Academic literature on the topic 'In situ zymography'

Aim. This study sets out to evaluate the antiproteolytic activity of copaiba oil-based emulsion at the resin/dentin adhesive interface union formed with conventional and self-etching adhesives systems. Methods. At in situ zymography, 30 teeth were sectioned 2 mm below the enamel-dentin junction; a smear layer was standardized and subdivided into four groups. Gelatin conjugated with fluorescein was used and taken to the fluorescence microscope for evaluation. In cytotoxicity, the Trypan Blue method was used at four different time points. The tested groups were (G1) control with distilled water; (G2) 2% chlorhexidine (CLX); (G3) emulsion based on copaiba oil (EC) 10% + X; (G4) 10% EC + Y; and (G5) EC 10% alkaline. The zymographic assay used the same groups described, but in 30 seconds and 10 and 20 minutes. HT1080 cells were incubated and submitted to electrophoresis. The gel was analyzed using ImageJ software. Mann–Whitney and Kruskal–Wallis tests were used in the statistical analysis ( p < 0.05 ). Results. ECs showed higher cell viability in the cytotoxicity test and showed a significant difference in 10 and 20 minutes. In the zymographic assay, alkaline EC reduced 67% of MMP-2 activity and 44% of MMP-9 compared to 2% chlorhexidine. At in situ zymography in qualitative evaluation, all groups tested showed inhibition of activity in metalloproteinases. Conclusion. EC showed activity in the inhibition of metalloproteinases in vitro and in situ, especially the alkaline one. The survey shows the possibility of using ECs, a product from Amazonian biodiversity, as a biomodifier in dentistry.

There are many methods for the study and quantification of enzymes in the body systems. Off-late, the method of zymography has assumed immense importance in diagnostic medicine. It is a method where enzymes are visualized using the substrate conversion technique. The product of reaction appears, or the substrate disappears and the mechanisms specific for the recognition of this measure the biochemical reaction. This procedure has many advantages, the most important ones being providing both qualitative and measurable data, such as molecular zymography methods for visualising hydrolytic enzymes and differentiating among whole molecules, degradation and complexes. There are three main types of zymography, namely- In-Gel zymography (IGZ), In-Situ zymography (ISZ) and In-vivo zymography (IVZ). There are many variations of this technique like transfer 2D and reverse zymography. This method is mainly used to study the expression of matrix metalloproteinases. Alternate to the conventional zymography techniques have been suggested, like usage of the new Ponceau S staining protocol that provides significant benefits in terms of assay usability and cost reduction and is comparatively faster and easier. Mild alterations in the actual procedure of zymography can take care of minor issues arising during the procedure.

This study aimed to investigate matrix metalloproteinase (MMP) activity in human dentin using in-situ and gelatin zymography, after at-home and in-office bleaching, related to their clinical exposure times. Dentin specimens (n = 5) were treated with 35% hydrogen peroxide (50 min per session/4 sessions), 10% carbamide peroxide (180 min/21 sessions), or no treatment. All were subjected to in-situ zymography. Dentin slices were, subsequently, obtained, covered with fluorescein-conjugated gelatin, and examined with confocal laser-scanning microscopy. The fluorescence intensity was quantified and statistically analyzed using one-way ANOVA and Bonferroni tests (α = 0.05). Furthermore, gelatin zymography was performed on protein extracts obtained from dentin powder (N = 8 teeth), treated with hydrogen peroxide or carbamide peroxide, with different exposure times (10/50 min for hydrogen peroxide; 252/1260 min for carbamide peroxide). The results of the in-situ zymography showed no statistical differences between the bleached specimens and the control group, with a medium level of gelatinolytic activity expressed in the dentin tubules. The results of gelatin zymography showed an increased expression of pro-MMP-9 in carbamide peroxide groups. The expression of pro-MMP-2 decreased in all the experimental groups. The bleaching treatments performed on the enamel of sound teeth do not influence dentinal enzymatic activity. However, when unprotected dentin tissue is bleached, matrix metalloproteinases are more expressed, particularly when carbamide peroxide is used, proportional to the exposure time.

Matrix metalloproteinases (MMPs) play an important role in tooth development and influence caries development and hybrid layer degradation. Literature is scant on the differences in the activity of MMPs between primary and permanent dentine. Accordingly, the aim of the present study was to investigate endogenous gelatinolytic activity in primary and permanent dentine. Separate batches of dentine powder were obtained from intact human primary and permanent molars (n = 6). Each batch was divided in two subgroups: (1) mineralised; and (2) demineralised with 10% H3PO4. After protein extraction, gelatine zymography was performed. Furthermore, in situ zymography was performed on dentine sections of the same groups (n = 3). The slices were polished, covered with fluorescein-conjugated gelatine and evaluated using a confocal microscope. In situ zymography data were analysed using two-way analysis of variance and post hoc Holm–Šidák statistics (α = 0.05). Primary dentine showed poorly defined bands in the zymograms that vaguely corresponded to the pro-form and active form of MMP-2 and the pro-form of MMP-9. In permanent dentine, demineralised powder demonstrated stronger gelatinolytic activity than mineralised powder. In situ zymography identified stronger enzymatic activity in primary etched dentine (p < 0.05). Stronger enzymatic activity recorded in primary dentine may be related to the differences in morphology and composition between primary and permanent dentine.

Respiratory tract lesions induced by the chemical warfare agent sulfur mustard (SM) are characterized by epithelial damages associated with inflammatory cell infiltration. Here we evaluated the imbalance between gelatinase and tissue inhibitors of metalloproteinases (TIMPs), and we tested pretreatment with the protease inhibitor doxycycline. Guinea pigs were intoxicated intratracheally with SM and evaluated 24 h after exposure. Matrix metalloproteinase (MMP) gelatinase activity of bronchial lavage (BL) fluid from SM-exposed guinea pigs was high compared with controls, as shown by both zymography and biotinylated substrate degradation, whereas TIMP-1 and -2 levels by immunoblotting were similar. Extensive areas of lysis were evidenced by in situ zymography, indicating imbalance between gelatinases and inhibitors towards net proteolytic activity. Doxycycline pretreatment resulted in 1) decreased gelatinase activity (zymography, free gelatinase activity assay, and in situ zymography); 2) decreased inflammation (BL fluid cellularity and protein level); and 3) dramatic decrease in histological epithelial lesions. Our results suggest inadequate levels of TIMP to counteract increased gelatinase activity and further support a role for MMP gelatinases in SM-induced respiratory lesions. They also suggest that doxycycline may hold promise as a therapeutic tool.

This thesis considers the possible mechanisms of haematuria in cases of thin basement membrane disease and mild forms of IgA nephropathy. The absence of inflammation in both diseases makes it difficult to explain the passage of erythrocytes to the urinary space. Two main approaches were used. The composition of glomerular basement membrane was evaluated using quantitative immuno-electron microscopy; this was successfully achieved with antibodies against collagen IV, laminin, fibronectin and collagen VI. Possible changes in degradation by proteolytic enzymes were assessed using quantitative in situ zymography, a novel technique where the ability of tissue sections to digest gelatin in a layer of photographic emulsion is measured. Initial experiments were performed to establish the techniques and to assess sources of variation in the final measurements. Reproducibility studies for immuno-electron microscopy and in situ zymography demonstrated a considerable amount of variation if processing of tissue was performed on separated days. Study groups included cases of thin basement membrane disease, IgA nephropathy and normal controls. To control for variation in the measurement methods, cases were arranged into triplet groups and handled as a single batch through immuno-electron microscopy and in situ zymography studies. Thin basement membrane disease cases demonstrated abnormalities in glomerular basement membrane composition, but not in glomerular gelatinolytic activity. Laminin was found to be lower than normal. Fibronectin and type IV collagen showed slight decreases; collagen IV appeared to be more compact in the thinner membranes. IgA nephropathy cases had higher glomerular gelatinolytic activity than normal cases. The glomerular basement membrane composition was not changed from normal.

Orientador: Jacks Jorge Júnior
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-20T03:09:29Z (GMT). No. of bitstreams: 1 SenaFilho_Marcondes_M.pdf: 9539347 bytes, checksum: ecd31e3eb890ecb3fcf26cc3dc064e25 (MD5) Previous issue date: 2012
Resumo: Sabe-se que a transição da mucosa normal para um carcinoma invasivo é um processo complexo, de múltiplas etapas e com etiologia multifatorial, onde as Lesões Potencialmente Malignas (LPMs) são de extrema importância. As LPMs são caracterizadas por uma maior possibilidade de malignização, quando comparadas à mucosa normal. Dentre estas, a Leucoplasia Bucal (LB) é a principal representante. Alguns estudos sugerem que há aumento da atividade de metaloproteinases (MMPs) 2 e 9 em mucosas com displasia epitelial e carcinomas espinocelulares (CECs) bucais, porém isso ainda não está totalmente claro e necessita de mais estudos. Os objetivos deste trabalho foram: 1) Avaliar a expressão imunoistoquímica das metaloproteinases 2 e 9; 2) Mensurar a atividade gelatinolítica total in situ; 3) Avaliar a integridade da lâmina basal através da expressão de colágeno IV; 4) Correlacionar e co-localizar a atividade gelatinolítica total in situ com a imunofluorescência de dupla marcação para colágeno IV e metaloproteinases 2 ou 9; 5) Correlacionar os achados anteriores com os aspectos clínicos e anatomopatológicos;. Foram realizadas 50 coletas (45 lesões e 5 controles) de biópsias oriundas de pacientes com suspeita clínica de lesões potencialmente malignas ou carcinomas bucais. Deste total, 30 (60%) receberam diagnóstico clínico de LB e 15 (30%) de CEC. Histopatologicamente, observou-se que 12 amostras tratavam-se apenas de hiperqueratose e acantose, 08 de displasia epitelial leve/moderada, 08 de displasia epitelial intensa, 10 CEC bem diferenciados e 04 carcinomas pouco diferenciados. Nas analises imunoistoquímicas, observou-se que a MMP-2 é fortemente expressa nos epitélios livres de displasia, diminuindo de acordo com a progressão da displasia epitelial ou indiferenciação da lesão. Ambas as MMPs foram expressas no estroma das lesões com displasia epitelial intensa e CEC's, porém a MMP-9 foi mais acentuada, principalmente nos CEC's indiferenciados. A atividade gelatinolítica in situ foi maior no epitélio dos controles e das hiperqueratoses e acantoses, se co-localizando apenas com a MMP-2. Todos os CEC's e 63% das displasias epiteliais intensas apresentaram descontinuidade da lâmina basal, onde houve co-localização das MMPs com atividade gelatinolítica. Nestas mesmas lesões, houve regiões de co-localização das MMP's, atividade gelatinolítica em lâminas basais ainda íntegras. A baixa expressão gelatinolítica das lesões displásicas e CEC's, correlacionado com as rupturas de lâmina basal e aumento da expressão das MMP's no estroma, além da co-localização das mesmas nas regiões de descontinuidade da lâmina basal, sugerem que as MMP's expressas no estroma das lesões provavelmente estão mais relacionadas com a progressão do tumor do que as expressas pelo parênquima
Abstract: The transition from normal oral mucosa to invasive carcinoma has a complex and multifactorial etiology. Oral leukoplakia (OL) is regarded as the most prevalent potentially malignant oral lesion, with a higher tendency for malignant transformation if compared to normal oral mucosa. Although an increased activity of matrix metalloproteinases (MMPs) 2 and 9 in oral mucosa presenting varying degrees of epithelial dysplasia and oral squamous cell carcinomas (OSCCs) has been suggested, but further investigation is necessary for confirming such association. Therefore, the aim of this research is to evaluate the immunohistochemical expression and the total in situ gelatinolytic activity of MMP-2 and MMP-9 in OL and OSCC, as well as to evaluate the integrity of the basal lamina through the expression of collagen IV, correlating the results obtained with clinical and pathological features. Immunohistochemical, immunofluorescent and in situ zymography reactions were carried out in 5 normal oral epithelium, 12 cases presenting hyperkeratosis and acanthosis with no epithelial dysplasia, 8 mild/moderate epithelial dysplasia, 8 severe epithelial dysplasia, 10 well differentiated and 4 poorly differentiated OSCC's. It was found that MMP-2 expression was higher in oral epithelium devoid of dysplasia, if compared to dysplastic and neoplastic oral epitheliums. In addition, despite both MMPs were expressed in the stroma of severe epithelial dysplasia and OSCC's samples, MMP-9 expression revealed to be more pronounced, especially in poorly differentiated OSCCs. in situ gelatinolytic activity was higher in normal epithelium and those presenting only hyperkeratosis and acanthosis, co-localizing only with MMP-2. All cases of OSCC and 63% of lesions with severe epithelial dysplasia showed discontinuity of the basal lamina, revealing co-localization of MMPs with in situ gelatinolytic activity in regions with continuous and discontinuous basal lamina. Taken together, the results obtained revealed that a low gelatinolytic activity in the parenchyma of dysplastic lesions and OSCC correlated with disruption of the basal lamina and with an increased expression of MMPs in the stroma, suggesting that MMP's expressed in the stroma of the lesions would be more related with tumor progression than MMP's expressed in the lesional parenchyma
Mestrado
Estomatologia
Mestre em Estomatopatologia

A longevidade das restaurações com compósitos está diretamente relacionada ao sucesso do tratamento estético. A vida útil das restaurações pode ser influenciada por vários fatores como o material empregado, tempo de uso clínico e a degradação da matriz dentinária por metaloproteinases da matriz (MMPs), que podem afetar a integridade da interface adesiva, fazendo com que a união dentina-­adesivo seja comprometida. Assim, buscando melhores resultados na durabilidade das restaurações adesivas, o objetivo deste estudo foi avaliar o efeito de substâncias inibidoras de MMPs (soluções de clorexidina -­ CHX a 0,2% e doxiciclina a 3%) e agente de ligação cruzada (extrato de semente de uva -­ grape seed extract - GSE a 15%) na Resistência de União (RU) de restaurações de resina composta submetidas à ciclagem mecânica. Cavidades de Classe I foram preparadas na área central da superfície exposta de 48 molares humanos hígidos, posteriormente separados em 4 grupos (n=12) de acordo com o tratamento de superfície recebido após o condicionamento de ácido: Grupo I -­ Controle (Sistema Adesivo -­ Adper Single Bond Plus -­ 3M ESPE); Grupo II -­ CHX 0,2% + Sistema Adesivo; Grupo III -­ GSE 15% + Sistema Adesivo e Grupo IV -­ Doxiciclina 3% + Sistema Adesivo. Após os procedimentos restauradores (Filtek Supreme Ultra -­ 3M ESPE), as amostras foram armazenadas em água destilada durante 24 horas a 37°C e subdividas em dois subgrupos: A -­ Controle (armazenado em saliva artificial) e B -­ Submetido à ciclagem mecânica (Coil Cycler, Proto-­tech, forca de 50 N, frequência de 1,5 Hz em 1 x 106 ciclos de aplicação). Após ciclagem mecânica, os corpos de prova foram cortados em palitos (0,8 x 0,8 mm; n = 5 por dente) e submetidos ao teste de microtração (Bisco) a velocidade de 1 mm/min. A atividade colagenolítica da MMP-­2 sob a ação dos primers bioativos foi analisada utilizando o kit EnzChek Gelatinase/Colagenase Assay (D-­12054, Molecular Probes, Eugene, OR, EUA). A atividade gelatinolítica das MMPs-­2 e -­9 após incubação com os primers bioativos foi avaliada através de zimografia convencional. Os resultados obtidos das duas análises foram apresentados na forma de porcentagem de inibição. Zimografia in situ foi realizada utilizando gelatina de fluorescência conjugada (Kit EnzChek Gelatinase/Collagenase Assay) como substrato de MMPs e avaliada com aumento de 40x através de microscopia de fluorescência. Posteriormente, os fótons fluorescentes foram analisados em software (ImageJ). Obtidos os resultados, os dados foram submetidos à análise estatística (2way ANOVA, Tukey, p>0,05) e demonstraram que os primers bioativos não exerceram influência significante sobre a RU dentina-­adesivo, independente da ação da ciclagem mecânica. A atividade colagenolítica, segundo zimografia convencional, demonstrou que os primers bioativos foram capazes de inibir as MMPs, sendo a CHX menos eficiente. Na análise de zimografia in situ, os primers bioativos diminuíram a atividade das MMPs localizadas na interface adesiva, com aumento da atividade gelatinolítica do grupo Controle sendo maior quando submetidos à ciclagem mecânica, efeito não encontrados no grupos tratados com primers bioativos. Concluiu-­se que, apesar de não apresentarem efeitos sobre a RU da interface adesiva, os primers bioativos apresentam ação sobre a degradação da camada híbrida.
The durability of composite resin restorations is one of the main points related to the success in aesthetic treatments. The longevity of adhesive restorations can be affected by various factors such as the materials, the clinical time of use and the dentin matrix degradation mediated by matrix metalloproteinases (MMPs). MMPs can affect the adhesive interface, compromising the bonding between dentin and adhesive. Thus, searching for better results on longevity of adhesive restorations, the aim of this study was to evaluate the effect of MMPs inhibitors (chlorhexidine -­ CHX 0.2% and doxycycline 3% ) and crosslinking agent (grape seed extract -­ GSE 15%) on resin composite bond strength (BS) of restorations submitted to load cycling. Class I cavities were performed in the central area of the exposed surface of 48 sound human molars. Then, it was divided into 4 groups (n=12) according to the surface treatment received after the acid etching: Group I -­ Control (Adhesive System -­ Adper Single Bond Plus -­ 3M ESPE); Group II -­ CHX 0.2% + Adhesive System; Group III -­ Doxycycline 3% + Adhesive System e Group IV -­ GSE 15% + Adhesive System. After restorative procedures (Filtek Supreme Ultra -­ 3M ESPE), the samples were stored in distilled water for 24 hours at 37°C and then subdivided into two subgroups (n=6): A -­ Control (stored in artificial saliva) and B -­ Submitted to load cycling (Coil Cycler, Proto-­tech, 50 N, 1.5 Hz, 1 x 106 cycles). After load cycling, the specimens were cut into sticks (0.8 x 0.8 mm; n = 5 per teeth) and submitted to the microtensile test (Bisco) at 1 mm/min. The collagenolytic activity of MMP-­2 under action of bioactive primers was analyzed using EnzChek Gelatinase/Colagenase Assay Kit (D-­12054, Molecular Probes, Eugene, OR, EUA). The gelatinolytic activity of MMPs-­2 and -­9 after incubation with the bioactive primers was evaluated by gelatin zymography. The results of these two analyzes were presented by percentage of inhibition. In situ zymography was performed using fluorescence conjugated gelatin (EnzChek Gelatinase/Collagenase Assay Kit) as MMP substrate and evaluated using 40x magnification under fluorescence microscopy. Then the fluorescent photons were analyzed with an appropriate software (ImageJ). The results were subjected to statistical analysis (2-­way ANOVA, Tukey, p<0.05) and the bioactive primers showed no significant influence on the dentin-­adhesive BS, regardless the load cycling action. According to gelatin zymography, the collagenolytic activity demonstrated that the bioactive primers were able to inhibit MMPs activity and CHX was less efficient. In situ zymography analysis, showed that the bioactive primers decreased the activity of MMPs located in adhesive interface. The data also demonstrated increased gelatinolytic activity on Control group, being higher when submitted to load cycling, and which effect was not found in the groups treated with bioactive primers. It could be concluded that although the bioactive primers did not influenced the BS of adhesive interface, they demonstrated positive results on hybrid layer degradation.

2013/2014
Restorative adhesive dentistry is based on the infiltration of dental adhesive resin within the dentin collagen network creating the hybrid layer. The stability and integrity of collagen fibrils within the hybrid layer are crucial for the maintenance of bond effectiveness over time. The matrix metallo-proteinases enzymes (MMPs), which are endogenous enzymes present in dentin tissue, are claimed to play a key role in affecting the integrity of the hybrid layer causing the hydrolysis and degradation of dentin collagen. The aim of this research project was to evaluate the efficacy of different cross-linking agent in preserving the adhesive/dentin layer. Two water soluble collagen cross-linking agents riboflavin, and the carbodiimide hydrochloride, 1-ethyl-3-[3-dimethylaminopropyl] (EDC) were initially tested, then additional cross-linking agents were included in the study: acrolein, (water soluble), and the N,N'-dicyclohexylcarbodiimide (DCC) (soluble in acetone and ethanol). The concentration of each solution was chosen according to the previous studies: 0.1% riboflavin/wt, 0.3M EDC/wt, 0,01% acrolein/wt, 0.5M DCC/ethanol, 0.5M/acetone. Cross-linkers were associated to different adhesive systems available on the market and different tests were performed to evaluate the ability of cross-linkers in preserving the hybrid layer: mechanical test (microtensile bond strength), qualitative test (nanoleakage analysis), and biochemical assays (zymographic analysis and in situ zymographic analysis). For the microtensile bond strength recently extracted third molars (N=5 for each group) were selected and restorative treatments were performed: in the experimental groups teeth were pretreated with the tested cross-linkers before the application of the adhesive systems (XP Bond, Scotch Bond 1XT, Optibond FL, Clearfil SE), while in the control groups the adhesives were applied according to the manufacturers’ instructions. The experimental groups were divided as follow: 1) 0.1% riboflavin/XP Bond; 0.3M EDC/Scotch Bond 1XT; 2) 0.3M EDC+XP Bond 3) 0.3M EDC+Scotch Bond 1XT; 4) 0.3M EDC+Optibond FL; 5) 0.01% acrolein+Scotch Bond 1XT; 6) 0.3M EDC+Clearfil SE. The bonded specimens were serially sectioned to obtain approximately 1-mm-thick beams in accordance with the microtensile non-trimming technique. Beams were stored at 37°C in artificial saliva to reproduce the oral cavity environment and then were stressed to failure after 24hours (T0), 6 months (T6) or 1 year (T12) using a simplified universal testing machine at a crosshead speed of 1 mm/min. Data were analyzed using Two-Way (variables: cross-linker pretreatment and storage time) analysis of variance (ANOVA) and post hoc Tukey test (p values < 0.05 were considered as statistically significant). The results of microtensile bond strength showed that the use of the tested cross-linkers tested does not affect the immediate bond strength, while at T12 bond strength values of the experimental groups are significantly higher compared to the control groups. For the qualitative interfacial nanoleakage analysis, additional molars (4 per group) were selected to evaluate the integrity of the tooth/resin interfaces. The experimental groups were divided as follow: 1) 0.1% riboflavin-XP Bond; 2) 0.3M EDC-Scotch Bond 1XT; 3) 0.3M EDC-Optibond FL. The specimens were prepared and stored as described for the microtensile test; after storage the beams were immersed for 24h in AgNO3, then rinsed in distilled water and immersed in photo-developing solution to reduce silver ions into metallic silver grain. Nanoleakage analysis was performed under light microscopy and the interfacial nanoleakage degree was scored on a scale of 0–4 based on the percentage of the adhesive surface showing silver nitrate deposition: 0: no nanoleakage; 1: <25% nanoleakage; 2: 25 to ≤50% nanoleakage; 3: 50 to ≤75% nanoleakage; and 4: >75% nanoleakage. Statistical differences among nanoleakage group scores were analyzed using the χ2 test. The results did not reveal differences in the interfacial nanoleakage expression between the experimental groups 2 (0.3M EDC-Scotch Bond 1XT) and 3 (0.3M EDC-Optibond FL) and their respective control group. After 12-month storage the treatment with the riboflavin significantly reduce the infiltration of AgNO3 within the hybrid layer created with XP Bond. For the biochemical assay of the enzymatic activity, zymographic analysis and in situ zymography were performed. The following groups were added to the previously described treatments and submitted to these tests: 0.5M DCC/ethanol, 0.5M DCC/acetone, 0.5M DCC/ethanol- Scotchbond 1XT, 0.5M DCC/acetone Scotchbond 1XT. For the zymographic analysis dentin powder was obtained from thirty human sound molars; the powder was treated with the cross-linking agents in association or not with the different adhesive systems. The extract proteins were submitted to electrophoresis on SDS-polyacrylamide gels copolymerized with gelatin as MMPs substrate. Zymograms showed that the use of cross-linkers before the adhesive application was able to reduce or totally inhibit the activity of MMPs (especially MMP-2 and MMP-9). The specimens submitted to in situ zymography were prepared as previous described for the microtensile test. Bonded specimens were cut in order to expose the adhesive/dentin interfaces and in situ zymography was performed applying a self-quenched fluorescein-conjugated gelatin on adhesive/dentin interface. The specimens were light-protected and incubated in humidified chambers at 37°C for 24 h, then the hydrolysis of quenched fluorescein-conjugated gelatin substrate (related to endogenous gelatinolytic enzyme activity) was assessed by examination with a confocal laser scanning microscope. Images revealed reduced green fluorescence in the hybrid layer of experimental groups (pretreated with the cross-linking agents) compared to the control group fluorescence. The results of all these studies suggest that the use of cross-linking agents before the bonding procedures improves the stability of the resin/dentin interface. We can speculated that cross-linkers are able to reinforced the dentin collagen network increasing the mechanical properties of the hybrid layer, as confirmed by the microtensile test. Furthermore the results showed that these agents are able to inhibit the MMP gelatinolytic activity, preserving the dentin collagen fibrils from the hydrolysis and avoiding the failure of the hybrid layer.
L’odontoiatria adesiva si basa sulla capacità delle resine adesive di penetrare all’interno del network del collagene dentinale portando alla formazione del cosiddetto strato ibrido; la stabilità e l’integrità delle fibrille collagene dello strato ibrido sono fondamentali per preservare le caratteristiche dell’adesione al tessuto dentinale nel tempo. È noto che le metallo-proteinasi della matrice (MMPs), enzimi endogeni della dentina, possono causare l’idrolisi e la conseguente degradazione dello strato ibrido dentinale. Scopo del presente progetto di ricerca è stata la valutazione dell’efficacia di diversi agenti cross-linkers del collagene sulla stabilità dell’interfaccia adesivo/dentinale. Inizialmente sono stati testati due diversi agenti cross-linkers, entrambi solubili in acqua: la riboflavina ed il carbodiimide hydrochloride, 1-Ethyl-3-[3-dimethylaminopropyl] (EDC), successivamente sono state incluse nello studio altre due sostanze: l’acroleina (solubile in acqua), e il N,N'-dicyclohexylcarbodiimide (DCC) (solubile in etanolo o acetone). La concentrazione di ogni soluzione testata è stata scelta in accordo con la letteratura: 0.1% riboflavina/wt, 0.3M EDC/wt, 0,01% acroleina/wt, 0.5M DCC/etanolo, 0.5M/acetone. Gli agenti cross-linkers sono stati testati in associazione a diversi sistemi adesivi disponibili sul mercato. Al fine di valutarne la capacità nella conservazione dello strato ibrido sono state scelte diverse tipologie di analisi: meccanica (microtensile bond strength), qualitativa (analisi del nanoleakage), e biochimiche (zimografia e zimografia in situ). L’analisi meccanica è stata effettuata mediante il test del microtensile per il quale sono stati scelti denti molari umani (5 per gruppo) su cui sono state effettuate le procedure per un restauro adesivo: nei gruppi sperimentali la dentina è stata trattata con l’agente cross-linker scelto prima dell’applicazione del sistema adesivo; nei gruppi controllo gli adesivi (XP Bond, Scotch Bond 1XT, Optibond FL, Clearfil SE) sono stati utilizzati seguendo le istruzioni del produttore. I gruppi sperimentali sono stati suddivisi nel seguente modo: 1) 0.1% riboflavina+XP Bond; 0.3M EDC+Scotch Bond 1XT; 2) 0.3M EDC+XP Bond 3) 0.3M EDC+ScotchBond 1XT; 4) 0.3M EDC+Optibond FL; 5) 0.01% acrolein+ScotchBond 1XT; 6) 0.3M EDC+Clearfil SE. I campioni sono stati poi tagliati per ottenere sticks delle dimensioni di 1 mm circa di spessore in accordo con la “non-trimming technique”. I campioni ottenuti sono stati conservati, in saliva artificiale a 37°C per simulare l’ambiente del cavo orale. Dopo 24 ore (T0), 6 mesi (T6), e 1 anno (T12) i campioni sono stati sottoposti a test di trazione usando una macchina universale alla velocità di 1 mm/min. I dati sono stati analizzati con il test di ANOVA a due vie (trattamento con il cross-linker/tempo di storaggio) e con il post hoc Tukey test (valori di p < 0.05 indicano differenza statisticamente significativa). I risultati del test di microtensile hanno mostrato che il pretrattamento con cross-linkers del collagene non influisce sui valori di adesione a T0, mentre i valori a T12 evidenziano che l’uso dei cross-linkers aumenta significativamente l’adesione rispetto ai rispettivi gruppi controllo. L’analisi qualitativa dell’interfaccia adesiva è stata effettuata mediante valutazione del nanoleakage. A tale scopo, sono stati aggiunti altri 4 molari per ciascun gruppo. I gruppi sperimentali sono stati divisi come segue: 1) 0.1% riboflavina+XP Bond; 2) 0.3M EDC+Scotch Bond 1XT; 3) 0.3M EDC+Optibond FL. I campioni sono stati preparati come precedentemente descritto per il test di microtensile e dopo i medesimi tempi di storaggio i campioni sono stati immersi in una soluzione acquosa di nitrato di argento (AgNO3) per 24 ore, sciacquati, e successivamente immersi in liquido di sviluppo al fine di ridurre gli ioni di argento. I campioni sono stati quindi analizzati al microscopio ottico, ed il nanoleakage è stato quantificato con valori da 0 a 4, basandosi sulla percentuale di superficie dello strato ibrido infiltrata dal nitrato d’argento, come segue: 1: <25% nanoleakage; 2: 25 a ≤50% nanoleakage; 3: 50 a ≤75% nanoleakage; and 4: >75% nanoleakage. I dati sono stati analizzati utilizzando il χ2 test. I risultati mostrano differenze significative a T12 solo per il gruppo 1 (0.1% riboflavina-XP Bond), in cui il gruppo sperimentale trattato con agente cross-linkers presenta all’interno dello strato ibrido una minore quantità di AgNO3 se comparato al gruppo controllo. Al contrario gli altri due cross-linkers presi in analisi non hanno mostrato differenze statisticamente significative in relazione al loro uso a T0 o T12. In relazione ai tests biochimici, sono state effettuati test si zimografia e la zimografia in situ al fine di valutare l’attività enzimatica delle MMPs dentinali in relazione al trattamento con agenti cross-linkers. Ai gruppi precedentemente testati sono stati aggiunti ulteriori 4 gruppi sperimentali: 0.5M DCC/etanolo, 0.5M DCC/acetone, 0.5M DCC/etanolo-Scotchbond 1XT, 0.5M DCC/acetone-Scotchbond 1XT. Per la zimografia sono stati scelti trenta molari sani dai quali è stata isolata la dentina e polverizzata. La polvere di dentina è poi stata trattata con i diversi cross-linkers e adesivi come precedentemente descritto. Le proteine estratte dalla polvere dentinale, trattata secondo i diversi gruppi di appartenenza, sono state quindi sottoposte a corsa elettroforetica su gel di SDS-page co-polimerizzato con gelatina, come substrato delle MMPs. I gels ottenuti mostrano che il trattamento della dentina con agenti cross-linker prima dell’applicazione degli adesivi dentinali è in grado di diminuire o inibire quasi completamente l’attività gelatinolitica delle MMPs (MMP-2 -9 in particolare). Per quanto riguarda la zimografia in situ i campioni sono stati preparati come descritto per il test di microtensile, successivamente sono stati tagliati in modo da esporre l’interfaccia adesiva. A livello della stessa interfaccia è stata posta della gelatina coniugata con fluorescina. L’idrolisi della gelatina causata dagli enzimi endogeni dentinali determina l’attivazione della fluorescenza evidenziando quindi in situ l’attività enzimatica. I campioni sono stati osservati mediante microscopio confocale ed hanno evidenziato una minore fluorescenza all’interno dello strato ibrido di tutti gruppi sperimentali presi in analisi se confrontate con i rispettivi gruppi controllo. In conclusione possiamo affermare che l’uso degli agenti cross-linkers del collagene prima delle procedure adesive migliora le caratteristiche generali dello strato ibrido. È possibile suppore che i cross-linkers siano in grado di rinforzare il network del collagene dentinale aumentandone le proprietà meccaniche, come dimostrato dal test del microtensile; inoltre i tests enzimatici mostrano come questi agenti siano in grado di inibire l’attività enzimatica delle MMPs preservando in questo modo l’idrolisi delle fibrille collagene ed evitando la conseguente disgregazione dello strato ibrido.
XXVII Ciclo
1986