| Objective:To investigate the effect of extraction time, concentration of acetic acid and solid-liquid ratio on the extraction of rat tail tendon collagen,establish and optimize the extraction process of rat tail tendon collagen. The objective of this experiment was preparing a chitosan/collagen hydrogel with gelatin microsphere(GMs) dual delivery system for sequential release of recombinant human bone morphogenetic protein-2(rh BMP-2) to induce bone regeneration. Methods:The treatment of mandibular defects result from accident, cysts, tumors, trauma, infection and fractures is the most urgent issue for patients and a great challenge to orthopedic doctors, tissue engineering provides a new way for the treatment of bone regeneration induced by bone defect area. Recombinant human BMP-2(rh BMP-2) is one of the most potent osteoinductive growth factors and has been widely used in various clinical orthopedic fields and clinical therapeutic trials in the dental. Although rh BMP-2 has significant osteogenic effect, rh BMP-2 always appears burst-release at early period and suffer a remarkable loss of activity in vivo when they are simple mixed with bone grafts due to their short half-life, which may impair the bone regeneration. Therefore this study selected good biological activity of collagen and chitosan as raw materials of prepared hydrogel carrier, and preparation of a sustained-release effect good carrier of gelatin microspheres, microspheres embedded in hydrogel to form a dual release system. Select healthy SD rats tail, extraction rat tail tendon collagen by acid-soluble method, calculation of different extraction time, acetic acid concentration and solid-liquid ratio on the extraction ratio of collagen, the optimal extraction parameters were explored by orthogonal test to analyzing factors extraction time, the concentration of acetic acid and solid-liquid ratio, etc.In order to make the rh BMP-2 can be better used in bone tissue engineering, we construct the CC-GMS sustained release of rh BMP-2 collagen / chitosan stent, and by in vitro experiments to the support of the micro structure, gel time, degradation, release properties and support the biological activity was evaluated. For further characterization of sustained release of rh BMP-2 CC-GMS scaffold for repairing bone defect ability, we adopt new Zealand white rabbits mandibular defects experimental model, and respectively by means of soft X-ray and spiral CT 3D reconstruction, micro CT, bone mineral content of bone mineral density of quantitative analysis and microstructure examination of bone defect repair was evaluated. Results:Univariate analysis showed that the extraction time, the concentration of acetic acid and liquid ratio within a certain range of parameters were positively correlated with the collagen extraction rate. The optimal parameters are extracted time 72 h, the concentration of acetic acid 0.5M, solid-liquid ratio of 1:40. SDS-PAGE electrophoresis showed that the samples are mainly collagen typeⅠ.Tannic acid precipitation test shows a low degree of degradation of collagen. The extract was confirmed by infrared spectral analysis to be typeⅠcollagen with intact structure. After cross-linking of collagen, SEM figure shows the internal interconnected to form a porous honeycomb structure, the pore size suitable. The SEM images revealed that GMs had a polyporous surface and were spherical in shape. The complex scaffold exhibited an ideal releasing profile in vitro. The enzyme stability of CC was good, and good release effect, the cumulative rh BMP-2 release rate was 75.11 ±4.36 % in 30 day. Isolation of rat BMSCs growth in good condition, to detect the cell cycle of most cells in a quiescent relatively inactive, flow cytometry, no expression of CD34, CD45, high expression of CD90. BMSCs was successfully induced to differentiate into osteoblasts and adipocytes, Alizarin red, Oil red O and alkaline phosphatase staining positive. The biological activity test showed that BMSCs grew well in the CC-GMS scaffold, and the number of cells increased significantly.The x-ray analysis indicated that the group of chitosan/collagen hydrogel with rh BMP-2-loaded GMS not only bridged the defects as early as 4 weeks, but also healed the defects at 8 weeks. These results were confirmed by micro-computed tomographic. When compared with other control groups, the composite scaffold group exhibited significantly greater new bone formation, as evidenced by bone mineral content evaluation, histological observations. Moreover, the biocompatibility and appropriate degradation of the composite scaffold could be obtained. Conclusions:The successful establishment and optimize the extraction process rat tail tendon collagen. get the optimum process parameters of extraction time, acetic acid concentration and the material liquid ratio. Effectively improve the extraction efficiency of rat tail tendon collagen. The CC-GMS scaffold has good biocompatibility and can be sustained release activity of rh BMP-2. Rh BMP2/ CC-GMS scaffold has good ability to induce bone regeneration in vivo, as tissue engineering bone substitute has a certain application prospect. |
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