Oxidative stress is responsible for genotoxicity of camphorquinone in primary human gingival fibroblasts.

OBJECTIVES: The photoinitiator camphorquinone (CQ), used in dental restorative materials, was found to be cytotoxic in cell cultures. Previously, we have shown that CQ induces alkali labile sites and DNA strand breaks in human gingival fibroblasts (HGF) associated with an increase of intracellular reactive oxygen species (ROS). Therefore, the objective of our study was to evaluate if DNA damage in HGF cells is caused by the generation of ROS. MATERIAL AND METHODS: HGF cells were treated with different concentrations (0.5-2.5 mM) of CQ. The cell viability was assessed using propidium iodide (PI) assay. Oxidative DNA damage was evaluated by an enzyme-modified comet assay using human 8-hydroxyguanine DNA-glycosylase 1 (hOGG1), which converts oxidized 7,8-dihydro-8-oxoguanine (8-oxoguanine) into DNA strand breaks and functions as a marker for oxidative modified DNA. RESULTS: The results showed that CQ induced DNA damage in HGF cells without cytotoxic effects for the chosen treatment time. CQ treatment led to the generation of 8-oxoguanine in DNA, which can be shown by a significant increase in tail moment after CQ treatment by the enzyme-modified comet assay. CONCLUSION: It may be concluded that DNA damage due to CQ is caused by oxidative stress in gingival fibroblasts. CLINICAL RELEVANCE: A more detailed insight into genotoxic mechanisms in oral cells can be of great importance for a better understanding of the biocompatibility of CQ.

Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth.

Human deciduous teeth have been proposed as a promising source of mesenchymal stem cells for application in bone and dental tissue engineering. We established cultures of mesenchymal stem cells from the pulp of human deciduous teeth (deciduous teeth stem cells, DTSCs) and analyzed their morphologic, growth, immunophenotypic, and osteo/odontogenic differentiation characteristics using different isolation methods and culturing environments. We compared the biologic behavior of DTSCs isolated either by enzymatic dissociation (DTSCs-ED) or by direct outgrowth from pulp tissue explants (DTSCs-OG). We found that different isolation methods give rise to different populations/lineages of cells with respect to their phenotypic and differentiation characteristics. DTSCs-ED cultures comprised heterogeneous cell populations, whereas DTSCs-OG comprised more homogenous spindle-shaped cells. We have characterized DTSCs as STRO-1(+)/CD146(+)/CD34(+)/CD45(-) cells. However, the percentage of STRO-1(+) and CD34(+) cells was higher in DTSCs-ED (STRO-1, 17.01 ± 5.04%; CD34, 19.79 ± 4.66%) compared to DTSCs-OG cultures (STRO-1, 5.18 ± 2.39%; CD34, 9.94 ± 3.41%), probably as a result of a higher release of stem/progenitor cells from the perivascular niche during enzymatic dissociation. DTSCs isolated using either method displayed an active potential for cellular migration and biomineralization, giving rise to 3D mineralized structures when challenged with dexamethasone, monopotassium phosphate, and ß-glycerophosphate. These cellular aggregates progressively expressed differentiation markers of functional odontoblasts, including dentin sialophosphoprotein, bone sialoprotein, osteocalcin, and alkaline phosphatase, having the characteristics of osteodentin. However, in DTSCs-ED, the mineralization rate and the amount of mineralized matrix produced was higher compared to DTSCs-OG cultures. Therefore, DTSCs-ED cells display enhanced biomineralization potential, which might be of advantage for application in clinical therapy.

Evaluation of stemness properties of cells derived from granulation tissue of peri-implantitis lesions.

OBJECTIVES: Peri-implantitis (PI) is an inflammatory disease associated with peri-implant bone loss and impaired healing potential. There is limited evidence about the presence of mesenchymal stromal cells (MSCs) and their regenerative properties within the granulation tissue (GT) of infrabony peri-implantitis defects. The aim of the present study was to characterize the cells derived from the GT of infrabony PI lesions (peri-implantitis derived mesenchymal stromal cells-PIMSCs). MATERIAL AND METHODS: PIMSC cultures were established from GT harvested from PI lesions with a pocket probing depth ≥6 mm, bleeding on probing/suppuration, and radiographic evidence of an infrabony component from four systemically healthy individuals. Cultures were analyzed for embryonic (SSEA4, NANOG, SOX2, OCT4A), mesenchymal (CD90, CD73, CD105, CD146, STRO1) and hematopoietic (CD34, CD45) stem cell markers using flow cytometry. PIMSC cultures were induced for neurogenic, angiogenic and osteogenic differentiation by respective media. Cultures were analyzed for morphological changes and mineralization potential (Alizarin Red S method). Gene expression of neurogenic (NEFL, NCAM1, TUBB3, ENO2), angiogenic (VEGFR1, VEGFR2, PECAM1) and osteogenic (ALPL, BGLAP, BMP2, RUNX2) markers was determined by quantitative RT-PCR. RESULTS: PIMSC cultures demonstrated high expression of embryonic and mesenchymal stem cell markers with inter-individual variability. After exposure to neurogenic, angiogenic and osteogenic conditions, PIMSCs showed pronounced tri-lineage differentiation potential, as evidenced by their morphology and expression of respective markers. High mineralization potential was observed. CONCLUSIONS: This study provides evidence that MSC-like populations reside within the GT of PI lesions and exhibit a multilineage differentiation potential. Further studies are needed to specify the biological role of these cells in the healing processes of inflamed PI tissues and to provide indications for their potential use in regenerative therapies.

HEMA modulates the transcription of genes related to oxidative defense, inflammatory response and organization of the ECM in human oral cells.

OBJECTIVES: 2-Hydroxyethyl methacrylate (HEMA) is a widely used monomer of dental resin composite materials. Incomplete curing of resins leads to elution of HEMA, which may come in contact with different cells in oral tissues. We aimed to analyze the impact of HEMA on the transcription of genes participating in detoxification of oxidative stress, inflammatory response and organization of the extracellular matrix (ECM) using human gingival fibroblasts (HGFs) and human oral keratinocytes (OKF6/TERT2). METHODS: Cells were grown in monolayer cultures and treated with different HEMA concentrations (0.5-10mM). H33342 and LDH assays were used to determine HEMA-caused cytotoxicity. Quantitative RT-PCR was used to analyze mRNA expression of four genes related to oxidative stress and five genes each related to inflammation and organization of the ECM. RESULTS: HEMA caused similar concentration-dependent cytotoxicity in fibroblasts and keratinocytes. Analysis of the transcription showed that genes were regulated in both cell types after HEMA treatment. Genes related to defense against oxidative stress were transcriptionally induced, genes related to inflammation were mainly reduced and genes related to the organization of the ECM were differentially modulated. SIGNIFICANCE: We analyzed concurrent and HEMA-dependent differential expression of 14 important genes, which have a special significance for cellular processes that are linked to redox and tissue homeostasis. The results suggest that HEMA has an impact on cellular redox-homeostasis with potential impairment of inflammatory responses and of the organization of the ECM in human gingival fibroblasts and oral keratinocytes as first target cells of eluted HEMA.

Impaired angiogenic differentiation of dental pulp stem cells during exposure to the resinous monomer triethylene glycol dimethacrylate.

OBJECTIVE: Dental pulp stem cells (DPSCs) can differentiate into tissue specific lineages to support dental pulp regeneration after injuries. Triethylene glycol dimethacrylate (TEGDMA) is a widely used co-monomer in restorative dentistry with adverse effects on cellular metabolism. Aim of this study was to analyze the impact of TEGDMA on the angiogenic differentiation potential of DPSCs. METHODS: DPSCs were characterized by flow cytometry. Short-term (max. 72h) cytotoxicity of TEGDMA was assessed by MTT assay. To evaluate TEGDMA effects on angiogenic differentiation, DPSCs were cultivated in angiogenic differentiation medium (ADM) in the presence or absence of short-term non-toxic TEGDMA concentrations (0.1mM and 0.25mM). Subsequently, angiogenic differentiation was analyzed by qRT-PCR analysis of mRNA markers and in vitro spheroid sprouting assays. RESULTS: DPSCs treated with 0.25mM TEGDMA revealed downregulation of angiogenesis-related marker genes PECAM1 (max. 3.8-fold), VEGF-A (max. 2.4-fold) and FLT1 (max. 2.9-fold) compared to respective untreated control. In addition, a reduction of the sprouting potential of DPSCs cultured in the presence of 0.25mM TEGDMA was detectable. Larger spheroidal structures were detectable in the untreated control in comparison to cells treated with 0.25mM TEGDMA. In contrast, TEGDMA at 0.1mM was not affecting angiogenic potential in the investigated time period (up to 28 days). SIGNIFICANCE: The results of the present study show that TEGDMA concentration dependently impair the angiogenic differentiation potential of DPSCs and may affect wound healing and the formation of granulation tissue.

Cytotoxic and genotoxic potential of the type I photoinitiators BAPO and TPO on human oral keratinocytes and V79 fibroblasts.

OBJECTIVES: Phenylbis(acyl) phosphine oxide (BAPO) and diphenyl(acyl) phosphine oxide (TPO) are alternative photoinitiators to camphorquinone (CQ) in dental resinous materials. Aim of this study was to investigate their cytotoxic/genotoxic potential in human oral keratinocytes (OKF6/Tert2) and Chinese hamster lung fibroblasts (V79) in comparison to CQ. METHODS: Cells were exposed to different concentrations of BAPO and TPO (1-50µM). Cytotoxicity was evaluated using H33342 and MTT assay, cell proliferation by BrdU proliferation assay and microscopy. Effects on cellular redox homeostasis were assessed by detecting intracellular levels of reactive oxygen/nitrogen species (ROS/RNS) using the DCFH2 assay and by quantification of mRNA expression of oxidatively regulated, cyto-protective enzymes. Genotoxic potential was determined by use of micronucleus (MN) assay. RESULTS: BAPO and TPO induced a concentration-dependent decrease of cell number. BAPO and TPO showed 50- to 250-fold higher cytotoxicity than CQ. In contrast to CQ, both photoinitiators revealed no increase of intracellular ROS/RNS. However, BAPO (10µM) at least significantly induced mRNA-expression of redox-regulated proteins after 24h similar to 2.5mM CQ. Additionally, BAPO significantly raised the number of micronuclei, but only in V79 cells (10µM: 12±1, 2.5mM CQ: 15±1, medium control: 6±3). However, it also significantly decreased proliferation of these cells (10µM BAPO: 19.8%±7.3% compared to controls). SIGNIFICANCE: BAPO and TPO revealed concentration-dependent cytotoxic effects in human oral keratinocytes and V79 cells. However, in contrast to CQ, no generation of intracellular ROS/RNS was found. Only BAPO induced genotoxicity in V79 cells.

Additive effects of TEGDMA and hydrogenperoxide on the cellular glutathione content of human gingival fibroblasts.

OBJECTIVES: Only few data are available about cytotoxic effects of leachable dental resin compounds in combination with hydrogen peroxide (H(2)O(2)) segregated from dental bleaching agents. Therefore, the purpose of this study was to evaluate the effects of various concentrations of triethylene-glycol dimethacrylate (TEGDMA) and H(2)O(2) on intracellular glutathione levels (GSH) and viability of human gingival fibroblasts (HGF) that are primary target cells of cytotoxic actions of these substances. METHODS: HGF were grown in 96-well plates for 24h, treated with various concentrations of TEGDMA (0.5-5.0mM) for 24h and subsequently for 90min with 0.2mM H(2)O(2) or culture medium (control). The relative intracellular GSH concentration was determined using a fluorescence assay with monobromobimane. Readings were normalized to cell numbers, which were determined by a propidium iodide assay. Data were statistically analyzed by t-test and ANOVA with Tukey's post test. A significance level of p<0.05 was used. RESULTS: Exposure to TEGDMA reduced the viability of HGF at concentrations > or =1.0mM. TEGDMA induced a decrease of the GSH pool in a concentration-dependent manner (p<0.05). The depletion of GSH was correlated with a reduction of viability (p<0.05) and the total cell number. Furthermore, a significant decrease of the intracellular GSH content was found when cells were exposed to TEGDMA in combination with H(2)O(2), compared to experiments without H(2)O(2). SIGNIFICANCE: We conclude from our findings that TEGDMA and H(2)O(2) have additive adverse effects on GSH metabolism and cell viability.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

BACKGROUND: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. METHODS: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (ß-galactosidase (SA-ß-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. RESULTS: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions. CONCLUSIONS: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

Human treated dentin matrices combined with Zn-doped, Mg-based bioceramic scaffolds and human dental pulp stem cells towards targeted dentin regeneration.

OBJECTIVE: This study aimed to investigate the potential of Mg-based bioceramic scaffolds combined with human treated-dentin matrices (hTDMs) and dentinogenesis-related morphogens to promote odontogenic differentiation and dentin-like tissue formation by Dental Pulp Stem Cells-DPSCs. METHODS: DPSC cultures were established and characterized by flow cytometry. Experimental cavities were prepared inside crowns of extracted teeth and demineralized by EDTA (hTDMs). Zn-doped, Mg-based bioceramic scaffolds, synthesized by the sol-gel technique, were hosted inside the hTDMs. DPSCs were spotted inside the hTDMs/scaffold constructs with/without additional exposure to DMP-1 or BMP-2 (100ng/ml, 24h). Scanning Electron Microscopy-SEM, live/dead fluorescence staining and MTT assay were used to evaluate cell attachment and viability; Real time PCR for expression of osteo/odontogenic markers; Inductively Coupled Plasma-Atomic Emission Spectrometry-ICP/AES for scaffold elemental release analysis; ELISA for hTDM growth factor release analysis; SEM and X-ray Diffraction-XRD for structural/chemical characterization of the regenerated tissues. RESULTS: Scaffolds constantly released low concentrations of Mg(2+), Ca(2+), Zn(2+) and Si(4+), while hTDMs growth factors, like DMP-1, BMP-2 and TGFß-1. hTDMs/scaffold constructs supported DPSC viability, inducing their rapid odontogenic shift, indicated by upregulation of DSPP, BMP-2, osteocalcin and osterix expression. Newly-formed Ca-P tissue overspread the scaffolds partially transforming into bioapatite. Exposure to DMP-1 or BMP-2 pronouncedly enhanced odontogenic differentiation phenomena. SIGNIFICANCE: This is the first study to validate that combining the bioactivity and ion releasing properties of bioceramic materials with growth factor release by treated natural dentin further supported by exogenous addition of key dentinogenesis-related morphogens (DMP-1, BMP-2) can be a promising strategy for targeted dentin regeneration.

Curcumin in Combination with Piperine Suppresses Osteoclastogenesis In Vitro.

INTRODUCTION: The dietary pigment curcumin is a natural polyphenol extracted from the Curcuma longa rhizomes native to South Asia. The antioxidative, antimicrobial, and anti-inflammatory activities besides its unknown side effects suggest that curcumin could be a promising antiresorptive agent to prevent replacement resorption in replanted teeth after traumatic avulsion. Piperine, an alkaloid present in black pepper, seems to enhance the bioavailability and activity of curcumin. Therefore, this study evaluated the biocompatibility of curcumin and piperine in cultures of periodontal ligament cells as well as their effects in an in vitro osteoclastogenesis model of RAW 264.7 macrophages. METHODS: The cytotoxicity in human periodontal ligament fibroblasts, human osteogenic sarcoma cells (SAOS-2), and murine osteoclastic precursors (RAW 264.7) was analyzed by using cell number determination and proliferation assays. The ability of curcumin and its conjugate to suppress the receptor activator of nuclear factor kappa B ligand-induced osteoclastogenesis was assessed by tartrate-resistant acid phosphatase (TRAP) staining and activity as well as real-time polymerase chain reaction. RESULTS: Curcumin at concentrations ≥ 10 µmol/L was cytotoxic in all cell types tested, whereas piperine showed only slight cytotoxicity at 30 µmol/L in RAW and SAOS cultures. Although curcumin caused already significant effects, the combination with piperine completely suppressed the osteoclastogenesis by decreasing the TRAP activity and inhibiting the expression of the specific osteoclast markers TRAP, cathepsin K, and calcitonin receptor. CONCLUSIONS: We demonstrated that curcumin combined with piperine suppressed the osteoclastogenesis in vitro without causing cytotoxic effects in periodontal ligament cells. These findings suggest its potential therapeutic application for the prevention and treatment of replacement resorption in replanted avulsed teeth.

Effects of alendronate on osteoclast formation and activity in vitro.

INTRODUCTION: Root resorption is a common complication after replantation following traumatic dental avulsion. Endodontic therapy combined with local and intracanal medications aims to avoid osteoclastic activity. In such cases, the application of alendronate (ALN), a bisphosphonate widely used for the treatment of bone disorders, could be of clinical relevance. This study evaluated alendronate biocompatibility on periodontal ligament cells as well as its effects on an in vitro osteoclastogenesis model. METHODS: Alendronate cytotoxicity (10(-3) to 10(-9) mol/L) in human periodontal ligament fibroblasts, human osteogenic sarcoma cells, and murine osteoclastic precursors (RAW 264.7) was analyzed using cell number determination, cell viability, and proliferation assays. ALN (10(-6) to 10(-12) mol/L) effects on RANKL-induced osteoclastogenesis of RAW cells were assessed by tartrate-resistant acid phosphatase (TRAP) staining and activity and real-time polymerase chain reaction. RESULTS: ALN at higher concentrations was cytotoxic for all cell types, inhibiting significantly the proliferation of human osteogenic sarcoma cells and human periodontal ligament fibroblasts (≥10(-5) mol/L). TRAP activity and expression of the osteoclast markers TRAP and cathepsin K by RAW-derived osteoclasts decreased significantly with ALN at low concentrations, reaching the maximum effect at 10(-10) mol/L. CONCLUSIONS: We showed that ALN at very low concentrations is an effective inhibitor of RANKL-generated osteoclasts, without causing cytotoxic effects on their precursors or periapical cells. ALN at such concentrations might be useful to prevent replacement resorption in avulsed teeth.

Genotoxic effects of camphorquinone and DMT on human oral and intestinal cells.

OBJECTIVE: Released components of oral biomaterials can leach into the oral cavity and may subsequently reach the gastrointestinal tract. Camphorquinone (CQ) is the most common used photoinitiator in resinous restorative materials and is often combined with the co-initiator N,N-dimethyl-p-toluidine (DMT). It has been shown that CQ exerts cytotoxic effects, at least partially due to the generation of reactive oxygen species (ROS). Objective of this study was to examine the cytotoxic and genotoxic potential of CQ in human oral keratinocytes (OKF6/TERT2) and immortalized epithelial colorectal adenocarcinoma cells (Caco-2). Furthermore, the effects of visible-light irradiation and the co-initiator DMT were investigated as well as the generation of ROS, the potential protective effect of glutathione (GSH) and a recovery period of CQ-treated Caco-2 cells. METHODS: The alkaline comet assay was used to determine DNA damage. Additionally, an enzyme modified comet assay was applied, which detects 7,8-dihydro-8-oxoguanine (8-oxoguanine), a reliable marker for oxidative stress. RESULTS: Our data revealed that high concentrations of CQ induced DNA lesions in OKF6/TERT2 cells. This DNA damage is at least partly caused by the generation of 8-oxoguanine. In addition, CQ and DMT increased ROS formation and induced DNA damage in Caco-2 cells. CQ-treatment resulted in generation of 8-oxoguanine. The antioxidant GSH efficiently prevented CQ-associated DNA damage. Furthermore, a recovery following CQ-treatment significantly reduced DNA damage. SIGNIFICANCE: We conclude that CQ-induced DNA damage is caused by oxidative stress in oral and intestinal cells. These lesions can be prevented and possibly repaired by GSH-treatment and recovery of cells after the photoinitiator is removed from cultures.

Angiogenic Potential and Secretome of Human Apical Papilla Mesenchymal Stem Cells in Various Stress Microenvironments.

Stem cells from the apical papilla (SCAP) of human adult teeth are considered an accessible source of cells with angiogenic properties. The aims of this study were to investigate the endothelial transdifferentiation of SCAP, the secretion of pro- and antiangiogenic factors from SCAP, and the paracrine effects of SCAP when exposed to environmental stress to stimulate tissue damage. SCAP were exposed to serum deprivation (SD), glucose deprivation (GD), and oxygen deprivation/hypoxia (OD) conditions, individually or in combination. Endothelial transdifferentiation was evaluated by in vitro capillary-like formation assays, real-time polymerase chain reaction, western blot, and flow cytometric analyses of angiogenesis-related markers; secretome by antibody arrays and enzyme-linked immunosorbent assays (ELISA); and paracrine impact on human umbilical vein endothelial cells (HUVECs) by in vitro transwell migration and capillary-like formation assays. The short-term exposure of SCAP to glucose/oxygen deprivation (GOD) in the presence, but mainly in deprivation, of serum (SGOD) elicited a proangiogenesis effect indicated by expression of angiogenesis-related genes involved in vascular endothelial growth factor (VEGF)/VEGFR and angiopoietins/Tie pathways. This effect was unachievable under SD in normoxia, suggesting that the critical microenvironmental condition inducing rapid endothelial shift of SCAP is the combination of SGOD. Interestingly, SCAP showed high adaptability to these adverse conditions, retaining cell viability and acquiring a capillary-forming phenotype. SCAP secreted higher numbers and amounts of pro- (angiogenin, IGFBP-3, VEGF) and lower amounts of antiangiogenic factors (serpin-E1, TIMP-1, TSP-1) under SGOD compared with SOD or SD alone. Finally, secretome obtained under SGOD was most effective in inducing migration and capillary-like formation by HUVECs. These data provide new evidence on the microenvironmental factors favoring endothelial transdifferentiation of SCAP, uncovering the molecular mechanisms regulating their fate. They also validate the angiogenic properties of their secretome giving insights into preconditioning strategies enhancing their therapeutic potential.

Wnt/ß-catenin signaling regulates Dental Pulp Stem Cells' responses to pulp injury by resinous monomers.

OBJECTIVES: Aim of this study was to investigate whether Dental Pulp Stem Cells-DPSCs responses to pulp injury caused by resinous monomers is be mediated through activation of Wnt/ß-catenin signaling. METHODS: DPSCs cultures were established from third molars of healthy donors and characterized for stem cell markers with flow cytometry. Cells were exposed to TEGDMA (T: 0.5-2mM) with or without presence of the Wnt-1 ligand (W:25-100ng/ml) or the GSK3ß inhibitor Lithium (L:1-10mM), used both as activators of Wnt/ß-catenin signaling. Cell viability was evaluated by MTT assay, cell cycle profiles by flow cytometry and expression of key molecules of Wnt/ß-catenin signaling by Real-time PCR and Western Blot. RESULTS: DPSC exposure to TEGDMA caused a concentration-dependent cytotoxicity, accompanied by G1 arrest at lower and G2/M arrest at higher concentrations or after prolonged exposure. Lithium caused a dual effect, by stimulating/inhibiting cell proliferation at lower/higher concentrations respectively and causing a G2/M arrest in a concentration-dependent manner. Wnt signaling could be activated in DPSCs after Lithium or Wnt-1 treatment, as shown by accumulation of ß-catenin, its translocation into the nucleus and enhanced expression of key pathway players, like LEF1 and Cyclin D1. Importantly, exposure to TEGDMA caused a more pronounced activation of the pathway, whereas cumulative effects were observed after T/L or T/W co-treatment, indicating a very strong activation of Wnt signaling after treatment of already "activated" (by Lithium or Wnt-1) cells with TEGDMA. SIGNIFICANCE: These findings highlight the important role of Wnt canonical signaling in pulp repair responses to common injuries.

Effects of BisGMA on glutathione metabolism and apoptosis in human gingival fibroblasts in vitro.

Aim of this study was to investigate the effects of the resin monomer BisGMA on the glutathione concentration (monobromobimane assay) and apoptosis (Annexin V/PI-assay) of cultured primary human gingival fibroblasts. Cells were treated for up to 24h with 0.001-0.25 mM BisGMA to determine growth curves using the DNA stain H33342. Subsequent Annexin V/PI-assays revealed that fibroblasts exposed to concentrations of 0.005-0.01 mM (non-cytotoxic) and 0.05 mM (ED(10)-concentration) showed no increase of the share of apoptotic cells compared to non-treated controls (5-8%), while 0.1 mM BisGMA (approximately ED(50)-concentration) caused a significant increase of the percentage of apoptotic cells (50%). Simultaneously to the induction of apoptosis, 0.1 and 0.25 mM of BisGMA caused a significant depletion of the intracellular GSH content after 18 h of incubation. Our results indicate that BisGMA at concentrations >0.1 mM causes an extreme depletion of the intracellular GSH pool as well as apoptosis.

Reduced glutathione prevents camphorquinone-induced apoptosis in human oral keratinocytes.

OBJECTIVES: Camphorquinone (CQ) is a widely used photoinitiator in dental visible light (VL)-cured resinous materials. However, little is known about the toxicity of CQ in human cells. This study was designed to investigate CQ induced oxidative strain and apoptosis in cultured human oral keratinocytes (OKF6/TERT 2). Furthermore, the effects of visible-light (VL)-irradiation and the reducing agent N,N-dimethyl-p-toluidine (DMT) were investigated. In addition, the preventive potential of the antioxidant glutathione (GSH) against CQ induced toxicity was analyzed as well. METHODS: The fluorescent DNA-staining dye Hoechst 33342 was used to quantify total cell numbers. Intracellular levels of reactive oxygen species (ROS) were measured by the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). Apoptosis was determined by FACS analysis (Annexin V-FITC/propidium iodide), by measuring caspase-3/7 activity (ELISA) and by DNA laddering. RESULTS: Our data show that CQ was dose-dependent cytotoxic and caused oxidative stress by inducing reactive oxygen species (ROS). The redistribution of phosphatidylserine (PS) to the outer layer of the plasma membrane, induction of caspase-3 enzyme activity and DNA fragmentation were also observed in CQ exposed cells. Interestingly, CQ-induced ROS generation enhanced by VL irradiation or a simultaneous treatment with DMT showed no quantitative effect on apoptosis. However, co-exposure of cells with GSH significantly reduced the intracellular ROS generation as well as apoptosis caused by CQ. SIGNIFICANCE: This is the first report showing that ROS-induced apoptosis, which is caused by CQ, is prevented by GSH.

Dental pulp stem cells' secretome enhances pulp repair processes and compensates TEGDMA-induced cytotoxicity.

OBJECTIVES: Aim of this study was to investigate the effects of dental pulp stem cells' (DPSCs) secretome, expressed through their culture conditioned medium (CM), on biological endpoints related to pulp repair and on TEGDMA-induced cytotoxicity. METHODS: DPSCs cultures were established and characterized for stem cell markers with flow cytometry. CM was collected from DPSCs under serum deprivation conditions (SDC) and normal serum conditions (NSC) at various time-points. CM effects on DPSCs viability, migration and mineralization potential were evaluated by MTT assay, transwell insert and in vitro scratch assay and Alizarin Red staining/quantification respectively. TEGDMA (0.25-2.0mM) cytotoxicity regarding the same biological endpoints was tested in the presence/absence of CM. TGF-ß1 and FGF-2 secretion in CM was measured by ELISA. RESULTS: CM collected under SDC (4d) was able to increase cell viability by 20-25% and to reduce TEGDMA cytotoxicity by 20% (p<0.05). CM positive effects were not obvious when collected under NSC. Transwell assay showed significant increase (26%, p<0.05) of DPSCs' migration after CM exposure, whereas both migration assays could not support a migration rate improvement in TEGDMA-treated cultures exposed to CM compared to TEGDMA alone. CM significantly (p<0.01) increased DPSCs mineralization potential and completely counteracted TEGDMA cytotoxicity on this process. ELISA analysis showed a time-dependent increase of TGF-ß1 and a TEGDMA concentration-dependent increase of both TGF-ß1 and FGF-2 in CM. SIGNIFICANCE: These findings suggest that DPSCs secretome increases their viability, migration and mineralization potential and counteracts TEGDMA-induced cytotoxicy, revealing a novel mechanism of DPSCs autocrine signaling on pulp repair processes.

Glutathione level and genotoxicity in human oral keratinocytes exposed to TEGDMA.

Triethylene-glycol dimethacrylate (TEGDMA) is an important matrix comonomer used in many resin-modified dental materials. As the monomer-polymer conversion of these biomaterials is up to 80% at best, TEGDMA may leach into the oral cavity and the pulp in millimolar concentrations. Objective of this study was to evaluate whether TEGDMA is genotoxic in immortalized human oral keratinocytes (OKF6/TERT2), for example, due to formation of oxidative DNA-lesions. OKF6-TERT2 cells were exposed to TEGDMA at concentrations ranging from 0.5 mM to 5.0 mM. Cell viability was analyzed by the fluorescent probe propidium iodide (PI), intracellular levels of reactive oxygen species (ROS) were measured by 2',7'-dichlorofluorescein diacetate, whereas glutathione concentrations (GSH) were read using monobromobimane. Genotoxicity was determined quantitatively by the alkaline comet assay. To explore the presence of oxidized bases that could be produced by oxidative events during short-term treatment with TEGDMA, the 8-hydroxyguanine DNA-glycosylase 1 (hOGG1)-modified comet assay was used. TEGDMA induced an early and rapid GSH-depletion in a concentration-dependent manner (p < 0.05). A total of 5 mM TEGDMA reduced GSH to 57.8% ± 8.6% of control values already after 30 min. There was no significant reduction in cell viability during 6 h of incubation, and only moderate ROS-formation was detected after 4 h of treatment with TEGDMA. But after 24 h, TEGDMA-concentrations of ≥2.5 mM induced a significant reduction of total cell numbers and cells' viability. Furthermore, TEGDMA caused a concentration-dependent DNA damage in OKF6/TERT2 cultures, which was not associated with a detectable formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the cellular genome. In conclusion, our results show that TEGDMA influences the intracellular redox metabolism and may exhibit pronounced cyto- and genotoxic effects in human immortalized oral keratinocytes. However, it may be concluded that oxidative stress is not causative for TEGDMA-dependent genotoxicity in these cells.

Effects of resinous monomers on the odontogenic differentiation and mineralization potential of highly proliferative and clonogenic cultured apical papilla stem cells.

OBJECTIVE: The aim of this study was to investigate the effects of resinous monomers on the odontogenic differentiation and mineralization potential of apical papilla stem cells (SCAP). METHODS: Cultures were established from developing third molars of healthy donors aged 14-18 years-old and were extensively characterized for proliferation rate, colony forming unit efficiency and expression of stem cell markers (STRO-1, CD146, CD34, CD45, CD105, CD117-c-Kit, CD24, CD90, Nanog, Oct3/4), in order to select those with enhanced stem cell and odontogenic differentiation properties. SCAP enriched cultures were then induced for odontogenic differentiation in the continuous presence of low concentrations (0.05-0.5 mM) of the monomers 2-hydroxy-ethyl-methacrylate-HEMA and triethylene-glycol-dimethacrylate-TEGDMA for 3 weeks (long-term exposure). Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) were evaluated. RESULTS: The results showed that both types of monomer-exposure significantly delayed the odontogenic differentiation and mineralization processes of SCAP cells. A down-regulation followed by recovery in the expression of differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP was recorded. This was accompanied by reduction of the mineralized matrix produced by monomer-treated-compared to non-treated contol cultures. Furthermore, a concentration-dependence was observed for both monomers during long-term exposure, whereas the effects of HEMA were evident at much lower concentrations compared to TEGDMA. SIGNIFICANCE: These findings suggest that resinous monomers can delay the odontogenic differentiation of SCAP cells, potentially disturbing the physiological repair and/or developmental processes of human permanent teeth.

Intracellular glutathione: a main factor in TEGDMA-induced cytotoxicity?

OBJECTIVE: To evaluate whether the reduction/prevention of triethylene glycol dimethacrylate (TEGDMA)-induced decrease of intracellular glutathione (GSH) protects human periodontal ligament fibroblasts (HPLF) against cell death. METHODS: HPLF were preincubated for 30 min with exogenous GSH and then treated with TEGDMA (2.5 mM) with/without GSH (0.5-2.5-5 mM) for the following incubation exposure types: 6h (GI); 6h followed by 18 h recovery time in presence (GII) or absence (GIII) of exogenous GSH; 24 h without recovery time (GIV). TEGDMA-cytotoxicity and intracellular glutathione were assessed by Hoechst 33342 and monobromobimane (MBBr) assays. Data were statistically analyzed with Bonferroni ANOVA (p<0.05). RESULTS: Preincubation with exogenous GSH increased the intracellular GSH-concentration. TEGDMA was cytotoxic at all treatment times except at 6h (GI) (94±7% of control). In GII the treatment with TEGDMA alone (59±7%) showed no different results to cultures exposed to TEGDMA and GSH. Exogenous GSH had no effect on the TEGDMA-induced cytotoxicity also in the GIII and GIV. Thus, a combined incubation with GSH did not prevent the cytotoxicity of TEGDMA, despite of a significant increase of intracellular GSH-concentration in the presence of exogenously supplied GSH. SIGNIFICANCE: The glutathione-decreasing effect of TEGDMA is not the major cause of TEGDMA-induced cytotoxicity, indicating more complex mechanisms, which are causative for TEGDMA-cytotoxicity in HPLF.