Analysis of cell surface markers within immature bovine articular cartilage

Previous studies have shown that articular cartilage grows by apposition from the joint surface, driven by proliferation of a progenitor cell sub-population residing in the superficial zone. To date, there is no individual marker for this progenitor sub-population; however, markers located to mesenchymal stem cells have been identified in articular cartilage. Using immunofluorescence, this study demonstrated the localisation of the stem cell markers, CD44, CD49e, CD105 and CD166 to the superficial zone of bovine immature articular cartilage. CD29 and the developmental markers, Notch1, Delta1, Jagged1, Jagged2 and Msx1 were located in cells throughout the tissue. The restricted expression of the majority of these stem cell markers to predominantly the superficial zone in immature bovine complements the appositional growth model notion and greatly suggests a resident stem cell population. To further investigate and quantify expression of these differentiation and stem cell markers, superficial zone cells were isolated, immunolabelled and analysed using flow cytometry. In addition, cells were cultured for 24 hours in monolayer for comparison and to enable epitope recovery. Stem cell marker expression was absent or reduced following cell isolation and upregulated following monolayer culture. Developmental markers displayed expression comparable to that seen in tissue following cell isolation, but expression was absent after monolayer culture. The differences observed suggest cell surface marker cleavage during cell isolation and subsequent cell adhesion and proliferation. To assess the changes in expression, superficial zone cells were cultured for 14 days, to provide a proxy for dedifferentiated cells. Superficial zone chondrocytes were immunolabelled at day 3, 7 and 14 and analysed using flow cytometry. The majority of cell surface receptors exhibited a unimodal increase in expression indicative of a homogeneous population. The number of cells expressing CD44 increased with time in culture, from 3 to 14 days, characteristic of cells adhering to plastic. Bimodal distributions were observed with CD105 and CD117, after 14 days in culture. This expression has not previously been reported and demonstrates a distinct and discrete subset of cells equating to 1-2% of the total superficial zone population analysed, comparable to chondroprogenitor percentages previously reported. The use of specific markers to isolate chondroprogenitors will allow for further characterisation, including a more in-depth understanding of the mechanisms of proliferation and differentiation within articular cartilage. This has the potential to lead to an improved understanding of the role of these markers and, as such, may provide us with a more beneficial cell type that could significantly contribute to the field of articular cartilage repair