Análise da atividade proteolítica das MMPS -2 e -9 em dentina humana sadia e sua interação na interface adesiva

Abstract

MMPs are enzymes that can degrade exposed collagen in dentin/resin interface causing serious damage to maintaining the integrity of the adhesive interface. The present study is divided in three chapters, whose aims: 1) To evaluate the effect of demineralization by different acid solutions in MMP-2 activation on human dentin (Chapter 1); 2) To evaluate the effect of a one-step adhesive system on dentinal MMP-2 and -9 activation using in situ zymography and an enzymatic activity assay (Chapter 2); and 3) To evaluate the ability of MMPs inhibition by 1-ethyl-3-(3- dimethylaminopropryl) carbodiimide (EDC) EDC cross-linker on dentin (Chapter 3). As for the methodology approaches, 3 in vitro studies were performed to evaluate enzymatic expression of human dentin. Dentin protein was extracted after demineralization by 1% phosphoric acid, 10% phosphoric acid and 10% citric acid for 1, 5 and 10 minutes, and subjected to gelatin zymographic and activity assay (Chapter 1). Dentin powder/slabs were treated with one-step adhesive Adper Easy Bond (3M ESPE) and MMP-2 and -9 activities were evaluated using in situ zymography and quantified by means of an specific enzymatic assay (Chapter 2). Dentin powder/slabs were treated with one of those adhesive systems: Optibond FL or Scotchbond 1XT with or without pre-treatment using EDC. The enzymatic activity was analyzed using gelatin zymography and in situ zymography (Chapter 3). MMP-2 activity was present in all tested groups and increased after demineralization by 10% phosphoric acid and 10% citric acid (Chapter 1). After treated with one-step adhesive, enzymatic activity increased. In situ evaluation showed that MMPs action is associated with the exposed and unprotected collagen promoted by adhesive monomers (Chapter 2). Zymograms revealed increased expression of dentin endogenous MMP-2 and -9 after adhesives systems application, while the use of 0.3M EDC as a primer, inactivated dentin gelatinizes (Chapter 3). It can be concluded that both acid solutions (Chapter 1) and self-etch adhesives (Chapter 2) are able to activate dentin MMPs and that solutions like EDC cross-linker can inhibit the activation of those enzymes (Chapter 3).