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Synthesis and Characterization of Grafted Nanohydroxyapatites Using Functionalized Surface Agents Saba Haque,† Ihtesham Rehman,† and Jawwad A. Darr*,‡ Department of Materials, Queen Mary UniVersity of London, Mile End Road, London, United Kingdom E1 4NS, and Department of Chemistry, UniVersity College London, Christopher Ingold Laboratories, 20 Gordon Street, London WC1H 0AJ ReceiVed December 5, 2006. In Final Form: March 14, 2007 Synthetic hydroxyapatite, HA [Ca10(PO4)6(OH)2], is a bioactive material that is chemically similar to biological apatite, the mineral phase of bone (a nanocomposite material). Synthetic biocomposites, comprising a polymer and hydroxyapatite that are used for bone replacement, have limitations when loaded under fatigue in that the weak mechanical bond between the two phases can result in failure at the interface. Chemical coupling of the HA and polymer matrix may provide a means of improving the interfacial bonding between the polymer and HA phases. Herein, we report our first steps toward developing chemically coupled nano-biocomposites via a two-step process. We describe the synthesis and characterization of surface-grafted hydroxyapatite (SG-HA), which possesses a reactive CdC functional group. In future work, we will report on the second step, namely the coupling of this functional group to a polymer by a copolymerization reaction to give a chemically coupled nano-biocomposite. The SG-HA reported herein was characterized by a range of methods including 31P and 13C magic-angle spinning (MAS)-NMR, Fourier transform infrared (FTIR), and Raman spectroscopy.
Introduction Bone is a specialized tissue comprising mineral substances, organic tissues, and water.1 Synthetic bone substitutes have been developed to restore bone that has been lost due to disease or fracture, since there is insufficient availability of autografts (bone harvested from the patient).2-4 Cortical bone is largely a composite of collagen I fibers and biological apatite. Synthetic hydroxyapatite, HA [Ca10(PO4)6(OH)2], is a bioactive material that is chemically similar to biological apatite.5 HA has also been used as a bioactive phase in composites, coatings on metal implants, and granular filler for direct incorporation into human tissues.6 The inorganic component of bone (bone mineral) is an ill-defined calcium phosphate that contains up to 8 wt % carbonate. Substitution of carbonate or other ions into HA can occur in two distinct atomic sites in the lattice.7 These ions can partially substitute in the lattice for hydroxyl ions (OH-), known as the A-site, and/or for phosphate ions (PO43-), known as the B-site.1 Several HA-polymer biocomposites have been developed to mimic the biological and mechanical properties of bone.8 These include 40 vol % HA-reinforced high-density polyethylene (HDPE), commercially known as HAPEX, which was used as * To whom correspondence should be addressed. Telephone: +44 207 679 1003. Fax: +44 207 679 7463. E-mail:
[email protected]. Website: http://www.qmul.co.uk. † Queen Mary University of London. ‡ University College London. (1) Ravaglioli, A.; Krajewski, A. Bioceramics: Materials, Properties, Applications; Chapman & Hall: New York, 1992. (2) Fricain, J. C.; Bareille, R.; Ulysse, F.; Dupuy, B.; Amedee, J. J. Biomed. Mater. Res. 1998, 42, 96-102. (3) Moghadam, H. G.; Sandor, G. K. B.; Holmes, H. H. I.; Clokie, C. M. L. J. Oral Max. Surg. 2004, 62, 202-213. (4) Monchau, F.; Lefe`vre, A.; Descamps, M.; Belquin-Myrdycz, A.; Laffargue, P.; Hildebrand, H. F. Biomol. Eng. 2002, 19, 143-152. (5) Rehman, I.; Smith, R.; Hench, L. L.; Bonfield, W. J. Biomed. Mater. Res. 1995, 29, 1287-1294. (6) Suchanek, W. L.; Shuk, P.; Byrappa, K.; Riman, R. E.; TenHuisen, K. S.; Janas, V. F. Biomaterials 2002, 23, 699-710. (7) Gibson, I. R.; Bonfield, W. J. Biomed. Mater. Res. 2002, 59, 697-708. (8) Liu, Q.; de Wijn, J. R.; Bakker, D.; van Toledo, M.; van Blitterswijk, C. A. J. Mater. Sci.: Mater. Med. 1998, 9, 23-30.
a trimmable shaft in middle ear implants by Smith and Nephew in 1995.9-11 Other than HDPE, a range of polymers have been used in biomedical composites, including poly(methyl methacrylate), polyurethanes, polyethylene, and poly(lactic acid) (PLA).12 A key factor in the failure of composites occurs at the interface between the HA particles and polymer matrix, usually due to a weak mechanical bond between the two phases. The bonding mechanism is often simply a mechanical interlock, formed during cooling after manufacture.13 Chemical coupling of the HA to a polymer matrix provides a means of improving interfacial bonding in a composite.13,14 The principle is to generate reactive bonding sites on the surface of the filler particle, which can be used to form bonds between the filler and polymer. Tanaka et al.15 modified the surface of HA using alkyl phosphates, while D’Andrea and Fadeev16 used alkyl phosphonic acids. Bisphosphonates have been widely used for medical imaging and therapy, as well as for reducing prosthetic migration in knee prostheses.17,18 Silane coupling agents have (9) Dornhoffer, J. L. Laryngoscope 1998, 108, 531-536. (10) Tanner, K. E.; Davies, G. W.; Bonfield, W. In Ceramics, Cells and Tissues: Ceramic-Polymer Composites; Ravaglioli, A., Krajewski, A., Eds.; Istituto di ricerche tecnologiche per la ceramica del CNR: Faenza, Italy, 1997; pp 85-90. (11) Tanner, K. E.; McGregor, W. J.; Ton That, P. T.; Ward, I. M.; Bonfield, W. Fatigue From HAPEX - a Structural Bone Replacement Material, Structural Biomaterials for the 21st Century; TMS (The Minerals, Metals & Materials Society): London, 2001; pp 229-237. (12) Ramkrishna, S.; Mayer, J.; Wintermantel, E.; Leong, K. W. Compos. Sci. Technol. 2001, 61, 1189-1224. (13) Bonfield, W.; Wang, M.; Tanner, K. E. Acta Mater. 1998, 46, 25092518. (14) Phillips, M. J. Chemical coupling in biocomposites: surface modification of bioceramics creating chemically bound polymer composites with potential osteological applications. Ph.D. Thesis, Queen Mary University of London, U.K., 2004. (15) Tanaka, H.; Yasukawa, A.; Kandori, K.; Ishikawa, T. Langmuir 1997, 13, 821-826. (16) D’Andrea, S. C.; Fadeev, A. Y. Langmuir 2003, 19, 7904-7910. (17) Hilding, M.; Ryd, L.; Toksvig-Larsen, S.; Aspenberg, P. Acta Orthop. Scand. 2000, 71, 553-557. (18) Gibson, A. M.; Mendizabal, M.; Pither, R.; Pullan, S. E.; Griffiths, D. V.; Duncanson, P. Radiolabelled bisphosphonates and method. Eur. Patent WO 01/09146, 2001.
10.1021/la063517i CCC: $37.00 © 2007 American Chemical Society Published on Web 05/05/2007
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Figure 1. Coupling agents used for hydroxyapatite grafting: (a) methacrylic acid (MA), (b) 4-pentenoic acid (PA), and (c) vinyl phosphonic acid (VPA).
been used to bind polyethylene to HA, but such linkages are believed to be ultimately unstable in an aqueous environment.19 Yoshida and Greener20 investigated the use of adhesion promoters to improve the mechanical properties of ceramic-polymer composites but with limited success. Other coupling methods include polyacid adsorption [RCOOH],8 isocyanate grafting [NCO],21 and phosphonate coupling [RP(O)(OH)2].14,22 Our ultimate objective in this work was to use the controlled chemical binding of surface sites on bioactive ceramics to polymer backbones to produce bioactive composites comparable to cortical bone. Eventually, a large part of the implant will be replaced by new bone and will maintain its mechanical integrity over the duration of the transformation. Herein, we describe our strategy to prepare a credible synthetic bone substitute via a two-step process. Herein, we report the synthesis and characterization of surface-grafted hydroxyapatite (SG-HA), using a surface or coupling agent, which possesses an unsaturated CdC functional group. Three different coupling agents were surface-grafted onto HA particles, namely, methacrylic acid (MA, C4H6O2), 4-pentenoic acid (PA, C5H8O2), and vinyl phosphonic acid (VPA, C2H5O3P). These are shown in Figure 1. In our subsequent work, we intend to try and utilize this CdC functional group in a polymerization step to produce HA that is chemically coupled to a methacrylate polymer (this will be reported in due course). Experimental Section Methacrylic Acid (MA) Grafted Apatite (MA-HA). The basic reaction was a modified coprecipitation reaction similar to that reported previously for HA synthesis.14,22 Carboxylate groups (from MA) were expected to partially substitute phosphate groups on the surface of the calcium apatite; thus, reactant quantities were adjusted to account for this substitution, such that the Ca/(PO4 + RCO2-R) molar ratio was 1.67, using a similar calculation to that described by Gibson et al.7 The following amounts were measured before mixing: 59.0 g (0.25 mol) of calcium nitrate tetrahydrate [CNT, Ca(NO3)2‚4H2O], 13.2 g (0.1 mol) of diammonium hydrogen phosphate [DAHP, (NH4)2HPO4], and 4.24 mL (0.05 mol) of MA. The CNT suspension was prepared in 450 mL of deionized water (dH2O) and stirred for 10 min. The pH of the suspension was adjusted to 11 using 1 mL of aqueous ammonium hydroxide (NH4OH). The DAHP solution was prepared in 800 mL of dH2O and stirred for 10 min. The pH of the solution was adjusted to 11 using a total of 45 mL of aqueous NH4OH, followed by the addition of MA. Under stirring, the DAHP/MA mixture was added dropwise to the CNT suspension from a glass dropping funnel over a period of 120 min. The pH was continuously monitored throughout and was maintained at pH 11 using a total of 10 mL of aqueous NH4OH, added dropwise over this time period. The suspension was then stirred for 2 h before being left to age for a further 18 h. The resulting precipitate was filtered and then washed with 4 × 250 mL aliquots of dH2O before being freeze-dried at ∼1.3 × 10-4 bar for 18 h to obtain a very fine, flowing powder. This sample is hereafter referred to as “MA-grafted (19) Harper, E. J.; Braden, M.; Bonfield, W. J. Mater. Sci.: Mater. Med. 2000, 11, 491-497. (20) Yoshida, K.; Greener, E. H. J. Dent. 1994, 22, 57-62. (21) Liu, Q.; de Wijn, J. R.; van Blitterswijk, C. A. J. Biomed. Mater. Res. 1998, 40, 358-364. (22) Darr, J. A.; Phillips, M. J.; Wilson, K.; Rehman, I.; Griffiths, V.; Duncanson, P. AdV. Appl. Ceram. 2005, 104, 261-267.
Figure 2. X-ray diffraction (XRD) patterns in the 2θ range 20-40° of (a) vinyl phosphonic acid grafted HA (sample VPA-HA), (b) 4-pentenoic acid grafted HA (sample PA-HA), and (c) methacrylic acid grafted HA (sample MA-HA). An apatite-like phase was observed in each case with a good match to the line pattern for hydroxyapatite (JCPDS 09-0432). Miller indices of the diffraction peaks are given in parentheses. HA” (MA-HA). The MA-HA powder was then heat-treated in an air furnace [Carbolite Furnaces 1600] at 400, 500, 600, 700, and 800 °C for 2 h, with a heating rate of 2.5 °C min-1 in each case. Hereafter, these samples are referred to as MA-HA(X) where X ) heat-treatment temperature in °C. 4-Pentenoic Acid (PA) Grafted Apatite (PA-HA). Briefly, aqueous solutions of 13.2 g (0.1 mol) of DAHP and 5.08 mL (0.05 mol) of PA were mixed for 10 min and then dropwise added to 59.0 g (0.25 mol) of CNT in solution. Aqueous NH4OH was added, as for MA-HA, to adjust the pH to 11. After filtering and washing as described above, the fine, flowing powder after freeze-drying is hereafter referred to as “PA-grafted HA” (PA-HA). Vinyl Phosphonic Acid (VPA) Grafted Apatite (VPA-HA). Briefly, the pH of 59.0 g (0.25 mol) of aqueous CNT was adjusted to 11 using 1 mL of aqueous NH4OH. Aqueous solutions of 13.2 g (0.1 mol) of DAHP and 5.4 g (0.05 mol) of VPA were mixed for 10 min and, without any pH adjustment, were dropwise added to the CNT suspension. The pH was continuously monitored throughout the dropwise addition and maintained at pH 9 using NH4OH (20 mL in total was added). After filtering and washing as described above, the fine, flowing powder after freeze-drying is hereafter referred to as “VPA-grafted HA” (VPA-HA). Reference Hydroxyapatite. HA (27.0 g) was prepared according to a modified method of Sung et al.23 At the end of the precipitation reaction, the suspension was stirred for 2 h before being left to age for a further 18 h. The resulting precipitate was filtered and then washed with 4 × 250 mL aliquots of dH2O before being freeze-dried at 1.3 × 10-4 bar for 18 h to obtain a fine, flowing powder. Hereafter, the powder is referred to as “as-prepared HA powder”. Characterization. The phase composition and purity of the samples were investigated by powder X-ray diffraction (XRD) using a Siemens/Bruker D5000 diffractometer. Sample phases were identified by comparing their XRD patterns to the Joint Committee on Powder Diffraction Standards (JCPDS) database. Samples were also characterized using Fourier transform infrared (FTIR) and Raman spectroscopies, 31P and 13C magic-angle spinning nuclear magnetic resonance (MAS-NMR) spectroscopy, simultaneous thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The specific surface area (SSA) of the powders was determined using Brunauer-EmmettTeller (BET) nitrogen adsorption.
Results and Discussion The X-ray diffraction (XRD) patterns of the surface-grafted hydroxyapatites (SG-HA, Figure 2) displayed broad peaks, (23) Sung, Y. M.; Lee, J. C.; Yang, J. W. J. Cryst. Growth 2004, 262, 467472.
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Figure 3. Fourier transform infrared (FTIR) spectra in the regions (a) 3800-3200 cm-1 and (b) 1800-400 cm-1 showing comparisons of (i) hydroxyapatite (HA), (ii) vinyl phosphonic acid grafted HA (sample VPA-HA), (iii) 4-pentenoic acid grafted HA (sample PA-HA), and (iv) methacrylic acid grafted HA (sample MA-HA).
suggesting an apatite structure. After heat treatment, the XRD data for sample MA-HA(600) suggested that a phase-pure apatite structure had been retained (Figure S1 in the Supporting Information). Samples MA-HA(700) and MA-HA(800) revealed increasing amounts of calcium oxide phase impurity (CaO) were formed, as shown by a peak at 2θ ) 37.4° (Figure S1(e-f) in the Supporting Information). This suggested that the materials were calcium rich as expected after removal of the surface organics. The Fourier transform infrared (FTIR) spectra of as-prepared HA and all three as-prepared SG-HAs are shown in Figure 3. The spectra were similar in that all four showed phosphate stretching vibrations (υ3 and υ1) and bending modes (υ4 and υ2). The peaks for asymmetric stretching (υ3) due to phosphate bonds (P-O) were centered at 1093 and 1023 cm-1, the peak for the symmetric stretching of phosphate (υ1) was centered at 961 cm-1, and the peaks for the O-P-O bending of phosphate (υ4) were centered at 602 and 560 cm-1, as well as a weak peak due to phosphate bending (υ2) [at 470 cm-1] (for all SG-HAs).5 Also, a low-intensity peak corresponding to the stretching of hydroxyl groups centered at 3573 cm-1 was observed (Figure 3a), suggesting substitution of hydroxyl groups by carboxylate groups (A-type substitution) had not occurred. The peak for CdC stretching from the coupling agents was observed at ∼1640 cm-1 in the SG-HAs, and it overlapped with the peak for the bending of molecular water, usually seen at ∼1650 cm-1. Since both vibrations occurred in the same wavenumber region and both were of low intensity, it was difficult to definitely assign this peak to either mode. In the FTIR data for MA-HA and PA-HA (Figure 3b), there were additional peaks due to the symmetric (υ3) and asymmetric (υ8) stretching of the C-O bonds from the carboxylate groups, centered at ∼1433, 1460, and 1531 cm-1.24 Another peak due to the stretching of the C-O bond (that is conjugated to CdC bond) was observed at 1332 cm-1.25,26 The O-C-O bend (υ2) was centered at 868 cm-1.5 As expected, there were no vibrations from the carboxylate groups in the FTIR spectrum for VPA-HA. Nevertheless, there were subtle differences in the vibrations of the phosphate group for VPA-HA (24) Mehrotra, R. C.; Bohra, R. Metal Carboxylates; Academic Press: London, 1983. (25) Nakanishi, K.; Solomon, P. H. Infrared Absorption Spectroscopy; HoldenDay, Inc.: San Francisco, CA, 1977. (26) Qiu, X.; Hong, Z.; Hu, J.; Chen, L.; Chen, X.; Jing, X. Biomacromolecules 2005, 6, 1193-1199.
Figure 4. Raman spectra in the range 1150-870 cm-1 for (a) asprepared HA powder, (b) vinyl phosphonic acid grafted HA (sample VPA-HA), (c) 4-pentenoic acid grafted HA (sample PA-HA), and (d) methacrylic acid grafted HA (sample MA-HA). The spectra have been normalized such that the υ1 PO43- peak (centered at ∼963 cm-1) remains constant.
(usually 1100 cm-1 for HA), such as the peak for phosphate stretching (υ3), which was located at 1120 cm-1. This suggested that the phosphonate groups from the VPA had been incorporated into the surface phosphate sites of the HA particles. Figure 4 compares the Raman spectra of as-prepared HA and all three SG-HAs in the region of 1150-870 cm-1. In this spectral region, two main bands corresponding to the asymmetric (υ3) and symmetric (υ1) stretching of P-O bonds are usually observed for HA, centered at 1048 and 963 cm-1, respectively. The spectra were normalized such that the υ1 PO43- intensity remained constant.27,28 It can be clearly seen that the relative intensity of the υ1 PO43- band decreased in comparison to the υ3 PO43band, for the MA-HA and PA-HA samples, respectively, suggesting a reduced or at least restricted phosphate vibration, most likely due to its substitution by and/or interaction with carboxylate groups. The corresponding relative intensities for the VPA-HA samples were still comparable to that of HA, since the P-O bonds (from phosphate groups) were effectively substituted by a similar species (from phosphonate groups). However, this change caused a shift in the peak position of υ1 PO43- from 963 cm-1 in HA to 959 cm-1 in VPA-HA. In the (27) Penel, G.; Leroy, G.; Rey, C.; Sombret, B.; Huvenne, J. P.; Bres, E. J. Mater. Sci.: Mater. Med. 1997, 8, 271-276. (28) Penel, G.; Delfosse, C.; Descamps, M.; Leroy, G. Bone 2005, 36, 893901.
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Figure 5. 31P magic-angle spinning nuclear magnetic resonance (MAS-NMR) spectrum of vinyl phosphonic acid grafted HA (sample VPA-HA) showing phosphate (PO43-) and phosphonate [R-P(O)(O)22-] environments.
Raman spectra of all three SG-HAs (Figure S2 in the Supporting Information), a single sharp peak due to υ(CdC) stretching was observed at 1647, 1644, and 1636 cm-1 in MA-HA, PA-HA, and VPA-HA, respectively, but was absent in HA, as expected. Peaks associated with the stretching and bending of υ(C-H) in the dCH, dCH2, and sCH2 groups were observed between 3100 and 2800 cm-1 for all SG-HAs. The presence of the coupling agent for each SG-HA was further validated using solid-state 31P and 13C magic-angle spinning nuclear magnetic resonance (MASNMR) spectroscopy. The 31P MAS-NMR spectrum obtained for VPA-HA (Figure 5, Table 1) revealed two distinct phosphorus environments: a strong peak at δ ) 2.9 ppm due to the phosphate phophorus (PO43-) of HA14,29,30 and a weaker, broad peak at δ ) 11.9 ppm due to the phosphonate phophorus. The free phosphonic acid group [RsP(O)(OH)2, where R is CH2dCH] has been reported elsewhere in the range δ ) 16.0-18.6 ppm,31,32 and its upfield shift to δ ) 11.9 ppm indicated the movement of electrons toward the phosphonate phosphorus, causing a higher shielding effect. This was attributed to the bonding of VPA to the calcium ions in HA.14,33 Bakhmutova-Albert et al.34 also reported an upfield shift (of δ ) 2.0 ppm) in the 31P MAS-NMR spectrum for the free phosphonic acid, upon the formation of a metal-phosphonate compound. The 13C MAS-NMR trace for MA-HA (Figure S3 in the Supporting Information, Table 1) displayed peaks at δ ) 17.4, 139.5, and 182.6 ppm due to the CH3, CH2dC(CH3)COO-, and COO- carbons (C’s in italic), respectively, as expected.35-38 The carboxylate C peak was located approximately δ ) 9.1 ppm downfield compared to that observed in solution,39 suggesting (29) Isobe, T.; Nakamura, S.; Nemoto, R.; Senna, M.; Sfihi, H. J. Phys. Chem. B 2002, 106, 5169-5176. (30) Legrand, A. P.; Sfihi, H.; Bouler, J. M. Bone 1999, 25, 103S-105S. (31) Braybrook, J. H.; Nicholson, J. W. J. Mater. Chem. 1993, 3, 361-365. (32) Greish, Y. E.; Brown, P. W. Biomaterials 2001, 22, 807-816. (33) Christian, G. D.; O’Reilly, J. E. Instrumental Analysis; Allyn and Bacon: London, 1986. (34) Bakhmutova-Albert, E. V.; Bestaoui, N.; Bakhmutov, V. I.; Clearfield, A.; Rodriguez, A. V.; Llavona, R. Inorg. Chem. 2004, 43, 1264-1272. (35) National Institute of Advanced Industrial Science and Technology (AIST). Spectral database for organic compounds. SDBS Web. http://www.aist.go.jp/ RIODB/SDBS/ (accessed October 15, 2006). (36) Apperley, D. Personal Communication, 2005. (37) Levy, G. C.; Lichter, R. L.; Nelson, G. L. 13C NMR Spectroscopy; WileyInterscience: New York, 1980. (38) Dı´ez-Pen˜a, E.; Quijada-Garrido, I.; Barrales-Rienda, J. M.; Wilhelm, M.; Spiess, H. W. Macromol. Chem. Phys. 2002, 203, 491-502. (39) Breitmaier, E.; Voelter, W. 13C NMR Spectroscopy: Methods and Applications; Verlag Chemie: Weinheim, Bergatr, 1974.
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that bonding to HA had occurred. This shift was not due to the dimerization of carboxylic acids, since the infrared peak for a COOH dimer (an intense peak usually observed at 1695 cm-1) was not observed in the FTIR spectrum of MA-HA (Figure 3). The 13C MAS-NMR peak due to the CH2dC(CH3)COO- carbon atom after the binding of MA on HA had shifted downfield by δ ) 3.4 ppm.35 The CH2dC(CH3)COO- carbon was rather weak, and an assignment could not be definitely made from the background noise. The 13C MAS-NMR trace for PA-HA (Figure S4 in the Supporting Information, Table 1) revealed peaks at δ ) 186.3, 137.9, 112.7, 29.2, and 36.5 ppm due to the COO-, dCH-, dCH2, CH2CH2COO-, and CH2COO- carbons, respectively.35,37 Grafting onto the surface of HA was again suggested by a downfield shift of the carboxylate carbon (by δ ) 6.5 ppm) to δ ) 186.3 ppm and a smaller downfield shift (δ ) 3.0 ppm) of the CH2COO- carbon (Figure S4 in the Supporting Information, Table 1). These downfield shifts of the carboxylate carbons and neighboring carbon atoms (in samples MA-HA and PA-HA) can be correlated with 13C MAS-NMR studies40-42 of various carboxylic acids, in which the carboxylate carbon also shifted downfield upon deprotonation. Scanning electron microscopy (SEM) showed that MA-HA and PA-HA particles (Figure 6a and b) were generally angular and “blocklike”. BET surface areas of the MA-HA and PA-HA powders were found to be 185 and 113 m2 g-1, respectively. In comparison, the VPA-HA particles (Figure 6c) were spherical and microporous, with very fine, needlelike crystallites with SSA ) 199 m2 g-1 and an average particle size of 67 ( 12 nm (Figure 7c). The average MA-HA and PA-HA particle sizes were 40 ( 6 nm and 32 ( 5 nm, respectively, as observed from the transmission electron microscopy (TEM) images (Figure 7a and b). The thermal behavior of HA and all three surface-grafted HAs was assessed using simultaneous thermal analysis (STA) in the range 30-1100 °C (in air). The thermogravimetric analysis (TGA) weight loss plot for as-prepared HA (Figure S5 in the Supporting Information; shown in the range 200-800 °C) showed a gradual weight loss (8.7 wt % between 200 and 800 °C) due to the lattice water and carbonate (a small amount of carbonate was possibly incorporated into the HA lattice during precipitation). Lattice water can be lost by 550 °C, and carbonate decomposition begins at ∼650 °C in HA. The TGA plot for as-prepared MA-HA (Figure S6 in the Supporting Information) showed weight loss in the range 251-365 °C (3.3 wt % loss of the MA grafting agent). A sharp exotherm centered at 273 °C was observed in the differential scanning calorimetry (DSC) plot, corresponding to this weight loss. The weight loss for sample MA-HA in the range 365-700 °C (5.2 wt %) was also attributed to the loss of lattice water and carbonate. Above 700 °C, the weight loss was due to the gradual decomposition into HA and CaO (as shown by the XRD patterns of heat-treated MA-HA; see Figure S1 in the Supporting Information). The TGA plot for as-prepared PA-HA (Figure S7 in the Supporting Information) showed a small weight loss in the range 239-370 °C (2.2 wt % loss of the PA grafting agent). A sharp exotherm centered at 265 °C was observed in the DSC plot, which corresponded to this weight loss. The weight loss from sample PA-HA above 370 °C was attributed to the loss of lattice water and carbonate. In contrast to the heat treatment of sample MA-HA, the PA-HA sample did not appear to phase separate into HA and CaO until at least 1020 °C (as shown by (40) Gu, Z.; McDermott, A. J. Am. Chem. Soc. 1993, 115, 4282-4285. (41) Rabenstein, D. L.; Sayer, T. L. J. Magn. Reson. 1976, 24, 27-39. (42) Surprenant, H. L.; Sarneski, J. E.; Key, R. R.; Byrd, J. T.; Reilley, C. N. J. Magn. Reson. 1980, 40, 231-243.
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Table 1. NMR Peak Shifts for the 31P Nucleus in Sample VPA-HA and the 13C Nucleus in Samples MA-HA and PA-HA nucleus
sample
relevant environment
δ (ppm)c
reported δ (ppm)d
VPA-HA
phosphate (PO4 ) organophosphonate [R-P(O)(O)22-]
2.9 11.9
2.8 to 2.9 16.031, 18.632
0 -4.1 to -6.7
13
MA-HA
CH2dC(CH3)COOCH2dC(CH3)COOCH2dC(CH3)COOCH2dC(CH3)COO-
182.6 139.5 17.4 sf
173.535-37,39 136.135-38 17.935-38 127.935-38
+9.1 +3.4 0 s
13
PA-HA
CH2dCHCH2CH2COOCH2dCHCH2CH2COOCH2dCHCH2CH2COOCH2dCHCH2CH2COOCH2dCHCH2CH2COO-
186.3 36.5 29.2 137.9 112.7
179.835,37 33.535,37 28.635,37 136.435,37 115.735,37
+6.5 +3.0 0 0 0
P
Cb
Cb
3-
14,29,30
∆δ (ppm)e
31 a
a Positive shifts indicate deshielding relative to H3PO4. b Positive shifts indicate deshielding relative to TMS. c Observed NMR peak shifts. d Reported NMR peak shifts of free acid in the literature. e ∆δ ) difference between c and d. Negative ∆δ values indicate an upfield shift, while positive ∆δ values indicate a downfield shift. f This peak was too weak to be observed under background (despite long run times).
Figure 6. Scanning electron microscopy (SEM) images showing the powder morphology of (a) methacrylic acid grafted HA (sample MA-HA) [bar ) 100 µm], (b) 4-pentenoic acid grafted HA (sample PA-HA) [bar ) 10 µm], and (c) vinyl phosphonic acid grafted HA (sample VPA-HA) [bar ) 10 µm].
Figure 7. Transmission electron microscopy (TEM) images showing needlelike crystals for (a) methacrylic acid grafted HA (sample MA-HA), (b) 4-pentenoic acid grafted HA (sample PA-HA), and (c) vinyl phosphonic acid grafted HA (sample VPA-HA) [all bars ) 100 nm].
the XRD patterns of heat-treated PA-HA in Figure S9 in the Supporting Information). The TGA weight loss plot for asprepared VPA-HA (Figure S8 in the Supporting Information) showed a small weight loss in the range 285-567 °C (3.8 wt % loss of the VPA grafting agent). A broad exotherm centered at 411 °C was observed in the DSC plot, which corresponded to this weight loss. The weight loss from sample VPA-HA at 567700 °C (2.1 wt %) was attributed to the loss of carbonate. Above 700 °C, VPA-HA began to decompose, eventually forming crystalline β-tricalcium phosphate, β-TCP [Ca3(PO4)2], by 1070 °C (as shown by the XRD patterns of heat-treated VPA-HA; see Figure S10 in the Supporting Information). An exotherm corresponding to this transition was observed in the DSC plot centered at 706 °C. The TGA plots for MA-HA, PA-HA, and
VPA-HA were used to calculate the grafting concentrations in each sample, and they were found to be 1.25, 1.17, and 1.06 molecules nm-2 (equates to 1.8, 1.3, and 0.8 wt % C, respectively), respectively. This compares to a grafting density of ∼2.4 groups nm-2 as reported by D’Andrea and Fadeev16 for alkylphosphonic acids.
Conclusions Three different coupling agents were successfully grafted onto the surface of HA using a modified coprecipitation reaction. Analytical data confirmed the bonding of the coupling agent to the apatite. The presence of unsaturated CdC groups on the coupling agents was confirmed; these are potentially available for further reactions toward forming chemically coupled nano-
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biocomposites with the SG-HAs. In subsequent work, we will endeavor to manufacture such materials, paying particular attention to measuring their mechanical properties such as strength and fracture toughness. Such work is currently being studied in our laboratory and will be reported in due course. Acknowledgment. We thank the following for technical assistance: Dr. Z. Luklinska, R. Whitenstall and M. Willis (EM unit) and V. Ford (CADCAM), J. Caulfield (Technical assistance), and Dr. M. Phillips (Experimental officer). EPSRC is thanked for an Advanced Research Fellowship (J.A.D., Grant No. GR/ A11304) and funding (IR and IRC core grant). The Materials Department at Queen Mary is thanked for a scholarship (S.H.) and funding. We also wish to thank EPSRC for access to the Varian 300 spectrometer and Dr. D. Apperley (Durham University) for MAS-NMR data collection.
Haque et al.
Supporting Information Available: Details of materials and of characterization equipment used. X-ray diffraction patterns of methacrylic acid grafted hydroxyapatite (MA-HA): as-prepared and heated between 400 and 800 °C. Raman spectra in the range 1680-1630 cm-1 for hydroxyapatite (HA) and grafted HA samples. 13C magicangle spinning nuclear magnetic resonance (MAS-NMR) spectrum of sample MA-HA showing the different carbon environments. 13C MASNMR spectrum of 4-pentenoic acid grafted hydroxyapatite (sample PAHA) showing the different carbon environments. Simultaneous thermal analysis (STA) of as-prepared sample MA-HA carried out in air. STA of as-prepared sample PA-HA carried out in air. STA of as-prepared vinyl phosphonic acid grafted hydroxyapatite (sample VPA-HA) carried out in air. This information is available free of charge via the Internet at http://pubs.acs.org. LA063517I
