RESUMO
Matrix metalloproteinases (MMPs) in dentin are believed to participate in various physiological and pathological events in coronal dentin, but their exact source and location is not clear. The purpose of this study was to evaluate the activity of gelatinases in decalcified rat molars crowns by in situ zymography. Hemi-mandibles of five male Wistar rats were fixed in paraformaldehyde, decalcified in EDTA and glycerol solution and embedded in paraffin. Sections from the region of molar teeth were incubated with or without DQ gelatin in 50mM Tris-CaCl2 at 37°C for 2h and observed by means of confocal microscopy. Gelatinolytic activity was observed throughout the coronal dentin with varying intensities in different locations. High gelatinase activity was observed in the dentinal tubules, dentin-enamel junction (DEJ) and predentin, and it was weaker and less uniform in the intertubular dentin. This study shows that the location of gelatinase and relative activity can be detected by means of in situ zymography and confocal microcopy, and this methodology may provide a useful tool in studies on the role of gelatinases in tooth development, maturation and in pathological conditions.
Assuntos
Dentina/enzimologia , Gelatinases/metabolismo , Dente Molar/enzimologia , Desmineralização do Dente/enzimologia , Animais , Dentina/metabolismo , Ativação Enzimática , Masculino , Dente Molar/citologia , Dente Molar/metabolismo , Ratos , Ratos Wistar , Desmineralização do Dente/metabolismoRESUMO
This proof-of-concept study assessed whether the reduction of the degradation of the demineralized organic matrix (DOM) by pre-treatment with protease inhibitors (PI) is effective against dentin matrix loss. Bovine dentin slices were demineralized with 0.87 M citric acid, pH 2.3, for 36 hrs. In sequence, specimens were treated or not (UT, untreated) for 1 min with gels containing epigallocatechin 3-gallate (EGCG, 400 µM), chlorhexidine (CHX, 0.012%), FeSO(4) (1 mM), NaF (1.23%), or no active compound (P, placebo). Specimens were then stored in artificial saliva (5 days, 37°C) with the addition of collagenase (Clostridium histolyticum, 100 U/mL). We analyzed collagen degradation by assaying hydroxyproline (HYP) in the incubation solutions (n = 5) and evaluated the dentin matrix loss by profilometry (n = 12). Data were analyzed by ANOVA and Tukey's test (p < 0.05). Treatment with gels containing EGCG, CHX, or FeSO(4) led to significantly lower HYP concentrations in solution and dentin matrix loss when compared with the other treatments. These results strongly suggest that the preventive effects of the PI tested against dentin erosion are due to their ability to reduce the degradation of the DOM.
Assuntos
Cariostáticos/uso terapêutico , Colágeno/metabolismo , Colagenases/metabolismo , Dentina/metabolismo , Inibidores de Proteases/uso terapêutico , Erosão Dentária/prevenção & controle , Análise de Variância , Animais , Proteínas de Bactérias/metabolismo , Catequina/análogos & derivados , Catequina/uso terapêutico , Bovinos , Clorexidina/uso terapêutico , Dentina/ultraestrutura , Matriz Extracelular/metabolismo , Compostos Ferrosos/uso terapêutico , Fluoreto de Sódio/uso terapêutico , Desmineralização do Dente/enzimologia , Desmineralização do Dente/prevenção & controle , Erosão Dentária/enzimologiaRESUMO
Matrix metalloproteinases (MMPs) are present in sound coronal dentin and may play a role in collagen network degradation in bonded restorations. We investigated whether these enzymes can also be detected in root dentin. Crown and root sections of human teeth were powderized, and dentin proteins were extracted by using guanidine-HCl and EDTA. Extracts were analyzed by zymography and Western blotting for matrix metalloproteinases detection. Zymography revealed gelatinolytic activities in both crown and root dentin samples, corresponding to MMP-2 and MMP-9. MMP-2 was more evident in demineralized root dentin matrix, whereas MMP-9 was mostly extracted from the mineralized compartment of dentin and presented overall lower levels. Western blot analysis detected MMP-8 equally distributed in crown and root dentin. Because MMPs are also present in radicular dentin, their contribution to the degradation of resin-dentin bonds should be addressed in the development of restorative strategies for the root substrate.
Assuntos
Dentina/enzimologia , Metaloproteinases da Matriz/análise , Raiz Dentária/enzimologia , Adulto , Western Blotting , Corantes , Eletroforese em Gel de Poliacrilamida , Humanos , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 8 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , Peso Molecular , Coroa do Dente/enzimologia , Desmineralização do Dente/enzimologia , Adulto JovemRESUMO
This review focuses specifically on matrix metalloproteinases (MMPs) and their role in physiological and pathological extracellular matrix (ECM) remodeling and degradation processes in the oral environment. A group of enzymes capable of degrading almost all ECM proteins, MMPs contribute to both normal and pathological tissue remodeling. The expression of different MMPs may be upregulated in pathological conditions such as inflammation and tumor invasion. The balance between activated MMPs and tissue inhibitors of metalloproteinases (TIMPs) controls the extent of ECM remodeling. Prior to mineralization, MMPs may participate in the organization of enamel and dentin organic matrix, or they may regulate mineralization by controlling the proteoglycan turnover. There is evidence indicating that MMPs could be involved in the etiology of enamel fluorosis and amelogenesis imperfecta. They seem to play a part in dentinal caries progression, since they have a crucial role in dentin collagen breakdown in caries lesions. MMPs have been identified in pulpal and periapical inflammation and are strongly correlated with periodontal diseases, since they are the major players in collagen breakdown during periodontal tissue destruction. The use of MMP inhibitors could help the prevention and treatment of many MMP-related oral diseases.