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Materials Science and Engineering C 27 (2007) 855 864 www.elsevier.

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Biomimetic synthesis of hydroxyapatite/bacterial cellulose nanocomposites for biomedical applications


Y.Z. Wan a,, Y. Huang a , C.D. Yuan b , S. Raman c , Y. Zhu b , H.J. Jiang d , F. He a , C. Gao a
c

School of Materials Science and Engineering, Tianjin University, Tianjin 300072, PR China b School of Chemical Engineering, Tianjin University, Tianjin 300072, PR China Department of Community Health and Epidemiology, Queen's University, Kingston, Ontario, Canada K7L 3N6 d Wendeng Hospital of Orthopaedics, Shandong 264400, PR China Received 20 November 2005; received in revised form 23 July 2006; accepted 5 October 2006 Available online 15 November 2006

Abstract Hydroxyapatite (HAp) and bacterial cellulose (BC) are both excellent materials for use in biomaterial areas. The former has outstanding osteoconductivity and bioactivity and the latter is a high-strength nano-fibrous and extensively used biomaterial. In this work, the HAp/BC nanocomposites with a 3-dimensional (3-D) network were synthesized via a biological route by soaking both phosphorylated and unphosphorylated BCs in 1.5 simulated body fluid (SBF). Scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) were employed to characterize the HAp/BC nanocomposites. SEM observations demonstrated that HAp crystals were uniformly formed on the phosphorylated BC fibers after soaking in 1.5 SBF whereas little HAp was observed on individual unphosphorylated BC fibers. Our experimental results suggested that the unphosphorylated BC did not induce HAp growth and that phosphorylation effectively triggered HAp formation on BC. Mechanisms were proposed for the explanation of the experimental observations. XRD and FTIR results revealed that the HAp crystals formed on the phosphorylated BC fibers were carbonate-containing with nanosized crystallites and crystallinities less than 1%. These structural features were close to those of biological apatites. 2006 Elsevier B.V. All rights reserved.
Keywords: Bacterial cellulose; Hydroxyapatite; Nanocomposite; Surface treatment; Biomineralization

1. Introduction The most striking features of bacterial cellulose (BC) are its high mechanical strength and modulus and biodegradability. BC microfibrils have a density of 1600 kg m 3, Young's modulus of 138 GPa, tensile strength of at least 2 GPa, which are almost equal to those of aramid fibers [1]. In addition, it has sufficient porosity, 3-dimensional (3-D) network structure, water holding capability, and biocompatibility. Compared with plant cellulose that has found many applications in the biomedical field as tissue engineering materials due to their good biocompatibility, mechanical properties similar to those of hard and soft tissue and easy fabrication into a variety of shapes with adjustable interconnected porosity [2,3], BC possesses
Corresponding author. Tel./fax: +86 22 27405056. E-mail address: yzwantju@yahoo.com (Y.Z. Wan). 0928-4931/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2006.10.002

higher water holding capacity, higher crystallinity, higher tensile strength, and a finer web-like network. Compared with other natural biodegradable polymers such as collagen, chitosan, chitin, and gelatin, BC presents much higher mechanical properties, which are required in most cases when used as scaffold in tissue engineering [4]. Compared with animal-derived polymers, BC is free of any occurrence of crossinfection likely associated with collagen [5]. Despite the aforementioned advantages, BC has not been used as a biomaterial in tissue engineering though it has been widely used in foods, in acoustic diaphragms for audio speakers or headphones, for making unusually strong paper, and has medical applications such as wound dressings and artificial skin, artificial blood vessels, and specialty membranes [611]. Only very recently have Svensson et al. declared that BC is a promising material for a potential scaffold for tissue engineering of cartilage [12]. Nevertheless, as with plant cellulose, its

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Table 1 Designation of samples prepared in this study Sample no. 7-HAp/BC 14-HAp/BC 7-P-HAp/BC 14-P-HAp/BC Phosphorylation (Y/N) N N Y Y Surface treatment in CaCl2 (Y/N) Y Y Y Y Soaking time in 1.5 SBF (day) 7 14 7 14

2.2. Surface modification of BC In the present study, two surface conditions were created. In the first method, pieces of BC were immersed in a 0.1 M CaCl2 solution at 37 C for 3 days prior to biomimetic mineralization. The CaCl2 solution was renewed every 24 h. In the second approach, the BC samples were first phosphorylated prior to immersion in the CaCl2 solution as described above. The phosphorylation procedures were similar to those previously described [27]. Briefly, circular BC pellicles were placed into a round-bottomed flask equipped with a condenser, thermometer, and nitrogen gas inlet. 200 ml of dimethyl formamide (DMF) and 15 g of urea were added to the flask. The flask was then heated to 110 C and immediately a solution of 19 ml of 98% phosphoric acid (H3PO4) in 50 ml DMF was added to the flask. The suspension was further heated to 136 C and left to reflux for an hour under gentle stirring using a magnetic stirrer and a nitrogen stream. The reaction mixture was cooled under flowing nitrogen gas. The resulting BC pellicles were removed and thoroughly rinsed with deionized water to wash out the excess H3PO4. 2.3. Preparation of HAp/BC nanocomposites In order to induce apatite formation, surface treated BC samples were soaked in a 1.5 SBF solution at 37 C for 7 or 14 days. The supersaturation conditions were maintained by periodic replacement with a fresh solution. The obtained samples were taken out from the solution, rinsed with deionized water, and finally freeze-dried. The designation of the resulting samples is given in Table 1. 2.4. SEM analysis Scanning electron microscopy (SEM) was conducted to observe sample morphology and microstructure. Samples were sputter coated with gold and examined using either a JEOL JSM-6360LV or a Philips XL 30 environmental SEM.

inability to bond directly to bone is a concern when BC is used as a bone substitute material [13]. In the search for a new generation of bone substitutes that are bioactive and biodegradable, composites consisting of collagen and calcium phosphate minerals have received much attention because they mimic the basic composition of bone. In vivo tests have proven that such composites are bioactive and biodegradable to a certain extent, and hence they enhance new bone growth [14,15]. In spite of their promising bioactivity, the collagen/ calcium phosphate composites are mechanically weak and are consequently limited to non-loading applications [16]. Addition of strong synthetic polymers such as polylactic acid and crosslinking of collagen by glutaraldehyde were reported to improve the mechanical properties of such composites [17,18]. Choosing a polymer network template that has high mechanical properties on which the hydroxyapatite (HAp) precipitates is another method of increasing composite applicability. From this point of view, BC shows a promise due to its high mechanical performance. Inspired by natural bone and shell that are composed of natural HAp and collagen and by artificial combinations of HAp and collagen [1921], chitosan [22], chitin [23], gelatin [24], cotton [25], and silk fibril [26], used for various tissue engineering applications, preparation of HAp/BC composites has been proposed in an attempt to make a new class of biomaterials with high mechanical performance and good osteoconductivity and biodegradation for tissue engineering and orthopaedic surgery. The objective of the present study was to initiate biomimetic apatite formation from simulated body fluid (SBF) on BC fibers in order to synthesize porous HAp/BC nanocomposites with 3-D network structure. For optimized deposition of HAp crystals, surface modification of BC was conducted. The emphasis was placed on the characterization of the resulted materials. 2. Materials and methods 2.1. Preparation of BC pellicles The bacterial strain used in this work was Acetobacter xylinum X-2 that was incubated for 7 days in a static culture containing 0.3% (w/w) green tea powder and 5% (w/w) sucrose. The pH of the medium was adjusted to 4.5 by acetic acid. The obtained gel-like BC pellicles were purified by immersion in deionized water at 90 C for 2 h and then boiling in a 0.5 M aqueous solution of NaOH for 15 min. The BC pellicles were then washed with deionized water several times and soaked in 1% NaOH for 2 days. Finally, the BC pellicles were washed free of alkali. The washing was repeated.

Fig. 1. Thin-film XRD patterns of various samples studied in this work (a) BC (b) 7-HAp/BC (c) 14-HAp/BC (d) 7-P-HAp/BC (e) 14-P-HAp/BC.

Y.Z. Wan et al. / Materials Science and Engineering C 27 (2007) 855864 Table 2 Average crystallinity and crystallite size of the HAp crystals in the HAp/BC nanocomposites Sample no. 7-HAp/BC 14-HAp/BC 7-P-HAp/BC 14-P-HAp/BC Crystallinity (%) 0.156 0.266 0.561 0.787 Crystallite size (nm) 20 25 37 46

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2.5. TEM observation TEM examination of the samples was carried out in a Tecnai F20 G2 (FEI Company) transmission electron microscope operated at 200 kV. Energy dispersive X-ray microanalysis (EDX) was also conducted for selected areas. The TEM samples were prepared by depositing a few drops of the HAp/BC composite powders ultrasonically dispersed in ethanol for 20 min on a carbon-coated copper grid. 2.6. XRD analysis The structure of the samples was studied using a Rigaku D/max 2500 X-ray diffractometer with a thin film attachment. The samples were scanned from 10 to 60 with a scan speed of 6/min. The XRD technique was also employed to determine the crystallite size and crystallinity of the HAp crystals in the nanocomposite samples. The procedures were similar to those described by Cao et al. [28] and Rusu et al. [29]. The data were obtained with the help of a MDI/JADE6 software package attached to the Rigaku XRD instrument. 2.7. FTIR analysis The surface properties of the BC and nanocomposite samples were examined by a Perkin-Elmer S2000 Fourier transform infrared spectrometer (FTIR, Nicolet Magna IR 560) in an attenuated total reflectance (ATR) mode. FTIR spectra were
Fig. 3. Scanning electron micrographs of freeze-dried BC samples (a) asprepared (b) phosphorylated.

recorded in a spectral range of 4000400 cm 1 at a resolution of 4 cm 1. 3. Results 3.1. XRD analysis Fig. 1 gives XRD patterns of various samples obtained in this work. In the case of the pure BC (curve a), three broad peaks located at 14.2, 16.6, and 22.4 were observed. The broadness of the three characteristic peaks was due to the partial crystallinity of BC [30]. The three peaks assigned to BC became weak but were still observed in curves b and c, corresponding to the 7-HAp/BC and 14-HAp/BC, respectively. In addition, extra peaks located at 25.9, 31.9, 39.5, 46.7, and 49.6 were noted. These bands were characteristic peaks of HAp crystals [31], suggesting that HAp crystals can be obtained on BC via a biomimetic process. A similar pattern was noticed for the 7-P-HAp/BC samples as shown in curve d. The peaks attributed to the HAp were also observed in curve d. This indicated that HAp crystals were formed on BC when it was subsequently phosphorylated and CaCl2-treated prior to soaking in 1.5 SBF. In curve d, peaks assigned to BC were still obvious whereas these peaks became extremely weak in curve e for the

Fig. 2. FTIR spectra of various samples studied in this work (a) BC (b) 7-HAp/ BC (c) 14-HAp/BC (d) 7-P-HAp/BC (e) 14-P-HAp/BC.

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14-P-HAp/BC. It was found that the characteristic peaks assigned to HAp crystals were stronger in curve e for the 14P-HAp/BC than in curve d as shown in Fig. 1. This is an indication of larger thickness of HAp crystals on BC in the 14P-HAp/BC samples than the 7-P-HAp/BC samples. Table 2 lists the average crystallinity and crystallite size of the HAp crystals formed on BC fibers based on (002) reflection peaks from the XRD measurements. The crystallite sizes of the

HAp crystals were 20, 25, 37, and 46 nm, respectively for the 7-HAp/BC, 14-HAp/BC, 7-P-HAp/BC, 14-P-HAp/BC samples, respectively. The HAp crystals formed on the phosphorylated BC showed larger crystallite sizes than those on the unphosphorylated BC. These sizes were comparable to that (about 50 nm in length) of the HAp nanocrystals in natural bone [20] and to those obtained by other researchers such as Rusu et al. who observed a crystallite size range of 15 to 50 nm for the HAp

Fig. 4. Scanning electron micrographs of unphosphorylated BC samples after soaking in 1.5 SBF. (a) A top view with a low magnification; (b) a top view with a moderate magnification; (c) a top view with a high magnification showing microstructure of HAp crystals; (d) a cross-section view with a low magnification; (e) a cross-section view showing HAp particles entangled with BC fibers.

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crystals formed on a chitosan matrix [29] and Pang et al. who noted a size range of 20 to 53 nm for their as-prepared HAp nanoparticles obtained by chemical precipitation [32]. As shown in Table 2, the HAp deposits on the phosphorylated BC exhibited crystallinities of 0.561% and 0.787%, respectively, after 7 and 14 days soaking in 1.5 SBF. The corresponding values for the HAp crystals on the unphosphorylated BC were 0.156% and 0.266%. This means that both the crystallinity and crystallite size of the HAp crystals formed on the phosphorylated BC were higher in comparison to those on the unphosphorylated BC. It was noted that the observed low crystallinities were comparable to those (below 1%) found in biological apatites [29]. The low crystallinities were consistent with the broadness of the diffraction peaks of HAp shown in Fig. 1. Data in Table 2 showed that both the crystallinity and crystallite size improved with soaking time. An increasing trend of crystallite size and/or crystallinity of HAp with soaking time was also reported by Furuzono et al. [26], Yin et al. [33], and Rusu et al. [29]. 3.2. FTIR analysis Fig. 2 shows the FTIR spectra of as-prepared BC and various HAp/BC nanocomposites. The FTIR spectrum of BC was typical where the absorption band assigned to the hydroxyl group and hydrogen bond was observed at 32003500 cm 1 [34]. Similarities were observed among the four HAp/BC nanocomposite samples. Firstly, the 32003500 cm 1 peak was sharply weakened (nearly disappeared) in the four spectra of the HAp/BC composites. Secondly, peaks at 1030 and 962 cm 1 were observed in curves b to e (see Fig. 2). The two bands could 3 be attributed to the stretching mode of PO4 vibration [35]. 1 Lastly, two weak bands at 1418 and 873 cm corresponding to 2 the stretching mode of CO3 ions [36] were noted for the four nanocomposite samples. The existence of the two peaks indicated that PO4 sites of the HAp structure were partially substituted by carbonate ions [37]. It was noted that the peak at 962 cm 1 was located on the shoulder of the 1029 cm 1 peak, instead of a very sharp band found in typical HAp spectra. This shape seemed to indicate the presence of a poorly crystalline HAp [38], which was in accordance with the XRD results. 3.3. SEM analysis Fig. 3 shows SEM micrographs of the BC samples. The asprepared and phosphorylated BCs showed 3-D network structure and interconnected pores. When the unphosphorylated BC samples were soaked in 1.5 SBF, the surface of BC appeared to be covered with HAp particles as shown in Fig. 4ac. Note that round shaped particles were formed on the unphosphorylated BC samples. Moreover, these spherical HAp particles covered the entire BC surface and the spaces among fibers were fully covered. A representative SEM micrograph with a higher magnification (Fig. 4c) revealed that the spheres were composed of HAp crystals with needle-like shape morphology. Similar needle-like HAp crystals were also observed by other researchers [31,39]. It should be mentioned
Fig. 5. Scanning electron micrographs of phosphorylated BC samples after soaking in 1.5 SBF (cross-section views). (a) A low magnification; (b) a moderate magnification; (c) a high magnification showing microstructure of HAp crystals.

that the surface of the BC fibers inside the BC fabric was barely covered with HAp crystals (Fig. 4d and e). As shown in Fig. 4e, the apatite was not formed on the surface of individual inner BC fibers; instead, the HAp spheres seemed to be entangled with BC fibers. This suggested the inhomogeneous formation of HAp throughout the entire BC fabric.

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Fig. 6. TEM images of the microstructure of HAp/BC samples. (a) A relatively low magnification image showing several BC microfibrils; (b) EDX spectrum taken from a typical microfibril and its surrounding area in (a); (c) a high magnification image showing a single microfibril and its surrounding area; (d) EDX spectrum taken from the single BC microfibril and in the vicinity of the microfibril.

Fig. 5 shows SEM photographs of the phosphorylated BC with HAp precipitates. The surface feature differed from that shown in Fig. 4. Unlike the unphosphorylated BC samples, the deposits were uniformly distributed throughout the phosphorylated BC fabric, indicating that phosphorylation is necessary for the synthesis of the HAp/BC composites. The HAp particles were observed on the surfaces of all BC fibers both on the top and inside the BC fabric, indicating that HAp formation was equally carried out throughout the BC fabric. Moreover, each BC fiber was wrapped with HAp crystals and interconnected pores were still evident. Similar to the unphosphorylated BC samples, the coatings on the phosphorylated BC samples were porous and consisted of needle-like fragments (see Fig. 5c). It should be noted that the HAp layers were too thick due to the long immersion time. More investigation is needed to find HAp induction period and to obtain HAp crystals with proper thickness in our further studies.

3.4. TEM images For TEM analysis, only the HAp/BC composite samples with the phosphorylated BC fibers were chosen. Morphologies were observed and chemical components of the HAp/BC composite specimens were analyzed using TEM with EDX. Typical TEM images are shown in Fig. 6. In Fig. 6a, several BC microfibrils were observed and each of them was surrounded by several nano-sized particles. This might suggest that the two components in the composites are uniformly dispersed. The EDX analysis result shown in Fig. 6b suggested that these particles were HAp deposits. In an image with a higher magnification (Fig. 6c), a BC microfibril was observed with a diameter of ca. 8 nm, which agrees closely with a previously reported value of 7 nm [40]. Beside this BC microfibril were HAp nanoparticles. The corresponding EDX spectrum (Fig. 6d) suggested that all these precipitates

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Fig. 6 (continued ).

contained Ca and P, further confirming the existence of HAp crystals. 4. Discussion Biomineralization involves initial nucleation and subsequent growth of apatites from aqueous solutions. Surface functional groups of the substrate materials play a decisive role in both nucleation and growth. Modification of surface properties is thus paramount for biomedical materials since in some cases inhibition of apatite formation is preferred, such as on the blood-contacting surface of an implanted artificial organ; while in the case of tissuecontacting surface, enhancement of apatite formation is required. To this end, much research has been conducted to elucidate mechanisms of apatite formation on a foreign surface and to determine its substrate dependence [4143]. It is believed that such groups as amine group [43], silanol group [44], sodium titanate hydrogel layer [45], hydroxyl group [46], carboxyl group

[47], and phosphate group [46] can induce apatite formation. In the present study, two surface conditions were compared in terms of the capability of inducing HAp growth. As revealed by SEM images shown in Fig. 4, hydroxyl groups abundant on BC fibers appeared to trigger little HAp formation. From the morphological feature of the HAp/BC composites with the unphosphorylated BC (shown in Fig. 4), it is proposed that the incorporation of calcium ions is mainly through physical entrapment, i.e., absorption due to the 3-D network structure and that interaction between calcium ions and hydroxyl groups is weak. Furthermore, it is considered that formation of HAp crystals in the outer surface of the BC fabric prevents calcium ions from migrating into inner BC fibers as a result of physical hindrance, resulting in thick HAp crystals on the top surface and far less HAp formation inside the BC fabric. This finding reveals that the hydroxyl groups on BC fibers do not have enough reactivity to induce HAp formation, which agrees well with the findings concerning plant cellulose [35,48,49]. On the contrary, phosphate groups created by

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Fig. 7. Schematic diagrams of various interaction processes (a) HAp growth on unphosphorylated BC; (b) phosphorylation of BC; (c) HAp growth on phosphorylated BC.

phosphorylation were found to effectively stimulate apatite growth on BC fibers. This is apparently displayed in Fig. 5 where surface of each BC fiber is fully covered by a layer of HAp. This finding regarding the biomineralization of BC is similar to those reported by other researchers concerning the biomineralization of cotton, chitosan, and chitin [27,5052]. The observed homogeneous HAp growth on the phosphorylated BC fibers demonstrates that strong ionic interaction between the calcium ions and the phosphate groups is considered dominant and that physical absorption is negligible. The uniform formation of a HAp layer throughout the entire phosphorylated BC indicates that HAp crystals are formed simultaneously throughout the BC fabric. The different responses between the unphosphorylated and phosphorylated BC fibers may be interpreted by the following mechanisms (shown in Fig. 7). It is believed that the nonionic hydroxyl groups on the unphosphorylated BC may firstly bind the calcium ions through ionicdipolar interaction, and then HAp crystals grow around these trapped ions. The process is

schematically displayed in Fig. 7a. However, a different process is involved for the phosphorylated BC. As shown in Fig. 7b, esterification takes place during phosphorylation and thus anionic phosphate groups are bonded to the cellulose chain through strong covalent bonds. The negatively charged phosphate groups are capable of trapping calcium ions, forming calcium phosphate complexes that act as nuclei of HAp and HAp grows by further complexation with phosphate ions in SBF as shown in Fig. 7c. Note that the bonding between the calcium ions and BC chain is via strong ionic bonds for the phosphorylated BC while the calcium ions complexation with nonionic hydroxyl groups proceeds via ionicdipolar interaction for the unphosphorylated BC (Fig. 7a). The weak ionicdipolar interaction is responsible for the low apatite formation capability of the unphosphorylated BC. The weak induction capability of nonionic hydroxyl groups has also been reported by Tanahashi and his coworkers [43]. It is believed that the different mechanisms described above are responsible for the different morphologies of HAp particles observed on the

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unphosphorylated and phosphorylated BC fibers as interaction between organic and inorganic components controls the morphology [53]. A similar result has been reported by Mller et al. with respect to apatite growth on plant-derived cellulose [49]. HAp is one of the frequently used bioceramics for bone and dental tissues reconstitution. It has excellent biocompatibility with hard tissues, and high osteoconductivity and bioactivity. However, the rate of substitution in artificially made HAp is as slow as 1 mm/year [54]. The FTIR findings in our work show that the HAp crystals formed on BC fibers are carbonatecontaining. From clinical point of view, carbonate-containing HAp is more favourable as biological apatites also contain a few 2 percent carbonate ions (CO3 ) and show a faster biodegradation rate than neat HAp. Furthermore, as shown in Table 2, the low crystallinities of the HAp crystals formed on the phosphorylated BC fibers resemble biological apatites, which is beneficial to the control of in vivo resorbability rates [29]. Another key issue in tissue engineering is the development of suitable biodegradable scaffolds with high porosity, suitable pore size and highly interconnected pore structure. The observed 3-D porous network structure and interconnected pores that can be adjusted in a wide range make the HAp/BC composites promising in tissue engineering. In addition, it is expected that the high mechanical properties of BC allows the design of a wide range of HAp/BC composites with mechanical properties from that analogous to the soft tissues to that analogous to the hard tissues by controlling HAp to BC ratio and their 3-D structure. It is, therefore, believed that the HAp/BC nanocomposites prepared by the present biomimetic process are attractive in the biomedical field as, for example, tissue engineering scaffolds and even bone replacement implants. The investigation of the control of mechanical properties, porosity and volume and assessment of biological responses of the HAp/BC nanocomposites is under way. 5. Conclusions Despite the abundance in hydroxyl groups on BC, few HAp crystals were found on the surface of individual BC fibers after soaking in 1.5 SBF for up to 14 days when phosphorylation was not performed, indicating that the hydroxyl groups are not reactive enough to trigger HAp growth. Phosphorylation treatment of BC greatly enhanced its capability of inducing HAp formation. SEM revealed the homogeneous precipitation of HAp crystals on the surface of the phosphorylated BC fibers. As for the unphosphorylated BC, HAp crystals were observed only on the outer BC fibers but inner BC fibers were barely coated with HAp. XRD analysis confirmed that the HAp crystals in the HAp/BC nanocomposites had low crystallinities (less than 1%) and that the crystallite sizes were nano-sized. FTIR results showed that the HAp crystals obtained were partially carbonate substituted. Therefore, the HAp prepared by the current biomimetic process was similar to natural bone in structure, crystallinity and crystal size. TEM analysis confirmed that each nano-sized BC microfibril was surrounded by HAp nanocrystals. It was found that the phosphorylated BC could act

as potent substrates for apatite nucleation. Our preliminary results suggest that the HAp/BC nanocomposites are promising for applications in tissue engineering though further studies are required. Acknowledgements This study was financially supported by the Tianjin Municipal Science and Technology Committee through Grants No. 05YFSYSF01800 and No. 043111511. Grateful thanks are also due to the National Natural Science Foundation (Grant No. 50673076 and Grant No. 50539060). References
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