Biological Characteristics and Odontogenic Differentiation Effects of Calcium Silicate-Based Pulp Capping Materials

We compared calcium silicate-based pulp capping materials to conventional calcium hydroxide in terms of their biological properties and potential effects on odontogenic differentiation in human dental pulp stem cells (hDPSCs). We cultured hDPSCs on disks (7-mm diameter, 4-mm high) of ProRoot MTA (Dentsply Tulsa Dental Specialties), Biodentine (Septodont), TheraCal LC (Bisco), or Dycal (Dentsply Tulsa Dental Specialties). Cell viability was assessed with cell counting (CCK) and scanning electron microscopy (SEM). Odontogenic activity was assessed by measuring alkaline phosphatase (ALP) activity and gene expression (quantitative real-time PCR). CCK assays showed that Dycal reduced cell viability compared to the other materials (p < 0.05). SEM showed low and absent cell attachment on TheraCal LC and Dycal disks, respectively. hDPSCs exposed to TheraCal LC and Dycal showed higher ALP activity on day 6 than the control group (p < 0.05). The day-9 Runx2 expression was higher in the ProRoot MTA and TheraCal LC groups than in the control group (p < 0.05). On day 14, the ProRoot MTA group showed the highest dentin sialophosphoprotein levels (not significant; p > 0.05). In conclusion, various pulp capping materials, except Dycal, exhibited biological properties favorable to hDPSC viability. ProRoot MTA and TheraCal LC promoted higher Runx2 expression than Biodentine. Future studies should explore the odontogenic potential of pulp capping materials.     

Effect of Potassium Aluminum Sulfate Application on the Viability of Fibroblasts on a CAD-CAM Feldspathic Ceramic before and after Thermocycling

Potassium aluminum sulfate (alum) is a known adjuvant, which has been used as a mordant in textile industry for color fixation. This material has potential to be incorporated into dentistry for color stability, yet its toxicity first needs to be evaluated. The present study aimed to evaluate the cytotoxic potential of potassium aluminum sulfate (alum) on fibroblasts when applied onto feldspathic ceramic before and after thermocycling. Forty-eight feldspathic ceramic specimens were divided into four groups (FC: no alum application or thermocycling; FCT: thermocycling without alum application; FA: alum application without thermocycling; FAT: alum application and thermocycling) (n = 12). Cell viability was assessed by using a tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphnyltetrazolium bromide assay at 24 and 72 h, and cell cultures without any ceramic specimens served as control (C). One sample from each material group was further analyzed with energy dispersive X-ray spectroscopy (EDX). Cell viability at different time intervals within each group was analyzed with Friedman tests, while Kruskal–Wallis tests were used to compare the test groups within each time interval. Pairwise comparisons were further resolved by using Wilcoxon tests (a = 0.05). C had lower (p = 0.01) and FA had higher (p = 0.019) cell viability after 72 h. After 24 h, the highest cell viability was observed in C (p ≤ 0.036). After 72 h, the differences between C and FA, C and FAT, FC and FA, and FCT and FAT were nonsignificant (p > 0.05). Cell viability was not affected by alum application or thermocycling at any time interval (p ≥ 0.631). EDX analysis showed an increase in potassium concentration in FA and FAT when compared with FC and FCT. Regardless of the time interval, alum application onto feldspathic ceramic and thermocycling did not influence the cell viability.     

Behaviour of Human Oral Epithelial Cells Grown on Invisalign® SmartTrack® Material

Invisalign aligners have been widely used to correct malocclusions, but their effect on oral cells is poorly known. Previous research evaluated the impact of aligners’ eluates on various cells, but the cell behavior in direct contact with aligners is not yet studied. In the present study, we seeded oral epithelial cells (cell line Ca9-22) directly on Invisalign SmartTrack material. This material is composed of polyurethane and co-polyester and exhibit better mechanical characteristics compared to the predecessor. Cell morphology and behavior were investigated by scanning electron microscopy and an optical cell moves analyzer. The effect of aligners on cell proliferation/viability was assessed by cell-counting kit (CCK)-8 and 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay and live/dead staining. The expression of inflammatory markers and proteins involved in epithelial barrier function was measured by qPCR. Cells formed cluster-like structures on aligners. The proliferation/viability of cells growing on aligners was significantly lower (p < 0.05) compared to those growing on tissue culture plastic (TCP). Live/dead staining revealed a rare occurrence of dead cells on aligners. The gene expression level of all inflammatory markers in cells grown on aligners’ surfaces was significantly increased (p < 0.05) compared to cells grown on TCP after two days. Gene expression levels of the proteins involved in barrier function significantly increased (p < 0.05) on aligners’ surfaces after two and seven days of culture. Aligners’ material exhibits no cytotoxic effect on oral epithelial cells, but alters their behavior and the expression of proteins involved in the inflammatory response, and barrier function. The clinical relevance of these effects has still to be established.     

Study of Cytotoxic Properties of an Experimental Preparation with Features of a Dental Infiltrant

Microinvasive dentistry is based on the treatment of early carious lesions with the use of dental infiltrants. The commercially available Icon dental infiltrant does not contain any bacteriostatic component. An experimental preparation enriched with the missing component was synthesised. The aim of this study was to evaluate the cytotoxicity of the experimental preparation. Mouse fibroblasts of the L-929 lineage were used for the in vitro study. Cell morphology and viability were assessed. In the cytotoxicity analysis, it was shown that the experimental preparation (42.8 ± 10.3) after 24 h at two-fold dilution showed similar cytotoxicity to Icon (42.7 ± 8.8) (p > 0.05), while at four-fold dilution experimental preparation (46.7 ± 3.1), it was less toxic than Icon (34.2 ± 3.1) (p < 0.05). The experimental preparation has the potential to provide an alternative to the Icon commercial preparation. Further research is needed to evaluate the cytotoxicity of the experimental preparation over a longer period of time.     

The Cytocompatibility of Silver Diamine Fluoride on Mesenchymal Stromal Cells from Human Exfoliated Deciduous Teeth: An In Vitro Study

Silver diamine fluoride (SDF) has been used for many years for the treatment of caries, and minimally invasive dentistry concepts have made it popular again. The fact that its application does not require the administration of anesthesia makes its use in children more desirable. The aim of this study was to determine the cytotoxicity of two new commercial SDF products: Riva Star (SDI Dental Limited) and e-SDF (Kids-e-Dental) on mesenchymal stromal cells from human exfoliated deciduous teeth (SHEDs). SHEDs were exposed to SDF products at different concentrations (0.1%, 0.01% and 0.005%). Then different assays were performed to evaluate their cytocompatibility on SHEDs: IC50, MTT, cell migration (wound healing), cell cytoskeleton staining, cell apoptosis, generation of intracellular reactive oxygen species (ROS), and ion chromatography. Statistical analyses were performed using one-way ANOVA and Tukey’s post hoc test (p < 0.05). Riva Star Step 2 showed the same cell metabolic activity when compared to the control condition at any time and concentration. Meanwhile, e-SDF displayed high cytotoxicity at any time and any concentration (*** p < 0.001), whereas Riva Star Step 1 displayed high cytotoxicity at any time at 0.1% and 0.01% (*** p < 0.001). Only e-SDF showed a statistically significant decreased cell migration rate (*** p < 0.001) at all times and in all concentrations. At 0.1%, e-SDF and Riva Star Step 1 only showed 4.37% and 4.47% of viable cells, respectively. These results suggest that Riva Star has better in vitro cytocompatibility on SHEDs than does e-SDF. Riva Star Step 1 was found to be as cytotoxic as e-SDF, but it had better biological properties when mixed with Riva Star Step 2. Our findings suggest that Riva Star is more suitable when used in deciduous teeth due to its lower cytotoxicity compared to e-SDF.     

Cytotoxicity of Acrylic Resins,Particulate Filler Composite Resin and Thermoplastic Material in Artificial Saliva with and without Melatonin

There is limited information on the effect of melatonin on the cytotoxicity of dental materials. The study evaluated the cytotoxic effects of heat- and auto-polymerized acrylic resin, particulate filler composite resin and a thermoplastic material on L-929 fibroblast cell viability at different incubation periods in artificial saliva without and with melatonin. Disk-shaped specimens were prepared according to each manufacturer’s instructions and divided into two groups to be stored either in artificial saliva (AS) and AS with melatonin (ASM). The measurements were performed using an MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide) assay, in which the L-929 mouse fibroblasts cell culture was used. For the MTT test, extracts were examined at 1, 24, 72 h and 1 and 2 weeks. Data were analyzed using 3-way ANOVA and Tukey’s tests. No significant difference was found between groups AS and ASM (F = 0.796; p = 0.373). Incubation period significantly affected all materials tested (p < 0.001). Storing resin-based materials in artificial saliva with melatonin solution for 24 h may reduce cytotoxic effects on the fibroblast cells for which the highest effect was observed. Soaking resin prosthesis or orthodontic appliances in artificial saliva with melatonin at least 24 h before intraoral use or rinsing medium containing melatonin may be recommended for decreasing the cytotoxicity of dental resin materials.     

Using cholecystokinin to facilitate endoscopic clearance of large common bile duct stones

AIM:To evaluate the effect of cholecystokinin(CCK)during extracorporeal shockwave lithotripsy(ESWL)in the clearance of common bile duct(CBD)stones in endoscopic retrograde cholangiopancreatography(ERCP).METHODS:Between January 2007 and September2012,patients with large CBD stones who were treated with ESWL and ERCP were identified retrospectively.Patients were randomized in equal numbers to cholecystokinin(CCK)and no CCK groups.For each CCK case,a dose(3 ng/kg per min for 10 min)of sulfated octapeptide of CCK-8 was administered intravenously near the beginning of ESWL.ERCP was performed 4 h after a session of ESWL.The clearance rate of the CBD was assessed between the two groups.RESULTS:A total of 148 consecutive cases(CCK group:74,no CCK group:74)were tallied.Overall there were 234 ESWLs and 228 ERCPs in the 148 cases.The use of CCK showed a significantly higher rate of successful stone removal in the first ESWL/ERCP procedure(71.6%vs 55.4%,P=0.035),but resulted in similar outcomes in the second(42.8%vs 39.4%)and third(41.7%vs 40.0%)sessions,as well as total stone clearance(90.5%vs 83.8%).The use of mechanical lithotripsy was reduced in the CCK group(6.8%vs17.6%,P=0.023),and extremely large stone(≥30mm)removal was higher in the CCK group(72.7%vs41.7%,P=0.038).CONCLUSION:CCK during ESWL can aid with the clearance of CBD stones in the first ESWL/ERCP session.Mechanical lithotripsy usage was reduced and the extremely large stone(≥30 mm)clearance rate can be raised.     

Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy

AIM:To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action.METHODS:HepG2 cells were treated with different concentrations of cinobufacini.Cell viability was measured by methylthiazolyl tetrazolium(MTT) assay.Cell cycledistribution was analyzed by flow cytometry(FCM).Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope.Changes in morphology and ultrastructure of cells were detected by atomic force microscopy(AFM) at the nanoscale level.RESULTS:MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dosedependent manner.With the concentration of cinobufacini increasing from 0 to 0.10 mg/m L,the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1%(P 0.05).FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini.The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment,the regular reorganization of actin filaments in HepG2 cells become chaotic,while the nuclei were not damaged seriously.Additionally,high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini.It appeared as significant shrinkage and deep pores in the cell membrane,with larger particles and a rougher cell surface.CONCLUSION:Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity.     

Mapping Bone Marrow Cell Response from Senile Female Rats on Ca-P-Doped Titanium Coating

Chemical and topographical surface modifications on dental implants aim to increase the bone surface contact area of the implant and improve osseointegration. This study analyzed the cellular response of undifferentiated mesenchymal stem cells (MSC), derived from senile rats’ femoral bone marrow, when cultured on a bioactive coating (by plasma electrolytic oxidation, PEO, with Ca²⁺ and P⁵⁺ ions), a sandblasting followed by acid-etching (SLA) surface, and a machined surface (MSU). A total of 102 Ti-6Al-4V discs were divided into three groups (n = 34). The surface chemistry was analyzed by energy dispersive spectroscopy (EDS). Cell viability assay, gene expression of osteoblastic markers, and mineralized matrix formation were investigated. The cell growth and viability results were higher for PEO vs. MSU surface (p = 0.001). An increase in cell proliferation from 3 to 7 days (p < 0.05) and from 7 to 10 days (p < 0.05) was noted for PEO and SLA surfaces. Gene expression for OSX, ALP, BSP, and OPN showed a statistical significance (p = 0.001) among groups. In addition, the PEO surface showed a higher mineralized matrix bone formation (p = 0.003). In conclusion, MSC from senile female rats cultured on SLA and PEO surfaces showed similar cellular responses and should be considered for future clinical investigations.     

An enteral therapy containing medium-chain triglycerides and hydrolyzed peptides reduces postprandial pain associated with chronic pancreatitis

Background/Aim: Pain in patients with chronic pancreatitis is difficult to manage. We examined if an enteral formulation containing medium-chain triglycerides (MCT) and hydrolyzed peptides would (1) minimally stimulate the exocrine pancreas by blunting cholecystokinin release and (2) decrease pain in patients with chronic pancreatitis. Methods: In the first part of the study, on separate days, 6 healthy controls consumed a standard enteral formulation, an enteral formulation containing MCT and hydrolyzed peptides, and a high-fat meal. Baseline and postprandial plasma cholecystokinin (CCK) concentrations were analyzed. Subsequently, 8 patients with chronic pancreatitis were enrolled and instructed to complete a visual analog pain assessment for a baseline period of 2 weeks followed by three cans per day of the enteral formulation containing MCT and hydrolyzed peptides for 10 weeks. Results: Mean CCK levels for our control subjects were 0.46 ± 0.29 pM at baseline, 10.75 ± 0.45 pM in response to the high-fat meal, and 7.9 ± 1.25 pM in response to the standard enteral formulation. Of note, CCK levels were 1.43 ±0.72 pM in response to the enteral supplement containing MCT and hydrolyzed peptides. In patients with chronic pancreatitis, the average improvement in pain scores from baseline to the conclusion of the study was 61.8% (p = 0.01). This corresponded to a clinical improvement in 6 of the 8 patients. Conclusions: A complete enteral supplement containing MCT and hydrolyzed peptides minimally increases plasma CCK levels. This therapy may be effective in reducing postprandial pain associated with chronic pancreatitis.     

CIP2A facilitates apoptotic resistance of fibroblast-like synoviocytes in rheumatoid arthritis independent of c-Myc expression

The aim of this study is to investigate the effects of CIP2A (Cancerous inhibitor of protein phosphatase 2A) on the apoptosis of RA FLS. Proliferation and apoptotic activity of RA FLS following treatment with CIP2A siRNA or control siRNA were analyzed using MTT assays and Cell Death Detection kit. RA FLS was treated with CIP2A siRNA or control siRNA in 3-, 6-, and 9-day intervals for a Western blot analysis to determine C-Myc expression. To evaluate the signal transduction pathways engaged in apoptosis, caspase-3 activity, caspase-9 activity, PARP, and phosphorylation of the Akt kinase were analyzed by Western blot. Cell viability of RA FLS was significantly lower in the CIP2A siRNA-treated group compared with the control after 7 days (p = 0.022). Apoptosis of RA FLS was significantly higher in the CIP2A siRNA-treated group compared with the control when incubated for 3, 6, and 9 days (p = 0.029, p = 0.021, p = 0.043, respectively). C-Myc expression did not change with the different incubation periods. CIP2A siRNA-treated FLS expressed higher level of activated caspase-3, caspase-9, and PARP (p = 0.014, p = 0.020, p = 0.021, respectively) and lower level of phosphorylated Akt (p = 0.001) compared with those treated with the control siRNA. Our data show that CIP2A expression in RA FLS is an important mediator of dysfunctional apoptosis independent of c-Myc stabilization. Expression of CIP2A may contribute to apoptotic resistance of RA FLS through the activation of Akt and deactivation of caspase-3, caspase-9, and PARP. Inhibition of CIP2A may therefore constitute a novel, promising therapeutic target in RA.     

In vitro SCREENING ANTIBACTERIAL ACTIVITY OF Bidens pilosa LINNé AND Annona crassiflora MART. AGAINST OXACILLIN RESISTANT Staphylococcus aureus (ORSA) FROM THE AERIAL ENVIRONMENT AT THE DENTAL CLINIC

Currently multiresistant Staphylococcus aureus is one common cause of infections with high rates of morbidity and mortality worldwide, which directs scientific endeavors in search for novel antimicrobials. In this study, nine extracts from Bidens pilosa (root, stem, flower and leaves) and Annona crassiflora (rind fruit, stem, leaves, seed and pulp) were obtained with ethanol: water (7:3, v/v) and their in vitro antibacterial activity evaluated through both the agar diffusion and broth microdilution methods against 60 Oxacillin Resistant S. aureus (ORSA) strains and against S. aureus ATCC6538. The extracts from B. pilosa and A. crassiflora inhibited the growth of the ORSA isolates in both methods. Leaves of B. pilosa presented mean of the inhibition zone diameters significantly higher than chlorexidine 0.12% against ORSA, and the extracts were more active against S. aureus ATCC (p < 0.05). Parallel, toxicity testing by using MTT method and phytochemical screening were assessed, and three extracts (B. pilosa, root and leaf, and A. crassiflora, seed) did not evidence toxicity. On the other hand, the cytotoxic concentrations (CC₅₀ and CC₉₀) for other extracts ranged from 2.06 to 10.77 mg/mL. The presence of variable alkaloids, flavonoids, tannins and saponins was observed, even though there was a total absence of anthraquinones. Thus, the extracts from the leaves of B. pilosa revealed good anti-ORSA activity and did not exhibit toxicity.     

Quercetin as an Auxiliary Endodontic Irrigant for Root Canal Treatment: Anti-Biofilm and Dentin Collagen-Stabilizing Effects In Vitro

Bacterial reinfection and root fracture are the main culprits related to root canal treatment failure. This study aimed to assess the utility of quercetin solution as an adjunctive endodontic irrigant that does not weaken root canal dentin with commitment anti-biofilm activity and bio-safety. Based on a noninvasive dentin infection model, dentin tubules infected with Enterococcus faecalis (E. faecalis) were irrigated with sterile water (control group), and 0, 1, 2, 4 wt% quercetin-containing ethanol solutions. Live and dead bacteria percentages in E. faecalis biofilms were analyzed by confocal laser scanning microscopy (CLSM). Elastic modulus, hydroxyproline release and X-ray photoelectron spectroscopy (XPS) characterization were tested to evaluate the irrigants’ collagen-stabilizing effect. The cytotoxicity was tested by CCK-8 assay. Quercetin increased the proportion of dead bacteria volumes within E. faecalis and improved the flexural strength of dentin compared to control group (p < 0.05). Quercetin-treated dentin matrix had less elasticity loss and hydroxyproline release after collagenase degradation (p < 0.05). Moreover, quercetin solutions revealed an increase in the C-O peak area under both C1s and O1s narrow-scan spectra of XPS characterization, and no cytotoxicity (p > 0.05). Quercetin exhibited anti-biofilm activity, a collagen-stabilizing effect with cytocompatibility, supporting quercetin as a potential candidate for endodontic irrigant.     

Clinical and Microbiological Evaluation of a Chlorhexidine-Modified Glass Ionomer Cement (GIC-CHX) Restoration Placed Using the Atraumatic Restorative Treatment (ART) Technique

The aims of this study were to investigate the clinical effectiveness and patient acceptability of a modified glass ionomer cement placed using the atraumatic restorative treatment (ART) technique to treat root caries, and to carry out microbiological analysis of the restored sites. Two clinically visible root surface carious lesions per participant were restored using ART. One was restored with commercial glass ionomer cement (GIC) (ChemFil^® Superior, DENTSPLY, Konstonz, Germany) which acted as the control. The other carious root lesion was restored with the same GIC modified with 5% chlorhexidine digluconate (GIC-CHX; test). Patient acceptability and restoration survival rate were evaluated at baseline and after 6 months. Plaque and saliva samples around the test and control restorations were collected, and microbiological analysis for selected bacterial and fungal viability were completed at baseline, and after 1, 3, and 6 months. In total, 52 restorations were placed using GIC and GIC-CHX in 26 participants; 1 patient was lost to follow-up. After reviewing the restorations during their baseline appointments, participants indicated that they were satisfied with the appearance of the restorations (n = 25, 96%) and did not feel anxious during the procedure (n = 24, 92%). Forty-eight percent (n = 12) of the GIC-CHX restorations were continuous with the existing anatomic form as opposed to six for the GIC restorations (24%), a difference which was statistically significant (p = 0.036). There was no statistically significant reduction in the mean count of the tested microorganisms in plaque samples for either type of restorations after 1, 3, or 6 months. Restoration of carious root surfaces with GIC-CHX resulted in higher survival rates than the control GIC. ART using GIC-CHX may therefore be a viable approach for use in outreach dental services to restore root surface carious lesions where dental services are not readily available, and for older people and special needs groups.     

Synergistic cytotoxicity of acidity and 3,4-Dideoxyglucosone-3-ene under the existence of lactate in peritoneal dialysis fluid

Of the non-physiological compounds in glucose-rich peritoneal dialysis fluid, we investigated the synergistic cytotoxicity of acidity and 3,4-Dideoxyglucosone-3-ene(3,4-DGE) under the existence of lactate using human peritoneal mesothelial cells (HPMC). The effect of pH on cell viability at various levels of pH (5.5, 6.7, 7.15), with or without lactate was examined by adding 1N-HCl to phosphate buffer solution. We also examined the cytotoxic effects of 3,4-DGE and pH (5.5, 6.7 or 7.15). Additionally, we compared the cytotoxic effects of 3,4-DGE and pH (5.5, 6.7 or 7.15) under existence of lactate (40 meq/L) or absence of lactate. The cells were exposed to these solutions for 2 or 4 h. Cell viability was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenylterazolium bromide) assay. 3,4-DGE or acidic solution alone had no significant effects on MTT viability under the absence of lactate. However, acidic solutions containing 3,4-DGE significantly decreased MTT viability under the existence of lactate. The MTT viability of HPMC was not decreased by 3,4-DGE or acidity alone under the absence of lactate. However, the combination of acidity and 3,4-DGE markedly decreased MTT viability under the existence of lactate, strongly suggesting the synergistic cytotoxicity of 3,4-DGE and acidity under the existence of lactate.     

The Effect of Liquid Rubber Addition on the Physicochemical Properties,Cytotoxicity, and Ability to Inhibit Biofilm Formation of Dental Composites

The aim of this study was to evaluate the effect of modification with liquid rubber on the adhesion to tooth tissues (enamel, dentin), wettability and ability to inhibit bacterial biofilm formation of resin-based dental composites. Two commercial composites (Flow-Art–flow type with 60% ceramic filler and Boston–packable type with 78% ceramic filler; both from Arkona Laboratorium Farmakologii Stomatologicznej, Nasutów, Poland) were modified by addition of 5% by weight (of resin) of a liquid methacrylate-terminated polybutadiene. Results showed that modification of the flow type composite significantly (p < 0.05) increased the shear bond strength values by 17% for enamel and by 33% for dentine. Addition of liquid rubber significantly (p < 0.05) reduced also hydrophilicity of the dental materials since the water contact angle was increased from 81–83° to 87–89°. Interestingly, modified packable type material showed improved antibiofilm activity against Steptococcus mutans and Streptococcus sanguinis (quantitative assay with crystal violet), but also cytotoxicity against eukaryotic cells since cell viability was reduced to 37% as proven in a direct-contact WST-8 test. Introduction of the same modification to the flow type material significantly improved its antibiofilm properties (biofilm reduction by approximately 6% compared to the unmodified material, p < 0.05) without cytotoxic effects against human fibroblasts (cell viability near 100%). Thus, modified flow type composite may be considered as a candidate to be used as restorative material since it exhibits both nontoxicity and antibiofilm properties.     

Effect of interferon-γ and tumor necrosis factor-α on hepatitis B virus following lamivudine treatment

AIM: To evaluate anti-hepatitis B virus (HBV) activity and cytotoxicity of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) following lamivudine treatment of HepG2.2.15 cells.METHODS: HepG2.2.15 cells were treated with 2 μmol/L lamivudine for 16 d (lamivudine group), cultured for 10 d, followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (cytokine group), or treated with 2 μmol/L lamivudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (sequential group), or cultured without additions for 16 d (control group). Intracellular DNA was extracted from 3 × 10⁵ HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circular DNA that may have remained. Both HBV covalently closed circular DNA (cccDNA) and HBV DNA were examined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were quantified with enzyme-linked immunosorbent assay. Cell viability was measured with the cell counting kit-8 assay.RESULTS: Compared to lamivudine alone (22.63% ± 0.12%), both sequential (51.50% ± 0.17%, P = 0.034) and cytokine treatment (49.66% ± 0.06%, P = 0.041) showed a stronger inhibition of HBV cccDNA; the difference between the sequential and cytokine groups was not statistically significant (51.50% ± 0.17% vs 49.66% ± 0.06%, P = 0.88). The sequential group showed less inhibition of HBV DNA replication than the lamivudine group (67.47% ± 0.02% vs 82.48% ± 0.05%, P = 0.014); the difference between the sequential and cytokine groups was not statistically significant (67.47% ± 0.02% vs 57.45% ± 0.07%, P = 0.071). The levels of HBsAg and HBeAg were significantly decreased in the sequential treatment group compared to the other groups [HBsAg: 3.48 ± 0.04 (control), 3.09 ± 0.08 (lamivudine), 2.55 ± 0.13 (cytokine), 2.32 ± 0.08 (sequential), P = 0.042 for each between-group comparison; HBeAg: 3.48 ± 0.01 (control), 3.08 ± 0.08 (lamivudine), 2.57 ± 0.15 (cytokine), 2.34 ± 0.12 (sequential), P = 0.048 for each between-group comparison]. Cell viability in the cytokine group was reduced to 58.03% ± 8.03% compared with control cells (58.03% ± 8.03% vs 100%, P = 0.000). Lamivudine pretreatment significantly reduced IFN-γ + TNF-α-mediated toxicity of HepG2.2.15 cells [85.82% ± 5.43% (sequential) vs 58.03% ± 8.03% (cytokine), P = 0.002].CONCLUSION: Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-γ and TNF-α by decreasing the viral load.     

Genetic Polymorphism of the CCK Gene in Patients With Alcohol Withdrawal Symptoms

Background: Cholecystokinin (CCK) is considered to play an important role in the central nervous system via its interaction with other neurotransmitters such as dopamine, serotonin, gamma-aminobutyric acid, substance P, and enkephalins. We investigated the relationship between the C to T substitution in the Spl binding cis-element of the CCK gene promoter region (at position −45 numbered from initiation codon) and alcohol withdrawal symptoms. Methods: We examined 214 Japanese men with alcoholism (93 with delirium tremens, 49 with hallucination, 38 with seizure, and 93 with none of these symptoms) and 98 age-matched Japanese male controls by using a polymerase chain reaction-based single strand conformational polymorphism analysis. Results: Patients who displayed hallucination were significantly more likely to possess the C allele than control subjects (X² = 8.17, p = 0.017, Bonferroni correction: p = 0.064). In addition, we investigated the influence of CCK gene polymorphism on alcohol consumption among the control subjects but found no significant relationship. Conclusions: Our data suggested that the C allele at −45 locus of the CCK gene was higher in patients with hallucination than the control group at a rate that was not quite significant after Bonferroni correction for multiple testing.     

Space Maintainers Used in Pediatric Dentistry: An Insight of Their Biosecurity Profile by Applying In Vitro Methods

Space maintainers have presented an increased interest due to their chemical composition which influences the electrochemical and electrolytic processes of the oral cavity, leading to important biological activity. The present study was purported to evaluate the biological in vitro activity of three types of space maintainers (S1, S2, and S3, differing from each other in terms of metal composition) used in pediatric dentistry, in terms of their antimicrobial effect and biosecurity profile using two types of keratinocytes (PGK: primary gingival keratinocytes, and HaCaT: human immortalized keratinocytes) by assessing the morphology, viability, cytotoxicity, and gene expression of the cells. Statistical differences were calculated by the one-way ANOVA test, followed by Tukey’s post-test. Antimicrobial screening highlighted a dilution-dependent influence that, in the case of all strains tested, did not show inhibition or stimulation of bacterial growth. The in vitro evaluations revealed that the test samples did not induce important cytotoxic potential on both keratinocyte cell lines (HaCaT and PGK), with the cells manifesting no morphological alteration, a good viability rate (above 90%: PGK–S1, * p < 0.05), and a low cytotoxic activity (less than 11%: PGK, S1 *** p < 0.001 and S3 * p < 0.05; HaCaT, S1 ** p < 0.01). The data obtained in this study highlight the fact that the samples analyzed are biocompatible and do not develop the growth of the studied bacteria or encode the gene expression of primary and immortalized keratinocytes.     

The Influence of Hyaluronic Acid Biofunctionalization of a Bovine Bone Substitute on Osteoblast Activity In Vitro

Bovine bone substitute materials (BSMs) are used for oral bone regeneration. The objective was to analyze the influence of BSM biofunctionalization via hyaluronic acid (HA) on human osteoblasts (HOBs). BSMs with ± HA were incubated with HOBs including HOBs alone as a negative control. On days 3, 7 and 10, cell viability, migration and proliferation were analyzed by fluorescence staining, scratch wound assay and MTT assay. On days 3, 7 and 10, an increased cell viability was demonstrated for BSM+ compared with BSM− and the control (each p ≤ 0.05). The cell migration was enhanced for BSM+ compared with BSM− and the control after day 3 and day 7 (each p ≤ 0.05). At day 10, an accelerated wound closure was found for the control compared with BSM+/− (each p < 0.05). The highest proliferation rate was observed for BSM+ on day 3 (p ≤ 0.05) followed by BSM− and the control (each p ≤ 0.05). At day 7, a non-significantly increased proliferation was shown for BSM+ while the control was higher than BSM− (each p < 0.05). The least proliferation activity was observed for BSM− (p < 0.05) at day 10. HA biofunctionalization of the BSMs caused an increased HOB activity and might represent a promising alternative to BSM− in oral bone regeneration.