should also be considered as essential key factors. Fig. 6 displays a correlation between the dissociation weight and the sample immersion time. The graph shows a higher rate of weight loss for HCAp, indicating its higher soluble nature. By contrast, the rate of weight loss of HA is relatively low as compared to HCAp, indicating its stability. As there was no impurity present in the pure HA, the nature of its stability is obvious during the period of study. Thus, variation observed in their solubility could be attributed to the presence of CO2− 3 ions, which further confirms the resorbable nature of HCAp. The resorbable nature of HCAp would be beneficial for the new bone generation upon implantation.
4. Conclusions The present investigation demonstrated an easy, cost effective approach to a reproducible processing of HCAp
References [1] C.J. Damien, J.R. Parsons, J. Appl. Biomater. 2 (1991) 187. [2] H. Aoki, Science and Medical Applications of Hydroxyapatite, Ishiyaku EuroAmerica Press, St Louis, 1994. [3] R. Murugan, S. Ramakrishna, in: H.S. Nalwa (Ed.), Encyclopedia of Nanoscience and Nanotechnology, American Scientific Publishers, California, 2004, p. 595. [4] D.M. Roy, S.K. Linnehan, Nature 247 (1974) 220. [5] R. Murugan, K.P. Rao, T.S.S. Kumar, Bioceramics 15 (2002) 51. [6] R. Murugan, S. Ramakrishna, Biomaterials 25 (2004) 3073. [7] R. Murugan, S. Ramakrishna, J. Appl. Biomater. Biomech. 3 (2005) 93. [8] R. Murugan, T.S.S. Kumar, K.P. Rao, Mater. Lett. 57 (2002) 429. [9] R. Murugan, S. Ramakrishna, J. Cryst. Growth 274 (2005) 209. [10] R.Z. LeGeros, in: P.W. Brown, B. Constantz (Eds.), Hydroxyapatite and Related Materials, CRC press, Boca Raton, 1994. [11] R.Z. LeGeros, Nature 206 (1965) 403. [12] J. Barralet, S.S. Best, W. Bonfield, J. Biomed. Mater. Res. 41 (1998) 77. [13] I. Mayer, J.D.B. Featherstone, J. Cryst. Growth 219 (2000) 98. [14] R. Murugan, S. Ramakrishna, Acta Biomaterialia 2 (2006) 201. [15] PDF File #9-432, International Centre for Diffraction Data (ICDD), Newtown Square, PA. [16] J.C. Elliot, Structure and Chemistry of the Apatites and Other Calcium Orthophosphates, Elsevier, Amsterdam, 1994. [17] R. Murugan, S. Ramakrishna, Cryst. Growth Des. 5 (2005) 111. [18] R. Murugan, S. Ramakrishna, Biomaterials 25 (2004) 3829. [19] J.C. Merry, I.R. Gibson, S.M. Best, W. Bonfield, J. Mater. Sci., Mater. Med. 9 (1998) 779. [20] S. Joschek, B. Nies, R. Krotz, A. Gopferich, Biomaterials 21 (2000) 1645. [21] R. Murugan, S. Ramakrishna, in: H.S. Nalwa (Ed.), Handbook of Nanostructured Biomaterials and Their Applications in Nanobiotechnology, American Scientific Publishers, California, 2005, p. 141. [22] R. Murugan, S. Ramakrishna, Comput. Sci. Tech. 65 (2005) 2385. [23] T.J. Webster, R.W. Siegel, R. Bizios, Biomaterials 21 (2000) 1803. [24] W.E. Brown, L.C. Chow, Cryst. Growth 53 (1981) 31. [25] R. Murugan, K.P. Rao, T.S.S. Kumar, Bull. Mater. Sci. 26 (2003) 523. [26] M.T. Fulmer, I.C. Ison, C.R. Hankermayer, B.R. Constantz, J. Ross, Biomaterials 23 (2002) 751.
Nanoporous hydroxy-carbonate apatite scaffold made of natural bone R. Murugan a,⁎, S. Ramakrishna a , K. Panduranga Rao b a
NUS Nanoscience and Nanotechnology Initiative (NUSNNI), Division of Bioengineering, Faculty of Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore b Spark Biotech, 3720 Sunburst Lane, Naperville, IL 60564, USA Received 7 September 2005; accepted 31 January 2006 Available online 20 February 2006
Abstract A processing method of hydroxy-carbonate apatite (HCAp) from the natural bone and their phase purity, crystal structure, chemical functionality, thermal stability, morphology, and solubility are described in this investigation. The X-ray diffraction analysis suggested that the processed bone corresponds characteristically to carbonate (CO2− 3 ) containing hydroxyapatite. Fourier transform infrared spectroscopic study 3− − confirmed the presence of CO2− 3 in addition to hydroxyl (OH ) and phosphate (PO4 ) functional groups, indicating the formation of HCAp. Environmental scanning electron microscopy was employed to identify the surface morphology of HCAp, which obviously shows a nanoporous structure throughout the matrix. The in vitro solubility study performed under physiological conditions confirms the resorbable nature of HCAp. These findings indicate the feasibility of processing nanoporous HCAp scaffolds from the natural bone by a straightforward method. © 2006 Elsevier B.V. All rights reserved. Keywords: Hydroxy-carbonate apatite; Calcination; Bone scaffold; Nanoporous structure
1. Introduction Clinical treatment of bone defects arising from traumatic or non-traumatic events often requires artificial bone grafts. A variety of biomaterials so far have been investigated as bone graft substitutes. Among them, hydroxyapatite (HA) is frequently used in orthopedic, dental and maxillofacial applications owing to its biocompatibility, osteoconductivity and bioactivity, meaning that it supports bone growth and osteointegration [1–3]. Multiple techniques have been employed for the preparation of HA either from natural or from synthetic sources, which chiefly include coral exoskeleton, animal bone and chemical synthesis [4–9]. It is known that most of the synthetic HA are stoichiometric with a chemical composition of Ca10(PO4)6(OH)2. By contrast, human bones do not have a pure or a stoichiometric HA, which has other ions mainly of CO32− and trace of Na+, Mg2+, Fe2+, Cl−, F− [10]. Although
⁎ Corresponding author. Tel.: +65 6874 6593; fax: +65 6874 3346. E-mail address: [email protected] (R. Murugan). 0167-577X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2006.01.104
presence of these ions is significantly low, they play a vital role in the biochemical reactions associated with bone metabolism. As a result, CO32− containing HA has gained much attention, not only due to its ionic composition but also due to its functional properties favorable for bone growth. Further, it can be easily resorbed by the living cells compared to stoichiometric HA, which might lead to faster bone regeneration. There are several reports that describe the substitution of CO32− into apatite phase [11–13]. It can be substituted for either OH− (A-type) or PO43− (B-type) or it can exist on the crystal surface (labile). Sometimes, both A- and B-type substitutions can also occur [14]. The problem of substitutional site of CO32− has not yet been solved by a direct measurement and work is under process in this direction as well. Further, such ionic substitution can affect the crystal structure, crystallinity, surface charge, solubility and other vital properties, leading to major changes in the biological performance upon implantation. As an alternative, HCAp can be processed from the natural bone without any extraneous ionic substitution but leaving only the natural trace elements, which is reported in this investigation in a detailed fashion.
R. Murugan et al. / Materials Letters 60 (2006) 2844–2847
2. Experimental 2.1. Processing of HCAp The HCAp was derived from chemically and thermally processed bovine bone. Briefly, the cortical portion of bovine bone samples were sliced into required size under running water. The macroscopic impurities of soft tissues and bone marrow associated with the bone were removed by cleaning with a 2% NaCl solution followed by degreasing in acetone– ether mixture (3 : 2) for 24 h. The degreased bone was then treated with 4% NaOH aqueous solution under hydrothermal condition. The chemically treated bone samples were calcined overnight at 500 °C in a muffle furnace under atmospheric pressure and ambient humidity. The resultant product was washed with deionized water and dried in a vacuum oven. 2.2. Characterization The phase purity and crystallographic structure of HCAp was identified with a powder X-ray diffractometer (XRD) (Seifert XRD 3000, Germany). The measurement was carried out with a Cu Kα1 radiation at the wavelength of 1.5406 Å. The chemical functionality was determined by spectroscopic method using a Fourier transform infrared (FTIR) spectrophotometer (ThermoNicolet Avatar 360, USA) over the range between 4000 and 400 cm− 1 at 2 cm− 1 resolution averaging 100 scans. The CO32− content was qualitatively determined from the infrared spectrum of HCAp by comparing the extinction coefficient (E) of the 1450 cm− 1 (carbonate) and 569 cm− 1 (phosphate) peaks using the formula: % CO32− =13.5(E1450/E569)− 0.2, where (E1450/ E569)=Ei =log(T2,i/T1,i). Here, i is 1450 or 569 and T2,i and T1,i are the transmission intensities at the peak maximum and local baseline, respectively. The CO32− content was also quantitatively determined as carbon using a CHN analyzer (Medac, UK). Thermal stability was studied using a thermogravimetric analyzer (TGA) (PerkinElmer, USA). The thermogram was recorded from 50 to about 800 °C at 10 °C/min heating rate under nitrogen atmosphere in order to avoid oxidation and other unwanted side reactions with atmospheric air. The characteristic surface morphology was studied by an environmental scanning electron microscopy (ESEM) (Quanta 200F, FEI) with an accelerating voltage of 20 kV. The in vitro solubility test of HCAp was performed in phosphate buffer with pH 7.2. The following protocol was used in the experiment: 50 mg of powder sample was added to 50 ml of buffer and kept at 37 °C in a thermostatic water bath. The change in pH of the buffer was quantitatively measured at pre-determined time intervals. The dissolution of HCAp was also determined on a palletized sample, prepared by a hot-pressing method, in the same buffer used for the solubility study at room temperature. The weight loss of the pellet was measured at pre-determined time intervals and plotted in a graph against immersion time.
human calcified tissue concerning crystallographical, chemical, thermal, porous morphological and resorbable properties, and, at the same time, to use as a scaffold for bone tissue engineering. In this regard, cortical portion of bovine bone was thoroughly analyzed at various calcination temperatures, ranging from 200 to 900 °C, and the temperature at 500 °C was found to be suitable for the removal of organic impurities associated with the native bone. Therefore, this temperature was chosen for further studies intended. The XRD technique was employed to evaluate the phase purity and the crystallographic structural properties of HCAp (Fig. 1). The result indicates all the characteristic Bragg peaks pertaining to HCAp without any structural deformation or extraneous phase substitution. The Bragg peaks appearing approximately at 26°, 30–34°, 40° and 46° (2θ) corresponds to the characteristic peaks of HA with reference to ICDD file #9-432 [15]. The intense peak around 12° (2θ) corresponds to the background peak of silicon that was used as an adhesive for the powder grains. The crystallographical geometry of HCAp processed in this study has a resemblance to that of human bone apatite [16] and shows a poor crystallinity compared to pure HA [17,18] owing to the CO2− 3 substitution into apatite, suggesting the formation of HCAp phase. The chemical functionality of HCAp was determined from its FTIR spectrum (Fig. 2). It can be seen from the result that all the absorptional peaks corresponding to apatite phase are detectable. Briefly, a band at 3450 cm− 1 is due to the presence of surface hydroxyl groups. The 1043 cm− 1 band can be assigned to a −1 stretching vibration of PO3− are 4 ions and bands at 590–610 cm 3− due to deformation vibration of PO4 ions. In addition, it can be easily seen that the bands pertain to the CO2− 3 functional group at 1500 and 870 cm− 1, indicating the substitution of CO2− 3 ions into the apatite and thus confirms the formation of HCAp. The CO2− 3 peaks at 1550 and 1480 cm− 1 are assigned to the A- and B-types and at 870 cm− 1 it is associated with the labile surface CO2− 3 . The low intense peaks noticed at about 2900 cm− 1 may be attributed to H–C–O functional group; thereby it is obvious that the processed HCAp has a close chemical structure with that of biominerals of human calcified tissue. The amount of CO2− 3 content was estimated by a spectroscopic method using an equation given in the Experimental and it was found to be 2 wt.%. As human calcified tissue also contains CO2− 3 to about 3– 6 wt.% depending upon age factor, its presence in the HCAp is highly beneficial. Further, it is reported that the lower concentration of CO2− 3 ions substantially improves the mechanical strength [19]. The CO2− 3 content was also determined by CHN analyzer and was found to be 1.8 wt.%, which corroborates well with the results of spectroscopic
3. Results and discussion A major intention of this investigation is to process a bone graft substitute from bovine bone with quite similar biominerals of the
2845
Fig. 1. X-ray powder diffraction pattern of HCAp.
2846
R. Murugan et al. / Materials Letters 60 (2006) 2844–2847
Fig. 2. FTIR spectrum of HCAp, arrow denotes substitution of carbonate ions (A) A-type, (B) B-type.
method. Accordingly, qualitative and quantitative analyses proved the presence of CO2− 3 into the apatite phase and noticed that the total amount of the CO2− 3 content falls near to the preferred range. Fig. 3 shows a representative TGA trace of HCAp in terms of weight loss as a function of temperature. There is no significant weight loss pertaining to endothermic or exothermic reactions observed from the thermogram, indicating its thermal stability upon calcination. The initial weight loss from 50 to 200 °C could be responsible for the evaporation of surface adsorbed water molecules and the weight loss that occurred between 580 and 620 °C could be due to the 2− decomposition of trace CO2− 3 . The endothermic dissociation of CO3 is reported to occur at the temperatures between 400 and 600 °C in air and between 500 and 890 °C in nitrogen atmosphere [20], which concur well with our results. Therefore, TGA analysis also confirms the formation of HCAp and supports further FTIR analysis. The surface morphological structure of HCAp is shown in Fig. 4. The result indicates that the HCAp has a porous architecture throughout the matrix. As it can be seen from the gross morphology, almost all the pores are in the order of nanometer size range. The average pore size was estimated as 12 nm. Here, the average pore size denotes the number of pores within a single micrograph of processed bone. These pores were created due to the removal of organic constituents associated with the native bone; thereby its native structure was disturbed, but, however, quite analogous to the morphological
Fig. 3. TGA trace of HCAp, which shows the weight loss of carbonate due to its decomposition.
Fig. 4. ESEM image of HCAp, which shows nanoporous architecture throughout the matrix.
structure of human trabecula. It has been reported that nanoporous structure of the biomaterials tremendously enhance the cells adhesion, proliferation and differentiation required for tissue functions [21–23]. Therefore, it can be expected that HCAp would have the capability to support the bone tissue in-growth upon implantation. However, the mechanical integrity of the processed material might be lost due to the removal of organic matters; thereby this kind of HCAp can only be used for low-weight bearing bone grafting and coating on bioimplants. Further, the porous architecture with nanopores could enhance the efficacy of HCAp if we use them for bone tissue engineering applications. It is also worth mentioning that the cells live in a complex mixture of pores, ridges, and fibers of extracellular matrix in a nano-featured environment; thereby the processed nanoporous HCAp may be highly beneficial for bone tissue engineering as a scaffold. The dissolution rate was determined by using two different approaches: (i) pH method and (ii) weight loss method. First method is a direct method, in which pH was determined using an electrode. In second method, the weight loss was determined by calculating weight of the pellet before and after immersion into the physiological medium. This is a common method in which the surface observed water was carefully removed and dried to a weight. All the experiments were carried out for one dissolution run and the results were compared with a pure HA. The in vitro solubility of HCAp performed in phosphate buffer (a common buffering agent used in cell/tissue cultures) under
Fig. 5. In vitro solubility of HCAp under physiological conditions.
R. Murugan et al. / Materials Letters 60 (2006) 2844–2847
2847
scaffold from the natural bone. The overall results indicated that the HCAp has characteristic resemblance with the biominerals of human calcified tissues. The retention of nanoporous architecture, chemical constituents, and the resorbable nature of HCAp would be beneficial if we use them for bone tissue engineering. Acknowledgements The financial support of the National University of Singapore and the Singapore Millennium Foundation are gratefully acknowledged. Fig. 6. Dissociation weight loss of HCAp in phosphate buffer.
physiological conditions is depicted in Fig. 5, which obviously shows the pH change on soaking of HCAp. The solubility test was also conducted for a pure HA, without carbonate substitution, and included in the same figure for comparison. The pH of the buffer medium without sample was found to be stable throughout the experimental period, which act as a control, but the addition of apatite has more or less alkaline effect depending on its solubility. The pH of the medium containing HA was found to have an almost unvarying trend as it did not undergo a considerable resorption, indicating its stability during the period of study. By contrast, HCAp shows a decreasing trend in the pH as it has a significant amount of CO2− 3 ions, indicating its resorbable nature. Moreover, a poorly crystalline phase of the HCAp is also responsible for its higher solubility than pure HA. The initial rapid increase in the pH may be attributed to the thermodynamical changes of apatite [24]. These results are consistent with previous findings [25,26] in which they reported that the solubility mainly depends on the preparation methodology, ionic substitution, in particular, carbonate, and crystallinity of the apatite. As solubility is highly sensitive to structural and chemical compositions of the apatite, the functional composition and crystallinity should also be considered as essential key factors. Fig. 6 displays a correlation between the dissociation weight and the sample immersion time. The graph shows a higher rate of weight loss for HCAp, indicating its higher soluble nature. By contrast, the rate of weight loss of HA is relatively low as compared to HCAp, indicating its stability. As there was no impurity present in the pure HA, the nature of its stability is obvious during the period of study. Thus, variation observed in their solubility could be attributed to the presence of CO2− 3 ions, which further confirms the resorbable nature of HCAp. The resorbable nature of HCAp would be beneficial for the new bone generation upon implantation.
4. Conclusions The present investigation demonstrated an easy, cost effective approach to a reproducible processing of HCAp
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