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Biomimetic Mineralization Induced by Fibrils of Polymers Derived from a Bile Acid Xu Zhang,† Zhanyong Li,† and X. X. Zhu*,†,§ Key Laboratory of Functional Polymer Materials, Institute of Polymer Chemistry, Nankai University, Tianjin, 300071, China, and De´partement de Chimie, Universite´ de Montre´al, C.P. 6128, Succ. Centre-ville, Montre´al, QC, H3C 3J7, Canada Received February 22, 2008; Revised Manuscript Received June 27, 2008

A polymer containing bile acid pendant groups was prepared by free-radical polymerization of the 3R-methacrylate derivative of cholic acid. The polymer formed fibrils of about 1 nm in diameter in aqueous media and further assembled into bundles or lamella plates, as observed by transmission electron microscopy. After immersing the fibrils in simulated body fluid with ion concentrations equivalent to those in human plasma, plate-like crystals formed on the surface of the fibril assembly. The diffraction pattern corresponding to the (001) plane of hydroxyapatite was identified by transmission electron microscopy and selected area electron diffraction. The plate-like, single crystal hydroxyapatite formed on such a polymeric assembly may be useful in the matrix design for biomimetic mineralization and in the development of scaffold materials for hard tissue engineering.

Introduction The properties of mineralized tissues of plants and animals have inspired scientists to explore new materials which can mimic natural mineralization processes.1-5 There may still be many ambiguities on the details of biomineralization, but it is known that all organisms provide intrinsic organic matrices, or organic templates, in the first step of biomineralization.6-8 The key components of matrices generally include proteins, lipids, polysaccharides, or combinations thereof. The shapes of matrices may vary because they are assembled from different biomacromolecules under different conditions. Nevertheless, they can be approximately divided in two types as membrane-bound compartments or macromolecular frameworks with typical fibril structures. It not only provides the specific boundary spaces to nucleate and affect the shape and size of the mineral phase in local regions, but also play decisive roles in the construction of multiple-level hierarchical organization. Therefore, the design and synthesis of matrix material with a fibril structure is important to mimic a biomineralization system. There have been reports on the strategies to form a human bone-like hydroxyapatite phase on the surface of polymeric scaffolds9-12 or specific protein scaffolds13,14 in bone tissue engineering. New hydroxyapatite phases with various appearances such as “cauliflowers” were observed.15,16 The morphologies were often similar to those formed on the surfaces of ceramics and metal substrates17,18 but different from those of biological hydroxyapatite, which are thin platelets, regularly organized on the surface of collagen fibrils.19-23 Stupp and coworkers used self-assembled scaffold of peptide-amphiphilic molecules to direct plate-shaped polycrystalline mineral of hydroxyapatite in vitro.24,25 It seems possible to control the structure and arrangement of artificial fibers to modulate the morphology of minerals in vitro just as what may occur in vivo. The morphology and composition of the minerals can determine whether or not the scaffold could mimic the function of a fibril * To whom correspondence should be addressed. Tel.: (514) 340-5172. E-mail: [email protected]. † Institute of Polymer Chemistry, Nankai University. § Universite´ de Montre´al.

matrix in organisms, the interfacial reaction with the organic matrix on the molecular level, and the potential to construct an integrated material. Bile acids are natural compounds synthesized biologically in the liver. Cholic acid (Scheme 1), the most common bile acid in humans, is known for its facial amphiphilicity with nonpolar groups on the one side of the steroid skeleton and polar hydroxyl groups and carboxylic group located on the other side.26 One noteworthy character of bile acids is their selfassembling behavior. In 1958, two research groups found that sodium deoxycholate aggregated in solution to form a gelatinous complex of macromolecular dimensions.27,28 X-ray diffraction studies of these complex fibers showed that the molecules assumed an elongated helical configuration.27-29 The influence of the structure of bile salts on their self-association was studied by Hofmann et al.30,31 Later on, Ahlheim et al. prepared polymers from bile acid derivatives and studied the formation of organized structures of the polymers with lipids.32,33 We have used cholic acid and other bile acids in the development of new polymeric materials.34-38 The monomers and polymers derived from bile acids often form crystalline materials in organic solvents and water. The linear polymers with bile acid pendant groups afford an interesting structural advantage as scaffold with the main chain as hydrophobic “wick” and pendant carboxyl groups as hydrophilic “epidermis”. This led to our work on the study of the mineralization behavior of fibrils formed by a cholic acid-containing polymer in simulated body fluid (SBF) and the results indicate that such polymers may serve as matrices in the formation of inorganic crystalline materials mimicking a biomineralization process.

Experimental Section Materials. All chemicals including cholic acid, methacrylic acid, benzoyl chloride, the inorganic salts, and organic solvents were purchased from Tianjin Chemicals Co., China. Methacryloyl chloride (MAC) was prepared from methacrylic acid and benzoyl chloride.39 The initiator 2,2-azo-bis(isobutyronitrile) (AIBN) was recrystallized in methanol prior to use. Selected solvents were treated before distillation: chloroform was washed with water and dried with anhydrous CaCl2;

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Scheme 1. Preparation of the Polymer Containing Cholic Acid Moieties Used for the Biomineralization Experiments

Figure 1. FTIR spectra of sodium cholate (A), the hydrolyzed polymer (B), and poly(MECAME) (C) showing the band close to 1718 cm-1 corresponding to CdO stretching vibration of the methacrylate ester groups (mostly intact after hydrolysis), the band at 1740 cm-1 corresponding to the CdO stretching vibration of methyl ester group (diminished after hydrolysis), and the CsO stretching band at 1567 cm-1 for the carboxylate group (enhanced after hydrolysis).

triethylamine (Et3N) was refluxed over CaH2 for 8 h; toluene was washed with concentrated sulfuric acid to remove thiophene. Preparation of the Polymer. 36 The monomer, methyl 3R-methacryloyl-7R,12R-dihydroxy-5β-cholan-24-oate (MECAME, Scheme 1), synthesized by reacting freshly prepared methacryloyl chloride with the 3-hydroxyl group of cholic acid methyl ester (CAME),32 was dissolved in toluene in an ampule equipped with a magnetic stirrer. After purging with an inert gas (N2), AIBN (6 mol %) was added before sealing the ampule and placing it in an oil bath. The temperature was raised to about 70 °C over 2 h and then maintained for another 24 h. The polymer was precipitated from methanol and then filtered, washed, and dried. The weight-average MW was about 34000 (PDI ) 2.42). The selective hydrolysis of the methyl ester group of the polymer was accomplished by refluxing in NaOH solution (2 M) mixed with tetrahydrofuran (THF; volume ratio 1:2, not completely miscible) for about 3 h.36,40 After separating the two phases, the aqueous phase was dialyzed in high purity water (resistivity 18.0 MΩ · cm) to remove residual NaOH until the pH of the fluid was neutral. The sample was then centrifuged and freeze-dried after the removal of the deposit. Simulated Body Fluid (SBF) and Calcium Phosphate Formation. A protein-free acellular simulated body fluid with ion concentrations nearly equal to those in human plasma (142.0 mM Na+, 5.0 mM K+, 1.5 mM Mg2+, 2.5 mM Ca2+, 147.8 mM Cl-, 4.2 mM HCO3-, 1.0 mM HPO42-, and 0.5 mM SO42-) was prepared by dissolving reagent-grade chemicals of NaCl, NaHCO3, KCl,

K2HPO4 · 3H2O, MgCl2 · 6H2O, CaCl2, and Na2SO4 in high purity water with tris(hydroxymethyl)aminomethane ((CH2OH)3CNH2) and 1.0 M hydrochloric acid to adjust the pH to 7.25 at 36.5 °C.41 The hydrolyzed polymer (4 mg) was added into 25 mL of SBF at pH 7.25. The sample was filtered through a 0.22 µm Millipore filter to remove any deposit. After its immersion in an incubator for various time intervals, 3 mL of the sample were withdrawn and placed in a quartz cuvette maintained at 36.5 °C. The transparency of the SBF and the sample was monitored at two wavelengths, 220 and 500 nm, in comparison to the transparency of high purity water set as 100%. At the end of each mineralization experiment, the sample was dialyzed in high purity water for 2 days to remove any soluble inorganic ions until the pH of dialysate was near 7. The dialyzed fluid was then freezedried. Characterization of the Samples. The MW of the polymer was determined by size exclusion chromatography (Waters 410 system) at 35 °C with a 3 mg/mL solution in THF against polystyrene standards. Fourier transform infrared (FT-IR) spectroscopy was carried out on a Bruker Tensor 27 instrument with samples prepared with KBr in the pellet form. The transparency of the solution was measured by a Cary 100 UV-vis spectrophotometer (Varian). High-resolution transmission electron microscopy (TEM) and selected area electron diffraction (SAED) were performed on a Philips EM400ST instrument operated at 200 kV. Samples were prepared by placing a drop of the monomer or polymer solution (0.2 mg/mL) onto the holey carbon-coated TEM grid followed by complete drying in the air.

Results and Discussion Preparation of the Hydrolyzed Polymer. To induce mineral formation in an aqueous environment, it is necessary to increase the hydrophilicity of the polymer containing cholic acid. The methyl ester protecting groups on the polymer were selectively hydrolyzed by the use of a previously reported procedure40 in an aqueous solution of NaOH mixed with THF. This liberated the carboxyl group at position 24 on the alkyl chain of cholic acid (Scheme 1) without affecting the methacrylate ester bond at position 3 of cholic acid on the polymer side chain. FTIR spectroscopy was used to monitor the selective hydrolysis (Figure 1). The conversion of the methyl ester to the carboxylic acid group at position 24 of cholic acid was evidenced by the diminished band at 1740 cm-1 corresponding to the CdO stretching vibration of the methyl ester group and the enhanced

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Figure 3. Transparency of SBF (solid) and the hydrolyzed poly(MECAME) in SBF (dashes) as a function of time monitored at a wavelength of 220 nm. All solutions remained 100% transparent when monitored at 500 nm. The hydrolyzed poly(MECAME) sample shows oscillations in transparency over time beyond experimental errors, indicating the spontaneous organization process in SBF.

Figure 2. TEM images of poly(MECAME) (A) and the selectively hydrolyzed polymer (B) showing that the diameter of the fibril was about 1.5 nm before hydrolysis (A) and shrank to about 1.0 nm after hydrolysis (B). The insets (horizontal divisions at 1 nm for A and 2 nm for B) show the relative intensity of the electron beam transmitted through the bundle of fibrils as a reference of the diameter of the fibrils.

CsO stretching band at 1567 cm-1. The methacrylate ester groups remained intact in the hydrolyzed polymer as shown by the strong band for CdO stretching vibration in the neighborhood of 1718 cm-1. It is important to control the reaction time within 3 h to avoid hydrolysis of the methacrylate ester bond linking the cholic acid residue. Figure 2 provides a comparison of the TEM images of the polymer fibrils formed before and after the selective hydrolysis. The diameter of the fibrils formed by the original polymer (ca. 1.5 nm) in THF is slightly larger than that of the fibrils formed by the hydrolyzed polymer (ca. 1.0 nm). The fibrils increase in length with the MW of the polymer. The shorter fibrils can arrange into bundles or plates while the longer ones can form ribbon-like structures. The diameter of fibril is close to the dimension of one steroid unit of cholic acid and to the diameter of a thin fiber in an organogel.42 Hydroxyapatite Formation in SBF. To investigate the hydroxyapatite formation on the surface of hydrolyzed polymer fibril, a protein-free acellular SBF with ion concentrations nearly equal to those in human blood plasma was used. The SBF was shown to be stable with no change in ion concentration and pH up to 8 weeks of storage even though the ionic activity product (IP ≈ 10-94.3) indicates high supersaturation for hydroxyapatite (Ksp ≈ 10-118).43,44 Many materials showing the formation of

hydroxyapatite in SBF also showed the formation bone-like apatite in vivo.41 The constant solution composition is important to investigate the crystallization process. The concentration of polymer in the experiment was kept low enough (the molar ratio of the monomer units, calcium and phosphate ions was kept at ca. 1:80:32) to ensure the composition of the ionic species remain approximately constant during the mineralization. After the mineralization experiment, dialysis in high purity water was carried out to remove any soluble residual from the mineralized polymer. UV-vis spectrophotometry was used to monitor the precipitates from supersaturated calcium phosphate solutions.45 In fact, the transparency of the solution is determined by both the concentration and the dimension of suspended particles invisible to the naked eye. A decrease in the transparency of the solution may be due to an increased concentration or an increased dimension of the suspended particles, or both. The freshly prepared polymer samples were not suitable for immediate measurement of transparency because of initial fluctuations. It is to be noted that all solutions shown in Figure 3 remained 100% transparent when observed at a wavelength of 500 nm, indicating that only small nanoparticles may exist. Therefore, only the data observed at 220 nm are shown. On day 2, the transparency of the blank SBF decreased from about 80 to 76% at 220 nm (Figure 3). From day 3 and onward, the transparency remained quite constant within experimental error. This indicates the spontaneous formation of crystallites due to homogeneous nucleation in the SBF. The SBF remained quite stable over a period of 60 days, even though it might contain some crystallites in the nanometer scale under the supersaturation conditions. This is also consistent with the dissolution model of Nancollas et al. for sparingly soluble crystallites.44 For the SBF system containing hydrolyzed poly(MECAME), its transparency shown in Figure 3 has the following characteristics: (1) It oscillates between 70 and 75% beyond the experimental error of the measurements; (2) The frequency of the oscillation here may not reflect the frequency of organization process that occurs in the SBF due to the frequency of sampling in the experiments, but it is reasonable to expect a gradual decrease of the frequency over time due to a decreased level of saturation of the medium; and (3) The lower limits of the

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Figure 4. Illustration of the mineralization process showing the structural mismatch and alignment with mineralized polymers in the self-assembly process. Structural mismatch: A > C; transparency: B > A > C.

oscillation show a gradual decrease over time. The zigzag shape of the curve is an indication that heterogeneous nucleation and growth quickly occurred after placing the hydrolyzed polymer into SBF. The lower transparency (than that of the pure SBF) at the beginning may indicate a high level of the total concentration of all species, including the formation of nanocrystallites in the bulk of SBF. Such crystallites may tend to adhere onto the larger surfaces of polymeric assemblies to minimize the surface energy as the fibril structure of the polymer may be compatible with the nucleation of hydroxyapatite. The approaching of the crystallites to the polymer surface should lead to a decreased transparency (state A in Figure 4), but the alignment of polymer surface may not be always easy because of possible structural mismatch. The adjacent species may separate into the bulk of SBF (state B), which should lead to a higher transparency of the system. A change in the orientation of the two lattices should prompt to an orderly packing (state C), which leads to a decreased transparency. Therefore, the zigzag shape of the transparency curve is a good indication of the mineralization process in the system. Overall, the system shows a gradual decrease of the lower limits of the oscillation and a less transparent SBF system along with the formation of larger crystals. The results indicate that mineralization is a selforganization process that can be induced by the polymer containing cholic acid moieties and that the hierarchical structures may be a result of repetition of the self-organization process at different scale levels. TEM and SAED Experiments. To study the composition and morphology of the crystallites in SBF, high-resolution TEM and SAED have been carried out on selected samples. For the hydrolyzed polymers containing cholic acid, the TEM

Figure 5. High-resolution TEM images of the hydrolyzed poly(MECAME) samples incubated in SBF at day 1 (A), day 2 (B), and day 7 (C), showing the formation of single crystalline hydroxyapatite with layer structure and its evolution over time. The SAED pattern of insert C shows the [001] direction of hydroxyapatite is vertical to the surface of the plate-like fibril assembly.

image shows that the fine fibrils of the polymer already become thicker after soaking in SBF for 1 day (Figure 5A). The TEM image and the SAED are consistent with the decrease in the UV transparency observed for the samples (to ca. 75%) at 220 nm, indicating the quick nucleation and growth of hydroxyapatite on the polymer fibrils. The crystalline nanoparticles scatter like single crystals but they clearly form nanoparticle superstructures.46 For TEM observation, we selected and compared the fibril bundles both before and after hydrolysis and the dialyzed samples after mineralization in SBF. In the single layer of the plate-like fibril bundle or

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the ribbon-like structure, alignment in the images has been observed. The planes of fibril bundles must also be adjusted to be perpendicular to the electron beam to obtain highresolution TEM images and exact SAED patterns. After 48 h, some fibril bundles and plates were observed (Figure 5B). The fibril plates showed laminar structure with defined orientation in each layer and some sheet-like plates protruded from the laminar structure (arrow). The electron diffraction patterns in the inserts are caused by crystalline hydroxyapatite formed on the bundles or lamellas that are amorphous before mineralization. The single crystals formed on/between the polymer surfaces gradually become thicker with mineralization, while the image of polymer fibrils underneath becomes blurry. The results confirm the advantage of the hydrolyzed polymer as a matrix material on the morphology of crystals, polymorphs, and growth rate. It is difficult to say whether the polymer fibrils are really less aligned after immersion in SBF. The electron beam in the TEM experiments passed through layers of mineralized fibrils with slightly different orientations (bundles of a single layer were not always easy to be found), which may affect the image. The TEM images were obtained in the dry state and the sample preparation might have an effect on the image of the samples. George and co-workers13 used dentin matrix protein 1 to examine the apatite crystallization process in vitro, their highresolution lattice image showed the emergence of nanocrystalline arrays from the amorphous precursor in the samples incubated for 2 days. In our experiment, the growth rate of crystal phase on the polymer was as fast as the dentin matrix protein 1. Woo et al.47 used TEM to examine the mineralization in nanofibrous scaffolds and found that large amounts of small globular mineral deposits were attached to nanofibers within 14 days of cell culture in vitro, indicating that the morphologies of minerals might respond to the difference between scaffold structures. The hydrolyzed polymer sample soaked in SBF for 7 days revealed the formation of a crystalline phase of larger size (100∼200 nm) on the surface of the sample. For the fringe spacing observation, the focus must be placed on the most superficial crystalline phase with a thickness at higher magnification. Consequently, the polymer fibril underlayer becomes invisible. The fringe spacing from the crystalline phase can be easily observed and measured to be 0.468 nm, corresponding to the interplanar distance of the (110) planes of hydroxyapatite (Joint Committee on Powder Diffraction Standards, JCPDS 090432). To further discern the hydroxyapatite crystal formation and relative orientation of the hydroxyapatite crystallographic c-axis with respect to fibril long axis, SAED was performed. The SAED pattern taken from the plate-like crystal phase indicates that it is a single crystal of hydroxyapatite (P63/m) and the [001] direction of hydroxyapatite is vertical to the surface of the plate-like fibril assembly (Figure 5C, inset). Therefore, the preferential alignment of the hydroxyapatite crystallographic c-axis is vertical to the surface of the fibril assembly here. This is another example of the formation of plate-like hydroxyapatite on the surface of amphiphilic fibrils with a structure of hydrophobic “wick” and hydrophilic “epidermis”, but its orientation is different from that observed by Stupp and co-workers.24 There are also reports on different orientations of the natural hydroxyapatite crystallographic c-axis with respect to collagen fibril axis.48,49 Further study is needed to relate the nucleation sites to the growth direction of hydroxyapatite on such polymer fibrils. The structure, the

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alignment of polymeric fibrils, and the dynamic processes should be considered.50

Conclusion The amphiphilic polymer made from the methacrylic derivative of cholic acid can assemble into fibrils in aqueous media. The comparison with pure SBF in the absence of the polymer shows that the ordered fibril assemblies of the cholic acidcontaining polymer can induce mineralization similar to a biomineralization process. The amphiphilic moieties in fibrils and the conditions used may affect the morphology of crystalline materials. The SAED pattern taken from the definite plane of the crystals showed the alignment of the crystallographic axis to the long axis of the fibril. It would be interesting to elucidate the determining factors that may affect the orientation of the crystalline phase. Even though preliminary, this study shows that an amphiphilic polymer derived from a natural compound such as cholic acid can help in the understanding of the biomineralization process. The finding may lead to the further exploration of polymeric fibrils as the matrix materials to induce the formation of inorganic crystalline materials that can mimic a biomineralization process. Acknowledgment. Financial support from the Ministry of Science and Technology of China (2002CCA02500) and the Canada Research Chair program is gratefully acknowledged. X.X.Z. also thanks Fonds Barre´ of Universite´ de Montre´al for travel support during a sabbatical leave.

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