Analysis of the action of catechins derived from green tea in erosively demineralized human dentine: in vitro and in situ studies

• Main Author: Maria Denise Rodrigues de Moraes Bezerra
• Other Authors: SÃrgio Lima Santiago
• Format: Others
• Language: Portuguese
• Published: Universidade Federal do Cearà 2016
• Subjects: ODONTOLOGIA
• Online Access: http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=18736

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior === O presente estudo avaliou a aÃÃo de catequinas do chà verde em dentina humana sob situaÃÃo de desafio erosivo e originou trÃs capÃtulos. Nos dois primeiros, blocos de dentina humana (n=10) coronÃria ou radicular (4x4x2mm) foram imersos em saliva (2h) para formaÃÃo de pelÃcula adquirida. Foram submetidos de forma cÃclica (3x/dia-3 dias) ao desafio erosivo pela imersÃo em Ãcido cÃtrico (60s), seguido pelos tratamentos: G1-Ãgua destilada (AD), G2-SoluÃÃo de gluconato de clorexidina 0,12% (CLX), G3-InfusÃo de chà verde (CV). Os espÃcimens permaneciam em saliva artificial entre os desafios e sob agitaÃÃo (37ÂC). A microdureza de superfÃcie e perfilometria dos blocos foram analisadas diariamente. O estudo in situ foi randomizado, cego, cruzado e 20 voluntÃrios utilizaram dispositivo intra-oral, contendo 4 blocos de dentina humana, por trÃs fases de 5 dias cada. Foi realizada imersÃo extra-oral do dispositivo em 50 mL de Coca-Cola (4x/dia-1min), seguida por gotejamento (1mL, 4x/dia) sobre os blocos: G1-soluÃÃo de fluoreto de sÃdio (NaF) 0,05%, G2-epigalocatequina-3-galato (EGCG) 0,1%, G3-CV. Foram realizadas anÃlises de microdureza de superfÃcie, perfilometria e microscopia eletrÃnica de varredura. Para anÃlise da inibiÃÃo enzimÃtica, blocos de dentina humana foram congelados, moÃdos, submetidos à erosÃo por Ãcido cÃtrico (24h) e extraÃÃo de proteÃnas solÃveis. O pool de proteÃnas extraÃdo foi utilizado como fonte de metaloproteinases (MMPs), foi tratado: G1-sem tratamento, G2-CLX 0,12%, G3-NaF 0,05%, G4-CV, G5-EGCG 0,1% e foi submetido ao ensaio colorimÃtrico de inibiÃÃo enzimÃtica. Foi realizada a eletroforese das enzimas extraÃdas em gel de acrilamida sob condiÃÃes nÃo redutoras. O gel foi encubado por 3h em tampÃo de renaturaÃÃo modificado pelo acrÃscimo das soluÃÃes: G1-sem modificaÃÃo, G2-CLX 0,12%, G3-NaF 0,05%, G4-CV e G5-EGCG 0,1%, e corado em soluÃÃo contendo 0,025% Comassie blue e descorado com soluÃÃo de Ãcido acÃtico (10%). O padrÃo de normalidade dos dados foi analisado pelo teste de Kolmogorov-Smirnov, seguido por AnÃlise de variÃncia e teste de Tukey (&#945;=5%). Foi observada reduÃÃo na perda de dureza da dentina coronÃria in vitro com o uso de CLX e CV em comparaÃÃo com o controle. O tratamento com CV reduziu significativamente o desgaste e a rugosidade da dentina coronÃria in vitro. O tratamento in vitro com CV reduziu estatisticamente o desgaste e a rugosidade da dentina radicular quando comparado ao controle e à CLX. NÃo houve diferenÃa estatisticamente significante entre os grupos em relaÃÃo à perda de dureza de superfÃcie de dentina radicular in vitro. Quanto ao estudo in situ, os tratamentos com EGCG e CV reduziram a perda de dureza da dentina significativamente. Por outro lado, nÃo houve diferenÃa estatÃstica nos valores de desgaste e rugosidade. A zimografia demonstrou que a CLX 0,12%, o CV e o EGCG 0,1% apresentaram aÃÃo inibitÃria sobre as MMPs, enquanto que no ensaio colorimÃtrico o CV apresentou inibiÃÃo enzimÃtica. A soluÃÃo de CV reduz in vitro o desgaste e a rugosidade da dentina coronÃria e radicular erosivamente desmineralizada. O CV e a EGCG 0,1% reduzem os danos causados pela erosÃo em dentina coronÃria in situ e contribuem para inativaÃÃo de MMPs extraÃdas da dentina. === The objective of this study was to evaluate and compare the action of different enzyme inhibitors on human dentin in cyclical erosion challenging situation. To this were carried out 3 projects that yielded the following chapters: In the chapter 1 and 2, coronary or root human (n=10) dentin blocks (4x4x2 mm) were submitted cyclically (3x/ day/3 days) to erosive challenge by immersion in acid [dehydrated citric acid (C6H8O7), pH 3.75, 60 s] followed by treatments depending on the group: G1-distilled water; G2- 0.12% Chlorhexidine digluconate (CHX); G3- Green tea infusion (GT). The blocks were analyzed daily by surface profilometry and hardness. In the chapter 3, a randomized, blinded, crossover, in situ study in which 20 volunteers used palatal intra oral device for three phases of 5 days each, containing 4 blocks of human dentin. They were immersed in human saliva for 2 hours to acquired pellicle formation. The erosive challenge was performed by the device immersion in 50 mL of Cokeâ (pH 2.6, 4x/ day /1 min, extraorally) followed by treatment (4x/ day) by dropping on blocks 1 mL of the following solutions: G1- 0.05% Sodium fluoride, G2- 0.1% Epigallocatechin-3-gallate (EGCG), G3- Green tea infusion. At each phase the volunteer used only one substance. Quantitative analyzes were performed, such as percentage loss of surface hardness, roughness and wear, as well as qualitative analysis by scanning electron microscopy. Complementing the studies to analysis of enzyme inhibition, human dentin blocks were milled, subjected to erosion by citric acid and subjected to extraction of soluble proteins. Electrophoresis was performed and then the gel was incubated for 3 h in renaturation buffer modified according to the groups G1: without modification, G2- 0.12% chlorhexidine digluconate, G3- 0.05% NaF, G4- Green tea and G5- 0.1% EGCG. Gels were stained in a solution containing 0.025% Coomassie blue and destained with acetic acid 10% solution to verify the possible inhibitory action of the substances evaluated on the collagenolytic enzymes. Then were subjected to colorimetric enzyme inhibition test, in which the absorbance was measured. After extraction of dentin proteins, protein quantification by Bradfordâs method was performed, which showed 0.15 Âg soluble proteins. The results were analyzed using the Kolmogorov-Smirnov test to evaluate the normal range of results, followed by analysis of variance (ANOVA) and Tukey test, using a significance level of 5%. Results: A significant reduction in the dentin hardness loss in coronary dentin in vitro with the use of CHX and GT in comparison to the control (p <0.05). Treatment with GT significantly reduced wear and roughness of dentin in vitro (p <0.05). In relation to in vitro study on root dentin, the treatment with GT reduced the wear and roughness (p <0.05). There was no statistically significant difference between the groups regarding the loss of surface hardness of root dentin in vitro (p> 0.05). As for the in situ study, treatment with EGCG and GT reduced the loss of dentin hardness significantly (p <0.05). On the other hand, there was no significant difference in relation to wear and roughness values (p> 0.05). For zymography analysis, 0.12% CHX, green tea and 0.1% EGCG showed inhibitory action on the extracted metalloproteinases dentin and, the colorimetric assay green tea has enzimatic inhibition similar to standard inhibitor. Conclusion: Green tea solution reduces in vitro wear and roughness of coronary and root dentin erosively demineralized. The 0.1% EGCG and green tea reduce erosion damage on coronal dentin in situ and contribute to inactivate MMPs extracted dentin.