METHODS: NIH 3T3 mouse fibroblasts were cultured in Dulbecco's Modified Eagle's Medium and incubated for 3 days. The cells (3×104) were seeded on the pulpal side of dentine discs and the occlusal side of the discs were treated with different cavity disinfectants: Group 1: de-ionized water (control); Group 2: 2% chlorhexidine (CHX); Group 3: 2% QAS; Group 4: 5% QAS, and Group 5: 10% QAS. Cell morphology of NIH 3T3 cells was examined using scanning electron microscopy (SEM) and cell viability was assessed using Trypan blue assay. The eluates were collected and applied on cells seeded in 24-well plates. The total protein production, alkaline phosphatase activity and deposition of mineralized nodules were evaluated after 7 and 14 days. Immunofluorescence staining was performed on the samples with primary antibodies of CD68+, CD80+, and CD163+ assessing the macrophage M1/M2 phenotypes. The macrophages were imaged using a confocal scanning light microscope with an excitation wavelength of 488nm.
RESULTS: No significant difference in cell viability (p<0.0001), total protein production (p<0.01) and mineralized nodule production (p<0.05) was found between 2% QAS and the control, which was significantly higher than 2% CHX, 5% and 10% QAS after 14 days. Alkaline phosphatase production of 2% QAS was significantly lower than the control (p<0.001), but higher than 2% CHX at 14 days. The M1/M2 macrophage ratio was also significantly lower in the 2% and 10% QAS groups (p<0.05) compared to the control and 2% CHX groups.
SIGNIFICANCE: The 2% QAS cavity disinfectant does not have cytotoxic effects on 3T3 NIH mouse fibroblast cells and the predominance of the anti-inflammatory phenotype after its application may stimulate healing and tissue repair.
MATERIALS AND METHODS: Root discs (2 mm thickness) were cut apical to CEJ and sectioned into quadrants. HIFU setup with bowl-shaped piezo ceramic transducer submerged in a water tank was used for exposure on each specimen for 15 s, 30 s or 60 s. The specimens of the control group were left without any HIFU exposure. HIFU was generated with a continuous sinusoidal wave of 120Vpp amplitude, 250 KHZ resonance-frequency and highest ultrasonic pressure of ∼10 bar at the focus. Specimens for SEM were viewed, and micro-topography characterization performed, using AFM and Ra parameter and surface area (SA) calculated by specialized SPM surface analysis software. For nano-indentation testing, experiments were carried out using AFM. Macrophage cell isolation and culturing was performed on cementum to receive the HIFU treatment at different time periods. Raman spectroscopy were scanned to create spectra perpendicular to the cementum substrate to analyze generation of standard spectra for Raman intensity ratio of hydroxyapatite normalized to the peaks ν1 960 cm-1. Data was expressed as means ± standard deviations and analyzed by one-way ANOVA in term of Ra, SA, H and Er. Different points for fluorescence intensity ratio were analyzed by Raman using Wilcoxon rank sum test.
RESULTS: HIFU exposure at 60 s removed the smear layer and most of cementum appeared smoothened. AFM characterisation, showed a slight decrease in the irregularity of the surface as exposure time increased. Intact macrophages can be identified in control and all experimental HIFU groups. The level of fluorescence for the control and HIFU 15 and 30 s were low as compared to HIFU 60 s.
CONCLUSION: If HIFU can be successfully implemented, it may be a possible alternative to current methods used in periodontal therapy to achieve smooth root surfaces.
METHODS: HA having nanorods structure were synthetized using ultrasonication with precipitation method. HA nanorods were characterized by TEM for average-size/shape. Following phosphoric acid demineralization, dentine specimens were treated with HA-nanorods with/without subsequent HIFU exposure for 5 s, 10 s and 20 s then stored in artificial saliva for 1-month. Dentine specimens were characterized using different SEM and Raman spectroscopic techniques. In addition, the biochemical stability and HA-nanorods were examined using ATR-FTIR to observe attachment of nanoparticles. Also, surface nanoindentation properties were evaluated using AFM in tapping-mode.
RESULTS: HA-nanorods displayed well-defined, homogenous plate-like nanostructure. TEM revealed intact collagen-fibrils network structure with high density due to obliteration of interfibrillar spaces with clear evidence of remineralization in combined HA/HIFU treatment. With HA-nanorods treatment collagen-network structure was visible, consisting of fibrils interlaced into a compact pattern with evidence of minerals deposition. AFM investigation revealed clear mineral formation with the increase of HIFU exposure time. Bands associated with inorganic phase dominate well in HIFU exposed specimens with PO stretching within dentine mineral identified at 960 cm-1. Characteristic dentine structure for control and HIFU 20 s specimens is reflected as oscillatory mean Amide-I intensity with measurement giving a precise sinusoidal response of polarization angle β within dentinal tissue. Nanoindentation testing showed a gradual significant increase in elastic-modulus with the increase in HIFU exposure time after 1-month storage. FTIR spectrum of the HIFU exposed dentine displayed bands at 1650 cm-1, 1580 cm-1 and 1510 cm-1 that can be attributed to Amide-I, II and III.
SIGNIFICANCE: The synergetic effect of HIFU exposure on remineralization potential of demineralized dentine-matrix following nano-hydroxyapatite treatment was revealed. This synergetic effect is dependent on HIFU exposure time.
MATERIAL AND METHODS: Eighty-seven individuals (42 individuals consuming NW and 45 controls) were included. Clinical (plaque index, bleeding on probing, probing depth and clinical attachment loss) and radiographic (marginal bone loss) periodontal parameters were compared among NW and control groups. Gingival specimens were taken from subjects in NW and control groups, assessed for ICTP and CTX levels (using ELISA) and analyzed using micro-Raman spectroscopy. The significance of differences in periodontal parameters between the groups was determined using Kruskal-Wallis and Mann-Whitney U tests. The percent loss of dry mass over exposure time and the rate of release of ICTP and CTX from all groups were compared using the paired t-test to examine the effects of exposure time.
RESULTS: Clinical and radiographic periodontal parameters were significantly higher in the NW group than the control group (P
METHODS: VE-TPGS was added to RF-solution, at RF/VE-TPGS (w/w) ratios of 0.125/0.250 and 0.125/0.500. Demineralized dentine beams were used (10wt.% phosphoric acid), rinsed using deionized-water and analysed using ELISA (Human MMP2 ELISA; Human CTSK/Cathepsin-K for MMP2 and Cathepsin K analysis). AFM of dentine collagen-fibrils structure was done before and after dentine specimens' placement in mineralization solution and tested after 14days in artificial saliva/collagenase (AS/Co) solution. The specimens were tested after 24h in mineralization solution for surface/bulk elastic modulus. Nano-indentation was carried out for each specimen on intertubular-dentine with lateral spacing of 400nm. Reduced elastic-modulus and nano-hardness were calculated and collagen content was determined using hydroxyproline-assay. Micro-Raman were performed. TEM was carried out to study structural variations of dentine-collagen in artificial-saliva (collagenase). Data were presented as mean±standard deviation and analyzed by SPSS v.15, by analysis of variance.
RESULTS: Synergetic effect of VE-TPGS was observed with RF through higher structural integrity of dentine collagen-fibrils shown by TEM/AFM. Superior surface/bulk mechanical stability was shown by nano-indentation/mechanical testing. Improvement in collagenase degradation resistance for hydroxyproline release was observed and lower endogenous-protease release of MMP-2/Cathepsin-K. Raman-analysis analysed chemical interactions between RF and collagen confirming structural-integrity of collagen fibrils after crosslinking. After 24h mineralization, AFM showed mineral depositions in close association with dentine-collagen fibrils with RF/VE-TPGS formulations.
SIGNIFICANCE: Potential synergetic effect of RF/VE-TPGS was observed by reflection of higher structural integrity and conformational-stability of dentine-collagen fibrils.
METHODS: The interaction between HIFU waves and dentin-surface in terms of structural, mechanical and chemical variations were investigated by SEM, TEM, AFM, nano-indentation and Raman-analysis. The bonding between HIFU-treated dentin and two-step, etch-and-rinse, adhesive was preliminary explored by characterizing dentin-bound proteases activities, resin-dentin interfacial morphology and bond-durability with HIFU exposure at different time-points of 60, 90 and 120 s compared to conventional acid-etching technique.
RESULTS: With the increase in HIFU exposure-time from 60-to-120 s, HIFU waves were able to remove the smear-layer, expose dentinal-tubules and creating textured/rough dentin surface. In addition, dentin surfaces showed a pattern of interlocking ribbon-like minerals-coated collagen-fibrils protruding from the underlaying amorphous dentin-background with HIFU exposure for 90 s and 120 s. This characteristic pattern of dentin-surface showing inorganic-minerals associated/aligned with collagen-fibrils, with 90-to-120 s HIFU-treatment, was confirmed by the Raman-analysis. HIFU-treated specimens showed higher nano-indentation properties and lower concentrations of active MMP-2 and Cathepsin-K compared to the acid-etched specimens. The resin-dentin bonded interface revealed the partial/complete absence of the characteristic hybrid-layer formed with conventional etch-and-rinse bonding strategy. Additionally, resin-infiltration and resin-tags formation were enhanced with the increase in HIFU exposure-time to 120 s. Although, all groups showed significant decrease in bond-strength after 12 months compared to 24 h storage in artificial saliva, groups exposed to HIFU for 90 s and 120 s showed significantly higher μTBS compared to the control acid-etched group.
SIGNIFICANCE: The implementation of HIFU-technology for dental hard-tissues treatment could be of potential significance in adhesive/restorative dentistry owing to its ability of controlled, selective and localised combined tissue alteration/ablation effects.
METHODS: Databases (MEDLINE via PubMed; EMBASE; Cochrane Central Register of Controlled Trials and Cochrane Oral Health Group Trials Register databases) were searched from 1980 up to and including July 2016. The addressed PICO question was: "What effect does aPDT and/or LT as an adjunct to SRP have on the GCF inflammatory proteins in periodontal disease patients?"
RESULTS: Eight studies used aPDT while 10 studies used laser alone. Eight cytokines including tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, IL-8, IL-10, interferon gamma (IFN-γ), matrix metalloproteinase (MMP)-8 and granulocyte colony-stimulating factor (GM-CSF) were eligible for qualitative analysis for aPDT and LT studies. Four aPDT studies showed significant reduction in IL-1β while one study showed significant reduction in TNF-α levels after aPDT application at follow-up. One study showed significant reduction of IFN-γ, IL-8 and GM-CSF levels after aPDT at follow-up. IL-1β significantly reduced in 4 LT studies, while one study showed significant decrease for IL-6 and TIMP-1 levels. MMP-8 and TNF-α showed significant reduction in three and one study respectively.
CONCLUSION: It remains debatable whether adjunctive aPDT or LT is effective in the reduction of GCF inflammatory proteins in periodontal disease due to non-standard laser parameters and short follow up period. These findings should be considered preliminary and further studies with long-term follow up and standardized laser parameters are recommended.
MATERIALS AND METHODS: Single- (Streptococcus mutans or Lactobacillus acidophilus), dual- (Streptococcus mutans/Lactobacillus Acidophilus), and multi-species (Streptococcus mutans, Actinomyces naeslundii, and Streptococcus sanguis) biofilms were grown on acid-etched dentine discs. Biofilms were incubated (120 min/37 °C) and allowed to grow for 3 days anaerobically. Discs (no treatment) served as control (group 1). Groups II, III, IV, and V were then treated with 2% chlorhexidine, and 2%, 5%, and 10% QAS (20 s). Discs were returned to well plates with 300 μL of bacterial suspension and placed in anaerobic incubator at 37 °C and biofilms redeveloped for 4 days. Confocal microscopy, Raman, CFU, and MTT assay were performed.
RESULTS: Raman peaks show shifts at 1450 cm-1, 1453 cm-1, 1457 cm-1, 1460 cm-1, and 1462 cm-1 for control, 2% CHX, 2%, 5%, and 10% QAS groups in multi-species biofilms. There was reduction of 484 cm-1 band in 10% QAS group. CLSM revealed densely clustered green colonies in control group and red confluent QAS-treated biofilms with significantly lower log CFU for single/dual species. Metabolic activities of Streptococcus mutans and Lactobacillus acidophilus decreased with increasing QAS exposure time.
CONCLUSION: Quaternary ammonium silanes possess antimicrobial activities and inhibit growth of cariogenic biofilms.
CLINICAL SIGNIFICANCE: Available data demonstrated use of QAS as potential antibacterial cavity disinfectant in adhesive dentistry. Experimental QAS can effectively eliminate caries-forming bacteria, when used inside a prepared cavity, and can definitely overcome problems associated with present available cavity disinfectants.
METHODS: TPAu nanoparticles were fabricated from 0.31-g tetrachloroauric acid and 0.38-g of N-(2-mercaptopropionyl) glycine (2.4-mmol). Then co-dissolved using 35-mL of 6:1 methanol/acetic acid and mixed using NaBH4. EDC (0.3-M) was conjugated to TPAu nanoparticles at TPAU/EDC-0.25:1, and TPAU/EDC-0.5:1 treatment formulations ratios. Dentin specimens treated with 0.3-M EDC solution alone or left untreated were used as control. Nanoparticles formulations were characterized in term of particles morphology and size, Zeta potential, thermogravimetric analysis and small-angle X-ray scattering. Dentin substrates were characterized in term of TEM investigation, dentin proteases characterization, hydroxyproline liberation, elastic modulus measurement, Raman analysis and confocal microscopy viewing.
RESULTS: TEM evaluation of tiopronin protected gold nanoparticles dispersion revealed nano-clusters formations in both groups. However, based on our TEM measurements, the particle-size was ranging from ˜20 to 50 nm with spherical core-shape which were almost similar for both TPAu/EDC ratios (0.5:1 and 0.25:1). Zeta potential measurements indicate negative nanoparticles surface charge. SAXS profiles for both formulations, suggest a typical profile for uni-lamellar nanoparticles. Superior dentin collagen cross-linking effect was found with the TPAu/EDC nanoparticles formulations compared to the control and EDC treated groups.
SIGNIFICANCE: Cross-linking of dentin collagen using TPAu coupled with EDC through TPAu/EDC nanoparticles formulations is of potential significance in improving the biodegradation resistance, proteases inhibition, mechanical and structural stability of demineralized dentin substrates. In addition, the cross-linking effect is dependent on TPAu/EDC ratio, whereas higher cross-linking effect was found at TPAu/EDC ratio of 0.5:1.
MATERIALS AND METHODS: REVEAL dental fluorescence loupes and headlight system were used. Occlusal enamel was removed, and mid-coronal dentine was exposed. Carious artificial lesion was created. Streptococcus mutans, Actinomyces naeslundii, and Streptococcus sanguis were used. The assessment was performed using two diagnostic methods: naked eye and Design for Vision Glasses with inter examiner blinding using two calibrated examiners. After 7 days, Raman measurements were made on dentin disc specimens with 785 nm wavelength. The bacterial counts in colony-forming units (CFU) were used to examine the growth kinetics of biofilms. The collagen fibril structure within the discs was performed using Transmission Electron Microscope. Scanning Electron Microscope was used to image samples at various magnifications. FISH was performed with specimens fixed in 4% paraformaldehyde in phosphate-buffered saline. Reproducibility was measured by Cohen kappa scores, values of which range from 0 for less than chance agreement to 1 for almost perfect agreement (p
METHODS: Root canal preparation was performed using stainless steel K-files™ and F4 size protaper with irrigation protocols of 6% NaOCl + 2% CHX; 3.5% QIS; 2% QIS and sterile saline. Biofilms were prepared using E. faecalis adjusted and allowed to grow for 3 days, treated with irrigants, and allowed to grow for 7 days. AFM was performed and surface free energy calculated. MC3T3 cells were infected with endo irrigant treated E. faecalis biofilms. Raman spectroscopy of biofilms were performed after bacterial re-growth on root dentine and exposed to different irrigation protocols and collagen fibers analysed collagen fibers using TEM. Antimicrobial potency against E. faecalis biofilms and cytoxicity against 3T3 NIH cells were also. Resin penetration and MitoTracker green were also evaluated for sealer penetration and mitochondrial viability. Data were analysed using One-way ANOVA, principal component analysis and post-hoc Fisher's least-significant difference.
RESULTS: Elastic moduli were maintained amongst control (5.5 ± 0.9) and 3.5% QIS (4.4 ± 1.1) specimens with surface free energy higher in QIS specimens. MC3T3 cells showed reduced viability in 6%NaOCl+2%CHX specimens compared to QIS specimens. DNA/purine were expressed in increased intensities in control and 6% NaOCl + 2% CHX specimens with bands around 480-490 cm-1 reduced in QIS specimens. 3.5% QIS specimens showed intact collagen fibrillar network and predominantly dead bacterial cells in confocal microscopy. 3.5% QIS irrigant formed a thin crust-type surface layer with cytoplasmic extensions of 3T3NIH spread over root dentine. Experiments confirmed MitoTracker accumulation in 3.5% treated cells.
SIGNIFICANCE: Novel QIS root canal irrigant achieved optimum antimicrobial protection inside the root canals facilitating a toxic effect against the Enterococcus faecalis biofilm. Root dentine substrates exhibited optimum mechanical properties and there was viability of fibroblastic mitochondria.
MATERIALS AND METHODS: Sound extracted human molars were randomly divided into: manufacturer's instructions (MI), manual blend 2 mm (MB2), and manual blend 4 mm (MB4). Occlusal enamel was removed and flattened, dentin surfaces were bonded by Prime & Bond universal (Dentsply and Optibond FL, Kerr). For the MI group, adhesives were applied following the manufacturer's instructions then light-cured. For MB groups, SDR flow+ bulk-fill flowable composite resin was applied in 2- or 4-mm increment then manually rubbed by a micro brush for 15 s with uncured dentine bonding agents and the mixture was light-cured. Composite buildup was fabricated incrementally using Ceram.X One, Dentsply nanohybrid composite resin restorative material. After 24-h water storage, the teeth were sectioned to obtain beams of about 0.8 mm2 for 24-h and thermocycled micro-tensile bond strength at 0.5 mm/min crosshead speed. Degree of conversion was evaluated with micro-Raman spectroscopy. Contraction gaps at 24 h after polymerization were evaluated and atomic force microscopy (AFM) nano-indentation processes were undertaken for measuring the hardness across the interface. Depth of resin penetration was studied using a scanning electron microscope (SEM). Bond strength data was expressed using two-way ANOVA followed by Tukey's test. Nanoindentation hardness was separately analyzed using one-way ANOVA.
RESULTS: Factors "storage F = 6.3" and "application F = 30.11" significantly affected the bond strength to dentine. For Optibond FL, no significant difference in nanoleakage was found in MI/MB4 groups between baseline and aged specimens; significant difference in nanoleakage score was observed in MB2 groups. Confocal microscopy analysis showed MB2 Optibond FL and Prime & Bond universal specimens diffusing within the dentine. Contraction gap was significantly reduced in MB2 specimens in both adhesive systems. Degree of conversion (DC) of the MB2 specimens were numerically more compared to MS1 in both adhesive systems.
CONCLUSION: Present study suggests that the new co-blend technique might have a positive effect on bond strengths of etch-and-rinse adhesives to dentine.