The in vitro chondrogenic response of limb-bud mesenchyme to a water-soluble fraction prepared from demineralized bone matrix

Demineralized adult bone matrix initiates de novo ectopic endochondral ossification 2-3 weeks following its intramuscular implantation into adult animals. This phenomenon appears to be similar, in some ways, to inductive cell-matrix interactions which regulate cartilage and bone formation during development. In the present study, we used embryonic chick limb-bud mesenchymal-cell cultures to bioassay extracts of demineralized bone matrix for chondrogenic activity. Guanidinium-chloride (4 M) extracts of demineralized bovine bone were dialyzed against buffers of decreasing ionic strength and then cold water. The cold-water-soluble fraction was found to stimulate chondrogenesis in intermediate-density limb-bud cell cultures (2.2 X 10(6) cells per 35-mm dish), as revealed by visual inspection with phase optics, toluidine-blue staining of fixed plates, and [35S] sulfate incorporation in the cell layer. Further fractionation of this material by anion-exchange, carbohydrate-affinity, and molecular-sieve chromotography produced a semipurified preparation possessing chondrogenic-stimulating activity at doses ranging from 3 to 10 micrograms/ml. The in vitro chondrogenic response of limb-bud mesenchymal cells was dose-dependent, required a minimal initial plating density of 2.08 X 10(5) cells/mm2 of culture dish, and developed gradually over 8-10 days. At an optimal dose of extract, a continuous exposure period of at least 2-3 days was necessary to produce detectable chondrogenic stimulation. In addition, the amount of cartilage formed following an 8-day exposure was markedly influenced by the culture 'age' of the mesenchymal cells (i.e., the time between plating and the start of treatment).(ABSTRACT TRUNCATED AT 250 WORDS)