Co-distribution of cysteine cathepsins and matrix metalloproteases in human dentin

It has been hypothesized that cysteine cathepsin's (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. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe

Estabilidade de uniÃo de diferentes sistemas adesivos aplicados à dentina

There is a general consensus that resin-dentin bonds created by contemporary hydrophilic dentin adhesives deteriorate over time. One way to predict how resin-dentin interface would behave over time is aging the specimens in vitro by immersion in different solutions. The objective of this study was to evaluate the effect of immersion in NaOCl solution on the bond interface created by different adhesive systems. For this, it was used 28 human third molars that had superficial dentin exposed and composite build-ups were built on its surface using one of those adhesives, etch-and-rinse: Scothbond Multi-purpose and Single Bond, or self-etch: Clearfil SE and Adper SE. Specimens were cut into nontrimming dentin-composite beams to microtensile testing. After a period of 24 h in distilled water, sticks from each tooth were divided equally into two groups: control or aged in 10% NaOCl for 1 h. Beams were pulled until failure at crosshead speed of 1 mm/min and bond strength was calculated. Fractured sticks were analyzed and classified in: mixed fracture, cohesive in dentin and cohesive in composite; and expressed in percentage. Data from ÂTBS test were statistically analyzed using Kruskal-Wallis and Games-Howell tests. For comparison between control and aging groups was used the Mann-Whitney test (α=0.05). Two specimens from each adhesive were used to investigate the effect of NaOCl on the interfacial morphological characteristics. For that, specimens were cut in only one direction, dentin-resin discs were fixed in a glass-holder and ground with SiC papers under running water. Slices were treated by Massonâs trichrome acid staining technique to evidence collagen exposed zones and analyzed by optical microscopy. ÂTBS results showed that NaOCl solution significantly reduced bond strength comparing with the control groups for all adhesives tested. Scothbond MP (control: 39,95Â12,72; aged: 26,45Â9,90 MPa) showed the highest values and Adper SE (control: 13,21Â3,15; aged 4,95Â2,49) the lowest in both control and aging groups. Clearfil SE (control : 27,02Â6,84; aged: 16,17Â3,79) and Single Bond (control : 26,66Â8,35; aged: 11,77Â4,28) showed no difference in control groups, but after aging, Clearfil SE showed better results. Microscopic images analysis showed that NaOCl acts efficiently on degradation of collagen fibrils for all adhesive systems. Conclusion: Aging in 10% NaOCl solution reduces bond strength and modifies the interfacial morphological characteristics of the adhesive systems tested.Hà um consenso geral de que a uniÃo resina-dentina criada pelos adesivos dentinÃrios hidrofÃlicos contemporÃneos se deterioram com o tempo. Uma maneira de prever o comportamento da interface adesiva ao longo do tempo à atravÃs do envelhecimento in vitro por imersÃo em diferentes soluÃÃes. O objetivo deste trabalho foi avaliar o efeito da imersÃo em soluÃÃo de NaOCl na interface de uniÃo criada por diferentes sistemas adesivos. Para isso, foram usados 28 terceiros molares humanos que tiveram a dentina coronÃria exposta e um platà de resina composta confeccionado sobre sua superfÃcie apÃs a aplicaÃÃo de um dos seguintes sistemas adesivos, condicionamento total: Scothbond Multi-uso e Single Bond, ou autocondicionantes: Clearfil SE e Adper SE. Os espÃcimes foram cortados pela tÃcnica nontrimming de obtenÃÃo de palitos para o teste de microtraÃÃo. ApÃs um perÃodo de 24 horas em Ãgua destilada, os palitos de cada dente foram igualmente divididos em dois grupos: controle ou envelhecido em soluÃÃo de NaOCl a 10% por 1 hora. Os espÃcimes foram tracionados atà a ruptura da uniÃo a uma velocidade de 1 mm/min e sua forÃa de uniÃo mensurada. Os palitos fraturados foram analisados e classificados em: fratura mista, coesiva em dentina e coesiva em compÃsito, e os valores expressos em porcentagem. Os dados da resistÃncia de uniÃo foram estatisticamente analisados usando os testes Kruskal-Wallis e Games-Howell. Para comparaÃÃo entre os grupos controle e envelhecido foi usado o teste de Mann-Whitney (α=0.05). Dois espÃcimes de cada adesivo foram usados para investigar o efeito do NaOCl nas caracterÃsticas morfolÃgicas da interface de uniÃo. Para isso, espÃcimes foram cortados ao longo eixo em apenas uma direÃÃo e os discos de dentina-resina foram fixados em lÃmina de vidro e desgastados com lixas de SiC sob refrigeraÃÃo com Ãgua. As lÃminas foram tratadas pela tÃcnica de coloraÃÃo Ãcida de Masson para evidenciar as zonas de colÃgeno exposto e observadas atravÃs de microscopia Ãptica. Os resultados do teste de microtraÃÃo mostraram que a soluÃÃo de NaOCl reduziu significativamente a forÃa de uniÃo em comparaÃÃo aos grupos controle para todos os adesivos testados. Scothbond MU (controle: 39,95Â12,72; envelhecido: 26,45Â9,90 MPa) mostrou os maiores valores de uniÃo e Adper SE (controle: 13,21Â3,15; envelhecido 4,95Â2,49) os menores valores, tanto para o grupo controle quanto envelhecido. Clearfil SE (controle: 27,02Â6,84; envelhecido: 16,17Â3,79) e Single Bond (controle: 26,66Â8,35; envelhecido: 11,77Â4,28) nÃo mostraram diferenÃa estatÃstica nos grupos controle, mas apÃs envelhecimento, Clearfil SE mostrou melhores resultados. A anÃlise das imagens microscÃpicas mostrou que o NaOCl agiu efetivamente na degradaÃÃo das fibras de colÃgeno para todos os sistemas adesivos testados. ConclusÃo: O envelhecimento dos espÃcimes em soluÃÃo de NaOCl a 10% reduziu a resistÃncia de uniÃo e modificou as caracterÃsticas morfolÃgicas da interface adesiva para os sistemas adesivos testados

Adhesive performance of a multi-mode adhesive system: 1-Year in vitro study

Objectives The aim of this study was to investigate the adhesive stability over time of a multi-mode one-step adhesive applied using different bonding techniques on human coronal dentine. The hypotheses tested were that microtensile bond strength (\u3bcTBS), interfacial nanoleakage expression and matrix metalloproteinases (MMPs) activation are not affected by the adhesive application mode (following the use of self-etch technique or with the etch-and-rinse technique on dry or wet dentine) or by ageing for 24 h, 6 months and 1 year in artificial saliva. Methods Human molars were cut to expose middle/deep dentine and assigned to one of the following bonding systems (N = 15): (1) Scotchbond Universal (3M ESPE) self-etch mode, (2) Scotchbond Universal etch-and-rinse technique on wet dentine, (3) Scotchbond Universal etch-and-rinse technique on dry dentine, and (4) Prime&Bond NT (Dentsply De Trey) etch-and-rinse technique on wet dentine (control). Specimens were processed for \u3bcTBS test in accordance with the non-trimming technique and stressed to failure after 24 h, 6 months or 1 year. Additional specimens were processed and examined to assay interfacial nanoleakage and MMP expression. Results At baseline, no differences between groups were found. After 1 year of storage, Scotchbond Universal applied in the self-etch mode and Prime&Bond NT showed higher \u3bcTBS compared to the other groups. The lowest nanoleakage expression was found for Scotchbond Universal applied in the self-etch mode, both at baseline and after storage. MMPs activation was found after application of each tested adhesive. Conclusions The results of this study support the use of the self-etch approach for bonding the tested multi-mode adhesive system to dentine due to improved stability over time. Clinical significance Improved bonding effectiveness of the tested universal adhesive system on dentine may be obtained if the adhesive is applied with the self-etch approach

Effect of carbodiimide (EDC) on the bond stability of etch-and-rinse adhesive systems

Objective Recent studies supported the use of protein cross-linking agents during bonding procedures to inactivate endogenous dentin proteases, preventing dentin collagen degradation thus improving bond durability. The aim of this study was to evaluate the effect of a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-containing conditioner on the stability of the adhesive interface created by two etch-and-rinse adhesives. Methods Human dentin was etched with 35% phosphoric acid, treated with 0.3 M EDC-containing conditioner followed by a three-step or a two-step etch-and-rinse adhesive. Adhesives were applied to control specimens without EDC pre-treatment. Specimens were subjected to microtensile bond strength test and pulled to failure after 24 h or 1 year of storage and interfacial nanoleakage expression was evaluated and quantified by light microscopy. Additionally, to investigate endogenous dentin matrix metalloproteinase activity a zymographic assay was performed on protein extracts obtained from phosphoric-acid-etched dentin powder with or without EDC treatment. Results The use of the EDC-containing conditioner did not affect immediate bond strength to dentin but contributed to preserve the bond strength after 1 year (p < 0.05) for both tested adhesives. No difference was found in the interfacial nanoleakage expression that increased after aging irrespective from the treatment. EDC pre-treatment inhibited dentin endogenous MMPs as assayed with the zymography. Significance In conclusion, the results of the study provide proof that EDC can produce long-term inactivation of MMPs in acid-etched dentin matrices contributing to bond strength preservation over time. Future studies are needed to support the use of EDC in vivo

Evaluation of resin/dentin bonded interfaces formed by different adhesive strategies and exposed to NaOCl challenge

This study evaluated the importance of encapsulated collagen on resin/dentin interface created by different adhesive strategies. Composite build-ups were bonded to dentin using one of the following adhesive systems: Scotchbond Multi-Purpose (SBMP), Adper Scotchbond 2 (SB2), Clearfil SE (CSE) and Scotchbond SE Plus (SBSE), and cut into non-trimmed dentin-composite beams. Half of those beams were deproteinized using 10% NaOCl for 1 h and the other half was stored in water. Beams were pulled to failure and data were statistically analyzed by a two-way ANOVA and Tukey for multi-comparison test (\u3b1=0.05). Additional dentin disks were stained with Massons trichrome acid and processed with light microscopy in order to identify the exposed collagen zones. All groups showed a significant reduction on bond strength after proteolytic challenge (pSB2=CSE>SBSE (p<0.05) for control and treated groups. Microscopy analysis showed different collagen exposed zones in relation with the adhesive strategy used. It can be concluded that collagen encapsulation affects the quality of bond interface, which is related to the adhesive system used