Bone marrow stromal cells from embryonic, neo-natal and adult chickens were grown in vitro over a 21-day period. Marrow stromal cells from embryonic and neonatal chicks produced clonally derived chondrocytic colonies. The cells within the colonies were surrounded by a refractile, Alcian-blue-positive matrix and their cartilagenous nature was shown biochemically and immunocytochemically by the synthesis of collagen types II and X. The ability of chick bone marrow cells to form chondrocytic colonies decreased during development and was lost by adulthood. In addition to chondrocytic colonies, fat cells and fibroblasts were also observed in the cultures. Our data demonstrate that chick bone marrow stroma contains cells that are capable of differentiating along different pathways within the same culture, providing further evidence for the presence in bone marrow of a stromal stem cell.

A method is described for the cleavage of collagenous molecules by bacterial collagenase in the presence of sodium dodecyl sulphate (SDS) and urea. Comparison of three commercially available preparations of bacterial collagenase showed that the most efficient cleavage under these conditions was by the enzyme isolated from Achromobacter iophagus (E.C. 3.4.24.8). No non-specific proteinase activity was apparent in conditions where all collagen types showed some susceptibility to attack. This method represents a simple one-stage identification of collagenous molecules in complex mixtures of proteins and where limited amounts of a protein are available.

Enzyme dissociation of rabbit dental pulp enabled isolation of fibroblast-like cells. Confluent rabbit dental pulp fibroblasts maintained in Dulbecco's modification of Eagle's medium incorporate L-[3H]proline in a linear manner with time into non-diffusable macro-molecules for up to 24 h. Assays of 4-hydroxyl[3H]proline indicate that collagen synthesis was also linear over this period. The nature of the secreted collagen molecules was investigated by molecular-sieve chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The proportion of type-III collagen produced by the cells in culture was estimated from the pepsin-digest of the proteins in the medium before and after reduction and found to be 16 per cent by gel filtration and 15 per cent by SDS/gel electrophoresis.

Confluent dental pulp fibroblast cultures incorporated l-[3H]-fucose in a linear manner with time into non-diffusible macromolecules over 24 h. Ascorbate supplementation did not appear to alter the amount or type of macromolecules. Equilibrium CsCl-density-gradient centrifugation established that the [3H]-fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis showed that the major fucosylated glycoprotein had an apparent molecular weight of 230,000, but several other species were also seen. The major fucosylated glycoprotein was fibronectin by its affinity for gelatin and its immunoprecipitation with a specific anti-(cold-insoluble globulin).