Lu Caixia, Yao Zihua, Li Zhiyun, Wang Yunke
(Research Center of Physical and Chemistry Analysis, Hebei University; Key Laboratory of Analytical Science and Technology of Hebei Province, Baoding 071002, China)

Abstract Hydroxyapatite/Chitosan-sodium alginate nanocomposite materials were prepared through co-precipitation method. The mechanical performance, component and morphology of the composites were determined by means of UTM, XRD, TEM, SEM and TGA. Results indicated that nano-hydroxyapatite particles having similar chemical composition to nature bone in the composites were dispersed evenly in Chitosan and sodium alginate matrix. The mechanical property of the nanocomposite was well with maximum compressive strength value of 47.183 MPa at 50wt% HA of HA/CS-Alg. The composites can meet the demands of repairation and substitution of bone tissue and have potential application prospect in internal fixation of bone fracture. Furthermore, the research was helpful for the theoretical development of bone tissue engineering.
Keywords Hydroxyapatite; chitosan; sodium alginate; composite; co-precipitation method

1. INTRODUCE
Bone repair or regeneration is a common and complicated clinical problem in orthopaedic surgery. Hence ideal bone substituted material have been widely studied. Nature bone is actually an inorganic/organic composite mainly consisted of nano-structure hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂,HA) and collagen fibers^[1]. Hydroxyapatite is a major inorganic component of bone and one of excellent candidates for bone repair and regeneration, because of its similar chemical composition to that of the mineral phase of bone. It has been used extensively for biomedical implant applications and bone regeneration due to its bioactive, biodegradable and osteoconductive properties ^[2,3]. Owing to its inadequate mechanical properties and brittleness, the instability of the HA particulate is often encountered when the particles are mixed with saline or patient's blood, making it unable to be used as a load bearing implant.
    To solve the problem, incorporation of HA into polymer matrix has also been carried out to increase osteoconductivity and biodegradability with significant enhancement of mechanical strength ^[4]. Recently, a great attentions have been focused on the composite of HA in conjugation with natural polymers ^[5,6] and synthetic polymers ^[7,8]. Chitosan (CS), an inexpensive, inert, hydrophilic, biocompatible linear polymer, derived from the exoskeletons of insects and shells of crustaceans, is the second most abundant natural polysaccharide after cellulose. The chemical structure of chitosan is b-(1,4)-2-amino-2-deoxy-D-glucose which is obtained by partial deacetylating chitin. Due to its unique properties such as biodegradability, non-toxicity, anti-bacterial effect and biocompatibility ^[9], chitosan have been used widely in a variety of biomedical application, such as enzyme carriers, wound dressing, drug delivery carriers, tissue engineering, bone healing materials ^[10,11], now much attention has been paid to chitosan-based biomedical materials. Besides, alginate (C₅H₇O₄COONa, Alg) is another polysaccharide occurring in large amounts in nature, which is extracted from seaweeds. It is composed of a 1-4-linked block polymer of polyglucuronate (poly-G), polymannuronate (poly-M) acids or its copolymer bound in a random sequences^[12]. Sodium alginate and chitosan are ionic polymer of polysaccharides, which have good biocompatibility, high bioactivity and bonding properties.
   In this study, Hydroxyapatite, chitosan and sodium alginate were selected to formulate the composites, they have great impact on human health care systems due to its biocompatibility, bioresorbability, haemostatic, antiinfectivity, plasticity and adhesiveness ^[13,14], which makes them suitable binder and matrix for bone materials. The aim of this work is to prepare homogenous, high-strength and the perfect performances nano-HA/CS-Alg composites by co-precipitation method. The composites will be hoped to become bone substitutable material.

2.EXPERIMENTS
2.1 Materials and Instruments
Biomedical grade Chitosan(CS) with 99 per cent degree of the deacetylation; analytical grade calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O); Potassium di-hydrogen Phosphate (KH₂PO₄); sodium alginate was purchased from Tianlian Refined Co. Ltd. Shanghai, China; acetic acid; an injectable syringe with needle diameter of 0.7mm; a magnetic stirrer; Instron 1185 Universal Testing Machine (UTM); JEM-100SX (Japan electron) transmission electron microscopy (TEM); KYKY-2800B scanning electron microscopy (SEM) with a Thermo Noran-Vantage X-ray energy dispersive spectrometer; Y-2000 X-ray diffraction (XRD); Pyris6 thermogravimetry (TGA) made in PerkinElmer.
2.2 Preparation of hydroxyapatite/chitosan-sodium alginate composite materials
The solution of Ca(NO₃)₂ and KH₂PO₄ were prepared with near 1.67 Ca/P stoichiometric ratio. The 2 wt﹪chitosan solution is prepared by dissolving chitosan into 2 wt﹪ acetic acid with stirring on a magnetic stirrer for 4h to get a perfectly transparent solution. The 2wt﹪sodium alginate solution is prepared by dissolving the deionized water with stirring on a magnetic stirrer for 4h. The homogeneous sodium alginate solution was injected in the chitosan solution by a syringe with needle diameter of 0.7mm, the mass ratio of chitosan and sodium alginate is 2:1. The Ca(NO₃)₂ solution was added into the mixture of chitosan and sodium alginate solution with vigorous stirring for 10h, and pH was adjusted with ammonia solution to about 9-10 at 40ºC, then the KH₂PO₄ solution was dropped slowly into the mixture. The reaction temperature was maintained at 40ºC, the stirring was kept for 10h. The obtained white slurry was aged for 24h, and then the precipitate was filtered. In order to avoid serious aggregation of ultrafine powder during drying, the water in the precipitate was replaced by ethanol, the stirring was kept for 10h, filtered again, washed two to three times with distilled water. The precipitated composites were filled in a mould and then dried in a vacuum oven at 80ºC.
2.3 Characterization of the hydroxyapatite/chitosan-sodium alginate composite materials    
The cylindrical blocks of approximately10mm height and 10mm diameter were obtained. The blocks were then determined for its compressive strength by Universal Testing Machine. The composites were characterized by X-ray diffraction with Ni-filtered CuKa radiation generated at 25kV and 30mA at the wavelength of 1.54Å. The dispersion and particle size of composite were evaluated by a transmission electron microscopy. After the samples were cut as cross section by a sharp knife and sputtered with gold, the morphology of the composite was observed under scanning electron microscope. The organic and inorganic contents of composite were determined by using thermogravimetry analysis, about 10mg of samples were heated at the heating rate of 20ºC/min and the measurements were recorded from 50ºC to 800ºC.

Fig. 1 The maximum compressive strength of HA/CS-Alg composite

3.RESULTS AND DISCUSSION
3.1 Compressive strength
The equipment used for carrying out the test was universal testing machine with an operating head load of 5 KN. Cross-sectional area of the sample of known width and thickness was calculated. The measurements of the compressive strength of the composites were conducted to examine the mechanical performance. As shown in Figure 1 maximum compressive strength value of 47.183 MPa was obtained at 50wt﹪ HA of HA/CS-Alg composite , which indicating that such a novel bioabsorbable composite could be used as internal fixation of bone fracture in the view of application.
3.2 XRD analysis                   
Fig 2 shows the X-ray diffraction patterns for pure n-HA, chitosan and HA/CS-Alg composite. The characteristic peaks (Fig.2a) for chitosan were detected in the spectrum, two broad peaks of chitosan at 2q =10.1° and 19.8° are observed. The XRD pattern indicated that the inorganic phase was a single phase of HA (Fig.2c) with characteristic diffraction of (002) at 25.9° and with the overlapping diffraction of (300), (211), and (112) at 31.8°. Fig.2b showed the FWHM of a diffraction peak was very broad, which means that the crystallinity of the HA precipitated is lower. The diffraction from chitosan and sodium alginate were not detected from the XRD patterns, which means that the chitisan and sodium alginate obtained have amorphous structure.

Fig. 2 XRD patterns of (a) chitosan (b) HA/CS-Alg=50:50 composite (c) hydroxyapatite

Fig. 3 TEM photographs of (a) pure n-HA (b) HA/CS-Alg=50:50 composite