Study On The Key Technology Of Preparation Of Recombined Human-like Collagen â…¡/nano-hydroxyapatite/Chitosan Composite Used For Bone Repair

Objective: To prepare the composite of the recombined human-like collagen II (RHLC II)/ nano-hydroxyapatite (nHA)/ chitosan (CS) with a biomimetic method by the electrostatic self-assembly technique, which avoid virus-danger of animal-sourced collagen and osteogenesis-lack of nano-hydroxyapatite based-ceramic materials. Further, study the optimization mechanism of the scaffold molding technology and obtain a novel bone repair material which has a promising broad market. Farther, improve the biocompatibility and inducibility of the scaffold, and study the inducible effect of rabbit sourced BMSCs seeding on scaffold. These were to give preliminary experimental evidence of a novel bone repair materials or cell vehicle materials, and obtain an ideal bone substitution for clinic.Method:1.The preparation of nano-hydroxyapatite: HA were prepared from oyster shell or inorganic metallic salt by hydrothermal synthesis or coprecipitation method. The structure of HA was evaluated by the X-ray diffraction (XRD), scanning electron microscope (SEM) and ZETASIZER; Finally some useful strategies were chosed to prepare nature bone-like hydroxyapatite.2.The preparation of RHLC II/ nHA/ CS composites: In order to meet the needs of repair bone materials with high compressive strength, i.e. the binding between inorganic and organic phase is tight or compact, in this research, RHLC II/ nHA/ CS composites were prepared by mixing method and in situ synthesis at moderate temperature. After analyzing their mechanic properties and homogeneity, a better way was selected to prepare them. Moreover, to avoid the toxic danger of chemical crosslink agent such as glutaraldehyde and strengthen the composite, the natural crosslink agent-genipin was firstly chosed to fabricate the scaffold and a novel RHLC II/ nHA/ CS composite with good compatibility was obtained.3. Establishing a model for optimizing the preparation: It is a wise way to reduce the process cost and tailor the needs of board market. In this step, an optimized modeling technique of composite was obtained with response surface methodology (RSM) by Minitab soft. The micro-structure, mechanical properties and the biocompatible of the composite were studied by the X-ray diffraction (XRD), flourier transformation infrared spectra (FTIR), scanning electron microscope (SEM) and MTT colorimetric assay. At the same time, the degree of the cross-linking of the genipin and the biomimetic bone composite was determined by the Unico UV. In addition, we acquire stiffer scaffold by new crosslink method.4. The Evaluation of bone induction activity: Seeded the rabbit bone marrow stem cells on scaffold, then observed the percent of live cells,cell attachment,ALP activity and the appearance of mineralized nodules, we finally acquired a materials with bone induction activity.Result: 1. A novel preparation of RHLC II/nHA/CS bone repair materials was:(1)n-HA/ RHLC II powder was prepared with nHA/ RHLC II =1.53 by in situ synthesis at alkalescency environment;(2)2wt% chitosan was uniform mixed with n-HA/ RHLC II powder, then added genipin at 0.61% final concentration and in poured the stainless steel tooting .Cold preservation at 4℃for 24 h;(3)Precooled the scaffold in the -80℃ultra low temperature freezer for 3h and formed the shaping in vacuum freeze-drying for 48h.The scaffold was preserve at desiccator after soaking in PBS (pH=7.3) for many times.(4)The scaffold and BMSCs was cultured in vitro for several days until the scaffold showing the induction bone activity .Then this material could be a substitution for bone repair.2. The composite bone repair material of RHLC II/nHA/CS had much similarity in the compositions and the structure of the nature bone, which was prepared from the weak crystalline nHA deposited in the recombined human-like collagen and the polysaccharides. Three dimensional porous scaffolds was formed by the interaction among the aforementioned three and the strongly combination of the organic and inorganic surfaces(26∶74). Finally a composite with 75～100μm of pore diameter, above 90% of the porosity, 80.46 MPa of maximum compressive strength, 1 degree of the cytotoxicity and with strong bone induction activity (100% of attachment, higher ALP activity and the appearance of mineralized nodules et al. in induced media) was successfully synthesized, which was an excellent biomimetic bone scaffold in composition, structure, function and close to the nature bone in mechanical strength.Conclusion: We first time systematic study on the key technology of Preparation of recombined human-like collagen II/nano-hydroxyapatite/chitosan composite used for bone repair. After optimize the preparation, we acquired a novel composite, whose structure and function are close to the nature bone. Finally we obtained an ideal bone repair material with bone induction activity, which was seeded BMSCs in induced media. The compressive strength of these porous scaffolds could be up to 80.46 MPa and by adjusting the ratio of the compositions, scaffolds with different compressive strength could be composed. Thus, this kind of composite could satisfy the needs for the bone tissue repairing or substitute and be the promising material applied in clinic. It will tailor the mechanic properties to different social needs and solve shortage of bone repair materials.