Effect of chlorhexidine digluconate on antimicrobial activity, cell viability and physicochemical properties of three endodontic sealers.

OBJECTIVE: Assess the biological and physicochemical properties of AH Plus, BioRoot RCS and Pulp Canal Sealer (PCS) leachates with and without chlorhexidine (CHX). METHODS: The sealers were studied in no contact and 1-minute contact with CHX. For biological properties (antibacterial activity and cytotoxicity), leachates were formed in saline of freshly mixed, 1-, 7- and 28 days set sealers. The antibacterial properties of sealer leachates were investigated for planktonic and biofilm growth of E. faecalis, S. mutans, S.epidermidis and S.aureus. The 3-(4,5 dimethylthiazolyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate murine fibroblast cell viability after exposure to the leachates. The physical properties (water uptake, sorption, solubility, porosity, surface characteristics) of sealers and the pH of the immersion liquid (saline or distilled water) were also assessed over a 28-days period. RESULTS: CHX improved the antibacterial properties of the sealer leachates and reduced cell viability for all sealer leachates, except for freshly mixed PCS. BioRoot RCS leachates presented the highest antibacterial properties and cell viability with and without CHX contact. PCS was the material most affected by CHX in terms of physical properties, whereas for AH Plus, solubility was increased. CHX did not affect the physical properties of BioRoot RCS, except for solubility that was decreased. CHX contact did not change sealers' alkalinity in distilled water whereas it increased it for AH Plus and BioRoot RCS in saline. SIGNIFICANCE: CHX improved the antibacterial efficacy of sealer leachates and either compromised or did not affect cell viability. CHX affected to various extent sealers' physicochemical properties.

Cobalt-chromium alloys fabricated with four different techniques: Ion release, toxicity of released elements and surface roughness.

OBJECTIVE: To investigate the metal ion release, surface roughness and cytoxicity for Co-Cr alloys produced by different manufacturing techniques before and after heat treatment. In addition, to evaluate if the combination of materials affects the ion release. METHODS: Five Co-Cr alloys were included, based on four manufacturing techniques. Commercially pure titanium, CpTi grade 4 and a titanium alloy were included for comparison. The ion release tests involved both Inductive Coupled Plasma Optical Emission Spectrometry and Inductive Coupled Plasma Mass Spectrometry analyses. The surface analysis was conducted with optical interferometry. Cells were indirectly exposed to the materials and cell viability was evaluated with the MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) method. RESULTS: All alloys showed a decrease of the total ion release when CpTi grade 4 was present. The total ion release decreased over time for all specimens and the highest ion release was observed from the cast and milled Co-Cr alloy in acidic conditions. The cast and laser-melted Co-Cr alloy and the titanium alloy became rougher after heat treatment. All materials were within the limits of cell viability according to standards. SIGNIFICANCE: The ion release from Co-Cr alloys is influenced by the combination of materials, pH and time. Surface roughness is influenced by heat treatment. Furthermore, both ion release and surface roughness are influenced by the manufacturing technique and the alloy type. The clinical implication needs to be further investigated.

In vitro effects of dental monomer exposure - Dependence on the cell culture model.

Methacrylate monomers are major components of resin-based biomaterials. The polymerization of these materials is never complete, and methacrylates leaking from cured materials cause exposure of patients. Only some selected methacrylates have thoroughly been tested for possible interaction with living cells. In the current study, we compared the effects of 2-hydroxyethyl-methacrylate (HEMA; a carefully studied methacrylate) and hydroxypropyl-methacrylate (HPMA; a scarcely investigated methacrylate). Five cell lines differing in both source and cell type were used. The cells were exposed to methacrylates (1-8 mM). Cell viability, cell death, glutathione levels, reactive oxygen species (ROS), and cell growth pattern were measured. Both methacrylates reduced cell viability, and glutathione depletion was observed in all cell lines. The cell death pattern varied among the cell lines. The ROS levels and cell growth pattern also differed between the cell lines after exposure to methacrylate monomers. No difference between HEMA and HPMA exposures were observed in any of the cell lines. The variation between cell lines shows that the measured methacrylate toxicity depends heavily on the test system chosen. Further, the conformity between HEMA and HPMA effects suggests that the two methacrylates similarly affect living cells.

The dental monomer hydroxyethyl methacrylate (HEMA) counteracts lipopolysaccharide-induced IL-1ß release-Possible role of glutathione.

Methacrylate monomers, like 2-hydroxyethyl methacrylate (HEMA), are common components of resin based dental materials. Leakage of unpolymerized monomers after placement and curing leads to human exposure. HEMA is known to inhibit lipopolysaccharide (LPS) induced cytokine release. In this study we explore a possible role of the antioxidant glutathione (GSH) in this effect. In the RAW 264.7 murine macrophage cell line, HEMA (<2mM) did not induce cell death, but reduced cellular GSH levels, increased cellular ROS and decreased the IL-1ß release from LPS-stimulated cells. Moreover, the IL-1ß mRNA levels were reduced after 3-6h exposure, suggesting transcriptional effects of HEMA. The GSH modulators butylsulfoximine (BSO; inhibitor of GSH synthesis) and 2-oxothiazolidine-4-carboxylate (OTC; Cysteine precursor) caused a decrease and increase in the LPS-induced IL-1ß release, respectively, suggesting a role for GSH in negative regulation of LPS-induced IL-1ß release. However, the magnitude and dynamics of the effects of HEMA and BSO on LPS-induced IL-1ß release and GSH depletion differed considerably. Thus, GSH depletion alone could not explain the strong attenuation of LPS-induced IL-1ß release caused by HEMA. Formation of HEMA-protein conjugates due to the thiol reactivity of HEMA emerges as a likely candidate for the molecular mechanism accounting for this effect.

Cell toxicity of 2-hydroxyethyl methacrylate (HEMA): the role of oxidative stress.

2-Hydroxyethyl methacrylate (HEMA) is a methacrylate monomer used in polymer-based dental-restorative materials. In this study, the viability of human lung epithelial cells, BEAS-2B, was investigated after exposure to this monomer. Exposure to HEMA reduced the viability of the BEAS-2B cells as a result of increased apoptosis, interruption of the cell cycle, and decreased cell proliferation. Depletion of cellular glutathione and increased levels of reactive oxygen species (ROS) were seen after exposure of BEAS-2B cells to HEMA. The glutathione synthase inhibitor, L-buthioninesulfoximine (BSO), was used to study whether the reduced viability was caused by glutathione depletion and increased levels of ROS. Similarly to incubation with HEMA, incubation with BSO resulted in glutathione depletion and increased ROS levels, without increasing cell death or inhibiting cell growth. The results indicate that HEMA-induced cell damage is not caused exclusively by these mechanisms. Mechanisms other than glutathione depletion and ROS formation seem to be of importance for the toxic effect of HEMA on lung epithelial cells.

In vitro element release and biological aspects of base-metal alloys for metal-ceramic applications.

Objective: The aims of this study were to investigate the release of element from, and the biological response in vitro to, cobalt-chromium alloys and other base-metal alloys used for the fabrication of metal-ceramic restorations. Material and methods: Eighteen different alloys were investigated. Nine cobalt-chromium alloys, three nickel-chromium alloys, two cobalt-chromium-iron alloys, one palladium-silver alloy, one high-noble gold alloy, titanium grade II and one type III copper-aluminium alloy. Pure copper served as positive control. The specimens were prepared according to the ISO standards for biological and corrosion testing. Passive leaching of elements was measured by using Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) after incubation in cell culture media, MEM, for 3 days. Corrosion testing was carried out in 0.9% sodium chloride (NaCl) and 1% lactic acid for 7 days, and the element release was measured by Inductively Coupled Plasma - Optical Emission Spectroscopy (ICP-OES). The biological response from the extract solutions was measured though MTT cytotoxicity testing and the Hen's egg test-chorio-allantoic membrane (HET-CAM) technique for irritationt. Results: The corrosion test showed similar element release from base-metal alloys compared to noble alloys such as gold. Apart from the high-copper alloy, all alloys expressed low element release in the immersion test, no cytotoxic effect in the MTT test, and were rated non-irritant in the HET-CAM test. Conclusions: Minimal biological response was observed for all the alloys tested, with the exception of the high-copper alloy.

Dental monomers inhibit LPS-induced cytokine release from the macrophage cell line RAW264.7.

Methacrylate monomers have been identified in aqueous extracts of freshly cured dental fillings. The hypothesis tested presently was that low concentrations of triethyleneglycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) alone or in combination interfere with the LPS-induced release of cytokines from the macrophage cell line RAW264.7. The cells were exposed to 5-200 µM of monomers for 24 h followed by a 24 h combined exposure to monomers and LPS. TEGDMA reduced LPS-induced release of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), whereas HEMA only reduced IL-1ß release. Co-exposure to the two monomers indicated an additive effect. Moreover, the reduced cytokine release persisted for 24 h after termination of the monomer exposure. The LPS-induced activation of proteins in pre-transcriptional signaling pathways (CD14, p-ERK1/2, p-p38, p-JNK, p-IκB-α and p-NFκB-p65) was not altered by monomer exposure, neither were the levels of IL-1ß and TNF-α mRNA. However, the LPS-induced level of pro-IL-1ß was decreased by the monomer treatment. Thus, HEMA and TEGDMA may interfere with post-transcriptional regulation of synthesis and release of these cytokines. Overall, the results suggest that low concentrations of monomers may cause impaired macrophage responses, and that these effects can persist for up to 24 h after exposure.

In vitro photosensitization initiated by camphorquinone and phenyl propanedione in dental polymeric materials.

Documentation is scarce on the photobiological effects of photoinitiators present in dental light curable materials. The aim of this study was to determine cellular effects of the photoinitiators camphorquinone (CQ) and phenyl propanedione (PPD) and to investigate whether these substances produced reactive oxygen species after low and high doses of optical radiation (between 0 and 17J/cm(2)). Rat salivary gland cells in vitro were exposed to visible blue light and/or UVA. Hematoporphyrin (HP), a photosensitizer used in medicine, and the UVA-filter 2-methoxy-4-hydroxy-benzophenone (B-3) were used as reference substances. It was found that PPD produced hydrogen peroxide, but not singlet oxygen, upon light irradiation. CQ produced neither hydrogen peroxide nor singlet oxygen. Cell death by necrosis and apoptosis was induced by irradiation in the presence of CQ, PPD and HP. Doses higher than 6J/cm(2) UVA and blue visible light from a source similar to clinically applied sources, induced apoptosis even in the absence of photosensitizers added. A reciprocity relationship was found between radiant exposure (at constant irradiance) and concentration of photoinitiators. In conclusion, the oral cells under investigation were light sensitive, and the sensitivity increased in presence of photoinitiators. PPD acted by mechanisms that included reactive oxygen species and CQ probably by formation of free radicals.

Apoptosis induced by the monomers HEMA and TEGDMA involves formation of ROS and differential activation of the MAP-kinases p38, JNK and ERK.

OBJECTIVES: Cytotoxic methacrylate monomers have been identified in aqueous extracts of freshly cured compomers. Some of these compounds, including HEMA and TEGDMA, induce apoptosis and necrosis in vitro. The aim of the present study was to elucidate possible signaling pathways involved in apoptosis following exposure to HEMA or TEGDMA in a salivary gland cell line. METHODS: The cells were exposed to various concentrations of HEMA or TEGDMA. ROS formation was determined by dichlorofluorescein assay. Phosphorylated MAP-kinases ERK1/2, p38 and JNK, as well as specific caspases were identified by Western blotting. Apoptosis was assayed by fluorescence microscopy. RESULTS: HEMA or TEGDMA exposure resulted in ROS formation and concentration-dependent apoptosis as well as phosphorylation of ERK. Phosphorylation of JNK and p38 was induced by HEMA. Selective inhibitors of ERK and JNK modified the apoptotic response after HEMA and TEGDMA exposure, whereas p38 inhibition modified the apoptotic response only after HEMA exposure. Vitamin C reduced HEMA-induced apoptosis. SIGNIFICANCE: ROS formation and differential MAP kinase activation appear to be involved in the apoptotic response following exposure to HEMA and TEGDMA.

Pattern of cell death after in vitro exposure to GDMA, TEGDMA, HEMA and two compomer extracts.

OBJECTIVES: In vitro exposure to chemical compounds in dental materials may cause cell death by apoptosis, necrosis or a combination of both. The aim of this paper was to evaluate aqueous extracts of freshly cured compomers Freedom (SDI) and F2000 (3M ESPE), and constituents identified in the extracts, GDMA (glycerol dimethacrylate), TEGDMA (triethylene glycol dimethacrylate) and HEMA (2-hydroxyethyl methacrylate) for their ability to induce necrosis and apoptosis in primary rat alveolar macrophages and the J744A1 macrophage cell line. METHODS: The cells were exposed to either extracts of freshly cured samples of the products or to one of the constituents identified in the extracts. Cytotoxicity and necrosis were assayed by MTT test and fluorescence microscopy, respectively. Apoptosis was assayed by fluorescence microscopy and flow cytometry. RESULTS: Concentration-related apoptosis and necrosis were found in both cell types after exposure to extracts from Freedom and F2000. GDMA appeared to be the most cytotoxic of the tested constituents in the J744A1 cell line as evaluated by the MTT test. TEGDMA was more cytotoxic than HEMA using the MTT test and fluorescence microscopy, whereas HEMA caused a greater accumulation of apoptotic cells seen by fluorescence microscopy and flow cytometry. For various concentrations of HEMA and TEGDMA, the extent of apoptosis appeared inversely related to the cytotoxicity evaluated by the MTT test. SIGNIFICANCE: As an apoptotic response elicits less inflammatory response in the surrounding tissues than a necrotic process, the role of cell death pattern could be important for the evaluation of the biocompatibility of dental materials.

Irritation and cytotoxic potential of denture adhesives.

OBJECTIVE: The present study aimed to examine the in vitro biocompatibility of denture adhesives. BACKGROUND: Denture adhesives absorb water to become viscous and sticky, and by this process, other constituents like colouring, flavouring, wetting and preserving agents may be released. Some of these constituents may induce adverse reactions among users of denture adhesives. MATERIALS AND METHODS: Five commercially available denture adhesives; three different creams, a powder, and a cushion were included in the study. The irritation and cytotoxic potential was evaluated using the Hen's Egg Test Chorioallantoic Membrane (HET-CAM) method and three cell culture methods; filter diffusion, dimethylthiazol diphenyltetrazolium bromide (MTT) assay and agar diffusion. RESULTS: None of the tested denture adhesives showed a noteworthy acute irritation as evaluated by the HET-CAM method. None of the tested denture adhesives induced cytotoxicity in the filter diffusion test. One of the denture adhesives induced a severe cytotoxic reaction in both the MTT and agar diffusion assays. These tests employ longer exposure times than in both the filter diffusion and the HET-CAM test. CONCLUSION: Denture adhesives are commonly used throughout the day, and our results raise the concern that denture adhesives may contribute to mucosal inflammation in denture wearers.

Studies on curcumin and curcuminoids. XXIX. Photoinduced cytotoxicity of curcumin in selected aqueous preparations.

Natural curcumin was evaluated as a potential photosensitizer for oral applications. The photocytotoxicity of curcumin on salivary gland acinar cells (SM 10-12) was investigated in five aqueous preparations consisting of 5% DMSO, non-ionic micelles, cyclodextrin, liposomes, or a hydrophilic polymer. The difference in phototoxic effects between natural curcumin and synthetic curcumin was examined. Cytotoxicity in SM 10-12 cells exposed to curcumin in the concentration range 0.4-13.5 microM was investigated by MTT test, a fluorescence-staining microscopic test, and by Western immunoblotting techniques. The potential formation of a photoreaction product, hydrogen peroxide, was evaluated by a fluorescence assay. The light source was a halogen lamp used in the dental clinic, emitting mainly in the blue part of the spectrum. The phototoxic effect on SM 10-12 cells was dependent on curcumin concentration, the light dose and the type of preparation. Natural and synthetic curcumin induced phototoxicity to the same extent. Significant effects on the cells were obtained at low curcumin concentrations (< or =0.5 microM) and at a low light dose (< or =6 J cm(-2)), after 3 h incubation. Neither the activation of caspases-3, -7, -8 or -9, nor the formation of hydrogen peroxide could be detected in cells exposed to curcumin and light. The liposome preparation was the most efficient vehicle for curcumin to induce cell death. The phototoxic effect induced by curcumin is highly dependent on the type of preparation. Curcumin might be a potential photosensitizer in the treatment of oral lesions and cancers provided careful selection of the vehicle.

In vitro cell death induced by irradiation and chemicals relevant for dental applications; dose-response and potentiation effects.

Resin-based dental materials polymerized using blue light are frequently used in dental practice and may come in contact with the oral mucosa. Remnants from oral hygiene product ingredients, such as sodium lauryl sulfate (SLS), add to the chemical exposure of the mucosa. The aim of the present in vitro study was to elucidate the cytotoxic effects in terms of apoptosis and necrosis after exposures to combinations of an adhesive (0.5% and 0.6%), SLS (concentration range 0.0025%-0.0075%), and irradiation from a dental curing lamp (radiant exposure of 8 J cm(-2)). The test system chosen was rat submandibular salivary gland acinar cells, and the cytotoxic effects were measured by fluorescence microscopy and flow cytometry methods. Cytotoxicity was observed as a result of irradiation. The most pronounced cytotoxic effects were seen in cells exposed to a combination of adhesive and SLS compared with those exposed to either agent alone. Necrosis was the dominating form of cell death for all exposures, except for the highest concentration of SLS. Apoptosis was dose-dependent on SLS in the rat submandibular acinar cells. Cytotoxic considerations of dental materials should include contributions from irradiation and other chemicals that might be present in the oral cavity.