Extraction and Characterization of Dentin Matrix Proteins From Human and Bovine Teeth

Objectives: The aim of this study was to extract and evaluate the presence of dentin matrix proteins from human and bovine teeth.
Methods: Enamel and pulp tissue were removed from 10 human teeth and 5 healthy bovine teeth. The dentin was sectioned, and the sections grounded in a cryogenic mill (CryoMill, Retsch GmbH, Germany). Subsequently, the pulverized dentin was solubilized in 10% EDTA (pH 7.2) containing the phenylmethyl sulfonyl fluoride (5mM) protease inhibitors and the irreversible cystine inhibitor (10mM N-ethylmalemide) at 4 °C. The dentin-EDTA mixture was centrifuged at 827 RCF (xg) for 10 minutes, and the supernatant was dialyzed and lyophilized. A 12% polyacrylamide gel electrophoresis was performed with different concentrations (3%, 1.5%, 0.75%) of the lyophilized extracts diluted in PBS (Sigma Aldrich, USA). After application of the Sample Buffer (TruPageTM LDS Sample Buffer, Sigma Aldrich, USA), denaturation was performed at 95 °C for 10min before electrophoresis at 150V and 45mA for 1h30min in the Mini-PROTEAN® Electrophoresis System (Bio-Rad, USA). The ProteoMassTM Peptide and Protein MALDI-MS Calibration Kit (Sigma-Aldrich, USA) was used for identification of peptide sequences by mass spectrometry.
Results: Electrophoresis showed the presence of proteins in the 20-35 kDa range. The possible proteins present in the extract having molecular weight corresponding to that obtained from polyacrylamide gel, such as FGF-2, BMP- 2, BMP-4, BMP-7, VEGF, IGF-1, PDGF -A, BDNF, TGF-β2, TGF-β3.
Conclusions: It was possible to extract and characterize a wide range of bioactive dentin proteins in healthy human and bovine teeth.