Evaluation of different irrigation techniques on dentin erosion and smear layer removal: A scanning electron microscopy study.

Aim: The purpose of the present study is to evaluate the effectiveness of different irrigation techniques EndoVac, RinsEndo, Canal CleanMax (CCMax), SonicMax, Passive Ultrasonic Irrigation (PUI), and Manual Needle Irrigation (MNI) on dentin erosion and smear layer removal using Scanning electron microscopy (SEM). Materials and Methods: Seventy extracted single-rooted teeth were instrumented with ProTaper rotary instruments. The roots were randomly divided into six experimental groups (n = 10): EndoVac, RinsEndo, CCMax, SonicMax, PUI, and MNI. Final irrigation was performed with NaOCl followed by ethylenediaminetetraacetic acid and NaOCl. Six roots served as control and were irrigated with only NaOCl. Next, the roots were sectioned in a buccolingual direction and the halves were examined by SEM. Smear layer and erosion scores were recorded in the cervical, middle and apical third of the roots. Data analysis was performed using the Kruskal-Wallis and Mann-Whitney U-tests with a level of significance α = 0.05. Results: PUI had the greatest amount of smear layer compared to other groups (P < 0.001). However, SonicMax had the greatest degree of erosion compared to RinsEndo, MNI, PUI, and EndoVac groups (P < 0.001) and CCMax group (P < 0.05). Conclusions: RinsEndo and EndoVac techniques left the least amount of smear layer while causing erosion the least compared to CCMax, PUI, and MNI techniques.

In vitro Study of the Role of Human Neutrophil Enzymes on Root Caries Progression.

The role of the host immune system in caries progression is mainly speculative, and it is believed that it entails the enzymatic degradation of the dentin organic matrix. The aim of this study was to evaluate the proteolytic effect of human neutrophil enzymes on root caries progression. For this, specimens of bovine root dentin were divided into 4 groups (n = 30): caries (C), caries + neutrophils (C + N), no caries (Control), and no caries + neutrophils (Control + N). Streptococcus mutans biofilm (105 CFU/mL) was grown on the root surface to artificially induce root carious lesions (C and C + N groups). Specimens were then exposed to neutrophils (5 × 106 cells/mL) for 48 h (C + N and Control + N groups). Caries development and neutrophil exposures were repeated a 2nd and 3rd time. Caries depth (CD) and dentin demineralization (DD) were assessed by infiltration of rhodamine B using fluorescence microscopy. Collagen fibril ultrastructure was characterized under a polarized microscope with Picrosirius red staining. There were no significant differences (p > 0.05) in CD and DD between the C and C + N groups for 1, 2, and 3 caries-neutrophil exposures. Immature collagen was significantly less present in the carious groups (C, p = 0.003; C + N, p = 0.01) than in the noncarious groups in the most superficial 200 µm. We thus concluded that human neutrophil enzymes did not influence short-term root caries progression, and immature collagen fibrils were more susceptible to degradation during S. mutans-induced root caries progression.

Evidence to the role of interflavan linkages and galloylation of proanthocyanidins at sustaining long-term dentin biomodification.

OBJECTIVES: The interactivity of proanthocyanidins (PACs) with collagen modulates dentin matrix biomechanics and biostability. Herein, PAC extracts selected based on structural diversity were investigated to determine key PAC features driving sustained effects on dentin matrices over a period of 18months. METHODS: The chemical profiles of PAC-rich plant sources, Pinus massoniana (PM), Cinnamomum verum (CV) and Hamamelis virginiana (HV) barks, as well as Vitis vinifera (VV) seeds, were obtained by diol HPLC analysis after partitioning of the extracts between methyl acetate and water. Dentin matrices (n=15) were prepared from human molars to determine the apparent modulus of elasticity over 18months of aging. Susceptibility of the dentin matrix to degradation by endogenous and exogenous proteases was determined by presence of solubilized collagen in supernatant, and resistance to degradation by bacterial collagenase, respectively. Data were analyzed using ANOVA and Games-Howell post hoc tests (α=0.05). RESULTS: After 18months, dentin matrices modified by PM and CV extracts, containing only non-galloylated PACs, were highly stable mechanically (p<0.05). Dentin matrices treated with CV exhibited the lowest degradation by bacterial collagenase after 1h and 18months of aging (p<0.05), while dentin matrices treated with PM showed the least mass loss and collagen solubilization by endogenous enzymes over time (p<0.05). SIGNIFICANCE: Resistance against long-term degradation was observed for all experimental groups; however, the most potent and long-lasting dentin biomodification resulted from non-galloylated PACs.

Centrifugal partition chromatography enables selective enrichment of trimeric and tetrameric proanthocyanidins for biomaterial development.

Proanthocyanidins (PACs) find wide applications for human use including food, cosmetics, dietary supplements, and pharmaceuticals. The chemical complexity associated with PACs has triggered the development of various chromatographic techniques, with countercurrent separation (CCS) gaining in popularity. This study applied the recently developed DESIGNER (Depletion and Enrichment of Select Ingredients Generating Normalized Extract Resources) approach for the selective enrichment of trimeric and tetrameric PACs using centrifugal partition chromatography (CPC). This CPC method aims at developing PAC based biomaterials, particularly for their application in restoring and repairing dental hard tissue. A general separation scheme beginning with the depletion of polymeric PACs, followed by the removal of monomeric flavan-3-ols and a final enrichment step produced PAC trimer and tetramer enriched fractions. A successful application of this separation scheme is demonstrated for four polyphenol rich plant sources: grape seeds, pine bark, cinnamon bark, and cocoa seeds. Minor modifications to the generic DESIGNER CCS method were sufficient to accommodate the varying chemical complexities of the individual source materials. The step-wise enrichment of PAC trimers and tetramers was monitored using normal phase TLC and Diol-HPLC-UV analyses. CPC proved to be a reliable tool for the selective enrichment of medium size oligomeric PACs (OPACs). This method plays a key role in the development of dental biomaterials considering its reliability and reproducibility, as well as its scale-up capabilities for possible larger-scale manufacturing.

The effect of ultrasonically activated irrigation and laser based root canal irrigation methods on debris removal.

PURPOSE: To test the efficacy of debris removal of 5 in vitro irrigation protocols: conventional irrigation, irrigation activated by ultrasound, Er:YAG laser with photon-induced photoacoustic streaming (PIPS) tip, and 2 diode laser techniques. METHODS: The root canals of 75 extracted human canine teeth were enlarged and bisected longitudinally. Standardized grooves were prepared 2-6 mm from the root apex and filled with 0.5 mm of dentine debris. The teeth were reassembled and irrigated using 1 of the following techniques (n = 15/technique): (1) conventional irrigation, (2) diode laser irrigation - tip placed 2 mm from working length, (3) diode laser irrigation - tip placed at root canal orifice, (4) Er:YAG laser - PIPS, and (5) ultrasonically activated irrigation. The root segments were disassembled, and the amount of remaining debris was evaluated by 3 examiners under a stereomicroscope at ×20 magnification using a 4-grade scoring system. The data were statistically analyzed using Kruskal-Wallis and Dunn's multiple comparison tests, with a 95% confidence level (p = 0.05). Cohen's kappa test was used to evaluate inter-observer and intra-observer agreement. RESULTS: Ultrasonically activated irrigation removed significantly more dentinal debris than conventional irrigation (p = 0.016), but there were no significant differences between the other groups. CONCLUSIONS: Ultrasonically activated irrigation was more effective than conventional irrigation in the removal of apically placed dentine debris.

Encapsulation of grape seed extract in polylactide microcapsules for sustained bioactivity and time-dependent release in dental material applications.

OBJECTIVE: To sustain the bioactivity of proanthocyanidins-rich plant-derived extracts via encapsulation within biodegradable polymer microcapsules. METHODS: Polylactide microcapsules containing grape seed extract (GSE) were manufactured using a combination of double emulsion and solvent evaporation techniques. Microcapsule morphology, size distribution, and cross-section were examined via scanning electron microscopy. UV-vis measurements were carried out to evaluate the core loading and encapsulation efficiency of microcapsules. The bioactivity of extracts was evaluated after extraction from capsules via solvent partitioning one week or one year post-encapsulation process. Fifteen human molars were cut into 7mm×1.7mm×0.5mm thick mid-coronal dentin beams, demineralized, and treated with either encapsulated GSE, pristine GSE, or left untreated. The elastic modulus of dentin specimens was measured based on three-point bending experiments as an indirect assessment of the bioactivity of grape seed extracts. The effects of the encapsulation process and storage time on the bioactivity of extracts were analyzed. RESULTS: Polynuclear microcapsules with average diameter of 1.38µm and core loading of up to 38wt% were successfully manufactured. There were no statistically significant differences in the mean fold increase of elastic modulus values among the samples treated with encapsulated or pristine GSE (p=0.333), or the storage time (one week versus one year storage at room temperature, p=0.967). SIGNIFICANCE: Polynuclear microcapsules containing proanthocyanidins-rich plant-derived extracts were prepared. The bioactivity of extracts was preserved after microencapsulation.

Assessing Collagen and Micro-permeability at the Proanthocyanidin-treated Resin-Dentin Interface.

PURPOSE: To establish a fluorescence-based method to simultaneously assess micro-permeability and collagen cross-linking induced by chemical agents at the resin-dentin interface. MATERIALS AND METHODS: Three chemical agents were investigated (proanthocyanidin-rich grape seed extract: GSE; carbodiimide hydrochloride/N-hydroxysuccinimide: EDC/NHS; glutaraldehyde: GD) along with a control (distilled water) as primers applied on flat occlusal dentin surfaces of 48 teeth and restored with two commercially available etch-and-rinse adhesives. Resin-dentin interfaces were polished and infiltrated with rhodamine-B solution for confocal laser scanning microscopy analysis. Parameters were chosen that would allow acquisition of a simultaneous appearance of collagen and interfacial micro-permeability (rhodamine-B). Fluorescence emission intensity (FEI) was converted into numerals and values were calculated for each group. Data were statistically analyzed using one-way ANOVA and post-hoc Scheffe's and multiple comparisons tests (α = 0.05). T-tests with Pearson correlations were used to investigate correlations between collagen cross-linking and micro-permeability. RESULTS: The FEI of collagen was the highest for GD, followed by GSE, with no significant differences between EDC/ NHS and the control group (p > 0.05). Micro-permeability was significantly affected by the adhesives (p < 0.05). Micro- permeability was the lowest for GSE groups, regardless of the adhesives (p < 0.001). Weak correlations were found between micro-permeability and collagen auto-fluorescence. CONCLUSIONS: Non-enzymatic collagen cross-linking induced by GSE and GD can be detected by increased collagen auto-fluorescence, and results in reduced interfacial micro-permeability. Increased collagen auto-fluorescence was correlated with fluorescent collagen cross-links and decreased micro-permeability at the resin-dentin interface. Collagen auto-fluorescence is a useful tool to detect auto-fluorescent exogenous cross links and their potential impact on the quality of the resin-dentin interface.

Collagen-collagen interactions mediated by plant-derived proanthocyanidins: A spectroscopic and atomic force microscopy study.

UNLABELLED: Collagen cross-linkings are determinant of biological tissue stability and function. Plant-derived proanthocyanidins (PACs) mimic different hierarchical levels of collagen cross-links by non-enzymatic interactions resulting in the enhancement to the biomechanics and biostability of collagen-rich tissues such as dentin. This study investigated the interaction of PACs from Vitis vinifera grape seed extract with type I collagen in solubilized form and in the demineralized dentin matrix (DDM) by fluorescence spectral analysis; collagen-collagen binding forces in presence of cross-linking solutions by atomic force microscopy (AFM); and spectroscopic analysis of the DDM using attenuated total reflectance Fourier transform-infrared spectroscopy (ATR-FTIR). Glutaraldehyde (GA) and carbodiimide hydrochloride (EDC) with known cross-linking mechanisms were selected for comparative analyses. Changes in fluorescence upon interaction of solubilized type I collagen with PACs, EDC and GA reflected pronounced modifications in collagen conformation. PACs also promoted stronger collagen-collagen fibrils interaction than EDC and GA. A new feature was observed using ATR-FTIR spectroscopic analysis in PACs-treated collagen and DDM. The findings suggest covalent interactions between collagen and PACs. The mechanisms of interaction between PACs-collagen hold attractive and promising tissue-tailored biomedical applications and the binding forces that potentially drive such interaction were characterized. STATEMENT OF SIGNIFICANCE: Connective tissues such as skin, bone and dentin are mainly composed of type I collagen, which is cross-linked to promote tissue stability, strength and function. Novel therapies using substances that mimic cross-links have been proposed to promote repair of collagen-based-tissues. In dentistry, naturally occurring proanthocyanidins (PACs) have the potential to enhance dentin mechanical properties and reduce its enzymatic degradation, but their mechanisms of cross-linking are unclear. The present study investigated the specific interactions between PACs-type I collagen in purified and dentin collagen and compared to the well described cross-linking mechanisms promoted by synthetic chemical substances. Findings reveal that covalent-like bonds are induced by plant PACs in type I collagen as well as in complex dental native tissue, promoting strong collagen-collagen interactions.

Time-dependent effectiveness of the intracanal medicaments used for pulp revascularization on the dislocation resistance of MTA.

BACKGROUND: The aim of the present study was to evaluate the time-dependent effectiveness of the intracanal medicaments used in pulp revascularization on the dislocation resistance of mineral trioxide aggregate (MTA). METHODS: One hundred ninety-two extracted human maxillary incisor teeth were sectioned apically 12 mm below and coronally 2 mm above the cemento-enamel junction. Roots were enlarged to size 40 (Protaper F4). Next, Peeso reamers from #1 to #5 were used sequentially. Sodium hypochlorite (2.5 %), EDTA (17 %), and distilled water were used in final irrigation. The specimens were randomly divided into four groups (n = 48): Group 1, in which triple antibiotic paste (TAP) (ciprofloxacin + metronidazole + minocycline) was prepared and delivered into the canals using a lentulo spiral; Group 2, in which double antibiotic paste (DAP) (ciprofloxacin + metronidazole) was placed into the canals; Group 3, in which calcium hydroxide paste (CH) (calcium hydroxide + distilled water) was introduced into the roots; and Group 4 (control), in which no medicament was applied into the root canals. Then, the samples were kept in saline solution for 2, 4, and 12 weeks, after which time 16 roots were selected randomly from each group, representing the samples of each time point. After removal of the medicaments, MTA was placed into the coronal third of the roots, and the samples were incubated for 7 days. A push-out test was used to measure the dislocation resistance (DR) of MTA. The data were analyzed using a two-way ANOVA followed by Tukey's pairwise comparisons (p = 0.05). RESULTS: The time factor displayed a significant effect on the DR of MTA (p < 0.05). All medicaments resulted in significantly smaller DR values after 12 weeks compared to after 1 week (p < 0.05). A significant unfavorable effect of TAP and DAP was observed as early as 2 weeks after the application, while 2 and 4 weeks after the application of CH there was no effect on the DR of MTA. No significant differences were found between the time points in the control group (p > 0.05). CONCLUSION: The type and the intracanal duration of medicaments used for pulp revascularization should be chosen carefully to provide maximum antimicrobial effect while creating a favorable environment both for stem cell attachment and MTA adhesion.

Effect of spreader size on microleakage of roots filled with cold lateral compaction technique.

OBJECTIVES: To evaluate the effect of spreader size on apical leakage of maxillary incisor teeth. MATERIALS AND METHODS: A total of 75 permanent human teeth with no carious and no fracture or crack were used for this study. After removing the crown from the cementoenamel junction and the standardization of the root lengths, the specimens were randomly divided into five groups: Group 1 - Roots were not instrumented. Group 2 - Root canals were enlarged using the step-back technique to a #40 file and filled using cold lateral compaction (CLC) of gutta-percha (GP). Group 3 - During the filling procedure, the first spreader used was size 40. Group 4 - The first spreader used was size 35. Group 5 - The initial spreader used was size 25. The amount of leakage through the filled root canals was evaluated by computerized fluid filtration model. Statistical analyzes were done using Kruskal-Wallis test and Mann-Whitney test (P < 0.05). RESULTS: There were statistically significant differences among the groups (P < 0.05). While the uninstrumented group (Group 1) had no leakage, instrumented but not filled roots (Group 2) demonstrated the highest leakage values. There were no differences between Group 3 and 4. Group 5 showed significantly less leakage than Group 3 and 4. CONCLUSION: Spreader size used during CLC of GP appeared to be a significant factor on apical leakage of roots. Using smaller size spreader during CLC may provide relatively less leakage.

Effect of storage solutions on microhardness of crown enamel and dentin.

OBJECTIVE: The aim of this study was to determine alterations in microhardness of crown dentin and enamel, after 2 and 12-month storage in de-ionized water, 0.2% glutaraldehyde, Hanks' Balanced Salt Solution (HBSS), 0.1% sodium hypochlorite (NaOCl) or 0.1% thymol. MATERIALS AND METHODS: Freshly extracted, nonsterile 60 intact human premolars were distributed to five groups. Six teeth from each group were evaluated after two, and other six teeth were evaluated after 12 months storage. After grinding and polishing of teeth, Vickers hardness was evaluated with making indentations on enamel and dentin, using a pyramid diamond indenter tip exerting 100 g load for 15 s. RESULTS: After 2 months storage in solutions, range of the hardness values (HV) of enamel and dentin were in between 315-357 and 64-67, respectively. However, 12 months storage of the teeth resulted in a statistically significant decrease in microhardness when compared to microhardness of teeth stored for 2 months (P = 0.001). Although the differences were not significant regarding solutions, all solutions decreased the microhardness both in enamel and dentin (P > 0.05). However, decrease in microhardness was relatively less in de-ionized water and thymol solutions while glutaraldehyde decreased microhardness the most: 63% for enamel and 53% for dentin. CONCLUSIONS: Microhardness of enamel and dentin was in an acceptable range when teeth were stored for 2 months in de-ionized water, glutaraldehyde, HBSS, NaOCl or in thymol; thus, teeth kept up to 2 months in these solutions can be used for mechanical in vitro tests. However, 12 months storage significantly decreased the microhardness of enamel and dentin.

Quantification of antibiotic in biofilm-inhibiting multilayers by 7.87 eV laser desorption postionization MS imaging.

The potential of laser desorption postionization mass spectrometry (LDPI-MS) imaging for small molecule quantification is demonstrated here. The N-methylpiperazine acetamide (MPA) of ampicillin was adsorbed into polyelectrolyte multilayer surface coatings composed of chitosan and alginate, both high molecular weight biopolymers. These MPA-ampicillin spiked multilayers were then shown to inhibit the growth of Enterococcus faecalis biofilms that play a role in early stage infection of implanted medical devices. Finally, LDPI-MS imaging using 7.87 eV single-photon ionization was found to detect MPA-ampicillin within the multilayers before and after biofilm growth with limits of quantification and detection of 0.6 and 0.3 nmol, respectively. The capabilities of LDPI-MS imaging for small molecule quantification are compared to those of MALDI-MS. Furthermore, these results indicate that 7.87 eV LDPI-MS imaging should be applicable to quantification of a range of small molecular species on a variety of complex organic and biological surfaces. Finally, while MS imaging for quantification was demonstrated here using LDPI, it is a generally useful strategy that can be applied to other methods.

Identification and imaging of peptides and proteins on Enterococcus faecalis biofilms by matrix assisted laser desorption ionization mass spectrometry.

The heptapeptide ARHPHPH was identified from biofilms and planktonic cultures of two different strains of Enterococcus faecalis, V583 and ATCC 29212, using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). ARHPHPH was also imaged at the boundary of cocultured, adjacent E. faecalis and Escherichia coli (ATCC 25922) biofilms, appearing only on the E. faecalis side. ARHPHPH was proteolyzed from κ-casein, a component in the growth media, by E. faecalis microbes. Additionally, top down and bottom up proteomic approaches were combined to identify and spatially locate multiple proteins within intact E. faecalis V583 biofilms by MALDI-MS. The resultant tandem MS data were searched against the NCBInr E. faecalis V583 database to identify thirteen cytosolic and membrane proteins which have functional association with the cell surface. Two of these proteins, enolase and GAPDH, are glycolytic enzymes known to display multiple functions in bacterial virulence in related bacterial strains. This work illustrates a powerful approach for discovering and localizing multiple peptides and proteins within intact biofilms.

Apical microleakage of root-end cavities prepared by Er, Cr: YSGG laser.

The aim of this study is to assess the apical microleakage of the composite filled root-end cavities prepared by an Erbium, Chromium: Yttrium-Scandium-Gallium-Garnet laser. Fifty-five maxillary incisor teeth were enlarged and filled. Following the apical resection, root-end cavities were prepared using conventional methods: either using a bur (n = 30) or an ultrasonic retrotip (n = 15). Root-end cavities of the 15 teeth in the bur group were finished with the laser at 3.5 W. All root-end cavities were filled using two-step self-etching primer and composite resin. After 4 months of storage, apical microleakage was measured by a fluid filtration method. Microleakage of composite filled root-end cavities that were prepared by Er, Cr: YSGG was significantly larger than those made by conventional methods (p < 0.05). In conclusion, using the Er, Cr: YSGG laser has no advantages over conventional root-end cavity preparation methods when a composite filling material is used to seal root-end cavities.