Study On The Preparation Of Hydroxyapatite/collagen/alginate Composites

Since a single material could not meet the functional requirements of bone, biomedical composite materials have received extensive attention. Combination of two or more components leads to better properties over that of individual component, providing a new route for biomaterials used for defected tissues of the human body. Biomedical composite materials have been the most flourishing field of biomaterials.Nanohydroxyapatite/collagen (Nano-HA/COL) composites have been extensively investigated due to their composition and structure similar to natural bone. Alginate, an anionic polysaccharide, has been widely used as an instant gel for bone tissue engineering for its good biocompatibility and biodegradation under normal physiological conditions. The presence of calcium is believed to be formation of an "egg-box" structure, with Ca2+ ions forming junctions between blocks of guluronic acid residues on adjacent chains, accordingly resulting in an elastic hydrogel. Since there exist some polysaccharides in natural bone, the addition of some structural polysaccharide may be helpful. Some studies showed that hydroxyapatite/collagen composite had good biocompatibility and that addition of alginate would result in a material with better properties. However, the preparation methods of these composites were mostly limited to a simple blend of collagen/HA composites and alginate solution. Maybe just for this reason, up to the present, little attention has been paid to the interactions between alginate and collagen molecular. In this study, we studied the molecular interactions between alginate and collagen by collagen fibril reconstruction in vitro and subsequent preparation of collagen/alginate composites. A composite gel composed of collagen, alginate and hydroxyapatite was prepared via in situ synthesis and co-mixing, using collagen assembly, and then freeze-drying of the composite gel to obtain collagen-alginate-HA porous materials. Scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and atomic force microscope, differential scanning calorimetry were used to characterize the physiochemical properties of the prepared materials and to reveal the possible interactions between molecular species. The swelling behavior was also tested to investigate the stability of two types of composites. The biological compatibility of H-C-A1(in situ),M1(co-mix) and CA(collagen and alginate composite) was evaluated by fluorescence, MTT and alkaline phosphates activity(ALP).The results indicated that the introduction of alginate, to some extent, influenced the collagen assembly process. H-bonding was the main interaction under experimental condition. The in-situ composites had interconnected porous structure with typical pores ranging between 200-600μm, and inorganic phase, poorly crystallized hydroxyapatite, uniform dispersion of inorganic phase was achieved with the organic substances. DSC-TG results showed that the thermal stability of in-situ materials were better than co-mix materials. Swelling results suggested that the in-situ composite was more stable than co-mix composite.The biological evaluation of the composites showed that the cell viability and proliferation was very good in all the materials prepared, H-C-A1,M1 and CA. The MTT test results showed that H-C-A1 favored the differentiation and proliferation of the cells better than M1 and CA. The ALP results showed that H-C-A1 had the best ability to forming new bone. The results of SEM showed the growth of cell was good on the surface of composite, and the morphology of cells was integrated, the pseudopod of cells spread outside obviously, the cells grew on the surface of materials overlaply, proliferated cells were active. These showed that the materials have good biocompatibility with H-C-A1 giving the best results.