人脐带Wharton胶间充质干细胞的生物学特性及雪旺细胞分化研究

[目的]研究人脐带Wharton胶间充质干细胞(WJMSCs)的生物学特性及向雪旺细胞诱导分化的可能性,为神经组织工程提供新的种子细胞.[方法]去除新鲜人脐带动、静脉和脐带外膜组织,获得Wharton胶,采用组织块培养法获得脐带Wharton胶中的干细胞,免疫荧光鉴定WJMSCs的细胞表面特异标记CD44,CD105,CD34,CD45,Stro-1,Vimentin和Nestin,评价干细胞的生物学特性;采用b-FGF和Forskolin等诱导WJMSCs向雪旺细胞分化,光镜观察诱导后细胞形态的变化;免疫荧光染色鉴定雪旺细胞特异性标记物p75和GFAP的表达;Western分析诱导前后雪旺细胞特异性标记物GFAP的表达.[结果]分离培养的WJMSCs免疫荧光染色CD44、CD105、Stro-1和Vimentin表达阳性,而CD34、CD45和Nestin表达阴性,具有间充质干细胞的生物学特性;WJMSCs经过胶质细胞生长因子的作用,光镜下发现诱导的细胞形态与雪旺细胞相似;免疫荧光染色p75和GFAP阳性;Western结果显示诱导的雪旺细胞标记物GFAP表达阳性.[结论]人脐带Wharton胶间充质干细胞可诱导分化成为雪旺样细胞,其表型和分子特征与雪旺细胞相似,诱导分化的人脐带Wharton胶间充质干细胞是一种理想的神经组织工程的种子细胞.     

软骨细胞外基质对脐带Wharton胶间充质干细胞生物学性状的作用

[目的]探讨软骨细胞外基质微环境对源于人脐带Wharton胶的间充质干细胞增殖的影响,观察该微环境促进干细胞向软骨细胞方向分化的潜能.[方法]①取3％ (w/v)猪关节软骨细胞外基质和2.4％ (w/v)海藻酸盐为原料,按体积比1∶1的比例充分混合,将其用带16G针头的1 ml注射器匀速滴入氯化钙溶液制作软骨细胞外基质/海藻酸盐混合凝胶微球,通过扫描电镜观察该微球内部结构.②将人脐带Wharton胶中间质干细胞(WJMSCs)混于软骨细胞外基质/海藻酸混合液,滴入氯化钙溶液形成复合干细胞的混合凝胶微球进行培养,设为实验组;同理WJMSCs混合于单纯藻酸盐凝胶并形成微球作为对照组.③通过扫描电镜和FDA-PI染色观察上述两组微球内细胞的生长粘附情况,体外培养4周,经组织学和DNA测定观察干细胞的表型及增殖情况.[结果]①构建的软骨细胞外基质/海藻酸盐混合凝胶微球呈半透明乳白色,内部具有较多不规则的孔隙结构,具有三维立体培养细胞的环境特点.②比较实验组和对照组不同细胞培养时间段细胞增殖量,发现实验组细胞增殖率高于对照组,第28 d时两组细胞DNA含量的差异有统计学意义,(P＜0.05).③实验组中细胞呈圆形或椭圆形,形成明显的软骨陷窝样的结构,有大量细胞外基质分泌,对照组中细胞呈圆形,陷窝样的结构不明显.④组织学染色观察发现:实验组中培养的干细胞明显有向软骨细胞方向分化的趋势.[结论]软骨细胞外基质微环境不仅利于人脐带Wharton胶中间质干细胞的增殖,而且具有将WJMSCs向软骨细胞方向诱导分化的潜能.     

TGF－β1诱导成体干细胞向软骨分化的研究进展

软骨缺损的修复在临床上一直以来都是一个难题,主要是因为软骨自身修复能力很弱,而目前较常应用的软骨细胞移植法,又受到细胞来源的限制.因此人们对软骨缺损修复的研究开始转向了成体干细胞向软骨细胞诱导分化上.近年来应用于诱导成体干细胞向软骨细胞分化的生长因子有很多,TGF-β1就是其中重要的一种.作为一种多功能的缩氨酸,TGF-β1是一种应用广泛的生长因子,它在诱导软骨分化和维持软骨表型上起着重要作用[1],而且,Dickinson等的研究指出,在软骨发生过程中,有高浓度的TGF-β1mRNA或蛋白的表达[2].本文就TGF-β1在诱导成体干细胞向软骨细胞分化方面的作用综述如下.     

不同培养条件下脂肪干细胞与软骨细胞共培养的研究

探讨病理状态下软骨细胞能否诱导脂肪干细胞(ADSC)向软骨细胞分化以及可能的最佳条件,以便为临床修复关节软骨损伤提供可能的新途径.[方法]分离共培养新西兰大白兔骨关节炎模型的ADSC和软骨细胞,根据不同血清浓度(10% FBS和2% FBS)和不同培养空间(纤维蛋白凝胶支架和无支架单层培养)分组,共培养14 d后,胰酶消化终止.倒置显微镜观察和透射电镜观察共培养后ADSC的形态变化.甲苯胺蓝染色法和免疫细胞化学检测共培养后ADSC的蛋白多糖和Ⅱ型胶原的表达水平.RT-PCR检测蛋白多糖和Ⅱ型胶原的基因转录水平.[结果]共培养7 d后部分ADSC变圆,14d时ADSC形态高度分化与成熟软骨细胞相似,其蛋白多糖和Ⅱ型胶原的基因转录和蛋白表达均增高,尤以10%FBS支架共培养组最为明显.[结论]与病理状态下软骨细胞共培养后,ADSC可以被诱导成软骨样细胞.高浓度血清三维培养可以增强这种诱导作用.     

SPIO标记骨髓间充质干细胞和软骨细胞共培养的研究

[目的]探讨SPIO标记骨髓间充质干细胞和软骨细胞共培养的可行性,为临床修复关节软骨损伤寻找新途径.[方法] 分离、扩增新西兰大白兔骨髓间充质干细胞(BMSCs)和软骨细胞,根据SPIO标记(50μg/ml)和不同培养环境(共培养、单独培养)分4组,每天在倒置显微镜观察共培养后BMSCs的形态变化,共培养14 d后,免疫组化检测Ⅱ型胶原表达情况,阿利新蓝法检测蛋白多糖(GAG)表达水平.[结果] 共培养7 d后标记的部分BMSCs变圆,14 d时BMSCs形态高度分化与成熟软骨细胞相似,其蛋白多糖和Ⅱ型胶原的基因转录和蛋白表达均增高,明显优于对照组,差异具有显著性意义(P<0.01).[结论]1.软骨细胞微环境能有效诱导BMSCs向软骨细胞分化;2.SPIO可以安全、有效地标记BMSCs.     

脐带间充质干细胞分化为内皮细胞促进血管新生

目的 探讨脐带间充质干细胞能否在体内外分化为内皮细胞,参与血管新生.方法 体外实验采用内皮细胞生长因子和碱性成纤维细胞生长因子对脐带间充质于细胞进行诱导,观察其形态变化.体内实验将脐带间充质干细胞移植到小鼠后肢缺血模型中,采用免疫组织化学鉴定细胞在体内的迁移和分化,激光多普勒血流成像仪鉴定缺血局部血流恢复情况.结果 在体外脐带间充质干细胞能够形成血管网样结构.在体内脐带间充质干细胞能够分化为内皮细胞,表达CD31抗体,参与实验组的动物血管重建.与小鼠的血管网络发生整合,实验组的动物后肢血流灌注明显好于对照组.结论 脐带间充质干细胞能够分化为内皮细胞,参与血管新生,为治疗性血管新生提供新的细胞选择.     

外源性TGF-β1及IGF-1诱导大鼠脂肪干细胞向软骨细胞分化的实验研究

目的探讨大鼠脂肪干细胞(ADSCs)的分离、培养、扩增方法及单层培养条件下外源性转化生长因子TGFβ-1和胰岛素样生长因子IGF-1诱导ADSCs向软骨细胞定向分化的可能性及两者的协同作用分析。方法取大鼠大网膜及肾周脂肪囊等处脂肪组织,酶消化法分离培养脂肪干细胞;免疫荧光测定CD29和CD44抗原的表达;分别用4组诱导培养基定向软骨方向诱导;诱导2周后细胞爬片行免疫荧光鉴定Collagen II表达;RT-PCR、Western blot对定向诱导后的细胞的表达进行检测。结果自大鼠脂肪组织中可分离出脂肪干细胞,体外生长形态类似成纤维细胞,可长期增殖,CD29和CD44抗原表达阳性,定向诱导后IGF-1组、TGFβ-1组及TGFβ-1 IGF-1组细胞可表现出软骨细胞的特性,TGFβ-1和IGF-1共同作用组软骨特异性基质的分泌明显高于单独诱导组。结论从大鼠脂肪组织中可分离出脂肪干细胞,生物学特性稳定,TGFβ-1和IGF-1均能定向诱导ADSCs向软骨方向分化,两者共同诱导时具有协同作用。     

人脐带间充质干细胞向心肌样细胞分化的显微形态学研究

目的 观察人脐带间充质干细胞(MSCs)向心肌样细胞分化过程中的显微形态学改变及其意义,为心肌细胞的移植探索新的细胞来源.方法 从人脐带华尔通氏胶分离、培养MSCs,倒置相差显微镜、扫描电镜和透射电镜观察细胞分化前后细胞形态的变化及超微结构,用免疫组化的方法检测分化的心肌样细胞是否表达心肌肌钙蛋白I(troponin I)、心肌肌钙蛋白T(troponin T)和心肌结蛋白(desmin). 结果 扫描电镜观察到人脐带MSCs经5-aza和DMSO诱导后体积变大变长,呈杆状细胞且细胞浆中可见肌丝样结构出现;透射电镜可以观察分化的细胞浆中有肌丝的存在.同时分化的细胞表达心肌细胞的标记troponin I、troponin T和desmin. 结论 人脐带间充质干细胞可以分化为心肌样细胞,可以作为心肌细胞移植的来源.     

骨髓间质干细胞移植梗死心肌后细胞因子分泌及对血管新生的影响

目的研究骨髓间质干细胞(MSCs)移植梗死心肌后细胞因子的分泌及其对血管新生的作用.方法贵州香猪24只,按照计算器随机数法随机分为对照组、实验组.抽取骨髓液3 ml,按照Wakitani的方法培养出骨髓间质干细胞,经5-氮胞苷(5-aza)诱导后,5-溴脱氧尿苷(BrdU)标记备用.开胸结扎左冠状动脉前降支,自体骨髓间质干细胞分别经局部注射和结扎的左冠状动脉前降支远端灌注移植入自体急性心肌梗死区域,对照组以DMEM作对照.术后3周、6周取标本,免疫组织化学法、计算机图象分析检测组织血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)和Ⅷ因子表达.结果实验组梗死区3周、6周bFGF、VEGF灰度值明显高于对照组(P<0.01),微血管计数明显增多(P<0.01).结论自体骨髓间质干细胞移植梗死心肌后能分泌VEGF、bFGF,诱导血管新生.     

人脐带Wharton胶间质干细胞的免疫特性研究

目的 对人脐带Wharton胶间质干细胞(human umbilical cord Wharton's Jelly-derived MSC,hWJMSC)进行免疫特性的体外和体内研究,探讨其应用于临床的可行性.方法 取人脐带并分离Wharton胶,组织块法培养hWJMSC,通过细胞表面干细胞标志和多向分化实验对其进行鉴定;通过流式细胞术、免疫组织化学及RT-PCR等实验方法体外检测hWJMSC的主要组织相容性抗原(HLA-ABC和HLA-DPDQDR,即MHC-Ⅰ和MHC-Ⅱ)、表面共刺激分子(CD40、CD80、CD86)和免疫抑制分子(HLAC、IDO、PGE2)等的表达;通过细胞因子蛋白芯片和Western blot实验检测并验证hWJMSC中与免疫抑制相关的细胞因子(IL-10、TGF-β、VEGF、HGF)的表达;另外,还构建了hWJMSC-支架复合体,将其埋人家兔背部皮下,观察其在体内的免疫反应.结果 从人脐带Wharton胶中分离、培养获得长梭形细胞,经流式细胞术及多向分化实验鉴定其为间质干细胞;流式细胞术及免疫组化等检测显示所分离的hWJMSC不表达MHC-Ⅱ,表达MHC-Ⅰ,阳性表达免疫抑制因子HLA-G、IDO、PGE2以及免疫抑制相关细胞因子IL-10、TGF-β、VEGF、HGF,表明其具有低免疫原性和诱导免疫耐受的能力;hWJMSC-支架埋人家兔皮下未引起免疫反应.结论 hWJMSC的免疫原性低,具有抑制免疫排斥、诱导宿主免疫耐受的能力,可应用于异体移植.

Abstract：

Objective To probe the immunological traits of mesenchymal stem cells derived from umbilical cord Wharton's jelly (WJMSCs). Methods The diced Wharton's jelly which was from healthy fullterm birth human umbilical cord was cultured. The mesenchymal stem cells were identified with mesenchymal stem cells markers expression by flow cytometry and multiple differentiation ability. The expression of MHC- Ⅰ / Ⅱ, costimulatory molecules (CD40, CD80 and CD86) was detected with flow cytomctry, immunocytochemistry, and RT-PCR. The expression of immune inhibitors like HLA-G, IDO, and PGE2 was detected by immunocytochemistry and RT-PCR. The expression of immune-related molecules as IL-10, TGF-β, FGF and VEGF was detected with antibody microarray and western blot. Further more, to clarify the in vivo immune reaction of hWJMSCs, we fabricated the hWJMSC-scaffold constructs and implanted them into the rabbit backs. The lymphocyte infiltration and implanted cell survival observed with immunofluorescence. Results After culturinge of diced Wharton's jelly tissue, we obtained spindle-shaped cells. With differentiation medium, the cells can differentiate into osteoblasts, chongdrocytes, adipose cells and schwann cells. Expression of MHC, costimulatory molecules, and a series of immune suppressive-related molecules was found. Immune inhibitors as HLA-G, 1DO, PGE2, and immune suppressive related molecules as HGF, VEGF, TGFand IL-10 were positively expressed. But the cells did not express MHC-Ⅱ. No immune rejection was observed in vivo after implantation of hWJMSC-scaffold constructs. Conclusion It can be concluded that hWJMSCs have very low immunogenicity, which means the cells have potential to induce immune tolerance.The hWJMSCs do not provoke immune rejection in vivo.     

人脐带Wharton胶细胞外基质对软骨种子细胞的作用

目的 观察人脐带Wharton胶细胞外基质（hWJECM）对兔软骨细胞增殖的影响.方法用差速离心法制作hWJECM,制成0.5%浓度浆料分别铺于六孔细胞培养板和96孔细胞培养板上,形成1 mm厚的薄膜,包被有此膜的培养板作为试验组,未铺膜单纯培养液组作为空白对照组,在培养板上培养兔关节软骨细胞.通过倒置显微镜观察,甲苯胺蓝染色观察两个组的兔软骨细胞5,10,15天的生长形态和增殖情况,用CCK8细胞增殖实验比较两组的1、3、5、7 d的增殖情况,来评估软骨种子细胞的增殖和表型维持情况.结果倒置显微镜观察5、10、15 d的细胞增殖情况好于单纯培养液组,5、15 d的甲苯胺蓝染色的结果也证明这个观点,CCK8细胞增殖试验1,3天时试验组和对照组的增殖没有明显的统计学差异（P=0.7142; P=0.0657）,第5,7天显示hWJECM组在细胞增殖方面明显地优于单纯培养液的对照组（P=0.0001;P=0.0006）.结论 hWJECM免疫原性低,无细胞毒性作用,能够很好地促进软骨细胞的增殖.     

脐带Wharton胶来源MSCs生物学特性及其优越性的研究进展

目的总结脐带Wharton胶来源MSCs(Wharton’s jelly-MSCs,WJ-MSCs)生物学特性及其优越性的研究进展。方法查阅近年来关于WJ-MSCs的生物学特性及其与脐带血MSCs(umbilical cord blood MSCs,UBMSCs)和BMSCs相比较的文献,进行综述分析。结果从脐带Wharton胶中可分离获得大量具有自我复制、自我更新、高度增殖和多向分化潜能的MSCs。与UBMSCs和BMSCs相比较,WJ-MSCs在分离时间、分离成功率、倍增时间、传代数量和扩增潜能等方面均具有优越性。结论 WJ-MSCs具有取材简便、来源丰富、相对纯净、无伦理问题等优点,是细胞移植治疗、基因治疗和组织工程器官构建的理想种子细胞,为组织再生修复与原位重建提供了新思路。     

非接触式共培养体系对脐带间充质干细胞向类髓核细胞的诱导分化效应

目的探讨人脐带间充质干细胞向类髓核细胞的分化潜能,为椎间盘退变性疾病的生物学治疗探索新的细胞来源。方法取人正常足月产婴儿脐带及成人椎间盘,分离消化华通胶组织和髓核组织,收集培养脐带间充质干细胞及成人髓核细胞。利用带有插入层的Transwell 6孔培养板进行细胞非接触式共培养,细胞比例为1∶1,以脐带间充质干细胞单独培养作为对照组。共培养1周后,提取脐带间充质干细胞总RNA,利用Real-Time PCR检测其Ⅱ型胶原、蛋白多糖及SOX9基因的相对表达改变。结果成功分离提取脐带间充质干细胞;Real-Time PCR检测结果显示实验组脐带间充质干细胞Ⅱ型胶原、蛋白多糖和SOX9基因相对表达较对照组显著增加,差异有统计学意义（P〈0.01）。结论人脐带间充质干细胞能够被髓核细胞诱导分化为类髓核细胞,有可能为椎间盘退变性疾病的生物学治疗提供一种新的细胞来源。     

优化底物酶及冻存剂组成对软骨细胞增殖力及活力的影响

[目的]本文研究的目的在于找到软骨细胞分离的合适方法及优化冰冻保存软骨组织的配方。[方法]来源于骨关节炎膝关节置换术的软骨标本被用于本次试验,采用300或400 u/ml II型胶原酶(CTII-1或2)进行软骨细胞体外分离,保存在不同组成的冷冻剂中,进行梯度冷却并保存在液氮中48 h,随后进行软骨细胞活力及增殖潜力的测定。[结果]CTII-1较CTII-2更能分离出高活力的软骨细胞(P<0.05),并且300或400 u/ml的底物酶浓度比较合适。10%DMSO+90%FCS是一种比较合适的冰冻保护剂,能够最大程度保留软骨细胞的增殖潜力与活力,并且毒副作用小。[结论]通过相关的优化措施诸如软骨细胞体外分离底物酶以及冰冻保护剂的组成,从关节软骨组织中分离具有高增殖潜力的活力软骨细胞是一种可行的方法。     

转BMP-7软骨细胞在Matrigel胶支架中修复兔膝关节软骨缺损的实验研究

[目的]研究骨形态发生蛋白(bone morphogenic protein,BMP-7)在构建 Matrigel 胶支架软骨膜块修复关节软骨缺损中的作用.[方法]将软骨细胞种植于 Matrigel 胶支架上,体外培养,在培养系统中加入 BMP-7,观察软骨细胞在支架中的繁殖以及载体降解情况;将培养出的软骨膜块植入于动物关节软骨缺损模型中进行定期观测(4周).[结果]Matrigel 胶支架可满足组织工程软骨修复需要,BMP-7 在用于构建膜块修复关节软骨缺损模型中可促进软骨细胞增殖,并在植入 4 周动物试验中发现与对照组比较在关节软骨缺损修复质量上有显著性差异(P<0.01).[结论]BMP-7 在 Matrigel 胶软骨膜块中可明显促进组织工程软骨膜块构建以及关节软骨缺损的修复.     

Chemotaxis of human articular chondrocytes and mesenchymal stem cells

Migration of chondrocytes and mesenchymal stem cells (MSCs) may be important in cartilage development, tissue response to injury, and in tissue engineering. This study analyzed growth factors and cytokines for their ability to induce migration of human articular chondrocytes and bone marrow‐derived mesenchymal stem cells in Boyden chamber assays.In human articular chondrocytes serum induced dose‐ and time‐dependent increases in cell migration. Among a series of growth factors and cytokines tested only PDGF induced a significant increase in cell migration. The PDGF isoforms AB and BB were more potent than AA. There was an aging‐related decline in the ability of chondrocytes to migrate in response to serum and PDGF. Human bone marrow MSC showed significant chemotaxis responses to several factors, including FBS, PDGF, VEGF, IGF‐1, IL‐8, BMP‐4, and BMP‐7. In summary, these results demonstrate that directed cell migration is inducible in human articular chondrocytes and MSC. PDGF is the most potent factor analyzed, and may be useful to promote tissue integration during cartilage repair or tissue engineering. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1407–1412, 2008     

Control of extracellular matrix homeostasis of normal cartilage by a TGFbeta autocrine pathway. Validation of flow cytometry as a tool to study chondrocyte metabolism in vitro

OBJECTIVE: To validate flow cytometry as an experimental technique for the study of the homeostasis of the extracellular matrix (ECM) of human articular cartilage. METHODS: Given the established insights in the relation between the transforming growth factor (TGF)-beta type II Receptor (TGF-betaRII)/TGF-beta auto/paracrine pathway, the intracellular levels of matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), and the accumulation of ECM molecules in the ECM of articular cartilage, this metabolic pathway was used as a reference model to fulfill the objective. Chondrocytes were liberated from visually intact femoral condyle cartilage and cultured in gelled agarose to maintain their differentiated phenotype. After 2 weeks of culture, the chondrocytes were isolated from the agarose and flow cytometry was used to analyse the expression of TGF-betaRII on the plasmamembrane, the expression of TGFbeta1, MMP-1, MMP-3, TIMP-1 and TIMP-3 inside the cells, as well as the amounts of aggrecan, type II collagen and hyaluronan in the cell-associated matrix (CAM). The expression of the different substances was analysed with flow cytometry and reported as mean fluorescence intensity (MFI), which is due to the binding of FITC-labeled antibodies to their specific antigens. In addition, the effects of exogenous TGFbeta1 on the expression of these proteins was investigated on chondrocytes cultured in serum-free media. Enzyme Linked Immunosorbent Assay (ELISA) was performed to evaluate the MMP-1, MMP-3, TIMP-1 and MMP-1/TIMP-1 complex in the culture medium collected after the last 3 days of the culture period. The correlations between the data were analysed with the Spearman's test. RESULTS: Exogenous TGF-beta1 increased the accumulation of aggrecan and hyaluronan in the CAM of chondrocytes and down-regulated the intracellular levels of MMP-1 and -3. TIMP-1 and -3 were increased after exposure to TGF-beta1. Baseline expression of TGF-betaRII on the plasmamembrane of normal human articular chondrocytes significantly correlated with the intracellular levels of TGFbeta1, TIMP-1 and TIMP-3. TGFbeta1 was correlated with TIMP-1, TIMP-3 and MMP-1. Aggrecan in the CAM was inversely correlated with the ratio of MMP-1 to TIMPs. In addition, there were correlations between TIMP-1 and TIMP-3, aggrecan and hyaluronan. ELISA also revealed the correlation between MMP-1 and TIMP-1 secreted by the chondrocytes into the nutrient medium. MMP-1/TIMP-1 complex was hardly found in the medium. CONCLUSIONS: Some aspects of ECM metabolism of normal cartilage were evaluated by flow cytometry. Chondrocytes from normal human cartilage, when cultured in gelled agarose, showed correlations between the expression of TGF-betaRII/TGF-beta1 and the intracellular levels of TIMPs, indicating that TGF-beta autocrine pathway may contribute to homeostasis of the ECM in the normal cartilage. The relations between MMPs, TIMPs and the ECM molecules support that a physiological balance between MMPs and TIMPs results in a well-controlled matrix turnover in normal cartilage.     

Allograft of cultured chondrocytes into articular cartilage defects in rabbits--experimental study of the repair of articular cartilage injuries

Articular cartilage defects were created by dill holes, 2 mm wide and 3 mm deep, through the articular cartilage into the subchondral bone in the patellar groove of the femur in mature rabbits. The defects received graft of cultured chondrocytes and the matrix obtained from the primary culture of chondrocytes isolated from the articular cartilage or auricular cartilage in immature rabbits. The isolated cells were cultured for 10 to 14 days. For graft, the cultured chondrocytes together with the matrix were detached from the culture chamber using rubber policemen and centrifuged. The repair of the grafted defects or defects without graft (control) was histologically studied 2 to 12 weeks after operation. The defects without the graft were progressively filled with fibrous tissue containing spindle shaped cells, fibers perpendicular to the surface, and matrix showing weak metachromasia with toluidin blue at 8 weeks. The defects received articular cartilage cell graft were occupied by new cartilage tissue consisting colonylike crumps of chondrocytes 2 weeks after operation. The crumps showed strong metachromasia with toluidin blue and strong stainability for safranin-O. By 4-8 weeks, the defects were filled with homogeneous cartilage. At 12 weeks, arrangement of the chondrocytes of the superficial layer of the new cartilage became columnar as seen in the normal articular cartilage. The defects received elastic cartilage cell graft were filled by reformed cartilage with chondrocytes surrounded by elastic fibers 2-12 weeks after operation. The results indicate that allograft of cultured chondrocytes with matrix into the articular cartilage defects accerated the repair process of the defects by formation of the new cartilage derived from the grafted chondrocytes.