Preparation and characterization of hydroxyapatite/chitosan-sodium alginate composite materials

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.

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(PO(OH),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-(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 (CCOONa, 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.

2.1 Materials and Instruments
Biomedical grade Chitosan(CS) with 99 per cent degree of the deacetylation; analytical grade calcium nitrate tetrahydrate (Ca(NO·4HO); Potassium di-hydrogen Phosphate (KHPO); sodium alginate was purchased from Tianlian Refined Co. LtdShanghai, China; acetic acid; an injectable syringe with needle diameter of 0.7mm; amagnetic 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-2000X-ray diffraction (XRD); Pyris6 thermogravimetry (TGA) made in PerkinElmer.
2.2 Preparation of hydroxyapatite/chitosan-sodium alginate composite materials
The solution of Ca(NO and KHPO 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(NO 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 KHPO 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 CuKradiation 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 50ºCto 800ºC

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

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 ofHA/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 2=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.
08805302.gif (12794 bytes)
Fig. 2
XRD patterns of (a) chitosan (b) HA/CS-Alg=50:50 composite (c) hydroxyapatite
08805303.jpg (44648 bytes)
Fig. 3 TEM photographs of (a) pure n-HA (b) HA/CS-Alg=50:50 composite

3.3 TEM measurements observations
In order to confirm the XRD results concerning thedistribution of HA crystallites in the composite, TEM measurements were performed. TEM photographs of n-HA and hydroxyapatite/chitosan-sodium alginate composite were shown in Fig.3. The TEM micrographs revealed the structural features of the composites. By shape, the HA nano-crystallites were found to be rod-like, with the addition of chitosan, the composite powders show a slimly shuttle-like morphology with an average of about 80nm in length and 20 nm in width[15]. This infers that the growth of n-HA crystals in the (002) direction of -axis may have been promoted. The HA were dispersed in the chitisan and sodium alginate matrix homogeneously. This advantage may be helpful for forming uniform block materials, especially when using colloidal processing along with slip casting [16]
08805304.jpg (70655 bytes)
Fig. 4 SEM of (a) pure HA (b) HA/CS-Alg=50:50 composite

3.4 Surface profiling and SEM
The surface morphological changes of the HA/CS-Alg=50:50 composites were studied by scanning electron microscope. Fig 4 shows the microstructures of the specimens. Fig 3a exhibited nano-size HA with almostuniform size particle, though having a number of micro-cracks and flaws. Fig 3b shows the composite filled with chitsan and sodium alginate, the HA particles are embedded in the matrix to form tight composite, which redound to the increase of compressive strength.
08805305.gif (8055 bytes)
Fig. 5 TGA curves of (a) pure HA; (b) The composite of 30% HA ; (c) The composite of 50% HA

3.5 TGA analysis
The TGA thermograms of prepared samples are shown in Fig 5. It can be seen from the trace of HA (Fig 5a) that there is no phase transformation taken place upon heating, which indicates its thermal stability at high temperature.The observed mass loss at about 120ºCcan be due to the dehydroxylation of nano-HA[17]. There was no notable occurrence of mass loss found for HA. Fig 5b shows the TGA curves of HA/CS-Alg=70/30 composite. An initial decrease in the onset of decomposition temperature corresponds to the loss of water, because the mass loss observed here is most pronounced up to 100℃.The relatively small mass loss is assigned to the removal of absorbed water between 80ºCand 120ºC. From the above discussion it could be inferred that HA does not interfere with the weight loss. The mass loss in the rang of 260-600ºCcan be due to thermal decomposition of chitosan and sodium alginate. The total mass loss was found to be around 30% and 50% from Fig.5b and 5c respectively, the thermograms for both samples showed the similar trends due to dehydroxylation and decomposition of organic macromolecules. Obviously, there was no mass loss occurred after 600ºC, which indicated that all the organic macromolecules were decomposed at below 600ºC, The composition of residue in the thermogram was found to be almost consistent with the HA content as initially added during the preparation, so it is believed that n-HA was perfectly incorporated into the composites

In the paper, the composites of HA/CS-Alg have been prepared by a co-precipitation method. There is an excellent miscibility between the matrix of chitisan, sodium alginate and n-HA, nano-size HA identified by TEM, SEM and XRD was perfectly incorporated into the composites. The analysis of TGA indicated the composition of composite were consistent with as initially added during the preparation, stronger interactions among the composition have been occurred, which endows the composite with well mechanical strength. The compressive strength can meet the need of bone carrying capacity. Based on the excellent properties, the composite of HA/CS-Alg can meet the demand of bone repairation of osteo-tissue engineering and substitute, and have extensive potentials to be used as substituted materials in bone tissue engineering and will contribute to the guided regeneration of new bone.