纳米磷酸钙在可注射组织工程骨中的应用研究

发布时间：2018-02-05 23:59

  本文关键词： 纳米磷酸钙 壳聚糖 可注射 成骨分化 组织工程　出处：《浙江理工大学》2013年硕士论文　论文类型：学位论文

【摘要】：随着人口老龄化现象的加剧和交通/运动创伤的增加，人们对骨移植材料的需求也日益增多。利用组织工程骨来实现硬组织的修复替代正成为本领域一种最为理想的治疗手段。从临床角度来看，可注射组织工程骨的使用减少了患者的痛苦，避免了手术感染的风险，不易留疤且缩减了治疗的费用，因而受到越来越多地重视。 本文以温敏性壳聚糖水凝胶为流动相，以纳米磷酸钙颗粒(nCP)为药物载体，以骨髓间充质干细胞(MSCs)为种子细胞，构建可注射组织工程骨，利用纳米磷酸钙颗粒优异的吸附和载药性能实现对药物和生长因子的可控释放，进而促进可注射组织工程骨对骨缺损的治疗和修复。具体研究内容和结果如下： 1.利用化学共沉淀方法制备了纳米磷酸钙颗粒，并对其形貌、结晶度和孔隙率等性能进行表征。通过原核表达制备了人重组骨形态发生蛋白2（rhBMP-2），并利用所制备的颗粒为载体负载了rhBMP-2和地塞米松（Dex），研究了药物负载量、释放速度及周期。结果表明：所制备的nCP颗粒呈均匀球状，结晶性较好，平均粒径为56.2±5.8nm，平均孔径为11.96nm，孔隙率和比表面积分别达到47.2%和89.337m~2/g，有利于后续实验药物的负载及释放；rhBMP-2基因可在大肠杆菌BL21中成功表达，纯化后蛋白产量为27mg每升培养基，尿素梯度透析复性产率为35.9%；每50mg颗粒上rhBMP-2的负载率为56.1±0.493%，Dex的负载率为12.8±0.372%，负载的Dex和rhBMP-2可以较缓慢的速度持续释放半个月左右，证明nCP是较理想的药物载体，满足骨组织工程的应用要求。 2.制备了壳聚糖/β-甘油磷酸钠/纳米磷酸钙（CS/GP/nCP）水凝胶，对水凝胶的凝胶时间、形貌、低温下的储存性能进行了表征。结果表明，随着GP浓度的提高，CS/GP/nCP体系的pH随之增高，凝胶时间逐渐变短，4℃下保持液态的时间更长。CS/GP/nCP水凝胶呈连续多孔的海绵状结构，孔径在100μm左右，适宜细胞的增殖，以及营养物质和生长因子的传递和运输。 3.成功地提取大鼠的骨髓间充质干细胞，，并与CS/GP/nCP水凝胶和载药nCP颗粒复合构建可注射组织工程骨，对其生物相容性进行表征。结果表明制备的可注射组织工程骨材料具有良好的生物相容性，MSCs能够在材料中正常增殖。当CS/GP/nCP体系中的Dex浓度为10~(-9)M左右时，对MSCs的增殖有一定的促进作用；当Dex浓度过高时，会抑制MSCs的增殖。当rhBMP-2的浓度达到30μg/mL时，对体系中MSCs的成骨分化有显著的促进作用。Dex和rhBMP-2共同作用时，相对于不含药物或仅含单种药物的体系，CS/GP/nCP凝胶中的MSCs的增殖速度更快，且促成骨分化的效果更显著。
[Abstract]:As population ageing intensifies and traffic / sports trauma increases. The demand for bone graft materials is also increasing. The use of tissue engineering bone to achieve the replacement of hard tissue is becoming the most ideal treatment in this field. From the clinical point of view. The use of injectable tissue engineered bone reduces the pain of patients, avoids the risk of surgical infection, does not scar easily and reduces the cost of treatment, so more and more attention is paid to it. In this paper, the injectable tissue engineered bone was constructed using thermo-sensitive chitosan hydrogel as mobile phase, nano-calcium phosphate (CPN) as drug carrier and bone marrow mesenchymal stem cells (MSCs) as seed cells. The controllable release of drugs and growth factors can be realized by the excellent adsorption and drug loading of nano-calcium phosphate particles. In order to promote the treatment and repair of bone defects with injectable tissue engineered bone, the specific research contents and results are as follows: 1. Nanometer calcium phosphate particles were prepared by chemical coprecipitation method and their morphology was analyzed. The crystallinity and porosity were characterized. The recombinant human bone morphogenetic protein 2rhBMP-2 was prepared by prokaryotic expression. The rhBMP-2 and dexamethasone Dexo were loaded with the prepared particles as the carrier. The drug loading, release rate and cycle were studied. The results showed that the prepared nCP particles were spherical in shape. The average particle size is 56.2 卤5.8 nm, the average pore size is 11.96 nm, and the porosity and specific surface area are 47.2% and 89.337 mg / g, respectively. It is beneficial to the loading and release of the following experimental drugs; RhBMP-2 gene was successfully expressed in Escherichia coli BL21. The protein yield was 27mg / L and urea gradient dialysis renaturation yield was 35.9mg / l. The loading rate of rhBMP-2 on 50mg granules was 56.1 卤0.493% and 12.8 卤0.372% respectively. The sustained release of Dex and rhBMP-2 at a slower rate for about half a month proved that nCP was an ideal drug carrier and could meet the requirements of bone tissue engineering. 2. Chitosan / 尾 -glycerophosphate / nano-calcium phosphate / CS / GP / nCP-based hydrogels were prepared. The gel time, morphology and storage performance at low temperature were characterized. With the increase of GP concentration, the pH of CS / GP / nCP system increases and the gel time becomes shorter. At 4 鈩

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