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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.

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

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.

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.

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 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.

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.