Cartilage differentiation in micro-mass cultures of chicken limb buds

In micro-mass cultures of stage 23-24 chicken embryos the expression of cartilage phenotype was density dependent and it always took place practically to the same extent at the density of 2 X 10(5) cells per culture. The cells formed aggregates on day 1 and cartilage nodules appeared on day 2. By day 3, 17% of the culture surface consisted of areas showing cartilage differentiation and these regions represented more than 50% on day 6. By day 14, the centre of th cultures contained a mass of cartilage. The progression of cartilage differentiation was indicated by the increasing amount of toluidine blue fixed by the cultures. By day 14 the DNA and protein contents increased 8.7 fold and 16.7 fold, respectively. Uronic acid and OH-proline were detected from the 2nd day. From day 3 to 14, the microgram uronic acid and microgram OH-proline per microgram DNA gradually increased 25 fold and 14 fold, respectively. A decreased density of the inoculum reduced the probability of cartilage formation and it failed to occur at the density of 1.25 X 10(4) cells per culture. Stage 19-20 limb cultures with identical density had the same morphological characteristics. Aggregates appeared but nodules failed to do so in cell cultures of stage 28 limb bud soft tissue region. It is supposed that an oxygen gradient may be present in the cell aggregates.