Inhibition of in vitro Mineralization by DMP1

Objective: Dentin matrix protein-1 (DMP1) is a minor but important constituent of dentin and bone extracellular matrices. DMP1 was predicted based on its primary structure to have a high affinity for apatite (HA). Previous studies in our laboratory had shown that without post-translational modification recombinant DMP1 (rhDMP1) made in E. coli could nucleate HA in vitro, while rephosphorylated rhDMP1 had no effect. The reported immunolocation of DMP1 around the odontoblast periphery suggested that this protein might serve in vivo to prevent mineralization. Hence the purpose of these experiments was to compare the in vitro function of « native » DMP1 to that previously observed for rhDMP1. Methods: Bovine DMP1 (bDMP1) was made by infecting human bone marrow stromal cells with replication-deficient DMP1-adenovirus constructs and purifying the protein from the media under nondenaturing conditions. Three concentrations (1, 10 and 25 µg/mL) were studied in triplicate using a gelatin- gel diffusion system. The bDMP1 was included at the site in the gel where apatite formation occurs in the absence of any protein. Gels were maintained at room temperature with a constant Ca and P diffusion flux through the pH 7.4-buffered gels. Ca and P content of the opaque precipitant bands were monitored at 3.5 days. Comparisons were made to controls without protein, and to single diffusion gels. Data were processed and statistical analysis was done with the Dunnet test Results: At 1µg/ml bDMP1 there was no effect ; the ratio of experimental to control was 0.96±0.01 :1. In contrast, at 10 and 25µg/ml, the ratios were 0.80±0.047 :1 and 0.70±0 :1, respectively. The observed inhibition was statistically significant. Conclusions: These results indicate that bDMP1 is an in vitro inhibitor of mineralization, and suggest that in situ bDMP1 may have a similar effect.