Liposomes embedded with differentiating factors as a new strategy for enhancing DPSC osteogenic commitment.

Human dental pulp stem cell (DPSC) differentiation toward the osteoblastic phenotype is enhanced when culture media are supplemented with differentiating factors, i.e. ascorbic acid, ß-glycerophosphate and dexamethasone. Liposomes, spherical vesicles formed by a phospholipid bilayer, are frequently used as carriers for drugs, growth factors and hydrophobic molecules. The aim of this work was to speed up DPSC commitment to the osteogenic lineage by embedding differentiating factors within liposomes. Firstly, liposomes were prepared by rehydrating a phospholipidic thin film and characterised in terms of dimensions. Secondly, liposome-exposed DPSCs were characterised by their immunophenotypic profile. Levels of CD90 were significantly decreased in the presence of liposomes filled with ascorbic acid, ß-glycerophosphate and dexamethasone (Lipo-Mix) with respect to normal differentiation medium (DM), while CD73 and CD29 expression were enhanced, suggesting osteogenic commitment. Additionally, an appreciable extracellular matrix deposition is detected. Thirdly, the Lipo-Mix formulation better increases alkaline phosphatase activity and levels of Collagen I secretion with respect to DM. In parallel, the new liposome formulation is capable of decreasing the release of H2O2 and of triggering a precocious antioxidant cell response, redressing the redox balance required upon mesenchymal stem cell commitment to osteogenesis. It can be therefore hypothesised that Lipo-Mix could represent a suitable tool for clinical regenerative purposes in the field of tissue engineering by speeding up DPSC osteogenic commitment, mineralised matrix deposition and remodelling.

Interactive effects of LPS and dentine matrix proteins on human dental pulp stem cells.

AIM: To investigate the combinatorial effects of lipopolysaccharide (LPS) and extracted dentine matrix proteins (eDMP) on regenerative and inflammatory responses in human dental pulp stem cells (DPSCs). METHODOLOGY: Culture media were supplemented with several concentrations of LPS, eDMP and combinations of both. Cell viability was assessed over 1 week by MTT assay; cell survival was evaluated after 24 h and 7 days by flow cytometry. The expression of mineralization-associated marker genes was determined by real-time quantitative polymerase chain reaction (RT-qPCR). To analyse the inflammatory response, secretion of interleukin 6 (IL-6) was quantified in the initial and the late phase of cell culture by enzyme-linked immunosorbent assay (ELISA). Data were treated nonparametrically and Mann-Whitney U-tests were performed to compare all experimental groups (α = 0.05). RESULTS: Whereas LPS had no impact on viability, eDMP led to a concentration-dependent decrease, which was significant after 7 days (P ≤ 0.024). A moderate decline of cell survival induced by LPS was detected after 48 h (P ≤ 0.026), whereas eDMP was able to reverse this effect. eDMP alone caused increased expression of tested marker genes, LPS had no regulatory effect. Combined eDMP and LPS induced an upregulation of collagen type I and osteocalcin, whereas expression levels of dentine matrix acidic phosphoprotein and dentine sialophosphoprotein were similar to the control. IL-6-secretion was increased by LPS over time. eDMP markedly elevated initial production of IL-6 (P ≤ 0.002), but suppressed LPS-induced cytokine production in the later phase. CONCLUSIONS: Lipopolysaccharide did not affect cell viability but interfered with odontoblast-like cell differentiation of DPSCs. Proteins from the dentine matrix may have a protective effect, attenuate the detrimental impact of LPS and thus play an important role during pulp repair.

Dentine matrix proteins: isolation and effects on human pulp cells.

AIM: To establish a simplified and efficient protocol for the isolation and concentration of matrix proteins from human dentine, and to assess the effects of extracted dentine matrix proteins (eDMP) on the behaviour of human pulp cells. METHODOLOGY: Matrix proteins were isolated from human dentine, purified, concentrated and characterized with protein and enzyme-linked immunosorbent assays (ELISA). Culture media were supplemented with eDMP in different concentrations, referred to as eDMP 1-10 000, to assess viability and proliferation of human pulp cells by DNA and MTT assays; apoptotic events were quantified by flow cytometry. Chemotactic effects of eDMP were assessed in a modified Boyden chamber assay. Expression levels of odontoblastic marker genes in pulp cells cultured with eDMPs were determined by real-time quantitative PCR, and the ability to induce mineralization was demonstrated by alizarin red staining. Nonparametric statistical analysis was performed to pairwise compare different groups at all time-points (Mann-Whitney U-test, α = 0.05). RESULTS: High concentrations of eDMP exhibited significant antiproliferative effects (P ≤ 0.023) after 5 (eDMP 1000) and 7 days (eDMP 500) without affecting cell viability. Apoptosis was barely influenced (P ≥ 0.089). eDMP exerted a concentration-dependent chemotactic stimulus on dental pulp cells with statistical significance already at low dosage (P = 0.006 at eDMP 10). Changes in gene expression indicated a differentiation into odontoblast-like cells, which was corroborated by findings of mineral nodule formation. CONCLUSIONS: A novel, effective and time-saving protocol for isolation and concentration of dentine matrix proteins is presented. As eDMP stimulates chemotaxis, differentiation and mineralization without affecting viability, endogenous dentine matrix proteins might be valuable for approaches to regenerate or engineer dental pulp.

Critical role of superoxide anions and hydroxyl radicals in HEMA-induced apoptosis.

OBJECTIVE: Resin monomers such as 2-hydroxyethyl methacrylate (HEMA) induce apoptosis because of the excess formation of reactive oxygen species (ROS). The portion of ROS including superoxide anions, hydrogen peroxide or hydroxyl radicals in monomer-induced apoptosis is unknown. Here, the effectiveness of superoxide anions or hydroxyl radicals was analyzed using tempol or sodium formate as radical scavengers. METHODS: RAW264.7 mouse macrophages were exposed to HEMA (0-6-8mM) in the presence of tempol (0-0.05-0.5-5.0mM) or sodium formate (0-1-5-10mM). The formation of ROS using DCFH2-DA or dihydrorhodamine 123 (DHR123) as fluorescent dyes and the induction of apoptosis was determined by flow cytometry after 1h or 24h exposure periods. Expression of enzymes related to ROS metabolism was detected by Western blotting. RESULTS: DCF fluorescence significantly increased after short exposure (1h) while DHR123 fluorescence was enhanced after a long exposure period (24h) in cells treated with HEMA. Although no influence was detected on the formation of ROS, tempol or sodium formate protected cells from HEMA-induced apoptosis. The number of cells in late apoptosis or necrosis induced with 6 or 8mM HEMA was reduced in the presence of tempol or low concentrations of sodium formate. HEMA-induced expression of catalase, indicating oxidative stress, decreased in the presence of tempol. SIGNIFICANCE: Superoxide anions and hydroxyl radicals contribute to HEMA-induced apoptosis. The current findings support the development of strategies based on the pharmacological inhibition of enzymes producing superoxide anions finally converted to hydroxyl radicals to compensate for potential adverse tissue reactions associated with dental composites.

Toll-like receptors, LPS, and dental monomers.

Unreacted monomers released from dental resin-based composites at non-cytotoxic concentrations cause a depletion of glutathione and an increase of reactive oxygen species (ROS), leading to, e.g., DNA damage and apoptosis. ROS-sensitive MAP-kinases are activated by HEMA and TEGDMA. MAP-kinases are also involved in the bacteria-triggered cell responses of the innate immune system, e.g., after bacterial lipopolysaccharide (LPS) binding to the Toll-like receptor (TLR) 4. Therefore, both bacteria and monomers imply environmental stress to pulp tissue, and they may influence the target cell reactions in a combined way. In macrophages, cell-surface antigens and cytokines were up-regulated after exposure to LPS, but TEGDMA caused a significant down-regulation. Regulation was dependent on exposure time, indicating that LPS and TEGDMA act differently on MAP-kinases. Furthermore, the cell type played a decisive role. Inhibition of the immune response may result in a decrease in inflammatory symptoms and/or a reduced defense capacity against bacteria.

TEGDMA reduces mineralization in dental pulp cells.

Direct application of dentin bonding agents onto the exposed pulp has been advocated, but in vivo studies indicate a lack of reparative dentin formation. Our objective was to investigate the role of triethylene glycol dimethacrylate (TEGDMA), a commonly used compound in dentin bonding agents, as a potential inhibitor of mineralization. Human pulp cells were exposed to different concentrations of TEGDMA, and expression of the mineralization-related genes collagen I, alkaline phosphatase, bone sialoprotein, osteocalcin, Runx2, and dentin sialophosphoprotein was analyzed. Gene expression studies by real-time polymerase chain-reaction revealed a concentration- and time-dependent decrease of mineralization markers. A subtoxic TEGDMA concentration (0.3 mM) reduced expression levels by 5 to 20% after 4 hrs and by 50% after 12 hrs. Furthermore, alkaline phosphatase activity and calcium deposition were significantly lower in dental pulp cells treated with TEGDMA over 14 days. These findings indicate that even low TEGDMA concentrations might inhibit mineralization induced by dental pulp cells, thus impairing reparative dentin formation after pulp capping with dentin bonding agents.

Cytotoxicity and genotoxicity of pulp capping materials in two cell lines.

AIM: The aim of this study was to evaluate the cytotoxicity and genotoxicity of the new castor oil bean cement (COB) material in comparison to commonly used pulp capping materials. METHODOLOGY: Specimens of COB, calcium hydroxide (Hydro C), and mineral trioxide aggregate (white and gray MTA) were extracted in culture medium (91.6 mm(2) sample surface mL(-1)). Transfected human pulp cells (tHPCs) were exposed to dilutions of the extracts for 1 h, and the generation of reactive oxygen species (ROS) was determined by flow cytometry (FACS) using H(2)DCF-DA as a dye. Survival of tHPCs was measured photometrically using a crystal violet assay after a 24-h exposure period. Genotoxicity as indicated by the formation of micronuclei in V79 cells, and the modification of the normal cell cycle by extracts of the materials was analysed by FACS. RESULTS: Clear cytotoxic effects were detected only with extracts of Hydro C under the current experimental conditions. The two MTA preparations induced an insignificant reduction in the number of cells. In contrast, the extracts of COB slightly induced cell proliferation. Extracts of Hydro C caused a twofold increase in ROS production, whilst the other tested materials were ineffective. An increase in the number of micronuclei was not detected with any material tested; Hydro C slightly increased the number of cells in G1 and G2. CONCLUSIONS: The COB and the two MTA preparations did not negatively influence cell survival or ROS production and may thus be further considered for pulp capping studies.

The toxicokinetics and distribution of 2-hydroxyethyl methacrylate in mice.

The cytotoxicity of dental composites has been attributed to the release of residual monomers from polymerized resin-based composites due to the degradation processes or the incomplete polymerisation of materials. 2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental resin-based composites. It was shown in vitro that HEMA was released into the adjacent biophase from such materials during the first days after placement. In this study uptake, distribution, and excretion of 14C-HEMA applied via gastric tube or subcutaneous administration at dose levels well above those encountered in dental care were examined in mice to test the hypothesis that HEMA can reach cytotoxic levels in mammalian tissues. 14C-HEMA was taken up rapidly from the stomach and intestines after gastric administration and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as (14)CO(2). Two metabolic pathways of 14C-HEMA can be described. The peak HEMA levels in all tissues examined after 24h were lower than known toxic levels. Therefore the study did not support the hypothesis.

The induction of oxidative stress, cytotoxicity, and genotoxicity by dental adhesives.

OBJECTIVES: Polymerized dental resin materials release residual monomers that may interact with pulp tissues. We hypothesized that dental adhesives might cause cytotoxicity in pulp cells via the generation of reactive oxygen species (ROS), which may also contribute to genotoxic effects in vitro. METHODS: For cytotoxicity testing, transformed human pulp-derived cells were exposed to extracts of primers and bonding agents of Clearfil SE bond, Clearfil Protect bond, AdheSE, Prompt L-Pop, and Excite for 24h. The cytotoxicity of the same materials was also analyzed in a dentin barrier test device using three-dimensional pulp cell cultures. The generation of ROS in monolayer cultures was measured after a 1h exposure period by flow cytometry (FACS), and genotoxicity as indicated by the formation of micronuclei was determined in V79 cells after a 24h exposure period. RESULTS: The dentin primers and bonding agents decrease cell survival in a dose-related manner. Cytotoxicity of bonding agents based on concentrations which caused 50% cell death (EC50) were ranked as follows: Excite (0.16 mg/ml)>AdheSE bond (0.30 mg/ml)>Clearfil Protect bond (0.35 mg/ml)>Clearfil SE bond (0.37 mg/ml), and Prompt L-Pop bond (0.68 mg/ml). Dentin primers were about 10-fold less effective. In contrast, no cytotoxic effects of the dental adhesives were observed in a dentin barrier test device. Yet, all dental adhesives increased the amounts of ROS about fivefold in pulp cells in a dose-related manner, and, again, the bonding agents were more efficient than the dentin primers. Finally, the number of micronuclei was increased about sixfold by extracts of the AdheSE primer. SIGNIFICANCE: Our results suggest that the cytotoxic potencies demonstrated by these materials might be of clinical relevance, since all dental adhesives disturbed the cellular redox state of pulp cells in monolayer cultures. As a result, the concentrations of biologically active ingredients of some of the agents may be high enough to modify pulp cell metabolism when the materials are used in deep cavities or directly contact pulp tissue.

Adhesion of eukaryotic cells and Staphylococcus aureus to silicon model surfaces.

Silicon wafers modified by silanisation with different functional groups are used to study the bioactivity of surfaces with varying physicochemical properties. Oxidation of the wafers created very hydrophilic surfaces, and moderately wettable surfaces were produced by coating with poly(ethylene glycol) (PEG). Immobilization of hydrocarbon chains to the wafers produced hydrophobic surfaces, and hydrophobicity was further increased by fluorocarbon coatings. The oxidized and the hydrocarbon-modified surfaces supported the adhesion of human MG-63 osteoblasts and 3T3 mouse fibroblasts as well as Staphylococcus aureus 8325-4. Adhesion of osteoblasts and fibroblasts, however, was decreased on highly hydrophobic fluorocarbon surfaces, whereas adhesion of S. aureus was supported. Coating of the fluorocarbon surface with fibronectin increased the number of attached eukaryotic cells, but the accumulation of bacteria remained unchanged. In contrast, surface coatings with PEG-groups inhibited the binding of S. aureus; however, the adhesion of the eukaryotic cells was high. The number of S. aureus on PEG-modified surfaces covered with fibronectin increased about twofold, yet it was still decreased to 25-30% related to the number of bacteria on other surfaces. These findings provide evidence that the PEG-modified surfaces showed selective bioactivity, preventing the attachment of a microbial pathogen but supporting the adhesion of eukaryotic cells.

Inhibition of TEGDMA and HEMA-induced genotoxicity and cell cycle arrest by N-acetylcysteine.

OBJECTIVES: Dental resin monomers like triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) are able to cause an imbalance of the redox state in mammalian cells. The resulting oxidative stress originating from reactive oxygen species (ROS) has been associated with cytotoxicity. We hypothesized that ROS might contribute to the generation of genotoxicity by TEGDMA and HEMA as well. Therefore, we examined the formation of micronuclei in V79 cells by both resin monomers in the presence of the antioxidant N-acetylcysteine (NAC), which scavenges ROS. In addition, we analyzed the effects of TEGDMA and HEMA on the normal cell cycle in the presence of NAC. METHODS: V79 fibroblasts were exposed to increasing concentrations of TEGDMA and HEMA in the presence and absence of NAC for 24h. Genotoxicity was indicated by the formation of micronuclei. The modification of the normal cell cycle was analyzed by flow cytometry (FACS). RESULTS: A dose-related increase in the number of micronuclei in V79 cells-induced by TEGDMA and HEMA indicated genotoxicity of both chemicals. However, the formation of micronuclei was reduced in the presence of 10 mmol/L NAC, indicating its protective role. A cell cycle delay in G2 phase caused by TEGDMA was absent when cells were co-treated with NAC. Similarly, the presence of NAC led to a reversion of the cell cycle delay in HEMA-treated cell cultures. SIGNIFICANCE: Our results suggest that genotoxic effects and the modification of the cell cycle caused by TEGDMA and HEMA are mediated, at least in part, by oxidative stress.

Genetic and cellular toxicology of dental resin monomers.

Monomers are released from dental resin materials, and thus cause adverse biological effects in mammalian cells. Cytotoxicity and genotoxicity of some of these methacrylates have been identified in a vast number of investigations during the last decade. It has been well-established that the co-monomer triethylene glycol dimethacrylate (TEGDMA) causes gene mutations in vitro. The formation of micronuclei is indicative of chromosomal damage and the induction of DNA strand breaks detected with monomers like TEGDMA and 2-hydroxyethyl methacrylate (HEMA). As a consequence of DNA damage, the mammalian cell cycle was delayed in both G1 and G2/M phases, depending on the concentrations of the monomers. Yet, the mechanisms underlying the genetic and cellular toxicology of resin monomers have remained obscure until recently. New findings indicate that increased oxidative stress results in an impairment of the cellular pro- and anti-oxidant redox balance caused by monomers. It has been demonstrated that monomers reduced the levels of the natural radical scavenger glutathione (GSH), which protects cell structures from damage caused by reactive oxygen species (ROS). Depletion of the intracellular GSH pool may then significantly contribute to cytotoxicity, because a related increase in ROS levels can activate pathways leading to apoptosis. Complementary, cytotoxic, and genotoxic effects of TEGDMA and HEMA are inhibited in the presence of ROS scavengers like N-acetylcysteine (NAC), ascorbate, and Trolox (vitamin E). Elevated intracellular levels of ROS can also activate a complex network of redox-responsive macromolecules, including redox-sensitive transcription factors like nuclear factor kappaB (NF-kappaB). It has been shown that NF-kappaB is activated probably to counteract HEMA-induced apoptosis. The induction of apoptosis by TEGDMA in human pulp cells has been associated with an inhibition of the phosphatidylinositol 3-kinase (PI3-K) cell-survival signaling pathway. Although the details of the mechanisms leading to cell death, genotoxicity, and cell-cycle delay are not completely understood, resin monomers may be able to alter the functions of the cells of the oral cavity. Pathways regulating cellular homeostasis, dentinogenesis, or tissue repair may be modified by monomers at concentrations well below those which cause acute cytotoxicity.

NF-kappaB protection against apoptosis induced by HEMA.

The cytotoxicity of dental monomers has been widely investigated, but the underlying mechanisms have not been elucidated. We studied the molecular mechanisms involved in cell death induced by HEMA. In human primary fibroblasts, HEMA induced a dose-dependent apoptosis that was confirmed by the activation of caspases-8, -9, and -3. We found an increase of reactive oxygen species (ROS) and NF-kappaB activation after HEMA exposure. Blocking of ROS production by anti-oxidants had no direct influence on apoptosis caused by HEMA, but inhibition of NF-kappaB increased the fraction of apoptotic cells. Accordingly, mouse embryonic fibroblasts (MEF) from p65-/- mice were more susceptible to HEMA-induced apoptosis than were wild-type controls. Our results indicate that exposure to HEMA triggers apoptosis and that this mechanism is not directly dependent upon redox signaling. Nevertheless, ROS induction by HEMA activates NF-kappaB, which exerts a protective role in counteracting apoptosis.

Inhibition of phosphatidylinositol 3-kinase amplifies TEGDMA-induced apoptosis in primary human pulp cells.

Cytotoxicity of triethylene glycol dimethacrylate (TEGDMA), a co-monomer of dental resinous restorative materials, is firmly established in vitro, but the molecular mechanisms are unknown. Here we examined apoptosis and necrosis induced by TEGDMA in human primary pulp cells. The levels of apoptotic and necrotic cell populations differentially increased after exposure to increasing concentrations of TEGDMA. A two-fold increase in the percentage of apoptotic cells was induced by 1 mmol/L TEGDMA. However, a population shift among cells in apoptosis and necrosis was detected when cell cultures were exposed to 2 mmol/L TEGDMA. Inhibition of the MAP Kinase/ERK pathway had no influence on cell survival, but inhibition of phosphatidylinositol 3 kinase (PI3-Kinase; Akt/protein kinase B) by LY294002 amplified TEGDMA-induced apoptosis. Moreover, Akt phosphorylation was inhibited in the presence of TEGDMA. These results suggest that depression of PI3K signaling may be a primary target in TEGDMA-induced apoptosis.

The induction of gene mutations and micronuclei by oxiranes and siloranes in mammalian cells in vitro.

Oxiranes and siloranes are candidate molecules for the development of composite materials with low shrinkage. Since some of these molecules are highly reactive, they could lead to adverse biological effects from underlying genetic mechanisms. Therefore, we analyzed the formation of micronuclei (chromosomal aberrations) and the induction of gene mutations (HPRT assay) in mammalian cells. The numbers of micronuclei induced by the oxirane di(cyclohexene-epoxidemethyl)ether (Eth-Ep) at low concentrations (10 micro M) were about five-fold higher than controls. The related compound epoxy cyclohexyl methyl-epoxy cyclo-hexane carboxylate (Est-Ep) was less effective. The activity of diglycidylether of bisphenol A (BADGE) was even lower but similar to the most reactive silorane, di-3,4-epoxy cyclohexylmethyl-dimethyl-silane (DiMe-Sil). No induction of micronuclei was detected in the presence of a rat liver homogenate (S9). Est-Ep and Eth-Ep also induced gene mutations. Our analyses indicated low mutagenic potentials of siloranes; however, some oxiranes induced strong effects at two genetic endpoints.

Responses of L929 mouse fibroblasts, primary and immortalized bovine dental papilla-derived cell lines to dental resin components.

OBJECTIVE: The use of adequate target cells for cytotoxicity testing of dental restorative materials has often been experimentally assessed with respect to the clinical relevance of the test results. In the present study, the responses in primary bovine dental papilla-derived cells (pulp cells) were compared with those in transformed dental papilla-derived cell lines and L929 mouse fibroblasts after exposure to various dental resin compounds. METHODS: Primary bovine dental papilla-derived cells (CPC), tCPC B (CPC cells transformed with SV40 T-antigen), tCPC E (CPC cells transformed with E6/E7 oncogen), and L929 mouse fibroblast cells were exposed to various compounds of dental resin materials for 24 h, and cytotoxicity was determined using the MTT assay. Bis-GMA, UDMA, 1,6 hexane diol dimethacrylate (HDDM), TEGDMA, HEMA, MMA, camphorquinone (CQ), bisphenol A (BPA), and glycidyl methacrylate (GMA) were tested. Concentrations leading to 50% cell survival (TC50 values) were calculated from fitted dose-response curves. RESULTS: The simple ranking of the cytotoxic effects of the dental resin compounds in the four cell types was identical, and TC50 values determined in L929 cells here were consistent with findings by other authors using continuous cell lines. However, the concentrations of the resin compounds necessary for eliciting cytotoxic responses in the various cells were clearly different. The analyses of TC50 values of the resin compounds revealed a linear correlation between cell lines, and the overall sensitivities increased as follows: CPC=tCPC B

The induction of micronuclei in vitro by unpolymerized resin monomers.

Components of resin materials may damage DNA, leading to genetic alterations in mammalian cells. Here, monomers were analyzed for the induction of chromosomal aberrations indicated by micronuclei induced in V79 cells. A dose-related increase in the numbers of micronuclei was observed with triethyleneglycol dimethacrylate (TEGDMA), 2-hydroxyethyl methacrylate (HEMA), and glycidyl methacrylate (GMA). These effects were reduced, however, by a metabolically active microsomal fraction from rat liver. The very low activity of Bis-GMA and UDMA and the elevated numbers of micronuclei caused by high concentrations of methyl methacrylate and bisphenol A were associated with cytotoxicity. Our findings provide evidence for the induction of micronuclei by TEGDMA, HEMA, and GMA under physiological conditions, indicating clastogenic activity of these chemicals in vitro. Since it has been shown that TEGDMA also caused gene mutations and DNA sequence deletions in mammalian cells, the activity of this substance should be analyzed in vivo.

Release of prostaglandin E2, IL-6 and IL-8 from human oral epithelial culture models after exposure to compounds of dental materials.

A three-dimensional human tissue model based on TR146 cells isolated from a squamous cell carcinoma of the buccal mucosa was used to test for the release of the proinflammatory molecules prostaglandin E2 (PGE2), interleukin 6 (IL-6), and interleukin 8 (IL-8) after exposure to nickel chloride (NiCl2), cobalt chloride (COCl2), palladium chloride (PdCl2), and triethylene glycol dimethacrylate (TEGDMA). These compounds have documented adverse biological effects in vitro. The release of PGE2 from the tissue culture models was inversely correlated with cell viability (MTT assay). Toxic concentrations of NiCl2 and CoCl2 induced the release of PGE2 by factors of about 200-300 compared to controls, but PdCl2 which was nontoxic enhanced PGE2 levels about 10-fold. TEGDMA, however, did not stimulate PGE2 release. None or weakly toxic concentrations of Ni and Co chloride induced IL-6 and IL-8 release by a factor of 5-10 compared to controls. The amounts of IL-6 were induced 25- to 30-fold by PdCl2 under physiological conditions, and IL-8 levels were also slightly enhanced. Nontoxic TEGDMA concentrations induced IL-6 levels 5-fold, but IL-8 amounts increased only slightly. We conclude that a steep rise of PGE2 is closely associated with cytotoxicity. On the other hand, the specific induction of IL-6 occurs at much lower concentrations. Therefore, the measurement of this cytokine may be included as another parameter in evaluating the biological activity of dental materials under nontoxic experimental conditions in vitro.

The induction of micronuclei in V79 cells by the root canal filling material AH plus.

The in vitro micronucleus test (MNT) is an alternative to the chromosomal aberration assay for the detection of chromosomal mutations. Here, the epoxy resin-based root canal filling material AH Plus, the compounds paste A and paste B were tested for cytotoxicity and the induction of micronuclei in V79 cells. Paste A is the epoxy-resin-containing compound. AH Plus was tested immediately after mixing and after a setting time of 24 h. The materials were eluted in dimethyl sulfoxide (DMSO) and physiologic saline for 24 h. DMSO eluates of the mixed material, paste A and paste B clearly reduced the viability of V79 cells, but eluates prepared with physiological saline were at least 10-100-fold less toxic. Likewise, freshly mixed AH Plus was mutagenic in a dose-dependent manner in V79 cells after elution in dimethyl sulfoxide only. The numbers of micronuclei were about 7-fold higher in treated cell cultures compared with untreated controls. No mutagenicity was observed with DMSO and physiological saline eluates of mixed AH Plus set for 24 h. DMSO eluates of paste A induced effects similar to the freshly mixed AH Plus, and paste B was not effective. Therefore, we provide evidence for the induction of chromosomal mutations by freshly mixed AH Plus under experimental conditions.

Influence of albumin and collagen on the cell mortality evoked by zinc oxide-eugenol in vitro.

Zinc oxide-eugenol (ZOE) is cytotoxic in vitro but does not cause pulp damage. Various dentin proteins may play a protective role in vivo. We investigated the influence of bovine serum albumin (BSA) and collagen on ZOE cytotoxicity in the Millipore filter test and a dentin barrier test. Agar or agarose with 1% or 5% BSA or calf collagen (1% and 2%) were introduced between L-929 fibroblasts and test specimens. ZOE was severely toxic in the absence of BSA. However BSA (1% and 3%) reduced cell damage to 50% and 35%. In contrast, collagen did not provide protection from ZOE cytotoxicity. When dentin was loaded with 3% BSA and the "pulpal" side of a dentin barrier test chamber was perfused with cell culture medium containing 3% BSA, cytotoxicity of ZOE was completely inhibited. Therefore we conclude that albumin may be one factor responsible for the absence of ZOE toxicity in pulp studies.