Background Chondrocytes resist proliferation and terminal differentiation in healthy articular cartilage. Under disease condition, the chondrocytes change phenotype. Moreover, cartilage degradation, vascularization and calcification of the cartilage are initiated. Prehypertrophic chondrocytes located in the deep zone of articular cartilage are believed to be a hallmark in the pathogenesis of osteoarthritis (OA). The change in phenotype changes the secretion of extracellular matrix proteins as well as collagenolytic proteases. A simple ex vivo model where prehypertrophy can be induced my be valuable tool in investigating the different chondrocyte phenotypes.
Objectives To validate the robustness and utility of an ex vivo full-depth cartilage explant model for studying the different chondrocyte phenotypes.
Methods Full depth cartilage explants, incl. the superficial layer, middle and deep zone, were extracted from the medial femoral condyle of cattle age 12±2 months. The explants weighted 12-16 mg. The explants were incubated with DMEM/F-12 + P/S without FCS for 4 days. Then the explants were cultured for additional 8 days in following conditions; 1)DMEM/F-12 + P/S medium (W/O); 2) Tri-iodothyrone hormone (T3) [100ng/mL]; 3) T3 [50ng/mL]; 4) T3 [10ng/mL]; 5) T3 [100ng/mL] + ascorbic acid (AA) [100μg/mL] + b-glycerolphosphate (b-GP) [10mM]; 6) T3 [50ng/mL] + AA [100μg/mL] + b-GP [10mM]; 7) T3 [10ng/mL] + AA [100μg/mL] + b-GP[10mM].
The medium was changed every 2nd or 3rd day, and the supernatant were kept and stored at -20°C. Safranin O/Fast Green staining was used to visualize the morphology of the explants. Representative pictures were taken from each zone with a magnification of 20X. Within each picture the total number of cells was counted along with the number of cells associated with prehypertrophy; column, cluster and clonal cells. Column cells are ≥3 cells in a straight line within close proximity. Cluster cells are ≥3 cells within close proximity but with no organization. Clonal cells are 2 cells within the same lacuna. AlarmarBlue was used to measure the viability of the explants.
Results There was no significant decrease in the cell viability of the explants during the experiment. No major morphological changes were observed in the upper and middle zone. We therefore focused the study on the deep zone. Significant morphological changes were observed at day 13. Table 1 shows the cell count for the different chondrocytes phenotypes. The total number of cells was highest in group 1. The lowest concentrations of T3 induce proliferation in the form of clonal activity. What is evident for the model is that several phenotypes could be induced.
Conclusions Using this ex vivo model it is possible to study the different phenotypes of the chondrocyte in all the zones of the cartilage. These data suggest that chondrocytes will become prehypertrophic when cultured in medium and left untreated for a period of 12 days.
Disclosure of Interest None Declared