RESUMO
Organotypic cultures of embryonic mouse tooth germs were used to investigate the developmental expression and roles of MMPs in the formation and mineralization of dentin and enamel matrices. The spatially and temporally regulated expression of MMP-2, MMP-9 and MMP-20 in developing mouse tooth germs in vivo was maintained in culture. The inhibition of metalloproteinases activity by marimastat altered the morphogenesis and mineralization of the tooth germs associated with an inhibition of the activation of both MMP-20 and MMP-2. MMP inhibition deregulated the molecular processing of two major dental matrix proteins, amelogenin and dentin sialoprotein (DSP). This coincided with their accumulation and the loss of their normal distribution within the extracellular matrix, resulting in a defective mineralization of dentin and enamel matrices. These findings demonstrate the critical role of MMPs in the processing and maturation of the dental matrix.
Assuntos
Calcificação Fisiológica/fisiologia , Inibidores Enzimáticos/farmacologia , Metaloproteinases da Matriz/metabolismo , Dente Molar/embriologia , Dente Molar/enzimologia , Odontogênese/fisiologia , Amelogênese/fisiologia , Amelogenina , Animais , Esmalte Dentário/citologia , Esmalte Dentário/embriologia , Esmalte Dentário/metabolismo , Proteínas do Esmalte Dentário/metabolismo , Dentina/citologia , Dentina/embriologia , Dentina/enzimologia , Dentinogênese/fisiologia , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular , Feminino , Ácidos Hidroxâmicos/farmacologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 20 da Matriz , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Camundongos , Dente Molar/citologia , Técnicas de Cultura de Órgãos , Fosfoproteínas , Precursores de Proteínas , Sialoglicoproteínas/metabolismoRESUMO
In order to study the involvement of matrix metalloproteinases (MMPs) on dentin formation and mineralization, day 18 embryonic mouse tooth germs were cultured for 10 d in the presence or absence of Marimastat, a general MMP inhibitor, or CT(1166), a more selective inhibitor of gelatinases (MMP-2 and MMP-9) and stromelysin-1 (MMP-3). With Marimastat a dose-dependent increase in thickness of the predentin layer and a decreased mineralization of dentin were observed. At the highest concentration of the inhibitor used, enamel formation had ceased. With CT(1166), these effects were already apparent at the lowest concentration used. Western blot analyses demonstrated that the two inhibitors inhibited the expression of enamelysin (MMP-20). These observations indicate that MMPs (possibly MMP-2, -3, -9 and/or -20) play a role in the onset of dentin mineralization. The lack of enamel formation was possibly due to diffusion of amelogenin from its normal site of apposition. The protein clearly was not retained at the surface of the non-mineralized dentin layer, and immunopositive amelogenin accumulated in the odontoblast compartment. The diffusion of enamel proteins and the accumulation revealed by immunolabeling of two small leucine-rich proteoglycans, decorin and biglycan, in the predentin may have contributed to impaired dentin mineralization.