Aggrecan catabolism during mesenchymal stromal cell in vitro chondrogenesis

Abstract

During skeleton formation, mesenchymal cells condense and differentiate into chondrocytes in a process known as chondrogenesis. Glycosaminoglycans (GAGs), main components of aggrecan in the extracellular matrix (ECM), have an important role in this process. An in vitro simplified system has been devised to study chondrogenesis using mesenchymal progenitor cells. Although the capacity of mesenchymal stromal cells to differentiate into the chondrogenic lineage is well established, there is a lack of knowledge with respect to lysosomal enzyme activity during the chondrogenic process. To further understand GAG's catabolic activities during in vitro chondrogenesis, we evaluated three lysosomal enzymes. Chondrogenic differentiation was demonstrated by Alcian blue positive stain quantified by a grading system using ImageJ. Enzyme activity for N-acetylgalactosamine-6-sulfate-sulfatase during chondrogenic induction decreased significantly with time of culture; β-galactosidase enzyme activity had a similar tendency of temporal activity. On the contrary, β-glucuronidase enzyme activity decreased from the first to second week of induction, but remained the same during the third week of culture. Aggrecan's immunohistochemistry values for aggregates under chondrogenic induction revealed a similar temporal pattern to that of N-acetylgalactosamine-6-sulfate-sulfatase and β-galactosidase enzyme activity. This work has contributed to the evaluation of enzyme activities associated with GAG degradation, critical component of cartilage ECM. These findings are relevant in understanding the role of enzymes responsible for degradation of molecules predominantly synthesized in the chondrogenic differentiation process. A better understanding of the roles of these enzymes during development could help elucidate further association of deficiencies of these enzymes in skeletal pathologies, primarily chondrodysplasias.