Academic literature on the topic 'Collagen cross-linkers'

The achievement of a strong and stable bond between composite resin and dentin remains a challenge in restorative dentistry. Over the past two decades, dental materials have been substantially improved, with better handling and bonding characteristics. However, little attention has been paid to the contribution of collagen structure/stability to bond strength. We hypothesized that the induction of cross-linking in dentin collagen improves dentin collagen stability and bond strength. This study investigated the effects of glutaraldehyde-and grape seed extract-induced cross-linking on the dentin bond strengths of sound and caries-affected dentin, and on the stability of dentin collagen. Our results demonstrated that the application of chemical cross-linking agents to etched dentin prior to bonding procedures significantly enhanced the dentin bond strengths of caries-affected and sound dentin. Glutaraldehyde and grape seed extract significantly increased dentin collagen stability in sound and caries-affected dentin, likely via distinct mechanisms.

Collagen-based biomaterials constitute one of the most widely studied types of materials for biomedical applications. Low thermal and mechanical parameters are the main disadvantages of such structures. Moreover, they present low stability in the case of degradation by collagenase. To improve the properties of collagen-based materials, different types of cross-linkers have been researched. In recent years, phenolic acids have been studied as collagen modifiers. Mainly, tannic acid has been tested for collagen modification as it interacts with a polymeric chain by strong hydrogen bonds. When compared to pure collagen, such complexes show both antimicrobial activity and improved physicochemical properties. Less research reporting on other phenolic acids has been published. This review is a summary of the present knowledge about phenolic acids (e.g., tannic, ferulic, gallic, and caffeic acid) application as collagen cross-linkers. The studies concerning collagen-based materials with phenolic acids are summarized and discussed.

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

The subject of the thesis is the stability of the adhesive interface created at the level of the dental substrate. The connection between polymerized adhesives and the remaining mineralized dentin occurs through the collagen fibrils extending from the underlying mineralized matrix towards the hybrid layer. The collagen fibrils contain bound, non-collagenous proteins such as growth factors and matrix proteases. These proteases play an important role during dentin maturation, but they become trapped and inactive after the collagen matrix mineralized. However, during adhesive procedures, the enzymes of the collagen matrix are exposed and activated, irrespective of the E&R or SE procedure employed, resulting in the progressive degradation of the collagen fibril anchoring the restorative material to tooth structure. This leads to solubilization of collagen and loss of retention of the adhesive restoration. Recent studies supported the use of protein cross-linking agents during bonding procedures to prevent dentin collagen degradation caused by endogenous enzymes to improve bond durability. Acrolein (ACR),1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and dicyclohexylcarbodiimide (DDC) are compounds claimed to be potent cross-linkers. The present series of studies aimed at investigating the effect of various collagen crosslinkers on dentin protease activity and their effect on bond-strength to the dentinal substrate. Thus, selected collagen cross-linkers were tested to determine their effect on MPa values on coronal and radicular dentin, the marginal infiltration at the resin dentin interfaces, the presence of endogenous enzymatic activity after treatment and the localization of the gelatinase activity on dentin. Additionally, the effect of the crosslinkers and the effect of treatment period (duration) on the degradation of collagen matrices were examined. The results obtained suggest that collagen crosslinkers can inactivate e the collagen degradation through the inactivation of dentin proteases. However, in vivo studies are essential to better understand the feasibility of the tested molecules as dentin conditioning.

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Os objetivos desse estudo foram investigar o efeito da aplicação de diferentes agentes promotores de ligações cruzadas (cross-linkers) sobre (1) a rigidez do colágeno dentinário e (2) sobre a degradação da união resinadentina produzida pela técnica dry-bonding. Para responder ao primeiro objetivo, 40 discos de dentina com 0,3 mm de espessura foram obtidos de pré-molares hígidos. Após completa desmineralização em ácido fosfórico 10%, os discos foram aleatoriamente divididos em 5 grupos (n=8): água deionizada (controle), carbodiimida (EDC) 0,5 mol/L, proantocianidina (PA) 5% ou glutaraldeído (GA) 5% ou 25%. Os discos permaneceram imersos nas soluções por 60s. A rigidez do colágeno foi indiretamente avaliada com base na contração após desidratação, por meio de transformador linear diferencial variável (LVDT). Para responder ao segundo objetivo, 64 pré-molares humanos hígidos foram desgastados para remoção total do terço coronário, obtendo desta forma superfícies planas de dentina. Estas foram condicionadas com ácido fosfórico a 37% por 15s, seguido do tratamento com as mesmas soluções descritas acima (n=16), exceto para o GA 25%. Após um tempo pré-determinado de tratamento, a dentina foi mantida úmida em metade dos dentes (n=8), e desidratada com jatos de ar nos dentes restantes (n=8). Em seguida, em todos os dentes, foi aplicado o adesivo Prime & Bond 2.1 e foi construído um bloco de resina. Espécimes de 0,81 mm² de área adesiva foram submetidos ao ensaio mecânico de microtração, após 24h ou 6 meses de envelhecimento em saliva artificial. Também foram analisados os tipos de fratura após o teste de microtração e oito espécimes por grupo/período de envelhecimento foram preparados para análise da nanoinfiltração. Para ambos os experimentos, os dados foram submetidos aos testes de ANOVA e Tukey (p<0,05). Na ausência de tratamento...(Resumo completo, clicar acessO letrônico abaixo)
The aims of this study were to investigate the effect of the application of cross-linkers on (1) the stiffness of dentin collagen and (2) the resin-dentin bond degradation produced by the dry-bonding technique. To respond the first objective, forty 0.3 mm-thick dentin disks were obtained from sound premolars. After complete demineralization in 10% phosphoric acid, the discs were randomly divided into 4 groups according to the treatment (n=8): deionized water (control), 0.5 mol/L carbodiimide (EDC), 5% proanthocyanidin (PA) and 5% or 25% glutaraldehyde (GA). The discs were immersed in the solutions for 60s. Collagen stiffness was assessed indirectly based on the shrinkage after dehydration, measured in a linear variable differential transformer (LVDT). For second objective, flat dentin surfaces were produced in 64 sound pre-molars. The dentin was etched with phosphoric acid 37% for 15s, followed by treatment with the same solution described previously (n=16), except for the 25%GA. After the 60s treatment, the dentin was kept moist in half of the teeth (n=8) and dehydrated by air jets in the remaining teeth (n=8). Prime & Bond 2.1 adhesive was applied in all teeth followed by the built up of a composite block. Specimens of 0.81mm² adhesive area were obtained for the microtensile bond strength test, performed after 24h or 6 months of aging in saliva-like solution. After the mechanical test, the failure type was analyzed. Eight specimens by group were prepared for nanoleakage analysis. For both experiments, data were submitted to ANOVA and Tukey tests (p<0.05). In the absence of treatment with cross-linkers (control group), the percentage of shrinkage of the dentin matrix was 60%. Only EDC (23.9%) and glutaraldehyde 25% applied for 10 min (21.7%) were able to significantly reduce dentin shrinkage after dehydration. The highest bond strength values were found for EDC and 10% GA...(Complete abstract eletronic access below)

Orientador: Fernanda Gonçalves Basso
Banca: Elisa Maria Aparecida Giro
Banca: Rita de Cássia Loiola Cordeiro
Banca: Hérica Adad Ricci
Banca: Adriano Fonseca de Lima
Resumo: Os objetivos desse estudo foram investigar o efeito da aplicação de diferentes agentes promotores de ligações cruzadas (cross-linkers) sobre (1) a rigidez do colágeno dentinário e (2) sobre a degradação da união resinadentina produzida pela técnica dry-bonding. Para responder ao primeiro objetivo, 40 discos de dentina com 0,3 mm de espessura foram obtidos de pré-molares hígidos. Após completa desmineralização em ácido fosfórico 10%, os discos foram aleatoriamente divididos em 5 grupos (n=8): água deionizada (controle), carbodiimida (EDC) 0,5 mol/L, proantocianidina (PA) 5% ou glutaraldeído (GA) 5% ou 25%. Os discos permaneceram imersos nas soluções por 60s. A rigidez do colágeno foi indiretamente avaliada com base na contração após desidratação, por meio de transformador linear diferencial variável (LVDT). Para responder ao segundo objetivo, 64 pré-molares humanos hígidos foram desgastados para remoção total do terço coronário, obtendo desta forma superfícies planas de dentina. Estas foram condicionadas com ácido fosfórico a 37% por 15s, seguido do tratamento com as mesmas soluções descritas acima (n=16), exceto para o GA 25%. Após um tempo pré-determinado de tratamento, a dentina foi mantida úmida em metade dos dentes (n=8), e desidratada com jatos de ar nos dentes restantes (n=8). Em seguida, em todos os dentes, foi aplicado o adesivo Prime & Bond 2.1 e foi construído um bloco de resina. Espécimes de 0,81 mm² de área adesiva foram submetidos ao ensaio mecânico de microtração, após 24h ou 6 meses de envelhecimento em saliva artificial. Também foram analisados os tipos de fratura após o teste de microtração e oito espécimes por grupo/período de envelhecimento foram preparados para análise da nanoinfiltração. Para ambos os experimentos, os dados foram submetidos aos testes de ANOVA e Tukey (p<0,05). Na ausência de tratamento...(Resumo completo, clicar acessO letrônico abaixo)
Abstract: The aims of this study were to investigate the effect of the application of cross-linkers on (1) the stiffness of dentin collagen and (2) the resin-dentin bond degradation produced by the dry-bonding technique. To respond the first objective, forty 0.3 mm-thick dentin disks were obtained from sound premolars. After complete demineralization in 10% phosphoric acid, the discs were randomly divided into 4 groups according to the treatment (n=8): deionized water (control), 0.5 mol/L carbodiimide (EDC), 5% proanthocyanidin (PA) and 5% or 25% glutaraldehyde (GA). The discs were immersed in the solutions for 60s. Collagen stiffness was assessed indirectly based on the shrinkage after dehydration, measured in a linear variable differential transformer (LVDT). For second objective, flat dentin surfaces were produced in 64 sound pre-molars. The dentin was etched with phosphoric acid 37% for 15s, followed by treatment with the same solution described previously (n=16), except for the 25%GA. After the 60s treatment, the dentin was kept moist in half of the teeth (n=8) and dehydrated by air jets in the remaining teeth (n=8). Prime & Bond 2.1 adhesive was applied in all teeth followed by the built up of a composite block. Specimens of 0.81mm² adhesive area were obtained for the microtensile bond strength test, performed after 24h or 6 months of aging in saliva-like solution. After the mechanical test, the failure type was analyzed. Eight specimens by group were prepared for nanoleakage analysis. For both experiments, data were submitted to ANOVA and Tukey tests (p<0.05). In the absence of treatment with cross-linkers (control group), the percentage of shrinkage of the dentin matrix was 60%. Only EDC (23.9%) and glutaraldehyde 25% applied for 10 min (21.7%) were able to significantly reduce dentin shrinkage after dehydration. The highest bond strength values were found for EDC and 10% GA...(Complete abstract eletronic access below)
Doutor

2012/2013
The subject of this thesis is the stability of the adhesive interface in dentistry. Success in adhesive dentistry means long lasting restorations. However, there is substantial evidence that this ideal objective is not achieved. Current research in this field aims at increasing the resin-dentin bond durability. This doctoral research examines the fundamental processes responsible for the aging mechanisms involved in the degradation of resin-bonded interfaces, as well as some potential approaches to prevent and counteract this degradation. Resin-dentin bond degradation is a complex process that is not completely understood, involving the hydrolysis of both the resin and the collagen component of the hybrid layer. The hydrophilic and acidic characteristics of current dentin adhesives have made hybrid layers highly prone to water sorption, which causes polymer degradation and results in decreased resin-dentin bond strength over time. These unstable polymers inside the hybrid layer may result in an incomplete encapsulation of collagen fibers, which become vulnerable to mechanical and hydrolytical fatigue, as well as degradation by host-derived proteases with collagenolytic activity. These enzymes, such as matrix metalloproteinases (MMPs) and cysteine cathepsins, have a crucial role in the degradation of type I collagen, the organic component of the hybrid layer. The first part of this thesis aims to review the current knowledge regarding adhesion to the tooth substrate (Chapter 1), focusing on the fundamental processes that are responsible for the degradation of the adhesive interface (Chapter 2). Since the permeability of adhesives to water is particularly evident in simplified adhesive formulations, the research activity was focused on self-etch and universal adhesive systems’ behavior. Thus, the research study reported in Chapter 3 showed that the bond strength and nanoleakage expression of two-step and one-step self-etch tested bonding systems were affected by storage for 6 month and 1 year in artificial saliva. Although it is generally accepted that the permeability of adhesives to water is particularly evident in simplified adhesive formulations, the stability over time was not related to the number of steps of bonding systems, but to their chemical formulations. The performance of a new universal (or multi-mode) adhesive system through storage in artificial saliva was also investigated. The original results presented in Chapter 4 found that improved bonding effectiveness of the tested universal adhesive system on dentin was obtained when the adhesive was applied with the self-etch approach. Indeed, the etch-and-rinse approaches tested (both on wet and dry dentin) resulted in immediate bond strength comparable to the self-etch mode but expedited long-term aging resulted in reduced bond strength and increased nanoleakage expression, irrespective of dentin wetness. Moreover, the results of the zymographic analysis showed evident changes in dentinal MMP-2 and -9 enzyme activities after the application of the tested adhesives, revealing differences in the extent of enzyme activation. These findings exhibit that the activation of endogenous MMPs is not related to the adhesive system or the strategy employed. Thus, regardless of the approach and the material used in bonding procedures, a stable and durable bond is not achieved. Therefore, experimental strategies that aim to enhance the adhesive interface, particularly improving the durability of the resin-dentin bond strength by inhibiting intrinsic collagenolytic activity and increasing the resistance of dentin collagen matrix to enzymatic degradation are needed. The last part of the thesis is focused on both the strategies to inhibit the proteolytic and collagenolytic activity of the endogenous proteases and the methods to increase the mechanical strength of collagen network and its resistance to enzymatic degradation (Chapter 5). Chlorhexidine (CHX) has been used as a non-specific MMP inhibitor to prevent degradation of hybrid layers. However, CHX is water-soluble and may leach out of hybrid layers, compromising its long-term anti-MMP effectiveness. An entirely different approach is to treat the acid-etched dentin containing activated matrix-bound MMPs with cross-linking agents that inactivate the catalytic site of proteases. In particular, the ability of a cross-linker agent, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), to prevent collagen degradation was evaluated under occlusal cycle loading. Previous research successfully utilized EDC to increase the durability of resin-dentin bonds by increasing the mechanical properties of the collagen matrix; however, the 1 to 4 hrs required for that procedure was clinically unacceptable. For this reason, the purpose of the last part of the research, presented in Chapter 6, was to evaluate the ability of 0.5 M EDC short-time (1 min) pre-treatment to improve the stability of demineralized dentin collagen matrices by quantifying the release of telopeptide fragments over time. The results showed that EDC application for 1 min may be a clinically relevant and effective means for stabilizing the collagen network not only by strengthening the fibrils, but also by reducing the enzymatic degradation rate. Thus, dentin collagen reinforcement and strengthening through EDC cross-linking might be of importance to improve the bond strength and structural integrity of the resin-dentin interface over time against the enzymatic and hydrolytic degradation.
La tesi qui presentata riguarda la stabilità dell'interfaccia adesiva in odontoiatria. Il successo delle moderne terapie conservative è rappresentato dalla longevità dei restauri adesivi. Tuttavia, vi è una sostanziale evidenza che questo obiettivo ideale non sia raggiunto. La stabilità dell’interfaccia adesiva dipende dalla formazione di uno strato ibrido, compatto e omogeneo, durante l’impregnazione del substrato dentinale da parte dei monomeri adesivi. Poiché lo strato ibrido rappresenta un’entità complessa, in cui interagiscono componenti biologiche diverse (matrice dentinale collagenica e cristalli d’idrossiapatite residui) e non (monomeri resinosi e solventi), i fenomeni d’invecchiamento interessano in maniera sinergica sia la porzione resinosa che quella dentale. L’articolato processo che porta alla degradazione dell’interfaccia adesiva coinvolge infatti la componente resinosa, attraverso l’idrolisi della resina negli spazi interfibrillari, e quella organica, attraverso la disorganizzazione delle fibre collagene dovuta ad un incompleto incapsulamento delle stesse, nonché alla degradazione da parte di proteasi intrinseche con attività collagenolitica. È stato dimostrato come questi enzimi, le metalloproteinasi della matrice (MMP) e le catepsine, abbiano un ruolo cruciale nella degradazione del collagene di tipo I, la principale componente organica dello strato ibrido. Inoltre le caratteristiche idrofile e acide degli attuali sistemi adesivi dentinali hanno reso lo strato ibrido molto suscettibile all'assorbimento di acqua, comportando, attraverso l’idrolisi, la degradazione dello stesso e andando così a contribuire ad una diminuzione della forza di legame nel tempo. Attualmente l’interesse della comunità scientifica mira ad aumentare la durata del legame adesivo con il substrato dentinale. Dopo un’attenta analisi delle attuali conoscenze riguardanti adesione al substrato dentale (Capitolo 1), la prima parte della tesi si propone di valutare i processi fondamentali che sono responsabili della degradazione dell'interfaccia adesiva (Capitolo 2). Poiché la permeabilità all’acqua degli adesivi è particolarmente evidente nelle formulazioni semplificate, l'attività di ricerca si è concentrata sull’analisi del comportamento dei sistemi adesivi self-etch e dei recenti sistemi adesivi universali. I risultati riportati nel Capitolo 3 ha dimostrato come la forza di legame e l’espressione del nanoleakage dei sistemi adesivi self-etch two-step e one-step testati sia negativamente influenzata dall’invecchiamento in saliva artificiale per 6 mesi e 1 anno. Sebbene sia generalmente accettato che la permeabilità degli adesivi all'acqua è particolarmente evidente in formulazioni di adesivi semplificati, la stabilità nel tempo non è stata correlata al numero di passaggi dei sistemi adesivi, bensì alle loro composizioni chimiche. Sono state in seguito analizzate anche le prestazioni di un nuovo sistema adesivo universale (o multimodale). I risultati presentati nel Capitolo 4 hanno stabilito una migliore efficienza adesiva del sistema universale, testato sul substrato dentinale, quando l'adesivo è stato applicato con l'approccio self-etch. Infatti, la tecnica etch-and-rinse, testata sia su dentina umida che secca, ha comportato una forza di adesione immediata paragonabile alla modalità self-etch, ma a tempi di invecchiamento incrementali si è evidenziata una diminuzione della forza di legame e una maggiore espressione del nanoleakage, a prescindere dalla condizione di umidità dentinale. Inoltre, i risultati dell'analisi zimografica hanno mostrato evidenti variazioni dell’attività enzimatica delle metalloproteinasi MMP-2 e -9 dopo l'applicazione degli adesivi testati. Questi risultati dimostrano come l'attivazione delle MMP endogene non sia correlata al sistema adesivo o alla strategia adottata. Ne evince che, indipendentemente dal metodo e dal materiale utilizzato nelle procedure adesive, non si è in grado di stabilire un legame affidabile e duraturo. Pertanto si avverte l’esigenza di strategie sperimentali che mirino a migliorare la stabilità dell’interfaccia adesiva, in particolare incrementando la durata della forza di legame in dentina inibendo l'attività collagenolitica intrinseca e aumentando la resistenza del collagene alla degradazione enzimatica. L'ultima parte della tesi è focalizzata quindi sulle strategie per inibire l'attività proteolitica e collagenolitica delle proteasi endogene e sui metodi per aumentare la resistenza meccanica del collagene alla degradazione enzimatica (Capitolo 5). Un potente agente antibatterico, la clorexidina (CHX), è stato usato come inibitore non specifico delle MMP al fine di impedire la degradazione dello strato ibrido. Tuttavia la CHX, essendo solubile in acqua, può dissolversi nello strato ibrido, compromettendo la sua efficacia anti-MMP a lungo termine. Un approccio completamente diverso è quello di trattare la dentina mordenzata con agenti cross-linker. In particolare, simulando il carico occlusale, è stata valutata la capacità di un agente cross-linker, l’1-etil-3-(3-dimetilammino-propil) carbodiimmide (EDC), per prevenire la degradazione del collagene. Precedenti ricerche hanno utilizzato con successo l’EDC con lo scopo di aumentare la durata dell’interfaccia adesiva, aumentando le proprietà meccaniche della matrice di collagene; tuttavia, il tempo necessario (da 1 a 4 ore) richiesto per tali procedure è clinicamente inaccettabile. Per questo motivo, lo scopo dell’ultima parte della ricerca, presentata nel Capitolo 6, è stato quello di valutare la capacità di 0,5 M EDC nel breve periodo di pretrattamento (1 min), andando a quantificare il rilascio di frammenti di telopeptidi di collagene nel corso del tempo. I risultati hanno dimostrato che l'applicazione di EDC per 1 min può essere un approccio clinicamente rilevante ed efficace nello stabilizzare il collagene, non solo rafforzando le fibrille, ma anche riducendo la velocità di degradazione enzimatica. Di conseguenza, l’utilizzo di questo cross-linker può garantire una valida strategia per migliorare la forza di legame e l'integrità strutturale dell'interfaccia adesiva nel tempo contro l’attività enzimatica intrinseca del collagene e la degradazione idrolitica.
XXVI Ciclo
1985