MMPs Extraction Assays in Healthy and Carious Dentin

Objectives: Degradation of dentin extracellular matrix (ECM) and caries lesion progression has been attributed to increased activity of endogenous enzymes like matrix metalloproteases (MMPs). The validation and standardization of reproducible protein extraction protocols will stimulate further research to better understand the roles of MMPs in specific mechanisms of ECM degradation. This study compared and optimized in vitro extraction protocols for MMPs and investigated the proteolytic profiles of healthy and carious dentin.
Methods: Dentin powder obtained from human healthy and carious molar teeth underwent protein extraction via three different protocols that included incubations with: 4 M Guanidine-hydrochloride (HCl) followed by 0.5M EDTA and a second incubation with guanidine-HCl pH 7.4 (protocol G-EDTA); 1% phosphoric acid demineralization and incubation with buffer containing 50 mM Tris-HCl, 5 mM CaCl₂, 100 mM NaCl, 0.1% Triton X-100, 0.1% nonionic detergent P-40, 0.1 mM ZnCl₂ and 0.02% NaN₃ pH 6.0 (protocol PA); or 50 mM Tris-HCl (pH 7.5) containing 4M guanidine-HCl and 0.5 M EDTA pH 7.4 (protocol E). Experiments were run in triplicate. The amount of total protein extracted and the gelatinolytic activity was compared among the protocols and in healthy versus caries dentin. The dentin MMP-2 and -9 activities were quantified using ImageJ and expressed in arbitrary units according to the activity of recombinant MMPs.
Results: The total protein content extracted from healthy dentin was higher for G-EDTA protocol, followed by E and PA. However, protocols G-EDTA and PA were more effective in extracting active gelatinases. Initial results have shown increased gelatinolytic activity/µg of total protein extracted with guanidine incubation and PA. Preliminary findings have shown higher levels of gelatinolytic activity in carious versus healthy dentin.
Conclusions: Dentinal active MMPs are efficiently extracted using G-EDTA and PA protocols. Increased activity of MMPs in carious than healthy tissue supports the role of MMPs in caries progression.