【作者】 杨自权；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 外科学， 2008， 博士

【摘要】 关节软骨属于透明软骨,覆盖在关节的表面,起着缓冲和吸收震荡,减少关节运动时的摩擦阻力等重要作用。关节软骨结构较为特殊,组织内只有软骨细胞一种细胞成分,软骨细胞处于自身分泌的基质成分形成的软骨陷窝内。由于没有血管组织的分布,组织血液供应差,因而关节软骨自身修复能力极差。关节软骨损伤一旦发生即很难修复,继而会发生不可逆的病理变化,造成关节的退行性改变。目前,由关节软骨病变造成的关节疼痛和骨性关节炎已经成为老年人导致残疾和生活受限的主要原因之一,同时由运动造成的关节软骨损伤在年轻的人群中也很常见。过去的几十年中,尽管有许多方法尝试修复关节软骨损伤,但是到目前为止尚无一种方法能产生结构和功能与正常软骨相似的透明软骨组织。关节软骨损伤的治疗一直是骨科研究领域内的难题之一。组织工程学是近年发展起来的一门将生命科学和工程学原理相结合的新兴交叉学科,通过利用少量种子细胞经过体外培养扩增,并附着在一定的支架材料上移植到体内而形成新的有生命力的组织。目前利用组织工程方法修复组织器官损伤的研究已取得了较快的进展,也为关节软骨损伤的治疗开辟了新途径。软骨细胞作为软骨组织的唯一细胞成分首先被用来修复软骨损伤,已有许多动物实验证实软骨细胞移植可以促进关节软骨损伤的修复。而瑞典医生Brittberg等更是率先将组织工程学原理应用于临床关节软骨损伤的修复,开辟了软骨损伤治疗的新途径。但是随着研究的深入,发现以软骨细胞作为种子细胞修复软骨损伤存在许多问题,如软骨细胞的来源有限,自体软骨细胞移植需要采集正常部位的软骨组织,对机体造成新的损伤,而异体移植则存在疾病传播、免疫排斥等风险;而且由于软骨细胞属于分化终末的细胞,体外培养传代后会出现“失分化”现象,逐渐丧失其表型,不再产生软骨特异性的基质成分。因此需要寻找更为理想的种子细胞。干细胞是一类处于“不成熟”和未分化状态的细胞,具有较强的自我更新能力并具备多向分化能力。由于胚胎干细胞的应用受到伦理学的限制,目前干细胞的应用主要集中在成体干细胞上。间充质干细胞(Mesenchymal stem cells,MSCs)是一类广泛分布于多种组织中的成体干细胞,增殖能力强,具有多向分化能力,可以分化为骨、软骨、肌肉、脂肪等多种组织,存在于骨髓中的骨髓间充质干细胞(Bone Marrow-derived Mesenchymal Stem Cells , BMSCs)更具有采集方便、对机体损伤小的特点,因而成为组织工程理想的种子细胞。基因治疗为骨骼肌肉系统损伤的治疗开辟了新途径,在软骨损伤的治疗中,可以通过基因转染技术修饰软骨细胞或软骨前体细胞,在损伤区域局部持续产生生物活性分子,促进间充质干细胞的分化和软骨基质成分的产生,从而促进软骨损伤的修复。研究显示转化生长因子β(Transforming growth factorβ,TGF-β)、骨形态发生蛋白(Bone Morphogenetic Proteins,BMPs)、胰岛素样生长因子(insulinlike growth factor,IGF)、成纤维细胞生长因子(fibroblast growth factor,FGF)等生长因子可以刺激和诱导间充质干细胞分化为软骨细胞。但由于外源性生长因子半衰期较短,进入体内后被机体迅速降解而失去其生理功能,不能满足细胞需要生长因子持续刺激的情况。随着分子生物学技术的发展和成熟,许多研究将基因治疗应用于组织器官的修复和重建。基因治疗和和组织工程学的结合,产生了基因强化组织工程学(genetically enhanced tissue engineering)。基因强化组织工程学通过基因转染技术将编码特定功能因子的基因转入种子细胞或生物活性基质材料,使转染细胞或基因活化基质表达目的产物,可以促进组织和器官的修复和重建。目前,已有多项研究利用间充质干细胞做为种子细胞,将基因强化组织工程学原理应用于骨、软骨、肌腱等组织的修复,并取得了良好的效果。目前软骨基因强化组织工程研究中大多采用TGF-β, BMPs, IGF, FGF等生长因子的基因转染软骨细胞或间充质干细胞,虽然取得了一定的成功,但是研究显示过量表达的生长因子会导致关节滑膜增生、关节内骨赘形成和关节周围肌肉水肿和纤维化等并发症,转入基因的调控以及基因产物的表达等问题还有待深入研究。Sox9是软骨发育形成过程中的关键转录因子,对软骨的发育成熟等过程起着重要的调节作用。研究显示在胚胎软骨发育过程中,Sox9决定间充质干细胞的聚集和向软骨细胞的分化。Sox9可以与II型胶原、aggrecan等基因的增强子元件相结合,激活II型胶元和aggercan等基因的表达,从而促使间充质细胞向软骨方向分化。但是目前对Sox9基因的研究主要集中于胚胎软骨形成方面,对于体外培养的间充质干细胞转染Sox9基因后能否促进间充质干细胞向软骨方向分化尚无明确报道。基于Sox9在胚胎软骨形成中的作用,我们设想Sox9基因可能对出生后个体的间充质干细胞也产生类似的作用,通过Sox9基因转染,使骨髓间充质干细胞高表达Sox9蛋白,直接启动和激活MSCs的软骨分化过程,然后利用Sox9基因修饰的BMSCs修复关节软骨损伤,从而避免由于生长因子过度作用而产生的副作用,如果此设想能被验证,将会为MSCs向软骨方向定向分化找到新的方法,为软骨损伤修复探索新途径。本研究的目的在于①建立一种骨髓间充质干细胞的分离培养方法,从骨髓中快速分离纯化出间充质干细胞并进行体外培养扩增;②通过比较不同代次BMSCs的多向分化能力,明确体外传代培养对BMSCs分化能力的影响,确定收集种子细胞的最佳时期;③扩增纯化重组真核表达Sox9质粒,用脂质体做为载体转染骨髓间充质干细胞,观察Sox9基因在骨髓间充质干细胞中的表达;④采用基因转染技术将Sox9基因转入兔骨髓间充质干细胞中,观察Sox9基因高表达对BMSCs软骨分化的影响;⑤以藻酸盐做为载体材料,负载Sox9基因修饰的骨髓间充质干细胞移植于兔关节软骨损伤区,观察损伤修复的效果,旨在为组织工程学方法修复关节软骨损伤探索新途径,为临床治疗关节软骨损伤提供研究基础。本研究共分为五个部分第一部分兔骨髓间充质干细胞体外分离培养及生物学性状观察目的建立一种骨髓间充质干细胞快速分离纯化的方法,优化体外培养体系,体外扩增培养并初步鉴定骨髓间充质干细胞,同时观察体外培养的骨髓间充质干细胞的生物学性状,旨在建立和优化BMSCs的分离纯化体系,为后续的组织工程软骨损伤修复研究提供研究基础和足够的种子细胞。方法采用密度梯度离心和差速贴壁培养相结合的方法从兔骨髓组织中分离间充质干细胞,进行体外传代培养扩增,绘制细胞生长增殖曲线,测定细胞倍增时间,MTT法测定细胞代谢能力。流式细胞术测定细胞表面抗原,分析细胞周期,对分离细胞初步鉴定。结果密度梯度离心和差速贴壁筛选后所分离的细胞大多为圆形的单核细胞,体外培养3次换液后,血细胞已基本去除。传代培养后细胞生长较原代细胞明显加快,传代8代以内细胞生长良好,增殖迅速,传代10代后细胞呈现衰老征象。第10代细胞与第3、5、8代细胞相比,细胞倍增时间明显延长。流式细胞术鉴定体外培养传代三代后细胞已较为纯化,表达间充质细胞表面抗原CD44的细胞可达91.7%,而表达造血细胞表面标志CD45的细胞仅为1.5%。结论利用密度梯度离心和差速贴壁培养法可迅速高效地分离纯化兔骨髓间充质干细胞,体外培养8代以内的兔骨髓间充质干细胞均具有较强的增殖能力,可以作为软骨组织工程研究的种子细胞。第二部分体外传代培养对骨髓间充质干细胞多向分化能力的影响目的定向诱导骨髓间充质干细胞向成骨、成脂肪、成软骨方向分化,以进一步鉴定所分离的细胞;同时观察比较不同代次细胞的多向分化能力,研究体外培养传代对MSCs多向分化能力的影响。方法选取第3代和第8代细胞,分别加入成骨诱导培养液,成脂肪诱导培养液,成软骨诱导培养液进行定向诱导培养,通过组织学观察,生物化学测定,半定量RT-PCR和Western blot等对细胞的多向分化能力进行比较。结果经过体外成骨诱导,成脂肪诱导,成软骨诱导后,第3代和第8代骨髓间充质干细胞均可以定向分化成成骨细胞、脂肪细胞和软骨细胞。第3代细胞和第8代细胞的成骨分化能力和成脂肪分化能力无明显差异。第8代细胞的成软骨能力较第3代细胞明显下降。结论本部分实验显示上一节实验中分离得到的CD44阳性/CD45阴性细胞可以向成骨、成脂肪和成软骨方向分化,进一步证实了这些细胞是具有多向分化能力的MSCs。对第8代细胞和第3代BMSCs的多向分化能力的比较实验显示,虽然体外传代培养对细胞的成骨和成脂肪分化能力无明显影响,但是成软骨分化的能力出现下降。进行软骨组织工程及基因治疗等研究时应当采用体外培养时间较短的,分化能力较强的种子细胞,以保证移植治疗的效果。第三部分重组真核表达Sox9质粒的扩增鉴定及转染骨髓间充质干细胞目的扩增纯化鉴定Sox9质粒。利用脂质体做为载体,介导重组真核表达Sox9质粒转染骨髓间充质干细胞,观察Sox9基因在骨髓间充质干细胞中的表达。方法将含有Sox9基因的真核重组表达质粒转化大肠杆菌对质粒进行扩增,采用碱裂解法和树脂吸附法分离提纯质粒,通过限制性核酸内切酶酶切和基因测序的方法对质粒进行鉴定。脂质体载体介导重组Sox9质粒和EGFP质粒转染兔骨髓间充质干细胞,利用RT-PCR和Western blot检测目的基因产物的表达。同时利用流式细胞术和荧光显微镜测定基因转染效率。结果DNA纯度检测结果显示所提取的质粒较为纯化,OD260/OD280值在1.7~1.8之间。限制性内切酶酶切和基因测序结果证实Sox9质粒正确,未发生突变。RT-PCR和Western blot检测结果显示Sox9质粒转染组细胞开始表达目的分子。流式细胞检测显示MSCs的转染效率为26.8%,倒置荧光显微镜观察转染效率为22%±3.8%。结论利用脂质体介导重组Sox9质粒可以成功转染兔骨髓间充质干细胞,外源性Sox9基因可以在BMSCs中表达,并产生其编码的目的蛋白。第四部分Sox9基因转染对BMSCs软骨分化影响的实验研究目的观察Sox9基因转染对间充质干细胞向软骨方向分化的影响,为基因强化软骨组织工程探索新途径。方法利用脂质体介导重组Sox9质粒转染兔骨髓间充质干细胞,Geneticin筛选稳定表达细胞。通过细胞生长代谢和细胞周期分析观察基因转染对细胞的影响。通过RT-PCR,Western blot,免疫组织化学染色及GAG含量测定等检测Sox9基因转染对BMSCs成软骨分化的影响。结果Sox9基因转染组与对照EGFP基因转染组细胞在细胞生长代谢和细胞周期分布上均无明显差异。Sox9基因转染后,BMSCs出现向软骨方向的分化,细胞表达软骨特异性分子II型胶原和Aggrecan,GAG含量明显增加。免疫组织化学显示Sox9转染组II型胶原染色阳性,两对照组均为阴性。结论外源性Sox9基因转染未对BMSCs的生长造成明显影响。Sox9基因转染后可以启动BM SCs向软骨细胞方向的分化。第五部分Sox9基因修饰BMSCs修复兔膝关节软骨损伤目的利用藻酸盐做为载体材料,将经过Sox9基因修饰的BMSCs植入兔关节软骨损伤区,观察修复效果,探索软骨损伤修复的新途径。方法在兔膝关节股骨内侧髁负重区关节面制作直径4mm全层关节软骨损伤模型。24兔48膝随机分为3组,以藻酸盐做为载体材料,I组(实验组)植入经Sox9基因修饰的BMSCs-藻酸钙复合物,II组(实验对照组)植入未经过基因转染的BMSCs-藻酸钙复合物,III组(空白载体对照组)植入不含细胞的藻酸钙珠。分别于术后6周、12周时处死动物,做大体观察,HE染色和免疫组织化学染色,使用Wakitani半定量评分系统对修复组织进行评分,观察关节软骨损伤的修复情况。结果术后6周,I组关节软骨损伤区大多由透明样软骨样修复组织填充,色泽与周围正常软骨组织基本接近,表面光滑,与周围软骨组织结合良好;II组标本缺损区多数由纤维样软骨组织填充,表面光滑,与周围正常组织之间结合较好但界限清晰;III组标本缺损区由无细胞的藻酸盐材料填充,多数材料填充区表面凹陷不光滑,修复区与周围软骨之间结合差,二者间界限清晰。术后12周,I组标本软骨缺损区域由透明样软骨填充,修复组织内细胞呈现出柱状排列的趋势,修复组织表面光滑,与周围正常软骨组织的结合良好,软骨下骨板修复良好,与周围软骨间界限已不清晰;II组标本大多由纤维软骨组织填充,修复组织表面不光滑,与周围软骨出现明显裂隙,多数标本软骨下骨板仅部分修复;III组标本与6周时无明显变化。II型胶元免疫组织化学染色显示实验组修复组织II型胶原免疫组化染色阳性,证实产生的修复组织为透明样软骨。组织学评分结果显示实验组软骨损伤修复效果明显优于两对照组。结论以藻酸盐做为载体材料负载经体外Sox9基因修饰的BMSCs可有效地修复兔关节软骨损伤,与对照组相比Sox9基因修饰组软骨缺损得到了良好的填充,修复组织为透明样软骨,修复组织与周围正常软骨之间结合良好,为临床治疗关节软骨损伤提供了新的方法和途径。总结利用密度梯度离心和差速贴壁培养法可迅速高效地分离和纯化兔骨髓间充质干细胞,体外培养8代以内的兔骨髓间充质干细胞均具有较强的增殖能力强,可以作为软骨组织工程研究的种子细胞。体外传代培养对BMSCs的成骨和成脂肪分化能力无明显影响,但是其成软骨分化的能力出现下降。进行软骨组织工程及基因治疗等研究时应当采用体外培养时间较短的,分化能力较强的种子细胞,以保证移植治疗的效果。利用脂质体介导重组Sox9质粒可以成功转染兔骨髓间充质干细胞,外源性Sox9基因可以在BMSCs中表达,并产生其编码的目的蛋白。外源性Sox9基因转染未对BMSCs的生长造成明显影响。Sox9基因转染后可以启动BMSCs向软骨细胞方向的分化。以藻酸盐做为载体材料负载经体外Sox9基因修饰的BMSCs可有效地修复兔关节软骨损伤,与对照组相比Sox9基因修饰组软骨缺损得到了良好的填充,修复组织为透明样软骨,修复组织与周围正常软骨之间结合良好,为临床治疗关节软骨损伤提供了新的方法和途径。更多还原

【Abstract】 Articular cartilages are hyaline cartilage covering the surface of joints, absorbing mechanic shocks and providing almost frictionless motion between the articular surfaces of diarthrodial joints. Chondrocytes are the only type of cells residing in cartilage and are trapped in their lacuna formed by extracellular matrix. Because of their avascular nature, cartilage shows very poor capacity of self-repair; most of the injuries are maintained for years and can eventually lead to further degeneration. Joint pain caused by cartilage degeneration is one of the major reasons of disability in the population of elder populations, while cartilage trauma is one of the most common sport injuries in young patients. Although many repair techniques have been proposed over the past decades, none has successfully regenerated long-lasting hyaline cartilage tissue to replace damaged cartilage. The repair of articular cartilage remains one of the most challenging areas in orthopaedic researches.Tissue engineering is a newly developed technique which combines the life science and engineering approaches, using in vitro amplification cells loaded onto biomaterials to build new live tissues. Recently, with more successful reports, tissue engineering demonstrated tremendous clinical potential for regeneration of hyaline-like cartilage tissue and treatment of chondral lesions.As the only type of cell in the cartilage, chondrocytes were firstly researched for repairing cartilage injuries. Brittberg et al first report that they successfully repair the full thickness defects of knee joint with hyaline-like cartilage using patients’autologous chondrocytes. However more researches show that chondrocytes are not ideal for tissue engineering, for the concerns of limited donor cells for transplantation and the dedifferentiation when cultured in vitro. So it is still necessary to find alternative cells for cartilage tissue engineering.Stem cells are cells in an“immature”or undifferentiated situation. They have high self renewal capacity and are capable of differentiate into several type of cells. The ethics concerns with ES cells limited their researches and usage. As there are few ethics concerns, more researches are now focusing on adult stem cells. Mesenchymal stem cells (MSCs) are one of the adult stem cells residing in many tissues. MSCs have multi-lineage potentials and can differentiate into osteoblasts, chondrocytes and adipocytes etc. Among MSCs, the bone marrow derived mesenchymal stem cells (BMSCs), which reside in the bone marrow tissue, can be easily obtained with minor injuries, sounds be an ideal source of MSCs.Gene transfer technology has opened novel treatment avenues toward the treatment of damaged musculoskeletal tissues. Gene transfer provides the capability to achieve sustained, localized presentation of bioactive molecules or gene products to sites of cartilage damage that could improve cartilage healing. Many investigators are investigating the feasibility of using genetically engineering chondrocytes or chondrocyte precursors for tissue-engineering applications. By using transgenic techniques, the cells can produce essential growth factors to stimulate chondrogenesis of mesenchymal stem cells or promote the synthesis of cartilageous matrix. As growth factors have limited bioavailability and stability in vivo, transfection of the cells with target gene encoding the growth factors should be a sound way to improve tissue engineering. Initial experiments have showed that overexpression of growth factors include TGF-β, IGF-1, and BMP-2 etc by transduced chondrocytes or mesenchymal cells have enhanced the repair of cartilage injuries. But more researches showed that the overexpression of growth factors may have some adverse effects such as degeneration of cartilage and forming of osteoarthritis.Sox9 is the key transcription factor during chondrogenesis. During the embryo skeleton development, Sox9 play an important role for the aggregate and differentiation of mesenchymal stem cells. Sox9 is essential for chondrocyte differentiation and cartilage formation. SOX9 enhances the promoter activity of type II collagen and aggrecan thus promote the chondrogenesis of MSCs. Although there are many researches on the structure and function of Sox9, most of them are focused on the cartilage formation. There is limited knowledge of Sox9 on the adult MSCs cultured in vitro. Based on the previous studies of Sox9, our hypothesis is that the Sox9 gene may have same chondrogenesis effect on adult MSCs in vitro. Using Sox9 overexpressing MSCs may promote the repair of cartilage injuries without the adverse effects of growth factors.The purpose of our study are aimed at①to establish a high efficacy method to isolate the BMSCs from bone marrow tissues and expand the BMSCs in vitro;②to investigate the influence of extensive subcultivation on the multiple differentiation potentials of BMSCs;③to amplify and purify the recombinant Sox9 plasmid and transfected BMSCs using a liposome vector;④to observe the influence of Sox9 overexpression on the chondrogenesis of BMSCs;⑤to repair articular cartilage defects with Sox9 gene modified BMSCs embedded in alginate.The whole project was divided into five partsChapter I Isolation and observation of rabbit bone marrow stem cellsObjective To establish a high efficacy method to isolate the rabbit BMSCs from bone marrow tissues and observe the biological characteristics of the cells cultured in vitro.Methods Bone marrow stem cells of New Zealand Rabbit were obtained and purified by gradient centrifuge and adhesion culture in vitro. The cells were analysised for their proliferation ability and growth kinetics. Flow cytometry were used for the analysis of surface antigens and cell cycle distributions.Results After the density gradient isolation and selective adhere culture procedure; Most of the isolated cells were round mononuclear cells with over 95% of viability After 3 times of medium changing, most of the unattached haematopoietic cells were washed away. After passaged into new flasks, the sub-cultured cells proliferated much more quickly than the primary culture During the first 8 subcultivation passaging period, the morphology of the cells are similar, most of the cells are uniformly spindle-like shaped. The expanded cells of the first 8 passages grow faster than the primary culture and experienced similar growth kinetics. The growth speed was much slower for late passaged cells of passage 10. The doubling time of the cells from Passage 10 are longer that that of Passage 3, 5, 8. Flow cytometry assay revealed that after 3 passaging process, the BMSCs were quite homogenous, with 91.7% of the cultured cells positive for mesenchymal adhesion molecules CD44 and only 1.5% of the cells expressing the haematopoietic antigens CD45.Conclusions The combination of density gradient centrifugation and selective adhere culture is a highly efficacy method to isolate BMSCs from bone marrow. The first 8 passaged cells have great capacity of proliferation and could be used for the further tissue engineering study.Chapter II The study of influence of extensive subcultivation on the multiple differentiation potentials of BMSCsObjective To further confirm the BMSCs by multilineage differentiation potentials and investigate the influence of extensive subcultivation on the multiple differentiation potentials of BMSCs.Methods The passage 3 and passage 8 of the isolated cells were used for multiple differentiations into osteoblasts, chondrocyte and adipocyte with different induction mediums respectively. Histology, immunohistology, biochemistry assay, RT-PCR and Western blot were used for the tests of multilineage differentiations.Results The cells from both passage 3 and passage 8 could be induced into osteoblasts, adipocytes and chondrocytes in vitro. There were no differences on the osteogenic and adipogenic potentials between two passages, but there was a decrease on the chondrogenic potential of BMSCs from passage 3 to passage 8.Conclusions Despite there were no changes of osteogenic and adipogenic potentials during the in vitro subcultivation, there was a decrease of the chondrogenic potential in late passaged cells comparing to the early passaged cells. So, for cartilage repairing, to achieve good results, it is better to use the early passage BMSCs than the later ones.Chapter III The preparation recombinant eukaryotic expression Sox9 plasmids and the transfection of BMSCsObjective To amplify and purify the recombinant Sox9 plasmid and transfected BMSCs using a liposome method.Methods Recombinant eukaryotic expression Sox9 plasmid were amplified, purified and then confirmed by restriction digestion and gene sequencing. Rabbit BMSCs were transfected by Sox9 plasmids and control EGFP plasmids. The expression of the exogenous gene was confirmed by RT-PCR and Western blot. The gene transfection efficiency was tested by GFP expressing cells using flow cytometry assay and fluoresce microscopic observation.Results The plasmids were purified after the preparation process with a consistent OD260/OD280 between 1.7~1.8. BMSCs were successfully transfected with Sox9 gene and verified by RT-PCR and Western blot analysis. The transfection efficiency was about 26%.Conclusions BMSCs can be transfected by recombinant eukaryotic expression Sox9 plasmids using the liposome method. The Sox9 gene could express in BMSCs and produced their coding proteins.Chapter IV The influence of overexpression Sox9 on the chondrogenesis of BMSCs Objective To observe the influence of Sox9 overexpression on the chondrogenesis of BMSCsMethods BMSCs were transfected by recombinant eukaryotic expression Sox9 plasmid using liposome vectors and the stable expressing cells were screened by geneticin selections. Gene transfected cells were analysised by the growth kinetic and cell cycle assay. The chondrogenesis of gene modified BMSCs were analysised by RT-PCR, western blot, immunohistology and GAG assay.Results The overexpression of Sox9 gene had no effects of cell proliferation and cell cycle distribution. The BMSCs went into chondrogenic differentiation after transfected by Sox9 gene. The transfected cells began to express chondrogenic marker molecules and synthesis more GAG than the control groups.Conclusions Overexpression of exogenous Sox9 gene has no effect on the growth kinetics of BMSCs while trigger the chondrogenic differentiation process of BMSCs.Chapter V Repair cartilage defects with Sox9 gene modified MSCs embedded in AlginateObjective To repair articular cartilage defects with Sox9 gene modified BMSCs embedded in alginate.Methods Cartilage defects were made in the medial condylar of femur.48 knee joints of 24 rabbits were randomly divided into 3 groups. In the experiment group (group I), the defects were fill with Sox9 gene modified MSCs embedded in alginate, in the experiment control group (group II) the defects were filled with MSCs embedded in alginate and the defects of empty control group (group III) were filled with alginate alone. The animals were sacrificed at 6 weeks or 12 weeks after the implantation. The samples were evaluated macroscopically and histologically. A semi-quantity scoring system was also used to evaluate the repairing. Results The defects in group I were filled with hyaline like cartilage tissue which showed smooth surface and good integrating with the adjacent cartilage, both at 6 and12 weeks postoperatively; while most of the defects in group II were filled with fibrocartilage regenerate tissue and most of the defects in group III were filled with fibrous tissues. The histological scoring system showed that the cartilage repair of experiment groups are better than the two control groups with statistical significances.Conclusions Sox9 gene modified MSCs can enhance the repair of cartilage defects. Gene enhanced tissue engineering may be a promising way for the treatment of cartilage injuries.SummariesThe combination of density gradient centrifugation and selective attachment culture may be an efficient way to isolate and purify BMSCs from bone marrow. The BMSCs cultured in vitro shows multi-lineage potentials of differentiation. The chondrogenic potential of late passage BMSCs are poor than the early passage cellsBMSCs can be transfected by recombinant Sox9 plasmid using liposome vectors. The overexpression of exogenous Sox9 gene can trigger the chondrogenic differentiation of BMSCs.Sox9 gene modified BMSCs embedded in alginate may regenerate hyaline like cartilage. Gene enhanced tissue engineering may be a promising way to repair cartilage defects.更多还原