【作者】 王玮；

【导师】 高长有；

【作者基本信息】 浙江大学 ， 材料学， 2010， 博士

【摘要】 本文构建了纤维蛋白凝胶填充聚乳酸-乙醇酸(PLGA)支架用于关节软骨缺损的修复。采用明胶微球为致孔剂制备了平均孔径为350μm、孔隙率为87%的PLGA多孔支架。利用纤维蛋白凝胶的溶胶-凝胶过程,将纤维蛋白凝胶负载于PLGA多孔支架中构建复合体系。纤维蛋白凝胶能够均匀地填充PLGA支架的孔隙,在PLGA大孔中充满了直径200nm左右的纳米纤维填充物。体外软骨细胞共培养表明,虽然填充支架与PLGA支架在细胞增殖上无明显差别,但填充支架能够更好地维持软骨细胞的正常表型,促进细胞分泌粘多糖(GAGs)。评价了PLGA支架在37℃的磷酸盐缓冲液(PBS, pH=7.4)和兔关节软骨缺损处的降解性能。体外降解表明PLGA分子量半衰期为6周；24周后,PLGA的分子量降低到初始分子量的7%。植入体内的PLGA表现出更快的降解速率,12周后,分子量降低到初始分子量的4%。将异体骨髓间充质干细胞(BMSCs)负载于PLGA/纤维蛋白凝胶复合支架中,植入新西兰大白兔关节软骨全层缺损(直径3mm,深度4mm)处,以PLGA/BMSCs作为对照组。12周后取样,切片后进行苏木素-伊红染色(H-E)、过碘酸雪夫碱(PAS)染色GAGs、Ⅱ型胶原免疫组化染色。结果表明实验组新生软骨表面连续,软骨层厚度甚至稍厚于正常关节软骨厚度；新生软骨组织和周围正常软骨组织连接较好；软骨层中细胞密度接近正常关节软骨,深层细胞呈现明显陷窝状；宿主接近的新生组织中富含GAGs和Ⅱ型胶原,但着色稍弱于周围正常软骨,缺损中心区域有较少的GAGs、未见Ⅱ型胶原沉积。对照组主要生成纤维组织,新生组织PAS和Ⅱ型胶原染色的着色强度明显弱于实验组。表明纤维蛋白凝胶填充的PLGA多孔支架比单一的PLGA支架有更好的软骨修复能力。采用CM-Dil标记异体BMSCs,负载于PLGA/纤维蛋白凝胶中植入兔关节软骨缺损处。小动物成像系统发现即使在植入12周后依然可见红色荧光位于缺损区域,冰冻切片结果也表明植入的BMSCs在12周后依然存活。为了进一步提高关节软骨的修复效果,将转化生长因子-β1 (TGF-β1)负载于该复合支架中,构建PLGA/纤维蛋白凝胶/BMSCs/TGF-β1复合体系,将该复合体系植入到兔关节软骨全层缺损(直径4mm,深度4mm),以PLGA/纤维蛋白凝胶/BMSCs为对照组。12周后,实验组新生组织填充整个缺损区域,软骨层厚度接近正常软骨,软骨下骨和潮线恢复良好,软骨层和软骨下骨连接一致；整个新生软骨层中富含GAGs和Ⅱ型胶原,其中的细胞为典型软骨细胞的表型,细胞密度接近正常软骨。不含TGF-β1的对照组,宿主附近的新生组织中富含GAGs和Ⅱ型胶原,着色稍弱于周围正常软骨,缺损中心主要生成纤维组织；荧光定量聚合酶链式反应(qRT-PCR)的结果也证实了实验组软骨特异性基因的表达显著高于对照组。说明负载TGF-β1的复合支架能更好地促进关节软骨缺损的修复。为了克服生长因子易失活价格昂贵等缺点,将基因治疗引入PLGA/纤维蛋白凝胶复合支架。采用季铵化壳聚糖(TMC)作为质粒DNA (pDNA)的载体,通过TMC和pDNA之间的静电作用,将pDNA压缩形成纳米复合粒子。在2D培养系统中,TMC/pDNA对BMSCs的转染效率为9%。采用能够表达TGF-β1的质粒DNA (pDNA-TGF-β1)转染BMSCs能够在10天内持续表达TGF-β1将构建的PLGA/纤维蛋白凝胶/BMSCs/(TMC/pDNA-TGF-β1)植入兔关节软骨全层缺损处(直径4mmm,深度4mm),以无pDNA或无BMSCs为对照组。实验组2周和4周取样,western blotting和qRT-PCR检测到异种的TGF-β1的表达,但随时间延长TGF-β1的量减少。实验组12周取样发现缺损处填满半透明的组织,表面平整,与周围组织的界限已不明显。组织学染色发现,新生组织厚度接近正常软骨,与宿主组织连接较好,较难找出缺损位置；整个新生软骨层中PAS和Ⅱ型胶原着色均匀,且和正常软骨差别；细胞为典型软骨细胞的表型,有明显的软骨陷窝出现；软骨下骨和潮线恢复良好,软骨层和软骨下骨连接一致；qRT-PCR的结果也证实了软骨特异性基因的表达显著上调。不含pDNA组,宿主附近的新生组织中富含GAGs和Ⅱ型胶原,着色稍弱于周围正常软骨,但缺损中心仍有1mm左右的区域未修复；未加入BMSCs组,则主要生成了纤维组织,基本未见软骨样组织。负载功能DNA能够原位转染BMSCs、表达TGF-β1,能促进BMSCs向软骨细胞分化及软骨特异性基质的合成,促进缺损关节软骨的修复。更多还原

【Abstract】 A composite scaffold was fabricated by fibrin gel filled poly(lactide-co-glycolide) (PLGA) sponge for cartilage tissue engineering. The PLGA sponge with an average pore size of 350μm and a porosity of 87% was fabricated by a gelatin porogen leaching method. Via a process of sol-gel of fibrin gel, it was filled into the PLGA sponge. The fibrin gel evenly distributed in the composite scaffold with visible fibrinogen fibers with a diameter about 200nm after drying. In vitro co-culture with chondrocytes found that in the PLGA/fibrin gel the chondrocytes distributed more evenly and kept a round morphology as that in the normal cartilage. Although the chondrocytes seeded in the PLGA sponges showed similar proliferation behavior with that in the PLGA/fibrin gel, they were remarkably elongated, forming a fibroblast-like morphology. Moreover, a larger amount of glycosaminoglycans (GAGs) was secreted in the PLGA/fibrin gel than that in the PLGA sponges after 4wk. The results suggest that the fibrin/PLGA may be more favorably applied for cartilage tissue engineering than the PLGA sponge.Degradation of the PLGA sponges was investigated in PBS (pH=7.4) at 37℃and in cartilage defects, respectively. In vitro, the number-average molecular weight (Mn) of the scaffold decreased almost exponentially along with the incubation time. After 24wk, the Mn decreased from 76kDa to 5.6kDa. Meanwhile, Mn of the sponges decreased to 3.3kDa at 12wk post-implantion in cartilage defect, showing a faster degradation rate.BMSCs were employed as seed cell for the animal experiment. The PLGA/fibrin gel/BMSCs was implanted into the full-thickness cartilage defects made in New Zealand white rabbit joints (3mm in diameter and 4mm in thickness), while the PLGA/BMSCs served as the control. At 12wk post-implantation, the generated neo-cartilage integrated well with its surrounding normal cartilage and subchondral bone in the experimental group, whereas only a little bit of cartilage-like tissue and fibrous tissue was observed in the group absent from fibrin gel. These results imply that the PLGA/fibrin gel may be a better choice for cartilage restoration than the PLGA sponge too, when the BMSCs are used as the seed cells.The effectiveness of any cellular repair approach depends on the retention of cell viability after implantation. To evaluate the cell viability, allogenic BMSCs were labeled with CM-Dil fluorochrome, seeded in PLGA/fibrin gel scaffolds and implanted into the full-thickness cartilage defects (4mm in diameter and 4mm in thickness). The red fluorescence in the defects zone and in BMSCs was observed by small animal in vivo fluorescence imaging system and laser scanning confocal microscope after frozen section, respectively. The results showed that even after 12wk post-implantation, the transplanted BMSCs still localized and kept alive in the defects.Then the composite scaffold was upgraded by incorporating with transforming growth factor-β1 (TGF-β1). The PLGA/fibrin gel/BMSCs/TGF-β1 composite constructs were implanted into the full-thickness cartilage defects (4mm in diameter and 4mm in thickness), while the constructs absent from TGF-β1 served as the control. At 12wk post-implantation, the generated neo-cartilage integrated with its surrounding normal cartilage and subchondral bone in the experimental group, whereas only a little bit of cartilage-like tissue was observed in the group absent from TGF-β1. Immunohistochemical and GAGs staining confirmed the similar distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) data also showed that the cartilage special genes expressed in the neo-tissue were higher than those of the control group. The composite scaffold incoporated with TGF-β1 improved cartilage restoration substantially.Growth factors are expensive and generally have a short-half life in the order of minutes because of rapid clearance via the lymphatic system. Another way to solve the problem is the use of gene therapy, which was incorporated into this composite system in the next study. A cationized chitosan derivative N,N,N-trimethyl chitosan chloride (TMC) was employed as a carrier to condense DNA forming nano-complexes. In vitro, BMSCs were transfected by the TMC/DNA complexes with an efficiency of 9% and showed heterogeneous TGF-β1 expression in a 10 day culture period after transefected by TMC/pDNA encoding TGF-β1 (pDNA-TGF-β1). The PLGA/fibrin gel/BMSCs/(TMC/pDNA-TGF-β1) constructs were implanted into the full-thickness cartilage defects (4mm in diameter and 4mm in thickness), while the scaffolds absent from pDNA-TGF-β1 or BMSCs served as the control. In vivo heterogeneous TGF-(31 was expressed in the experimental group at least lasting for 4wk detected by western-blotting and qRT-PCR. At 12wk post-implantation, the generated neo-cartilage integrated well with its surrounding normal cartilage and subchondral bone in experimental group, whereas only a little bit of cartilage-like tissue and fibrous tissue was observed in the group absent from pDNA-TGF-β1 and BMSCs, respectively. Immunohistochemical and GAGs staining confirmed the similar distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The qRT-PCR data also showed that cartilage special genes expressed in the neo-tissue were comparable to those of the normal cartilage and were much higher than those of the control groups. The successful repair thus evinces the potentiality of using this composite construct for cartilage regeneration.更多还原