【作者】 戴文达；

【导师】 董健； 陈国平； 姚振均；

【作者基本信息】 复旦大学 ， 外科学， 2010， 博士

【摘要】 第一部分PLGA/Collagen复合材料结构设计与制备目的：设计和制备新型结构的PLGA/Collagen复合材料,使之更适合作为组织工程支架用于再生医学研究。方法：通过模具设计,控制PLGA/Collagen复合材料立体结构,利用冷冻干燥技术将胶原多孔海绵复合于PLGA编织网膜上。材料按不同结构分为三组：(1)THIN型,多孔胶原海绵形成于PLGA编织网膜的网孔中；(2) SEMI型,多孔胶原海绵形成于PLGA编织网膜的网孔中和单侧；(3) SANDWICH型,多孔胶原海绵形成于PLGA编织网膜的网孔中和两侧。材料制备完成后行扫描电镜观察材料表面和横截面微观结构,利用图像软件统计孔径大小,测定吸水率和膨胀率测定。结果：成功构建THIN、SEMI、SANDWICH型PLGA/Collagen复合材料,表面孔径分别为136.4±11.8μm、142.9±15.3μm和139.6±12.5μm; SEMI和SANDWICH型PLGA/Collagen复合材料吸水率分别为12.9±1.71和13.6±2.3倍,明显优于THIN型材料(4.4±0.4)(P<0.001),结构符合设计要求。结论：新构建的SEMI、SANDWICH型PLGA/Collagen复合材料结构和表面孔径满足软骨组织工程研究的需要；能为软骨再生提供更大的细胞容纳能力和组织再生空间。第二部分PLGA/Collagen复合材料用于软骨再生的体外研究目的：观察和比较不同结构的THIN、SEMI、SANDWICH型PLGA/Collagen复合材料在体外对牛软骨细胞增殖分泌的效果。方法：分离与培养牛膝关节软骨细胞(BACs),倒置相差显微镜观察细胞生长情况。扩增至第2代时接种至三种PLGA/Collagen复合材料,(1) THIN/BACs组,细胞接种于THIN型PLGA/Collagen复合材料；(2)SEMI/BACs组,细胞接种于SEMI型PLGA/Collagen复合材料；(3)SANDWICH/BACs组,细胞接种于SANDWICH型PLGA/Collagen复合材料,检测细胞接种效率。扫描电镜观察细胞在材料表面和内部的生长情况；体外培养一周后检测BACs/材料复合内DNA和GAG含量,Real-time PCR检测Ⅰ型胶原、Ⅱ型胶原、Aggrecan mRNA表达强度。牛膝关节软骨组织和体外单层培养一周的PI BACs做对照。结果：BACs成功地分离与扩增,生长与增殖情况良好；P2 BACs接种至PLGA/Collagen复合材料,平均接种效率在SEMI、SANDWICH组分别为87.8±1.6%、95.6±2.2%,显著高于THIN组(P<0.05)。扫描电镜观察BACs在PLGA/Collagen复合材料表面和中心生长活跃,分布均匀；一周后标本检测DNA含量SEMI、SANDWICH组显著高于THIN组(P<0.05),但GAG含量与无明显差异。三组细胞／材料复合物Ⅱ型胶原和Aggrecan mRNA表达强度均显著高于体外单纯培养的BACs,但SEMI、SANDWICH组与THIN组相比亦无显著统计学差异。结论：PLGA/Collagen复合材料具备优秀的生物相容性；SEMI、SANDWICH组的结构设计显著地提高BACs在PLGA/Collagen复合材料上的接种效率；SEMI、SANDWICH组较THIN组明显促进透明软骨样ECM成分分泌。第三部分PLGA/Collagen复合材料用于软骨再生的体内研究目的：观察和比较不同结构的THIN、SEMI、SANDWICH型PLGA/Collagen复合材料在体内对牛软骨细胞成软骨的效果。方法：P2 BACs接种至THIN、SEMI、SANDWICH型PLGA/Collagen复合材料,(1)THIN/BACs组,细胞接种于THIN型复合材料；(2)SEMI/BACs组,细胞接种于SEMI型复合材料；(3)SANDWICH/BACs组,细胞接种于SADWICH型复合材料；(4)对照组,随机选择空白THIN、SEMI、SANDWICH型复合材料,体外软骨细胞培养基培养1周后植入裸鼠背部皮下。2、4、8周后取出行标本大体观察,厚度测量。检测新生软骨样组织内DNA和GAG含量,Real-time PCR检测Ⅰ型胶原、Ⅱ型胶原、Aggrecan mRNA表达强度；对标本纵切面和横切面行组织学和免疫组织化学检测；并以生物力学方法评估标本的弹性模量和刚度。牛膝关节软骨组织做对照。结果：2、4、8周后取出标本,三组BACs/材料复合物均呈透明软骨样外观而空白对照组至第8周时基本被吸收。SEMI/BACs、SANDWICH/BACs组新生软骨的厚度分别为THIN/BACs组的1.66和1.73倍(P<0.05)。三组标本的DNA含量非常接近,但SEMI/BACs、SANDWICH/BACs组每μg DNA的GAG含量显著地高于THIN/BACs组(P<0.05),分别为牛膝关节软骨标本的60.1%、52.1%和26.6%。Real-time PCR检测发现标本内Ⅱ型胶原、Aggrecan mRNA表达强度随移植时间进行性上升,而Ⅰ型胶原mRNA表达强度进行性下降；组织学和免疫组化染色发现细胞分布均匀,大量透明软骨样ECM形成。SEMI/BACs、SANDWICH/BACs组标本的弹性模量分别达到了牛膝关节软骨的54.8%、49.3%,刚度达到了68.8%、62.7%。结论：PLGA/Collagen复合材料植入裸鼠体内后呈现了优秀的生物相容性；新设计的SEMI和SANDWICH材料较THIN相比,更能促进体内软骨新生；SEMI和SANDWICH材料在体内成软骨方面无显著的差异；新构建的组织工程软骨在化学组成和力学强度上已经非常接近于天然膝关节软骨。更多还原

【Abstract】 Part.1 Design and fabrication of novel structure PLGA/collagen hybrid scaffoldsObjective:To design and fabricate PLGA/collagen scaffolds with novel structures for the sake of future application in the research of regeneration medicine.Methods:Prepare the PLGA/collagen hybrid scaffolds with different structures by forming collagen microsponges in the openings or the sides of a PLGA knitted mesh. 3 kinds of structures were achieved using special designed moulds. (1) THIN: collagen micro-sponge formed in interstices of PLGA mesh; (2) SEMI:collagen micro-sponge formed on one side of PLGA mesh; (3) SANDWICH:collagen sponge formed on both sides of PLGA mesh. Surface and cross-section morphology were observed by scanning electron microscopy. Pore sizes were calculated with image analyzing software. Water uptake and swelling ratio was also evaluated.Results:THIN, SEMI and SANDWICH PLGA/collagen scaffolds with designed structures were successfully fabricated. The surface pore sizes in HIN, SEMI and SANDWICH scaffolds were 136.4±11.8μm、142.9±15.3μm and 139.6±12.5μm respectively. The swelling ratio of SEMI and SANDWICH scaffolds were 12.9±1.71 and 13.6±2.3, much higher than THIN scaffold which was 4.4±0.4 (P<0.001).Conclusion:THIN, SEMI and SANDWICH PLGA/collagen scaffolds with novel structures were prepared. The micro-structure and pore sizes, increased collagen micro-sponges were expected to facilitate cell adhesion, tissue accommodation and regeneration. Part.2 Effect of novel structure PLGA/Collagen hybrid scaffolds on chondrogenesis in vitroObjective:To investigate and compare the effect of THIN, SEMI, SANDWICH PLGA/Collagen hybrid scaffolds on the chondrogenesis in vitro.Methods:Bovine articular chondrocytes (BACs) were isolated, cultured and examined by phase contrast microscopy. P2 BACs were seeded onto the scaffolds and seeding efficiency was investigated. (1) THIN/BACs:BACs were seeded onto THIN PLGA/Collagen scaffolds; (2) SEMI/BACs:BACs were seeded onto SEMI PLGA/Collagen scaffolds; (3) SANDWICH/BACs:BACs were seeded onto SANDWICH PLGA/Collagen scaffolds. Cell growth on the surface and inside the scaffolds were observed by SEM. Quantification of DNA and GAG was carried out after the samples were cultured in vitro for 1 week. Real-time PCR was done to evaluate the expression of type I collagen, type II collagen and Aggrecan mRNA. Compare was done between the THIN, SEMI, SANDWICH group, BACs cultured in flasks for 1 week and natural cartilage.Results:BACs were successfully isolated and multiplied. The seeding efficiency of BACs on SEMI and SANDWICH group were 87.8±1.6% and 95.6±2.2% respectively, much higher than THIN group (P<0.05). Homogenous cell distribution and active cell growth were achieved throughout the scaffolds when checked by SEM. DNA volume in SEMI and SANDWICH groups were significant higher than THIN group (P<0.05), while no difference in GAG volume and lower than those of natural cartilage. The typeⅡcollagen and Aggrecan mRNA expression showed no difference between 3 groups.Conclusion:Excellent biocompatibility was shown in PLGA/Collagen hybrid scaffolds to BACs. The novel design of SEMI and SANDWICH increased the cell accommodation capability by building thicker collagen micro-sponges on the PLGA knitted mesh. The seeding efficiency of BACs on SEMI and SANDWICH scaffolds were greatly improved. Hyaline cartilage-like ECM excretion was enhanced in SEMI and SANDWICH scaffolds much more than THIN group. Part.3 Effect of PLGA/Collagen novel structure hybrid scaffolds on chondrogenesis in vivoObjective:To investigate and compare the effect of THIN, SEMI, SANDWICH PLGA/Collagen hybrid scaffolds on the chondrogenesis in vivo.Methods:P2 BACs were seeded onto 3 kinds of PLGA/Collagen hybrid scaffolds, and after cultured for 1 week in vitro, all the samples were transplanted subcutaneously in the dorsum of nude mice. (1) THIN/BACs:BACs were seeded onto THIN PLGA/Collagen scaffolds; (2) SEMI/BACs:BACs were seeded onto SEMI PLGA/Collagen scaffolds; (3) SANDWICH/BACs:BACs were seeded onto SANDWICH PLGA/Collagen scaffolds; (4) Control:empty PLGA/Collagen scaffold of each group was selected randomly. Samples were harvested at 2,4 and 8 weeks after transplantation and the thickness were valued. Quantification of DNA and GAG was carried out and expression of type I collagen, type II collagen and Aggrecan mRNA were evaluated by Real-time PCR. Histological and immune-histological staining was done for the transverse and cross-section of the samples. The biomechanical properties of all the 3 groups were also examined.Results:Gross view of THIN/BACs, SEMI/BACs and SANDWICH/BACs samples were cartilage-like and ivory white when harvested at 2,4,8 weeks after transplantation, and the original shapes were retained after 8 weeks. The thickness of the new formed cartilage in SEMI/BACs and SANDWICH/BACs was 1.66 and 1.73 times higher than that of THIN/BACs (P<0.05). DNA volume in 3 groups were quite similar while the GAG quantity perμg DNA was much higher in SEMI/BACs and SANDWICH/BACs groups (P<0.05), about 60.1% and 52.1% of that of natural cartilage. Real-time PCR revealed that the expression of type II collagen and Aggrecan mRNA increased with the transplantation time while the expression of type I collagen mRNA decreased. Histological and immune-histological staining showed a homogenous cell distribution and abundant hyaline cartilage-like ECM excretion. When compared to natural cartilage, mechanical strength of the engineered cartilage in SEMI/BACs and SANDWICH/BACs groups reached 54.8%,49.3% in Young’s modulus and 68.8%,62.7% in stiffness respectively.Conclusion:Excellent biocompatibility was shown in PLGA/Collagen hybrid scaffolds after being transplanted in nude mice. The novel design of SEMI and SANDWICH scaffolds enhanced the chondrogenesis in vivo compared to THIN scaffold. No significant difference was observed between the effects of chondrogenesis by SEMI or SANDWICH scaffolds. The engineered cartilages are quite similar to the natural articular cartilage in the aspects of chemical compositions and biomechanical properties.更多还原