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.

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.

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.

The Effect of Autophagy Induction in Oncolytic Reovirus Replication in Mesenchymal Stem Cells

Background and Aims: Oncolytic reoviruses can infect and kill malignant cells while sparing their normal counterparts. Reoviral infection can induce or activate autophagy, even though metformin can induce autophagy. Identifying and regulating the cellular pathways important for reovirus replication and oncolysis can improve targeted-biological therapies for cancer. Here, the autophagic process was triggered via metformin, and we investigated the effect of autophagy activation on oncolytic reovirus replication in mesenchymal stem cells as primary cells and L929 cell lines. Materials and Methods: Adipose derived mesenchymal stem cells (AD-MSCs) and L929 cells were treated with metformin and reovirus type-3 strain Dearing (T3D). Twenty-four hours after infection, the viability of AD-MSCs and L929 cells were examined by MTT assay. Also, the effect of metformin-induced autophagy in the reovirus replication in these cells was determined by real-time polymerase-chain-reaction. Results: Our results show that treatment with metformin and reovirus reduced the viability of the cells compared to treatment with metformin or reovirus alone in both cells. Also, coadministration of metformin and reovirus significantly decreased the relative expression level of the Beclin-1 gene compared to treatment with metformin in both cells. However, the expression level of the reovirus L3 gene after treatment with metformin and reovirus in L929 cells increased significantly compared to AD-MSCs. Conclusion: Our data suggest that metformin-induced autophagy enhances reoviral replication in AD-MSCs and L929 cells. These findings represent the role of autophagy induction in facilitating reovirus replication and contribute to a better understanding of reovirus-host interactions.

The A179L Gene of African Swine Fever Virus Suppresses Virus-Induced Apoptosis but Enhances Necroptosis

A179L, a non-structural protein of African swine fever virus (ASFV), is capable of suppressing apoptosis by binding the BH3 domain of the pro-apoptotic Bcl-2 family proteins via a conserved ligand binding groove. Our present study aims to determine if A179L affects necroptosis, the second form of programmed cell death induced by DNA and RNA viruses. Here we report that A179L enhanced TNF-α or TSZ (TNF-α, Smac, and Z-Vad)-induced receptor-interacting protein kinase (RIPK1), RIPK3, and mixed lineage kinase domain like peudokinase (MLKL) phosphorylation in L929 cells, a murine fibrosarcoma cell line. Sytox green staining revealed that A179L significantly increased the number of necroptotic cells in TSZ-treated L929 cells. Using human herpes simplex virus 1 (HSV-1) to model DNA virus-induced cell death, we found that A179L blocked the HSV-1-induced cleavage of poly (ADP-ribose) polymerase (PARP), caspase 8, and caspase 3 and decreased the number of apoptotic cells in HSV-1-infected IPEC-DQ cells, a porcine intestinal epithelial cell line. In contrast, A179L transfection of IPEC-DQ cells enhanced HSV-1-induced RIPK1, RIPK3, and MLKL phosphorylation and increased the number of necroptotic cells. Consistently, A179L also suppressed apoptosis but enhanced the necroptosis induced by two RNA viruses, Sendai virus (SeV) and influenza virus (IAV). Our study uncovers a previously unrecognized role of A179L in regulating cell death and suggests that A179L re-directs its anti-apoptotic activity to necroptosis.

Real-Time Analysis of Antiproliferative Effects of Mouthwashes Containing Alcohol, Sodium Fluoride, Cetylpyridinium Chloride, and Chlorhexidine In Vitro

Objectives. In this study, the cytotoxic responses of six different over-the-counter mouthwashes on L929 cells were analyzed by two different techniques: the traditional colorimetric tetrazolium-based reduction assay (MTT) and the modern impedance-based real-time cell analysis (RTCA) system to investigate their biocompatibility in vitro. Thus, the investigation of the antiproliferative effects of the specified materials via different techniques is vital to reach this goal. Materials and Methods. First, L929 mouse fibroblasts were exposed to the dilutions of mouthwashes for 2 minutes. After incubation, the tetrazolium reduction method was used to assess the metabolic viability of cells measured by colorimetric MTT assay and morphological inspection of cells was performed via phase-contrast microscopy. Furthermore, the effect of each mouthwash on the proliferation, morphology, and adhesion of L929 cells was monitored continuously by a noninvasive and label-free RTCA system for 140 h. Results. Our data showed that all of the mouthwashes had varying cytotoxic effects on fibroblasts compared to the control group in MTT assay. In addition to that, RTCA technology has provided the growth kinetic profiles that can be used to analyze if the treatment is causing antimitotic or DNA-damaging effect on cells. Thus, analysis via this system can tell us the mechanism of toxicity behind the cell growth inhibition in vitro. Here, we found that only mouthwash 1 moderately maintained the viability of the L929 cells, yet displaying antimitotic effects and the other mouthwashes (mouthwash 2-mouthwash 6) showed toxicity via DNA-damaging effects. Conclusions. Of the six types of mouthwash tested, the most biocompatible result was obtained from a mouthwash containing alcohol (i.e., mouthwash 1). On the other hand, sodium fluoride- (NaF-) and cetylpyridinium chloride- (CPC-) containing mouthwash (i.e., mouthwash 2) showed the most cytotoxic effect.

Cytotoxicity and pro-inflammatory effect of GaSb thin films in L929 cells

Gallium antimonide (GaSb)-based devices operate efficiently in the infrared region. Investigating the toxicity of GaSb thin film is necessary for using embedded GaSb-based devices in living organisms. In this study, viability, oxidative stress, inflammatory responses, apoptosis induction and genotoxicity of GaSb were assayed using L929 cells following a 24 h exposure to GaSb. GaSb thin films were deposited on a quartz substrate using radio frequency (RF) magnetron sputtering. These films were soaked in cell culture medium to prepare test solutions. The viability of cells treated with the GaSb extract was lower than that of control cells. GaSb elicited little reactive oxygen species (ROS) generation. Tumor necrosis factor (TNF)-[Formula: see text] and interleukin (IL)-1[Formula: see text] levels were elevated in GaSb-treated cell culture supernatants. Apoptosis and genotoxicity were not evident following GaSb treatment. Overall, these results demonstrate the low toxicity of GaSb compared with previous studies examining arsenic-containing III–V materials, which is desirable for biological devices.

Physicochemical and Biological Properties of Graphene-Oxide-Coated Metallic Materials

In this article, we present graphene oxide (produced by a modified Hummers’ method) coatings obtained using two different methods: electrophoretic deposition on 316L stainless steel and chemical modification of the surface of gold applied to the steel. The coating properties were characterized by microscopic and spectrometric techniques. The contact angle was also determined, ranging from 50° to 70°. Our results indicated that GO coatings on steel and gold were not toxic towards L929 cells in a direct cell adhesion test—on all tested materials, it was possible to observe the growth of L929 cells during 48 h of culture. The lack of toxic effect on cells was also confirmed in two viability tests, XTT and MTT. For most of the tested materials, the cell viability was above 70%. They showed that the stability of the coating is the crucial factor for such GO coatings, and prove that GO in the form of coating is non-toxic; however, it can show toxicity if detached from the surface. The obtained materials also did not show any hemolytic properties, as the percentage of hemolysis was on the level of the negative control, which is very promising in the light of future potential applications.

Aluminum particles generated during millisecond electric pulse application enhance adenovirus-mediated gene transfer in L929 cells

Gene electrotransfer is an attractive method of non-viral gene delivery. However, the mechanism of DNA penetration across the plasma membrane is widely discussed. To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/low-amplitude electric pulses to L929 cells, mixed with a human adenovirus vector expressing GFP. We observed a transgene expression increase, both in the number of GFP-converted cells and GFP levels, when we added a low-voltage/millisecond-pulse treatment to the adenovirus/cell mixture. This increase, reflecting enhanced virus penetration, was proportional to the applied electric field amplitude and pulse number, but was not associated with membrane permeabilization, nor to direct cell modifications. We demonstrated that this effect is mainly due to adenovirus particle interactions with aggregated aluminum particles released from energized electrodes. Indeed, after centrifugation of the pulsed viral suspension and later on addition to cells, the activity was found mainly associated with the aluminum aggregates concentrated in the lower fraction and was proportional to generated quantities. Overall, this work focused on the use of electrotransfer to facilitate the adenovirus entry into cell, demonstrating that modifications of the penetrating agent can be more important than modifications of the target cell for transfer efficacy.