Preparation of Bioactive Chitosan-hydroxyapatite Nanocomposites for Bone Repair through Mechanochemical Reaction

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1 Materials Transactions, Vol. 45, No. 4 (2004) pp. 994 to 998 Special Issue on Frontiers of Smart Biomaterials #2004 The Japan Institute of Metals Preparation of Bioactive Chitosan-hydroxyapatite Nanocomposites for Bone Repair through Mechanochemical Reaction Akihiko Yoshida 1; * 1, Toshiki Miyazaki 1; * 2, Eiichi Ishida 2 and Masahiro Ashizuka 1 1 Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu , Japan 2 Department of Materials Science, Faculty of Engineering, Kyushu Institute of Technology, Kitakyushu , Japan Natural bone is a kind of nanocomposites composed of orientated hydroxyapatite (HAp) along c-axis and fibrous collagen. Therefore, composites exhibiting composition and structure analogous to those of natural bone have been expected to be useful bone substitute materials. Organic polymer-hap composites have been attracted much attention since they have interesting features such as bone-bonding ability, i.e. bioactivity and flexibility. In the present study, chitosan-hap nanocomposites were prepared through mechanochemical reaction using conventional ball mill and subsequent aging. The obtained composites contained carbonate-containing HAp, and HAp nanocrystals in the composites aged at 25 C for 24 h showed a needle-like structure. They can form bone-like HAp on their surfaces after soaking in simulated body fluid (SBF), indicating potential for bioactivity in living body. The prepared chitosan-hap composites are expected to be one of the useful bone substitute materials. (Received October 17, 2003; Accepted December 19, 2003) Keywords: chitosan, hydroxyapatite (HAp), nanocomposite, mechanochemical reaction, bioactivity, simulated body fluid (SBF) 1. Introduction Natural bone is well known as a kind of three-dimensionally woven organic-inorganic nanocomposite in which hydroxyapatite (HAp, Ca 10 (PO 4 ) 6 (OH) 2 ) crystals are deposited on fibrous collagen. This specific structure leads to combined advantages, i.e. flexibility owing to fibrous collagen and high mechanical strength owing to bone apatite. 1,2) For repair of damaged bone tissues, ceramic biomaterials are clinically used in orthopedic fields. Among them, certain types of ceramics can bond to living bone in the body, while artificial materials are normally encapsulated by fibrous tissue, when implanted in bony defect. 3) The ceramics having such a specific biological affinity are called bioactive ceramics. Sintered HAp, glasses in the system Na 2 O-CaO- SiO 2 -P 2 O 5, named Bioglass Ò, and glass-ceramic A-W, containing crystalline apatite and wollastonite are known as typical bioactive ceramics. They have been clinically used as artificial bone. Bone-bonding ability of bioactive ceramics have received much attraction, since it enables a tight fixation between bone and artificial materials. However, bioactive ceramics known so far have limitation on clinical application, because of their mechanical properties such as a high Young s modulus, a low toughness and a brittle character. We expect that organic-inorganic nanocomposites give a novel design of bioactive materials with various mechanical performance. Furthermore, novel bone-repairing materials which can be shaped into desirable shapes during operation have been desired in medical fields. Recently developments of nanocomposites consisting of organic polymer and HAp have been researched by investigators. 4 12) Collagen 6) - gelatin 7) - and chitosan 8,9) -HAp nanocomposites were prepared through coprecipitation method. The prepared nanocomposites have shown success in * 1 Graduate Student, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology * 2 Corresponding author, tmiya@life.kyutech.ac.jp formation of HAp nanocrystals with orientation along c-axis, and collagen-hap nanocomposites particularly showed both bioactivity and as high mechanical strength as 40 MPa. Among organic polymers, chitosan has attractive features as bone substitutes such as low toxicity, biodegradability and high flexibility. 8,12) In addition, chitosan has sites for formation of a complex with calcium. Nemoto et al. documented that silk fibroin-hap nanocomposites can be successfully synthesized through mechanochemical reaction using a multi-ring mill, and that HAp nanocrystals in the composites showed an anisotropy and a preferred orientation along their c-axes at high concentration of silk fibroin. They consequently suggested that the mechanochemical method is useful for synthesis of organic polymer-hap nanocomposites with strong interaction between inorganic components and organic matrix. 11) If this type of technique can be applied to preparation of chitosan- HAp composites, novel bone substitutes with structure analogous to natural bone can be obtained. This study aimed to prepare chitosan-hap composites through mechanochemical reaction and subsequent aging at various temperatures for 24 h in order to induce specific structures of HAp nanocrystals. 13) The structure of the prepared composites was characterized by several methods. Their bioactivity was examined in simulated body fluid (SBF) that has similar concentrations to human blood plasma, proposed by Kokubo and his colleagues ) 2. Materials and Methods 2.1 Preparation of chitosan-hap composites Reagent grade chemicals of calcium hydrogen phosphate dihydrate (CaHPO 4 2H 2 O) and calcium nitrate tetrahydrate (Ca(NO 3 ) 2 4H 2 O) that were product of Wako Pure Chemical Industries, Ltd., Japan, were used as a precursor of HAp, while chitosan powder (average molecular weight 35 kda, deacetylation degree 90%) that was product of Kimica Co., Japan, was used as organic polymer. All the chemicals were

2 Preparation of Chitosan-HAp Nanocomposites through Mechanochemical Reaction 995 used without further purification. One gram of chitosan powder was dissolved in 120 ml of 0.2 mol/l CH 3 COOH solution, and then stirred for further 30 min. The ph of the solution was then adjusted at 10.0 to 10.1 with NH 3 aqueous solution to form a sol. Calcium hydrogen phosphate dihydrate and calcium nitrate tetrahydrate were mixed at a Ca/P molar ratio of The mixture was added to the chitosan sol at chitosan: HAp mass ratio of the resultant composite of 20:80. The solution was stirred for further 5 min to form a slurry. The prepared slurry was ballmilled at ambient temperature for 9 h. After the milling, it was poured into a Teflon Ò vessel, and gently stirred at various temperatures ranging 25 to 60 C for 24 h. The aged slurry was filtered and the residue was washed with ultrapure water until the ph of the filtrate reached neutral. The formed precipitate was dried at 60 C for 24 h in an oven and shaped into cylindrical specimens under uniaxial pressure of 30 MPa. Intensity (a. units) : HAp 2.2 Soaking in SBF It s known that artificial materials exhibiting bioactivity essentially form bone-like apatite layer on their surfaces when implanted in body defects. 18,19) Previous reports showed that in vivo bioactivity can be reproduced in vitro in SBF. Therefore, the cylindrical specimens 12 mm in diameter and 3 mm in length were immersed in 30 ml of SBF (Na þ 142.0, K þ 5.0, Mg 2þ 1.5, Ca 2þ 2.5, Cl 147.8, HCO 3 4.2, HPO and SO mmol/l) for 7 days. The solution was buffered at ph 7.40 with 50 mmol/l trishydroxymethyl-aminomethane ((CH 2 OH) 3 CNH 2 ) and appropriate amounts of hydrochloric acid (HCl), and its temperature was kept at 36.5 C. 16,17) After soaking for 7 d, the specimens were taken out of SBF, washed with ultrapure water and then dried in a desiccator. 2.3 Characterization Structure of the specimens were analyzed by using powder X-ray diffraction (XRD; RINT-Ultima þ /XRD-DSC-XII, Rigaku Co., Japan), thin-film X-ray diffraction (TF-XRD; JDX-3500K, JEOL, Ltd., Japan), Fourier-transform infrared spectroscopy (FT-IR; Spectrum GX2000R, Perkin Elmer Ltd., England), transmission electron microscopy (TEM; H- 9000NAR, Hitachi Co., Japan) and field-emission scanning electron microscopy (FE-SEM; JSM-6320F, JEOL, Ltd., Japan) equipped with an energy dispersive X-ray spectroscope (EDX; Link ISIS, Oxford Instruments, England). In TF-XRD, the angle of the incident beam was fixed at 1 against the surface of the specimen, and the measurements were performed with continuous scanning mode. In FT-IR spectroscopy, samples were pulverized and mixed with potassium bromide (KBr) at sample to KBr mass ratio of 1:100, and uniaxially pressed to form a transparent disk. The obtained disk was subjected to the measurement. Changes in element concentrations and ph of SBF after soaking of the specimens were measured by inductively coupled plasma (ICP) atomic emission spectroscopy (Optima 4300DV, Perkin Elmer Ltd., England) and a ph meter (F23IIC, Horiba Co., Japan), respectively. 3. Results θ Fig. 1 XRD patterns of the composites aged at various temperatures for 24 h. Figure 1 shows XRD patterns of the composites aged at various temperatures. Broad peaks assigned to crystalline HAp were detected around 26 and 32 in 2, which indicated 002 plane and envelop of 211, 112 and 300 planes, respectively. Crystallinity of HAp in the composites increased according to increase in aging temperature. Figure 2 shows FT-IR spectra of the composites aged at various temperatures, as well as original chitosan powder. Peaks assigned to methylene (CH 2 ) group were observed at 1383 and 2900 cm 1, that assigned to amide I (C=O) at 1656 cm 1 and that assigned to amide II (NH) band at 1552 cm 1, for all of the specimens. 10) These peaks are attributed to chitosan in the composites. On the other hand, peaks assigned to phosphate (PO 4 ) group were observed at 565, 603, 963 and 1036 cm 1, and that assigned to hydroxyl (OH) group at 3570 cm 1 for the specimens containing HAp. 10,13,20) In addition, peaks assigned to carbonate (CO 3 ) were observed at 875, 1422 and 1452 cm 1. This means that the condition of the carbonation of the formed HAp is B-type, where some phosphate ions are substituted by carbonate ions in the crystal lattice of HAp. 13) Figure 3 shows TEM images and electron diffraction patterns of the composites. HAp nanocrystals in the composites aged at 25 C took a form of aggregations of needle-like particles about 120 nm in size. The electron diffraction patterns of the HAp crystals exhibited a diffused-ring pattern. Such needle-like particles were not observed for the composites aged at 40 and 60 C, and their electron patterns exhibited clear patterns.

3 996 A. Yoshida, T. Miyazaki, E. Ishida and M. Ashizuka PO4 PO4 Transmittance (a. units) OH Original chitosan powder amino CH2 PO4 PO4 amide II CH2 amidei Wave number, W / cm -1 Fig. 2 FT-IR spectra of the composites aged at various temperatures for 24 h as well as that of original chitosan powder. Figure 4 shows FE-SEM images of the surfaces of the composites aged at various temperatures before and after soaking in SBF at ph 7.40 and 36.5 C for 7 days. The surfaces of all the composites were covered with deposits after the soaking within 7 days. Morphology of the deposites was quite similar to those of HAp formed on bioactive glasses and glass-ceramics in SBF. 21) EDX spectra showed that the deposits consisted of calcium and phosphorous, and both TF-XRD patterns before and after soaking in SBF gave broad peaks assigned to low crystalline HAp, although both EDX spectra and TF-XRD patterns were not shown. These results suggest that the deposits formed on the composites are low crystallline HAp. Figure 5 shows changes in element concentrations and ph of SBF after soaking of the composites for 7 days. Both of calcium and phosphorous concentrations and ph decreased after soaking. This indicates that calcium and phosphate ions in SBF were consumed due to formation of deposits on the surfaces of the composites. 4. Discussion We can see from the results in the present study that nanocomposite with specific structure of HAp crystals can be synthesized from HAp and chitosan through conventional ball-milling, when temperature of aging is appropriately controlled. The composites aged at 25 C formed aggregations of needle-like HAp nanocrystals, while those aged at 40 and 60 C aggregations of elliptic HAp nanocrystals. This means that aging at 25 C improves the specific structure of Fig. 3 TEM images and their electron diffraction patterns of the composites aged at various temperatures for 24 h. HAp nanocrystals. The results from Figs. 4 and 5 suggest that the HAp-chitosan composites can make deposition of HAp after soaking in SBF. The low crystallinity of the formed HAp layer is characteristic of bone apatite. 22) These mean that the obtained composites have potential to form HAp layer on their surfaces when implanted in the body, and make tight bond to living bone through the formed HAp. It is interesting to further investigate effect of the complex

4 Preparation of Chitosan-HAp Nanocomposites through Mechanochemical Reaction 997 Fig. 4 FE-SEM images of the surfaces of the composites aged at various temperatures for 24 h before and after soaking in SBF for 7 days. Original concentration Ca Concentration, C / mm Original concentration Concentration,C / mm Original ph ph Fig. 5 Changes in element concentrations and ph of SBF arising from soaking the composites for 7 days. P structure composed from inorganic HAp and organic chitosan on mechanical properties of the composites. In near future, we should reveal this point by quantitatively examining mechanical properties of the composites, in order to optimize synthetic conditions. 5. Conclusion Chitosan-HAp nanocomposites were synthesized by conventional mechanochemical reaction using ball-mill and subsequent aging. HAp in the nanocomposites showed a needle-like structure when aged at 25 C for 24 h. The nanocomposites can form HAp layer in SBF within 7 days. This type of the nanocomposites is expected to be useful as bone substitute materials, e.g., a bone spacer and a bone filler, owing to both flexibility and bioactivity. Acknowledgement One of the authors (T.M.) acknowledges the research grant by the Naito Foundation, Japan. REFERENCES 1) L. L. Hench: J. Am. Ceram. Soc. 74 (1991) ) T. Kokubo, H.-M. Kim and M. Kawashita: Biomaterials 24 (2003) ) L. L. Hench and J. Wilson: Introduction to Bioceramics, (World Scientific, Singapore, 1993) pp ) Q. Liu, J. R. de Wijin and C. A. van Blitterswijk: Biomaterials 18 (1997) ) X. Wang, Y. Li, J. Wei and K. de Groot: Biomaterials 23 (2002) ) M. Kikuchi, S. Itoh, S. Ichinose, K. Shinomiya and J. Tanaka: Biomaterials 22 (2001) ) M. C. Chang, C.-C. Ko and W. H. Douglas: Biomaterials 24 (2003)

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