Ustunel Ismail, Ozenci Alpay Merter, Sahin Zeliha, Ozbey Ozlem, Acar Nuray, Tanriover Gamze, Celik-Ozenci Ciler, Demir Ramazan
Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, Antalya, Turkey.
Acta Histochem. 2008;110(5):397-407. doi: 10.1016/j.acthis.2007.12.005. Epub 2008 Feb 12.
The presence of progenitor/stem cells in human articular cartilage remains controversial. Therefore, we attempted to isolate and culture progenitor/stem cells and to investigate their phenotypic characteristics. Biopsies were obtained (with consent) from patients undergoing arthroscopic surgery. Full depth explants were fixed and cryosectioned or enzymatically digested and the resulting cells cultured and plated on fibronectin-coated dishes. Chondrocytes were cultured until colonies of >32 cells were present. Colonies were trypsinized and expanded in monolayer for pellet culture. Immunolocalization of Notch and its ligands were detected in vivo and in vitro using immunocytochemistry. In vitro studies investigated differences in immunolocalization of Notch and its associated ligands in colony-forming cells and small clusters of non-colony-forming cells. The ultrastructure of the chondroprogenitors was examined by scanning and transmission electron microscopy. Results revealed that the immunolocalization of Notch-1 and its ligand Delta were concentrated in regions closest to the articular surface. Notch-1 was also densely localized in the deeper zone of articular cartilage. Notch-2 immunolabeling was densely localized in all zones of articular cartilage. Jagged-1 was concentrated in the deeper regions of articular cartilage. Notch-1, Delta and Jagged-1 were more abundant in colony-forming cells than non-colony-forming chondrocytes in vitro. Notch-3, Notch-4 and Jagged-2 were absent from all regions of the articular cartilage tissues and cultured cartilage cells in vitro. Ultrastructurally, chondrocytes cultured in monolayer dedifferentiated to fibroblast-like cells with cell surface processes of varying lengths, pellet cultured cells varied in morphology, as flattened and rounded. In conclusion, we propose that adult human articular cartilage may contain cells having progenitor cell features.
人关节软骨中祖细胞/干细胞的存在仍存在争议。因此，我们试图分离和培养祖细胞/干细胞，并研究它们的表型特征。在获得患者同意后，从接受关节镜手术的患者身上获取活检样本。将全层组织块固定并进行冷冻切片，或进行酶消化，然后将所得细胞培养并接种到纤连蛋白包被的培养皿上。培养软骨细胞直至出现大于32个细胞的集落。将集落用胰蛋白酶消化并在单层中扩增用于成球培养。使用免疫细胞化学方法在体内和体外检测Notch及其配体的免疫定位。体外研究调查了Notch及其相关配体在集落形成细胞和非集落形成细胞小簇中的免疫定位差异。通过扫描电子显微镜和透射电子显微镜检查软骨祖细胞的超微结构。结果显示，Notch-1及其配体Delta的免疫定位集中在最靠近关节表面的区域。Notch-1在关节软骨的较深区域也有密集定位。Notch-2免疫标记在关节软骨的所有区域都有密集定位。Jagged-1集中在关节软骨的较深区域。在体外，集落形成细胞中Notch-1、Delta和Jagged-1比非集落形成软骨细胞更丰富。Notch-3、Notch-4和Jagged-2在关节软骨组织的所有区域和体外培养的软骨细胞中均不存在。在超微结构上，单层培养的软骨细胞去分化为具有不同长度细胞表面突起的成纤维细胞样细胞，成球培养的细胞形态各异，有扁平的和圆形的。总之，我们认为成人关节软骨可能含有具有祖细胞特征的细胞。
