【作者】 余敏；

【导师】 王万春；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 第一部分兔骨髓间充质干细胞的分离、培养、鉴定及体外诱导分化目的：探索兔骨髓间充质干细胞的分离、培养、鉴定及体外诱导分化的方法,研究其体外增殖,分化能力。方法：取兔骨髓冲洗液,采用密度梯度离心与全骨髓培养结合的方法分离、纯化骨髓间充质干细胞,体外培养；流式细胞仪鉴定第4代骨髓间充质干细胞表面CD29、CD34和CD44抗原；使用MTT法测定第4、8代细胞的增殖情况,绘制生长曲线；分别体外诱导第3代细胞向软骨细胞和骨细胞分化,对诱导分化为骨细胞进行碱性磷酸酶染色、VonKossa染色、Ⅰ型胶原免疫细胞化学染色和Ⅰ型胶原RT-PCR检测鉴定,对诱导分化为软骨细胞进行甲苯胺蓝染色、番红O染色、Ⅱ型胶原免疫细胞化学染色和Ⅱ型胶原RT-PCR检测鉴定。结果：经密度梯度离心结合全骨髓培养法分离所得到的细胞形态均一,呈长梭形或纺锤形,接近融合状态时可呈现旋涡样生长。所分离的细胞表达CD44、CD29,不表达CD34。生长曲线显示第4、8代细胞均有较强的增殖能力；向骨细胞诱导分化后,细胞碱性磷酸酶染色呈现阳性,VonKossa染色可见钙结节,Ⅰ型胶原免疫细胞化学染色呈现阳性,RT-PCR出现Ⅰ型胶原条带；向软骨细胞诱导分化后,细胞甲苯胺蓝染色被染成紫红色,呈现出异染性,番红O染色见细胞聚集部位呈红色,表明细胞合成和分泌糖胺多糖,Ⅱ型胶原免疫细胞化学染色为阳性,RT-PCR可见Ⅱ型胶原条带。结论：密度梯度分离法与全骨髓培养法结合能理想的分离、纯化BMSCs; BMSCs在诱导剂作用下能表现出成骨细胞和软骨细胞生物学特性,能用于组织工程中修复关节软骨及软骨下骨损伤；BMSCs在体外具有良好的增殖、自我更新和定向分化潜能,是组织工程理想的种子细胞。第二部分PLGA-PCL/PLGA-HA-βTCP双相支架的制备及生物学特性研究目的：研究制备出一种新型双相PLGA支架,并进行各项结构性能和生物学特性检测,探讨其作为组织工程支架的可行性。方法：支架制备：采用粒子沥滤致孔法结合浇注成型法制备双相PLGA支架,再对整个支架表面进行透明质酸真空吸附；结构观察：观察支架大体外观,并将制备好的支架切面进行扫描电子显微镜观察；生物相容性检测：将支架植入活体兔皮下组织内,观察机体对支架的反应及支架的降解情况；分别对植入细胞的支架进行扫描电子显微镜和石蜡切片HE染色观察。结果：制备好的支架为白色海绵样物质,肉眼可见分为上下两层。扫描电子显微镜观察两层的孔径大小符合设计要求；植入活体内的支架没有引起异物炎症反应,并逐步降解,12周时已基本降解完全；扫描电子显微镜和石蜡切片HE染色均发现细胞与支架结合良好。结论：实验设计制备出的双相PLGA支架制备方法简单,各项性能参数易于控制,且两层支架之间结合紧密,改进了双层支架容易开裂的缺陷；支架与BMSCs的生物相容性良好,有利于BMSCs吸附、增殖；对支架两层进行相应的改性后,能更好的促进BMSCs分化为目的细胞；双相PLGA支架与关节软骨及软骨下骨的正常生理结构更加接近,且降解时间理想,能更好的修复软骨和软骨下骨损伤。第三部分PLGA-PCL/PLGA-HA-βTCP双相支架结合BMSCs修复兔膝关节骨软骨缺损目的：研究双相PLGA支架结合BMSCs修复兔膝关节骨软骨缺损的效果,探讨双相PLGA支架做为组织工程支架承载BMSCs修复骨软骨缺损的可行性。方法：将30只健康新西兰大白兔,60个膝关节股骨滑车部位造成直径5mm,深4-5mm的骨软骨缺损,并随机分为三组(A组、B组、C组),A组为自发修复,不植入任何材料,B组仅植入双相支架修复,C组植入支架与BMSCs的复合物。术后6周、12周分批随机处死,取标本进行大体形态观察和组织学观察其修复效果并评分比较；对B组、C组和正常关节标本进行生物力学测试,并将各项参数与正常关节组织比较。结果：术后6周,A组大体观察评分4.3±0.9,组织学评分0.8±0.2；B组大体观察评分6.8±0.7,组织学评分5.1±0.8；C组大体观察评分8.1±0.4,组织学评分12.4±0.9。术后12周,A组大体观察评分5.2±1.2,组织学评分3.7±0.5；B组大体观察评分7.7±0.8,组织学评分10.3±0.6；C组大体观察评分9.2±0.3,组织学评分17.2±0.3。总的修复效果C组最理想,A组最差。生物力学测试得出：正常关节蠕变时间2763±649s,应力松弛时间2142±402s,杨氏模量0.36±0.22MPa;B组标本蠕变时间2189±533s,应力松弛时间1647±381s,杨氏模量0.29±0.17MPa；C组标本蠕变时间2314±592s,应力松弛时间1936±334s,杨氏模量0.32±0.13MPa。B组和C组标本的蠕变时间和应力松弛时候较正常关节均缩短,杨氏模量较正常关节偏小,但差异均无统计学意义。结论：同种异体BMSCs是良好的组织工程种子细胞,能用于修复骨软骨缺损；双相PLGA支架能承载BMSCs至缺损区,为其提供结构支撑,诱导其分化,再生出新生组织,修复组织缺损,是良好的生物工程支架；双相PLGA支架结合BMSCs修复兔膝关节软骨及软骨下骨缺损,修复效果理想,各项生物学特性接近正常关节组织。更多还原

【Abstract】 Part I. The isolation, cultivation, identification and differentiation of rabbit bone mesenchy mal stem cells invitroObjective:To explore the method of isolating, cultivating and identification of rabbit bone mesenchymal stem cells (BMSCs), and study their capacity of proliferation and potential of differentiation in vitro.Method:Rabbit BMSCs in rinse solution of bone marrow was isolated by density gradient centrifugation and bone marrow culture, cultured in vitro. Flow cytometry was used to examine the expression of CD29, CD34 and CD44 cell surface antigens. The proliferation ability of the 4th and 8th passage cell was determined by MTT assay and the growth curve was drawed based on it. The 3rd passage cell was induced osteogenesis and chondrogenesis respectively, then alkaline phosphatase and VonKossa staining, immunocytochemical stain and RT-PCR of collegen I were used to examined the osteogenesis cells and toluidin blue and safranin O staining, immunocytochemical stain and RT-PCR of collegen II were used to examined the chondrogenesis cells.Results:The morphous of BMSCs obtained by density gradient centrifugation and bone marrow culture are spindle-shaped or polygon-shape uniformly, when the cells reached almost confluence, they presented colony whirlpool. Flow cytometry showed that more than 90% cells expressed CD44 and CD29, while only 5% of the cells expressed CD34. The growth curve showed that cells of the 4rd and 8th generation have high proliferation. For osteogenesis cells, the alkaline phosphatase staining and collegen I immunocytochemical stain was positive, VonKossa staining showed the calcium nodule, and mRNA of collegen I was observed in RT-PCR. For chondrogenesis cells, the toluidin blue staining showed the metachromasia of the cells, safranin O staining and collegen II immunocytochemical stain was positive, and the mRNA of collegen II was observed in RT-PCR.Conclusion:Density gradient centrifugation combined with bone marrow culture can isolate and purify the BMSCs ideally. Under the effect of inducer, BMSCs can represent specific cyte phenotype of chondrocytes or osteocytes, and can be used in tissue engineering to restore the osteochondral defect. BMSCs have satisfactory proliferation, self-renewal and cell differentiation potentiality in vitro, which are ideal seed cells of tissue engineering. Part II. The study of preparation and biological characteristics of PLGA-PCL/PLGA-HA-βTCP biphasicscaffoldObjective:To research and prepare a novel biphasic PLGA scaffold, and examine the structure and biological characteristics, explore the feasibility to be a tissue engineering scaffold.Method:Scaffold preparation:particle leaching and casting method were used to prepare the biphasic PLGA scaffold, hyaluronic acid was vac-sorb on the surface of the scaffold. Structure observe:observe the scaffold appearance in general and scanning electron microscopy examine the incisal surface. Biocompatibility:implant the scaffold into the subcutaneous tissue, observe the reaction of the local tissue and the speed of scaffold degradation. Examine the sells implanted scaffold with scanning electron microscopy and HE staining of paraffin section.Results:The appearance of the scaffold is white spongioid material, include two layers. Scanning electron microscopy showed the aperture consistent with the design. The implanted scaffold has not induce the inflammation, and degradation gradually. Until 12 weeks, the scaffold has degraded premodinantly. Both scanning electron microscopy and HE staining of paraffin section indicate the biphasic scaffold has satisfactory biocompatibility.Conclusion:In our study, the method to prepare the scaffold is simple, and the scoffold parameters are easy to control. The two layers are integrated tightly, which improve the flaw of the common biphasic scaffold. The biocompatibility of the biphasic scaffold is satisfactory, which facilitate the adsorption and proliferation of BMSCs. With the modification of the both layer, the BMSCs can differentiate to the purpose cells. The structure of the biphasic PLGA scaffold is more similar to the normal joint, the degradation time is satisfactory, so the biphasic PLGA scaffold can restore the osteochondral defect ideally. PartⅢ. Restore the osteochondral defect of rabbit knee joint with the PLGA-PCL/PLGA-HA-βTCP biphasic scaffold combined with BMSCsObjective:To investigate the reparation effect of osteochondral defect with biphasic PLGA scaffold combined with BMSCs. Explore the feasibility that the biphasic PLGA scaffold, loaded with BMSCs, work as tissue engineering scaffold to restore the osteochondral defect.Method:Thirty healthy rabbit, sixty knees are randomly devided into three groups (A, B, C group). On femoral trochlea of each knee joint, a diameter 5mm, depth 4-5mm cylindrical hole was made to cause osteochondral defect. A group is blank, which spontaneous restoration; and implant only biphasic scaffold in the osteochondral defect in B group, and the compomers of scaffold and BMSCs in C group. After 6w and 12w, sacrifice the rabbit respectively, compare the score of appearance and histological staining of specimen. Biomechanics test was proceed to examine the sample of B group, C group and normal joint. All the properties are compred with the normal joint tissue respectively.Result:6 weeks postoperative, A group the score of appearance in general is 4.3±0.9, score of histology is 0.8±0.2; B group the score of appearance in general is 6.8±0.7, score of histology is 5.1±0.8; C group the score of appearance in general is 8.1±0.4, score of histology is 12.4±0.9.12 weeks postoperative, A group the score of appearance in general is 5.2±1.2, score of histology is 3.7±0.5; B group the score of appearance in general is 7.7±0.8, score of histology is 10.3±0.6; C group the score of appearance in general is, score 9.2±0.3 of histology is 17.2±0.3. In the whole, the restore effect of C group is the best, and A group is the worst. Biomechanics test show:the creep time of normal joint is 2763±649s, stress relaxation time 2142±402s, Young’s modulus 0.36±0.22MPa; the creep time of B group is 2189±533s, stress relaxation time 1647±381s, Young’s modulus 0.29±0.17MPa; the creep time of C group is 2314±592s, stress relaxation time 1936±334s, Young’s modulus 0.32±0.13MPa. The creep time and stress relaxation time of B group and C group is shorter than the normal joint, and Young’s modulus is smaller the normal joint but there is no significance difference between them.Conclusion:The xenogenous BMSCs are satisfactory seed cells of tissue engineering, which can be used in restoration of osteochondral defect. The biphasic scaffold is a satisfactory scaffold, which can carry BMSCs to the defect area, provide structure support, induce the regeneration of new tissue, and restore the tissue defect. The restore effect of biphasic scaffold combined with BMSCs is ideal, and the biological properties of the new tissues are close to the normal.更多还原