The isolation and partial characterization of a rat incisor dentin matrix polypeptide with in vitro chondrogenic activity

Abstract

In vivo implants of demineralized dentin matrix into muscle induce the formation of bone within the muscle. As with bone matrix implants, the bone induction appears to follow a chondrogenic pathway. Outgrowth cells from explants of neonatal rat muscle respond to bone matrix, in vitro, by expressing a heightened synthesis of sulfated proteoglycans and type II collagen, phenotypic of cartilage. The in vitro cell culture system has been used as an assay to monitor the isolation of the factor responsible for expression of this phenotypic transformation. Soluble proteins extracted from rat incisor dentin matrix during demineralization with EDTA, and not precipitable with 1.0 M CaCl2, were active in the in vitro system. The active extract was fractionated by Sephacryl S-100 chromatography in 6 M guanidine HCl, isoelectric focusing in Immobilines, and by reverse phase high performance liquid chromatography. All fractions were assayed for activity at every stage. The final active fraction from the reverse phase chromatography on a Zorbax Poly-F column was purified to homogeneity, and yielded a single spot on two-dimensional gel electrophoresis. The component, RP-4, had pI 5.4-5.5, and an apparent Mr 6,000-10,000, based on globular protein standards. Maximal activity with respect to both sulfate incorporation into proteoglycan and production of type II collagen was in the 1.0-10 ng/ml range. The RP-4 had a unique amino-terminal amino sequence and was rich in Gly, Pro, Glx, and Ala residues. It was different from transforming growth factor-beta and the bone morphogenetic protein family of proteins in these essential features.