The evaluation of cytotoxicity and cytokine IL-6 production of root canal sealers with and without the incorporation of simvastatin: an invitro study

Background Freshly mixed root canal sealers when proximate the periapical tissues, trigger varying degrees of cytotoxicity/inflammatory reactions. Simvastatin, a class of the drug statin, is a widely used cholesterol-lowering agent with additional anti-inflammatory activities. This study assessed the effects of simvastatin on cytotoxicity and the release of IL-6 (Interleukin-6) production when incorporated in zinc oxide eugenol and methacrylate resin-based sealers. Methods Experimental groups consisted of conventional zinc oxide eugenol and methacrylate based-EndoREZ sealers (ZE & ER respectively) and 0.5 mg/mL simvastatin incorporated sealers (ZES & ERS). L929 mouse fibroblast cells were exposed to freshly mixed experimental sealers and evaluated for cytotoxicity (MTT assay) and inflammation levels (inflammatory marker IL-6 for ELISA) at various time intervals (0h, 24h and 7th day). The values were compared to the cell control (CC; L929 cells alone) and solvent control (SC; L929 cells + DMSO) groups. All the experiments were conducted in triplicates and subjected to statistical analysis using IBM SPSS Statistics software. Non parametric tests were conducted using Kruskal-Wallis and Friedman tests for inter-group and intra-group comparisons respectively. Pairwise comparison was conducted by post hoc Dunn test followed by Bonferroni correction. P values < 0.05 were considered statistically significant. Results All the experimental groups (ZE, ER, ZES, ERS) exhibited varying degree of cytotoxicity and IL-6 expression compared to the control groups CC and SC. The cell viability for ZE and ER decreased on day 7 as compared to 24 h. ZES and ERS had higher viable cells (75.93% & 79.90%) compared to ZE and ER (54.39% & 57.84%) at all time periods. Increased expression of IL-6 was observed in ZE & ER (25.49 pg/mL & 23.14 pg/mL) when compared to simvastatin incorporated ZE & ER (ZES-12.70 pg/mL & ERS-14.68 pg/mL) at all time periods. Highest level of cytotoxicity and inflammation was observed in ZE compared to all the other groups on day 7. Conclusions Addition of 0.5 mg/mL of simvastatin to the sealers (ZES and ERS) decreased the cytotoxicity in the freshly mixed state and reduces their inflammatory effect.

Effect of Lemongrass Essential Oil based Mouthwash against Microflora Associated with Dental Plaque

Mouthwash is one of the most convenient and effective method employed for dental plaque management. The aim of the undertaken study was to establish the antimicrobial and anti-biofilm properties of lemongrass essential oil (LGEO) based mouthwash on microbial flora from dental plaque and also check cytotoxicity of mouthwash formulation. Five main colonizers of dental plaque representing dental microflora and three different bacterial species mainly responsible for the formation of biofilm were selected in this study. LGEO based mouthwash was developed and its stability was also determined. The antimicrobial and anti-biofilm activity of LGEO based mouthwash has been evaluated against the representative dental microflora as per CLSI guidelines. Cytotoxicity of mouthwash was checked by globally used MTT assay employing NIH 3T3 mouse fibroblast cell line. The mouthwash has been found to exhibit the stability in its major component, citral and also found exhibit antimicrobial and anti-biofilm activity against dental microflora. No cytotoxic effect was observed on mouse fibroblast cell line. LGEO in formulated mouthwash being a natural, herbal material isolated from traditional medicinal plants appears as a good and effective substitute to control the microflora linked with dental plaque.

Regulatory Effect of Irresistin-16 on Competitive Dual-Species Biofilms Composed of Streptococcus mutans and Streptococcus sanguinis

Based on the ecological plaque hypothesis, suppressing opportunistic pathogens within biofilms, rather than killing microbes indiscriminately, could be a biofilm control strategy for managing dental caries. The present study aimed to evaluate the effects of irresistin-16 (IRS-16) on competitive dual-species biofilms, which consisted of the conditional cariogenic agent Streptococcus mutans (S. mutans) and oral commensal bacteria Streptococcus sanguinis (S. sanguinis). Bacterial growth and biofilm formation were monitored using growth curve and crystal violet staining, respectively. The microbial proportion was determined using fluorescence in situ hybridization. A 2, 5-diphenyltetrazolium bromide assay was used to measure the metabolic activity of biofilms. Bacterial/extracellular polysaccharide (EPS) dyeing, together with water-insoluble EPS measurements, were used to estimate EPS synthesis. A lactic acid assay was performed to detect lactic acid generation in biofilms. The cytotoxicity of IRS-16 was evaluated in mouse fibroblast L929 cells using a live/dead cell viability assay and cell counting kit-8 assay. Our results showed that IRS-16 exhibited selective anti-biofilm activity, leading to a remarkable survival disadvantage of S. mutans within competitive dual-species biofilms. In addition, the metabolic activity, EPS synthesis, and acid generation of dual-species biofilms were significantly reduced by IRS-16. Moreover, IRS-16 showed minimal cytotoxicity against mouse fibroblast L929 cells. In conclusion, IRS-16 exhibited remarkable regulatory effects on dual-species biofilms composed of S. mutans and S. sanguinis with low cytotoxicity, suggesting that it may have potential for use in caries management through ecological biofilm control.

Cell-free chromatin particles released from dying cells inflict mitochondrial damage and ROS production in living cells

Several hundred billion to a trillion cells die in the body every day and release cell free chromatin particles (cfChPs) which enter into the circulation, or are released locally into extracellular compartments of the body. We have reported that cfChPs from the dying cells can readily enter into living cells and damage their DNA. To test the hypothesis that internalised cfChPs might also inflict mitochondrial damage, we treated NIH3T3 mouse fibroblast cells with cfChPs isolated from sera of healthy individuals (10ng), or co-cultured the cells with hypoxia induced dying NIH3T3 cells. Abundant cfChPs could be detected in the cytoplasm of the treated cells by 4h. The latter was associated with evidence of mitochondrial damage in the form of ultra-structural changes, increased mitochondrial mass, alterations in mitochondrial shape, upregulation of the mitochondrial outer membrane protein TOM20, and changes in mitochondrial membrane potential. We also detected increased fluorescence signals of gamma-H2AX and p-ATM signifying double-strand breaks in mitochondrial DNA. There was marked increase in production of mitochondrial superoxide (ROS) as detected by MitoSOX Red, and activation of the intracellular antioxidant enzyme superoxide dismutase-1. Mitochondrial damage and ROS production could be inhibited by a cfChPs deactivating agent viz. anti-histone antibody complexed nanoparticles. Given that 1x109-1x1012 cells die in the body every day, we propose that cfChPs are major physiological triggers for mitochondrial damage and ROS production with an important bearing on human health and disease. Deactivation of cfChPs may provide a novel therapeutic approach to retard ageing and associated degenerative conditions that have been linked to oxidative stress.

25-Hydroxycholesterol-Induced Oxiapoptophagy in L929 Mouse Fibroblast Cell Line

25-hydroxycholesterol (25-HC) is an oxysterol synthesized from cholesterol by cholesterol-25-hydroxylase during cholesterol metabolism. The aim of this study was to verify whether 25-HC induces oxiapoptophagy in fibroblasts. 25-HC not only decreased the survival of L929 cells, but also increased the number of cells with condensed chromatin and altered morphology. Fluorescence-activated cell sorting results showed that there was a dose-dependent increase in the apoptotic populations of L929 cells upon treatment with 25-HC. 25-HC-induced apoptotic cell death was mediated by the death receptor-dependent extrinsic and mitochondria-dependent intrinsic apoptosis pathway, through the cascade activation of caspases including caspase-8, -9, and -3 in L929 cells. There was an increase in the levels of reactive oxygen species and inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2 in L929 cells treated with 25-HC. Moreover, 25-HC caused an increase in the expression of beclin-1 and microtubule-associated protein 1A/1B-light chain 3, an autophagy biomarker, in L929 cells. There was a significant decrease in the phosphorylation of protein kinase B (Akt) in L929 cells treated with 25-HC. Taken together, 25-HC induced oxiapoptophagy through the modulation of Akt and p53 cellular signaling pathways in L929 cells.

Cytotoxic Effects of Bulk-Fill Composites on L929 Fibroblast Cells

Objective: Unlike traditional composite resins, bulk-fill composite resins could be polymerized as thicker layers. This study aims to contribute to the field by investigating the cytotoxic effects of various bulk-fill composite resins on L929 mouse fibroblast cells in vitro. Material and Methods: In our study, six bulk fill and one conventional composite resin were used. Composite resin samples (8×4 mm) were prepared in a sterile cabinet by using a glass mod and polymerizing with a led light device (DTE LUX E, Germany). Composite samples (n:3) of which surface area was calculated according to ISO 10993-12: 2012 standards (3 cm2/ml), were kept in media for 24 h and 72 h in 37 oC incubator, their extracts were filtered in 1:1 and 1:2 proportion and were added on L929 mouse fibroblast cells. Cell viability was examined by the MTT assay and cell death by the LDH test. Cell viability results were evaluated using one-way analysis of variance (ANOVA) test (p<0.05). Results: When the 1:1 extracts from 4 mm thick bulk-fill composite samples were applied on L929 mouse fibroblast cells, cell viability rates showed significant differences compared to the control group at the end of 24 h and 72 h (except for Estelite Bulk Fill Flow). Although the extracts of the tested composite samples at 1:1 and 1:2 ratio at the end of 72 hours caused a decrease in L929 mouse fibroblast cell viability, the cell viability rate of only PRG-containing bulk fill composite and conventional composite remained below the cell viability ratio (70%) specified in ISO standards. Bulk fill composites did not produce toxic effects (except Beautifil Bulk Restorative) according to the LDH test. Conclusions: Despite decreasing in general the cell viability, bulk-fill composite resins used in 4 mm thick layers provided cell viability rates over the acceptability level, except PRG-containing bulk fill composite (Beautifil Bulk Restorative), which was cytotoxic to L929 mouse fibroblasts. Keywords Bulk fill composite; Cytotoxicity; L929 cells; LDH assay.

Freeform 3D Bioprinting Involving Ink Gelation by Cascade Reaction of Oxidase and Peroxidase: A Feasibility Study Using Hyaluronic Acid-Based Ink

Freeform bioprinting, realized by extruding ink-containing cells into supporting materials to provide physical support during printing, has fostered significant advances toward the fabrication of cell-laden soft hydrogel constructs with desired spatial control. For further advancement of freeform bioprinting, we aimed to propose a method in which the ink embedded in supporting materials gelate through a cytocompatible and rapid cascade reaction between oxidase and peroxidase. To demonstrate the feasibility of the proposed method, we extruded ink containing choline, horseradish peroxidase (HRP), and a hyaluronic acid derivative, cross-linkable by HRP-catalyzed reaction, into a supporting material containing choline oxidase and successfully obtained three-dimensional hyaluronic acid-based hydrogel constructs with good shape fidelity to blueprints. Cytocompatibility of the bioprinting method was confirmed by the comparable growth of mouse fibroblast cells, released from the printed hydrogels through degradation on cell culture dishes, with those not exposed to the printing process, and considering more than 85% viability of the enclosed cells during 10 days of culture. Owing to the presence of derivatives of the various biocompatible polymers that are cross-linkable through HRP-mediated cross-linking, our results demonstrate that the novel 3D bioprinting method has great potential in tissue engineering applications.

Evaluation in Cellulose Nanocrystals Effectiveness on Composite Film based Wound Dressing from Poly(vinyl alcohol) and Gum Tragacanth

Biomaterial-based wound dressings were fabricated using cellulose nanocrystals (CNCs) as nano-filler in a polymeric mixture of poly(vinyl alcohol) (PVA) and gum tragacanth (GT) via solution casting. Physical and chemical characteristics of neat PVA, PVA/GT and PVA/GT/CNC films with varying concentrations (2 to 10%) of CNCs were observed. Initial analysis of CNCs showed nanosized particles of 104 nm length and 7 nm width. Scanning electron microscopy (SEM) illustrated cluster formations of CNCs in the polymer matrix. Fourier transform infrared (FTIR) spectrometry was used to confirm the chemical functional groups in the material. The presence of GT and CNCs in the polymer matrix improved water uptake and prolonged stability for 7 days. The CNCs enhanced tensile strength from 54.63 MPa to 80.39MPa. Biological properties of PVA/GT/CNC films were analyzed. Results showed that the dressing material was nontoxic to mouse fibroblast cells L929, while film loaded with betel leaf extract exhibited excellent antibacterial activities against Staphylococcus aureus DMST 8840 and Pseudomonas aeruginosa TISTR 781, indicating that composite film was suitable for application in wound dressing.

The Expression and Activity of Rhodanese, 3-Mercaptopyruvate Sulfurtransferase, Cystathionine γ-Lyase in the Most Frequently Chosen Cellular Research Models

This paper provides information concerning the activity and expression levels of three sulfurtransferases (STRs): rhodanese (TST, EC: 2.8.1.1), 3-mercaptopyruvate sulfurtransferase (MPST, EC: 2.8.1.2) and cystathionine γ-lyase (CTH, EC: 4.4.1.1) in various cell lines. Since very limited data are available in the scientific literature on this subject, the available data are included in this paper. These shortages often force the researchers to carry out their own screening tests that allow them to choose an appropriate model for their further studies. This work supplements the existing deficiencies in this area and presents the activity and expression of STRs in the eight most frequently chosen cell lines: the mouse mammary gland cell line (NMuNG, ATCC: CRL-1636), mouse mammary gland tumor (4T1, ATCC: CRL-2539), mouse fibroblast (MEF, ATCC: SCRC-1008), mouse melanoma (B16-F1, ATCC: CRL-6323), human colorectal adenocarcinoma (Caco-2, ATCC: HTB-37), human embryonic kidney (HEK-293, ATCC: CRL-1573), human osteosarcoma (MG-63, ATCC: CRL-1427) and rat myocardium (H9c2, ATCC: CRL-1446). Changes in STRs activity are directly related to the bioavailability of cysteine and the sulfane sulfur level, and thus the present authors also measured these parameters, as well as the level of glutathione (its reduced (GSH) and oxidized (GSSG) form) and the [GSH]/[GSSG] ratio that determines the antioxidant capacity of the cells. STRs demonstrate diverse functionality and clinical relevance; therefore, we also performed an analysis of genetic variation of STRs genes that revealed a large number of polymorphisms. Although STRs still provide challenges in several fields, responding to them could not only improve the understanding of various diseases, but may also provide a way to treat them.