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Nanocrystalline Hydroxyapatite: Micelle Templated Synthesis and Characterization Yujie Wu and Susmita Bose* School of Mechanical and Materials Engineering, Washington State University, Pullman, Washington 99164 Received December 30, 2004. In Final Form: February 18, 2005 Nanocrystalline calcium phosphate based inorganic, hydroxyapatite (HAp), was synthesized using the dodecyl phosphate micelle system. The surfactant concentration during synthesis played an important role on the final properties of these HAp nanoparticles. A surfactant concentration close to the critical micelle concentration produced the nanoparticles with the highest surface area, with porous less agglomerated morphology. Compacts made of these nanopowders showed between 97 and 98% theoretical density of phase-pure HAp and promoted cell-material interaction when cytotoxicity tests were performed.
Nanocrystalline calcium phosphate based inorganic hydroxyapatite (Ca10(PO4)6(OH)2, HAp) was synthesized using dodecyl phosphate micelle system, where surfactant concentration was found to have an influence on nanoparticle surface area as well as morphology. Compacts made of these nanopowders showed about 98% theoretical density of phase-pure HAp and promoted cell-material interaction. Calcium phosphate based inorganics, especially bioactive HAp, are used as bone substitute material in orthopedic, dental, and maxillofacial applications, due to their chemical and structural similarity to the mineral part of bone.1,2 The major phase in bone is HAp with a calcium-to-phosphorus ratio of 1.67. Although these materials are highly biocompatible, they show very poor strength, as they do not sinter well. As the natural bone is made of nanoscale features, having primary constituent collagen protein and HAp inorganic, it is believed that making nanostructured HAp can improve the properties of synthetic bone, such as bioactivity, densification kinetics due to higher surface area. Hydroxyapatite supported palladium complexes have also been reported as efficient heterogeneous catalysts for the oxidation of alcohol, where the efficiency of a catalyst can be improved by making the catalyst support with nanoscale features with a high surface area.3 Thus, nanoscale HAp is an important inorganic from various applications point of view. Surfactant based template systems have a lot of promise to synthesize nanocrystalline material. They are being explored to synthesize nanoscale inorganics, as they are assumed to be very efficient templates for controlling particle size and shape.4-7 The emulsion technique provides an alternative to other nonconventional synthesis * Corresponding author. E-mail:
[email protected]. (1) (a) Jarcho, M. Clin. Orthop. Relat. Res. 1981, 157, 257. (b) Jarcho, M.; Kay, J. F.; Gumaer, K. I.; Duremus, R. H.; Drobeck, H. P. J. Bioeng. 1977, 1, 79-92. (2) Hench, L. L., Wilson, J., Eds. An Introduction to Bioceramics; Advanced Series is Ceramics, Vol. I; World Scientific: Singapore, 1993. (3) Mori, K.; Yamaguchi, K.; Hara, T.; Mizugaki, T.; Ebitani, K.; Kaneda, K. J. Am. Chem. Soc. 2002, 124, 11572. (4) (a) Pileni, M. P. Nat. Mat. 2003, 2, 145. (b) Pileni, M. P. Langmuir 1997, 13, 3266. (c) Pileni, M. P. J. Phys. Chem. 1993, 97, 6961. (d) Filankembo, A.; Pileni, M. P. J. Phys. Chem. 2000, 104, 5865. (e) Pileni, M. P. Langmuir 2001, 17, 7476-7486. (f) Lisiecki, L.; Filankembo, A.; Sack_Kongehl, H.; Weiss, K.; Pileni, M. P.; Urban, J. Phys. Rev. 2000, B61, 4968-4974. (5) Zarur, A. J.; Hwu, H. H.; Ying, J. Y. Langmuir 2000, 16, 30423049. (6) Althues, H.; Kaskel, S. Langmuir 2002, 18, 7428-7435. (7) Narayan, K. R.; Anderson, M. T.; Brinker, C. J. Chem. Mater. 1996, 8, 1682-1701.
methods of inorganics. Ahn et al. have reported nanostructure processing of hydroxyapatite based bioceramic in the presence of yttria stabilized zirconia, prepared by chemical precipitation.8 From our lab, we have reported reverse micelle mediated synthesis of hydroxyapatite nanopowders with controlled morphology, where the nanoparticle morphology was changed from needle shape to spherical depending on synthesis parameters.9 We have also reported the synthesis of other inorganic nanoparticles with different morphology, such as free-standing versus agglomerated, by controlling different synthesis parameters.10,11 In the present study, we are reporting synthesis and processing of hydroxyapatite nanopowder using the surfactant molecular templating approach and effect of surfactant concentration on the final properties of HAp nanoparticles. Hydroxyapatite (HAp, Ca10(PO4)6(OH)2) nanopowders were synthesized from the aqueous solutions of calcium nitrate and phosphoric acid using the micelle templated route in which mono-dodecyl phosphate was used as the surfactant. The surfactant concentration was varied to study its influence on surface area and densification behavior of HAp nanoparticles. Bioactivity studies in presence of modified human osteoblast (HOB) cells showed that these inorganics supported osteoblast cell growth. HAp nanopowders were synthesized using the micelle as a template system where mono-dodecyl phosphate ((C12H25O)P(O)(OH)2, Alfa Aesar) was used as the surfactant. The effect of the surfactant on the BET specific average surface area of the nanoparticle was studied by varying the surfactant concentration as 0.01, 0.02, and 0.03 M. Calcium nitrate ((Ca(NO3)24H2O, Alfa Aesar) and phosphoric acid (H3PO4 85%, Fisher) were used as calcium and phosphorus sources, respectively. Aqueous solutions of calcium and phosphorus precursors were prepared by dissolving 0.01 mol (2.3615 g) of Ca(NO3)24H2O and 0.006 mol (0.685 g) of H3PO4 into 2 mL of deionized water. The calcium and phosphorus precursor solutions were added into the surfactant solution, and the pH of the solution was adjusted to 7 by dropwise addition of NH4OH solution. The resulting mixture was aged overnight and dried on a hotplate for 2-3 h to obtain a white mass. This mass was grinded and heat-treated at (8) Ahn, E. S.; Gleason, N. J.; Nakahira, A.; Ying, J. Y. Nano Lett. 2001, 1 (3), 149. (9) Bose, S.; Saha, S. K. Chem. Mater. 2003, 15 (23), 4464-4469. (10) Banerjee, A.; Bose, S. Chem. Mater. 2004, 16 (26), 5610-5615. (11) Bose, S.; Banerjee, A. J. Am. Ceram. Soc. 2004, 87 (3), 487-489.
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450 °C for 1 h which produced white HAp nanopowders. As-synthesized nanopowders were characterized using X-ray diffraction analysis for their phase purity, BET analysis for their specific average surface area, and transmission electron microscopy (TEM) for their particle size and morphology. Densification of any material depends on the powder properties such as particle size, distribution, and morphology.12 The compacts (1.85 mm thick × 13 mm diameter) for densification studies were prepared by pressing 0.45 g of HAp nanopowders using a dry pressing die. They were densified in the furnace air environment at 1250 °C for 1, 2, 3, and 5 h and at 1000, 1050, 1100, 1150, and 1200 °C for 3 h to study the effect of time and temperature on densification, respectively. Some of these compacts were used for cytotoxicity tests using modified human osteoblast (HOB) cell lines. X-ray diffraction (XRD) analysis showed that the synthesized nanopowders had a phase-pure HAp crystalline structure, as shown in Figure 1. By using Scherrer’s equation, from the line-broadening data, the calculated crystallite size was 18 nm.13 Little broadness of the peaks in plots B and C compared to plot A can explain the smaller particle and crystallite size in the former. The specific average surface area of nanopowders was measured from N2 adsorption isotherms using a fully automated micromeritics Tristar 3000 BET surface area analyzer. Prior to the analysis, the adsorbed moisture on the fine powder surface was eliminated by preheating the powders at 350 °C for 3 h. Figure 2 shows the BET surface area in meter square per gram and the effect of various surfactant concentrations during synthesis on the final BET surface area of HAp nanoparticles. It was observed that a 0.02 M
surfactant concentration gave the highest surface area in the final HAp powders, 52 m2/g. Due to the addition of NH4OH, the HAp precipitation occurred according to the following reaction: 10Ca(NO3)2 + 6H3PO4 + 20NH4OH ) Ca10(PO4)6(OH)2 + 20NH4NO3 + 18 H2O. This resulting HAp precipitate interacts with the micelles to form a gel-like mass by electrostatic interaction. This leads to the formation of an organized structure, where the HAp precipitate is templated by micelles and solidified to form a matrix. During heat treatment, the micelle template decomposes into gases, such as CO2 and H2O, and leaves pores in the product. This type of mechanism is known in the silica based inorganic system.14 The surface area of the final powders is controlled by the particle size as well as the pore size and shape, which are determined by the micelle structure during powder preparation. In general, the micelle geometry is controlled by the concentration of the surfactant in solution and by the processing conditions. At different concentrations, the surfactant molecules aggregate into different forms, which influence the pore structure and surface area. It is reported that the critical micelle concentration (cmc) of a C12-chain anionic surfactant, such as sodium dodecanoate, is 2.2 × 10-2 M.15 This is close to the concentration 0.02 M of the present system studied, which is also a C12-chain anionic surfactant. Thus, once the concentration of surfactant reached the cmc, the micelle formed, which acted as a template for the HAp precipitate and provided the highest surface area in the final powders after calcination. A transmission electron microscope was used to support our findings on the BET surface area and to analyze the particle size and morphology of HAp nanopowder particles. Figure 3a shows a more porous morphology compared to Figure 3b which has a more agglomerated morphology. Porosity in nanoparticles is responsible for the higher surface area in HAp when synthesized with a surfactant concentration of 0.02 M. This porosity is also responsible for giving a lower green density in the compacts made of the former powder. The green densities of compacts made of HAp nanopowders synthesized using 0.01, 0.02, and 0.03 M surfactant were 2.1, 1.7, and 1.9 g/cm3, respectively. They represented BET surface areas of 21, 52, and 38 m2/g. The lowest green density in powders made with 0.02 M surfactant is attributed to inefficient compaction due to higher amount of porosity in the sample. TEM micrographs of HAp nanopowders synthesized using 0.02 M surfactant also showed the final particle size was between 60 and 80 nm with platelet morphology or lower aspect ratio (length-to-width ratio). Average aspect ratio of these nanoparticles varied between 1 and 2.2. Figure 3b shows the TEM micrograph of HAp nanopowders synthesized using 0.03 M surfactant. Both 0.01 and 0.03 M surfactant mediated synthesized nanoparticles showed apparent agglomerates and a higher aspect ratio morphology, aspect ratio between 2 and 4. Agglomeration in powders can result in lower densification ability and cause cracklike voids after densification. Thus, this difference in particle morphologies indicates that 0.02 M surfactant gave a better template effect on the HAp precipitate than the other two concentrations. Figure 3c shows the scanning electron microscopy (SEM) picture of the HAp compacts in presence of modified human osteoblast (HOB) cells after 4 days, showing the adhesion and cell-cell communication by the long extension on the HAp surface.
(12) Jarcho, M.; Bolen, C. H.; Thomas, M. B.; Bobick, J.; Kay, J. F.; Doremus, R. H. J. Mater. Sci. 1976, 11, 2027. (13) Moore, D. M.; Reynolds, R. C. X-ray diffraction and the identification and analysis of clay minerals; 1997; p 26.
(14) Narayan, K. R.; Anderson, M. T.; Brinker, C. J. Chem. Mater. 1996, 8, 1682-1701. (15) Kalyanasundaram, K. Photochemistry in microheterogeneous system; Academic Press: 1987; p 26.
Figure 1. X-ray plots for (A) commercial micron sized HAp powders and synthesized HAp nanopowders using a 0.02 M surfactant concentration calcined for 1 h at (B) 450 °C and (C) at 550 °C.
Figure 2. Surface area of HAp nanopowders synthesized using 0.01, 0.02, and 0.03 M surfactant concentrations after calcinations at 450 °C.
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Figure 3. TEM picture of as-synthesized HAp nanopowder (a) using 0.02 M surfactant and (b) using 0.03 M surfactant after heat treating at 450 °C for 1 h; (c) cytotoxicity test using HOB cells with the HAp compacts after 4 days.
Figure 4. Final density for compacts made of HAp nanopowders (a) vs surfactant concentration and (b) vs surface area, densified at 1250 °C for 3 h.
The compacts made of HAp nanopowders prepared using 0.01, 0.02, and 0.03 M surfactant were densified or sintered simultaneously in a muffle furnace at 1250 °C for 3 h. Both BET and sintering densities of samples were measured for at least three samples in each case, and the error range was between 1 and 5%. The standard temperature for densification of HAp without getting any phase transformation is 1250 °C. Figure 4a shows density of the HAp compacts made of nanopowders synthesized using different surfactant concentrations. The compacts made of powders using 0.02 M surfactant gave the highest density 97% (3.05 g/cm3) compared to the theoretical density (3.16 g/cm3) of HAp. Large particle size and wide particle size distribution along with hard agglomerates exhibits lower densification in HAp, and often achieving 80% densification in pure HAp is a critical challenge.16 Almost 98% densification was achieved after heat-treating these compacts made of synthesized HAp nanopowders at 1250 °C for 5 h. The relative density of the compact made with synthesized HAp nanopowders using 0.01 M surfactant had the lowest sintering density of 70% theoretical density. The difference in density is the result of the different surface areas of nanopowders, as shown in Figure 4b, which was primarily attributed to the surfactant concentration used during synthesis. HAp nanopowders made of 0.02 M surfactant as the template had the highest surface area, which served as the most (16) (a) Murray, M. G. S.; Wang, J.; Ponton, C. B.; Marquis, P. M. J. Mater. Sci. 1995, 30, 3061-3074. (b) Ruys, A. J.; Wei, M.; Sorrel, C. C.; Dickson, M. R.; Brandwood, A.; Milthpore, B. K. Biomaterials 1995, 16, 409-415.
efficient driving force during densification to give the highest sintering density. Thus, though these powders gave the lowest green density, the highest sintering density was due to higher surface area and lower aspect ratio in these powders. Lower aspect ratio and less agglomeration in nanoparticles help to achieve better densification ability. HAp nanopowders synthesized with 0.03 M surfactant showed better densification ability than those using 0.01 M surfactant because the former had a higher surface area. We have demonstrated the synthesis of phase-pure nanocrystalline hydroxyapatite inorganic using the dodecyl phosphate ((C12H25O)P(O)(OH)2) micelle system, where the surfactant concentration was found to have an influence on nanoparticle surface area as well as morphology. At the 0.02 M surfactant concentration, HAp nanoparticles showed the highest surface area 52 m2/g, which gave the highest density, 97-98% of the theoretical density, in the HAp compacts. They also supported cellmaterial interaction. TEM study showed that the final HAp nanoparticle size was between 60 and 80 nm with platelet morphology. Acknowledgment. The authors would like to thank the National Science Foundation for the financial support under the Presidential CAREER Award for Scientists and Engineers (PECASE) to Dr. S. Bose (CTS # 0134476) for this work. We also like to acknowledge the experimental help from Jessica Moore with the cytotoxicity tests. LA046754Z
