A rat incisor dentin matrix protein can induce neonatal rat muscle fibroblasts, in culture, to express phenotypic products of chondroblastic cells.

Abstract

Demineralized dentin matrix induces the ectopic formation of bone, in vivo, when implanted subcutaneously or in muscle pouches. In these situations the bone induction follows a chondrogenic pathway. As part of the strategy for the assay and isolation of the factors responsible for initiating induction, we have developed a cell culture system in which the addition of soluble factors extracted from the dentin matrix appears to initiate chondrogenesis. Indicators of chondrogenesis, relative to control cultures, were taken as an increase of 35S-sulfate incorporation into proteoglycan (PG), an altered size of the PG, production of type II collagen, and changes in cell morphology and matrix histochemistry. Our studies have taken two directions: the use of the cell culture system under standard conditions to select fractions inducing one or more of the above indica-tors; and, the purification and characterization of the in vitro chondrogenesis inducing factor(s). Here we report the identification of a peptide fraction which acts in culture to satisfy each of the above indicators of chondrogenesis. An EDTA extract of rat incisor dentin was fractionated by CaCl2 precipitation, Sephacryl S-100 chromatography, and reverse phase HPLC. A single peptide fraction from the HPLC, evidenced by the existence of a single spot on 2-D Gel Electrophoresis, was found to be a potent enhancer of 35S-sulfate incorporation during the standard assay, with maximal activity in the 1-10 ng/ml range. Further detailed studies showed that the heightened incorporation occurred without any increase in cell number. The neonatal rat muscle explant fibroblasts exposed to this fraction for 7 days in monolayer culture formed dense cell nodules which stained intensely with Alcian blue relative to controls.(ABSTRACT TRUNCATED AT 250 WORDS)