An ultrastructural report of human articular cartilage resident cells in correlation with their phenotypic characteristics

The recent discovery of progenitors based on their differential fibronectin-adhesion (FAA-CPs) and migratory-based (MCPs) assay has evoked interest due to their superiority in terms of their efficient chondrogenesis and reduced hypertrophic propensity. This study aims to isolate and enrich three articular cartilage subsets, chondrocytes, FAA-CPs, and MCPs, and compare their undifferentiated and chondrogenic differentiated status, using in-vitro phenotypical characterization in correlation with ultrastructural analysis using Transmission Electron Microscopy (TEM). Following informed consent, cartilage shavings were procured from a non-diseased human ankle joint and cultured to obtain the three subsets. Chondrocytes exhibited higher CD106 and lower CD49b and CD146 levels. Following chondrogenic differentiation, corroborative results were seen, with the MCP group showing the highest GAG/DNA ratio levels and uptake of extracellular matrix stain as compared to the FAA-CP group. TEM analysis of the chondrocytes revealed the presence of more autolytic cells with disintegrated cytoplasm and plasma membrane. The differentiated FAA-CPs and MCPs displayed higher collagen and rough endoplasmic reticulum. The results presented in this study provide novel information on the ultrastructural characteristics of cartilage resident cells, with the chondrocyte group displaying features of terminal differentiation. Both progenitor subtypes showed superiority in varied contexts, with greater collagen fibrils and greater GAG content in MCPs. The display of preferential and differentiation traits sheds insight on the necessity to enrich progenitors and coculturing them with the general pool of constituent cells to combine their advantages and reduce their drawbacks to achieve a regenerative tissue displaying genuine hyaline-like repair while limiting their terminal differentiation.

近期通过差异纤连蛋白黏附试验（FAA-CPs）与迁移特性试验（MCPs）所发现的祖细胞，因其高效的软骨生成能力与更低的肥大转化倾向，引发了学界的广泛关注。本研究旨在分离并富集三类关节软骨亚群：软骨细胞、差异纤连蛋白黏附祖细胞（FAA-CPs）与迁移特性祖细胞（MCPs），并通过体外表型表征结合透射电子显微镜（TEM）超微结构分析，比较其未分化与软骨分化状态。本研究经知情同意后，从无病变的人体踝关节获取软骨碎屑，经体外培养分离得到上述三类亚群。实验结果显示，软骨细胞表现出更高的CD106表达水平，以及更低的CD49b与CD146表达水平。软骨分化诱导后，该表型得到验证：与FAA-CPs组相比，MCPs组的糖胺聚糖/DNA比值最高，且细胞外基质染色摄取量更高。透射电子显微镜分析显示，软骨细胞组存在更多胞质与细胞膜崩解的自溶细胞；而分化后的FAA-CPs与MCPs则呈现出更高的胶原表达水平与更发达的粗面内质网。本研究结果为软骨驻留细胞的超微结构特征提供了全新的研究视角：软骨细胞组呈现出终末分化的特征。两类祖细胞亚群在不同研究维度均展现出优势：MCPs组拥有更为丰富的胶原纤维与更高的糖胺聚糖含量。两类祖细胞所展现出的特异性分化特性，提示我们有必要对祖细胞进行富集，并将其与软骨组成细胞的常规群体共培养，以整合二者优势、弥补各自缺陷，最终获得兼具真实透明样修复效果与可控终末分化的再生组织。