Accelerating Chondrogenic Differentiation of ATDC5 Cells

Objectives: During endochondral bone formation, chondrocytes undergo a process of maturation becoming hypertrophic, depositing a mineralized extracellular matrix and inducing the replacement of this calcified cartilage by bone. Cell culture systems have been devised to study chondrocyte function during the different stages of maturation. These systems are time consuming, labor intensive, involving primary and secondary culturing, and increased risk of contamination due to extensive manipulation. Recently, a chondrogenic cell line (ATDC5) was established from cultures of teratocarcinoma cells, that when treated with insulin for 6 weeks, undergo chondrocyte hypertrophy. While this system presents advantages over the use of primary cultures, 6 weeks is still a long culture period in the study of chondrocyte maturation. Therefore, the objective of this study is to establish a treatment protocol to accelerate ATDC5 chondrocyte differentiation and hypertrophy.

Methods: ATDC5 cells were cultured in a 1:1 mixture medium (Dulbecco's Modified Eagle's/ Ham's F12), containing 5% fetal bovine serum until confluent. Chondrogenic differentiation was induced by supplementing cultures at this time with transferrin, sodium selenite, and bovine insulin (ITS). To accelerate hypertrophy, ascorbic acid was added to the culture medium. At different time intervals, cells were collected and hypertrophy evaluated using biochemical and molecular biology techniques (protein measurements, alkaline phosphatase activity, and gene expression by RT-PCR).

Results: Treatment with ITS, caused a time-dependent increase in the expression levels of cbfa1, type II and type X collagen. In addition, alkaline phosphatase activity was elevated 2 fold, by the end of the second week. However, when ascorbic acid was added to media, all these markers of chondrogenesis and hypertrophy were further increased, some reaching the same levels just after one week.

Conclusions: Supplementation of ATDC5 culture media with ascorbic acid considerably reduced the culture period necessary to induce chondrogenic differentiation and hypertrophy.

Supported by NYU Dean's Research Award