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Corrosion and release of nickel ions from biomedical alloys are well documented, but little is still known about the effects of released nickel ions on cellular function with recurrent inflammatory challenges. Evidence suggests Ni(II) ions amplify LPS-induced secretion of several pro-inflammatory cytokines from monocytes. Exacerbating the inflammatory response, hyperglycemic conditions also affect monocytic function. This study investigated how Ni(II) and hyperglycemic conditions, both singly and in combination, alter monocyte proliferation, mitochondrial activity, inflammatory responses, and differentiation. Results showed that Ni(II) did not affect proliferation, but decreased mitochondrial activity in monocytic-cells and macrophages under normal conditions. However, hyperglycemic conditions negated the toxicity seen with Ni(II) exposure. Cytokine secretion in response to LPS was variable, with little effect on IL6 secretion, but significantly increased secretion of IL1β at intermediate Ni(II) concentrations. Hyperglycemic conditions did not alter these results significantly. Finally, exposure to eluants from nickel-based commercial alloys caused enhanced IL1β secretion from PMA-treated cells. These data suggest that corrosion products from nickel-containing dental alloys increased Ni(II)-induced changes in cytokine secretion by monocytes and macrophages. By better defining the effects of Ni(II) at these lower, biomedically relevant concentrations, we improve understanding of the biomedical alloy risk in the context of dental inflammation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2433-2439, 2018.

Recent studies have reported that sealers may alter the secretion of specific cytokines from THP1 monocytic cells in vitro. In this study, a cytokine array was used to determine if endodontic sealers changed secretion of 42 cytokines. White mineral trioxide aggregate (WMTA), MTA preparation (CS), AH-Plus (AHP), and Pulp Canal Sealer (PCS) were mixed, allowed to set for 72 h, then “aged” in buffered-saline for 12 weeks. Aged specimens were placed in direct contact with THP1 for 72 h and their cytotoxicity (MTT assay) was assessed. Materials that were not severely toxic were then exposed to THP1 with or without lipolysaccharide (LPS), and the culture medium was assayed for cytokine secretion. Secretion of cytokines was quantified using infrared scanning (Odyssey®); replicate pairs were averaged. PCS severely suppressed MTT activity and was not assessed for its influence on cytokine secretion. WMTA, CS, and AHP induced a broad-based increase in cytokine secretion (>20% vs. Teflon controls), but AHP induced the greatest increase (>100% in 17 of 42 cytokines). The effects of the sealers on LPS-activated THP1 were biphasic, with some increases and decreases cytokine secretion of >20%, but few larger effects. This work shows endodontic sealers may alter the secretion of a broad cross section of cytokines from monocytic cells. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2011.

The in vitro cytotoxicity of five endodontic sealers was measured >8-12 weeks using L929 mouse fibroblasts, osteoblastic cells (ROS) 17/2.8 rat osteoblasts, and MC3T3-E1 mouse osteoblasts. Discs (n = 6) of AH-plus Jet (AHP), two versions of Endo Rez (ER, ERx), Epiphany (EPH), and Pulp Canal Sealer (PCS) were prepared. The sealers and Teflon (Tf, negative control) were placed in direct contact with cells after immersion in phosphate-buffered saline for 1-12 wk. Cellular succinate dehydrogenase (SDH) activity was estimated using the MTT method (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole), and activities were normalized to Teflon® controls. The cellular responses to the materials were compared using analysis of variance with Tukey posthoc analyses (α = 0.05). Initially, all sealers suppressed normalized SDH activity of L929 fibroblasts by >90%. After 12 weeks of immersion in saline, AHP exhibited the SDH activity above Tf (120%), followed by ERx (78%), ER (58%), PCS (38%), and EPH (28%), all statistically distinct (p < 0.05). In general, the three cell lines responded similarly to the sealers. However, AHP caused unique responses: ROS cells were significantly (p < 0.05) less sensitive initially, and AHP was severely cytotoxic to MC3T3 cells (

Few published studies describe the biological properties of calcium phosphate cements (CPCs) for dental applications. We measured several biologically relevant properties of 3 CPCs over an extended (8 wk) interval. Monocalcium phosphate, calcium oxide, and synthetic hydroxyapatite were combined with either modified polyacrylic acid, light-activated modified polyalkenoic acid, or 35% w/w polymethyl vinyl ether maleic acid to obtain Types I, II, and III CPCs, respectively. Set cements were placed in direct contact with L929 fibroblasts for up to 8 weeks. Media Ca(+2) and pH were determined by atomic absorption spectroscopy and pH electrode respectively. Cell mitochondrial function was measured by MTT assay. Type I cements suppressed mitochondrial activity90% (vs. Teflon controls), but significantly (p0.05) improved to control levels over 8 weeks. Type II cements suppressed mitochondrial activity90% at all times. Type III cements elevated mitochondrial activity significantly after 7 wks. The pH profiles approached neutrality by 24 h, and all cements released calcium into the storage medium at all periods (24 h - 8 wk). We concluded that several types of cements had long-term biological profiles that show promise for dental applications.

Nickel-containing alloys are used in dentistry because of their low cost, but poor corrosion behavior increases their risk of causing adverse biological responses. Intraorally, nickel-containing alloys accumulate bacterial plaque that triggers periodontal inflammation via toxins such as lipopolysaccharide (LPS). Recent evidence suggests that in monocytes, Ni(II) amplifies LPS-induced secretion of several cytokines that mediate periodontal destruction. Thus, we investigated the effects of Ni(II), with or without LPS, on the secretion of a broader array of cytokines from monocytes. We then measured monocytic expression of two proteins, Nrf2 and thioredoxin-1 (Trx1), that influence the regulation of cytokine secretion. Cytokine arrays were used to measure the effects of 0-50 microM Ni(II) on cytokine secretion from human THP1 monocytes, with or without LPS activation. Immunoblots were used to estimate Nrf2 and Trx1 levels. Our results indicate that both Ni(II) alone and Ni(II) with LPS have broad-based effects on cytokine secretion. Ni(II) increased Nrf2 levels by threefold, and LPS amplified the effects of Ni(II) by 10-fold. Trx1 levels did not change under any condition tested. Our results suggest that Ni(II)-induced changes in cytokine secretion by monocytes are diverse and may be influenced by Nrf2 but are not likely influenced by changes in whole-cell Trx1 levels.

AIM: To test the hypothesis that extending the time of a traditional ex vivo cytotoxicity test helps to identify trends in the behaviour of root core materials and sealers, which could ultimately aid in predicting their clinical safety and performance. METHODOLOGY: Endodontic sealers and core specimens were initially tested in direct contact with L929 fibroblasts for 72 h. Cell response was estimated by measuring cellular succinate dehydrogenase activity relative to Teflon controls. Cytotoxicity (% of more active cells) was reassessed after 1, 3, 4 and 6 weeks, with the specimens stored in a physiologically balanced salt-solution between tests. RESULTS: Distinct trends in cytotoxicity among both core materials and sealers were observed over the 6-week test. Four of the six sealers and two of the three core materials showed cell viabilities of 70% cytotoxicity). CONCLUSIONS: The current results suggest that some endodontic materials have an elevated biological risk for extended intervals.

Angioplasty with stent placement is commonly used to treat coronary atherosclerosis. However, 20-40% of stainless steel stents restenose within 6 months via a prolonged inflammatory response mediated by monocytic infiltration and cytokine secretion. In the current study, we tested a hypothesis that blood flow and monocytes interact to alter stent corrosion. We assessed the effects of THP1 monocytes on the corrosion rate of 316L stainless steel (316LSS) under shear stress (0.5-50 dyn/cm(2)). In addition, THP1 cytokine secretion was determined using cytokine arrays and ELISA analyses. Data were compared using ANOVA and Tukey post hoc analysis (alpha = 0.05). Monocytes significantly lowered 316LSS corrosion rates without limiting current density. However, shear stress alone did not alter the corrosion rate of 316LSS. THP1 cells adhered to the 316LSS surface at all flow rates. Exposure to the 316LSS/corrosion test under high fluid flow rates increased (>twofold) the secretion of 7 of the 42 cytokines tested (angeogenin, GRO, I309, interleukin 8, interleukin 6, interleukin 1beta, and macrophage chemoattractant protein-1). Each of these cytokines play a role in wound healing, macrophage differentiation, and cell proliferation, all hallmarks of in-stent restenosis. Furthermore, only IL8 levels were significantly higher than any of the system controls during the 316LSS/corrosion test conditions. The IL8 levels from the 316LSS/corrosion tests were not significantly different from the +LPS control. Together, these data suggest that monocytic cells maybe activated by exposure to 316LSS stents and could contribute to in-stent restenosis and altered corrosion of the stent.

Statement of problem Alloys with high nickel content have been increasingly used for dental prostheses. These alloys have excellent hardness, elastic modulus, and strength, yet have high corrosion rates when exposed to chemical or physical forces that are common intraorally. Purpose The purpose of the current study was to measure the susceptibility of several types of nickel-based alloys to brushing abrasion relative to gold- and palladium-based alloys. Material and methods Au-Pt, Au-Pd, Pd-Ag, Ni-Cr, and Ni-Cr-Be dental alloys were brushed with a toothbrush (Oral-B Soft) and toothpaste (Ultrabrite) in a linear brushing machine, then the surface roughness was measured by profilometry (R a , R v , R p ). Specimens (n=4) were brushed for 48 hours in a saline solution (pH 7). The effect of brushing was determined using 2-sided t tests (α=.05), and roughness among alloys postbrushing was compared using 1-way ANOVA with Tukey post hoc analyses (α=.05). Results All polished alloy surfaces (before brushing) had roughnesses of ≤0.1 μm (R a ). The Pd-Ag alloy was the most resistant to brushing. The Ni-Cr-Be alloy abraded the most, with a postbrushing surface roughness of >1 μm (R a ). Ni-Cr alloys without Be had a postbrushing surface roughness of 0.25 μm (R a ). Postbrushing roughness of all other alloys ranged from 0.1-0.25 μm (R a ). R v and R p values behaved similarly to R a values for all alloys. Conclusions Although they have many excellent mechanical properties, Ni-Cr-Be alloys may be prone to degradation from brushing. (J Prosthet Dent 2008;99:455-460)

Objectives Human blood levels of mercury are commonly 10 nM, but may transiently reach 50–75 nM after dental amalgam placement or removal. Controversy persists about the use of mercury because the effects of these ‘trace’ levels of mercury are not clear. Concentrations of mercury ≥5000 nM unequivocally alter redox balance in blood cells including monocytes. In the current study, we tested a hypothesis that concentrations of mercury

Summary Blue light curing units (wavelengths of 400–500 nm) are a mainstay of restorative dentistry, and several high-intensity light sources have been developed to polymerize resin composites more rapidly. The biological safety of visible light has been assumed, but some reports of adverse biological effects of blue light in non-dental contexts support further evaluation of the biological safety of high-intensity blue light. The current study tested the hypothesis that blue light provokes cell stress responses resulting in the secretion of cytokines or expression of heat-shock proteins (HSP) in monocytes. Human monocytic cells were irradiated with three light sources (quartz-tungsten-halogen, plasma-arc and laser), then cellular proliferation, secretion of the inflammatory cytokine TNFα and induction of HSP72 were measured. Results indicated that although all three light sources significantly inhibited proliferation of monocytes, the secretion of TNFα was not induced following exposure to blue light and was not potentiated with administration of the activator lipopolysaccharide. Similarly, treatment with the plasma-arc light, which caused the largest temperature increase in previous studies, did not induce HSP72. The current results do not support activation of monocytes by blue light as an inflammatory risk factor in dental tissues during curing of composites. However, the results of the current study should be further verified in primary monocytes and an animal model before decisions about clinical risks are made.

Resin composites are widely used in dentistry, and are polymerized in situ using a blue-light activated, free-radical polymerization mechanism. Blue light (400–500nm) is used to activate camphoroquinone (CQ), which decomposes to form free radicals that are stabilized by dimethyl-p-toludine (DMPT). CQ and DMPT are applied near tooth pulpal tissues and are irradiated during restorative procedures, suggesting that pulpal cells are exposed to free radicals. Because glutathione is a major component of the cellular redox management system, we tested the hypothesis that blue light irradiation would shift cellular glutathione redox balance of cells exposed to CQ and DMPT. We also measured NFκB activation, a redox-sensitive transcription factor that regulates inflammatory responses and glutathione synthetic enzyme levels. THP1 human monocytes were exposed to sublethal levels of CQ (0.4 mM) or DMPT (1.0 mM), with or without blue light exposure (25 J/cm2) from a quartz-tungsten-halogen source. The ratio of reduced to oxidized glutathione was measured using as assay based on 5,5′-dithio-bis(2-nitrobenszoic acid). NFκB transactivation was measured by transfection of an NFκB-containing plasmid linked to a luciferase reporter. Our results showed that blue light, CQ, or DMPT alone had no significant effect on cellular glutathione redox balance, but that the combination of these agents induced a marked oxidative bias, and reduced total glutathione levels up to 50%. On the other hand, light, CQ, and DMPT alone or in combination suppressed NFκB transactivation by >70%. Our results suggest that CQ and DMPT pose risks to pulpal tissues with or without blue light irradiation, and that multiple, interacting mechanisms shape the response to these agents. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2007

The biological response to dental restorative polymer composites is mediated by the release of unpolymerized residual monomers. Several new composite formulations claim to reduce unpolymerized residual mass. The current study assessed the cytotoxic responses to several of these new formations and compared them with more traditional formulations. Our hypothesis predicted that if these new polymerization chemistries reduce unpolymerized residual mass, the cytotoxicity of these materials also should be reduced relative to traditional formulations. Methods: Materials (HerculiteXRV, Premise, Filtek Supreme, CeramxDuo, Hermes, and Quixfil) were tested in vitro in direct contact with Balb mouse fibroblasts, initially, then after aging in artificial saliva for 0, 1, 3, 5, or 8 weeks. The toxicity was determined by using the MTT assay to the estimate SDH activity. Knoop hardness of the materials also was measured at 0 and 8 weeks to determine whether surface breakdown of the materials in artificial saliva contributed to cytotoxic responses. Results: Materials with traditional methacrylate chemistries (Herculite, Premise, Filtek Supreme) were severely (>50%) cytotoxic throughout the 8-week interval, but materials with newer chemistries or filling strategies (Hermes, CeramXDuo, and Quixfil) improved over time of aging in artificial saliva. Hermes showed the least cytotoxicity at 8 weeks, and was statistically equivalent to Teflon® negative controls. Hardness of the materials was unaffected by exposure to artificial saliva. Conclusions: Newer polymerization and filling strategies for dental composites show promise for reducing the release of unpolymerized components and cytotoxicity. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006

Nickel is a component of biomedical alloys that is released during corrosion and causes inflammation in tissues by as yet unknown mechanisms. Recent data show that Ni(II) at concentrations of 10-50 microM amplifies lipopolysaccharide-triggered, NFkappaB-mediated cytokine secretion from monocytes. In the current study, we tested the hypothesis that Ni(II) amplifies cytokine secretion by activating the Nrf2 antioxidant pathway rather than by enhancing activity of the NFkappaB signaling pathway. Human THP1 monocytes were exposed to Ni(II) concentrations of 10-30 microM for 6-72 h, then immunoblots of whole-cell lysates or cytosolic and nuclear proteins were used to detect changes in Nrf2 or NFkappaB signaling. Our results show that Ni(II) increased (by 1-2 fold) whole-cell Nrf2 levels and nuclear translocation of Nrf2, and amplified lipopolysaccharide (LPS)-induction of Nrf2 (by 3-5 fold), but had no detectable effect on the initial activation or nuclear translocation of NFkappaB. Because Nrf2 target gene products are known regulators of NFkappaB nuclear activity, our results suggest that Ni(II) may affect cytokine secretion indirectly via modulation of the Nrf2 pathway.

Thioredoxin reductase (TrxR) reduces thioredoxin (Trx), thereby contributing to cellular redox balance, facilitating the synthesis of deoxy-ribose sugars for DNA synthesis, and regulating redox-sensitive gene expression. Auranofin is a gold compound that potently inhibits TrxR. This inhibition is one suspected mechanism of auranofin’s therapeutic benefit in the treatment of rheumatoid arthritis. The use of other gold compounds to treat cancer or inflammatory disease may rely on their ability to inhibit TrxR. In the current study, we tested the hypothesis that a variety of gold compounds may inhibit TrxR. Methods: We exposed rat-TrxR1 to auranofin, gold sodium thiomalate, sodium aurothiosulfate, triphenyl phosphine gold chloride, or gold acetate, and measured TrxR activity ex vivo. We then compared TrxR1 inhibitory levels of gold compounds to those that inhibited mitochondrial activity of THP1 monocytes and OSC2 epithelial cells, estimated by succinate dehydrogenase activity. Results: All gold compounds inhibited TrxR1 at concentrations ranging from 5 to 4000 nM (50% inhibitory concentration). The oxidation state of gold did not correlate with inhibitory potency, but ligand configuration was important. Au(I)-phosphine compounds (triphenyl phosphine gold chloride and auranofin) were the most potent inhibitors of TrxR. All TrxR1 inhibitory concentrations were sublethal to mitochondrial activity in both THP1 and OSC2 cells. Conclusions: Diverse types of gold compounds may be effective inhibitors of TrxR1 at concentrations that do not suppress cellular mitochondrial function. Inhibition may be optimized to some degree by altering the ligand configuration of the compounds. These results support future study of a variety of Au compounds for therapeutic development as inhibitors of TrxR1.

Blue light from dental photopolymerization devices has significant biological effects on cells. These effects may alter normal cell function of tissues exposed during placement of oral restorations, but recent data suggest that some light-induced effects may also be therapeutically useful, for example in the treatment of epithelial cancers. Reactive oxygen species (ROS) appear to mediate blue light effects in cells, but the sources of ROS (intra- versus extracellular) and their respective roles in the cellular response to blue light are not known. In the current study, we tested the hypothesis that intra- and extracellular sources of blue light-generated ROS synergize to depress mitochondrial function. Normal human epidermal keratinocytes (NHEK) and oral squamous cell carcinoma (OSC2) cells were exposed to blue light (380–500 nm; 5–60 J/cm2) from a dental photopolymerization source (quartz–tungsten–halogen, 550 mW/cm2). Light was applied in cell-culture media or balanced salt solutions with or without cells present. Intracellular ROS levels were estimated using the dihydrofluorescein diacetate (DFDA) assay; extracellular ROS levels were estimated using the leucocrystal violet assay. Cell response was estimated using the MTT mitochondrial activity assay. Blue light increased intracellular ROS equally in both NHEK and OSC2. Blue light also increased ROS levels in cell-free MEM or salt solutions, and riboflavin supplements increased ROS formation. Extracellularly applied ROS rapidly (50–400 μM

A goal of treatment in periodontal therapy is to regenerate a new fibroblastic attachment rather than to repair lost attachment with a long junctional epithelium. To date, there is no evidence that fibroblastic attachment formed during regeneration is stronger or less susceptible to periodontal breakdown than a long junctional epithelial attachment. We measured the rate and strength of attachment of epithelial cells (NHEK) and periodontal ligament fibroblasts (PDLF) cultured individually and cocultured to dentin surfaces to determine which cell type has a faster attachment rate and greater adhesive strength to human dentin, and whether the cell types attach independently. Longitudinal dentin slices were seeded with either PDLF or NHEK for 2 or 24 h. The specimens were placed into a parallel plate flow chamber and defined laminar shear stresses were applied. Shear stress was created by step increases in fluid flow rate. Effluent fluid was collected and cell numbers (detached) were counted using a hemocytometer. Cocultures of PDLF and NHEK at three seeding ratios (10:1, 1:1, 1:10) were also tested. Each cell type attached equally well to polystyrene or dentin. PDLF showed a stronger attachment to polystyrene and dentin at 24 versus 2 h. NHEK attached to polystyrene or dentin equally well at 2 and 24 h. NHEK were more strongly attached after 2 h when compared to PDLF. PDLF were more strongly attached after 24 h versus NHEK. When NHEK and PDLF were seeded together on dentin at a 1:1 ratio, PDLF appeared to be more strongly attached than NHEK at 2 but not 24 h. At a ratio of 10 PDLF:1 NHEK, PDLF appeared to be more strongly attached at 2 and 24 h. At a ratio of 1 PDLF:10 NHEK, NHEK appeared to be more strongly attached at 2 h, but PDLF showed a trend of stronger attachment at 24 h. We conclude that epithelial cells attach more quickly to dentin surfaces than PDLF, but do not demonstrate increased attachment strength over time (PDLF do show increased attachment strength overtime). The purported advantages of periodontal regeneration over periodontal repair are supported by our results. Furthermore, our results support the concept of guided tissue regeneration. On the basis of on cellular competition experiments, epithelial cells and PDLF do not act independently, because epithelial cells enhanced the attachment rate of PDLF.

The transcription factor NFkappaB plays a key role in the tissue inflammatory response. Metal ions released into tissues from biomaterials (e.g., Au, Pd, Ni, Hg) are known to alter the binding of NFkappaB proteins to DNA, thereby modulating the effect of NFkappaB on gene activation and, ultimately, the tissue response to biomaterials. Little is known about the effect of these metals on key signaling steps prior to NFkappaB-DNA binding such as transcription factor activation or nuclear translocation, yet these steps are equally important to modulation of the pathway. Oxidative stress is known to alter NFkappaB proteins and is suspected to play a role in metal-induced NFkappaB signaling modulation. Our aim in the current study was to assess the effects of sublethal levels of Ni, Hg, Pd, and Au ions on NFkappaB activation and nuclear translocation in the monocyte, which is acknowledged as an important orchestrator of the biological response to materials and the pathogenesis of chronic disease. Sublethal concentrations of Au(III), Ni(II), Hg(II), and Pd(II) were added to cultures of human THP1 monocytic cells for 72 h. In parallel cultures, lipopolysaccharide (LPS) was added for the last 30 min to activate the monocytic cells. Then cellular cytoplasmic and nuclear proteins were isolated, separated by electrophoresis, and probed for IkappaBalpha degradation (activation) and NFkappaB p65 translocation. Protein levels were digitally quantified and statistically compared. The levels of reactive oxygen species (ROS) in the monocytic cells were measured as a possible mechanism of metal-induced NFkappaB modulation. Only Au(III) activated IkappaBalpha degradation by itself. Au(III) and Pd(II) enhanced LPS-induced IkappaBalpha degradation, but Hg(II) and Ni(II) suppressed it. Au(III), Ni(II), and Pd(II) activated p65 nuclear translocation without LPS, and all but Ni(II) enhanced LPS-induced translocation. Collectively, the results suggest that these metal ions alter activation and translocation of NFkappaB, each in a unique way at unique concentrations. Furthermore, even when these metals had no overt effects on signaling by themselves, all altered activation of signaling by LPS, suggesting that the biological effects of these metals on monocytic function may only be manifest upon activation. None of the metal ions elevated levels of ROS at 72 h, indicating that ROS were probably not direct modulators of the NFkappaB activation or translocation at this late time point.

Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea, exerts chemopreventive effects by selectively inducing apoptosis in tumor cells. In contrast, EGCG accelerates terminal differentiation in normal human epidermal keratinocytes (NHEK) mediated partially by up-regulation of p57/KIP2, a cyclin-dependent kinase inhibitor that confers growth arrest and differentiation. However, it is unclear if EGCG modulates caspase 14, a unique regulator of epithelial cell terminal differentiation associated with cornification. Here, we examined the effect of EGCG on caspase 14 expression in NHEK and correlated the protein and mRNA expression of p57/KIP2 with those of caspase 14 in either normal keratinocytes or p57/KIP2-expressing tumor cells (OSC2, an oral squamous cell carcinoma cell line). Additionally, paraffin-embedded normal and untreated psoriatic (aberrant keratinization) skin sections from humans were assessed for caspase 14 by immunohistochemistry. In NHEK, EGCG induced the expression of caspase 14 mRNA and protein levels within a 24-h period. The expression of p57/KIP2 in OSC2 cells was adequate to induce caspase 14 in the absence of EGCG; this induction of caspase 14 was down-regulated by transforming growth factor-beta1. In human psoriatic skin samples, caspase 14 staining in the upper epidermis was reduced, especially in nuclear areas. These results suggest that, in addition to p57/KIP2, EGCG-induced terminal differentiation of epidermal keratinocytes involves up-regulation of caspase 14. Further understanding of how EGCG modulates cellular differentiation may be useful in developing green tea preparations for selected clinical applications.

Plant-derived phenolic compounds, including polyphenols (e.g., tannins), flavonoids, and phenolic acids, have been under investigation for their anticancer therapeutic and chemoprevention properties. Recently, certain mechanisms underlying the differential effects of green tea polyphenols (GTPPs) on tumor versus normal cells have been determined. These suggest that GTPPs may simultaneously activate multiple pathways. However, existing screening methods are insufficient for the identification of agents that possess both a cytotoxic effect on tumor cells and a protective effect on normal cells. The current study describes the establishment of an in vitro survival/apoptosis testing system based on detecting these mechanisms by a double-fluorescence method. This system is able to screen potential chemopreventive or therapeutic agents from (but not limited to) plant-derived compounds based on the pathways differentially activated by the agents. Tumor cell death and normal cell survival are detected simultaneously, in a device that co-cultures normal human cells adjacent to human tumor cells.

Green tea polyphenols (GTPPs) are considered beneficial to human health, especially as chemopreventive agents. Recently, cytotoxic reactive oxygen species (ROS) were identified in tumor and certain normal cell cultures incubated with high concentrations of the most abundant GTPP, (-)-epigallocatechin-3-gallate (EGCG). If EGCG also provokes the production of ROS in normal epithelial cells, it may preclude the topical use of EGCG at higher doses. The current study examined the oxidative status of normal epithelial, normal salivary gland, and oral carcinoma cells treated with EGCG, using ROS measurement and catalase and superoxide dismutase activity assays. The results demonstrated that high concentrations of EGCG induced oxidative stress only in tumor cells. In contrast, EGCG reduced ROS in normal cells to background levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromodeoxyuridine incorporation data were also compared between the two oral carcinoma cell lines treated by EGCG, which suggest that a difference in the levels of endogenous catalase activity may play an important role in reducing oxidative stress provoked by EGCG in tumor cells. It is concluded that pathways activated by GTPPs or EGCG in normal epithelial versus tumor cells create different oxidative environments, favoring either normal cell survival or tumor cell destruction. This finding may lead to applications of naturally occurring polyphenols to enhance the effectiveness of chemo/radiation therapy to promote cancer cell death while protecting normal cells.

The most abundant green tea polyphenol, epigallocatechin-3-gallate (EGCG), was found to induce differential effects between tumor cells and normal cells. Nevertheless, how normal epithelial cells respond to the polyphenol at concentrations for which tumor cells undergo apoptosis is undefined. The current study tested exponentially growing and aged primary human epidermal keratinocytes in response to EGCG or a mixture of the four major green tea polyphenols. EGCG elicited cell differentiation with associated induction of p57/KIP2 within 24 h in growing keratinocytes, measured by the expression of keratin 1, filaggrin, and transglutaminase activity. Aged keratinocytes, which exhibited low basal cellular activities after culturing in growth medium for up to 25 days, renewed DNA synthesis and activated succinate dehydrogenase up to 37-fold upon exposure to either EGCG or the polyphenols. These results suggest that tea polyphenols may be used for treatment of wounds or certain skin conditions characterized by altered cellular activities or metabolism.

Although pregnancy gingivitis is widely believed to result from elevated hormone concentrations, the mechanism(s) involved in the etiology of this condition remain unknown. Paradoxically, despite the apparent inflammation for a prolonged period, pregnancy gingivitis rarely progresses to periodontitis and usually resolves postpartum. We used several methods to test in vitro the hypothesis that the elevated progesterone levels of pregnancy might inhibit the production of some of the matrix metalloproteinases (MMPs) that are responsible for periodontal destruction.Cultured human gingival fibroblasts (GF) were tested in phenol red-free, serum-free medium with or without the progestogen, medroxyprogesterone acetate (MPA; 10(-6) M), using interleukin-1beta (IL-1beta) to initiate immune responses and MMP production. These MMP responses were examined by macroarrays, reverse transcription-polymerase chain reaction (RT-PCR), zymograms, and enzyme-linked immunosorbent assay (ELISA).Array analysis showed that pretreatment of GF with MPA reduced mRNA induction for MMPs-1, -3, and -10 in response to 6 to 8 hours incubation with IL-1beta. RT-PCR confirmed, that after 24 hours with IL-1beta , GF pretreated with MPA had undetectable levels of mRNA for MMPs-1, -2, -3, -7, -10, and -13. Zymograms of culture media from this 24-hour period showed reduction in several proteolytic activities. Examination of such 24-hour media using ELISA for MMP-3 and pro-MMP-13 confirmed that secretion of these enzymes was upregulated by IL-1beta and modulated downward by pretreatment with MPA.Production by GF of numerous MMPs in response to IL-1beta was significantly reduced by progesterone. This steroidal modulation of proteolytic enzymes could help to explain why pregnancy gingivitis typically is not characterized by progression to periodontitis.

Metals are components of a variety of biomaterials used in orthopedic and dental appliances; however, their biocompatibility with the surrounding tissues is not completely understood. Monocytes are important immune cells that respond to inflammatory stimuli by rapidly producing a variety of inflammatory proteins. Regulation of this response often involves activation of the transcription factor NF kappa B. The current study was designed to determine whether monocyte activation of NF kappa B in response to bacterial lipopolysaccharide (LPS) is affected by pretreatment with metal ions. Concentrations of metal ions that affected cell number after 24 h of exposure were first determined. Then THP-1 human monocytes were cultured for 2 h in media containing metal ions at concentrations below levels that altered cell growth. Parallel cultures were treated with 10 microg/mL Escherichia coli LPS, and all samples were cultured an additional 2 h. Nuclear proteins were extracted and normalized amounts were incubated with [(32)P]-end-labeled NF kappa B consensus oligonucleotide. NF kappa B-DNA complexes were identified and quantified by electrophoretic mobility shift analysis. The extent of NF kappa B-DNA complex formation after metal ion pretreatment with or without LPS induction was compared to no treatment or LPS-only treated controls. Finally, LPS-induced IL1 beta secretion was measured from palladium-treated and control cells. Concentrations were identified for each metal ion (Ag(+), Co(2+), Cu(2+), Hg(2+), Ni(2+), and Pd(2+)) that did not reduce cell number after 24 h of exposure (ranging from 5 microM for Ag(+) and Hg(2+) to 200 microM for Ni(2+)). Exposures of 2 h at these concentrations did not alter cell morphology, staining with trypan blue, or cell number. LPS exposure had no effect on cell number with or without metal ions after 2 h. When metal treatment alone was assessed, none of the metal ions had a significant effect on NF kappa B-DNA binding. However, pretreatment with Co(2+), Ni(2+), Ag(1+), Hg(2+), and Pd(2+) significantly decreased NF kappa B-DNA binding by 40-70% versus LPS alone. Only Cu(2+) had no effect on LPS-induced NF kappa B-DNA complex formation. Pd(2+) lowered, but did not abolish, IL1 beta secretion at concentrations comparable to those that altered NF kappa B-DNA binding. These results suggest that many commonly used metals alter monocyte function at concentrations that are not overtly toxic, and that protein levels controlled in part by NF kappa B also may be altered.

A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.

Smoking is clearly a high-risk behavior for the development of periodontal disease, and nicotine is the major vasoactive component in tobacco. Integrins are a large family of homologous transmembrane adhesion proteins serving as the principal receptors on animal cells to bind and communicate with many extracellular matrix proteins such as collagen, fibronectin, and laminin. This study's aim was to evaluate nicotine's effects on beta1 integrin expression as a function of either 1) the generalized effects on RNA/protein synthesis or 2) as a specific modulation of beta1 integrin synthesis.Pooled human gingival fibroblasts (HGFs) were cultured in nicotine concentrations commonly seen in smokers (0.025 to 0.8 microM), and relative incorporation of [35S]methionine into newly synthesized protein or [3H]uridine into newly synthesized RNA was measured. Cultures were harvested at various times, and duplicate cell aliquots were homogenized and fractionated to obtain cell membrane-enriched preparations or solubilized to obtain whole cell lysates. Radiolabeled RNA and proteins were quantitated by trichloroacetic acid (TCA) precipitation and liquid scintillation spectrometry. Beta1 integrin subunits were detected by SDS-PAGE and Western blotting, and the relative intensities of reactive bands were quantitated by scanning densitometry.After 17 hours of exposure, 0.4 and 0.8 microM nicotine resulted in a dose-dependent increase in beta1 integrin in whole cell lysates, and a decrease in beta1 integrin in the corresponding membrane-enriched fractions. There was also a statistically significant decrease (Por = 0.05) in radiolabeled proteins in culture. Although there appeared to be a mild, generalized reduction in radiolabeled RNA in nicotine-treated cultures compared to controls, a 1-way analysis of variance showed no statistical significance between values.Our results suggest that nicotine may induce an altered compartmentalization process in which beta1 integrin molecules are produced, but are not appropriately transferred to the membrane. Nicotine effects on cellular protein synthesis and its modulation of beta1 integrin expression may impair gingival fibroblast ability to adhere to and communicate with one another and with the extracellular matrix, which could impair wound healing and/or exacerbate periodontal disease.

Recent studies suggest that light in the UVA range (320-400 nm) activates signaling pathways that are anti-inflammatory, antioxidative and play a critical role in protection against cancer. These effects have been attributed to NF-E2-related factor (NRF2)-mediated up-regulation of 'phase 2' genes that neutralize oxidative stress and metabolize electrophiles. We had previously shown that small doses of blue light (400-500 nm) had selective toxicity for cultured oral tumor cells and increased levels of peroxiredoxin phase 2 proteins, which led to our hypothesis that blue light activates NRF2 signaling.A431 epidermoid carcinoma cells were treated in culture and as nude mouse xenografts with doses of blue light. Cell lysates and tumor samples were tested for NRF2 activation, and for markers of proliferation and oxidative stress.Blue light activated the phase 2 response in cultured A431 cells and reduced their viability dose dependently. Light treatment of tumors reduced tumor growth, and levels of proliferating cell nuclear antigen (PCNA), and oxidized proteins.Cellular responses to these light energies are worth further study and may provide therapeutic interventions for inflammation and cancer.

The release of metal ions from dental restorations has been well established, but the sublethal effects of these ions on oral tissues remain undefined. Metal ions are a potential cause of sublethal oxidative stress in cells. Oxidative stress is known to alter cellular processes which are important in the inflammation of tissues. The presence of oxidative stress is commonly assessed by measuring the oxidized (GSSG) and reduced (GSH) forms of cellular glutathione. Normally, the majority of cellular glutathione exists as GSH and the GSH–GSSG ratio is high. Since monocytes often orchestrate inflammatory responses, oxidative stress in monocytes is important. Our hypothesis for the current work was that metal ions induce oxidative stress in monocytes which is manifested by a lower GSH–GSSG ratio. Human THP-1 monocytes were exposed for 24 h to sublethal concentrations of ions of Ag, Be, Co, Cu, Hg, Ni, Pd, and Zn – all known to be released from dental biomaterials. GSH and GSSG were measured using colorimetric assays. Cu lowered the GSH–GSSG ratio as hypothesized, but the ratio could not be determined for other metals because other metal ions interfered with the GSSG assay. However, a statistically significant ( anova/Tukey) increase in GSH per cell was observed upon exposure to Ag (50%), Co (100%), Hg (250%) and Zn (10%). This increase may be indicative of an oxidative stress in its own right. Alterations in GSH levels may be important to how released metal ions alter cytokine secretion from monocytes and other cells which play a role in the inflammatory response to dental biomaterials.

Although pit and fissure sealants have been utilized extensively in dentistry as a way of preventing occlusal caries, results described by Olea et al. (1996) raised concerns about the safety of sealants and other resin-based dental materials due to the reported presence of bisphenol A (BPA) and its dimethacrylate ester (BPA-DM). Although the release of these compounds from dental materials has not been substantiated by two subsequent studies, we believed it was important to confirm or refute the report that BPA and BPA-DM have estrogenic activity in vitro. We grew breast cancer cells (MCF-7, T-47D, ZR-75-1) known to proliferate under estrogenic stimulation in phenol red-free DMEM containing human serum and concentrations of BPA or BPA-DM ranging from 10−8M to 5 × 10−6M. After 1 week, plates were harvested for crystal violet or sulforhodamine-B assays, and the optical densities of groups of treated cells were compared with values from control cells. At concentrations at or above 10−6M, both BPA and BPA-DM significantly increased cell proliferation (p < 0.05), comparable to the increase seen with 10−9M of estrogen. Flow cytometric methods demonstrated that these mitogenic effects occurred within 24 h of exposure to estrogen, BPA, or BPA-DM. The increase in DNA synthesis was analogous to that seen with estrogen stimulation. Thus, we confirmed that BPA and BPA-DM cause cell proliferation at micromolar concentrations that exceed the effective concentrations of estrogen by 1 to 10,000-fold. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 45, 192–197, 1999.

The ability of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) to repress host cell protein synthesis early in infection has been studied extensively and found to involve the activities of the UL41 gene product, the virion-associated host shutoff (vhs) protein. To date, UL41 homologs have been identified in the genomes of three other alphaherpesviruses: equine herpesvirus 1 (EHV-1), varicella-zoster virus, and pseudorabies virus, but very little is known about the putative products of these homologous genes. Our earlier observations that no rapid early host protein shutoff occurred in EHV-1-infected cells led us to test EHV-1 vhs activity more thoroughly and to examine the expression and function of the EHV-1 UL41 homolog, ORF19. In the present study, the effects of EHV-1 and HSV-1 infections on cellular protein synthesis and mRNA degradation were compared at various multiplicities of infection in several cell types under an actinomycin D block. No virion-associated inhibition of cellular protein synthesis or vhs-induced cellular mRNA degradation was detected in cells infected with any of three EHV-1 strains (Ab4, KyA, and KyD) at multiplicities of infection at which HSV-1 strain F exhibited maximal vhs activity. However, further analyses revealed that (i) the EHV-1 vhs homolog gene, ORF19, was transcribed and translated into a 58-kDa protein in infected cells; (ii) the ORF19 protein was packaged into viral particles in amounts detectable in Western blots (immunoblots) with monoclonal antibodies; (iii) in cotransfection vhs activity assays, transiently-expressed ORF19 protein had intrinsic vhs activity comparable to that of wild-type HSV-1 vhs; and (iv) this intrinsic vhs activity was ablated by in vitro site-directed mutations in which either the functionally inactive HSV-1 vhs1 UL41 mutation (Thr at position 214 replaced by Ile [Thr-214-->Ile]) was recreated within ORF19 or two conserved residues within the putative poly(A) binding region of the ORF19 sequence were altered (Tyr-190, 192-->Phe). From these results we conclude that EHV-1's low vhs activity in infected cells is not a reflection of the ORF19 protein's intrinsic vhs activity but may be due instead to the amount of ORF19 protein associated with viral particles or to modulation of ORF19 protein's intrinsic activity by another viral component(s).

Although glass ionomer cements are generally considered to be tissue-compatible, it has been suggested that unreacted components or setting reaction by-products can affect cell metabolism. The current study examined the effects of constituents leached out of three glass ionomer cements on growth and metabolism of oral epithelial cells. Aseptically prepared discs of Ketac-Cem Radiopaque (KCR), Ketac-Cem Maxicap (KCM) and Fuji I were incubated in Dulbecco's medium for 10 d, with daily medium changes. Cultures of hamster cheek pouch (HCP) cells, a line of hamster buccal pouch epithelial cells, were incubated in control or eluate-containing media for 24 h. Viable cell numbers were determined by the colorimetric MTS assay, and DNA and RNA syntheses were assessed using [3H]thymidine and [3H]uridine incorporation, respectively. Responses to materials were determined by comparison of cell numbers and radioisotope incorporation (counts per minute (cpm) per 1000 cells). Results were analysed by ANOVA and Duncan's multiple range test, then converted to percent control for comparison. The eluates of all three materials from the first 24 h of soaking inhibited HCP cell growth. The number of cells in cultures exposed to Fuji were 88% of control cultures, while those exposed to KCR and KCM were 58% and 59% of control, respectively. The difference between Fuji-exposed and control cultures was significant (P < 0.05). The two Ketac cements were different from Fuji-exposed and control cultures (P < 0.05) but not from each other. All of the materials caused significant increases in labelling of DNA compared to control cultures (P < 0.05) when calculated on a per cell basis, but the materials did not differ from each other. Both Ketac cements also significantly stimulated labelling of RNA per cell compared to control cultures (P < 0.05). All effects of the material decreased over time. Results suggest that leachable components of the materials may affect the rate of progression of HCP cells through the cell cycle, rather than overt toxicity that results in cell death.

The gingivitis associated with pregnancy has been attributed to increased concentrations of circulating estrogen and/or progesterone. However, the mechanism by which these steroids increase gingival inflammation is not known. Interleukin-6 (IL-6), a pleiotropic cytokine produced by many cell types including human gingival fibroblasts (hGF), is secreted in response to inflammatory challenges such as bacterial lipopolysaccharide and interleukin-1 (IL-1). This study tested the hypothesis that progesterone could modulate the local production of IL-6 by hGF. The effects of progesterone on IL-6 production were measured in vitro in serum-free, phenol red-free medium to eliminate possible effects of such medium additives. The concentration of IL-6 secreted into supernatant medium after a 24 hour challenge with IL-1 beta was estimated by radioimmunoassay. Total RNA from steroid-treated hGF was probed for IL-6 mRNA. In serum-free medium, progesterone dose-dependently and significantly (P < 0.05) inhibited IL-6 production by hGF, as did the glucocorticoids hydrocortisone (HC) and dexamethasone. At progesterone concentrations common in late pregnancy, IL-6 production was reduced to levels 40 to 50% of control. In addition, mRNA was significantly down-regulated by progesterone and HC, at both basal levels and after IL-1 beta challenge. These results suggest that high levels of progesterone during pregnancy affect the development of localized inflammation by down-regulation of IL-6 production, rendering the gingiva less efficient at resisting the inflammatory challenges produced by bacteria.

The in vitro cytotoxic response to endodontic sealers was assessed for one year. AH-Plus (AHP), Epiphany (EPH), EndoRez (ER), Guttaflow (GF), InnoEndo (IN), and Pulp Canal Sealer (PCS) were exposed to mouse osteoblasts and human monocytes after curing, 52 weeks of aging, and after resurfacing post-aging; cellular response was estimated by succinate dehydrogenase (SDH) activity. The effect of materials on TNFα secretion from activated (LPS) and inactivated monocytes also was measured. Cell responses were compared with ANOVA and Tukey post hoc analysis (α = 0.05). Initially, all materials except GF suppressed osteoblastic SDH activity compared with Teflon (Tf) controls. SDH activity in cells exposed to some aged sealers improved significantly; but IN and ER remained cytotoxic. When aged materials were resurfaced then tested, AHP, ER, GF, and IN did not change. EPH and PCS were more toxic. Monocytes responded similarly to the osteoblasts. No endodontic sealer activated monocytic TNFα secretion (p > 0.05 vs. −LPS Tf-controls). LPS-activated monocytes exposed to unresurfaced AHP and IN significantly suppressed TNFα secretion. When activated monocytes were exposed to the resurfaced sealers, differential suppression of TNFα secretion was observed for three of the four sealers tested (EPH, IN, and PCS). The results suggest that long-term aging may be a useful adjunct to in vitro assessment of these materials. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.

Transient expression assays measuring induction of an equine herpesvirus 1 (EHV-1) immediate early (IE) promoter construct, pIE beta gal, were used to examine trans-induction of the IE gene (IR1) promoter by superinfection with intact EHV-1 (Kentucky A strain), uv-inactivated EHV-1, or EHV-1 stocks highly enriched for defective interfering particles (DIPs), and by cotransfection with plasmids containing EHV-1 DNA fragments. Our results confirm reports by others in that the IE promoter can be induced by both intact and uv-inactivated EHV-1 and provide evidence that DIP-rich virus stocks also possess transinducing activity. Furthermore, IE promoter activity was induced by cotransfection of the promoter construct with either of two plasmids containing restriction enzyme DNA fragments that span ORF12, the putative EHV-1 homolog of the herpes simplex virus 1 (HSV-1) alpha-trans-inducing factor (alpha-TIF) gene, UL48, or by cotransfection with an isolated DNA fragment containing only ORF12, while no transactivation was associated with plasmids linearized by restriction enzyme digestion at a single site within ORF12. These data indicate that, despite its predicted lack of an acidic carboxy terminus (a region essential in HSV-1 alpha-TIF for trans-inducing activity), the EHV-1 ORF12 product is a functional alpha-TIF.

Background We determined recently that dentin sialophosphoprotein (DSPP), a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family of phosphoglycoproteins, is highly upregulated in human oral squamous cell carcinomas (OSCCs) where upregulation is associated with tumor aggressiveness. To investigate the effects of DSPP-silencing on the tumorigenic profiles of the oral cancer cell line, OSC2, short-hairpin RNA (shRNA) interference was employed to silence DSPP in OSC2 cells. Methodology/Principal Findings Multiple regions of DSPP transcript were targeted for shRNA interference using hDSP-shRNA lentiviral particles designed to silence DSPP gene expression. Control shRNA plasmid encoding a scrambled sequence incapable of degrading any known cellular mRNA was used for negative control. Following puromycin selection of stable lines of DSSP-silenced OSC2 cells, phenotypic hallmarks of oral carcinogenesis were assayed by western blot and RT-PCR analyses, MTT (cell-viability), colony-formation, modified Boyden-Chamber (migration and invasion), and flow cytometry (cell-cycle and apoptosis) analyses. DSPP-silenced OSC2 cells showed altered cell morphology, reduced viability, decreased colony-formation ability, decreased migration and invasion, G0/G1 cell-cycle arrest, and increased tumor cell sensitivity to cisplatin-induced apoptosis. Furthermore, MMP-2, MMP-3, MMP-9, VEGF, Ki-67, p53, and EGFR were down-regulated. There was a direct correlation between the degree of DSPP-silencing and MMP suppression, as indicated by least squares regression: MMP-2 {(y = 0.850x, p

Titanates are inorganic compounds with high affinity for specific metal ions or metal compounds, including gold. We have previously demonstrated that both monosodium titanate (MST) and amorphous peroxo-titanate (APT) alone do not suppress cellular metabolism of several cell types, and we have shown that MST and APT adsorb and release gold compounds in biological salt solutions. In the current study, we extend this work and show that MST and APT loaded with two gold compounds deliver sufficient levels of these compounds to alter the metabolism of mammalian cells. Fibroblasts (L929) or monocytes (THP1) were exposed to MST and APT loaded with either Au(III) or Auranofin®, a Au(I)-organic compound, for 24–72 h, after which succinate dehydrogenase (SDH) activity of the cells was measured using the MTT method. MST or APT alone did not suppress SDH activity of either cell type. AF and Au(III) alone suppressed SDH activity completely above 2 μM or 300 μM, respectively. APT and MST loaded with either gold compound suppressed L929 fibroblast SDH activity by 30–80% after 72 h, but Au(III)-loaded APT was more potent than AF-loaded APT. Monocyte SDH activity was not affected by any loaded titanate. Our results suggest that titanates could be used for solid phase delivery of metal compounds to affect mammalian cell function of some types of cells. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010

Metal-based drugs are largely undeveloped in pharmacology. One limiting factor is the systemic toxicity of metal-based compounds. A solid-phase, sequestratable delivery agent for local delivery of metals could reduce systemic toxicity, facilitating new drug development in this nascent area. Amorphous peroxotitanates (APT) are ion-exchange materials with high affinity for several heavy metal ions and have been proposed to deliver or sequester metal ions in biological contexts. In the current study, we tested a hypothesis that APTs are able to deliver metals or metal compounds to cells. We exposed fibroblasts (L929) or monocytes (THP1) to metal-APT materials for 72 h in vitro and then measured cellular mitochondrial activity (SDH-MTT method) to assess the biological impact of the metal-APT materials versus metals or APT alone. APT alone did not significantly affect cellular mitochondrial activity, but all metal-APT materials suppressed the mitochondrial activity of fibroblasts (by 30-65% of controls). The concentration of metal-APT materials required to suppress cellular mitochondrial activity was below that required for metals alone, suggesting that simple extracellular release of the metals from the metal-APT materials was not the primary mechanism of mitochondrial suppression. In contrast to fibroblasts, no metal-APT material had a measurable effect on THP1 monocyte mitochondrial activity, despite potent suppression by metals alone. This latter result suggested that "biodelivery" by metal-APT materials may be cell type-specific. Therefore, it appears that APTs are plausible solid-phase delivery agents of metals or metal compounds to some types of cells for potential therapeutic effect.

Dental endodontic sealers are in intimate contact with tissues around the root apex (periapical area) for extended periods. New endodontic sealers have been developed in the past decade, but the biological responses to many new products are not well documented. In this study, we assessed in vitro monocytic cytotoxic and inflammatory responses to several contemporary endodontic sealers. AH-Plus (AH), Pulp Canal Sealer (PC), Epiphany (EPH), Endo-Rez (ER), and an experimental Endo-Rez (ERx) were initially placed in buffered-saline for 12 weeks to simulate in vivo use. After "aging," specimens were placed in direct contact with THP1 monocytes for 72 h and their cytotoxicity (mitochondrial response; MTT) or ability to trigger or suppress cytokine secretion (ELISA; TNFalpha, IL1beta, IL=6; +/- lipopolysaccharide (LPS) exposure) were measured relative to Teflon (Tf) negative controls. Cellular responses among conditions were compared with ANOVA and Tukey post-hoc analysis (alpha = 0.05). Two of the five sealers, EPH and PC, still suppressed cell mitochondrial activity by 70% or more after 12 weeks of conditioning in saline. No sealer alone activated monocytic TNFalpha, IL1beta, or IL6 secretion (p > 0.05 vs. +LPS controls). When THP1 were activated by LPS after exposure to the sealers, differential suppression of TNFalpha, IL1beta, and IL6 secretion was observed for two of the five sealers tested. (EPH and PC) This data suggest that common endodontic sealers do not activate monocytic TNFalpha, IL1beta, and IL6 secretion in vitro by themselves, but degradation products of the sealers may suppress activation of monocytes.

Endosseous dental implants use is increasing in patients with systemic conditions that compromise wound healing. Manufacturers recently have redesigned implants to ensure more reliable and faster osseointegration. One design strategy has been to create a porous phosphate-enriched titanium oxide (TiUnite®) surface to increase surface area and enhance interactions with bone. In the current study, the corrosion properties of TiUnite® implants were studied in cultures of monocytic cells and solutions simulating inflammatory and hyperglycemic conditions. Furthermore, to investigate whether placement into bone causes enough mechanical damage to alter implant corrosion properties, the enhanced surface implants as well as machined titanium implants were placed into human cadaver mandibular bone, the bone removed, and the corrosion properties measured. Implant corrosion behavior was characterized by open circuit potentials, linear polarization resistance, and electrical impedance spectroscopy. In selected samples, THP1 cells were activated with lipopolysaccharide prior to implant exposure to simulate an inflammatory environment. No significant differences in corrosion potentials were measured between the TiUnite® implants and the machined titanium implants in previous studies. TiUnite® implants exhibited lower corrosion rates in all simulated conditions than observed in PBS, and EIS measurements revealed two time constants which shifted with protein-containing electrolytes. In addition, the TiUnite® implants displayed a significantly lower corrosion rate than the machined titanium implants after placement into bone. The current study suggests that the corrosion risk of the enhanced oxide implant is lower than its machined surface titanium implant counterpart under simulated conditions of inflammation, elevated dextrose concentrations, and after implantation into bone. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010

In this study, we examined the potassium requirements for sperm-egg fusion in mouse. Zona-free mouse eggs prepared by the method described by Boldt and Wolf were inseminated with capacitated sperm in culture media containing 0-6 mM extracellular K+, and scored for penetration. Penetration of zona-free eggs was dependent on extracellular K+, with no penetration observed under K(+)-free conditions. Media transfer experiments indicated that the lack of penetration observed was due to effects on fusion, and not on postpenetration events such as sperm head decondensation. To analyze whether the K+ effect was attributable to an effect on the sperm (i.e., occurrence of acrosome reactions), sperm were treated with the Ca2+ ionophore A23187 before insemination. Less than 5% of zona-free eggs were penetrated with ionophore-treated sperm under K(+)-free conditions, suggesting that K+ is required for fusion per se. Addition of ionophore to insemination cultures similarly did not overcome the block to fusion observed under K(+)-free conditions. The potassium channel blockers 4-aminopyridine (0.1-5 mM) and tetraethyl ammonium chloride (5-50 mM) had no inhibitory effect on fusion. These data indicate that extracellular K+ is required for sperm-egg fusion and that this requirement may not involve membrane K+ channels.

The effects of hyperglycemia, altered cell function, or inflammatory mediators on implant corrosion are not well studied; yet, these effects are critical to implant biocompatibility and osseointegration. Because implant placement is burgeoning, patients with medically compromising systemic conditions such as diabetes are increasingly receiving implants, and the role of other inflammatory diseases on implant corrosion also needs investigation. In the current study, the corrosion properties of commercially available, machined titanium implants were studied in blood, cultures of monocytic cells, and solutions containing elevated dextrose concentrations. Implant corrosion was estimated by open circuit potentials, linear polarization resistance, and electrical impedance spectroscopy (EIS) for 26 h. In selected samples, THP1 monocytic cells were activated for 2 h with Lipopolysaccharide prior to implant exposure, and IL-1beta secretion was measured to assess the affect of the implants on monocyte activation. Implants under conditions of inflammatory stress exhibited more negative E(corr) values, suggesting an increased potential for corrosion. Linear polarization measurements detected increased corrosion rates in the presence of elevated dextrose conditions over PBS conditions. EIS measurements suggested that implants underwent surface passivation reactions that may have limited corrosion over the short term of this test. This result was supported by cyclic polarization tests. IL-1beta secretion was not altered under conditions of corrosion or implant exposure. The results suggest that inflammatory stress and hyperglycemia may increase the corrosion of dental endosseous titanium-based implants, but that longer, more aggressive electrochemical conditions may be necessary to fully assess these effects.

Objectives The use of lithium disilicate dental ceramics is increasing in dentistry and previous reports have suggested that they may have greater biological risks than previously thought. We tested a hypothesis that composition and processing influence the biological properties of these ceramics. Methods The cytotoxicity of two machined and three pressed lithium disilicate materials ( n = 6) were tested in vitro using mouse fibroblasts in direct contact with the materials for 72 h. Cellular response was estimated by mitochondrial succinate dehydrogenase activity (MTT method). Mitochondrial activity was expressed as a percentage of Teflon ® controls, then compared to Teflon ® using 2-sided t -tests ( α = 0.05). Polished materials were aged in artificial saliva and tested for cytotoxicity periodically over 6 weeks, then were repolished (320 grit SiC paper), aged and tested again for 4 weeks. Results All materials significantly (50–70%) suppressed cellular mitochondrial activity in the initial week, but suppression decreased by 25–30% over the next 2 weeks. In weeks 4 and 6 some materials exhibited a cytotoxic ‘relapse’ of 10–20%. The cytotoxic response was no different for machined or pressed materials, but the presence of ZnO had at least an association with longer-term cytotoxicity and relapse. Repolishing to 320 grit did not increase cytotoxicity significantly. Significance Our results suggest that lithium disilicates are not biologically inert, and that many have a similar cytotoxicity dynamic regardless of small differences in composition or processing.

In this study, human MCF-7 breast cancer cells, which express functional estrogen and progesterone receptors, were used to compare the efficacy of combined antiestrogen plus antiprogestin therapy to antiestrogen monotherapy. Cells were treated with the antiestrogen 4-hydroxytamoxifen (4-OHT) and/or the antiprogestin mifepristone (MIF) and effects on cell proliferation (cytostatic action), cell cycle phase, the phosphorylation state of the tumor suppressor retinoblastoma protein (Rb), and induction of active cell death (cytotoxic action) were determined. Combination hormonal therapy showed both increased cytostatic and cytotoxic activity as compared to either monotherapy. The increased cytostatic action was mediated by Rb activation; whereas, the cytotoxic (pro-apoptotic) action of combined hormonal therapy correlated to a significant reduction in Rb protein levels. To test the apparent role of Rb protein loss in the pro-apoptotic action of combined hormonal therapy, Rb was downregulated in MCF-7 cells using siRNA-targeting. The siRNA-mediated knockdown of Rb combined with 4-OHT therapy resulted in a pro-apoptotic action similar to that resulting from 4-OHT and MIF combination treatment, which included increased cell detachment from the monolayer, high-molecular-weight genomic DNA fragmentation, and cleavage of poly ADP-ribose polymerase (PARP) and lamin A. From these studies, we conclude that Rb protein downregulation is required for 4-OHT-treated, estrogen receptor positive (ER+) breast cancer cells to undergo active cell death. We discuss the potential of using an antiprogestin such as MIF plus antiestrogen treatment to more effectively downregulate Rb in ER+ breast cancer cells to increase the overall cytotoxic action of hormonal therapy.

Monosodium titanate (MST) particles effectively bind specific metals and are therefore promising compounds for delivery or sequestration of metals in biological contexts. Yet, the biological properties of MST are largely unexplored. Our previous study showed that the cytotoxicity of these compounds was mild, but the nature of the dose response curves suggested that residual titanates in culture may have interfered with the assay. In the current study, we assessed the importance of these artifacts, and extended our previous results using fibroblasts for biological evaluation. We also assessed the biological response to a new type of titanate (referred to as amorphous peroxo-titanate or APT) that shows more promising metal binding properties than MST. Methods: The degree of titanate-induced interference in the MTT (mitochondrial activity assay) was estimated by means of cell-free assays with and without a final centrifugation step to remove residual titanate particulate. Cytotoxic responses to titanates were assessed by measuring succinate dehydrogenase activity (by MTT) in THP1 monocytes or L929 fibroblasts after 24–72 h exposures. Monocytic activation by APT was assessed by TNFα secretion (ELISA) from monocytes with or without lipopolysaccharide (LPS) activation. Results: We confirmed that residual titanate particulates may alter the SDH activity assay, but that this effect is eliminated by adding a final centrifugation step to the standard MTT procedure. Addition of MST or APT at concentrations up to 100 mg/L altered succinate dehydrogenase activity by < 25% in both monocytes and fibroblasts. Fibroblasts displayed time-dependent adaptation to the MST. APT did not trigger TNFα secretion or modulate LPS-induced TNFα secretion from monocytes. Conclusions: Although further in vitro and in vivo assessment is needed, MST and APT exhibit biological properties that are promising for their use as agents to sequester or deliver metals in biological systems. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2007

The green tea polyphenol epigallocatechin-3-gallate (EGCG) regulates gene expression differentially in tumor and normal cells. In normal human primary epidermal keratinocytes (NHEK), one of the key mediators of EGCG action is p57/KIP2, a cyclin-dependent kinase (CDK) inhibitor. EGCG potently induces p57 in NHEK, but not in epithelial cancer cells. In humans, reduced expression of p57 often is associated with advanced tumors, and tumor cells with inactivated p57 undergo apoptosis when exposed to EGCG. The mechanism of p57 induction by EGCG is not well understood. Here, we show that in NHEK, EGCG-induces p57 via the p38 mitogen-activated protein kinase (MAPK) signaling pathway. In p57-negative tumor cells, JNK signaling mediates EGCG-induced apoptosis, and exogenous expression of p57 suppresses EGCG-induced apoptosis via inhibition of c-Jun N-terminal kinase (JNK). We also found that restoration of p57 expression in tumor cells significantly reduced tumorigenicity in athymic mice. These results suggest that p57 expression may be an useful indicator for the clinical course of cancers, and could be potentially useful as a target for cancer therapies.

Monosodium titanate (MST) is an inorganic sorbent/ion exchanger developed for the removal of radionuclides from nuclear wastes. We investigated the ability of MST to bind Cd(II), Hg(II), Au(III), or the Au-organic compound auranofin to establish the utility of MST for applications in environmental decontamination or medical therapy (drug delivery). Adsorption isotherms for MST were determined at pH 7-7.5 in water or phosphate-buffered saline. The extent of metal binding was determined spectroscopically by measuring the concentrations of the metals in solution before and after contact with the MST. Cytotoxic responses to MST were assessed using THP1 monocytes and succinate dehydrogenase activity. Monocytic activation by MST was assessed by TNFalpha secretion (ELISA) with or without lipopolysaccharide (LPS) activation. MST adsorbed Cd(II), Hg(II), and Au(III) under conditions similar to those in physiological systems. MST exhibited the highest affinity for Cd(II) followed by Hg(II) and Au (III). MST (up to 100 mg/L) exhibited only minor (

The biological effects of camphorquinone (CQ), an initiator for light-polymerized resins, have been reported to relate to its ability to generate free radicals and cause radical-induced membrane damage via lipid peroxidation. However, the effects of CQ on lipids other than peroxidation may result in unfavorable tissue responses especially at concentrations that are not overtly toxic to cells. The purpose of the current study was to examine the effects of CQ on cell lipid metabolism at subtoxic concentrations, with or without visible light irradiation. HCP and THP-1 cells were exposed to CQ with or without light irradiation under clinically relevant conditions and lipid metabolism was analyzed using 14C-labeling and thin-layer chromatography. We found that CQ increased synthesis of neutral lipids, such as triglycerides, from 7 to nearly 15% of the total and diglycerides from 2% to about 3% of the total in HCP cells, while synthesis of phospholipids, such as sphingomyelin, was decreased by 1-1.5%. In THP-1 cells cholesterol synthesis increased more than 2-fold and cholesterol ester synthesis increased more than 5-fold. Light-activated CQ did not differ significantly in terms of its bioactivity compared to no-light conditions. We conclude that CQ significantly altered the metabolism of several important structural lipids in two cell types at sub-toxic concentrations that are clinically relevant. These changes in lipid metabolism may in turn affect membrane integrity and permeability and possibly lead to significant changes in cell responses.

The toxicity of anti-rheumatic gold compounds has limited their use and development, yet both the toxicological and therapeutic actions of these compounds remain unclear. In the current study, we tested the hypothesis that intracellular reactive oxygen species (ROS) induced by Au(I) or Au(III) compounds mediate their ability to suppress mitochondrial activity. Methods Human THP1 monocytes were exposed to HAuCl 4 · 3H 2 O (Au(III)), or the anti-rheumatic compounds auranofin (AF) or gold sodium thiomalate (GSTM) for 6–72 h, after which mitochondrial activity (succinate dehydrogenase) was measured. To assess the role of cellular redox status as a mediator of mitochondrial suppression, monocytes were pre-treated with a pro-oxidant ( t -butyl hydroquinone, t -BHQ) or antioxidant ( N -acetyl cysteine, NAC ). ROS levels were measured 0–24 h post-gold addition to determine their role as mediators of mitochondrial activity suppression. Results AF was the most potent inhibitor of mitochondrial activity, followed by Au(III) and GSTM. Only Au(III) induced intracellular ROS; no ROS formation was observed in response to AF or GSTM exposure. Although anti- and pro-oxidants had some effects on mitochondrial suppression of Au compounds, collectively the data do not support redox effects or ROS formation as major mediators of Au-compound mitochondrial suppression. Conclusions Our results do not indicate that ROS and redox effects play major roles in mediating the cytotoxicity of AF, GSTM or Au(III).

The perennial controversy about the safety of mercury in dental amalgams has adversely affected the availability and the quality of dental care. Chronic Hg(II) blood concentrations above 300 nM are known to alter function of the nervous system and the kidney. However, the effects of blood concentrations of 10 to 75 nM, far more common in the general population, are not clear and mechanisms of any effects are not known. The monocyte is an important potential target of Hg(II) because of its critical role in directing inflammatory and immune responses. In the current study we tested the hypothesis that concentrations of Hg(II) of 10 to 300 nM alter monocyte activity via a redox-dependent mechanism. Mitochondrial activity was used to establish inhibitory concentrations of Hg(II) following 6 to 72 h of exposures to THP1 human monocytic cells. Then subinhibitory concentrations were applied, and total glutathione levels and reactive oxygen species (ROS) were measured. Antioxidants [N-acetyl cysteine, (NAC); Na2SeO3, (Se)] and a pro-oxidant (tert-butylhydroquinone, tBHQ) were used to support the hypothesis that Hg(II) effects were redox-mediated. After 72 h of exposure, 20 microM of Hg(II) inhibited monocytic mitochondrial activity by 50%. NAC mitigated Hg(II)-induced mitochondrial suppression only at concentrations of greater than 10 microM, but Se had few effects on Hg-induced mitochondrial responses. tBHQ significantly enhanced mitochondrial suppression at higher Hg(II) concentrations. Hg(II) concentrations of 75 and 300 nM (0.075 and 0.30 microM, respectively) significantly increased total glutathione levels, and NAC mitigated these increases. Se plus Hg(II) significantly elevated Hg-induced total cellular glutathione levels. Increased ROS levels were not detected in monocytes exposed to mercury. Hg(II) acts in monocytic cells, at least in part, through redox-mediated mechanisms at concentrations below those commonly associated with chronic mercury toxicity, but commonly occurring in the blood of some dental patients.

The use of hydrophilic dental monomers in dentin bonding agents has vastly improved resin–dentin bond strengths, but incomplete polymerization of these monomers and their leaching into adjacent (pulpal) oral tissues has raised concerns about their biocompatibility. The sublethal effects of these resins are virtually unknown, but their electrophilic nature led to the hypothesis that they may alter cellular oxidative stress pathways. Glutathione balance between reduced (GSH) and oxidized (GSSG) is a major mechanism by which cells maintain redox balance and was therefore the focus of the current investigation. THP-1 human monocytic cells were exposed to hydroxyethyl methacrylate (HEMA), benzoyl peroxide (BPO), camphorquinone (CQ), or triethyelene glycol dimethacrylate (TEGDMA) for 24 h at sublethal doses, then GSH and GSSG levels were measured by means of Ellman's method adapted for cell culture. The results indicate that these dental resin compounds act at least partly via oxidative stress by increasing GSH levels at sublethal concentrations. However, the GSH-GSSG ratio was relatively unaffected. Only BPO altered the GSH-GSSG ratio at 24 h, again at sublethal levels (7.5–15 μmol/L). The results support the hypothesis that resin monomers act, at least in part, via oxidative stress, and that oxidative-stress pathways should be one focus of future investigations of monomer biocompatibility. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater

OBJECTIVE: Nickel and cobalt ions activate ICAM1 expression on endothelial cells and keratinocytes. Furthermore, these ions are released in vitro and in vivo from the types of alloys used for vascular stents, but the full biological consequences of this release is not known. In the current study, we determined if release of elements from vascular stent alloys that contained nickel and cobalt was sufficient to activate expression of key cellular adhesion molecules (CAMs) by endothelial cells. Expression of these CAMs is a critical step in the long-term inflammatory response to stent materials and possibly to in-stent restenonsis. METHODS: Stainless steel, NiTi, CoCrNi, and NiCr alloys were placed in direct contact with primary human microvascular endothelial cells for 72 hours after preparation at three roughnesses (120, 320, and 1200 grit). Expression of three CAMs--ICAM1, VCAM1, and e-selectin--was assessed using a modified ELISA procedure. Cytotoxicity of the alloys was assessed by measuring succinate dehydrogenase (SDH) activity and total protein content of the cells, and nickel release was measured by atomic absorption spectroscopy. RESULTS: None of the alloys suppressed SDH activity or total cellular protein significantly at any surface roughness, indicating little or no cytotoxicity. Ni release was measurable from all alloys, was greatest from the rougher surfaces, and was significantly different for the different alloy types. NiTi alloys exhibited the lowest nickel release. However, none of the alloys activated expression of the CAMs, regardless of surface roughness or nickel release level. Supplemental experiments using nickel ions alone confirmed that ICAM1 was inducible on the endothelial cells by Ni(II) concentrations above 100 microM. CONCLUSIONS: In this in vitro system, nickel or other elemental release from several common types of stent alloys was not sufficient to activate expression of CAMs on endothelial surfaces. Although these results indicate a low risk for direct activation of endothelial cells by ions released from stent alloys, other mechanisms, such as modulation of CAM expression by monocytes or smooth muscle cells, must be considered before ion-mediated influence on CAM expression can be dismissed.