Evaluation of the sensitive step of inhibition of chondrogenesis by retinoids in limb mesenchymal cells in vitro.

The sensitive step of inhibition of chondrogenesis in vitro by retinoids was investigated in modified micromass cultures of limb bud mesenchymal cells from mouse embryos of day 11 and 12. Evaluation of chondrogenesis was performed after alcian blue staining, using a simple random hit counting of cartilage nodules. All-trans-retinoic acid, 13-cis-retinoic acid, and a newly developed arotinoid, RO 13-6298, were tested for their ability to inhibit chondrogenesis. We found that inhibition of chondrogenesis depended on the dosage and the duration of treatment with the different retinoids. Further analysis showed that chondrogenesis in limb bud mesenchymal cells from the proximal part was irreversibly inhibited after one hour of treatment, whereas distal cells showed a reduction of cartilage development only after a treatment period of 12 and more hours. In respect to the doses of the retinoids, proximal cells were about one magnitude more vulnerable than distal cells. These proximo-distal differences were obtained with 13-cis-retinoic acid at 10 micrograms/ml, with all-trans-retinoic acid at 1 microgram/ml and with arotinoid RO 13-6298 with 10 ng/ml. It is supposed that the late blastemal stage of chondrogenic differentiation before the onset of matrix synthesis is the step which is most vulnerable to retinoid treatment.