Assessment of in vitro chondrogenesis after treatment with all-trans retinoic acid

Abstract

Retinoids, derivatives of Vitamin A, are valuable in the treatment of certain forms of skin disorders and cancer; however, their therapeutic application is limited by their potential teratogenic potency. To date, mechanisms involving retinoic acid’s teratogenicity are undetermined. Mesodermal cells grown in vitro differentiate into chondrocytes and produce cartilage-specific compounds such as proteoglycans. The purpose of this study was to determine the effects of all-trans retinoic acid (RA) on growth and differentiation of chondrocytes. Micromass cell culturing techniques were performed in which mesodermal limb bud cells were dispersed into a single cell suspension, placed into cell culture dishes, and incubated with all-trans RA for a 120hr period. After incubation, cells were fixed and stained for proteoglycan content with Alcian green and optical density (OD) determined at 600 nm. Subsequently, cultures were stained for cell proliferation using crystal violet (OD=570nm). The absorbances of stained cells were compared to vehicle controls (VC). Results indicated a dose dependent relationship of retinoic acid affecting proteoglycan content and cell proliferation at doses ranging from 10-9-10-6M. After treatment with 10-9M, 10-8M, 10-7M, 10-6M RA, proteoglycan content decreased to 49.8%, 39.3%, 28.6%, and 15.6% of VC, respectively. Cell proliferation decreased to 84.1%, 75.6%, 70.3%, and 69.3% of VC, respectively. Temporally, RA decreased cell proliferation earlier (83% of VC @ 48) than proteoglycan content (20% of VC @ 72hrs). Using an ELISA, TNF a was measured in medium upon cell culture treatments with 10-9-10-6M all-trans RA. Results from the ELISA assay show a minimal detectable level of TNFa within 120hr chick embryonic cell cultures. Research supported in part by Federal Hatch Funds to MAS.