Chondrogenic differentiation in cultures of embryonic rat mesenchyme

The morphology and fine structure of day 12 rat embryonic mesenchyme from forelimb bud, mandibular arches, and frontonasal prominence is described as the cells undergo chondrogenesis in high density, micromass culture. The cultures began as a multilayered "pavement" of flattened mesenchymal cells, 3-4 deep, with moderate intercellular space but little identifiable electron-dense extracellular matrix. Pre-cartilage condensations, which consisted of aggregates of cells which had rounded up, displaying little or no intercellular space, formed within the first 24 h in limb mesenchyme and after an additional 24 h in mandibular and frontonasal cultures. Gap junctions occur between these cells, indicating a phase of direct cell-cell communication. Chondrogenesis within these aggregates began within the next 24 h in limb cultures but was delayed an additional 24-48 h in the frontonasal and especially in mandibular cultures. The aggregates in both limb and mandibular mesenchyme form discrete nodules bordered by a perichondrium consisting of 2-3 layers of flattened cells. Evidence from late stage mandibular cultures suggests that chondroblasts are added to the nodules from the perichondrium, as occurs in vivo. By contrast, the frontonasal cartilage is initially unbordered and forms anastomosing trabecular arrays. Some of these arrays fuse into larger structures with time, but others become surrounded by a single, flattened perichondrium, resulting in the stacking of these trabeculae as chondrification proceeds. The sequence of cartilage matrix production, as revealed in long-term facial cultures, appears to occur in three stages, an early phase in which the extracellular matrix consists primarily of proteoglycans, followed by a phase of homogeneous collagen-proteoglycan matrix and a mature, territorial matrix. In all three cultures the cartilage ultimately produced resembles mature rat hyaline cartilage with chondroblasts surrounded by a territorial matrix of type II collagen and proteoglycan granules.