Crystallographic Study of Hydroxyapatite Bioceramics Derived from Various Sources R. Murugan* and S. Ramakrishna Nanobioengineering Laboratory, NUS Nanoscience and Nanotechnology Initiative, Division of Bioengineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore Received November 22, 2003;
CRYSTAL GROWTH & DESIGN 2005 VOL. 5, NO. 1 111-112
Revised Manuscript Received August 15, 2004
ABSTRACT: Hydroxyapatite (HA) is a calcium phosphate based bioceramic material rich in hard tissues such as bones and teeth. It is used as an artificial bone substitute in orthopaedic and dental applications. It can be derived from synthetic or natural resources. It has all the characteristic features of biomaterials, in particular, crystallographic similarity with natural bone minerals. However, crystallographic properties HA prepared from various resources are considerably different due to the material processing conditions and key ingredients used. In the present study, we report the comparative crystallographic analysis of HA derived from the chemical route, coral and xenogeneic bone. As observed from the powder X-ray diffraction (XRD) analysis, crystallinity of HA was also quite dissimilar with respect to preparation methodology. Each lattice cell parameters of prepared HA were calculated using the standard least-squares method and compared with JCPDS data as a reference model. The results showed that the lattice constants of HA have slight changes from batch to batch due to secondary ionic substitution. Introduction Hydroxyapatite (Ca10(PO4)6(OH)2) is a calcium phosphate salt and being used as an artificial bone material due to its biocompatibility, bioactivity, osteoconductivity, nontoxicity, noninflammatory behavior, and nonimmunogenicity.1 Current preparation methodology of HA chiefly includes conventional wet chemical, solid state, calcination of xenogenic bone, and hydrothermal conversion of calcium carbonate exoskeleton. The properties of HA differ with respect to preparation route, reaction ingredients, etc. Among many properties, crystallographic changes directly affect the bioactivity of HA; thus, it is necessary to study its phase purity and crystallography before intending clinical application. HA crystallizes in a hexagonal system of space group P63/m with cell parameters a ) b ) 9.432 Å and c ) 6.881 Å.2 With this intention, the present work deals with the preparation of HA from synthetic calcium and phosphorus precursors, natural coral exoskeleton, and xenogeneic bone, and their crystallographic and phase purity is compared. Experimental Procedures Method I. HA was prepared from calcium and phosphorus precursors by a wet chemical method as reported earlier.3 Briefly, a 0.3 M aqueous solution of (NH4)2HPO4 was slowly added drop by drop to a 0.5 M aqueous solution of CaCl2 at 60 °C. The minimum pH was adjusted to 10 by adding concentrated NH4OH and aged for 24 h under stirring. After aging, the white precipitated HA (PHA) was filtered, washed with distilled water, and microwave irradiated for 15 min. Method 2. HA was prepared from natural coral exoskeleton as reported earlier.4 Briefly, a stoichiometric amount of coral powder, dibasic calcium phosphate, and deionized water corresponding to HA was mixed homogeneously under hydrothermal conditions. The reaction slurry was microwave irradiated for a few minutes, followed by repeated washing with * To whom correspondence should be addressed.
deionized water, and dried under vacuum. The resultant product is referred as coralline HA (CHA). Method 3. HA was prepared from xenogeneic bone as reported earlier.5 After the macroscopic impurities of bovine cortical bones were cleaned, they were boiled in distilled water for 12 h and degreased by immersing in acetone-ether mixture at a ratio of 3:2 for 24 h. The chemical processed bone samples were then calcined at 900 °C, which is known as bovine HA (BHA). The phase purity and crystallographic studies were performed with a high-resolution powder X-ray diffractometer (Shimadzu XRD-600, Japan) in a Guiner geometry using a monochromatic CuKR radiation at the wavelength of 1.5406 Å. The XRD patterns were recorded between 20 and 60° (2θ) in steps of 0.01° intervals with a 1 s counting time at each step.
Results and Discussion The XRD technique is employed to assess the phase purity and crystallographic changes of the HA bioceramics derived from various sources as described previously. Figure 1 summarizes the XRD patterns of HA prepared in this study. All the samples were of typical apatite crystal structure but have different crystallinity with respect to preparation methodology. Figure 1a shows the XRD pattern of PHA, indicating undifferentiated broad peaks with poor crystallinity around the characteristic region near to 32° (2θ). Actually, it should be as three individual peaks each at ∼31.8, 32.1, and 32.9° (2θ). However, the crystallographic structure of PHA is quite similar to biological apatite.6 The structure of biological apatite is also included for comparison (Figure 1f). The possible reason for the poor crystalline nature of PHA must be emerged from the preparation methodology owing to the low-temperature procedure. A small amount of PHA was heated at 900 °C for 2 h to study its structural changes at higher temperature, and the result exhibits all characteristic diffracted peaks corresponding to the apatite phase with a higher degree of crystallinity (Figure 1b). The high-temperature treat-
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Crystal Growth & Design, Vol. 5, No. 1, 2005
Murugan and Ramakrishna Table 1. Crystallographic Parameters of Various Apatite Samples lattice constants (Å)
Figure 1. XRD patterns of HA derived from various sources. (a) PHA, (b) PHA heated to 900 °C, (c) CHA, (d) CHA heated to 900 °C, (e) BHA, and (f) biological apatite.
ment shapes the diffracted peaks more sharply, in particular, near 32° (2θ), which is a good sign for the improvement in the crystallinity of PHA. There were no extraneous substitutions observed for PHA, which means key ingredients reacted well and produced phase pure HA. The XRD pattern of CHA (Figure 1c) shows characteristic peaks pertaining to apatite phase with poor crystallinity due to microwave processing and resembles the structure of natural bone minerals. The result did not show any extraneous phases corresponding to CaCO3 or CaO, suggesting that the reaction has produced HA with phase purity. The crystallinity of CHA can be increased by being heated at 900 °C under air for 2 h. The respective XRD pattern is illustrated in Figure 1d and indicates well-resolved sharp diffraction peaks, which is a good sign for the improvement of crystallinity. The XRD pattern of BHA (Figures 1e) shows a highly crystalline phase of apatite as compared to Methods 1 and 2 owing to its chemical and thermal processing. However, it shows a peak shift corresponding characteristically to carbonate ions. The presence of carbonate ions must have emerged from the consequence of bovine trace minerals. The natural bone mineral contains a substantial amount of carbonate ions to strengthen bone mechanically. It also confirmed the existence of carbon-
materials
ao ) bo
co
co/ao
unit cell volume (Å3)
PHA by method 1 CHA by method 2 BHA by method 3 HA (JCPDS)
9.419 9.420 9.426 9.418
6.890 6.905 6.894 6.884
0.732 0.733 0.731 0.731
531.80 533.07 532.90 531.22
ate ions from the calculated lattice cell values. Apart from that, there were no additional phases observed for BHA. All the XRD peaks can be hkl indexed based on a hexagonal crystal system of space group P63/m with reference to the JCPDS standard.7 The lattice cell parameters were determined by refining the respective XRD data by the standard least-squares fitting method (Table 1). The calculated cell values of all HA were near to the JCPDS standard but have slight differences among them. The slight differences in the a- and c-axis of BHA can be due to the substitution of carbonate at trace level. The previous results imply that the crystallographic parameters and phase purity of HA depend upon the reaction ingredients and preparation methodology. Conclusions HA was prepared from three different routes, and their phase purity and crystallographical changes observed among them with respect to JCPDS standard were compared. The XRD patterns of all the HA samples prepared in this study show chiefly the apatite phase, and the high temperature treatment promotes their crystallinity considerably. This study suggests that phase purity of HA purely depends on preparation methodology and reaction key ingredients. References (1) LeGeros, R. Z. Calcium Phosphates in Oral Biology and Medicine; Karger: Basel, Switzerland, 1991. (2) Kay, M. I.; Young, R. A.; Posner, A. S. Nature 1964, 204, 1050. (3) Murugan, R.; Ramakrishna, S. Biomaterials 2004, 25, 3829. (4) Murugan, R.; Rao, K. P.; Kumar, T. S. S. Bioceramics 2002, 15, 51. (5) Murugan, R.; Kumar, T. S. S.; Rao, K. P. Mater. Lett. 2002, 57, 429. (6) Murugan, R.; Rao, K. P.; Kumar, T. S. S. Bull. Mater. Sci. 2003, 26, 523. (7) Powder Diffraction File #9-432, International Centre for Diffraction Data (ICDD): Newton Square, PA.
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