【作者】 吴巧凤；

【导师】 陈槐卿；

【作者基本信息】 四川大学 ， 生物医学工程， 2005， 硕士

【摘要】 骨组织工程学是利用组织工程学原理对骨缺损进行修复或重建的一门学科。近年来,将种子细胞与支架材料复合,在体外构建生物复合材料是骨组织工程学研究的热点。理想的生物复合材料应在保证材料力学性能的同时,具有良好的生物相容性和多孔相通性,以支持细胞的生长和功能的表达,为血管、神经的生长,营养物质的输送以及代谢产物的排出提供空间,促进骨组织的生长。目前应用较广泛的种子细胞为骨髓间充质干细胞(bone mesenchymal stem cells,MSCs),而支架材料多选用磷酸钙陶瓷与多聚物的复合物。本实验主要研究了成骨诱导后不同时期,大鼠骨髓间充质干细胞(rat mesenchymal stem cells, rMSCs)的成骨特性以及不同工艺参数的β-磷酸三钙(β-TCP)/聚L-乳酸(PLLA)多孔支架材料对种子细胞形态及功能的影响,从而选出处于最佳时期的种子细胞和最适工艺的β-TCP/PLLA多孔支架材料,以构建生物相容性更高的新型多孔生物复合材料。我们首先采用密度梯度离心法分离和培养大鼠骨髓间充质干细胞,并通过形态学观察和表面抗原测定等方法对细胞进行鉴定,然后将细胞向成骨方向诱导分化,比较诱导后不同时间细胞的形态及功能的差异,从而挑选出处于最佳时期的细胞作为种子细胞;用溶液浇铸—模压成型—沥滤法制备具有一定力学强度(抗弯强度≥5Mpa,抗压更多还原

【Abstract】 Bone tissue engineering is a subject which applying engineering and medicine principles to repairing bone defect and bone reconstruction. Recently, researchers are mainly focusing on fabricating bio-active compounds. Ideal bio-active compounds should possess good biocompatibility and three dimensions porous structure as well as assuring their mechanical properties. Only by this kind of structure can the composites support cells proliferation and differentiation, provide enough space and communicate ways for supplying adequate nutrients for blood vessels and nerves to grow in. At present, bone mesenchymal stem cells (MSCs) have been researched and used extensively as seeding cells for their low immunologic reaction, good ability of proliferation and easy to induced differentiation. Compounds of phosphate ceramics and polymers are usually used as scaffolds for their controllable biodegradability, good mechanical strength and osteogenesis in vivo. Traditional composites made by bioceramic β-tricalcium phasphate (β-TCP) and poly L-lactic acid (PLLA) are widely used in bioengineering. However, optimal ratio, porosity and pore size of this kind of scaffolds were not very clear yet. In this study, to explore new type compounds which possessed better osteoconductivity and osteoinductivity properties, we chose osteoblastic induced rat bone mesenchymal stem cells (rMSCs) as seeding cells, and cultured them on β-TCP/ PLLA scaffolds with different ratio, different pore size and different porosity. The aim was to investigate the optimum construction, which owned better properties for supporting cells growth, proliferation and differentiation. Firstly, we used the method of density gradient centrifugation to isolate and culture rMSCs. Through observing the cells morphology and assaying the cells surface antigen by flow cytometry (FCM), we found that the cells we cultured were positive for CD44 and negative for CD45, which were some characters of mesenchymal stem cells. Then, we detected the proliferation and differentiation of osteoblastic induced mesenchymal stem cells in different phases. Results suggested that the osteoblastic cells came into proliferative phase, matrix synthesis phase and mineralization phase after 10, 14 and 20 days induced respectively. Although these three phases were not apart strictly, we could choose the cells in the best phase to be target cells. To evaluate the biocompatibility of different technical parameters scaffolds and to build new type bio-active scaffolds, we chose passage 2 osteoblastic induced rMSCs as seeding cells in the latter study. Secondly, the process which consisted of solvent casting, com-pression molding and leaching stage had been used to fabricate scaffold of β-TCP/PLLA composites. Scaffolds were fabricated according to weight ratio of β-TCP/PLLA=1:1, 1:2 and 2:1; the porosity of the composites ranged from 50% to 75% when the porogen-NaCl added was 60wt%, 70wt% and 80wt% respectively; the pore size ranged from 200 to 950μm by adding different crystal porogen-NaCl. All the scaffolds had good mechanical strength (compressive strength ≥10MPa and bending strength ≥5MPa). We cultured osteoblastic induced rMSCs on the scaffolds of different ratio, different porosity and different pore size toscreen the suitable scaffolds. The results indicated that scaffolds of β-TCP/PLLA=2:1 could not only promote cells proliferation, but significantly increased osteocalcin production, which was the critical function of osteoblasts. In addition, scaffolds with NaCl=70 wt%,pore size=200-450 μm showed better compatibility to the target cells. Finally, we seeded passage 2 osteoblastic induced rMSCs on the scaffold with optimal parameters (β-TCP/PLLA=2:1, NaCl=70 wt%,pore size=200-450 μm) to fabricate new bio-active bone tissue engineering composite. We implanted this kind of composite, scaffold with rMSCs and scaffold without cells into the thigh muscles of nude mice respectively. Animals were sacrificed at the 8th and 12th week, SEM and histological sections showed that a significantly higher amount of bone in the osteoblastic induced rMSCs and β-TCP/PLLA scaffold composite as compared with rMSCs/scaffold and scaffold without cells. What’s more, rMSCs not induced showed multidifferential properties in vivo, which was not preferred in bone tissue engineering. This study showed that compounds of osteoblastic induced rMSCs and scaffold with β-TCP/PLLA=2:1, NaCl=70 wt%,pore size=200-450 μm had good properties as a kind of bone substitute.更多还原