Effect of Collagen Crosslinkers on Dentin Protease Activity

The enzymatic degradation of demineralized dentin matrices (DDM) is a challenge in the longevity of adhesive restorations. The activity of matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) was shown to be responsible for progressive degradation. The inhibition or inactivation of these enzymes is a strategy for increasing the durability of adhesive restorations. The aim of these studies was to evaluate the effect of various collagen crosslinkers on dentin protease activity and to provide detailed knowledge of the functional mode of collagen crosslinkers in the prevention of dentin degradation. Several plant-derived and synthetic collagen crosslinkers were selected, and their effect on MMP- and CC-mediated degradation was evaluated after short- and long-term incubation using direct or indirect methods. Gelatin zymography, in situ zymography, generic MMP activity assays, protein analysis and multiplex analysis were used to identify and quantify the activity in dentin. After short-term incubation, all collagen crosslinkers showed a significant reduction in MMP- and CC-mediated degradation. UVA-induced crosslinking with or without photosensitizers was found to be more effective in the inactivation of cathepsin K compared with the inactivation of MMPs. Total MMP activity, in situ zymography and protein analysis confirmed the reduction in MMP activity after crosslinker pretreatment of DDMs. After six-month incubation, only some collagen crosslinkers maintained their MMP and CC inactivation, confirming that the effect is collagen crosslinker specific. The series of studies provided insight about the effect and inactivation mechanisms of collagen crosslinker pretreatment on prevention of proteolytic degradation of dentin matrices. The use of plant-derived collagen crosslinker agents could offer a good solution to increasing the longevity of tooth-adhesive bonding.Ristisilloittajien vaikutus dentiinin proteaasien aktiivisuuteen Hampaan väriset resiinipaikat ovat yleisimmin käytettyjä paikka-aineita korjaavassa hammashoidossa. Kliininen vaste riippuu pääasiassa paikka-aineen ja hammaskudoksen välisen rajapinnan laadusta. Monet tutkimukset osoittavat, että rajapinta ei ole niin stabiili kuin sen pitäisi olla. Suurin ongelma on dentiinin kollageenin hajoaminen ajan myötä. Hajoaminen johtuu dentiinin proteaasien, matriksin metalloproteinaasien (MMP) ja kysteiini-katepsiinien (CC), toiminnasta. Tutkimuksen tavoitteena on arvioida ristisilloittajien soveltuvuutta dentiinin proteaasien inaktivointiin ja tuottaa tietoa ristisilloittajien toiminnasta dentiinin hajoamisen estämisessä. Tämän saavuttamiseksi valitut ristisilloittajat tutkittiin ja niiden vaikutusta dentiinistä löytyvien MMP:n ja CC:n inaktivointiin arvioitiin. Dentiinin proteaasien inaktivointia ristisilloittajilla tutkittiin sarjassa kokeita. Ensimmäisessä tutkimuksessa arvioitiin dentiinin kollageenimatriksin hajoamista ristisilloittajilla tehdyn esikäsittelyn jälkeen. Toinen tutkimus osoitti UVA-valon indusoiman uusien sidosten syntymisen inaktivoivan vaikutuksen proteaaseihin demineralisoidussa dentiinissä. Kolmannessa tutkimuksessa arvioitiin ristisillloittajien vaikutusta dentiinin MMP:n suoraan inaktivaatioon sekä ristisilloittajien kykyä muodostaa uusia sidoksia demineralisoidun dentiinin kollageeniosassa ja ei-kollageeniosassa. Dentiinin proteaasien inaktivaation käänteisyys testattiin pitkän inkubaation jälkeen. Tutkimukset antavat tietoa ristisilloittajien kyvystä estää dentiinin kollageenimatriksin proteolyyttistä hajoamista. Tämä auttaa parantamaan sidostusten kestävyyttä ja kehittämään uusia sidostustekniikoita, mikä on tärkeää kliinisen lopputuloksen kannalta.Siirretty Doriast

La contribución del espesor de la cerámica y el tipo de adhesivo a la resistencia de desunión de las carillas dentales de cerámica con láser Er, Cr: YSGG.

Purpose: De-bonding strength of ceramic veneers by laser use needs to be evaluated in detail. The aim of this study, is to determine the contribution of ceramic thickness and cementing agents to the de-bonding strength of ceramic veneers using Er,Cr:YSGG laser. Methods: A total of 120 maxillary central incisors specimens were randomly divided into twelve groups on the basis of disc thickness, cementing agent, and Er,Cr:YSGG laser use. Under laboratory conditions, 120 IPS Empress II system discs 0.5mm, 1mm, and 2mm in thickness were applied to the tooth surfaces, for laser use. An Er,Cr:YSGG laser system was applied to the central surface of the IPS Empress II discs on specimens in all laser groups (Groups 1,3,5,7,9,11). Then the shear bond strength (SBS) for all specimens were tested with a testing machine at a speed of 0.5mm/min. The SBS values were considered as the de-bonding strength. Results: The mean de-bonding strength values for Groups 9 and 11 (0,5 mm disc thickness + laser application) have the lowest median load (0.000 N), while Group 4 (2mm disc thickness + no laser) has the highest median load (573.885 N). The de-bonding strengths of all the groups without laser application were higher than those of all groups with laser use. When laser is applied, the mean de-bonding strength decreases with decreasing disc thickness, and it reaches zero at 0.5mm thickness of discs cemented by self- or total-etch adhesives. Conclusions: The de-bonding strength decreases with laser use, and decreasing disc thickness. In the absence of laser, the mean de-bonding values of discs cemented by a total etch adhesive system are always higher than those of discs cemented with a self-etch adhesive system. Without any extra load, all 0.5mm thick discs were dislodged from teeth while applying or testing the laser.Propósito: La resistencia de desunión de las carillas de cerámica mediante el uso del láser debe evaluarse en detalle. El objetivo de este estudio es determinar la contribución del espesor de la cerámica y los agentes de cementación a la resistencia de desunión de las carillas de cerámica utilizando el láser Er, Cr: YSGG. Métodos: Un total de 120 incisivos centrales maxilares se dividieron al azar en doce grupos según el grosor del disco, el agente de cementación y el uso del láser Er, Cr: YSGG. En condiciones de laboratorio, se aplicaron en las superficies de los dientes 120 discos del sistema IPS Empress II de 0,5mm, 1mm y 2mm de grosor, para uso con láser. Se aplicó un sistema láser Er, Cr: YSGG a la superficie central de los discos IPS Empress II en muestras de todos los grupos de láser (Grupos 1,3,5,7,9,11). Luego, la resistencia de la unión al cizallamiento (SBS) para todas las muestras se probó con una máquina de prueba a una velocidad de 0.5mm/min. Los valores de SBS se consideraron como la fuerza de desunión. Resultados: Los valores medios de resistencia de desunión para los Grupos 9 y 11 (espesor de disco de 0,5mm + aplicación de láser) demostró la carga media más baja (0,000 N), mientras que el Grupo 4 (espesor de disco de 2 mm + sin láser) tuvo la carga media más alta (573.885 N). Las fuerzas de desunión de todos los grupos sin aplicación de láser fueron superiores a las de todos los grupos con uso de láser. Cuando se aplica el láser, la fuerza media de desunión disminuye al disminuir el grosor del disco, y llega a cero con el grosor de 0,5mm de los discos cementados, para ambos adhesivos de grabado. Conclusiones: la fuerza de desunión disminuye con el uso del láser y disminuye con el grosor del disco. En ausencia de láser, los valores medios de desunión de los discos cementados con un sistema de adhesivo de grabado total son siempre más altos que los de los discos cementados con un sistema de adhesivo de autograbado. Sin ninguna carga adicional, todos los discos de 0,5mm de grosor se desprendieron de los dientes al aplicar el láser

The effect of phytic acid on enzymatic degradation of dentin

We evaluated the effect of phytic acid on matrix metalloproteinase (MMP)- or cysteine cathepsin (CC)-mediated dentin degradation. Demineralized dentin beams were divided into five groups (n = 12) and treated with 1%, 2%, or 3% phytic acid or with 37% phosphoric acid. Untreated demineralized beams served as controls. After incubation for 1 or 3 wk, dry mass loss was determined and aliquots of incubation media were analysed for cross-linked telopeptide of type I collagen (ICTP) fragments for MMP-mediated and c-terminal telopeptide of type I collagen (CTX) for cathepsin-k-mediated degradation. The direct effect of phytic acid was evaluated using MMP activity assay. Data were analysed using repeated-measures anova. ICTP releases with 1% and 2% phytic acid treatment were statistically significantly lower than those following phosphoric acid treatment at 3 wk. The CTX release for phytic acid-treated beams at 3 wk was not significantly different from that of untreated control beams, but it was significantly lower than that of phosphoric acid-treated beams. Their MMP activities at 3 wk were not significantly different from those of the controls but they were significantly lower than those seen for phosphoric acid-treated beams. Compared to phosphoric acid, phytic acid treatment resulted in a reduced dentinal host-derived endogenous enzymatic activity and collagen degradation

To etch or not to etch, Part II: On the hydrophobic-rich content and fatigue strength of universal adhesives

Objective: To determine whether smear layer management, via conservative etching pro-tocols, and the hydrophobic-rich content of hybrid layers would affect the fatigue strength of resin-dentin interfaces.Methods: Bar-shaped dentin beams obtained from sound third molars were wet-polished for 30 s. Dentin was etched with 32 % ortho-phosphoric acid for 3 or 15 s, 10 % meta -phosphoric acid for 15 s or by a prime-and-rinse application using a mild universal ad-hesive (Scotchbond Universal, 3M ESPE). Self-etch application served as control. Coating was performed with a solvent-free bisGMA-based resin. Composite buildups were made with a nanofilled composite. Resin-dentin beams with twin-bonded interfaces were sec-tioned and stored in deionized water for 24 h at 37 celcius before 4-point flexural quasi-static monotonic testing (n = 16). Stress-life fatigue behavior was evaluated under cyclic loading (n = 35) by the staircase method at 4 Hz. The tension side of cyclic-loaded unfractured beams were evaluated under SEM, along with the micro-morphology of etched dentin surfaces. Monotonic data was analyzed by two-way ANOVA followed by the Tukey Test and cyclic-loaded data by Kruskal-Wallis on Ranks (alpha = 0.05).Results: Etching protocols and higher hydrophobic-rich content produced significantly higher fatigue life distributions (p MPA 15 s > OPA 3 s > P + R > SE. Less aggressive etching and coating reduced crack formation at hybrid layers.Significance: Current oversimplification trends in resin-dentin bonding constitute a trade-off between hybridization quality and easier adhesive handling. Controlled dentin etching and increasing the hydrophobic-rich content of hybrid layers may be necessary to extend the longevity of mild universal adhesives. (c) 2022 Published by Elsevier Inc. on behalf of The Academy of Dental Materials. CC_BY_NC_ND_4.0</p

Biochemical and immunohistochemical identification of MMP-7 in human dentin

Objectives: Matrix metalloproteinases (MMPs) are dentinal endogenous enzymes claimed to have a vital role in dentin organic matrix breakdown. The aim of the study was to investigate presence, localization and distribution of MMP-7 in sound human dentin. Methods: Dentin was powdered, demineralized and dissolved in isoelectric focusing buffer. Resolved proteins were transferred to nitrocellulose membranes for western blotting (WB) analyses. For the zymographic analysis, aliquots of dentin protein were electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing fluorescently labeled gelatin. Further, the concentrations of dentinal MMPs were measured using Fluorescent Microsphere Immunoassay with a human MMP-MAP multiplex kit. Pre- and post-embedding immunolabeling technique was used to investigate the localization and distribution of MMP-7 in dentin. Dentin was cryo-fractured, the fragments partially decalcified and labeled with a primary monoclonal anti-MMP-7 and a secondary antibody conjugated with gold nanoparticles. MMP-7 labelings were identified in the demineralized dentin matrix as highly electron-dense dispersed gold particles. Results: WB and zymographic analysis of extracted dentin proteins showed presence of MMP-7 (similar to 20-28 KDa). Further, MMP-7 was found in the supernatants of the incubated dentin beams using Fluorescent Microsphere Immunoassay. FEI-SEM and TEM analyses established MMP-7 as an intrinsic constituent of the human dentin organic matrix. Conclusion: This study demonstrated that MMP-7 is an endogenous component of the human dentin fibrillar network. Clinical significance: It is pivotal to understand the underlying processes behind dentin matrix remodeling and degradation in order to develop the most optimal clinical protocols and ensure the longevity of dental restorations.Peer reviewe

Long-term effect of curcuminoid treatment on resin-to-dentin bond strength

Endogenous dentin proteases contribute to the degradation of collagen fibrils in the hybrid layer. Recently, inhibition of host-derived proteases by curcuminoids has shown promising results. The aim of this study was to evaluate the effect of curcuminoid treatment on the microtensile bond strength (μTBS) after 24 h or 12 months of storage. Fifty-four extracted sound human molars were flattened to mid-coronal dentin and divided into nine groups. After phosphoric acid-etching for 15 s, the dentin was experimentally treated for 60 s using 100 μM or 200 μM of curcumin, diflourobenzocurcumin, or demethoxycurcumin dissolved in 1% and 2% dimethyl sulfoxide (DMSO)/water solutions. Untreated and DMSO-treated groups served as controls. After bonding agent application, each tooth was restored with dental composite. The molars were sectioned into 0.9 × 0.9 × 6 mm beams. The μTBS testing was performed after 24 h and 12 months of storage in artificial saliva. Data were analyzed using regression analyses. Failure patterns were evaluated using scanning electron microscopy. Dentin treatment with curcuminoids did not adversely affect 24-h μTBS compared to controls. After 12 months, the μTBS of curcuminoid groups was statistically significantly higher than the controls. This study indicates the feasibility of using curcuminoids as protease inhibitors.</p

Long-term effect of curcuminoid treatment on resin-to-dentin bond strength

Endogenous dentin proteases contribute to the degradation of collagen fibrils in the hybrid layer. Recently, inhibition of host-derived proteases by curcuminoids has shown promising results. The aim of this study was to evaluate the effect of curcuminoid treatment on the microtensile bond strength (μTBS) after 24 h or 12 months of storage. Fifty-four extracted sound human molars were flattened to mid-coronal dentin and divided into nine groups. After phosphoric acid-etching for 15 s, the dentin was experimentally treated for 60 s using 100 μM or 200 μM of curcumin, diflourobenzocurcumin, or demethoxycurcumin dissolved in 1% and 2% dimethyl sulfoxide (DMSO)/water solutions. Untreated and DMSO-treated groups served as controls. After bonding agent application, each tooth was restored with dental composite. The molars were sectioned into 0.9 × 0.9 × 6 mm beams. The μTBS testing was performed after 24 h and 12 months of storage in artificial saliva. Data were analyzed using regression analyses. Failure patterns were evaluated using scanning electron microscopy. Dentin treatment with curcuminoids did not adversely affect 24-h μTBS compared to controls. After 12 months, the μTBS of curcuminoid groups was statistically significantly higher than the controls. This study indicates the feasibility of using curcuminoids as protease inhibitors.</p

A novel dry-bonding approach to reduce collagen degradation and optimize resin-dentin interfaces

In dentistry, the wet-bonding approach relies on water to maintain demineralized collagen expanded for proper resin infiltration; nevertheless, hydrolytic instability of the resin-dentin interface is inevitable with current bonding techniques. Considering dimethyl sulfoxide's (DMSO) ability to "biomodify" collagen and precipitate enzymes, the aim was to test whether the use of DMSO would permit adequate resin bonding to H3PO4-etched dehydrated dentin and assess its impact on collagen degradation by host-derived enzymes. Etched dentin surfaces from extracted sound human molars were randomly bonded in wet or dry conditions using aqueous or ethanolic DMSO solutions as pretreatments and bonding resins with or without DMSO. Bonded teeth were sectioned into resin-dentin slabs for confocal in situ zymography and beams for microtensile bond strength test. Demineralized powdered dentin was incubated in the tested DMSO -media and a hydroxyproline assay evaluated dissolution of collagen peptides. Zymography was performed on protein extracts obtained from dry and wet H3PO4-ecthed dentin powder treated with the DMSO- media. The correlative biochemical analysis demonstrated that reduction of water content during dentin hybridization by the innovative dry-bonding approaches with DMSO is effective to inactivate host-derived MMP-2 and MMP-9 and thus reduce collagen degradation while simultaneously optimizing resin-dentin bonding.Peer reviewe

Effect of pH on dentin protease inactivation by carbodiimide

A water-soluble crosslinking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), has been used as a pretreatment of acid-etched dentin to inactivate matrix-bound endogenous dentin proteases. The aim of this study was to evaluate the effect of pH on the inactivation capacity of EDC. Demineralized dentin beams (1 7 2 7 6 mm) were divided into six groups (n = 8 per group). Then, EDC (0.3 M) was solubilized in distilled water with pH of 2, 4, 6, 7, 9, or 11. Control EDC was solubilized in 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer and its pH was adjusted to 6. The dentin beams were pretreated for 1 min with EDC at each pH or with EDC in MES buffer at pH 6.0 and then incubated in 1 ml of simulated body fluid (pH 7.2) for 1, 3, 7, or 14 d. Untreated beams served as controls. At each study time-point, the dry mass of dentin beams was assessed and the incubation media were analyzed for carboxyterminal telopeptide of type-I collagen (ICTP) and C-terminal telopeptide of type I collagen (CTX) using specific ELISAs. Data were subjected to repeat-measures anova. The results of the study indicated that specimens pretreated with EDC in MES buffer showed the lowest collagen degradation in terms of mass loss and release of telopeptides, while specimens pretreated in alkaline media showed the highest collagen degradation. This study indicates that the pH of the EDC solution plays an important role in the stability of dentin protease inactivation

Targeting Cariogenic Streptococcus mutans in Oral Biofilms with Charge-Switching Smart Antimicrobial Polymers

Cariogenic biofilms produce strong acidic microenvironments, which is the primary cause of dental caries. Streptococcus mutans is a dominant species in cariogenic biofilms. Herein, we report a pH-responsive, charge-switching smart copolymer to selectively target and eradicate bacteria in cariogenic biofilms. To that end, the copolymer is designed to be activated in an acidic environment. The smart copolymer, Poly-1A, consists of ternary compositions of monomers with a cationic ethyl ammonium group, a carboxylic group, and a hydrophobic group in the side chains. The net charge of Poly-1A was charge neutral at neutral pH, but it switched to be cationic because the acidic carboxylate side chains were protonated and became neutral; however, the ammonium groups remained positive. Poly-1A with a net positive charge bound to the anionic surface of oral bacteria by electrostatic interactions and disrupted the bacterial membranes, causing bacterial death. Poly-1A reduced the cell viability of planktonic and biofilm S. mutans at pH 4.5, while it was not bactericidal at pH 7.4. Poly-1A did not reduce the cell viability of human gingival fibroblasts and periodontal ligament stem cells for a 1 h incubation