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Study of the proteolytic activity of human sound and carious dentin

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Author(s):
Cristina de Mattos Pimenta Vidal
Total Authors: 1
Document type: Doctoral Thesis
Press: Piracicaba, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
Defense date:
Examining board members:
Marcela Rocha de Oliveira Carrilho; Carlos Eduardo Francci; Ivarne Luis Santos Tersariol; Marcelo Giannini; Roberta Caroline Bruschi Alonso
Advisor: Marcela Rocha de Oliveira Carrilho
Abstract

There are just over 15 years since the matrix metaloproteases (MMPs) were considered responsible for the degradation of dentin organic matrix in caries progression. From then on, only few studies were published based on this premise. More recently, it was showed that other proteases, like the cysteine-cathepsins (CTs), could also participate of such degradation process. The objective of this study was to evaluate and compare the abundance and proteolytic activity of different MMPs (MMP-2, -8 and -9) and CTs (B, K e L) in sound and carious dentin. Firstly, the abundance and localization of enzymes in sound and carious dentin was performed by conventional immunohistochemistry. In addition, MMP-2, -9 and CTs B and K abundance associated with evaluation of collagen structure in sound and carious dentin was done by immunofluorescence. Different dentin enzyme-extraction methods were also tested: 1) using guanidine-hydrochloride associated with EDTA (G-EDTA); 2) phosphoric acid (AF); 3) acetic acid and 4) guanidine-hydrochloride associated with acetic acid (G-AC). The presence of MMP-2, -8 e -9 and CTs B and K was evaluated by western blot. Proteolytic activity in dentin extracts was analyzed by zymography and spectrofluorometrically. The organic matrix solubilization in sound and carious dentin was evaluated by hydroxyproline assay (HYP). Immunohistochemistry showed the presence of MMP-2, -8 and -9 and CTs B, K and L, especially in deep dentin and predentin, showing more abundance in caries. The same results were observed in immunofluorescence, which also showed that the collagen structure was modified in carious dentin. The presence of MMP-2, -8 and -9 and CTs B and K was showed in western blot in all extraction protocols evaluated, with more abundance in carious when compared to sound dentin. Gelatinolytic activity corresponding to MMP-2 was showed in zymography, but it was different according to the extraction protocol. Proteolytic activity for MMPs and CTs was observed spectrofluorometrically in sound dentin and some variation was observed according to the extraction protocol. Dentin organic matrix solubilization was confirmed by HYP, with higher HYP released in AC extraction protocol. When comparing sound and carious dentin, organic matrix degradation was higher in caries. It can be concluded that, besides MMP-2, -8 and -9, the CTs B, K and L are also present in sound and carious dentin, with higher abundance in caries. However, the presence and activity may vary according to the extraction method used. The results indicate that both MMPs and CTs may act in concert in dentin organic matrix degradation in caries progression and also support the physiopathology theory for caries in which a host-derived proteolytic mechanism would be responsible for dentin degradation (AU)

FAPESP's process: 09/13652-0 - Proteolytic Activity of Sound and Caries-Affected Human Dentin.
Grantee:Cristina de Mattos Pimenta Vidal
Support Opportunities: Scholarships in Brazil - Doctorate