Search

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) It has been hypothesized that cysteine cathepsins (CTs) along with matrix metalloproteases (MMPs) may work in conjunction in the proteolysis of mature dentin matrix. The aim of this study was to verify simultaneously the distribution and presence of cathepsins B (CT-B) and K (CT-K) in partially demineralized dentin; and further to evaluate the activity of CTs and MMPs in the same tissue. The distribution of CT-B and CT-K in sound human dentin was assessed by immunohistochemistry. A double-immunolabeling technique was used to identify, at once, the occurrence of those enzymes in dentin. Activities of CTs and MMPs in dentin extracts were evaluated spectrofluorometrically. In addition, in situ gelatinolytic activity of dentin was assayed by zymography. The results revealed the distribution of CT-B and CT-K along the dentin organic matrix and also indicated co-occurrence of MMPs and CTs in that tissue. The enzyme kinetics studies showed proteolytic activity in dentin extracts for both classes of proteases. Furthermore, it was observed that, at least for sound human dentin matrices, the activity of MMPs seems to be predominant over the CTs one. Univ Estadual Campinas, Fac Dent Piracicaba, BR-13414903 Piracicaba, Brazil Univ Sao Paulo, Bauru Sch Dent, Dept Biol Sci, BR-17012901 Bauru, SP, Brazil Univ Bologna, Dept Biomed & Neuromotor Sci, I-40126 Bologna, Italy Univ Trieste, Dept Med Sci, I-34128 Trieste, Italy Univ Iowa, Coll Dent, Dept Operat Dent, 801 Newton Rd, Iowa City, IA 52246 USA CNR, IGM, Unit Bologna, IOR, I-40136 Bologna, Italy Univ Fed Ceara, Dept Pharm Dent & Nursing, BR-60020181 Fortaleza, Ceara, Brazil Univ Carlo Bo, Dept SteVA, I-61029 Urbino, Italy Georgia Hlth Sci Univ, Dent Coll Georgia, Oral Biol, Augusta, GA 30912 USA Univ Oulu, Inst Dent, FI-90014 Oulu, Finland Oulu Univ Hosp, Med Res Ctr Oulu, FI-90014 Oulu, Finland Univ Oulu, FI-90014 Oulu, Finland Univ Helsinki, Dept Oral & Maxillofacial Dis, FI-00014 Helsinki, Finland Helsinki Univ Hosp, FI-00014 Helsinki, Finland Univ Fed Sao Paulo, Dept Biochem, BR-04021001 Sao Paulo, Brazil Univ Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, BR-08773520 Mogi Das Cruzes, Brazil Anhanguera Univ Sao Paulo UNIAN SP, Biomat Dent Program, BR-05145200 Sao Paulo, Brazil Anhanguera Univ Sao Paulo UNIAN SP, Biotechnol & Innovat Hlth Program, BR-05145200 Sao Paulo, Brazil Univ Fed Sao Paulo, Dept Biochem, BR-04021001 Sao Paulo, Brazil FAPESP: 2007/54618-4 FAPESP: 2009/14005-9 CNPq: 312768/2013-3 Web of Science

Orientadores: Fernanda Miori Pascon, Marcelo Giannini Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba Resumo: Os objetivos nesta tese foram: (1) investigar o efeito de FeCl3 na atividade da Catepsina K (CT-K) e capacidade de prevenir degradação do colágeno solúvel (CS) e insolúvel (CI); (2) comparar os efeitos do ácido fosfórico a 35% (AF) e soluções condicionantes contendo ácido cítrico (AC) e FeCl3 na resistência da união a dentina e degradação do colágeno. A tese foi apresentada em dois artigos: (1) para avaliar a atividade enzimática da CT-K e efeito de FeCl3 utilizou-se a espectrofluorimetria. Para avaliar a degradação do CS e CI (fibras de tendões de cauda de camundongo - FTCCs), os substratos foram incubados com CT-K e concentrações de 0,02% a 2% de FeCl3. O sobrenadante foi submetido à eletroforese de proteínas. Os experimentos foram feitos em triplicata. As FTCCs foram caracterizadas em microscopia eletrônica de varredura (MEV). A quantidade de Fe absorvida foi determinada pela espectroscopia de energia dispersiva de raios-X (EDX). Os dados foram submetidos à análise de variância (Brown-Forsythe), normalidade (Shapiro-Wilk ou Anderson-Darling), ANOVA um fator + Holm-Sidak ou Tukey (?=5%). (2) para avaliar a resistência da união a microtração (?TBS), 78 dentes foram distribuídos em 13 grupos de soluções contendo AC e/ou FeCl3 nas concentrações: 10%-1,8%, 10%-0,6%, 10% AC, 5%-1,8%, 5%-0,6%, 5% AC, 1%-1,8%, 1%-0,6%, 1% AC, 1,8% FeCl3, 0,6% FeCl3 (aplicadas por 15 segundos) e com AF (aplicado por 5 ou 15 segundos). Utilizou-se o sistema adesivo Adper Scotchbond Multi-Purpose, fotoativado por 20 segundos com o aparelho Bluephase G2 (1200Mw/cm2). A ?TBS foi avaliada em 24 horas e 9 meses de armazenamento. Para a degradação do colágeno dentinário, fatias de dentina (n=3) foram tratadas com e sem FeCl3 e incubadas com CT-K. O sobrenadante foi submetido à eletroforese de proteínas. A dentina foi caracterizada após a incubação utilizando MEV. Outras fatias foram submetidas às soluções condicionantes por 15 segundos e analisadas utilizando EDX. Os dados foram submetidos aos testes: normalidade, ANOVA dois fatores + Holm-Sidak, ANOVA um fator + Tukey ou Fisher (?=5%). A análise por espectrofluorimetria revelou que o FeCl3 inibiu a atividade de CT-K nas concentrações 0,005%-0,08%e suprimiu a atividade na concentração 0,08%. FeCl3 foi capaz de prevenir a degradação do CS e CI contra CT-K para as FTCCs e para a dentina desmineralizada. As micrografias revelaram desenovelamento das FTCCs na ausência de FeCl3 e integridade quando expostas a concentrações de 0,02%-0,8%de FeCl3. Observou-se interação Fe/colágeno. Os grupos 5-0,6 e 5-1,8 de AC/FeCl3 apresentaram maiores valores de ?TBS comparados ao AF, independentemente do tempo de armazenamento. Observou-se redução da ?TBS aos 9 meses para todos os grupos. A solução contendo 1,8% de FeCl3 preservou a estrutura da dentina. Observou-se pela análise de EDX que a imersão nas soluções condicionantes com FeCl3 por 15 segundos foi suficiente para que as fibrilas de colágeno adsorvessem Fe. Concluiu-se que o FeCl3 agiu como um indutor de reticulação do colágeno e como inibidor da atividade CT-K e estas duas habilidades atuaram em conjunto para a preservação do colágeno tipo I nas FTCCs e na dentina desmineralizada Abstract: The aim of this thesis was (1) to investigate the effect of FeCl3 on Cathepsin K (CT-K) activity of and the ability to prevent degradation of soluble (SC) and insoluble collagen (IC), and (2) to compare the effects of phosphoric acid 35% and conditioning solutions containing citric acid (CA) and/or FeCl3 in dentine bond strength and collagen degradation. The thesis was presented in two articles: (1) spectrofluorimetry was used to evaluate the FeCl3 effect against CT-K enzymatic activity. To evaluate SC and IC (mouse tail tendon fibers-MTTF) degradation, the substrates were incubating with CT-K with different concentrations of FeCl3 (0.02% to 2%). The supernatant was submitted to protein electrophoresis. The experiments were done in triplicate. The fibers were characterized after incubation by scanning electron microscopy (SEM). The amount of Fe absorbed was determined by X-ray dispersive energy spectroscopy (EDX). Data were submitted to analysis of variance (Brown-Forsythe), normality test (Shapiro-Wilk or Anderson-Darling), and one-way ANOVA (Holm-Sidak or Tukey post-test) (?=5%). (2) for the microtensile bond strength (?TBS), 78 teeth were distributed into 13 groups of solutions containing citric acid (AC) and/or FeCl3 in different concentrations: 10%-1,8%, 10%-0,6%, 10% AC, 5%-1,8%, 5%-0,6%, 5% AC, 1%-1,8%, 1%-0,6%, 1% AC, 1,8% FeCl3, 0,6% FeCl3 (applied for 15 seconds) e com AF (applied for 5 and 15 seconds). The Adper Scotchbond Multi-Purpose adhesive system was used and light/cured for 20 seconds with Bluephase G2 (1200Mw/cm2). The ?TBS was evaluated at 24 hours and 9 months of storage. For the dentine collagen degradation, dentine slices (n=3) were treated with and without FeCl3 were incubated with CT-K. The supernatant was submitted to protein electrophoresis. Dentine was characterized after enzymatic treatment by SEM, and other slices were submitted to conditioning solutions and analyzed by EDX. The data were submitted to the tests: normality, two¿way ANOVA and post-test Holm-Sidak, one¿way ANOVA and Tukey or Fisher tests (?=5%). Spectrofluorimetry analysis revealed that FeCl3 inhibited CT-K activity at concentrations 0.005%-0.08% and suppressed activity when using 0.08%. FeCl3 was able to prevent the degradation of soluble collagen and collagen fibers against CT-K for the MTTF and the demineralized dentine. The micrographs revealed unraveling of collagen fibers in the absence of FeCl3 and showed fiber integrity exposed to 0.02%-2% FeCl3 concentrations. The Fe/collagen interaction was observed. AC/FeCl3 5-0.6 and 5-1.8 solutions had higher ?TBS values ??compared to the AF, regardless of storage time. A reduction of ?TBS was observed at 9 months for all groups. The treatment solution 1.8% FeCl3 preserved the dentine structure. The EDX analysis indicated that the 15 second immersion in the FeCl3 conditioning solutions was sufficient for the collagen fibrils to adsorb Fe. It was concluded that FeCl3 acted both as an inducer of collagen cross-linking and as inhibitor of CT-K activity, both abilities acted together for the preservation of collagen type I on the MTTF and on the demineralized dentine Doutorado Materiais Dentários Doutora em Materiais Dentários CAPES 99999.010711/2014-07, 1777-2014