Extraction And Bio-characteristic Analysis Of Porcine Dentin Matrix Protein Components

Interactions between oral epithelial and ectoblastic mesenchymal regulate dental germ differentiation as well as dental debeloping. During the development progress of dentin, dentin matrix proteins'interactions directly affect odontoblast differentiation, maturation, secretion of matrix, and mediating hydroxyapatite crystal deposition. The organic matrix also determines the dentin formation and the proteins are basic materials for mineral formation. The organic matrix is mainly consisted of collagen, only 10% is noncollagen, which plays the roles as template and regulation in biological mineralization. The noncollagen proteins'researches mainly focus on single protein's bio-characteristic. This article applied tissue protein extraction method to get Dentin Matrix Protein Components (DMPCs) from dentin proteins, and examined the protein activity by stimulated hDPSC growth in vitro. 1 Extraction and purification of dentin matrix proteinDentin proteins were extracted from developing porcine molars using sequential 4M guanidine and 4M guanidine/0.5M EDTA extractions, marked separately then concentrated for next step. Purify the components according to liquid phase chromatography. After dialysis of proteins by PBS, DMPCs were concentrated and stored in 4℃.The components were analyzed by SDS-PAGE. Based on the relation between the relative molecular mass (RMM) and the relative mobility, most of the compoments were 96, 38, 20Kda, close to the RMM of ordinary DSP, DPP. The result shows that the extract method with EDTA can get out several different noncollagen proteins, and the RMM could confirm it is a stable way to extract DMPCs.2 The effect of hDPSC growth stimulated by DMPCsSet up 4 testing groups and 1 control group for the test of protein activity. In testing groups, make the end concentrations of DMPCs 50, 100, 150, 200μg/ml in culture medium, while in the control group 2% FCS was used as a control in culture medium. Then analyze the protein activity of cultured human Dental Pulp Stem Cells (hDPSC) by MTT and alkaline phosphatase (ALP). The results came out as follows:In MTT, compared with control group, in testing groups, 100 and 150μg/ml in 3d significantly stimulated the cell proliferation (P <0.05). ALP activity was increased by the components at 100μg/ml in 1d, 100 and 150μg/ml in 3d compared with control (P <0.05). So we can figure out that dentin matrix components extracted by guanidine and EDTA could stimulate the growth and increase ALP activity of hDPSC. It can be concluded DMPCs'function to hDPSC is progressive from linear relationship analysis, which means DMPCs could promote dentinogenesis and let dental pulp stem cells become into Odontoblast-like cells.