Seven aliquots (1 g each) of demineralized dentin powder were obtained and subjected to the above-mentioned biomodification. Then, specimens in each group were incubated in 1 mL of Adper Single Bond 2 (3M ESPE, St. Paul, MN, USA) for 24 h at 4 °C in the dark, thoroughly rinsed with acetone and repeatedly centrifuged (4 000g for 10 min each) at 4 °C. Enzyme extraction and zymographic analyses were carried out as previously described4 (link),21 (link) to evaluate the effect of PA on the gelatinolytic activity of host-derived MMPs on dentin.
Briefly, the specimens were suspended in the extraction buffer (50 mmol⋅L−l Tris-HCl, pH 6.0) to ultrasonically extract the enzyme proteins. The supernatants were collected by centrifugation. Then, proteins were precipitated at 4 °C by adding powdered ammonium sulphate, redissolved and further dialysed through a 30-kDa membrane overnight. Total protein concentrations in demineralized dentin powder extracts were determined by Bradford assay (Beyotime Biology, Haimen, China). Dentin proteins were electrophoresed under non-reducing conditions on 7.5% sodium dodecyl sulfate (SDS)–polyacrylamide gels copolymerized with 2 g⋅L−1 gelatin (GMS30071.1; Gemend, Shanghai, China). Gels were stained in 0.2% Coomassie Brilliant Blue R-250 and destained. Wet gelatine zymograms were scanned using an EagleEye II imaging system (Stratagene, Santa Clara, CA, USA).
Briefly, the specimens were suspended in the extraction buffer (50 mmol⋅L−l Tris-HCl, pH 6.0) to ultrasonically extract the enzyme proteins. The supernatants were collected by centrifugation. Then, proteins were precipitated at 4 °C by adding powdered ammonium sulphate, redissolved and further dialysed through a 30-kDa membrane overnight. Total protein concentrations in demineralized dentin powder extracts were determined by Bradford assay (Beyotime Biology, Haimen, China). Dentin proteins were electrophoresed under non-reducing conditions on 7.5% sodium dodecyl sulfate (SDS)–polyacrylamide gels copolymerized with 2 g⋅L−1 gelatin (GMS30071.1; Gemend, Shanghai, China). Gels were stained in 0.2% Coomassie Brilliant Blue R-250 and destained. Wet gelatine zymograms were scanned using an EagleEye II imaging system (Stratagene, Santa Clara, CA, USA).
