Assaying Endogenous MMP-2,-8 in Acid-etched Dentinal Cavity Walls
Location
Philadelphia Campus
Start Date
7-5-2014 1:00 PM
Description
Objective: Inhibition of matrix metalloproteinase (MMP) activity is expected to increase long-term stability and durability of resin-based restorations by preventing degradation of denuded collagen fibrils in the hybrid layer (HL). In order to better understand the role of endogenous MMP activity in HL degradation, quantification and assessment of MMPs and their activity are imperative. The objective of this study was to quantify MMP-2,-8 and to assess endogenous activity of MMP-8 in a single-tooth sample containing dentinal cavity walls. Methods: Occlusal surfaces of 22 extracted non-carious human molar teeth (-80ºC) were removed using low-speed diamond saw. A cavity (2x4x2mm in dimension) was prepared on dentinal surface, leaving surrounding dentinal walls of 1mm thickness. Cavity walls were then acid-etched with 38% phosphoric acid, and each sample was pulverized using analytic mill with liquid nitrogen, demineralized in 1% phosphoric acid, suspended at 1:2 ratio in extraction buffer (100mM Tris-HCl pH 7.6, 0.1mM ZnCl2, 100mM CaCl2, 200mM NaCl, 1% Triton X-100) containing protease inhibitor cocktail, and centrifugally concentrated approximately 2x.Total protein concentration was determined via Modified Lowry assay. MMP-2,-8 concentrations were determined via ELISA and endogenous MMP-8 activity by release of fluorescent substrate. Results: Molar1 Molar2 Molar3 Molar4 Average MMP-8 (pg/mg dentin) 0.179 0.465 0.1036 0.2036 0.238 ± 0.157 MMP-2 (pg/mg dentin) 12.85 7.26 3.27 - 7.79 MMP-8 activity (µM fluorescence) 2.1988 2.7494 1.4895 2.5841 2.255 ± 0.56 . Zymogram of dentin samples displayed an intense band at 72 kDa, corresponding to pro-form form of MMP-2. Conclusion: A reliable and consistent method of extracting MMPs from a single-tooth dentin sample has allowed for quantification and assessment of endogenous activity of MMP-2,-8 within dentinal cavity walls. This method will contribute to the growing body of knowledge regarding the role of endogenous MMP activity and its inhibition, which has the potential to prolong longevity of resin-based restorations.
Assaying Endogenous MMP-2,-8 in Acid-etched Dentinal Cavity Walls
Philadelphia Campus
Objective: Inhibition of matrix metalloproteinase (MMP) activity is expected to increase long-term stability and durability of resin-based restorations by preventing degradation of denuded collagen fibrils in the hybrid layer (HL). In order to better understand the role of endogenous MMP activity in HL degradation, quantification and assessment of MMPs and their activity are imperative. The objective of this study was to quantify MMP-2,-8 and to assess endogenous activity of MMP-8 in a single-tooth sample containing dentinal cavity walls. Methods: Occlusal surfaces of 22 extracted non-carious human molar teeth (-80ºC) were removed using low-speed diamond saw. A cavity (2x4x2mm in dimension) was prepared on dentinal surface, leaving surrounding dentinal walls of 1mm thickness. Cavity walls were then acid-etched with 38% phosphoric acid, and each sample was pulverized using analytic mill with liquid nitrogen, demineralized in 1% phosphoric acid, suspended at 1:2 ratio in extraction buffer (100mM Tris-HCl pH 7.6, 0.1mM ZnCl2, 100mM CaCl2, 200mM NaCl, 1% Triton X-100) containing protease inhibitor cocktail, and centrifugally concentrated approximately 2x.Total protein concentration was determined via Modified Lowry assay. MMP-2,-8 concentrations were determined via ELISA and endogenous MMP-8 activity by release of fluorescent substrate. Results: Molar1 Molar2 Molar3 Molar4 Average MMP-8 (pg/mg dentin) 0.179 0.465 0.1036 0.2036 0.238 ± 0.157 MMP-2 (pg/mg dentin) 12.85 7.26 3.27 - 7.79 MMP-8 activity (µM fluorescence) 2.1988 2.7494 1.4895 2.5841 2.255 ± 0.56 . Zymogram of dentin samples displayed an intense band at 72 kDa, corresponding to pro-form form of MMP-2. Conclusion: A reliable and consistent method of extracting MMPs from a single-tooth dentin sample has allowed for quantification and assessment of endogenous activity of MMP-2,-8 within dentinal cavity walls. This method will contribute to the growing body of knowledge regarding the role of endogenous MMP activity and its inhibition, which has the potential to prolong longevity of resin-based restorations.