Identification of the chondrogenic-inducing activity from bovine dentin (bCIA) as a low-molecular-mass amelogenin polypeptide

Dentin extracellular matrix has been shown to contain components capable of inducing chondrogenesis and osteogenesis at ectopic sites when implanted i n vivo, and chondrogenesis in cultures of embryonic muscle-derived fibrobla sts (EMF) in vitro. The polypeptide responsible, called the chondrogenic-in ducing agent (CIA), has been isolated from a 4.0-M guanidinium hydrochlorid e extract of demineralized bovine dentin matrix. Following Sephacryl S-100 chromatography, CIA activity was identified in fractions by assay for uptak e of [S-35]-SO4], into proteoglycan by the EMF after 24 hrs in culture. The active fraction induced the EMF to produce type TT collagen mRNA and decre ase production of type I collagen mRNA after 5 days in culture. The EMF + C IA, cultured for 4 to 7 wks, formed toluidine-blue- and alizarin-red-staina ble nodules, indicative of chondrogenic induction. In vivo implants in rat muscle with collagen carrier produced ectopic bone after 7 wks. The CIA was brought to near-homogeneity by reverse-phase high-performance liquid chrom atography, tested at each step by EMF [S-35]-SO4-incorporation assays. The CIA components had masses in the ranges of 6000 to 10,000 Da by both mass s pectroscopy and gel electrophoresis. The CIA amino acid composition, NH2-te rminal, and internal amino acid sequences were determined. These data showe d unequivocally that the CIA peptides were derived from bovine amelogenin. The peptides contain the amino-terminal portion of the bovine amelogenin. T he presence of these chondrogenic/osteogenic amelogenin-polypeptides in den tin matrix leads us to hypothesize that they may be involved in epithelial- mesenchymal signaling during tooth development interactions-the first time a function has been indicated for these molecules.