Objective This study aimed to investigate the effects ofin vitrocontinuous passaging on the morphological phenotype and differentiation characteristics of mouse hyaline chondrocytes, as well as on the balance of the extracellular matrix (ECM). Methods Enzymatic digestion was conducted to isolate mouse hyaline chondrocytes, which expanded over five passagesin vitro. Hematoxylin-eosin stain was used to show the changes in chondrocyte morphology. Semi-quantitative polymerase chain reaction was performed to analyze the mRNA changes in the marker genes, routine genes, matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) in chondrocytes. Zymography was carried out to elucidate changes in gelatinase activities. Results After continuous expansionin vitro, the morphology of round or polygonal chondrocytes changed to elongated and spindled shape. The expression of marker genes significantly decreased (PPP>0.05). Meanwhile, the ratio of MMPs/TIMPs was altered. At the protein level, the activities of gelatinases decreased after passaging, especially for P4 and P5 chondrocytes (Pin vitroexpansion culture. Simultaneously, the anabolism and catabolism of the cartilage ECM became uncontrollable and led to the imbalance of ECM homeostasis. When hyaline chondrocytes are applied in research on relevant diseases or cartilage tissue engineering, P0–P2 chondrocytes should be used.
