Effect of bleaching agent extracts on murine macrophages.

OBJECTIVES: The aim of this study was to evaluate the cytotoxicity and the influence of bleaching agents on immunologically cell surface antigens of murine macrophages in vitro. MATERIALS AND METHODS: RAW 264.7 cells were exposed to bleaching gel extracts (40% hydrogen peroxide or 20% carbamide peroxide) and different H2O2 concentrations after 1 and 24-h exposure periods and 1-h exposure and 23-h recovery. Tests were performed with and without N-acetyl cysteine (NAC) and buthionine sulfoximine (BSO). Cell viability was determined by MTT assay. The expression of surface markers CD14, CD40, and CD54 with and without LPS stimulation was detected by flow cytometry, while the production of TNF-α was measured by ELISA. Statistical analysis was performed using the Mann-Whitney U test (α = 0.05). RESULTS: Extracts of bleaching agents were cytotoxic for cells after a 1-h exposure; cells could not recover after 24 h. This effect can be mitigated by the antioxidant NAC and increased by BSO, an inhibitor of glutathione (GSH) synthesis. LPS stimulated expression of all surface markers and TNF-α production. Exposure to bleaching agent extracts and H2O2 leads to a reduction of TNF-α, CD14, and CD40 expression, while the expression of CD54 was upregulated at non-cytotoxic concentrations. Whereas NAC reduced this effect, it was increased in the presence of BSO. CONCLUSIONS: Extracts of bleaching agents were irreversibly cytotoxic to macrophages after a 1-h exposure. Only the expression of CD54 was upregulated. The reactions are mediated by the non-enzymatic antioxidant GSH. CLINICAL RELEVANCE: The addition of an antioxidant can downregulate unfavorable effects of dental bleaching.

Effectiveness and biological compatibility of different generations of dentin adhesives.

OBJECTIVES: Besides possessing good mechanical properties, dental materials should present a good biological behavior and should not injure the involved tissues. Bond strength and biocompatibility are both highly significant properties of dentin adhesives. For that matter, these properties of four generations of adhesive systems (Multi-Purpose/Single Bond/SE Plus/Easy Bond) were evaluated. MATERIALS AND METHODS: Eighty bovine teeth had their dentin exposed (500- and 200-µm thickness). Adhesive was applied on the dentin layer of each specimen. Following that, the microshearing test was performed for all samples. A dentin barrier test was used for the cytotoxicity evaluation. Cell cultures (SV3NeoB) were collected from testing materials by means of 200- or 500-µm-thick dentin slices and placed in a cell culture perfusion chamber. Cell viability was measured 24 h post-exposition by means of a photometrical test (MTT test). RESULTS: The best bonding performance was shown by the single-step adhesive Easy Bond (21 MPa, 200 µm; 27 MPa, 500 µm) followed by Single Bond (15.6 MPa, 200 µm; 23.4 MPa, 500 µm), SE Plus (18.2 MPa, 200 µm; 20 MPa, 500 µm), and Multi-Purpose (15.2 MPa, 200 µm; 17.9 MPa, 500 µm). Regarding the cytotoxicity, Multi-Purpose slightly reduced the cell viability to 92% (200 µm)/93% (500 µm). Single Bond was reasonably cytotoxic, reducing cell viability to 71% (200 µm)/64% (500 µm). The self-etching adhesive Scotchbond SE decreased cell viability to 85% (200 µm)/71% (500 µm). Conversely, Easy Bond did not reduce cell viability in this test, regardless of the dentin thickness. CONCLUSIONS: Results showed that the one-step system had the best bond strength performance and was the least toxic to pulp cells. In multiple-step systems, a correct bonding technique must be done, and a pulp capping strategy is necessary for achieving good performance in both properties. CLINICAL RELEVANCE: The study showed a promising system (one-step self-etching), referring to it as a good alternative for specific cases, mainly due to its technical simplicity and good biological responses.

Oxidative stress and cytotoxicity generated by dental composites in human pulp cells.

Dental composites are a source of residual monomers that are released into the oral environment. Since monomers act on cultured cells through reactive oxygen species (ROS), we hypothesized that composites generate ROS associated with cytotoxicity. Human pulp-derived cells were exposed to extracts of methacrylate-based materials including triethylene glycol dimethacrylate and 2-hydroxyethyl methacrylate-free composites (Tetric Ceram, Tetric EvoCeram, els, els flow, Solitaire 2) and a silorane-based composite (Hermes III). The materials were polymerized in the presence and absence of a polyester film and then extracted in culture medium. The generation of ROS was measured by flow cytometry, and cytotoxicity was determined as well. Methacrylate-based composites reduced cell survival but varied in efficiency. Undiluted extracts of Solitaire 2 specimens prepared in the absence of a polyester film reduced cell survival to 26% compared with untreated cultures. Cytotoxicity was reduced when specimens were covered with a polyester film during preparation. Cytotoxicity of the composites was ranked as follows: Solitaire 2 >> els flow > Tetric Ceram = Tetric EvoCeram = els > Hermes III. The generation of ROS followed the same pattern as detected with cytotoxic effects. A positive correlation was found between ROS production and cell survival caused by extracts made from materials not covered with a polyester film. These findings suggest that components released from composites affect cellular signaling networks through ROS formation. Regenerative and reparative capacities of the dentine-pulp complex may be impaired by biologically active resin monomers released from composite restorations.

Human and bovine pulp-derived cell reactions to dental resin cements.

OBJECTIVES: The objective of this study was to evaluate the cytotoxic reaction of transfected human pulp derived cells (tHPDC) and transfected bovine pulp derived cells (tBPDC) after exposure to resin cements [RelyX UnicemClicker (RX), MaxCem (MC), Panavia F 2.0 (PF), BisCem (BC), and Bistite II DC (BII)] and to compare it to the generation of reactive oxygen species (ROS). MATERIALS AND METHODS: Set materials were extracted in culture medium, cell survival as a measure of cytotoxicity was determined photometrically using crystal violet after cells were exposed to the extracts for 24 h. The generation of ROS was detected by flow cytometry after cells were exposed to extract dilutions for 1 h. RESULTS: The ranking of the least to the most cytotoxic material was: RX < BII < PF < BC < MC for both cell lines, but for tHPDC, only MC and PF eluates were different from untreated controls. Generally, tBPDC were more susceptible to materials than tHPDC, but only for RX and BC was this difference statistically significant. All undiluted extracts increased ROS production in both cell lines but to a higher amount in tHPDC than in tBPDC. CONCLUSIONS: tHPDC reacted less sensitive than tBPDC in the cytotoxicity test but with the same rank order of materials. In contrast, the cellular oxidative stress reaction was more pronounced in tHPDC than in tBPDC. CLINICAL RELEVANCE: Depending on the residual dentine layer in deep cavities, biologically active resin monomers or additives released from resin cements may influence the dentine­pulp complex, for instance, its regenerative and reparative capacities.

Inhibition of cell survival, viability and proliferation by dentin adhesives after direct and indirect exposure in vitro.

OBJECTIVES: The influence of dentin adhesive systems (Scotchbond Multi-Purpose, XP Bond, Xeno V, Clearfil Protect Bond, AdheSE) on cell survival, viability and proliferation was characterized after direct and indirect exposure using different cell culture techniques. MATERIALS AND METHODS: The primers and cured bonding parts were directly exposed to cells using cell culture inserts, and complete materials were analyzed in a dentin barrier test. Cell responses were examined in 3T3 mouse fibroblasts after 24- and 72-h exposure periods by the estimation of total cell numbers (survival), apoptosis (viability) and cell proliferation. RESULTS: Cell numbers were effectively reduced by the primers of AdheSE, Protect Bond, and Scotchbond Multi-Purpose as well as XP bond after direct exposure in a cell culture insert test device. Likewise, Scotchbond Multi-Purpose primer induced a rate of apoptosis (93.9%) even higher than detected with Protect Bond primer (91.6%). Cell proliferation was entirely inhibited by primers and by Xp Bond as well. The Scotchbond Multi-Purpose was most cytotoxic in a dentin barrier test device after a 24-h indirect exposure. It also increased the percentage of cells in apoptosis to 15.4% compared to untreated controls. CONCLUSION: Unpolymerized primers of dentin adhesives were more cytotoxic than polymerized bonding counterparts. Moreover, total etch dentin adhesives were more cytotoxic than self-etch adhesives. CLINICAL RELEVANCE: When dentin adhesives are used in deep cavities without a protective dentin barrier the leachable hydrophobic and hydrophilic component of dentin adhesive systems can penetrate to the pulp and may induce cytotoxic responses in pulp tissues.

Cytotoxicity of printed resin-based splint materials.

OBJECTIVES: Printed splints may be an alternative as a treatment of functional disorders in addition to physical, manual and physiological therapeutics. The objective is to investigate whether different 3D printed splint materials, which are fabricated with different fabrication orientation and post-processing (washing and post polymerisation) exhibit different in vitro cytotoxicity. MATERIAL AND METHODS: 600 discs (n = 25 per group, 5mmx1mm) were printed (P30+ DLP-printer, Straumann, CH; 100 µm layer) from splint materials (M1: Luxaprint OrthoPlus, DMG, G; M2: V-Print Splint, Voco, G). Printing was performed under 90° (A1), 45° (A2) or 0° (A3) alignment to the building platform. Specimens were either automatically washed (W1) (Straumann P Wash, Straumann, CH) or manually cleaned (W2) (Voco Pre-/Main-Clean protocol, Voco, G), and post polymerization was performed (P1: Cure, Straumann, CH; P2: Otoflash N171, Ernst Hinrichs Dental, G). RAW264.7 mouse macrophages were exposed to extracts of the specimens and cytotoxicity was determined as cell survival using a crystal violet assay. Optical density values obtained from exposed cell cultures were normalized to untreated controls (100%), summarized as means and statistically analyzed (ANOVA, α=0.05). RESULTS: Cell survival varied between 9.1+/-1.3% (alignment A2/post cure P2/material M2/wash system W2) and 58.5+/-5.9% (alignment A1/post cure P1/material M1/wash system W1). Univariate analysis of variance revealed significant differences between mean values for post cure (p = 0.000), wash system (p = 0.002) and materials (p = 0.000), but not for the alignment (p = 0.406). With standardised washing and adapted post cure, both tested materials provided lowest cytotoxicity even in all three printing alignments. CONCLUSIONS: The selection of the material as well as the post-processing (post-polymerization, washing procedure) show influence on the in vitro cytotoxicity. Alignment during manufacturing does not affect toxicity. CLINICAL RELEVANCE: Materials, washing and post-polymerization should be matched to reduce cytotoxic effects during additive manufacturing.

Influence of HEMA on LPS- and LTA-stimulated IL-6 release from human dental pulp cells.

OBJECTIVE: Dental pulp cells interact with immunogenic components such as LPS (lipopolysaccharide) or LTA (lipoteichoic acid) released from microorganisms in carious lesions. In the present investigation, the formation of the pro-inflammatory cytokines TNFα and IL-6 in LPS- or LTA-stimulated cells from the dental pulp interface and pulp fibroblasts was analyzed in the presence of the resin monomer 2-hydroxyethyl methacrylate (HEMA) under varying cellular redox conditions. METHOD: Human pulp fibroblasts (HPC) or cells from the dental pulp interface expressing an odontoblast phenotype (hOD-1) were exposed to LTA, LPS or HEMA for 1 h or 24 h. Redox homeostasis was modified by the prooxidant BSO (L-buthionine sulfoximine) or the antioxidant NAC (N-acetyl cysteine). Formation of TNFα or IL-6 was analyzed by ELISA, and cell survival was determined by a crystal violet assay. Statistical analyses were performed using the Mann-Whitney-U-test. RESULTS: Secretion of TNFα was not detected in LPS- or LTA-stimulated HPC or hOD-1, and IL-6 was not found after a short exposure (1 h). After a 24 h exposure, LPS induced a 3-fold increase in IL-6 formation in HPC, while LTA stimulated IL-6 release about 20-fold. Likewise, LTA was more effective than LPS in hOD-1 stimulating IL-6 levels about 50-fold. HEMA inhibited the LPS- and LTA-induced IL-6 release, and this effect was enhanced by BSO but counteracted by NAC in both cell types. IL-6 release was independent of cell survival rates. CONCLUSIONS: The protective immune response in odontoblasts and pulp fibroblasts is impaired by monomers such as HEMA through the disturbance of the redox homeostasis.

Relevance of Cellular Redox Homeostasis for Vital Functions of Human Dental Pulp Cells.

Odontogenic MSCs are vulnerable to LPS-triggered bacterial infections, and they respond by secreting inflammatory mediators, such as IL-6, and with mineralization. Since both processes might be prone to a disturbance of the redox homeostasis, the oxidative stress influence on vital functions of human dental pulp cells (HPCs) was investigated. With these aims, a model of LPS-stimulated primary HPCs was established, and anti- and pro-oxidant substances were administered up to 21 days to measure inflammation and mineralization parameters. LPS-stimulated HPCs retained mineralization potential, which was decreased with the antioxidants NAC and fisetin and the pro-oxidant BSO. The expression of surface markers related to odontogenic commitment was influenced accordingly but counteracted by the enhanced expression of BMP2 and ALP at the transcriptional level. LPS triggers an early IL-6 production in non-odontogenic conditions, while it can be measured only after 15 days in the presence of the differentiation medium. The present study shows that HPCs functions causally depend on a tightly regulated cellular redox balance. Our data demonstrate a redox control of pulp MSC odontogenic commitment along with a potential association between an IL-6 late secretion and mineralization. These findings lay the groundwork for investigations on the molecular role of IL-6 in dental hard tissue metabolism.

Distinguished properties of cells isolated from the dentin-pulp interface.

BACKGROUND: Dental odontoblasts produce dentin mineralized matrix, trigger immune responses and act as sensory cells. The understanding of the mechanisms of these functions has been particularly restricted due to the lack of odontoblasts being cultivable in vitro. Because of the lack of specific markers to identify cells of the odontoblastic lineage, properties of the cells isolated from the dentin-pulp interface were compared to dental pulp cells, periodontal ligament cells, osteoblasts, skin fibroblasts, epithelial cells (A549) and HeLa in the present study. METHODS: After surgical procedures, the pulp tissue was removed from the tooth crown, and cells were scrapped off the dentin-pulp interface. Explants from teeth of three patients were routinely cultivated, and cells were harvested after several weeks. Cell morphology and ultrastructure was studied by light microscopy (LM), scanning (SEM) or transmission electron microscopy (TEM). Expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), TRPV4, and S100 calcium binding protein A4 (S100A4) were analyzed at the protein level by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using specific antibodies. The differential expression of S100A4 in the various cell lines was further investigated at the gene level by semiquantitative real-time PCR. Mineralization in the various cell types was observed after alizarin red staining after a 28 days incubation period. The immunophenotype of the cells was examined by flow cytometry using monoclonal anti-human antibodies CD90-FITC, CD73-PE, CD105-PE, CD29-PE, CD140a-FITC, CD144-PE, CD45-FITC or CD34-FITC. Differences between median values were statistically analyzed (Mann-Whitney U-test). RESULTS: Cells from the dentin-pulp interface retain the polarity of odontoblast morphology in culture with an elongated, rounded cell body, and an extended cellular process. Ultrastructural analysis of the cells indicates high secretory activity including the extracellular deposition of fibrillar collagen. An extended rough endoplasmic reticulum is lined by a large number of ribosomes, and a vast number of secretory granules merges with the cell membrane. Protein expression of DSPP, DMP1, and TRPV4 as a transient receptor potential cation was detected in all cell lines. S100A4 was found differentially expressed in cultures of cells from tooth tissues. High expression of S100A4 was observed at the protein and gene level in two fractions of cells isolated from the dentin-pulp interface, but was absent or only weakly expressed in pulp cells. S100A4 expression in cells from the dentin-pulp interface and pulp cells is consistent with the intensity of the formation of mineralized nodules detected by alizarin red staining. Immunophenotyping revealed that a high percentage of CD73 (ecto-5-nucleotidase), an enzyme active on the surface of immune-competent cells, was expressed in cells of the dentin-pulp interface. While 72%-78% of positive cells were detected in dentin-pulp interface fractions, only 28-64% of the cells in pulp cell cultures were stained. CONCLUSIONS: The present findings obtained with a variety of cells of different origin provide experimental evidence that cells isolated from the dentin-pulp interface express unique properties different from dental pulp cells in particular. The differential expression of S100A4 is a relevant marker candidate for differentiating between dental pulp cells and cells of the odontoblast lineage.

Cytotoxic effects of different self-adhesive resin cements: Cell viability and induction of apoptosis.

PURPOSE: The effects of four different self-adhesive resin cement materials on cell viability and apoptosis after direct and indirect exposure were evaluated using different cell culture techniques. MATERIALS AND METHODS: Self-adhesive cements were applied to NIH/3T3 mouse fibroblasts by the extract test method, cell culture inserts, and dentin barrier test method. After exposure periods of 24 h and 72 h, the cytotoxicity of these self-adhesive materials was evaluated using the MTT assay (viability) and the Annexin-V-FITC/PI staining (apoptosis). RESULTS: The lowest cell viability was found in cells exposed to BeautiCem SA for 24 h in the extract test method. Cell viability was reduced to 70.6% compared to negative controls. After the 72 h exposure period, viability rate of cell cultures exposed to BeautiCem SA decreased more than 2- fold (29.5%) while cells exposed to RelyX U200 showed the highest viability rate of 71.4%. In the dentin barrier test method, BeautiCem SA induced the highest number of cells in apoptosis after a 24 h exposure (4.1%). Panavia SA Cement Plus was the material that caused the lowest number of cells in apoptosis (1.5%). CONCLUSION: The used self-adhesive cements have showed different cytotoxic effects based on the evaluation method. As exposure time increased, the materials showed more cytotoxic and apoptotic effects. BeautiCem SA caused significantly more severe cytotoxic and apoptotic effects than other cements tested. Moreover, cements other than BeautiCem SA have caused necrotic cell death rather than apoptotic cell death.

Redox Control of IL-6-Mediated Dental Pulp Stem-Cell Differentiation on Alginate/Hydroxyapatite Biocomposites for Bone Ingrowth.

Composites and porous scaffolds produced with biodegradable natural polymers are very promising constructs which show high biocompatibility and suitable mechanical properties, with the possibility to be functionalized with growth factors involved in bone formation. For this purpose, alginate/hydroxyapatite (Alg/HAp) composite scaffolds using a novel production design were successfully developed and tested for their biocompatibility and osteoconductive properties in vitro. Redox homeostasis is crucial for dental pulp stem cell (DPSC) differentiation and mineralized matrix deposition, and interleukin-6 (IL-6) was found to be involved not only in immunomodulation but also in cell proliferation and differentiation. In the present study, we evaluated molecular pathways underlying the intracellular balance between redox homeostasis and extracellular matrix mineralization of DPSCs in the presence of composite scaffolds made of alginate and nano-hydroxyapatite (Alg/HAp). Prostaglandin-2 (PGE2) and IL-6 secretion was monitored by ELISA assays, and protein expression levels were quantified by Western blotting. This work aims to demonstrate a relationship between DPSC capacity to secrete a mineralized matrix in the presence of Alg/HAp scaffolds and their immunomodulatory properties. The variation of the molecular axis Nrf2 (nuclear factor erythroid 2-related factor 2)/PGE2/IL-6 suggests a tight intracellular balance between oxidative stress responses and DPSC differentiation in the presence of Alg/HAp scaffolds.

Shotgun Proteomics of Human Dentin with Different Prefractionation Methods.

Human dentin is not only a composite material of a collagenous matrix and mineral to provide strength and elasticity to teeth, but also a precious reservoir full of bioactive proteins. They are released after demineralization caused by bacterial acids in carious lesions, by decalcifying irrigants or dental materials and they modulate tissue responses in the underlying dental pulp. This work describes a first-time analysis of the proteome of human dentin using a shotgun proteomic approach that combines three different protein fractionation methods. Dentin matrix proteins were extracted by EDTA and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), OFFGEL isoelectric focusing (IEF) or strong cation exchange chromatography (SCX). Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified 813 human proteins with high confidence, however, isoelectric focusing turned out to be the most beneficial prefractionation method. All Proteins were categorized based on the PANTHER system and representation analysis revealed 31 classes and subclasses to be overrepresented. The acquired knowledge provides a comprehensive insight into the number of proteins in human dentin as well as their physiological and pathological functions. Thus, the data presented paves the way to the analysis of specific functions of dentin matrix proteins in vivo and their potential in tissue engineering approaches to regenerate dental pulp.

Differential gene expression involved in oxidative stress response caused by triethylene glycol dimethacrylate.

Triethylene glycol dimethacrylate (TEGDMA) is a comonomer that is released from dental resin-based materials into hydrophilic solvents. The compound reduces cell vitality, and causes genotoxicity in mammalian cells in vitro. Here, we used gene expression profiling, combined with pathway analysis tools, to identify the molecular events associated with TEGDMA cytotoxicity in human fibroblasts using Affymetrix HG-U133A 2.0 GeneChip arrays. Increased ROS production and a cell cycle delay caused by 3mm TEGDMA after a 6h exposure were related to a cell response at the transcriptional level. The predominant biological processes associated with the genes that were differentially expressed in untreated and treated cell cultures included oxidative stress, cellular growth, proliferation and morphology, cell death, gene expression as well as DNA replication and repair. The most significantly upregulated genes were GEM (17-fold), KLHL24, DDIT4, TGIF, DUSP5 and ATF3, which are all related to the regulation of the cell structure, stress response, and cell proliferation. TXNIP was the most downregulated transcript (five-fold), whose gene product regulates the cellular redox balance. The downregulation of NRG1, ASPM, FBXO5, and PLK2 is linked to the regulation of cell proliferation and cell structure. The underlying mechanisms of the up- and downregulation of genes seem to be activated by the production of ROS, and the related regulation of the cellular redox balance disturbed in the presence of TEGDMA appears to be of the utmost importance. The coordinated induction of genes coding for oxidative stress response and antioxidant proteins is a critical mechanism of protection against TEGDMA-induced cell damage.

Functions of transcription factors NF-κB and Nrf2 in the inhibition of LPS-stimulated cytokine release by the resin monomer HEMA.

OBJECTIVE: Resin monomers like 2-hydroxyethyl methacrylate (HEMA) interfere with effects induced by stressors such as lipopolysaccharide (LPS) released from cariogenic microorganisms. In this study, mechanisms underlying monomer-induced inhibition of the LPS-stimulated secretion of inflammatory cytokines from immunocompetent cells were investigated. METHODS: Secretion of pro-inflammatory cytokines TNF-α, IL-6 and the anti-inflammatory IL-10 from RAW264.7 mouse macrophages exposed to LPS and HEMA (0-6-8mM) was determined by ELISA. The formation of reactive oxygen (ROS) and nitrogen species (RNS) was determined by flow cytometry (FACS) after staining of cells with specific fluorescent dyes. Cell viability was analyzed by FACS, and protein expression was detected by Western blotting. RESULTS: Secretion of TNF-α, IL-6 and IL-10 from LPS-stimulated cells increased after a 24h exposure. A HEMA-induced decrease in cytokine secretion resulted from the inhibition of LPS-stimulated NF-κB activation. Nuclear translocation of NF-κB was inhibited possibly as a result of enhanced levels of hydrogen peroxide (H2O2) and nitric oxide (NO) in HEMA-exposed cells. Oxidative stress caused by HEMA-induced formation of H2O2 and LPS-stimulated peroxynitrite (ONOO) also enhanced nuclear expression of Nrf2 as the major regulator of redox homeostasis, as well as Nrf2-controlled stress protein HO-1 to inhibit NF-κB activity. HEMA inhibited the LPS-stimulated expression of NOS (nitric oxide synthase) to produce NO but counteracted the expression of Nox2, which forms superoxide anions that combine with NO to peroxynitrite. CONCLUSIONS: Resin monomers like HEMA inhibit LPS-stimulated NF-κB activation essential for cytokine release as a crucial response of immunocompetent cells of the dental pulp to invading cariogenic pathogens.

Alginate/Hydroxyapatite-Based Nanocomposite Scaffolds for Bone Tissue Engineering Improve Dental Pulp Biomineralization and Differentiation.

Tissue engineering is widely recognized as a promising approach for bone repair and reconstruction. Several attempts have been made to achieve materials that must be compatible, osteoconductive, and osteointegrative and have mechanical strength to provide a structural support. Composite scaffolds consisting in biodegradable natural polymers are very promising constructs. Hydroxyapatite (HAp) can support alginate as inorganic reinforcement and osteoconductive component of alginate/HAp composite scaffolds. Therefore, HAp-strengthened polymer biocomposites offer a solid system to engineer synthetic bone substitutes. In the present work, HAp was incorporated into an alginate solution and internal gelling was induced by addition of slowly acid-hydrolyzing D-gluconic acid delta-lactone for the direct release of calcium ions from HAp. It has been previously demonstrated that alginate-based composites efficiently support adhesion of cancer bone cell lines. Human dental pulp stem cells (DPSCs) identified in human dental pulp are clonogenic cells capable of differentiating in multiple lineage. Thus, this study is aimed at verifying the mineralization and differentiation potential of human DPSCs seeded onto scaffolds based on alginate and nano-hydroxyapatite. For this purpose, gene expression profile of early and late mineralization-related markers, extracellular matrix components, viability parameters, and oxidative stress occurrence were evaluated and analyzed. In summary, our data show that DPSCs express osteogenic differentiation-related markers and promote calcium deposition and biomineralization when growing onto Alg/HAp scaffolds. These findings confirm the use of Alg/HAp scaffolds as feasible composite materials in tissue engineering, being capable of promoting a specific and successful tissue regeneration as well as mineralized matrix deposition and sustaining natural bone regeneration.

Cell responses to cariogenic microorganisms and dental resin materials-Crosstalk at the dentin-pulp interface?

OBJECTIVE: Resin monomers released from unpolymerized dental adhesives or composites and bacterial products like lipopolysaccharide (LPS) or lipoteichoic (LTA) are simultaneously present in specific applications following treatment of deep caries lesions. This review is focused on evidence concerning cell responses as a result of the interactions between adaptive mechanisms activated by resin monomers and signaling pathways of the immune response triggered by LPS or LTA originating from cariogenic microorganisms. METHODS: Current understanding of dental caries progression and pathways in eukaryotic cells in response to LPS stimulation in a clinical situation as well as cell reactions to oxidative stress caused by resin monomers is analyzed based on publications available through online databases. RESULTS: LPS and LTA activate the redox-sensitive transcription factor NF-κB as a major regulator in immunocompetent dental pulp cells. Cell reactions to LPS/LTA associated with oxidative stress are downregulated by the redox-sensitive transcription factor Nrf2. Thus, activation of Nrf2 through resin monomer-induced oxidative stress due to the increased formation of reactive oxygen species (ROS) could be a molecular mechanism underlying the inhibition of LPS-stimulated responses such as the release of pro- or anti-inflammatory cytokines. Likewise, crosslinking of NF-κB and Nrf2-regulated biocompatibility pathways regulates cell death induced by the interaction of LPS and resin monomers. SIGNIFICANCE: A multidimensional scenario through independent but linked NF-κB- and Nrf2-regulated pathways is activated in the clinical situation of caries treatment. Unfavorable or beneficial consequences strictly depend on a wide range of combinations and concentrations of bacterial products and resin monomers.

Flavin-containing enzymes as a source of reactive oxygen species in HEMA-induced apoptosis.

OBJECTIVE: Oxidative stress induced by compounds of dental composites like 2-hydroxyethyl methacrylate (HEMA) due to excess formation of reactive oxygen species (ROS) disturbs vital cell functions leading to apoptosis. The sources of ROS in cells exposed to resin monomers are unknown. The present study investigates functions of flavin-containing ROS and RNS (reactive nitrogen species) producing enzymes in cells exposed to HEMA. METHODS: The formation of oxidative stress in RAW264.7 mouse macrophages exposed to HEMA (0-6-8mM) was determined by flow cytometry (FACS) after staining of cells with 2'7'-dichlorodihydrofluorescin diacetate (H2DCF-DA), dihydroethidium (DHE) or dihydrorhodamine 123 (DHR123). Cells in apoptosis or necrosis were identified by annexin-V-FITC/propidium iodide labeling followed by FACS analysis. Expression of ROS/RNS producing enzymes was analyzed by Western blotting. RESULTS: DCF fluorescence increased in cells exposed to HEMA for 1h suggesting the production of hydroxyl radicals, H2O2, or nitric oxide and superoxide anions which form peroxynitrite (ONOO-). Increased DHR123 fluorescence after 24h indicated the formation of mostly H2O2. The induction of apoptosis in the presence of HEMA was decreased by low concentrations of diphenylene iodonium (DPI), an inhibitor of flavin-containing enzymes. Expression of p47phox, a regulatory subunit of the superoxide producing Nox2, was downregulated, and the expression of NOS which produces nitric oxide (NO) was possibly inhibited by feedback loop mechanisms in HEMA-exposed cultures. Inhibition of HEMA-induced apoptosis by VAS2870 or apocynin further suggested a crucial function of Nox2. SIGNIFICANCE: The present findings show the physiological relevance of flavin-containing enzymes in monomer-induced oxidative stress and apoptosis.

Effect of N-acetyl-L-cysteine on ROS production and cell death caused by HEMA in human primary gingival fibroblasts.

Previous investigations have shown that 2-hydroxyethyl methacrylate (HEMA) causes reactive oxygen species (ROS) production, which in turn affects cell survival and cell death. The purpose of this study was to evaluate the effects of the antioxidant N-acetyl-L-cysteine (NAC) on HEMA-induced toxicity in human primary gingival fibroblasts (HGF). HGF were treated with various concentrations of HEMA (0-12 mm) in the absence and presence of NAC (1, 5, and 10 mm). The 3-(4,5 dimethyiazol-2-1)-2-5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the mitochondrial dehydrogenase activity after HEMA exposure. Viability and cell death were determined by flow cytometry using Annexin V and PI staining. ROS production was detected by the increasing fluorescence of the oxidation-sensitive dye 2',7'-dichlorofluorescein diacetate (DCFH-DA) after HEMA treatment. After a 24h incubation period, HEMA concentrations higher then 10mm caused a decrease of cell viability, mitochondrial activity, and an increase of cell death. HEMA concentrations of 4-12 mm markedly increased ROS levels in a dose-dependent manner. High NAC concentrations (5 and 10 mm) significantly reduced cell death, and restored the mitochondrial activity after a 24 h co-treatment, but 1 mm NAC increased HEMA toxicity (p<0.05). All NAC concentrations significantly reduced ROS levels induced by HEMA after a 2 h exposure (p<0.05), but no such reduction was observed after a 4 h treatment. Furthermore, treatment with 10 mm HEMA and 1 mm NAC for 6h caused an increase in ROS levels compared to 10 mm HEMA alone (p<0.05). In conclusion, our results suggest that high NAC concentrations protect HGF against HEMA cytotoxicity by reducing the induced ROS levels.

Interaction between LPS and a dental resin monomer on cell viability in mouse macrophages.

OBJECTIVE: Lipopolysaccharide (LPS) from cariogenic microorganisms and resin monomers like HEMA (2-hydroxyethyl methacrylate) included in dentin adhesive are present in a clinical situation in deep dentinal cavity preparations. Here, cell survival, expression of proteins related to redox homeostasis, and viability of mouse macrophages exposed to LPS and HEMA were analyzed with respect to the influence of oxidative stress. METHODS: Cell survival of RAW264.7 mouse macrophages was determined using a crystal violet assay, protein expression was detected by Western blotting, and HEMA- or LPS-induced apoptosis (cell viability) was analyzed by flow cytometry. Cells were exposed to HEMA (0-8mM), LPS (0.1µg/ml) or combinations of both substances for 24h. The influence of mitogen-activated protein kinases (MAPK) was analyzed using the specific inhibitors PD98059 (ERK1/2), SB203580 (p38) or SP600125 (JNK), and oxidative stress was identified by the antioxidant N-acetylcysteine (NAC). RESULTS: Cell survival was reduced by HEMA. LPS, however, increased cell survival from 29% in cultures exposed to 8mM HEMA, to 46% in cultures co-exposed to 8mM HEMA/LPS. Notably, LPS-induced apoptosis was neutralized by 4-6mM HEMA but apoptosis caused by 8mM HEMA was counteracted by LPS. Expression of NOS (nitric oxide synthase), p47phox and p67phox subunits of NADPH oxidase, catalase or heme oxygenase (HO-1) was associated with HEMA- or LPS-induced apoptosis. While no influence of MAPK was detected, NAC inhibited cytotoxic effects of HEMA. SIGNIFICANCE: HEMA- and LPS-triggered pathways may induce apoptosis and interfere with physiological tissue responses as a result of the differential formation of oxidative stress.

The effect of triethylene glycol dimethacrylate on the cell cycle of mammalian cells.

The induction of DNA damage by a genotoxic agent is a signal leading to cell cycle delay, and thereby enables and induces DNA repair prior to cell cycle progression. Triethylene glycol dimethacrylate (TEGDMA), a monomer of dental resinous materials, caused mutagenic effects in mammalian cells probably as a consequence of DNA damage. Therefore, we hypothesized that TEGDMA will induce a cell cycle delay in mammalian cells. Here, cell lines deficient and proficient of a functional p53 tumor suppressor protein were used to study the effects of TEGDMA on the various phases of the cell cycle. V79 Chinese hamster lung fibroblasts (p53 deficient), N1 human skin fibroblasts (p53 proficient), and primary human pulp fibroblasts (p53 proficient) were exposed to increasing TEGDMA concentrations (0-3 mmol/l). Cell survival and vitality were determined after a 24-h exposure period and a 24-h recovery period, and the distribution of cells between the phases of the cell cycle in untreated and TEGDMA-treated cultures was analyzed by flow cytometry. The majority of the TEGDMA-treated V79 cells accumulated in G2 phase. In contrast, about 30% of human N1 fibroblasts were reversibly blocked in G1 phase by 0.5-3.0 mmol/l TEGDMA. The fraction of G2-phase cells was increased only by high TEGDMA concentrations. The percentage of human pulp cells in G1 phase increased very slightly with 1 mmol/l TEGDMA, but cell numbers in G1 phase were reduced by 10-20% by 1.5-3 mmol/l TEGDMA. The percentage of pulp cells in G2 phase increased about 2-fold without any obvious effect of a 24-h recovery period. Therefore, TEGDMA caused cell cycle delays through p53-dependent and independent pathways in the various cell lines. From these results, we conclude that TEGDMA may influence physiological processes like cell growth and differentiation of human pulp cells in vivo.