脂肪基质细胞体外分离培养及成软骨定向诱导

背景:组织工程技术是解决软骨等组织缺损的理想途径,但其种子细胞的来源问题仍未很好地解决。目的:观察脂肪基质细胞体外分离培养及其在体外向成软骨细胞诱导分化的能力。方法:取新西兰白兔皮下脂肪组织,以胶原酶等消化分离获得脂肪基质细胞,以成软骨诱导培养液对其进行成软骨诱导分化,倒置显微镜下观察脂肪基质细胞体外培养传代情况;免疫组织化学、阿尔辛蓝染色、天狼猩红染色等方法观察脂肪基质细胞成软骨诱导可行性;MTT法观察脂肪基质细胞与诱导培养的脂肪基质细胞体外增殖情况。结果与结论:脂肪基质细胞可于体外分离培养,在成软骨诱导后,可定向分化为软骨样细胞,其可分泌软骨细胞特有的Ⅱ型胶原及酸性黏多糖。原代培养的脂肪基质细胞呈长梭形,胞核椭圆,第3天进入指数增长期,群体倍增时间为55h左右细胞在第4天进入平台期,体外传代培养9代后,细胞开始出现衰老征象;成软骨诱导细胞增殖速度显著增快,随诱导培养时间的延长,细胞体积明显增大,形态由长梭形变为宽大、多伪足的多角形,胞核亦变大,核仁明显,培养第2天进入指数增长期,群体倍增时间为30h左右,在第8天进入平台期,经体外传代培养15代后,细胞增殖速度减慢,开始出现衰老征象。提示脂肪基质细胞在一定条件下可定向分化为软骨细胞,可作为软骨组织工程种子细胞来源。     

人骨髓间质干细胞作为骨、软骨组织工程种子细胞的实验研究

目的 :探讨经体外扩增、诱导分化的人骨髓间质干细胞(humanbonemarrowmesenchymalstemcells,hMSC)作为组织工程化骨、软骨种子细胞的可行性。方法 :体外分离、培养、扩增hMSC ,以流式细胞仪检测hMSC的表面抗原。诱导hMSC向成骨细胞、软骨细胞分化。以倒置显微镜和电子显微镜观察细胞的形态 ;以组织化学、免疫组织化学和RT PCR ,检测成骨细胞、软骨细胞的特异性标志物。结果 :分离得到的细胞可表达hMSC的特异抗原 ,在体外扩增 15代以上 ,其形态及表面抗原保持不变。成骨诱导培养的细胞上清液中ALP的含量高于对照组 (P <0 .0 5 )。成骨及成软骨诱导的细胞形态均由成纤维样梭形向多边形转变。透射电镜观察可见大量扩张的粗面内质网、高尔基体及线粒体。扫描电镜观察可见经成骨诱导后的细胞表面有钙盐沉积 ,成软骨诱导的细胞表面有胶原样突起。成骨诱导培养后 ,可见碱性磷酸酶 (ALP)染色、Ca结节染色、胶原 Ⅰ (COL Ⅰ )及骨钙素 (osteocalcin ,OC)免疫组化染色阳性 ,同时RT PCR检测COL Ⅰ、OCmRNA表达阳性。成软骨诱导后 ,甲苯胺蓝染色见细胞周围有大量的异染性基质 ,免疫组化和RT PCR检测COL Ⅱ表达阳性。结论 :hMSC在体外可大量扩增。在特定培养液诱导下 ,可向成骨细胞及软骨细胞转化 ,可作为骨、软骨组     

骨髓间充质干细胞定向分化为软骨细胞

目的:体外定向诱导成人骨髓间充质干细胞(MSC)分化为单一的软骨细胞,探索体外成软骨的必要条件。方法: 采用淋巴细胞分离液密度梯度离心法分离骨髓,体外培养扩增,在较高的细胞密度下加入转化生长因子β1(TGF-β1),以微团培养(micromass culture)方式,诱导MSC分化为软骨细胞。HE染色观察细胞形态,阿新蓝、甲苯氨蓝染色检测软骨基质的分泌,免疫组织化学检测软骨特异性Ⅱ型胶原表达。结果: HE染色呈典型的细胞性软骨结构;阿新蓝染色阳性、甲苯氨蓝异染性;Ⅱ型胶原免疫组织化学检测阳性。结论: 成人骨髓MSC在体外分化为单一的软骨细胞需要高细胞密度、诱导因子TGF-β1及合适的培养条件。     

骨髓间充质干细胞向软骨细胞分化过程中miR130α的表达

目的诱导骨髓间充质干细胞向软骨细胞分化,初步探索miR130a在骨髓间充质干细胞向软骨细胞分化过程中的调控作用。方法体外TGF-β1诱导骨髓间充质干细胞向软骨细胞分化,免疫荧光法和免疫组化染色鉴定Ⅱ型胶原,阿辛兰染色鉴定氨基葡聚糖。用Real-time PCR法检测细胞miR130a的表达。结果骨髓间充质干细胞在TGF-β1诱导下可分化为软骨细胞。培养7 d后,诱导培养组细胞miR130a表达的水平显著低于培养前的水平(P<0.05)。结论骨髓间充质干细胞体外诱导可以分化为软骨细胞,在向软骨细胞分化的早期,miR130a表达降低伴随着软骨细胞的分化,提示与软骨形成的过程有关。     

TGF-β1诱导分化的兔BMSCs复合左旋聚乳酸\β-磷酸三钙多孔支架材料体外研究实验

目的 研究体外培养rBMSCs经TGF-β1诱导分化的软骨细胞复合左旋聚乳酸\β-磷酸三钙(PLLA\β-TCP)多孔支架材料体外构建仿生人工软骨. 方法 低温挤出成形法制备成PLLA\β-TCP复合多孔支架材料,体外分离、培养rBMSCs至第3代,利用含有TGF-β1特殊诱导系统诱导其向软骨细胞分化,诱导14d后用甲苯胺蓝染色及Ⅱ型胶原免疫组化进行鉴定后与PLLA\β-TCP多孔支架材料体外复合培养,并取第7、14、21d细胞复合材料进行电镜扫描观察细胞贴附、生长、增殖状况,同时消化收集贴附支架第7、14、21d的细胞,行RT-PCR检测分化软骨细胞相关基因aggrecan、Co12A1在mRNA水平的表达,Western-bolt检测Ⅱ型胶原蛋白的分泌情况.结果 rBMSCs经诱导后向软骨细胞分化,甲苯胺蓝染色见分化软骨细胞分泌糖胺聚糖(glycosaminoglycan,GAG),Ⅱ型胶原免疫组织化学染色呈阳性;电镜扫描见分化细胞在支架材料分布均匀,黏附良好;RT-PCR及Westem-bolt检测示7、14、21daggrecan、Co12A1在mRNA水平、Ⅱ型胶原蛋白均有不同程度表达. 结论 利用含有TGF-β1特殊诱导系统诱导rBMSCs分化的软骨细胞复合到PLLA\β-TCP多孔支架材料上,细胞生长良好,并能正常分泌软骨细胞特异细胞外基质,体外成功构建了组织工程软骨.     

人类软骨糖蛋白39诱导关节软骨祖母细胞的成软骨分化

背景：有研究表明人类软骨糖蛋白39与骨关节软骨的退变与修复具有一定的关系,但其具体的作用机制并不十分明确。 目的：观察人类软骨糖蛋白39对成人膝关节软骨祖母细胞成软骨诱导分化的影响。 方法：取成人关节软骨,消化分离培养关节软骨祖母细胞;流式细胞仪检测传代细胞中能够表达CD105、CD166的细胞量并进行分离提纯。将分离的软骨祖母细胞采用单层培养法培养,传代培养至第2代后向分离培养所得的软骨祖母细胞中,分别经过含人类软骨糖蛋白39成软骨培养基及普通成软骨诱导培养基的诱导培养14 d后,通过免疫组织化学染色观察经诱导后细胞中Ⅱ型胶原的表达及通过大体组织学观察评估软骨的形成。 结果与结论：关节软骨组织中可以分离出能够表达CD105、CD166的关节软骨祖母细胞,软骨祖母细胞经过诱导分化后逐渐聚集并形成结节,经诱导后Ⅱ型胶原免疫细胞化学着色阳性,且经人类软骨糖蛋白39诱导细胞形成的结节更大,Ⅱ型胶原表达更多。结果表明,成人关节软骨中能够分离培养出具有成软骨分化能力的干细胞系细胞即软骨祖母细胞,且能够被定向诱导分化为软骨细胞,人类软骨糖蛋白39对其分化过程具有一定的促进作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

胎盘来源的间充质干细胞体外分离培养及向软骨细胞分化的研究

目的对人胎盘间充质干细胞体外分离培养及向软骨细胞分化进行研究,为软骨组织工程提供种子细胞。方法取人正常分娩后的胎盘,用组织块培养法分离获取胎盘间充质干细胞,对所获细胞进行体外培养和表面标志的流式细胞仪分析。对经过证实的骨髓间充质干细胞用含TGF-β_3、维生素C、胰岛素、地塞米松、转铁蛋白的诱导培养基处理7-28d,对诱导细胞进行甲苯胺蓝染色及Ⅱ型胶原染色,鉴定诱导后的软骨细胞表型。结果培养的人胎盘间充质干细胞均一表达CD_(29)和CD_(44),而CD_(31)、CD_(34)、CD_(45)和HLA-DR呈阴性。细胞经诱导液作用14d后,甲苯胺蓝染色及Ⅱ型胶原染色阳性。结论采用组织块培养法能分离获取高纯度的人胎盘间充质干细胞,该细胞经诱导液作用可定向分化为软骨细胞。     

成年比格犬骨髓间质干细胞体外软骨方向分化的实验研究

目的：体外诱导成年比格犬骨髓间质干细胞（BMSCs）定向分化为软骨细胞，探讨体外诱导成软骨的方法和条件。方法:比格犬股骨取骨髓10 mL，体外行原代和传代培养扩增，加入转化生长因子（TGF-β1），以高密度细胞团块培养，诱导BMSCs分化为软骨细胞。甲苯胺蓝染色检测软骨基质的分泌，免疫组织化学染色检测软骨特异性Ⅱ型胶原表达。结果:诱导的软骨样组织甲苯胺蓝染色阳性;Ⅱ型胶原免疫组织化学检测阳性。结论： 应用含TGF-β1的诱导液在体外可以诱导比格犬BMSC分化为软骨细胞，诱导的软骨细胞可作为软骨组织工程较理想的种子细胞。     

兔脂肪干细胞与软骨细胞微球共培养成软骨分化最适诱导比例的实验研究

通过间接共培养ADSC与软骨细胞微球,得出使ADSC发生软骨向分化的最佳比例。体外分离培养新西兰大白兔ADSC和软骨细胞,制成微球分置于Tranwell上下室,实验分为阳性对照组,阴性对照组,生长因子对照组及不同比例(0.5∶1; 1∶1; 1∶2; 1∶3; 1∶5;1∶7)的ADSC和软骨细胞共培养组,培养28天后分别行组织形态学观察,GAG、COL\|Ⅱ和COL\|Ⅹ的定性定量检测。阿利新蓝\|核固红染色及Ⅱ型胶原免疫组化染色显示比例为0.5∶1和1∶1的阳性较弱,而Ⅹ型胶原在生长因子对照组的表达最为强烈。DMMB法显示总GAG含量在共培养组较阴性对照组均有所增加(P<0.05),且增加量与软骨细胞的比例呈正相关,但是在ADSC:AC比例达到1∶5之后有所下降。OHP检测总胶原蛋白的变化同上,且1∶5组中沉积的胶原量比阴性对照组高出7.3倍。能够使ADSCs最大程度的表达软骨特异性细胞外基质,同时使COL\|Ⅹ等软骨肥大标志无明显上调的ADSC与软骨细胞的比例为1∶5,共培养诱导较生长因子能够抑制ADSC软骨向分化后的肥大问题。     

三维培养诱导人脂肪间充质干细胞微球的成软骨分化能力

背景：关节软骨损伤后修复结果不满意,需要新的手段,而脂肪间充质干细胞较适宜做种子细胞诱导软骨,然而怎么能够使诱导的软骨具有功能需要研究。 目的：采用三维培养体系诱导人脂肪间充质干细胞微球向软骨分化。 方法：无菌切取吸脂术后脂肪组织,分离培养人脂肪间充质干细胞,传至第3代进行流式细胞术分析,成骨成脂肪诱导等鉴定,同时也给予合适的培养条件用三维培养的方式向软骨细胞诱导,并行阿利辛蓝染色鉴定糖胺多糖的合成,苏木精-伊红染色进行组织学分析,免疫荧光检测Ⅱ型胶原表达,称质量测量软骨硬度。 结果与结论：分离的人脂肪间充质干细胞CD105,CD44,CD29均高表达,而 CD45,CD34低表达,并且成骨成脂诱导后细胞茜素红染色和油红O染色均为阳性。三维培养法诱导的软骨细胞可表达大量糖胺多糖及Ⅱ型胶原。结果证实,三维培养法诱导人脂肪间充质细胞向软骨分化后,具有软骨细胞的特性。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

人脂肪源干细胞分离培养及向成骨细胞的诱导分化

背景：脂肪源干细胞可分泌众多的免疫调节因子,不引起T细胞的细胞毒作用,并可通过调整T淋巴细胞的种类和数量。 目的：探讨人脂肪源干细胞在体外分离培养扩增的方法及向成骨细胞诱导分化的能力。 方法：以0.1%的Ⅰ型胶原酶通过组织消化的方法分离人脂肪组织中的干细胞,体外扩增培养至第2代后检测其表面抗原的表达,并在成骨诱导液中促进其向成骨细胞的分化,通过碱性磷酸酶染色、茜素红染色及对碱性磷酸酶的RT-PCR检测来明确其分化能力。 结果与结论：体外分离培养的脂肪源干细胞生长稳定,扩增速度快。流式细胞仪检测结果显示其高表达干细胞相关抗原。向成骨细胞诱导后经免疫组化染色可见矿化结节形成,RT-PCR检测发现碱性磷酸酶表达阳性。提示脂肪源干细胞在体外分离培养方法简单,扩增速度快,并具有定向分化的能力,是可靠的组织修复和细胞治疗的种子细胞来源。     

兔脂肪干细胞在含有转化因子和转铁蛋白培养基中向软骨细胞的分化

背景：成软骨的种子细胞的选择是软骨组织工程研究中的关键因素。 目的：观察脂肪干细胞在含有转化因子和转铁蛋白诱导条件下向软骨细胞分化的能力。 方法：取新西兰大白兔颈背部脂肪组织,机械分离及酶消化法获得脂肪干细胞,显微镜下观察细胞黏附及生长情况;加入含有转化因子β1和转铁蛋白的诱导培养基培养2周后以免疫组化方法检测Ⅱ型胶原的表达。 结果与结论：从兔脂肪组织中分离出的干细胞原代培养时24 h贴壁,96 h后达80%融合;于软骨诱导培养基内向软骨诱导7 d后形成软骨结节,14 d后Ⅱ型胶原免疫组化阳性。结果初步表明兔脂肪干细胞经诱导后可以向软骨细胞分化。     

Multidifferentiation potential of mesenchymal stem cells in three-dimensional collagen gel cultures

Mesenchymal stem cells (MSCs) have a great therapeutic potential resulting from their ability to differentiate into multiple tissues when cultured under specific conditions. However, it has not been clearly demonstrated whether or not MSCs exhibit a multidifferentiation potential in three-dimensional collagen gel cultures. This study was conducted to explore the multidifferentiation potential of MSCs cultured in three-dimensional collagen gels. Human MSCs were cultured in 0.3% collagen gel for 20 days in chondrogenic differentiation medium (CDM), and for 14 days in osteogenic differentiation medium (ODM). Increases in GAG deposits, intensity of toluidine blue staining, and mRNA expressions of chondrogenic markers (type II collagen and type X collagen) were found in human MSCs cultured in the collagen gel maintained in CDM. Positive staining for alkaline phosphatase (ALP) activity and alizarin red, and increases in mRNA expressions of osteogenic markers (type I collagen, bone sialoprotein and ALP) were noted in the MSCs maintained in ODM. These findings emphasize that human MSCs have an ability to differentiate into both bone and cartilaginous tissues in three-dimensional collagen gel cultures, indicating potential clinical applications of MSC transplant therapy with collagen gel as a scaffold for bone or cartilage regeneration in complicated tissue defects.     

Stromelysin 1, neutrophil collagenase, and collagenase 3 do not play major roles in a model of chondrocyte mediated cartilage breakdown.

AIMS: To determine the collective roles of stromelysin 1, neutrophil collagenase, and collagenase 3 in chondrocyte mediated cartilage proteoglycan and type II collagen degradation in tissue culture model systems. METHODS: Bovine nasal cartilage explants were cultured with and without recombinant human interleukin 1 alpha (IL-1 alpha), recombinant human tumour necrosis factor alpha, or retinoic acid. Proteoglycan and type II collagen release were determined by colorimetric assay and immunoassay, respectively, in the absence and presence of matrixin inhibitors. Potential toxic effects of the inhibitors were assessed by measuring rates of glycolysis. RESULTS: Loss of proteoglycan and type II collagen from nasal cartilage was inhibited by batimastat, a broad spectrum matrixin inhibitor. BB-3437, a selective inhibitor of stromelysin, neutrophil collagenase, and collagenase 3, at the concentrations used in this study, showed a weak but dose dependent inhibitory effect on the IL-1 stimulated degradation of type II collagen, but had virtually no effect on proteoglycan breakdown. Neither inhibitor affected rates of glycolysis. CONCLUSIONS: Stromelysin 1, neutrophil collagenase, and collagenase 3 are unlikely to contribute to chondrocyte mediated proteoglycan degradation in our model system. The modest effect of a selective inhibitor of these enzymes on IL-1 stimulated collagen breakdown suggests a minor role for one or more of these proteinases; potent inhibition by an inhibitor of interstitial collagenase and the gelatinases suggests that these enzymes play a major role in IL-1 stimulated, chondrocyte mediated type II collagen breakdown from nasal cartilage.     

Stromelysin 1, neutrophil collagenase, and collagenase 3 do not play major roles in a model of chondrocyte mediated cartilage breakdown.

AIMS: To determine the collective roles of stromelysin 1, neutrophil collagenase, and collagenase 3 in chondrocyte mediated cartilage proteoglycan and type II collagen degradation in tissue culture model systems. METHODS: Bovine nasal cartilage explants were cultured with and without recombinant human interleukin 1 alpha (IL-1 alpha), recombinant human tumour necrosis factor alpha, or retinoic acid. Proteoglycan and type II collagen release were determined by colorimetric assay and immunoassay, respectively, in the absence and presence of matrixin inhibitors. Potential toxic effects of the inhibitors were assessed by measuring rates of glycolysis. RESULTS: Loss of proteoglycan and type II collagen from nasal cartilage was inhibited by batimastat, a broad spectrum matrixin inhibitor. BB-3437, a selective inhibitor of stromelysin, neutrophil collagenase, and collagenase 3, at the concentrations used in this study, showed a weak but dose dependent inhibitory effect on the IL-1 stimulated degradation of type II collagen, but had virtually no effect on proteoglycan breakdown. Neither inhibitor affected rates of glycolysis. CONCLUSIONS: Stromelysin 1, neutrophil collagenase, and collagenase 3 are unlikely to contribute to chondrocyte mediated proteoglycan degradation in our model system. The modest effect of a selective inhibitor of these enzymes on IL-1 stimulated collagen breakdown suggests a minor role for one or more of these proteinases; potent inhibition by an inhibitor of interstitial collagenase and the gelatinases suggests that these enzymes play a major role in IL-1 stimulated, chondrocyte mediated type II collagen breakdown from nasal cartilage.     

IGF-1 and BMP-2 Induces Differentiation of Adipose-Derived Mesenchymal Stem Cells into Chondrocytes-Like Cells

Articular cartilage defects are common, causing significant morbidities. Tissue engineering using pluripotent stem cells is a new promising modality for cartilage repair. In the current study, we investigated the chondrogenesis of rabbit adipose-derived stem cells (ADSCs). We isolated rabbit ADSCs and transfected these cells with constructs encoding human insulin growth like factor 1 (IGF-1) and bone morphogenic protein 2 (BMP-2). We examined the growth and morphology of these transfected cells and their production of type II collagen and MMP-3. We found that IGF-1 and BMP-2 drove the chondrogenesis of ADSCs, which showed mature chondrocyte-like cells and formed cartilage nodules. These cells also produced type II collagen with a reduced production of MMP-3. Our findings suggested that human ADSCs could differentiate into chondrocyte-like cells driven by IGF-1 and BMP-2 and held promises as an abundant and ready source of stem cells for cartilage repair and regeneration.     

重组hTGF-β1基因转染人脂肪干细胞对其向软骨分化的影响

目的研究重组hTGF-β1腺病毒(AdhTGF-β1)转染人脂肪干细胞(ADSCs)对其向软骨分化的作用。方法重组AdhTGF-β1转染人ADSCs,对照组转染AdLacZ,腺病毒的量以200pfu/细胞计算,体外细胞团聚集连续诱导培养21d,酶联免疫吸附定量检测(ELISA)hTGF-β1蛋白的表达,然后分别从大体观察、组织学和II型胶原蛋白免疫组化的检测对形成组织进行评价。结果 hTGF-β1蛋白量在14d时达最高峰,随后逐渐降低。连续细胞团聚集诱导培养21d,细胞团收缩成近似小球形的组织块,外观成乳白色。HE染色可见细胞团外周为由数层扁平状成纤维样细胞组成的纤维软骨膜,下部区域有巢状软骨样细胞组成,有些区域可见软骨样细胞包埋在软骨陷窝内。Safranin'O染色显示,形成的软骨组织区域有被染成桔红色蛋白多糖类基质分泌。而对照组苏木素-伊红染色观察见无软骨样组织形成或有向软骨分化现象。Ⅱ型胶原免疫组化染色检测显示实验组细胞团出现较明显的阳性染色区域,可见棕黄色的颗粒分布于胞浆内。对照组Ⅱ型胶原免疫组化染色检测显示无明显的阳性染色区。结论重组hTGF-β1腺病毒转染人ADSCs诱导人ADSCs向软骨细胞表型分化形成软骨样组织,为hTGF-β1基因转染的人ADSCs在软骨组织工程应用中奠定了基础。     

人软骨细胞培养上清诱导脐血间充质干细胞向软骨细胞分化

背景：人脐血间充质干细胞作为软骨缺损修复的种子细胞日益受到关注,现有常用诱导方法无论是应用诱导培养基还是诱导因子,因其价格昂贵、用量大等因素限制了实际应用。 目的：验证人软骨细胞培养上清诱导人脐血间充质干细胞向软骨细胞分化的可行性。 方法：利用密度梯度离心法和贴壁培养法分离新生儿脐血,获取并培养人脐血间充质干细胞,流式细胞仪鉴定细胞表面抗原。切取股骨头置换或全髋关节置换患者透明软骨组织,体外分离培养软骨细胞,利用其培养上清培养人脐血间充质干细胞,培养2周后观察细胞表型外观变化,免疫组化染色检测Ⅱ型胶原表达结果。 结果与结论：密度梯度离心法与贴壁培养法可以分离获取人脐血间充质干细胞,流式细胞仪鉴定表面标记CD90,CD105高表达,不表达CD34,CD45。软骨细胞培养上清诱导2周后,人脐血间充质干细胞向多角形、圆形转变,Ⅱ型胶原免疫组化检测表达阳性。提示软骨细胞培养上清可诱导人脐血间充质干细胞表达Ⅱ型胶原,向软骨细胞分化。     

Young Adult Chondrocytes Proliferate Rapidly and Produce a Cartilaginous Tissue at the Gel-Media Interface in Agarose Cultures

Primary chondrocytes cultured in agarose can escape the gel, accumulate at the interface between agarose and the culture medium, and form an outgrowing tissue. These outgrowths can appear as voluminous cartilage-like nodules that have never been previously investigated. In the present study, bovine articular chondrocytes from three age groups (fetal, young adult, aged) were seeded and cultured in agarose to test the hypothesis that hyaline-like cartilage outgrowths develop at the interface by appositional growth, in an age-dependant manner. Macroscopic appearance, cell content, cell division, cytoskeletal morphology, and extracellular matrix (ECM) composition were analyzed. Fetal chondrocytes produced a fibrous interfacial tissue while aged chondrocytes produced ECM-poor cell clusters. In contrast young adult chondrocytes produced large cartilaginous outgrowths, rich in proteoglycan and collagen II, where cells in the central region displayed a chondrocyte morphology. Cell proliferation was confined to the peripheral edge of these outgrowths, where elongated cell morphology, cell-cell contacts, and cell extensions toward the culture medium were seen. Thus these voluminous cartilaginous outgrowths formed in an appositional growth process and only for donor chondrocytes from young adult animals. This system offers an interesting ability to proliferate chondrocytes in a manner that results in a chondrocyte morphology and a cartilaginous ECM in central regions of the outgrowing tissue. It also provides an in vitro model system to study neocartilage appositional growth.