In the serum-free culture medium of bovine odontoblasts, we detected active gelatinolytic metalloproteinases, MMP-2 and MMP-9 (gelatinase A and B). The activity of MMP-2, in particular, appeared suddenly around day 21 in the culture, coinciding with the development of odontoblastic cell processes and the loss of alkaline phosphatase. RT-PCR analysis of these odontoblasts demonstrated that messages of MMP-2 but not MMP-9 increased significantly between day 15 and day 21. This in vitro observation indicates that medium conditioned by these odontoblasts and containing significant amounts of MMP-2 mRNA, degrades not only the collagenous substrates but also purified dentin phosphophoryn as well.
We have also observed that dephosphorylated dentin phosphoprotein beccmes a better substrate for casein kinase II after limited proteolysis with MMP-2. These results support our working hypothesis that MMP-2 mediated proteolytic processing is an important step in accelerating the process of dentin matrix maturation, which includes phosphorylation and subsequent mineralization. The authors and others have suggested the significance of extracellular phosphorylation of matrix proteins in biomineralization both in bone and in dentin (Zhu et al., Biochem. J., 323 : 637-643, 1997 ; Mikuni-Takagaki et al., J.Bone Miner. Res., 10 : 231-241, 1995). Our present histochemical analysis in MMP-2 knockout mice confirms this idea, with the the delayed formation of mineralized tissues, dentin and bone.