Elisabet L. Kostoryz

In vitro biocompatibility of oxirane/polyol dental composites with promising physical properties

OBJECTIVES Visible light cure oxirane/polyol resins of Cyracure UVR-6105 with pTHF-250 has been previously shown useful for development of dental composites. This oxirane/polyol (4016) in combination with other oxiranes were formulated into composites (4016E, 4016G and 4016GB) containing 72.9-74.9% quartz filler. The main objective of the study was to evaluate some of the physical properties and the biocompatibility of the composites. RESULTS PhotoDSC analysis of composites demonstrated twice the enthalphy values of Z100 (31J/g). Composites 4016E and 4016G showed compressive strengths similar to Z100 (337+/-35Mpa), P>0.05. Discs of composite 4016E, containing Epon 825 oxirane (E), and composite 4016G containing Araldite GY 281 oxirane (G) were non-cytotoxic (-) while the composite 4016GB, containing G and Ebecryl 1830 (B), was mildly (+) cytotoxic to L929 cells in the agar diffusion assay. Seven-day extracts of 4016GB composite were cytotoxic while extracts of 4016E and 4016G were less cytotoxic to L929 cells in the MTT assay. Extracts were obtained from 7 day incubations of composite (3 cm(2) surface area/ml) in acetone or ethanol/saline (1:20) at 37 degrees C. All composite extracts were non-mutagenic to Ames strains TA100, TA98, TA97a and TA1535. The overall results with composite 4016GB suggest that leachable components were cytotoxic but non-mutagenic. With the exception of oxirane components, G and E, the oxirane Cyracure UVR-6105 and other components were non-mutagenic. From cytotoxicity studies, the photoinitiator, Sarcat CD 1012, was the most cytotoxic (TC(50)=14 microM) component. Components G (TC(50)=17 microM), E (TC(50)=50 microM) and B (TC(50)=151 microM) were significantly (p < 0.05) more cytotoxic than Cyracure UVR-6105 (1488 microM) and the polyol, pTHF-250 (TC(50)=6072 microM). SIGNIFICANCE Favorable results obtained with composites 4016G and 4016E indicates that suitable oxirane/polyol formulations can be designed and optimized for development of dental composites with acceptable mechanical properties and biocompatibility. However, leachable analysis of extracts obtained from longer incubation periods is needed before final conclusions could be drawn about the leachability of oxirane components.

In vitro cytotoxicity of solid epoxy-based dental resins and their components

OBJECTIVE The objective of this study was to evaluate the effect of adding a spiroorthocarbonate (SOC) or a polyol on the cytotoxicity of epoxy-based dental resins. METHODS Resins contained one of the epoxies: diglycidyl ether Bisphenol A (GY-6004); 3,4-epoxycyclohexanemethyl-3,4-epoxycyclohexane carboxylate (UVR-6105); vinyl cyclohexane dioxide (ERL-4206) or the three-epoxy mixture (Epoxy-M). The SOC was t/t-2,3,8,9-di(tetramethylene)-1,5,7,11-tetraoxaspiro[5.5]undecane (SOC). The polyols were polytetrahydrofuran (p-THF-250) and polycaprolactone triol (TONE-301). The photoinitiator (4-octylphenyl)phenyliodonium hexafluoroantimonate and camphorquinone were used for light curing the resins. Four types of resins (epoxy, SOC/epoxy, polyol/epoxy and SOC/polyol/epoxy) were evaluated for cytotoxicity as solids in the agar diffusion assay and as aqueous extracts in the MTT assay using L929 cells. RESULTS In agar diffusion analysis, ERL-4206 and UVR-6105 resins were severely cytotoxic (+3), but the addition of SOC changed them to non-cytotoxic (-). Addition of 1-3% SOC changed Epoxy-M from mild (+) to non-cytotoxic. Adding SOC changed GY-6004 from moderate (+2) to mild (-) cytotoxicity. Generally, addition of SOC did not change cytotoxicity when added to polyol/epoxy combinations. Either polyol produced resins with reduced cytotoxicity when added to UVR-6105, but the opposite occurred when added to Epoxy-M resins. In MTT analysis, percent cell survival from 100 microliters resin extracts were statistically compared (ANOVA, p < 0.05). Epoxy-M and GY-6004 resin extracts were significantly less cytotoxic than UVR-6105 and ERL-4206 resin extracts were. Overall, the SOC component reduced the cytotoxicity of all SOC/epoxy combinations, except SOC/ERL-4206, which was significantly more cytotoxic than ERL-4206 resin extract. This may be the result of cell fixative effects observed for SOC/ERL-4206 in agar diffusion analysis. Addition of SOC produced significantly less cytotoxic SOC/polyol/Epoxy-M resins when compared to its non-SOC counterpart. The contrary result was obtained with SOC/polyol/UVR-6105 resin combinations. Consistent with agar diffusion results, adding polyol significantly decreased cytotoxicity of UVR-6105 resins. The cytotoxicity of these resins may be related to the 50% cytotoxicity (TC50) of their components as leachates. The TC50 values of the individual components were compared to BISGMA. Polyols, epoxy monomers, SOC monomer and camphorquinone were significantly (p < 0.05) less cytotoxic than BISGMA. SIGNIFICANCE Addition of SOCs and polyols in the formulation of epoxy-based resins may contribute to development of biocompatible dental composites.

Lipid Vesicles as Membrane Models for Toxicological Assessment of Xenobiotics

Traditionally animals and cell cultures have been used to assess the toxic potential of xenobiotics on cell membranes. In search for more reproducible, quantitative, cost- and time-effective assays, toxicologists have recently become interested in biomimetic lipid vesicle-based test systems. Lipid vesicles (liposomes) have long been appreciated as simple cell membrane models in biochemical and biophysical studies providing a good understanding of the physicochemical properties of liposome systems. More recently a number of reports have been published on the interactions of toxic substances with vesicles. Literature reports on liposome assays have appeared for widely different classes of xenobiotics, such as dental materials, antibiotics, detergents, and peptides. In this review we focus on those reports that contain a quantitative and significant correlation with more established toxicological tests like cell culture assays. We provide an introduction to the structure and main characteristics of vesicles and related lipid aggregates. The two main assays presented are leakage of fluorescence dyes and differential scanning calorimetry (DSC) measurements of the solid-ordered/liquid-disordered main phase transition temperature (Tm).

Enzymatic Biodegradation of HEMA/BisGMA Adhesives Formulated With Different Water Content

Dentin adhesives may undergo phase separation when bonding to wet demineralized dentin. We hypothesized that adhesives exhibiting phase separation will experience enhanced biodegradation of methacrylate ester groups. The objective of this project was to study the effect of enzyme-exposure on the release of methacrylic acid (MAA) and 2-hydroxyethyl methacrylate (HEMA) from adhesives formulated under conditions simulating wet bonding. HEMA/bisGMA(2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane), 45/55 w/w ratio, was formulated with different water content: 0 Wt % (A00), 8 wt % (A08), and 16 wt % (A16). After a three day prewash, adhesive discs were incubated with/without porcine liver esterase (PLE) in phosphate buffer (PB, pH 7.4) at 37 degrees C for 8 days. Supernatants were collected daily and analyzed for MAA and HEMA by HPLC. For all formulations, daily MAA release in the presence of PLE was increased compared to MAA release in PB. HEMA release in the presence of PLE was not detected while HEMA release was consistently measured in PB. A08 and A16 released significantly larger amounts of HEMA compared to A00. Analysis of the cumulative release of analytes showed that the leachables in PLE was significantly increased (p < 0.05) as compared with that released in PB indicating that MAA release was not only formed from unreacted monomers but from pendant groups in the polymer network. However, the levels of analytes HEMA in PB or MAA in PLE were increased in A08 and A16 as compared with A00, which suggests that there could be a greater loss of material in HEMA/bisGMA adhesives that experience phase separation under wet bonding conditions.

Dynamic mechanical analysis and esterase degradation of dentin adhesives containing a branched methacrylate

A study of the dynamic mechanical properties and the enzymatic degradation of new dentin adhesives containing a multifunctional methacrylate are described. Adhesives contained 2-hydroxyethyl methacrylate, 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy) phenyl]-propane, and a new multifunctional methacrylate with a branched side chain-trimethylolpropane mono allyl ether dimethacrylate (TMPEDMA). Adhesives were photopolymerized in the presence of 0, 8, and 16 wt % water to simulate wet bonding conditions in the mouth and compared with control adhesives. The degree of conversion as a function of irradiation time was comparable for experimental and control adhesives. In dynamic mechanical analysis, broad tan delta peaks were obtained for all samples, indicating that the polymerized networks are heterogeneous; comparison of the full-width-at-half-maximum values obtained from the tan delta curves indicated increased heterogeneity for samples cured in the presence of water and/or containing TMPEDMA. The experimental adhesive showed higher T(g) and higher rubbery modulus indicating increased crosslink density when compared with the control. The improvement in esterase resistance afforded by adhesives containing the TMPEDMA is greater when this material is photopolymerized in the presence of water, suggesting better performance in the moist environment of the mouth. The improved esterase resistance of the new adhesive could be explained in terms of the densely crosslinked network structure and/or the steric hindrance of branched alkyl side chains.

Biocompatibility of Hydroxylated Metabolites of BISGMA and BFDGE

Unpolymerized dental monomers can leach out into the oral biophase and are bioavailable for metabolism. We hypothesize that metabolites would be less toxic than parent monomers. We first identified the formation of metabolites from bisphenol F diglycidyl ether (BFDGE) and Bisphenol A glycidyl methacrylate (BISGMA) after their exposure to liver S9 fractions. Then, the metabolites and parent compounds were subjected to in vitro cytotoxicity, mutagenicity, and estrogenicity studies. Bisphenol A bis(2,3-dihydroxypropyl) ether and bisphenol F bis(2,3-dihydroxypropyl) ether were the hydroxylated metabolites of BISGMA and BFDGE, respectively. Cytotoxicity against L929 cells showed that the metabolites were significantly (p < 0.05) less cytotoxic than the parent monomers. Only BFDGE was mutagenic in the Ames assay with strain TA100 of Salmonella typhimurium. Parent and metabolite compounds did not stimulate estrogen-dependent MCF-7 cell proliferation above solvent controls. These results indicated that the hydroxylated metabolites were non-mutagenic, non-estrogenic, and less cytotoxic than their parent monomers.

Oxidative mutagenesis of doxorubicin-Fe(III) complex

Doxorubicin has a high affinity for inorganic iron, Fe(III), and has potential to form doxorubicin-Fe(III) complexes in biological systems. Indirect involvement of iron has been substantiated in the oxidative mutagenicity of doxorubicin. In this study, however, direct involvement of Fe(III) was evaluated in mutagenicity studies with the doxorubicin-Fe(III) complex. The Salmonella mutagenicity assay with strain TA102 was used with a pre-incubation step. The highest mutagenicity of doxorubicin-Fe(III) complex was observed at the dose of 2.5nmol/plate of the complex. The S9-mix decreased this highest mutagenicity but increased the number of revertants at a higher dose of 10nmol/plate of the complex. On the other hand, the mutagenicity of the doxorubicin-Fe(III) complex at the doses of 0.25, 0.5, 1 and 2nmol/plate was enhanced about twice by the addition of glutathione plus H(2)O(2). This enhanced mutagenicity as well as of the complex itself, the complex plus glutathione, and the complex plus H(2)O(2) were reduced by the addition of ADR-529, an Fe(III) chelator, and potassium iodide, a hydroxyl radical scavenger. These results indicate that doxorubicin-Fe(III) complex exert the mutagenicity through oxidative DNA damage and that Fe(III) is a required element in the mutagenesis of doxorubicin.

Effects of Dental Resins on TNF-α-induced ICAM-1 Expression in Endothelial Cells

Many reports have demonstrated inflammation after the placement of dental restorations. To explain this side-effect, we studied a biomarker in the inflammatory response. The intercellular adhesion molecule-1 (ICAM-1) is a key mediator for recruitment of leukocytes to the site of inflammation. Therefore, we investigated whether methacrylates (a BISGMA-based dental resin, BISGMA, and MAA) and Cyracure™ UVR 6105, an epoxy monomer, could alter ICAM-1 expression in unstimulated and TNF-a-stimulated endothelial cells. Six-well plates with monolayers of human umbilical vein cells, ECV 304 (ATCC CRL 1998), were exposed to TNF-a (1 ng/mL) in the presence and absence of subtoxic and TC50 doses of chemicals for 24 hrs at 37°C/5% CO2. Several doses of TNF-a (0.5-2 ng/mL) were co-incubated with 100 μL of undiluted aqueous dental resin extracts. Cells were harvested and stained with mAB FITC-conjugated anti-human ICAM-1 (CD54). ICAM-1 expression was measured by flow cytometry. Cells expressed basal levels of ICAM-1, which was up-regulated by TNF-a but was not changed by all samples studied. Except for UVR 6105, the methacrylates significantly decreased ICAM-1 expression in TNF-α-stimulated cells. These findings suggest that methacrylates may decrease the recruitment of leukocytes to sites of inflammation.

Bisphenol A and its biomaterial monomer derivatives alteration of in vitro cytochrome P450 metabolism in rat, minipig, and human.

Bisphenol A (BPA) is a common structural component in a wide variety of biomaterial monomers. The effects of BPA and the following derivatives: bisphenol A glycidyl methacrylate (BisGMA), bisphenol A glycidyl diacrylate (BAGDA), bisphenol A ethoxylate dimethacrylate (BAEDM), bisphenol A dimethacrylate (BADM), and bisphenol A diglycidyl ether (BADGE) on mixed function oxidases (MFOs) are reported in this study. The rate of formation of metabolites from isoform-specific substrates for the MFOs (or cytochromes) CYP 1A, 2A, 2C, 2E, 3A, and 4A in the absence (control) and presence of BPA and derivatives was used to assess inhibition or stimulation of human, rat (male and female) liver, and minipig liver microsomal MFO activity. For human preparations the strongest inhibition by BPA was observed for CYP 2C. The inhibition was most prominent when a lower dose of BPA was used on the complete post-mitochondrial fraction. BPA inhibited rat microsomal CYP 1A isoform-specific metabolite production to 29 +/- 3% of control levels (100%). Biomaterial monomers exhibited mixed effects. For example, BPA stimulated CYP 4A in pooled human S9 to 129 +/- 1% of control. Also, BADM and BAGDA stimulated CYP 4A to 141% and 142% of control values, respectively.

In‐Vitro Stability, Metabolism, and Transport of Dental Monomers Made from Bisphenol A and Bisphenol F

The stability and bioavailability of the biomaterial monomers, bisglycidyl methacrylate (BISGMA), bisphenol F diglycidyl ether (BFDGE), and bisphenol A dimethacrylate (BPADM) were investigated using in-vitro techniques. A reverse-phase high-pressure liquid chromatographic/mass spectrometric (HPLC/MS) system was developed to quantitate each monomer and its primary metabolite. Each monomer (10 x 10 -6 M) was incubated at 37 °C under various conditions. Aliquots (N = 3) were removed at various time intervals and quantitated from a standard curve. The in-vitro transport of each parent monomer and its tetrahydroxy metabolite was measured in a Caco-2 system. BISGMA and BPADM were stable in aqueous solution at pH 1. However, BFDGE, was unstable. Plasma esterase of the rat rapidly hydrolyzed the ester compounds, but human esterase did not have a hydrolytic effect on BISGMA or BPADM. BFDGE disappeared in both rat and human plasma, but no tetrahydroxy metabolite was observed. All three parent compounds were unstable in human- and rat-hepatic fractions producing either tetrahydroxy metabolites or bisphenol A (BPA). The tetrahydroxy metabolites, however, were relatively stable under identical conditions, but BPA disappeared when incubated in hepatic-microsomal fractions. While BPADM metabolism produced BPA, an estrogen disrupter, BISGMA and BFDGE did not appear to produce BPA. These results suggest that the potential toxicity of leached dental monomers is more likely to be a result of the metabolite rather than the parent monomer. From Caco-2 transport studies, BFDGE and its tetrahydroxy metabolite both crossed the Caco-2 membrane at a low rate of transport in 2 h (approximately 3 and 5.2%, respectively). The BISGMA metabolite crossed at approximately 8%, indicative of a moderate rate of transport, and BPA crossed at approximately 10% in 1 h (high rate of transport). The transport of BPADM and BISGMA was unable to be determined due to nonspecific absorption to the acrylic vertical transport well. The transport of BFDGE tetrahydroxy metabolite is of particular interest as BFDGE is likely to be chemically hydrolyzed in stomach acid. It is well known that epoxies react with acids resulting in ring opening, so it is not surprising that BFDGE decomposes at pH 1. As a result, it is necessary to identify the decomposition (hydrolysis) products and test their bioavailability.