Preparation Of N, O-carboxymethyl Chitosan/Nano-β-tricalcium Phosphate Composite And Its Basic Research About Biological Properties

Biological materials provide a new choice for the reparation treatment of bone defects caused by various reasons, a variety of deficiencies restricted the development of a single material as biological materials. therefore, research on the development of novel composite materials that have the advantages of various materials has become hot topic in bone repair and bone tissue engineering scaffold material development.Chitosan is a kind of innately biodegradable positive ion polysaccharides, which is similar to extracellular polysaccharides.It can promote adhension and proliferation of histocytes. Carboxymethyl chitosan (N,O-CMC) is carboxymethylation product of chitosan and has an advantage over chitosan on water solubility and complexation with metal ions. So the latter is often used as matrix of composite materials and plays the role of organic phase in bionic bone.β-tricalcium phosphate (β-TCP) is a compound of great qualities mostly because its chemical composition is close to natural bone, as well as its good bioactivity and biodegradability.The degradation product such as calcium and phosphate ions can enter the circulatory system in vivo and promote new bone formation. Thus it has become the hot research topic in the bone repair and tissue engineering scaffold materials.Currently Institute with of beta-TCP powder body most in micron level at home and abroad, nano level beta-TCP (n-beta-TCP) of synthesis and composite research reported less; and n-beta-TCP biological ceramic because grain small, makes material in the of inherent stomatal or defects size greatly reduced, difficult caused wear Crystal fracture, conducive to improve material of fracture toughness; while, grain of refinement and great to increased has n-beta-TCP of Crystal territories number, makes Nano biological ceramic performance out unique of Super plastic and the elastic, mechanical performance; and nano material increases of than surface area and can improve its surface activity. N-beta-TCP and N,O-CMC complex research can make up for the micron level beta-TCP deficiency, and helped to give full play to the advantages of the two, experiments laid the Foundation for its clinical application. One standard of the main clinical successes of bone repair material is osseointegration between the defect and the material. The precondition of osseointegration is that the repair material can survive and function in the body. This requires the material has the ability to vascularize at the suitable time, because during the bone remodeling process , vascularization has a close relation to bone formation, and sufficient blood provision is the key of successful bone defect repair. VEGF is the most strong factor which can promote vasoformation,and is the critical regulatory factor during vasoformation and bone formation. The express level of VEGF has a direct influence on blood provision of new bone, and finally affects the speed and quality of bone formation. Consequently ,the study about enhancing the expressing of VEGF in bone-repair materials by various ways to promote the formation of vessels around defected bone tissue has become the emphasis on bone-repair and bone tissiue engineering investigations. It can indirectly reflect the biological activity of bone through the test of VEGF expressing level of the materials.The elementary structure frame of bone tissue is consist of nano-inorganic phase and nano- organic phase,both of them are arranged by suitable proportion and order . Because of this reason, The organic phase ,namely ,self-made nano-pureβ-Ca3(PO4)2 (n-β-TCP) powder were distribute evenly into the bio-degradable organic N,O-CMC to prepare porous N,O-carboxymethylchitosan/nano-β-tricalciumphosphate(N, O-CMC/n-β-TCP) composites with different mass proportions and have done research as follows:1. Calcium nitrate tetrahydrate and diammonium hydrogen phosphate were used to make nano-sized pureβ-TCP powder by microwave assisted co-precipitation method.2. Nano-sized pureβ-TCP powder and N,O-CMC were made into porous N,O-CMC/n-β-TCP composites in various mass proportions by means of mechanical mixing-bicarbonate drilling-press shaping method.3.In order to make the feasibility approach to the composites as bone repair material and to provide selection basis and experiment foundation for scaffold material in bone tissue engineering,the composites were observed by SEM and characterized and analyzed by XRD, FTIR, water absorption rate, open porosity and mechanics property test.4. The degradation property of the composites was studied in simulated body fluid in vitro. In order to evaluate whether the composites were suitable for bone repair and bone tissue engineering, the changes of pH, calcium concentration, phosphonium concentration in the simulated body fluid were tested and the changes of the weight, surface structure, components of the composites were also observed and detected.5. The experiment on the biological property of the composite and osteoblast-like cells.The osteoblast-like cells were cultivated in vitro on the composites with different mass proportion and artificial-bone. The cells'growth and attachment to the materials were observed by SEM; proliferation of the cells was estimated by MTT; the levels of VEGFmRNA and VEGF protein in above materials were tested and compared by Real-Time PCR and Western blotting.According to the experiment results, the composites which is suitable for osteoblast-like cells to attach, grow and proliferate was selected. The results provided basis for bone formation experiment in vivo and biological improvement as a bone scaffold material in the future.The experiment results show as follows:1. Rod-shape pureβ-TCP powder with 150-250 nm in length, 100 nm in width can be prepared by microwave assisted co-precipitation method using calcium nitrate tetrahydrate and diammonium hydrogen phosphate. This obviously offer a new method which producting unit simple ,efficient and quick .2. Multiporous composites of N,O-CMC/n-β-TCP with different mass ratios can be prepared by the way of"mechanical mixing-bicarbonate of soda producing pores-shaping". The XRD and FTIR analyses indicate that the strength and tenacity of composites are improved because of theβ-TCP nanometer characteristic and can raise the mechanical property, meanwhile overcoming the great brittleness and molding difficult defect .It offer a method and reference for preparing bong biomaterials with production unit simple and low carbon protection.3.The water absorption and porosity in these composites is over 65% and 45% respectively. There are irregular r pores with different sizes and shapes in the material. The composite with the mass ratio of 1/1 is of the biggest aperture. These kinds of materials can basicly meet the requirements of the bone repair , as their compressive strength and bending strength over 13.528MPa(higher than that of human cancellous bone) and 10.52MPa respectively.4. In terms of the degradation capability of the three proportions of N,O-CMC/n-β-TCP composites, the pH is over 6.9 in each group especially when the proportion is 1:1(pH:7.0-7.4). This condition is helpful in keeping the pH staying slightly alkaline condition and can avoid or reduce the chance of sterile inflammation. The XRD and FTIR analyses show that bone- like apatite formed on the surface of the composites after infused in the simulated body fluid. And there were the largest number of bone- like apatite formed when the proportion is 1:1. We can infer that these three groups of composites have great bone conduction bioactivity and the capability will be better when the ratio is 1:1.5. Through the SEM observation, MG63 cells live well when the cells were cultured together on the surface of the three different proportions matericals in vitro. From MTT test ,We see the proliferation results is similar to that of artificial bones when the mass ratio of N,O-CMC/n-β-TCP is 1:1, but it's significantly higher than that of the other two groups and there are statistics difference ; the rusults of the levels of VEGF mRNA and VEGF protein secreted by MG63 through Real-Time PCR and Western blotting are just the same as we can see from MTT test.We can get conclusions from the results above: these three proportions of composites can basicly meet the requirements of bone repair matericals, especially the group with the mass ratio of N,O-CMC/n-β-TCP is 1:1 owns the best biological performance of the groups in vitro.