- Email: [email protected]
Production of ultra-fine bioresorbable carbonated hydroxyapatite
Acta BIOMATERIALIA Acta Biomaterialia 2 (2006) 201–206 www.actamat-journals.com
Brief communication
Production of ultra-fine bioresorbable carbonated hydroxyapatite R. Murugan *, S. Ramakrishna NUS Nanoscience and Nanotechnology Initiative, Division of Bioengineering, Faculty of Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore Received 3 January 2005; received in revised form 15 September 2005; accepted 15 September 2005
Abstract Ionic-substituted hydroxyapatite (HAp) based materials may be a better choice than pure HAp owing to their similarity in chemical composition with biological apatite. The present study reports a process for the production of carbonated hydroxyapatite (CHAp) using microwaves. The CHAp was evaluated for its phase purity, chemical homogeneity, functionality, morphology, and solubility. The CHAp thus obtained was compared with a pure HAp and a biological apatite, which provides quite an interesting insight into the carbonate substitution. The in vitro ionic dissolution rates determined under physiological conditions clearly demonstrate the soluble nature of CHAp compared to HAp. The overall results indicate that the processed CHAp has increased resorption relative to pure HAp and has a chemical composition corresponding to some extent with that of biological apatite. Ó 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Carbonated hydroxyapatite; X-ray diffraction; FTIR; Elemental analysis; Solubility
1. Introduction Nature, always a paradigm, sets high standards for the researchers who design biomedical implants. This may be true, particularly in bone-related therapy, because there is no ideal material or implant available that mimics real bone. Despite the numerous attempts that have been made in the last few decades, there is still a substantial need for bone substitutes to stimulate research in a more dynamic way. Hydroxyapatite (HAp), Ca10(PO4)6(OH)2, has a long history of being used as a biomaterial in bone grafting, bone tissue engineering, and bone drug delivery, owing to its obvious properties of biocompatibility, bioactivity, osteoconductivity, non-toxicity, non-inflammatoriness and non-immunogenicity. Numerous techniques are also available for processing HAp at micro and nanoscale levels [1–12]. Although it is considered as a good bone substitute, HAp slightly differs from the biological apatite in terms of structure, composition, crystallinity, solubility,
*
Corresponding author. Tel.: +65 6874 6593; fax: +65 6874 3346. E-mail address: [email protected] (R. Murugan).
and biological reactivity. Most of the biological apatites are non-stoichiometric, poorly crystalline, and contain several foreign ions, mainly carbonate ðCO2 3 Þ and traces of Na+, Mg2+, Fe2+, Cl, HPO2 , F [13,14]. Among them, 4 CO2 ions play a vital role in the bone metabolism; they 3 occupy about 3–8 wt.% of the calcified tissue and may vary depending on the age factor. Therefore, CO2 3 substituted HAp may have a tremendous potential in bone-related therapy. Further, HAp is the least soluble and the most stable material among the calcium phosphates, which is an undesirable characteristic because it may impede the rate of bone regeneration upon implantation [15]. By contrast, biological apatite has a higher solubility due to the presence of the above said trace elements. It is always desirable that a bone substitute should be bioresorbable to some extent so that it can be replaced, over a period of time, with the regenerated bone. The resorbability of HAp can be improved with the help of some ceramic oxides, ionic doping agents, or by reducing its grain size to the nano level [16–20]. On the other hand, substitution of CO2 3 ions into the apatite may lead to a higher solubility and it is expected that it may also increase mechanical strength. Therefore, it is important to pay a
1742-7061/$ - see front matter Ó 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2005.09.005
202
R. Murugan, S. Ramakrishna / Acta Biomaterialia 2 (2006) 201–206
great deal of attention to the development of CHAprelated biomaterials; thereby this investigation presents an elegant process using microwaves, which allows the production of CHAp with improved characteristics. 2. Experimental section 2.1. Processing of carbonated hydroxyapatite The starting materials, CaCl2, (NH4)2HPO4, and (NH4)2CO3, were obtained from Sigma (USA) and used as Ca, P, and CO3 precursors, respectively. The CHAp was prepared in accordance with the method described earlier with some modifications, using the above precursors [20]. Briefly, 0.3 M aqueous solution of (NH4)2HPO4 was mixed with 0.15 M (NH4)2CO3 and added dropwise to 0.5 M aqueous solution of CaCl2 under stirring at 1000 rpm. The pH of the reaction medium was adjusted to 10 by adding concentrated NH4OH solution using a syringe. The solution was maintained at a constant tem perature of 60 °C. A suspension of CHAp seed particles precipitated from the reaction medium was aged for 24 h followed by microwave irradiation (2.5 GHz, 800 W) for 15 min. The suspension, after undergoing microwave irradiation, was filtered, washed with deionized water, and dried in a vacuum oven. 2.2. Characterization Fourier transform infrared (FTIR) spectroscopy (ThermoNicolet Avatar 360, USA) was employed to determine the chemical functionality of CHAp. The potassium bromide (KBr) disk technique was used for analysis using 2 mg of CHAp powder compacted with 200 mg of KBr under hydraulic pressure. The FTIR spectrum was recorded in the 4000–400 cm1 region with 2 cm1 resolution averaging 100 scans. The carbonate content present in the CHAp was quantitatively measured by using a CHN analyzer (Perkin Elmer, USA). The Ca and P contents of the CHAp were determined by an inductively coupled plasma atomic emission spectrometer (ICP-AE). The crystallographic structural analysis was carried out by X-ray diffraction (XRD) using a Shimadzu XRD-600 powder diffractometer (Japan) with a Cu Ka radiation over the 2h range of 20–60° at a scanning speed of 1°/min. The surface morphology of CHAp was investigated using a scanning electron microscope (SEM) (JSM 5600, JEOL, Japan). The material for SEM analysis was prepared by sprinkling the dried powders onto one side of a double adhesive tape, which was stuck to a copper stub. The stub was then gold coated using JEOL JFC-1200 fine coater (Japan) to a thickness of 20–30 nm and examination was performed using an accelerating voltage of 20 kV. An elemental distribution analysis was carried out to identify the ionic substitution using an energy dispersive X-ray analyzer (EDXA) (Kevex, San Carlos, USA), which was directly connected to the SEM. For EDXA analysis, the
CHAp was not coated with gold and the environmental mode was the same as for a SEM analysis. The particle size and distribution of CHAp were measured using a particle size analyzer (Malvern Inc., UK) employing a laser diffraction technique. Before taking measurements, the powders were excited ultrasonically for a few minutes to break-up loosely bound agglomerates and to facilitate more accuracy. The in vitro solubility test was performed in a Tris buffer of pH 7.4 at a ratio of 1 mg/ml in a thermostatic incubator regulated at 37 °C. The dissolution of CHAp was determined on a palletized sample, prepared by a hot-pressing method, in the same buffer used for the in vitro solubility study at room temperature. The weight loss of the pellet was measured at pre-determined time intervals and plotted against immersion time. 3. Results and discussion The present study demonstrates the production of CHAp using microwaves, which has characteristics that can be compared favorably with a biological apatite. The microwave technique was employed keeping in view that it may provide a homogeneous phase product at the atomic level. This technique has many advantages over conventional heating methods, such as time and energy saving, reduced processing time and temperature, rapid heating rates, minimization of grain growth, and eco-friendliness. Some of the physico-chemical properties of the microwave irradiated material (i.e. the CHAp) were analyzed with respect to its suitability in bone substitution and results thus obtained were compared with properties of pure HAp and biological apatite. The Ca/P molar ratio is one of the important characteristics of the biomaterial to be used for bone substitution, because of scaling its phase purity, chemical homogeneity, and solubility. Therefore, the Ca/P molar ratio of the CHAp was determined by ICP and it was found to be 1.69, which is quite similar to that of pure HAp (1.66). The slight increase in the Ca/P ratio can be attributed to the substitution of CO2 3 into apatite, indicating the formation of CHAp. It also shows that the amount of CO2 3 substitution is very low. The calculated Ca/P ratio of CHAp is appreciable and fits quite well with that of biological apatite, which ranges from 1.50 to 1.85 [21]. It is worth pointing out that the Ca/P ratio of biological apatite mainly depends on the type of species and their age factor. FTIR spectroscopy was employed to confirm the substitution of CO2 3 . A representative FTIR spectrum of the CHAp is shown in Fig. 1A. It shows all the characteristic absorption bands of the apatite in addition to CO2 3 bands, indicating the formation of CHAp; thereby it supports the result of ICP analysis. Briefly, in the middle infrared region, the spectrum displays all the peaks pertaining to hydroxyl 2 (OH), phosphate ðPO3 functional groups. 4 Þ, and CO3 A broad peak concerning H–O–H absorption is noticed around 3480 cm1. The characteristic peak of PO3 4 stretching vibration appears at 1040 cm1 and deformation vibration of PO3 appears at 610 and 570 cm1. In addition, 4
R. Murugan, S. Ramakrishna / Acta Biomaterialia 2 (2006) 201–206
203
Fig. 1. (A) FTIR spectrum of processed CHAp, indicating all characteristic peaks of HAp with carbonate ionic substitution. (B) XRD pattern of the CHAp, indicating well-resolved Bragg peaks pertaining to apatite phase. (C) EDXA spectrum of the CHAp, showing their characteristic elements.
absorption bands of CO2 3 associated with the symmetric stretching mode at 1440–1560 cm1 and out of plane bending mode at 870 cm1 are noticed in the spectrum. These results obviously indicate the substitution of CO2 into 3 the apatite and confirm the formation of CHAp. Furthermore, the spectrum reveals that two types of CO2 3 substitutions have occurred into the apatite phase with respect to 1 their exchanging sites. The CO2 can 3 band at 1445 cm 2 3 be ascribed to B-type CO3 substitution on PO4 sites and band at 1550 cm1 can be ascribed to A-type CO2 3 substitution on OH sites. Although the FTIR analysis provides proof for the A- and B-type CO2 3 substitutions, it shows incomplete A-type CO2 3 substitution because the absorption peak pertaining to OH can still be observed in the spectrum around 3500 cm1. It suggests that the majority 3 of the CO2 3 exchanged mainly for PO4 whereas it was par tially exchanged for OH , probably due to the effect of microwave irradiation involved in the preparation methodology. Interestingly, it has to be mentioned that most of the biological apatites contain both A- and B-type CO2 in 3
their lattice [21]. It can thereby be concluded that the prepared CHAp is chemically and structurally analogous to biological apatite. Since FTIR displayed clear evidence of CO2 3 substitution, it is worth determining the amount of substituted CO2 quantitatively. CO2 content estimated 3 3 by a FTIR spectroscopic analysis, as detailed in our earlier report [22], was found to be 3.0 wt.% (Table 1). In addition, the CHN analyzer was also employed to determine the amount of CO2 3 substitution and the results were compared with the results obtained from the spectroscopic method. The amount of CO2 3 present in the CHAp was found to be 2.9 wt.% from the CHN analysis, which is consistent with the FTIR results. It is worth pointing out that CO2 3 content of the biological apatite varies from 3 to 8 wt.% depending upon age and type of species. As the amount of CO2 present in the CHAp lay within this range, it 3 may be said that the CHAp mimics biological apatite in terms of ionic substitution, in particular, CO2 3 . The XRD technique was employed to examine phase purity and structure of the CHAp. The phase identification
Table 1 Physico-chemical properties of CHAp Samples
HAp CHAp BAp
Ca/P molar ratio
CO2 3 by CHN (wt.%)
CO2 3 by spec. (wt.%)
1.66 1.69 –
0.0 2.9 2.7
0.0 3.0 2.6
BAp: biological apatite.
˚) Lattice constants (A a0 = b0
c0
c0/a0
9.4180 9.4154 9.4190
6.8840 6.8880 6.8890
0.731 0.732 0.731
Unit cell volume ˚ 3) (A 528.781 528.797 529.323
204
R. Murugan, S. Ramakrishna / Acta Biomaterialia 2 (2006) 201–206
was carried out by comparing the diffraction peaks of the CHAp with a pure HAp (ICDD PDF #9-432) [23]. A representative pattern is shown in Fig. 1B. The pattern shows all the Bragg peaks corresponding characteristically to CHAp [18], showing its structural integrity. Though it is noticed that there is no major difference between CHAp and HAp, still there is a slight change observed in their unit cell parameters. The cell parameters were calculated by refining the XRD data using a standard least squares method and the corresponding lattice cell constants are listed in Table 1. A slight decrease in the lattice parameter Ôa0Õ and a slight increase in the lattice parameter Ôc0Õ were noticed for CHAp as compared to HAp. The reduction in the Ôa0Õ parameter of CHAp corresponding to HAp 2 could be attributed to the replacement of PO3 4 by CO3 . It can be noticed from the XRD data that there are no extraneous phases in the CHAp, indicating possible formation of a pure CHAp phase using microwave technique. Interestingly, the crystallographic behaviour of CHAp is also quite analogous to that of biological apatite [22]. Accordingly, these results suggest that the microwave technique is suitable for the production of CHAp, mimicking a biological apatite to some extent. A typical EDXA spectrum of CHAp is shown in Fig. 1C, displaying its elemental composition. The spectrum shows that CHAp is primarily composed of Ca and P with some amount of C and O. The presence of Ca and P is responsible for the apatite phase and the presence of C in the spectrum is primarily responsible for the carbonate phase. As there is no foreign element detected in the spectrum, the purity of CHAp could be easily assessed. The surface morphology of CHAp was characterized by SEM as shown in Fig. 2A. The microwave irradiated CHAp is composed of white tiny crystals, which is in the form of free flowing powder. This observation shows that most of the particles are fused together thereby forming a cluster. This phenomenon might be attributed to a high surface area to volume ratio of ultra-fine crystals related to microwave exposure. It should be also pointed out that the shape of CHAp grains is quite different from the biological apatite, which mostly exhibits a needle-like structure. However, we expect that it may be possible to obtain a grain shape similar to biological apatite by further optimizing microwave irradiation conditions. Nevertheless, a keen interest was paid to measuring the particle size of CHAp. As it is quite difficult to determine the particle size of the CHAp directly from the SEM analysis owing to agglomeration, a particle size analyzer was employed, which provided a better appraisal than SEM. The particle size distribution of CHAp is shown in Fig. 2B. The results indicate that the particles of CHAp are widely distributed, ranging from 0.1 to 15 lm in size, in which most of the particle sizes are between 0.5 and 5 lm with an average value of 1 lm. Further, particle size determines the surface area of materials, i.e. a smaller particle size tends to produce greater surface area. Accordingly, size analysis suggests that CHAp has a large surface area to volume ratio,
Fig. 2. (A) SEM photograph of the CHAp, indicating that most of the grains are agglomerated owing to their high surface area to volume ratio. (B) Histogram of particle size distribution of the CHAp, indicating that most of the particles are within the range of sub-micrometers to nanometers.
because most of the particles get fused and form a cluster-like morphology, which is also evident from the SEM observation. The wide distribution of ultra-fine particles, particularly those in the nanometer scale range, may provide better circumstances for good bioaffinity with living cells in the bone metabolism and may also be ideal to be used for coating biomedical implants. The in vitro solubility of CHAp was examined in Tris buffer under a physiological condition of pH 7.4 at 37 °C. The change in the pH of the buffer with and without apatite was recorded at pre-determined time intervals using a pH meter. Fig. 3A shows a graph of pH versus time, illustrating the level of solubility of CHAp. The variations of pH with time for a Tris buffer without HAp and with HAp were also determined and included in the same graph for comparison. The pH of Tris buffer without HAp is rather stable throughout the experimental period, whereas addition of apatite has a considerable alkalizing effect on the pH of the medium. CHAp showed a drastic change in the pH compared to HAp, suggesting that it dissolves much faster than HAp, which can be attributed to the dissociation of CO2 3 ions into the medium. As solubility is highly sensitive to structural and chemical compositions of the apatite and depends on the buffering condition, these
R. Murugan, S. Ramakrishna / Acta Biomaterialia 2 (2006) 201–206
to biological apatite. The in vitro solubility of CHAp under physiological conditions is appreciable and is found to be higher than HAp, which is a good sign of its bioresorbable nature. However, further characterization in vitro with biological systems and in vivo animal studies is needed to elucidate its suitability as a bioresorbable bone substitute. Based on the experimental results, CHAp may be a better material for bone substitution than pure HAp.
7.6
A without HAp or CHAp with HAp with CHAp pH
205
7.4
Acknowledgements 7.2 1
10
100
1000
Time (h)
B
100
Pellet wt%
HCAp HAp
References
95
90 0
100
200
300
The financial support of the National University of Singapore and the Singapore Millennium Foundation is gratefully acknowledged.
400
Immersion time (h)
Fig. 3. (A) The in vitro solubility of CHAp under physiological conditions. (B) Weight loss of CHAp and HAp in Tris buffer; both are indicating the soluble nature of CHAp compared to HAp.
factors should also be considered as essential key factors for in vitro resorption. The pH value is dependent on the solubility of apatite, wherein the pH decreases as the solubility increases. Accordingly, it is suggested from Fig. 3A that the rate of solubility of the CHAp is higher than that of HAp, which is a good sign for the resorbable nature of CHAp in vitro. Fig. 3B displays a correlation between weight loss of the samples and immersion time. The graph shows a higher rate of weight loss for CHAp, indicating its higher soluble nature. The content of CO2 3 should lead to a higher solubility compared to HAp, thereby a considerable amount of weight loss was noticed for the CHAp. As there was no impurity present in the pure HAp, the nature of its stability was obvious during the period of study. These results indicate that the CO2 3 ions play a vital role in determining the solubility of the apatite. Thereby considerable variations observed in the solubility of CHAp and HAp further confirms the resorbable nature of CHAp in vitro. 4. Conclusions This study reveals the feasibility of producing CHAp using microwaves. The overall results indicate that CHAp has structural and chemical functionalities quite similar
[1] Aoki H. Medical applications of hydroxyapatite. Tokyo: Ishiyaku EuroAmerica; 1994. [2] Hench LL, Wilson J. Surface-active biomaterials. Science 1984;226:630–6. [3] Bunker BC, Rieke PC, Tarasevich BJ, Campbell AA, Fryxell GE, Graff GL, et al. Ceramic thin-film formation on functionalized interfaces through biomimetic processing. Science 1994;264: 48–55. [4] Suchanek W, Yoshimura M. Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants. J Mater Res 1998;13:94–117. [5] Murugan R, Ramakrishna S. Porous bovine hydroxyapatite for drug delivery. J Appl Biomater Biomech 2005;3:93–7. [6] Murugan R, Ramakrishna S. Crystallographic study of hydroxyapatite bioceramics derived from various sources. Cryst Growth Des 2005;5:111–2. [7] Murugan R, Ramakrishna S. Nanostructured biomaterials. In: Nalwa HS, editor. Encyclopedia of nanoscience and nanotechnology. Stevenson Ranch, CA: American Scientific; 2004. p. 595–613. [8] Murugan R, Ramakrishna S. Coupling of therapeutic molecules onto surface modified coralline hydroxyapatite. Biomaterials 2004;25: 3073–80. [9] Murugan R, Rao KP. Grafting of glycidylmethacrylate upon coralline hydroxyapatite in conjugation with demineralized bone matrix using redox initiating system. Macromol Res 2003;11:14–8. [10] Murugan R, Rao KP. Graft polymerization of glycidylmethacrylate onto coralline hydroxyapatite. J Biomater Sci Polym Edn 2003;14: 457–67. [11] Murugan R, Rao KP. Controlled release of antibiotic from surface modified coralline hydroxyapatite. Trends Biomater Artif Organs 2002;16:43–5. [12] Murugan R, Ramakrishna S. Bioresorbable composite bone paste using polysaccharide based nano hydroxyapatite. Biomaterials 2004;25:3829–35. [13] Bigi A, Cojazzi G, Panzavolta S, Ripamonti A, Roveri N, Romanello M, et al. Chemical and structural characterization of the mineral phase from cortical and trabecular bone. J Inorg Biochem 1997;68:45–51. [14] LeGeros RZ. Effect of carbonate on the lattice parameters of apatite. Nature 1965;206:403–4. [15] Kivrak N, Tas AC. Synthesis of calcium hydroxyapatite–tricalcium phosphate composite bioceramic powders and their sintering behavior. J Am Ceram Soc 1998;81:2245–52. [16] Murugan R, Ramakrishna S. Effect of zirconia on the formation of calcium phosphate bioceramics under microwave irradiation. Mater Lett 2003;58:230–4.
206
R. Murugan, S. Ramakrishna / Acta Biomaterialia 2 (2006) 201–206
[17] Murugan R, Kumar TSS, Rao KP. Fluorinated bovine hydroxyapatite: preparation and characterization. Mater Lett 2002;57:429–33. [18] Murugan R, Rao KP, Kumar TSS. Microwave synthesis of bioresorbable carbonated hydroxyapatite using goniopora. Bioceramics 2002;15:51–4. [19] Murugan R, Ramakrishna S. Bioactive nanomaterials in bone grafting and tissue engineering. In: Nalwa HS, editor. Handbook of nanostructured biomaterials and their applications in nanobiotechnology. Stevenson Ranch, CA: American Scientific Publishers; 2005. p. 141–68.
[20] Murugan R, Ramakrishna S. Aqueous mediated synthesis of bioresorbable nanocrystalline hydroxyapatite. J Cryst Growth 2005;274: 209–13. [21] Elliot JC. Structure and chemistry of the apatites and the other calcium orthophosphates. Amsterdam: Elsevier Science; 1994. [22] Murugan R, Rao KP, Kumar TSS. Heat-deproteinated xenogeneic bone from slaughterhouse waste: physico-chemical properties. Bull Mater Sci 2003;26:523–8. [23] Powder diffraction file #9-432, International Centre for Diffraction Data, Newtown Square, PA, USA.
Brief communication
Production of ultra-fine bioresorbable carbonated hydroxyapatite R. Murugan *, S. Ramakrishna NUS Nanoscience and Nanotechnology Initiative, Division of Bioengineering, Faculty of Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore Received 3 January 2005; received in revised form 15 September 2005; accepted 15 September 2005
Abstract Ionic-substituted hydroxyapatite (HAp) based materials may be a better choice than pure HAp owing to their similarity in chemical composition with biological apatite. The present study reports a process for the production of carbonated hydroxyapatite (CHAp) using microwaves. The CHAp was evaluated for its phase purity, chemical homogeneity, functionality, morphology, and solubility. The CHAp thus obtained was compared with a pure HAp and a biological apatite, which provides quite an interesting insight into the carbonate substitution. The in vitro ionic dissolution rates determined under physiological conditions clearly demonstrate the soluble nature of CHAp compared to HAp. The overall results indicate that the processed CHAp has increased resorption relative to pure HAp and has a chemical composition corresponding to some extent with that of biological apatite. Ó 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Carbonated hydroxyapatite; X-ray diffraction; FTIR; Elemental analysis; Solubility
1. Introduction Nature, always a paradigm, sets high standards for the researchers who design biomedical implants. This may be true, particularly in bone-related therapy, because there is no ideal material or implant available that mimics real bone. Despite the numerous attempts that have been made in the last few decades, there is still a substantial need for bone substitutes to stimulate research in a more dynamic way. Hydroxyapatite (HAp), Ca10(PO4)6(OH)2, has a long history of being used as a biomaterial in bone grafting, bone tissue engineering, and bone drug delivery, owing to its obvious properties of biocompatibility, bioactivity, osteoconductivity, non-toxicity, non-inflammatoriness and non-immunogenicity. Numerous techniques are also available for processing HAp at micro and nanoscale levels [1–12]. Although it is considered as a good bone substitute, HAp slightly differs from the biological apatite in terms of structure, composition, crystallinity, solubility,
*
Corresponding author. Tel.: +65 6874 6593; fax: +65 6874 3346. E-mail address: [email protected] (R. Murugan).
and biological reactivity. Most of the biological apatites are non-stoichiometric, poorly crystalline, and contain several foreign ions, mainly carbonate ðCO2 3 Þ and traces of Na+, Mg2+, Fe2+, Cl, HPO2 , F [13,14]. Among them, 4 CO2 ions play a vital role in the bone metabolism; they 3 occupy about 3–8 wt.% of the calcified tissue and may vary depending on the age factor. Therefore, CO2 3 substituted HAp may have a tremendous potential in bone-related therapy. Further, HAp is the least soluble and the most stable material among the calcium phosphates, which is an undesirable characteristic because it may impede the rate of bone regeneration upon implantation [15]. By contrast, biological apatite has a higher solubility due to the presence of the above said trace elements. It is always desirable that a bone substitute should be bioresorbable to some extent so that it can be replaced, over a period of time, with the regenerated bone. The resorbability of HAp can be improved with the help of some ceramic oxides, ionic doping agents, or by reducing its grain size to the nano level [16–20]. On the other hand, substitution of CO2 3 ions into the apatite may lead to a higher solubility and it is expected that it may also increase mechanical strength. Therefore, it is important to pay a
1742-7061/$ - see front matter Ó 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2005.09.005
202
R. Murugan, S. Ramakrishna / Acta Biomaterialia 2 (2006) 201–206
great deal of attention to the development of CHAprelated biomaterials; thereby this investigation presents an elegant process using microwaves, which allows the production of CHAp with improved characteristics. 2. Experimental section 2.1. Processing of carbonated hydroxyapatite The starting materials, CaCl2, (NH4)2HPO4, and (NH4)2CO3, were obtained from Sigma (USA) and used as Ca, P, and CO3 precursors, respectively. The CHAp was prepared in accordance with the method described earlier with some modifications, using the above precursors [20]. Briefly, 0.3 M aqueous solution of (NH4)2HPO4 was mixed with 0.15 M (NH4)2CO3 and added dropwise to 0.5 M aqueous solution of CaCl2 under stirring at 1000 rpm. The pH of the reaction medium was adjusted to 10 by adding concentrated NH4OH solution using a syringe. The solution was maintained at a constant tem perature of 60 °C. A suspension of CHAp seed particles precipitated from the reaction medium was aged for 24 h followed by microwave irradiation (2.5 GHz, 800 W) for 15 min. The suspension, after undergoing microwave irradiation, was filtered, washed with deionized water, and dried in a vacuum oven. 2.2. Characterization Fourier transform infrared (FTIR) spectroscopy (ThermoNicolet Avatar 360, USA) was employed to determine the chemical functionality of CHAp. The potassium bromide (KBr) disk technique was used for analysis using 2 mg of CHAp powder compacted with 200 mg of KBr under hydraulic pressure. The FTIR spectrum was recorded in the 4000–400 cm1 region with 2 cm1 resolution averaging 100 scans. The carbonate content present in the CHAp was quantitatively measured by using a CHN analyzer (Perkin Elmer, USA). The Ca and P contents of the CHAp were determined by an inductively coupled plasma atomic emission spectrometer (ICP-AE). The crystallographic structural analysis was carried out by X-ray diffraction (XRD) using a Shimadzu XRD-600 powder diffractometer (Japan) with a Cu Ka radiation over the 2h range of 20–60° at a scanning speed of 1°/min. The surface morphology of CHAp was investigated using a scanning electron microscope (SEM) (JSM 5600, JEOL, Japan). The material for SEM analysis was prepared by sprinkling the dried powders onto one side of a double adhesive tape, which was stuck to a copper stub. The stub was then gold coated using JEOL JFC-1200 fine coater (Japan) to a thickness of 20–30 nm and examination was performed using an accelerating voltage of 20 kV. An elemental distribution analysis was carried out to identify the ionic substitution using an energy dispersive X-ray analyzer (EDXA) (Kevex, San Carlos, USA), which was directly connected to the SEM. For EDXA analysis, the
CHAp was not coated with gold and the environmental mode was the same as for a SEM analysis. The particle size and distribution of CHAp were measured using a particle size analyzer (Malvern Inc., UK) employing a laser diffraction technique. Before taking measurements, the powders were excited ultrasonically for a few minutes to break-up loosely bound agglomerates and to facilitate more accuracy. The in vitro solubility test was performed in a Tris buffer of pH 7.4 at a ratio of 1 mg/ml in a thermostatic incubator regulated at 37 °C. The dissolution of CHAp was determined on a palletized sample, prepared by a hot-pressing method, in the same buffer used for the in vitro solubility study at room temperature. The weight loss of the pellet was measured at pre-determined time intervals and plotted against immersion time. 3. Results and discussion The present study demonstrates the production of CHAp using microwaves, which has characteristics that can be compared favorably with a biological apatite. The microwave technique was employed keeping in view that it may provide a homogeneous phase product at the atomic level. This technique has many advantages over conventional heating methods, such as time and energy saving, reduced processing time and temperature, rapid heating rates, minimization of grain growth, and eco-friendliness. Some of the physico-chemical properties of the microwave irradiated material (i.e. the CHAp) were analyzed with respect to its suitability in bone substitution and results thus obtained were compared with properties of pure HAp and biological apatite. The Ca/P molar ratio is one of the important characteristics of the biomaterial to be used for bone substitution, because of scaling its phase purity, chemical homogeneity, and solubility. Therefore, the Ca/P molar ratio of the CHAp was determined by ICP and it was found to be 1.69, which is quite similar to that of pure HAp (1.66). The slight increase in the Ca/P ratio can be attributed to the substitution of CO2 3 into apatite, indicating the formation of CHAp. It also shows that the amount of CO2 3 substitution is very low. The calculated Ca/P ratio of CHAp is appreciable and fits quite well with that of biological apatite, which ranges from 1.50 to 1.85 [21]. It is worth pointing out that the Ca/P ratio of biological apatite mainly depends on the type of species and their age factor. FTIR spectroscopy was employed to confirm the substitution of CO2 3 . A representative FTIR spectrum of the CHAp is shown in Fig. 1A. It shows all the characteristic absorption bands of the apatite in addition to CO2 3 bands, indicating the formation of CHAp; thereby it supports the result of ICP analysis. Briefly, in the middle infrared region, the spectrum displays all the peaks pertaining to hydroxyl 2 (OH), phosphate ðPO3 functional groups. 4 Þ, and CO3 A broad peak concerning H–O–H absorption is noticed around 3480 cm1. The characteristic peak of PO3 4 stretching vibration appears at 1040 cm1 and deformation vibration of PO3 appears at 610 and 570 cm1. In addition, 4
R. Murugan, S. Ramakrishna / Acta Biomaterialia 2 (2006) 201–206
203
Fig. 1. (A) FTIR spectrum of processed CHAp, indicating all characteristic peaks of HAp with carbonate ionic substitution. (B) XRD pattern of the CHAp, indicating well-resolved Bragg peaks pertaining to apatite phase. (C) EDXA spectrum of the CHAp, showing their characteristic elements.
absorption bands of CO2 3 associated with the symmetric stretching mode at 1440–1560 cm1 and out of plane bending mode at 870 cm1 are noticed in the spectrum. These results obviously indicate the substitution of CO2 into 3 the apatite and confirm the formation of CHAp. Furthermore, the spectrum reveals that two types of CO2 3 substitutions have occurred into the apatite phase with respect to 1 their exchanging sites. The CO2 can 3 band at 1445 cm 2 3 be ascribed to B-type CO3 substitution on PO4 sites and band at 1550 cm1 can be ascribed to A-type CO2 3 substitution on OH sites. Although the FTIR analysis provides proof for the A- and B-type CO2 3 substitutions, it shows incomplete A-type CO2 3 substitution because the absorption peak pertaining to OH can still be observed in the spectrum around 3500 cm1. It suggests that the majority 3 of the CO2 3 exchanged mainly for PO4 whereas it was par tially exchanged for OH , probably due to the effect of microwave irradiation involved in the preparation methodology. Interestingly, it has to be mentioned that most of the biological apatites contain both A- and B-type CO2 in 3
their lattice [21]. It can thereby be concluded that the prepared CHAp is chemically and structurally analogous to biological apatite. Since FTIR displayed clear evidence of CO2 3 substitution, it is worth determining the amount of substituted CO2 quantitatively. CO2 content estimated 3 3 by a FTIR spectroscopic analysis, as detailed in our earlier report [22], was found to be 3.0 wt.% (Table 1). In addition, the CHN analyzer was also employed to determine the amount of CO2 3 substitution and the results were compared with the results obtained from the spectroscopic method. The amount of CO2 3 present in the CHAp was found to be 2.9 wt.% from the CHN analysis, which is consistent with the FTIR results. It is worth pointing out that CO2 3 content of the biological apatite varies from 3 to 8 wt.% depending upon age and type of species. As the amount of CO2 present in the CHAp lay within this range, it 3 may be said that the CHAp mimics biological apatite in terms of ionic substitution, in particular, CO2 3 . The XRD technique was employed to examine phase purity and structure of the CHAp. The phase identification
Table 1 Physico-chemical properties of CHAp Samples
HAp CHAp BAp
Ca/P molar ratio
CO2 3 by CHN (wt.%)
CO2 3 by spec. (wt.%)
1.66 1.69 –
0.0 2.9 2.7
0.0 3.0 2.6
BAp: biological apatite.
˚) Lattice constants (A a0 = b0
c0
c0/a0
9.4180 9.4154 9.4190
6.8840 6.8880 6.8890
0.731 0.732 0.731
Unit cell volume ˚ 3) (A 528.781 528.797 529.323
204
R. Murugan, S. Ramakrishna / Acta Biomaterialia 2 (2006) 201–206
was carried out by comparing the diffraction peaks of the CHAp with a pure HAp (ICDD PDF #9-432) [23]. A representative pattern is shown in Fig. 1B. The pattern shows all the Bragg peaks corresponding characteristically to CHAp [18], showing its structural integrity. Though it is noticed that there is no major difference between CHAp and HAp, still there is a slight change observed in their unit cell parameters. The cell parameters were calculated by refining the XRD data using a standard least squares method and the corresponding lattice cell constants are listed in Table 1. A slight decrease in the lattice parameter Ôa0Õ and a slight increase in the lattice parameter Ôc0Õ were noticed for CHAp as compared to HAp. The reduction in the Ôa0Õ parameter of CHAp corresponding to HAp 2 could be attributed to the replacement of PO3 4 by CO3 . It can be noticed from the XRD data that there are no extraneous phases in the CHAp, indicating possible formation of a pure CHAp phase using microwave technique. Interestingly, the crystallographic behaviour of CHAp is also quite analogous to that of biological apatite [22]. Accordingly, these results suggest that the microwave technique is suitable for the production of CHAp, mimicking a biological apatite to some extent. A typical EDXA spectrum of CHAp is shown in Fig. 1C, displaying its elemental composition. The spectrum shows that CHAp is primarily composed of Ca and P with some amount of C and O. The presence of Ca and P is responsible for the apatite phase and the presence of C in the spectrum is primarily responsible for the carbonate phase. As there is no foreign element detected in the spectrum, the purity of CHAp could be easily assessed. The surface morphology of CHAp was characterized by SEM as shown in Fig. 2A. The microwave irradiated CHAp is composed of white tiny crystals, which is in the form of free flowing powder. This observation shows that most of the particles are fused together thereby forming a cluster. This phenomenon might be attributed to a high surface area to volume ratio of ultra-fine crystals related to microwave exposure. It should be also pointed out that the shape of CHAp grains is quite different from the biological apatite, which mostly exhibits a needle-like structure. However, we expect that it may be possible to obtain a grain shape similar to biological apatite by further optimizing microwave irradiation conditions. Nevertheless, a keen interest was paid to measuring the particle size of CHAp. As it is quite difficult to determine the particle size of the CHAp directly from the SEM analysis owing to agglomeration, a particle size analyzer was employed, which provided a better appraisal than SEM. The particle size distribution of CHAp is shown in Fig. 2B. The results indicate that the particles of CHAp are widely distributed, ranging from 0.1 to 15 lm in size, in which most of the particle sizes are between 0.5 and 5 lm with an average value of 1 lm. Further, particle size determines the surface area of materials, i.e. a smaller particle size tends to produce greater surface area. Accordingly, size analysis suggests that CHAp has a large surface area to volume ratio,
Fig. 2. (A) SEM photograph of the CHAp, indicating that most of the grains are agglomerated owing to their high surface area to volume ratio. (B) Histogram of particle size distribution of the CHAp, indicating that most of the particles are within the range of sub-micrometers to nanometers.
because most of the particles get fused and form a cluster-like morphology, which is also evident from the SEM observation. The wide distribution of ultra-fine particles, particularly those in the nanometer scale range, may provide better circumstances for good bioaffinity with living cells in the bone metabolism and may also be ideal to be used for coating biomedical implants. The in vitro solubility of CHAp was examined in Tris buffer under a physiological condition of pH 7.4 at 37 °C. The change in the pH of the buffer with and without apatite was recorded at pre-determined time intervals using a pH meter. Fig. 3A shows a graph of pH versus time, illustrating the level of solubility of CHAp. The variations of pH with time for a Tris buffer without HAp and with HAp were also determined and included in the same graph for comparison. The pH of Tris buffer without HAp is rather stable throughout the experimental period, whereas addition of apatite has a considerable alkalizing effect on the pH of the medium. CHAp showed a drastic change in the pH compared to HAp, suggesting that it dissolves much faster than HAp, which can be attributed to the dissociation of CO2 3 ions into the medium. As solubility is highly sensitive to structural and chemical compositions of the apatite and depends on the buffering condition, these
R. Murugan, S. Ramakrishna / Acta Biomaterialia 2 (2006) 201–206
to biological apatite. The in vitro solubility of CHAp under physiological conditions is appreciable and is found to be higher than HAp, which is a good sign of its bioresorbable nature. However, further characterization in vitro with biological systems and in vivo animal studies is needed to elucidate its suitability as a bioresorbable bone substitute. Based on the experimental results, CHAp may be a better material for bone substitution than pure HAp.
7.6
A without HAp or CHAp with HAp with CHAp pH
205
7.4
Acknowledgements 7.2 1
10
100
1000
Time (h)
B
100
Pellet wt%
HCAp HAp
References
95
90 0
100
200
300
The financial support of the National University of Singapore and the Singapore Millennium Foundation is gratefully acknowledged.
400
Immersion time (h)
Fig. 3. (A) The in vitro solubility of CHAp under physiological conditions. (B) Weight loss of CHAp and HAp in Tris buffer; both are indicating the soluble nature of CHAp compared to HAp.
factors should also be considered as essential key factors for in vitro resorption. The pH value is dependent on the solubility of apatite, wherein the pH decreases as the solubility increases. Accordingly, it is suggested from Fig. 3A that the rate of solubility of the CHAp is higher than that of HAp, which is a good sign for the resorbable nature of CHAp in vitro. Fig. 3B displays a correlation between weight loss of the samples and immersion time. The graph shows a higher rate of weight loss for CHAp, indicating its higher soluble nature. The content of CO2 3 should lead to a higher solubility compared to HAp, thereby a considerable amount of weight loss was noticed for the CHAp. As there was no impurity present in the pure HAp, the nature of its stability was obvious during the period of study. These results indicate that the CO2 3 ions play a vital role in determining the solubility of the apatite. Thereby considerable variations observed in the solubility of CHAp and HAp further confirms the resorbable nature of CHAp in vitro. 4. Conclusions This study reveals the feasibility of producing CHAp using microwaves. The overall results indicate that CHAp has structural and chemical functionalities quite similar
[1] Aoki H. Medical applications of hydroxyapatite. Tokyo: Ishiyaku EuroAmerica; 1994. [2] Hench LL, Wilson J. Surface-active biomaterials. Science 1984;226:630–6. [3] Bunker BC, Rieke PC, Tarasevich BJ, Campbell AA, Fryxell GE, Graff GL, et al. Ceramic thin-film formation on functionalized interfaces through biomimetic processing. Science 1994;264: 48–55. [4] Suchanek W, Yoshimura M. Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants. J Mater Res 1998;13:94–117. [5] Murugan R, Ramakrishna S. Porous bovine hydroxyapatite for drug delivery. J Appl Biomater Biomech 2005;3:93–7. [6] Murugan R, Ramakrishna S. Crystallographic study of hydroxyapatite bioceramics derived from various sources. Cryst Growth Des 2005;5:111–2. [7] Murugan R, Ramakrishna S. Nanostructured biomaterials. In: Nalwa HS, editor. Encyclopedia of nanoscience and nanotechnology. Stevenson Ranch, CA: American Scientific; 2004. p. 595–613. [8] Murugan R, Ramakrishna S. Coupling of therapeutic molecules onto surface modified coralline hydroxyapatite. Biomaterials 2004;25: 3073–80. [9] Murugan R, Rao KP. Grafting of glycidylmethacrylate upon coralline hydroxyapatite in conjugation with demineralized bone matrix using redox initiating system. Macromol Res 2003;11:14–8. [10] Murugan R, Rao KP. Graft polymerization of glycidylmethacrylate onto coralline hydroxyapatite. J Biomater Sci Polym Edn 2003;14: 457–67. [11] Murugan R, Rao KP. Controlled release of antibiotic from surface modified coralline hydroxyapatite. Trends Biomater Artif Organs 2002;16:43–5. [12] Murugan R, Ramakrishna S. Bioresorbable composite bone paste using polysaccharide based nano hydroxyapatite. Biomaterials 2004;25:3829–35. [13] Bigi A, Cojazzi G, Panzavolta S, Ripamonti A, Roveri N, Romanello M, et al. Chemical and structural characterization of the mineral phase from cortical and trabecular bone. J Inorg Biochem 1997;68:45–51. [14] LeGeros RZ. Effect of carbonate on the lattice parameters of apatite. Nature 1965;206:403–4. [15] Kivrak N, Tas AC. Synthesis of calcium hydroxyapatite–tricalcium phosphate composite bioceramic powders and their sintering behavior. J Am Ceram Soc 1998;81:2245–52. [16] Murugan R, Ramakrishna S. Effect of zirconia on the formation of calcium phosphate bioceramics under microwave irradiation. Mater Lett 2003;58:230–4.
206
R. Murugan, S. Ramakrishna / Acta Biomaterialia 2 (2006) 201–206
[17] Murugan R, Kumar TSS, Rao KP. Fluorinated bovine hydroxyapatite: preparation and characterization. Mater Lett 2002;57:429–33. [18] Murugan R, Rao KP, Kumar TSS. Microwave synthesis of bioresorbable carbonated hydroxyapatite using goniopora. Bioceramics 2002;15:51–4. [19] Murugan R, Ramakrishna S. Bioactive nanomaterials in bone grafting and tissue engineering. In: Nalwa HS, editor. Handbook of nanostructured biomaterials and their applications in nanobiotechnology. Stevenson Ranch, CA: American Scientific Publishers; 2005. p. 141–68.
[20] Murugan R, Ramakrishna S. Aqueous mediated synthesis of bioresorbable nanocrystalline hydroxyapatite. J Cryst Growth 2005;274: 209–13. [21] Elliot JC. Structure and chemistry of the apatites and the other calcium orthophosphates. Amsterdam: Elsevier Science; 1994. [22] Murugan R, Rao KP, Kumar TSS. Heat-deproteinated xenogeneic bone from slaughterhouse waste: physico-chemical properties. Bull Mater Sci 2003;26:523–8. [23] Powder diffraction file #9-432, International Centre for Diffraction Data, Newtown Square, PA, USA.