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.

Evaluation of ghrelin as a distinguishing marker for human articular cartilage-derived chondrocytes and chondroprogenitors.

Purpose of the study: Cell-based therapeutics for articular cartilage repair primarily employed bone marrow-derived mesenchymal stem cells and chondrocytes. Research to overcome their limitation of formation of a functionally poor fibro-hyaline type of repair tissue led to the discovery of chondroprogenitors (CPCs), cartilage resident stem cells. These cells isolated by adhesion assay using fibronectin (FAA-CPs) and migration of progenitors from explants (MCPs) display higher chondrogenic and lower terminal differentiation potential. During in-vitro culture, chondrocytes tend to de-differentiate and acquire characteristics similar to stem cells, thus making it challenging to distinguish them from other cell groups. Ghrelin, a cytoplasmic growth hormone secretagogue, has been proposed to play a vital role in chondrogenesis, with reports of its higher expression in chondrocytes than BM-MSCs. The aim of this study was to compare the mRNA expression of Ghrelin between BM-MSCs, chondrocytes, FAA-CPs and MCP and the possibility of it serving as a distinguishing marker. Methods: The four populations isolated from three human osteoarthritic knee joints were characterised by CD marker expression for positive (CD 90, CD73 and CD105) and negative (HLA-DR, CD34 and CD45) MSC markers and trilineage differentiation (adipogenic, osteogenic and chondrogenic) and subjected to qRT-PCR to assess Ghrelin's gene expression. Results: This study showed that all groups exhibited similar expression of CD markers and multilineage potential. Though chondrocytes showed greater expression of Ghrelin, it was not statistically significant to classify it as a distinguishing marker between these cell populations. Conclusion: Ghrelin does not serve to differentiate the subpopulations in terms of their mRNA expression. Further evaluation using their associated enzymes and receptors could provide valuable information to uncover their potential as unequivocal biomarkers.

Comparison of methods for the isolation and culture of Migratory chondroprogenitors from Human articular cartilage.

PURPOSE: Resident articular stem cells isolated using a migratory assay called Migratory Chondroprogenitors (MCPs) have emerged as a promising cellular therapeutic for the treatment of cartilage pathologies. In-vivo studies using MCPs report their superiority over bone-marrow mesenchymal stem cells and chondrocytes for treating chondral defects. However, there is no consensus on their isolation protocol. This study aimed to compare four reported isolation methods of MCPs and identify the optimal and feasible protocol for future translational work. METHODS: Human MCPs isolated from osteoarthritic cartilage (n = 3) were divided into four groups: a) MCP1: 8-15 mm cartilage explants, b) MCP2: 8-10 mm explants digested in 0.1% collagenase for 2 hrs. and cultured c) MCP3: 1 mm cartilage explants and d) MCP 4: 25 mm explants with a X tear, 7-day culture, and trypsinization to release migrated cells. The MCPs were subjected to the following analysis: growth kinetics, surface marker expression, mRNA gene expression for markers of chondrogenesis and hypertrophy, and trilineage differentiation. RESULTS: MCPs isolated via the four methods showed similar surface marker profiles, chondrogenic (SOX-9, ACAN, COL2A1) and hypertrophic (COL1, RUNX2) gene expression. The migration time for the MCP3 group was the longest. The MCP1, MCP2, and MCP4 groups produced MCPs with comparable cellular expansion feasibility. CONCLUSIONS: MCPs can be preferably isolated by the any of the three above methods based on the investigator's discretion. In the case of small cartilage samples similar to the MCP3 group, the isolation of MCP is plausible, keeping in mind the additional time required.

Human fetal cartilage-derived chondrocytes and chondroprogenitors display a greater commitment to chondrogenesis than adult cartilage resident cells.

Obtaining regeneration-competent cells and generating high-quality neocartilage are still challenges in articular cartilage tissue engineering. Although chondroprogenitor cells are a resident subpopulation of native cartilage and possess a high capacity for proliferation and cartilage formation, their potential for regenerative medicine has not been adequately explored. Fetal cartilage, another potential source with greater cellularity and a higher cell-matrix ratio than adult tissue, has been evaluated for sourcing cells to treat articular disorders. This study aimed to compare cartilage resident cells, namely chondrocytes, fibronectin adhesion assay-derived chondroprogenitors (FAA-CPCs) and migratory chondroprogenitors (MCPs) isolated from fetal and adult cartilage, to evaluate differences in their biological properties and their potential for cartilage repair. Following informed consent, three human fetal and three adult osteoarthritic knee joints were used to harvest the cartilage samples, from which the three cell types a) chondrocytes, b) FAA-CPCs, and MCPs were isolated. Assessment parameters consisted of flow cytometry analysis for percentage expression of cell surface markers, population doubling time and cell cycle analyses, qRT-PCR for markers of chondrogenesis and hypertrophy, trilineage differentiation potential and biochemical analysis of differentiated chondrogenic pellets for total GAG/DNA content. Compared to their adult counterparts, fetal cartilage-derived cells displayed significantly lower CD106 and higher levels of CD146 expression, indicative of their superior chondrogenic capacity. Moreover, all fetal groups demonstrated significantly higher levels of GAG/DNA ratio with enhanced uptake of collagen type 2 and GAG stains on histology. It was also noted that fetal FAA CPCs had a greater proliferative ability with significantly higher levels of the primary transcription factor SOX-9. Fetal chondrocytes and chondroprogenitors displayed a superior propensity for chondrogenesis when compared to their adult counterparts. To understand their therapeutic potential and provide an important solution to long-standing challenges in cartilage tissue engineering, focused research into its regenerative properties using in-vivo models is warranted.