Aqueous Microgels for the Growth of Hydroxyapatite Nanocrystals

Mar 26, 2008 - Susann Schachschal, Andrij Pich,* and Hans-Juergen Adler. Department of Macromolecular Chemistry and Textile Chemistry, Technische ...
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Langmuir 2008, 24, 5129-5134

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Aqueous Microgels for the Growth of Hydroxyapatite Nanocrystals Susann Schachschal, Andrij Pich,* and Hans-Juergen Adler Department of Macromolecular Chemistry and Textile Chemistry, Technische UniVersita¨t Dresden, D-01062 Dresden, Germany ReceiVed December 3, 2007. In Final Form: February 11, 2008 In present article, we demonstrate that aqueous microgels can be used as containers for the in-situ synthesis of hydroxyapatite. The hydroxyapatite nanocrystals (HAp NCs) become integrated into microgels forming hybrid colloids. The HAp NCs loaded in the microgel can be varied over a broad range. The HAp NCs are localized within the microgel corona. The deposition of the inorganic nanocrystals decreases the colloidal stability of the microgels and leads to particle aggregation at high HAp NCs loading. Because of the strong interactions between HAp NCs and polymer chains, the swelling degree of microgels decreases, and temperature-sensitive properties disappear at high loading of the inorganic component. We demonstrate that hybrid colloids can be used as building blocks for the preparation of nanostructured films on solid substrates.

1. Introduction The biomimetic preparation of composite materials in synthetic systems received recently strong interest and has stimulated intensive research in this field. Hydroxyapatite (HAp, Ca10(PO4)6(OH)2) belongs to the most important biominerals that can be found in natural hard tissues.1,2 However, hydroxyapatite as well as other calcium phosphates exhibit poor mechanical properties (low elasticity and high brittleness). The combination of HAp with a polymeric matrix leads to hybrid materials exhibiting high flexibility, mechanical strength, and biocompatibility and good processing/shaping.3 The interaction of HAp with the polymeric matrix is realized by covalent bonds, hydrogen bonds,4 dipole-dipole interactions, or the complexation of Ca2+ ions by functional groups such as amine, acetylamine, or hydroxyl.5 In this way, the polymeric matrix plays an important role in the nucleation and growth of HAp crystals and determines their size, morphology, and orientation in the composite material. The most important application fields for hybrid materials containing HAp are tissue engineering,6 implants,3 drug delivery systems,7,8 catalysis,9,10 adsorbents,11 and protein chromatography.12,13 A large variety of polymeric materials have been used as templates for the synthesis of HAp such as protein collagen,14 poly(L-lactic acid),15 poly(aspargic acid),16 alginates,17 gelatine,18 * Corresponding author. E-mail: [email protected]. (1) Mann, S.; Webb, J.; Williams, R. J. P. Biomineralization: Chemical and Biological PerspectiVes; VCH Publishers: New York, 1989. (2) Dorozhkin, S. V.; Epple, M. Angew. Chem. 2002, 114, 3260-3277. (3) Neumann, M.; Epple, M. Eur. J. Trauma 2006, 2, 125-131. (4) Li, Z.; Yubao, L.; Aiping, Y.; Xuelin, P.; Xuejiang, W.; Xiang, Z. J. Mater. Sci.: Mater. Med. 2005, 16, 213-219. (5) Rusu, V. M.; Ng, C. H.; Wilke, M.; Tiersch, B.; Fratzl, P.; Peter, M. G. Biomaterials 2005, 26, 5414-5426. (6) Rodrı´guez-Lorenzo, L. M.; Ferreira, J. M. F. Mater. Res. Bull. 2004, 39, 83-91. (7) Kim, H.-W.; Knowles, J. C.; Kim, H.-E. J. Mater. Sci.: Mater. Med. 2005, 16, 189-195. (8) Barroug, A.; Glimcher, M. J. J. Orthop. Res. 2002, 20, 274-280. (9) Zahouily, M.; Bahlaouan, W.; Bahlaouan, B.; Rayadh, A.; Sebti, S. ArkiVoc 2005, 13, 150-161. (10) Wakamura, M. Fujitsu Sci. Tech. J. 2005, 41/2, 181-190. (11) Kawai, T.; Ohtsuki, C.; Kamitakahara, M.; Tanihara, M.; Miyazaki, T.; Sakaguchi, Y.; Konagaya, S. EnViron. Sci. Technol. 2006, 40, 4281-4285. (12) Felı´cio-Fernandes, G.; Laranjeira, M. Quı´mica NoVa 2000, 23, 441-446. (13) Jungbauer, A.; Hahn, R.; Deinhofer, K.; Luo, P. Biotechnol. Bioeng. 2004, 87, 364-375. (14) Hsu, F.; Tsai, S.; Lan, C.; Wang, Y. J. Mater. Sci.: Mater. Med. 2005, 16, 341-345. (15) Zhang, R.; Ma, P. X. J. Biomed. Mater. Res. 1999, 285-293.

chitosan,4,5,19 chitin,20 dendrimers,21,22 hydrogels,23-26 and so forth. The use of polymeric particles as templates for the growth of HAp nanocrystals has not been intensively studied. Polymeric particles as templates provide several advantages such as uniform size, extremely large surface area, and enormous possibilities for surface functionalization. In this way, well-defined hybrid particles can be prepared for use in chromatography columns or as catalysts in different technical systems. An applicability of polymeric particles coated with Pd0 for the growth of HAp nanocrystals has been demonstrated by Tamai et al.27 The homogeneous growth of the hydroxyapatite layer on the particle surface led to the formation of polystyrene core-HAp shell hybrids. Recently, Sukhorukov and co-workers28 demonstrated the utilization of pH-sensitive polyelectrolyte capsules for the growth of hydroxyapatite nanocrystals and the fabrication of hybrid hollow spheres. The aim of present study was to demonstrate that aqueous microgel particles can be used for the growth of HAp nanocrystals. Because of the porous microgel structure and the presence of functional groups, the HAp nanocrystals can be effectively stabilized in the microgel interior. Obtained hybrid colloids can be used for injectable tissue replacement or as building blocks for the preparation of scaffolds for tissue engineering in the form of nanostructured films or hydrogels. (16) Bigi, A.; Boanini, E.; Gazzano, M.; Rubini, K. Cryst. Res. Technol. 2005, 40, 1094-1098. (17) Malkaj, P.; Pierri, E.; Dalas, E. J. Mater. Sci.: Mater. Med. 2005, 16, 733-737. (18) Montemegro, R. V. D. Crystallization, Biomimetics and Semiconducting Polymers in Confined Systems; Ph.D. Thesis, Golm, 2003. (19) Kong, L.; Gao, Y.; Cao, W.; Gong, Y.; Zhao, N.; Zhang, X. J. Biomed. Mater. Res. A 2005, 75, 275-282. (20) Wan, A. C. A.; Khor, E.; Hastings, G. W. J. Biomed. Mater. Res. 1997, 38, 235-241. (21) Boduch-Lee, K. A.; Chapman, T.; Petricca, S. E.; Marra, K. G.; Kumta, P. Macromolecules 2004, 37, 8959-8966. (22) Chen, H.; Holl, M. B.; Orr, B. G.; Majoros, I.; Clarkson, B. H. J. Dent. Res. 2003, 82, 443-448. (23) Kaneko, T.; Ogomi, D.; Mitsugi, R.; Serizawa, T.; Akashi, M. Chem. Mater. 2004, 16, 5596-5601. (24) Song, J.; Saiz, E.; Bertozzi, C. R. J. Am. Chem. Soc. 2003, 125, 12361243. (25) Song, J.; Malathong, V.; Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 3366-3372. (26) Ho, E.; Lowman, A.; Marcolongo, M. J. Biomed. Mater. Res. A 2007, 249-256. (27) Tamai, H.; Yasuda, H. J. Colloid Interface Sci. 1999, 212, 585-588. (28) Schukin, D. G.; Sukhorukov, G. B.; Mo¨hwald, H. Angew. Chem., Int. Ed. 2003, 42, 4472-4475.

10.1021/la7037872 CCC: $40.75 © 2008 American Chemical Society Published on Web 03/26/2008

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Table 1. Reagents Used for the Synthesis of Hydroxyapatite within Microgels and Some Characteristics of Obtained Hybrid Particlesa N 0 1 2 3 4 5 6 7

M (g) 0.2725 0.2725 0.2725 0.2180 0.1635 0.1635

Ca(NO3)2‚4H2O (NH4)2HPO4 HApT HApM Rh (g) (g) (wt %) (wt %) Ca/P (nm) 0.0342 0.0683 0.2049 0.2733 0.3080 0.4099 0.236

0.0114 0.0229 0.0686 0.0915 0.1031 0.1372 0.79

5.1 9.7 24.2 34.7 44.5 51.6

4.7 7.9 23.6 34.2 44.4 52.2

1.54 1.52 1.90 1.65 1.86 1.58 1.71

475 366 388 331 agg agg agg agg

a M, microgel; T, theoretical value; M (superscript), measured value; agg, microgel aggregation.

2. Experimental Section Materials. N-Vinylcaprolactam (VCL) (Aldrich) was purified by conventional methods and then distilled under vacuum. Acetoacetoxyethyl methacrylate (AAEM), vinylimidazole (VIm), initiator 2,2′-azobis(2-methylpropyonamidine) dihydrochloride (AMPA), and cross-linker N,N′-methylenebisacrylamide (BIS) (Aldrich) were used as received. Calcium nitrate tetrahydrate (Ca(NO3)2‚4H2O) (Gru¨ssing, 99%), diammonium hydrogenphosphate ((NH4)2HPO4) (Riedel-de Hae¨n, 99%), and ammonia solution (Fisher Chemicals, 25%) were used as received. Microgel Synthesis.29 The polymerization procedure can be described as follows. Appropriate amounts of AAEM (0.321 g), VCL (1.783 g), VIm (0.071 g), and BIS cross-linker (0.06 g) were dissolved in 145 g of deionized water. A double-walled glass reactor equipped with a stirrer and reflux condenser was purged with nitrogen. The monomers and cross-linker were dissolved in water by stirring in the reactor for 1 h at 70 °C under continuous purging with nitrogen. After that, a 5 mL aqueous solution of APMA initiator (0.05 g) was added under continuous stirring, and the reaction was carried out for 8 h. The microgel dispersion was purified by dialysis with a Millipore dialysis system (cellulose membrane, MWCO 100000). The VIm content (4.91%) in the microgel has been determined by potentiometrical titration. Final solid content of the microgel dispersion was 1.59%. Preparation of Hydroxyapatite Nanocrystals. An appropriate amount of Ca(NO3)2‚4H2O was dissolved in a microgel dispersion, and the pH value was adjusted to 10 by the addition of 25% NH4OH. In a separate flask, an appropriate amount of (NH4)2HPO4 was dissolved in water, and the pH value was adjusted to 10 by the addition of 25% NH4OH. (Detailed amounts of ingredients are listed in Table 1.) The (NH4)2HPO4 solution has been added slowly under continuous stirring to the polymeric dispersion containing Ca(NO3)2 and mixture was stirred for 24 h. Samples were purified by dialysis (SpectraPor 3 membrane MWCO 3500, Millipore) to remove nonreacted reagents and byproducts. Characterization Methods. Dynamic light scattering DLS measurements were performed using commercial laser light scattering spectrometer (ALV/DLS/SLS-5000) equipped with an ALV-5000/ EPP multiple digital time correlator and laser goniometer system ALV/CGS-8F S/N 025 was used with a helium-neon laser (Uniphase 1145P, output power of 22 mW and wavelength of 632.8 nm) as the light source. The DLS experiments were carried out in the range of scattering angles θ ) 30-120°. All solutions were filtered using a 5.0 µm membrane filter before measurements. Stability measurements of microgel dispersions were performed with separation analyzer LUMiFuge 114 (LUM GmbH, Germany). Measurements were made in glass tubes at acceleration velocities from 500 to 3000 rpm. The slope of the sedimentation curves was used to calculate the sedimentation velocity and to get information about the stability of the samples. (29) Pich, A.; Tessier, A.; Boyko, V.; Lu, Y.; Adler, H. J. Macromolecules 2006, 39, 7701-7707.

Figure 1. (a) SEM image of VCL/AAEM/VIm microgel particles (inset shows a copolymer structure). (b) Hydrodynamic radius (Rh) as a function of temperature for VCL/AAEM/VIm microgels. SEM images were taken with Gemini microscope (Zeiss, Germany). Samples were prepared in the following manner. Microgel dispersions were diluted with deionized water, dropped onto cleaned glass supports, and dried at room temperature. Each sample was coated with a thin Au/Pd layer to increase the contrast and quality of the images. Pictures have been obtained at a voltage of 4 kV. TEM images have been obtained with a Hitachi HD 2000 instrument operating at 200 kV. Diluted microgel dispersions were placed on carbon-coated copper grids and dried at room temperature. To determine the hydroxyapatite content in composite particles, the TGA 7 Perkin-Elmer instrument (Pyris software, version 3.51) was used. Samples were analyzed in the temperature range of 25800 °C (heating rate 10 K/min in a nitrogen atmosphere). The XRD measurements have been performed with a Siemens P5005 powder X-ray diffractometer (source, Cu KR; λ ) 1.54 Å). IR spectra were recorded with a Mattson Instruments Research Series 1 FTIR spectrometer. Dried polymer samples were mixed with KBr and pressed to form tablets.

Results and Discussion The microgel particles used in the present study are based on a copolymer of vinylcaprolactam (VCL) and acetoacetoxyethyl methacrylate (AAEM). As described in our previous studies, the microgel particles possess a heterogeneous structure and consist of an AAEM-rich core and a VCL-rich shell as a result of some peculiarities in the polymerization process.30 The use of a small fraction of vinylimidazole (VIm) during VCL/AAEM microgel synthesis allows the selective incorporation of VIm units into the swollen VCL corona.29 The VCL/AAEM/VIm microgels are characterized by a narrow particle size distribution (Figure 1a) and superior colloidal stability. The temperature sensitivity of the microgels is provided by the phase-transition temperature (Ttr) of PVCL chains that occurs at approximately 33 °C. (The (30) Boyko, V.; Pich, A.; Lu, Y.; Richter, S.; Arndt, K.-F.; Adler, H. J. Polymer 2003, 44, 7821-7827.

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Scheme 1. Synthesis of HAp NCs in Microgel Particles

lower critical solution temperature (LCST) value for linear PVCL is around 32 °C.) This phenomenon occurs because of the different solvation of PVCL chains by water molecules at the temperatures below and above the phase-transition temperature. (Below Ttr, the polymer is swollen, and above Ttr, the polymer is in a shrunk coil conformation.) It is believed that the volume-phase transition in the microgel occurs as a result of reduced hydrogen bonding between water molecules and the polymer and hydrophobic aggregation of the polymer that leads to microgel shrinkage (Figure 1b). This process is reversible, and if the temperature decreases below a critical value, then the microgel swells. The porous microgel structure has been used in the present study to deposit hydroxyapatite nanocrystals (HAp NCs). The preparation of hybrid microgels containing HAp NCs is shown in Scheme 1. The first step is the addition of Ca2+ to the microgel dispersion, and the formation of HAp in the aqueous phase can be performed by the precipitation reaction of calcium and hydrophosphate ions in a basic aqueous medium.

Figure 2. (a) Hydrodynamic radius as a function of calcium nitrate concentration in water. (b) Sedimentation velocity of hybrid microgels with different HAp contents.

As shown in Scheme 1, the ionic strength in the reaction mixture increases considerably after the addition of calcium salt. The presence of ions in the aqueous phase strongly influences the solubility of the polymer chains in water. This effect induces a change in microgel size as well as the volume-phase transition temperature and colloidal stability. The complexity of such transformations is enhanced by different influences originating from the nature and charge of the ions. As shown in Figure 2a, the addition of a Ca salt to the microgel solution led to some microgel shrinkage. (The calcium nitrate concentrations used in this experiment correlate with that used for the synthesis of HAp NCs.) Figure 2a indicates that the decrease in the microgel hydrodynamic radius occurs after the addition of salt; however, no particle aggregation occurs in the investigated salt concentration range. The VCL/AAEM/VIm microgels are sterically stabilized and exhibit a weak charge at pH 7 (Figure S1a, Supporting Information).29 Therefore, the reduction of microgel size is probably not a consequence of charge compensation but can be explained by the partial reduction of the hydrogen bonds between polymer chains and water molecules. This leads to some “dehydratation” of polymer segments and a more coiled conformation that induces the shrinkage of the microgel shell. Another reason for microgel shrinkage could be the complexation of Ca ions by β-diketone groups of AAEM units. In this case, Ca2+ can form intermolecular complexes that also provoke the partial shrinkage of the microgels. However, it should be noted that the VCL/AAEM/VIm microgels do not shrink completely after the addition of salt. (In the collapsed state as shown in Figure 1b, microgels possess an Rh value of 150 nm.) The FTIR analysis did not provide strong evidence for the complexation of Ca ions in the microgel network. To address this issue, we performed a control experiment where the microgel and a calcium nitrate solution were mixed together, and after 12 h, we have dialyzed the sample to remove “free” Ca ions. After 3 days of dialysis, we used a purified sample and performed HAp synthesis (by adding ammonium phosphate and adjusting the pH to 9). As reference, another experiment has been carried out where HAp loading was performed under similar conditions but no dialysis step has been conducted. (The standard method used in this study is presented in Scheme 1.) The dialyzed sample had an inorganic phase content of around 7 wt %, and the nondialyzed sample contained 44 wt % HAp. Obviously, the large number of Ca ions has been removed by dialysis from the microgel dispersion. However, some fraction of metal ions was entrapped by the microgel network and further transformed to calcium phosphate. It is interesting that the TEM analysis confirmed that the morphology of the formed HAp is quite different in two cases (Figure S2, Supporting Information). The microgel particles prepared in the conventional way exhibit needlelike HAp nanocrystals. In the case of the dialyzed sample, we detected aggregated spherical amorphous particles in the microgel

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Figure 3. SEM images of PVCL/PAAEM/PVIm-HAp hybrid microgels prepared with different HAp contents: (a) microgel, (b) 4.7% HAp, (c) 7.9% HAp, (d) 23.6% HAp, (e) 34.2% HAp, (f) 44.4% HAp, and (g) 52.2% HAp.

structure. Probably the formation and controlled growth of crystalline HAp nanorods is hindered in the situation when in the reaction system only “immobilized” Ca ions are available. This may lead to the conclusion that free Ca ions are necessary to support crystal growth within microgels by diffusion from the aqueous phase. In further experiments, HAp deposition into microgels was performed in the way shown in Scheme 1 (no dialysis step). After the addition of diammonium hydrophosphate, the fast formation of HAp takes place in the reaction mixture. This process is followed by the strong reduction of the colloidal stability of the microgels. As shown in Figure 2b, the sedimentation velocity of the composite particles increases with the increase in HAp content. The samples with high HAp loading show clear phase separation after the reaction has been completed (Figure 2b). These results indicate that the presence of HAp increases the microgel affinity to form aggregates and precipitate from solution. The electrophoretic mobility measurements (Figure S1b, Supporting Information) indicate that microgels are positively charged at acidic pH and possess a weak charge at neutral and basic pH. The composites with deposited HAp exhibit similar behavior to that of the original microgel sample. This indicates that HAp nanocrystals are localized in the microgel interior and do not contribute to the surface charge. However, HAp nanocrystals interact with the microgel network and reduce the mobility of the polymer segments, and HAp loading increases the microgel density. Both effects can lead to the gradual loss of colloidal stability upon the deposition of HAp nanocrystals. The morphology of the composite particles is demonstrated in Figure 3. The SEM images indicate that the deposition of HAp NCs presumably occurs in the microgels; however, secondary HAp NCs can be also found. The increase in HAp content induces some morphology changes in the microgels. They appear as flattened discs with coronas filled with HAp NCs. The composite particles shrink after HAp loading. This has also been confirmed by DLS measurements for colloidally stable samples (Table 1). Some reduction of microgel size can be expected because HAp NCs deposited in the microgel shell reduce the hydration and mobility of the polymer chains due to the adsorption of polymer

segments on the inorganic particle surface. Because of the fact that microgel samples have been dried on solid supports for the microscopy investigations and water removal causes a strong reduction of the microgel size, the dimensions of microgel particles in micrographs appear smaller compared to hydrodynamic radii determined by DLS. The dark-field TEM images presented in Figure 4 give more detailed insight into the structure of composite microgels. Note that in these images only the compact microgel core is clearly visible. The HAp NCs possess needlelike morphology and are located in the outer microgel layer and are randomly distributed within the microgels. The increase in HAp loading does not considerably change the morphology and size of NCs. The results of the EDX line scan performed on a single microgel particle with deposited HAp NCs presented in Figure 5 indicate the presence of Ca and P in the microgel outer layer that confirms the effective deposition of HAp NCs into the microgels. It should be noted that the controlled deposition of HAp NCs takes place only in VCL/AAEM microgels functionalized by VIm units. The VCL/AAEM microgels (no additional functionalities) and VCL/AAEM microgels modified by primary or secondary amino groups led to the accumulation of HAp on the microgel surface and the nonhomogeneous distribution of inorganic NCs in the microgel network (Figures S3-S6, Supporting Information). Additionally, the formation of HAp aggregates in the aqueous phase was detected. For this reason, microgel aggregation was detected even at very low HAp loadings. These results indicate that the presence of imidazole groups in the microgel shell provides a suitable environment for HAp growth. On the basis of the experimental results discussed above, we assume that the controlled growth and stabilization of the HAp nanocrystals in the VCL-rich microgel shell occurs because of the effective interactions between polymer segments and HAp NCs surface (Figure 6). In basic media (reaction conditions), the hydroxyl groups on the HAp surface participate in the formation of hydrogen bonds with the carbonyls of VCL units. The VIm units can interact with Ca2+ on the HAp surface by the formation of coordination bonds by donating the electron pair available on the N atom. On the contrary, in the weakly acidic range we can

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Figure 4. TEM images of PVCL/PAAEM/PVIm-HAp hybrid microgels containing different amounts of HAp: (a) 0, (b) 7.9, (c) 34.2, and (d) 52.2%.

Figure 5. TEM image with an EDX line scan of a single microgel containing 52.2% HAp. The element distribution curves are shown as separate windows (with the colors of the lines in the image corresponding to the colors in the element distribution spectra).

Figure 6. Schematic representation of the interactions between HAp and the microgel network.

assume that there is an electrostatic attraction between the phosphonate groups of HAp and imidazolium ions located in the polymer chains. This indicates that the highly swollen functionalized shell of the VCL/AAEM/VIm microgels controls to

some extent the growth of the HAp NCs and provides their stabilization in the polymer network. The elemental analysis makes it possibile to determine the Ca/P ratio, which is an important characteristic of HAp. The experimental results summarized in Table 1 suggest that in all samples the Ca/P ratio oscillates between 1.5 and 1.9 (the theoretical value for HAp is 1.67). The Ca/P ratio for HAp prepared without microgels (sample 7, Table 1) was 1.71. A Ca/P ratio different from the theoretical value indicates that some substitutions of other ions are probably present in the crystal structure instead of calcium (substitution by other metal ions) or phosphate (substitution by CO32-).31 The presence of CO32is indicated in the FTIR spectrum of HAp at 1430 cm-1 in Figure 7a. Additionally, some traces of HPO42- were detected at 877 cm-1, which suggests that some PO43- ions have been substituted by HPO42-.31 The CO32- and HPO42- signals cannot be detected in the spectra of composite microgels containing HAp as a result of the overlap with specific bands of polymer chains, but we (31) (a) Dorozhkina, E. I.; Dorozhkin, S. V. Chem. Mater. 2002, 14, 42674272. (b) Tadic, D.; Veresov, A.; Putlaev, V. I.; Epple, M. Mat.-wiss. U. Werstofftech. 2003, 34, 1048-1051.

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materials that could be used as direct evidence of calciumpolymer interactions in microgel samples. The XRD data for selected samples presented in Figure 7b suggest that hybrid materials exhibit characteristic signals that correlate with the reference spectra for HAp. The signal intensity is strongly dependent on the amount of HAp in the sample, and this is the reason for the bad signal/noise ratio in the spectra of composite microgels. Obtained hybrid microgels form composite films upon drying on solid substrates. Even at high HAp loading, films remain transparent, which indicates that no strong phase separation occurs during the drying process (Figure S7, Supporting Information). The TEM investigations coupled with EDX analysis indicate that the HAp NCs remain embedded in the polymeric network and no migration of the inorganic component occurs. These results allow us to conclude that hybrid microgels can be used for the preparation of well-defined composite films. In this case, the amount of inorganic material and its localization in the bulk material are determined by the loading and morphology of the microgel particles, respectively. We assume that obtained hybrid microgels containing HAp NCs can be effectively used as injectable tissue replacement materials. The simple microgel functionalization allows additional modification of the particle surface and conjugation with bioactive molecules. HAp-containing microgels can be used as building blocks for the preparation of scaffolds for tissue engineering in the form of nanostructured thin films or hydrogels.

Conclusions

Figure 7. (a) FTIR and (b) XRD spectra of VCL/AAEM/VImHAp hybrid microgels. The vertical arrows in graph a indicate the increase in signal intensity for the bands related to HAp.

assume that HAp synthesized in the presence of microgels can contain similar substitutions, as indicated above for HAp prepared without microgels. The stronger deviation of the Ca/P ratio from the theoretical one in the case of composite samples as compared to HAp prepared by the same method (Table 1) is an indication that microgel particles can contain some additional impurities in the form of metals or ionic initiator residues that become incorporated into the HAp structure. The most important absorption bands of the phosphate group in the HAp structure appear at 565, 603, 1034, and 1094 cm-1 in the FTIR spectrum. Figure 7a shows that the increase in HAp content in the microgels induces the stepwise increase in band intensity for the inorganic component. However, we did not see any remarkable features in the FTIR spectra of composite

We demonstrate that aqueous microgels can be used for the growth of HAp nanocrystals (HAp NCs). By using a conventional precipitation method, the HAp NCs become incorporated into the microgel particles. The HAp NCs are localized in the microgel shell, and the loading of inorganic material in the composite particles can be varied in a broad range. The incorporation of HAp NCs reduces the colloidal stability of the microgels and decreases their ability to change size in response to the temperature variation. The chemical structure of HAp NCs incorporated into microgels has been confirmed with XRD and IR spectroscopy. The incorporation of HAp NCs into polymeric particles allows the preparation of nanostructured composite films on solid supports by a simple casting procedure. Acknowledgment. We thank the Deutsche Forschungsgemeinschaft (DFG) for financial support. Supporting Information Available: Colloidal properties of microgels, microscopy results, and film-forming properties. This material is available free of charge via the Internet at http://pubs.acs.org. LA7037872