Dendrimer-based extrafibrillar demineralization for optimizing resin-dentin bond stability.

Optimizing resin-dentin bond stability using a bioactive adhesive with concomitant antibacterial properties and anti-proteolytic activities

Instability of resin-dentin bonds is the Achilles’ heel of adhesive dentistry. To address this problem, a chelate-and-rinse extrafibrillar dentin demineralization strategy has been developed that keeps intrafibrillar minerals within collagen fibrils intact to prevent activation of endogenous proteases that are responsible for collagen degradation within hybrid layers. The objective of the present study was to evaluate the potential of using chitosan >40 kDa as an antimicrobial extrafibrillar dentin-chelating agent to enhance bond durability. Transmission electron microscopy provided evidence for retention of intrafibrillar minerals and smear plugs in dentin conditioned with 1 wt% chitosan. Analyzed by Kruskal-Wallis analysis of variance, Dunn’s statistic, and separate Mann-Whitney tests, tensile bond strengths to wet- and dry-bonded dentin indicated that chelating dentin with chitosan for 60 s prior to bonding did not result in a significant decline in resin-dentin bond strength when compared with that of phosphoric acid etching (P > 0.05). Gelatinolytic activity within the hybrid layers was examined via in situ zymography after 24-h storage or after thermomechanical cycling and analyzed with 3-factor analysis of variance. After 24 h, enzymatic activity was detected only within completely demineralized phosphoric acid–etched dentin, with values derived from dry bonding significantly higher than those derived from wet bonding (P < 0.05). Negligible fluorescence was detected within hybrid layers when dentin was conditioned with chitosan, even after thermomechanical cycling, as compared with the controls. Reduction in water permeability in chitosan-conditioned dentin, attributed to smear plug retention, also fostered long-term bond stability. Antibacterial testing performed with live/dead staining indicated that the acetic acid–solubilized chitosan possessed antibacterial activities against 3 single-species biofilms: Streptococcus mutans, Actinomyces naeslundii, and Enterococcus faecalis. Taken together, the new chitosan-based extrafibrillar demineralization strategy retains intrafibrillar minerals, reduces endogenous protease-initiated collagen degradation, prevents water permeation within hybrid layers, and kills bacteria on dentin surfaces, which are crucial factors for enhancing resin-dentin bond durability.

ObjectiveSecondary caries caused by oral microbiome dysbiosis and hybrid layer degradation are two important contributors to the poor resin–dentin bond durability. Cavity cleansers with long-term antimicrobial and anti-proteolytic activities are in demand for eliminating bacteria-induced secondary caries and preventing hybrid layers from degradation. The objectives of the present study were to examine the long-term antimicrobial effect and anti-proteolytic potential of poly(amidoamine) dendrimers with amino terminal groups (PAMAM-NH2) cavity cleanser.MethodsAdsorption tests by attenuated total reflectance–infrared (ATR-IR) spectroscopy and confocal laser scanning microscopy (CLSM) were first performed to evaluate whether the PAMAM-NH2 cavity cleanser had binding capacity to dentin surface to fulfill its relatively long-term antimicrobial and anti-proteolytic effects. For antibacterial testing, Streptococcus mutans, Actinomyces naeslundii, and Enterococcus faecalis were grown on dentin surfaces, prior to the application of cavity cleanser. Colony-forming unit (CFU) counts and live/dead bacterial staining were performed to assess antibacterial effects. Gelatinolytic activity within the hybrid layers was directly detected by in situ zymography. Adhesive permeability of bonded interface and microtensile bond strength were employed to assess whether the PAMAM-NH2 cavity cleanser adversely affected resin–dentin bonding. Finally, the cytotoxicity of PAMAM-NH2 was evaluated by the Cell Counting Kit-8 (CCK-8) assay.ResultsAdsorption tests demonstrated that the binding capacity of PAMAM-NH2 on dentin surface was much stronger than that of 2% chlorhexidine (CHX) because its binding was strong enough to resist phosphate-buffered saline (PBS) washing. Antibacterial testing indicated that PAMAM-NH2 significantly inhibited bacteria grown on the dentin discs as compared with the control group (p < 0.05), which was comparable with the antibacterial activity of 2% CHX (p > 0.05). Hybrid layers conditioned with PAMAM-NH2 showed significant decrease in gelatin activity as compared with the control group. Furthermore, PAMAM-NH2 pretreatment did not adversely affect resin–dentin bonding because it did not decrease adhesive permeability and microtensile strength. CCK-8 assay showed that PAMAM-NH2 had low cytotoxicity on human dental pulp cells (HDPCs) and L929.ConclusionsPAMAM-NH2 cavity cleanser developed in this study could provide simultaneous long-term antimicrobial and anti-proteolytic activities for eliminating secondary caries that result from a dysbiosis in the oral microbiome and for preventing hybrid layers from degradation due to its good binding capacity to dentin collagen matrix, which are crucial for the maintenance of resin–dentin bond durability.

Water-free transportation (WFT) causes shrimp (Penaeus vannamei) flesh quality deterioration. However, the roles of endogenous protease-induced protein hydrolysis have been neglected in the research. In the present study, calpain zymography, gelatinase zymography, the hematoxylin-eosin staining method, and other methods were applied to investigate the response of various endogenous proteases (cathepsin, calpain, and gelatinase), the myofibril fragmentation index (MFI), and the microscopic morphology of shrimp muscle during WFT in comparison with the shrimp under the conventional water transportation strategy (WT). The results showed that the total activity of proteases in shrimp muscle increased significantly (p ≤ 0.05) after simulated transportation. Cathepsins and gelatinases were activated during WFT. No significant (p > 0.05) changes of the activity of caspase-3 and the muscle cell apoptosis rate were detected in shrimp muscle cells after WFT. In addition, the MFI increased and the gap among muscle fiber bundles enlarged after WFT. Compared with WFT, no significant (p > 0.05) effect on the activities of calpain, gelatinase, and caspase-3 in the muscle of shrimp was found after WT, and only the activity of cathepsin L significantly increased (p ≤ 0.05). Based on the findings, we concluded that the activation of various endogenous proteases was induced during WFT.

Quaternary ammonium silane-based antibacterial and anti-proteolytic cavity cleanser

Application of poly(amidoamine)-inositol hexakisphosphate conjugates for improving resin-dentine bond longevity.

Polymer conjugation optimizes EDTA as a calcium-chelating agent that exclusively removes extrafibrillar minerals from mineralized collagen

BackgroundIntrafibrillar remineralization within the hybrid layers (HLs) has recently attracted extensive attention in achieving durable resin-dentin bonds. The polyhydroxy-terminated poly(amidoamine) dendrimer (PAMAM-OH) at fourth generation becomes a desirable candidate to induce intrafibrillar remineralization to protect exposed collagen fibrils within HLs based on the size exclusion effect of fibrillar collagen. However, the remineralization process in vivo is time-consuming, during which the exposed collagen fibrils are vulnerable to enzymatic degradation, resulting in unsatisfactory remineralization. Thereby, if PAMAM-OH itself possesses concomitant anti-proteolytic activity during the induction of remineralization, it would be very beneficial to obtain satisfactory remineralization.MethodsBinding capacity tests using adsorption isotherm and confocal laser scanning microscopy (CLSM) were performed to assess if the PAMAM-OH had adsorption capacity on dentin. Anti-proteolytic testings were detected by MMPs assay kit, in-situ zymography and ICTP assay. Adhesive infiltration of resin-dentin interface and tensile bond strength before and after thermomechanical cycling were implemented to assess if the PAMAM-OH adversely affected resin-dentin bonds.ResultsAnti-proteolytic testings performed using MMPs assay kit, in-situ zymography and ICTP assay indicated that PAMAM-OH inhibited exogenous soluble MMP-9 as well as had inhibitory effect on the endogenous proteases. Adhesive infiltration of resin-dentin interface and tensile bond strength before and after thermomechanical cycling were implemented to indicate that the PAMAM-OH pretreatment had no adverse effects on immediate dentin bonding and prolonged the durability of resin-dentin bonds.ConclusionsPAMAM-OH possesses anti-proteolytic activity and prevents exposed collagen fibrils within HLs from degradation, which lays the foundation for the satisfactory intrafibrillar remineralization induced by PAMAM-OH within HLs to achieve durable resin-dentin bonds in the next work.

beta-Glucan synthase activity in plant membranes can be markedly altered by a multiplicity of apparently unrelated factors. In pea epicotyl membranes it is enhanced by low and inhibited by high concentrations of added Ca(2+), trypsin or soluble pea protease. Ca(2+) stimulates preexisting synthase activity, particularly in the presence of polycations (spermidine), but protease treatments activate and, with time, inactivate synthase zymogen. Endogenous pea protease activity is also associated with washed pea membrane and appears to be responsible for the decay observed with time in the beta-glucan synthase activity. Endogenous pea protease activity is inhibited by thiol inhibitors, e.g. iodoacetamide and Hg(2+), and by a heat-stable peptide, molecular weight approximately 10,000, that is found in supernatants of pea extracts. These protease inhibitors have the capacity to protect beta-glucan synthase activity from denaturation or its zymogen from activation due to endogenous or added protease activity. Evidence is described which supports the proposal that 1,4-beta-glucan synthase is destroyed and possibly converted to 1,3-beta-glucan synthase activity by protease action, and that the latter may then be greatly enhanced by Ca(2+) and polycations.

Effects of calcium-phosphate, laser and adhesive on dentin permeability and bond strength

Influence of endodontic irrigants on bond strength between glass-fibre posts and dentin: A systematic review of in vitro studies

The role of endogenous serine proteinase on disintegration of collagen fibers from grass carp (Ctenopharyngodon idellus)

Inspired by mussel-adhesion phenomenon in wet environment, polydopamine (PDA) was tried in recent research reports to improve the dentin adhesive interface with better strength and durability. The aim of this study was to comparatively evaluate the effect of PDA incorporated dentin adhesive systems and conventional adhesive systems on degree of conversion (DC), resin dentin bond strength (SBS), durability and MMP inhibition activity. In this study, PDA was incorporated in total-etch adhesive system (TE) and self-etch adhesive systems (SE) and degree of conversion (DC%) was evaluated using Fourier transform infrared spectroscopy. Dentin slabs obtained from 160 non-carious human premolars were divided into the following: Group 1, TE; Group 2, PDA incorporated TE adhesive (TEP); Group 3, SE; Group 4, PDA incorporated SE adhesive (SEP) and bonding procedures were performed. Samples were stored for either 24 h or 6 months, after which shear bond strength and resin dentin interface was evaluated using universal testing machine and confocal laser scanning microscopy (CLSM), respectively. The zymographic assay was performed on protein extracts obtained from dentin powder treated with each of the adhesives or with or without PDA followed by functional activity assay for MMP-2 and -9. Statistical analysis was done with two-way analysis of variance and Student t-test at significance level as p < 0.05. There was statistically no significant difference in the DC% between PDA incorporated adhesives and control. No significant reduction in SBS was observed in TEP and SEP in pre- and post-aging while CLSM showed better penetration depth of resin and lesser degradation of hybrid layer on aging. Significantly lesser MMP activity was seen in PDA incorporated adhesives qualitatively and quantitatively on zymographic and functional MMP activity assay respectively. HIGHLIGHTS Catechol moieties present in polydopamine provide anti-collagenolytic activity, MMP inhibitory activity and enhances wet adhesion. Polydopamine incorporated dentin bonding systems attempts to provide stable resin dentin bond with increased bond durability. It can inhibit both proteolytic and hydrolytic breakdown of resin–dentin bond degradation.

Mechanism study of the gel-forming ability of heat-induced gel from Peruvian hake (Merluccius gayi peruanus) surimi

To investigate urushiol's potential as a dentin cross-linking agent, promoting remineralization of etched dentin and preventing activation of endogenous proteases causing collagen degradation within the hybrid layer. The goal is to improve bond strength and durability at the resin-dentin interface. Urushiol primers with varying concentrations were prepared using ethanol and dimethyl sulfoxide (DMSO) as solvents. Dentin from healthy molars underwent grinding and acid etching for 15s, followed by a 1min application of urushiol primer. After 14 and 28days of remineralization incubation and remineralization were usedtoassessby Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR), Micro-Raman spectroscopy, X-ray Diffraction (XRD), Atomic Force Microscopy (AFM), Vickers Hardness, Scanning Electron Microscopy (SEM), and Energy X-ray dispersive spectroscopy (EDS). The overall performance of urushiol primers as dentin adhesives was observed by microtensile bond strength (μTBS) testing and nanoleakage assessment. Investigated the inhibitory properties of the urushiol primers on endogenous metalloproteinases (MMPs) utilizing in situ zymography, and the cytotoxicity of the primers was tested. Based on ATR-FTIR, Raman, XRD, EM-EDS and Vickers hardness analyses, the 0.7%-Ethanol group significantly enhanced dentin mineral content and improved mechanical properties the most. Pretreatment notably increased the μTBS of restorations, promoted the stability of the mixed layer, and reduced nanoleakage and MMPs activity after 28days. The urushiol primer facilitates remineralization in demineralized dentin, enhancing remineralization in etched dentin, effectively improving the bonding interface stability, with optimal performance observed at a 0.7 wt% concentration of the urushiol primer.