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.

基于差异纤连蛋白黏附实验筛选的祖细胞（fibronectin-adhesion assay-selected progenitor cells, FAA-CPs）与趋迁移实验筛选的祖细胞（migratory-based assay-selected progenitor cells, MCPs）的最新研究发现，因其在高效软骨生成（chondrogenesis）与降低肥大倾向方面的优势而引发学界广泛关注。本研究旨在分离并富集三种关节软骨亚群：软骨细胞（chondrocytes）、FAA-CPs与MCPs，并通过体外表型表征结合透射电子显微镜（Transmission Electron Microscopy, TEM）超微结构分析，比较其未分化与软骨分化状态。本研究在获取知情同意后，从无病变的人类踝关节取材软骨刮屑，经体外培养获得上述三种亚群。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。软骨分化后，实验结果得到验证：与FAA-CPs组相比，MCPs组的糖胺聚糖/脱氧核糖核酸（glycosaminoglycan/DNA, GAG/DNA）比值最高，且细胞外基质染色摄取量更高。对软骨细胞的透射电镜分析显示，可见更多胞质与细胞膜崩解的自溶细胞。分化后的FAA-CPs与MCPs则表现出更高的胶原表达与更发达的粗面内质网（rough endoplasmic reticulum）。本研究结果为软骨驻留细胞的超微结构特征提供了新的认识：软骨细胞群呈现终末分化（terminal differentiation）的特征。两种祖细胞亚型在不同层面均展现出优势：MCPs组拥有更多的胶原纤维与更高的糖胺聚糖含量。两种祖细胞亚型所展现出的偏好性与分化特性，提示我们有必要对祖细胞进行富集，并将其与软骨组织的常规组成细胞共培养，以整合二者优势、弥补各自缺陷，从而构建具备真正透明样修复效果的再生组织，同时限制其终末分化进程。