Validation of a human saliva model for the determination of leachable monomers and other chemicals from dental materials.

This study aimed to prove the validity of a mixture of chemicals, including salts, small organic molecules, mucin, and α-amylase, as saliva surrogate ("artificial saliva") for assessing leakage of methacrylate monomers and other constituents from dental materials. To achieve this, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA), diurethane dimethacrylate (UDMA), bisphenol A glycerolate dimethacrylate (BisGMA), diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO), bisphenol A (BPA), and five homologues of ethoxylated bisphenol A dimethacrylate (BisEMA EO2-6) in unstimulated and artificial saliva, and compared their concentrations in the two saliva media following either spiking with a mixture of the compounds or incubation of test specimens of printed biomaterials. Test specimens were immersed in unstimulated/artificial saliva, incubated at 37 °C for 24 h, and saliva aliquots were extracted with methanol and subsequently analyzed by LC-MS/MS. The method was validated with regard to matrix effects, linearity, selectivity, lower limits of quantification (LLOQ), precision, bias and combined measurement uncertainty (u'). The performance characteristics of the method were comparable for unstimulated and artificial saliva samples. The combined u' for individual chemicals at a concentration of 10 × LLOQ were within the range of 5.3-14 % for unstimulated saliva and 6.9-16 % for artificial saliva, except for the BisEMA homologues. Combined u' for the latter were 27-74 % in unstimulated saliva, and 27-79 % in artificial saliva. There was no detectable release of BPA from the test specimens, and the TPO concentrations were mainly below the LLOQ. TEGDMA and UDMA were detected in the highest quantities, and at comparable concentrations in the unstimulated and artificial saliva. For all BisEMA homologues, the release was higher in unstimulated saliva than in artificial saliva. The study showed that the artificial saliva model can be a suitable replacement for native saliva, but might underestimate leakage of more lipophilic methacrylates.

Preparation of a fluorinated dental resin system and its anti-adhesive properties against S. mutans.

OBJECTIVES: The purpose of this study was to characterize physicochemical properties and investigate anti-bacterial adhesion effect of dental resins containing fluorinated monomers. METHOD: Fluorinated dimethacrylate FDMA was mixed with commonly used reactive diluent triethylene- glycol dimethacrylate (TEGDMA) and fluorinated diluent 1 H,1 H-heptafluorobutyl methacrylate (FBMA) separately at a mass ratio of 60 wt./40 wt. to prepare fluorinated resin systems. Double bond conversion (DC), flexural strength (FS) and modulus (FM), water sorption (WS) and solubility (SL), contact angle and surface free energy, surface element concentration, and anti-adhesion effect against Streptococcus mutans (S. mutans) were investigated according to standard or referenced methods. 2,2-bis[4-(2-hydroxy-3-methacryloy-loxypropyl)-phenyl]propane (Bis-GMA)/TEGDMA (60/40, wt./wt.) was used as control. RESULTS: Both fluorinated resin systems had higher DC than Bis-GMA based resin (p < 0.05); compared with Bis-GMA based resin (FS, FDMA/TEGDMA resin system had higher FS (p < 0.05) and comparable FM (p > 0.05), while FDMA/FBMA resins system had lower FS and FM (p < 0.05). Both fluorinated resin systems had lower WS and SL than Bis-GMA based resin (p < 0.05), and FDMA/TEGDMA resin system had the lowest WS (p < 0.05) in all experimental resin systems. Only FDMA/FBMA resin system showed lower surface free energy than Bis-GMA based resin (p < 0.05). When the surface was smooth, FDMA/FBMA resin system had lower amount of adherent S. mutans than Bis-GMA based resin (p < 0.05), while after the surface became roughness, FDMA/FBMA resin system had comparable amount of adherent S. mutans as Bis-GMA based resin (p > 0.05). SIGNIFICANCE: Resin system prepared exclusively with fluorinated methacrylate monomers reduced the S. mutans adhesion due to their increased hydrophobicity and decreased surface energy., while flexural properties of it should be improved.

Bond strength between dentine and a novel fast-setting calcium silicate cement with fluoride.

The aim of this study was to evaluate the dentine bond strength of a novel fast-setting calcium silicate cement (Protooth) versus a calcium hydroxide-based cement (Dycal), a calcium silicate cement (ProRoot MTA), and a glass ionomer cement (Ketac-Molar). Mid-root dentine slices of 1 mm thickness were obtained from human maxillary incisors. After enlarging the lumen of the canal to 1.3 mm, the cavities were randomly filled with test materials. Samples were immersed in physiological-like solution. The push-out bond strength was tested on days 1, 28, and 180 (n = 12). Failure types of bonding were determined using a stereomicroscope. We analysed the data using linear regression. Dycal and day 1 were considered as reference for cement type and assessment time, respectively. Protooth, Ketac-Molar, and ProRoot MTA demonstrated higher push-out bond strength than Dycal. The push-out bond strength in the Protooth group increased on day 28 and 180. The bond strength of Ketac-Molar was significantly reduced on day 28. Dycal showed a significant decrease in bond strength on day 180 compared with that on day 1 and 28. Mixed failure was the dominant failure type. Protooth bonding to dentine was increased with time, in contrast to that of ProRoot MTA, Dycal, and Ketac-Molar, as a function of time.

Investigation on the antimicrobial activity of chitosan-modified zinc oxide-eugenol cement.

Introduction: Coronal leakage and reinfection after root canal therapy is an important reason for endodontic failure. Zinc oxide-eugenol (ZOE) -based materials are often used as a coronal seal to prevent secondary infection. The antibacterial effect of ZOE cement is mainly due to leaching of eugenol from the material, but the effect is reported to decrease over time. Chitosan (CH) is a natural polymer with antibacterial properties. The aim of the study was to investigate if incorporation of (CH) and chitosan oligosaccharide (COS) in a ZOE-based material improved both the immediate and sustained antibacterial properties of the material. Methods: Enterococcus faecalis, Streptococcus mutans and Staphylococcus epidermidis was used to investigate the antibacterial effect of the materials in a modified direct contact test (MDCT) immediately after setting and after storage for 18 weeks in water. Leaching per week of eugenol from the materials was quantified using gas chromatography-mass spectrometry (GC-MS). The effect of eugenol on growth of bacteria was measured by reading of optical density at 600 nm after 18 h growth. Mechanical properties were investigated in a compressive strength test according to ISO 3107. Results: The present study showed that a ZOE-based material has antibacterial activity both as freshly prepared and after immersion in water for 18 weeks. Incorporating CH or COS may increase the antibacterial effect depending on the bacterial species investigated. The amount of leached eugenol did not differ between materials or during or after storage. S. mutans showed the highest susceptibility to eugenol of the three species investigated. Modification of the materials with CH or COS reduced the compressive strength, but the requirements in ISO 3017 were still met.

Effect of methacrylated chitosan incorporated in experimental composite and adhesive on mechanical properties and biofilm formation.

The lifespan of a resin-based restoration is limited, with the main reason for failure being secondary caries. Biofilm formation at the tooth-material interface is a necessary etiological agent for caries development. Dental materials with antimicrobial properties may reduce formation of biofilm and thus increase the longevity of restorations. This study aimed to investigate the effect of methacrylated chitosan (CH-MA), incorporated into the polymeric network of an experimental dental composite and adhesive, on biofilm growth of Streptococcus mutans and to assess the mechanical properties of the modified materials. The methacrylation of low-molecular-weight chitosan was achieved and biofilm studies confirmed the antibacterial effect of the modified polymer in solution. Methacrylated chitosan was incorporated into an experimental composite and adhesive, and the modified materials reduced the formation of S. mutans biofilm. The incorporation of CH-MA did not alter the bond strength of the adhesives. However, the amount of CH-MA in composite that is required to elicit an antibacterial response challenges the mechanical properties of the material. The hardness and flexural strength of the composite decreased with increasing amounts of CH-MA. However, flexural strength values still met the requirement in the ISO standard.

Presence and leaching of bisphenol a (BPA) from dental materials.

BPA has been reported to leach from some resin based dental restorative materials and materials used for orthodontic treatment. To confirm and update previous findings, especially in light of the new temporary lower threshold value for tolerable daily BPA intake, we have investigated the leaching of BPA from 4 composite filling materials, 3 sealants and 2 orthodontic bonding materials. The materials were either uncured and dissolved in methanol or cured. The cured materials were kept in deionized water for 24 hours or 2 weeks. Samples were subsequently analyzed by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS-MS). The composite filling material Tetric EvoFlow® and the fissure sealant DELTON® showed significantly higher levels of BPA leaching compared to control samples for all test conditions (uncured, 24 h leaching and 2 weeks leaching). There were no significant differences in amount of leached BPA for any of the tested materials after 24 hours compared to 2 weeks. These results show that BPA is still released from some dental materials despite the general concern about potential adverse effects of BPA. However, the amounts of BPA were relatively low and most likely represent a very small contribution to the total BPA exposure.

Preparation and characterization of Bis-GMA-free dental composites with dimethacrylate monomer derived from 9,9-Bis[4-(2-hydroxyethoxy)phenyl]fluorene.

OBJECTIVE: Synthesize a new BPA-free monomer for use in methacrylate-based materials and evaluate critical properties of resin and composite materials based on the monomer. METHODS: Bis-EFMA was synthesized through reaction between 9,9-bis[4-(2-hydroxyethoxy)-phenyl]fluorene and 2-(methacryloyloxy)ethyl isocyanate. Experimental Bis-EFMA-based resin (Bis-EFMA/TEGDMA=50/50, wt./wt.) and composite were prepared. Critical properties were investigated according to standard or referenced methods Bis-GMA/TEGDMA (50/50, wt./wt.) resin system, Bis-GMA-based composite and 3M ESPE Filtek™ Z250 were used as controls. RESULTS: FT-IR and 1H NMR spectra confirmed the structure of Bis-EFMA monomer. Cured resin materials: Bis-EFMA-based and Bis-GMA-based resins had nearly the same degree of conversion (p>0.05); Bis-EFMA-based resin had significantly lower shrinkage, water sorption and solubility, and cytotoxicity than Bis-GMA-based resin (p<0.05); flexural properties of Bis-EFMA-based resin were all higher than those of Bis-GMA-based resin (p<0.05). Cured composite materials: There was no significant difference in conversion (p>0.05); Bis-EFMA-based composite had significantly lower shrinkage and solubility (p<0.05); water sorption of Bis-EFMA-based composite and Z250 were similar (p>0.05), but lower compared to Bis-GMA-based composite (p<0.05); Bis-EFMA-based composite had the deepest curing depth (p<0.05); Before water immersion, there was no significant difference in flexural strength between Bis-EFMA-based composite and each control composite (p>0.05), while FS became lower than that of Z250 (p<0.05), but higher than that of Bis-GMA-based composite (p<0.05) after water immersion; Flexural modulus of Bis-EFMA-based composite and Z250 were nearly the same (p>0.05), higher than that of Bis-GMA-based composite (p<0.05); Bis-EFMA-based composite showed less cytotoxicity than Bis-GMA-based composite and Z250 (p<0.05). SIGNIFICANCE: Bis-EFMA has potential as a substitute for Bis-GMA to prepare Bis-GMA-free dental composites.

Water sorption and solubility of polyamide denture base materials.

Purpose: Some patients experience adverse reactions to poly(methyl methacrylate)-based (PMMA) dentures. Polyamide (PA) as an alternative to PMMA has, however, not been well documented with regard to water sorption and water solubility. The aim of this in vitro study was to measure water sorption and water solubility of two PA materials compared with PMMA, and to evaluate the major components released from the PA materials and the effect on hardness of the materials. Methods: Ten discs (40.0 mm diameter, 2.0 mm thick) of each material (PA: Valplast and Breflex; PMMA: SR Ivocap HIP) were prepared according to manufacturers' recommendations. The specimens were tested for water sorption and water solubility, according to a modification of ISO 20795-1:2008. Released substances were analysed by gas chromatography/mass spectrometry (GC/MS). Results: There were statistically significant differences among the materials regarding water sorption, water solubility and time to water saturation. Breflex had the highest water sorption (30.4 µg/mm3), followed by PMMA-material (25.8 µg/mm3) and Valplast (13.6 µg/mm3). Both PA materials had statistically significant lower water solubility than the PMMA. Both PA had a net increase in weight. Analysis by GC/MS identified release of the compound 12-aminododecanolactam from the material Valplast. No release was found from the Breflex material. Conclusions: The PA denture materials show differences in water sorption and solubility, but within the limits of the standard requirements. The PA showed a net increase in weight after long-term water sorption. The clinical implications of the findings are not elucidated.

Antibacterial and Antibiofilm Effect of Low Viscosity Chitosan against Staphylococcus epidermidis.

Aim. The aim of this study was to investigate the antibacterial and antibiofilm properties of low viscosity chitosan on S. epidermidis growth and biofilm formation. Methods and Results. The antibacterial and antibiofilm properties were investigated, during both planktonic growth and biofilm formation. This was performed using different concentrations in media and by coating on polystyrene surfaces. In addition, the bactericidal effect was investigated using a modified direct contact test. The results showed that low viscosity chitosan in media had both a bacteriostatic and bactericidal effect on planktonic growth and biofilm formation of S. epidermidis in a concentration dependent manner. Polystyrene discs coated with chitosan reduced both early biofilm formation (6 h) and late biofilm formation (18 h), as confirmed by scanning electron microscopy. The modified direct contact test showed a bactericidal effect. Conclusion. This study demonstrated that low viscosity chitosan has a bacteriostatic and bactericidal activity against S. epidermidis and that the activity is dependent on the amount of chitosan added. In addition, low viscosity chitosan reduced biofilm formation both when added to media and when coated on polystyrene surfaces. Significance and Impact of Study. Low viscosity chitosan could be a contribution to new treatment approaches of biofilm-related infections of S. epidermidis.

Interactions of liposomes with dental restorative materials.

The in vitro adsorption and retention of liposomes onto four common types of dental restorative materials (conventional and silorane-based resin composites as well as conventional and resin-modified glass ionomer cements (GIC)) have been investigated due to their potential use in the oral cavity. Uncoated liposomes (positively and negatively charged) and pectin (low- and high-methoxylated) coated liposomes were prepared and characterized in terms of particle size and zeta potential. The adsorption of liposomes was performed by immersion, quantified by fluorescence detection, and visualized by fluorescence imaging and atomic force microscopy. Positive liposomes demonstrated the highest adsorption on all four types of materials likely due to their attractive surface charge. They also retained well (minimum 40% after 60 min) on both conventional resin composite and GIC even when exposed to simulated salivary flow. Although an intermediate initial level of adsorption was found for the pectin coated liposomes, at least 70% high methoxylated-pectin coated liposomes still remained on the conventional resin composite after 60 min flow exposure. This indicates significant contribution of hydrophobic interactions in the prolonged binding of liposomes to resin composites. Based on these results, the present paper suggests two new possible applications of liposomes in the preservation of dental restorations.

The influence of bis-EMA vs bis GMA on the degree of conversion and water susceptibility of experimental composite materials.

OBJECTIVE: The aim of this work was to assess the influence of the bis-EMA content on the degree of conversion (DC) and its effect on the water sorption and solubility. MATERIALS AND METHODS: In a polytetrafluorethylene (PTFE) mould, 30 samples (Ø = 5 mm, height = 2 mm) of four experimental dental composite resins were cured for 10 s, 20 s and 40 s. The DC was analysed by Fourier Transform (FT)-Raman spectroscopy. To analyse sorption and solubility, six samples (Ø = 15 mm and thickness = 1 mm) of each composite (n = 72) were stored in water at 37°C for different storage periods: 24 h, 7 days and 30 days. RESULTS: When cured for 20 or 40 s the DC increased with the increasing content of bis-EMA. However, the presence of 15 wt% of bis-GMA did not affect the DC, except when cured with 10 s irradiation time. This study also found a correlation between the content of bis-EMA and the reduced values for sorption and solubility, for all storage times used, when the materials were cured with 20 s. CONCLUSIONS: The DC of mixtures with higher content of bis-EMA is affected by the presence of bis-GMA at lower energy density delivered from the curing device, suggesting that the restrictions caused by the presence of hydrogen bonds is dependent of the irradiation time used.

Detection of leachables and cytotoxicity after exposure to methacrylate- and epoxy-based root canal sealers in vitro.

Root canal sealing materials may have toxic potential in vitro depending on the cell line, cytotoxicity assay, material chemistry, and degree of polymer curing. The aims of the present study were to detect leaching components from epoxy- or methacrylate-based root canal sealers and to investigate the degree of cytotoxicity after exposure to extracts from these materials. Qualitative determination of substances released from the materials was performed by gas- and liquid chromatography/mass spectrometry. Submandibular salivary gland acinar cell death (apoptosis/necrosis) was determined using a fluorescence staining/microscopy technique. The major leachable monomer from the epoxy-based material was bisphenol-A diglycidyl ether (BADGE), whereas leachables from the methacrylate-based materials were mainly triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), hydroxyethyl methacrylate (HEMA), and polyethyleneglycol dimethacrylate (PEGDMA). Exposure to diluted extracts of cured methacrylate-based materials caused a postexposure time-dependent increase in cell death. This effect was not demonstrated as a result of exposure to undiluted extract of cured epoxy-based material. Extracts of all fresh materials induced apoptosis significantly, but at lower dilutions of the epoxy- than the methacrylate-based materials. The degree of leaching, determined from the relative chromatogram peak heights of eluates from the methacrylate-based sealer materials, corresponded with the degree of cell death induced by extracts of these materials.

Effect of short LED lamp exposure on wear resistance, residual monomer and degree of conversion for Filtek Z250 and Tetric EvoCeram composites.

OBJECTIVES: The latest LED dental curing devices claim sufficient curing of restorative materials with short curing times. This study evaluates mechanical and chemical properties as a function of curing time of two commercial composite filling materials cured with three different LED lamps. METHODS: The composites were Filtek Z250 (3M ESPE) and Tetric EvoCeram (Ivoclar Vivadent) and the LED curing devices were bluephase 16i (Ivoclar Vivadent), L.E.Demetron II (Kerr) and Mini L.E.D. (Satelec). Control samples were cured with a QTH-lamp (VCL 400, Kerr). The wear resistance after simulated tooth brushing, degree of conversion, curing depth, and amounts of residual monomers were measured after different curing times. RESULTS: The results of this study show that short curing time with high-intensity LEDs may influence the bulk properties of the materials, resulting in lower curing depth and increased residual monomer content. The measured surface properties of the materials, degree of conversion and wear resistance, were not affected by short curing times to the same extent. SIGNIFICANCE: This study demonstrates that reduced exposure time with high intensity LEDs can result in composite restorations with inferior curing depth and increased leaching of monomers. Dentists are recommended to use sufficient curing times even with high intensity LEDs to ensure adequate curing and minimize the risk of monomer leaching.

Detection and quantification of monomers in unstimulated whole saliva after treatment with resin-based composite fillings in vivo.

Resin-based dental restorative materials contain allergenic methacrylate monomers, which may be released into saliva after restorative treatment. Monomers from resin-based composite materials have been demonstrated in saliva in vitro; however, studies analyzing saliva after restorative therapy are scarce. The aim of this study was to quantify methacrylate monomers in saliva after treatment with a resin-based composite filling material. Saliva was collected from 10 patients at four start points--before treatment, and 10 min, 24 h, and 7 d after treatment--and analysed by combined chromatography/mass spectrometry. The monomers bisphenol-A diglycidyl methacrylate (Bis-GMA), 2-hydroxyethyl methacrylate (HEMA), and urethane dimethacrylate (UDMA) were detected and quantified in the samples collected shortly (10 min) after treatment. The amounts detected ranged from 0.028 to 9.65 µg ml(-1) for Bis-GMA, from 0.015 to 0.19 µg ml(-1) for HEMA, and from 0.004 to 1.2 µg ml(-1) for UDMA. Triethyleneglycol dimethacrylate (TEGDMA) was detected in four of the samples. Ethoxylated bisphenol-A dimethacrylate (Bis-EMA) was not detected. Monomers were not detected in saliva samples collected before treatment, or 24 h or 7 d after treatment, with the exception of one sample, 24 h after treatment, in which HEMA was detected. In conclusion, monomers from the investigated resin-based composite and adhesive system were present in saliva shortly after treatment. One week after treatment, no monomers could be detected in patients' saliva samples.

Identification and quantification of leachable substances from polymer-based orthodontic base-plate materials.

The aim of this study was to analyse leachable monomers, additives, and degradation products from polymer-based orthodontic base-plate materials. One heat-cured resin (Orthocryl), one light-cured (Triad VLC), and three thermoplastic materials (Biocryl C, Essix A+, and Essix Embrace) were investigated. Elution was performed in water at 37°C for 10 days. The extract medium was changed and analysed daily. Chromatographic methods were used to identify and quantify the leachables. In addition, the content of residual methyl methacrylate (MMA) was quantified in the poly(methyl methacrylate) (PMMA)-based materials. Statistical analysis of the quantitative results was performed using a t-test for comparison of two independent samples. Monomers and additives leached from the materials polymerized in situ and from the thermoplastic PMMA-based material. No leachable substances were found in the extracts from the other thermoplastic materials. Accumulated over 10 days, a larger amount of MMA leached from the powder-and-liquid material, Orthocryl (42 µg/cm(2)), than from the thermoplastic material, Biocryl C (0.49 µg/cm(2)). The accumulated amounts of monomers leached from Triad VLC were 91 µg/cm(2) of urethane dimethacrylate and 2.2 µg/cm(2) of 2-hydroxyethyl methacrylate. Formaldehyde was found to leach from methacrylate-based materials: 3.2 µg/cm(2) from Orthocryl and 0.16 µg/cm(2) from Triad VLC. However, formaldehyde was not detectable in extracts from Biocryl C. Residual MMA was 5.4 wt % in Orthocryl and 0.4 wt % in Biocryl C. No phthalates were detected in the tested materials. In this in vitro study, minimal leaching was found from the thermoplastic materials, while leaching of methacrylates and formaldehyde was observed from the powder-and-liquid type and the paste material. Within the limitations of this study, the results suggest that prefabricated thermoplastic plates should be preferred for patients with an allergy to methacrylates.

Elution of substances from a silorane-based dental composite.

Previous studies have shown that residual monomers, initiators, and additives are eluted from methacrylate-based dental composite materials. Recently, a composite material (Filtek Silorane), based on a new resin chemistry, was introduced. The purpose of this study was to investigate substances eluted from Filtek Silorane in water and ethanol. Polymerized specimen discs of the material were immersed in either distilled water or 75 vol% ethanol at 37 degrees C, and the solutions were analyzed using liquid chromatography-mass spectrometry after 1, 4, 24, and 72 h eluting time. No substances were found to leach from Filtek Silorane in water, whereas silorane monomers and an initiator component were eluted from the material into the ethanol solution.

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.