Publications by authors named "Werner Geurtsen"

63 Publications

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

Clin Exp Dent Res 2021 Feb 18. Epub 2021 Feb 18.

Department of Conservative Dentistry, Periodontology & Preventive Dentistry, School of Dentistry, Hannover Medical School (MHH), Hannover, Germany.

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.
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http://dx.doi.org/10.1002/cre2.406DOI Listing
February 2021

Influence of 2-hydroxyethyl methacrylate (HEMA) exposure on angiogenic differentiation of dental pulp stem cells (DPSCs).

Dent Mater 2021 03 10;37(3):534-546. Epub 2021 Feb 10.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, D-30625 Hannover, Germany. Electronic address:

Objective: The angiogenic differentiation of dental pulp stem cells (DPSCs) is important for tissue homeostasis and wound healing. In this study the influence of 2-hydroxyethyl methacrylate (HEMA) on angiogenic differentiation was investigated.

Methods: To evaluate HEMA effects on angiogenic differentiation, DPSCs were cultivated in angiogenic differentiation medium (ADM) in the presence or absence of non-toxic HEMA concentrations (0.1 mM and 0.5 mM). Subsequently, angiogenic differentiation was analyzed on the molecular level by qRT-PCR and protein profiler analyzes of angiogenic markers and flow cytometry of PECAM1. The influence of HEMA on angiogenic phenotypes was analyzed by cell migration and sprouting assays.

Results: Treatment with 0.5 mM HEMA during differentiation can lead to a slight reduction of angiogenic markers on mRNA level. HEMA also seems to slightly reduce the quantity of angiogenic cytokines (not significant). However, these HEMA concentrations have no detectable influence on cell migration, the abundance of PECAM1 and the formation of capillaries. Higher concentrations caused primary cytotoxic effects in angiogenic differentiation experiments conducted for longer periods than 72 h.

Significance: Non-cytotoxic HEMA concentrations seem to have a minor impact on the expression of angiogenic markers, essentially on the mRNA level, without affecting the angiogenic differentiation process itself on a detectable level.
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http://dx.doi.org/10.1016/j.dental.2020.12.008DOI Listing
March 2021

Camphorquinone alters the expression of extracellular proteases in a 3D co-culture model of the oral mucosa.

Dent Mater 2021 02 27;37(2):236-248. Epub 2020 Nov 27.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, D-30625 Hannover, Germany. Electronic address:

Objective: Objective of our investigation was to determine the influence of CQ on the expression of antioxidant proteins and extracellular proteases in a 3D co-culture model (3DCCM) of the oral mucosa and to analyze the distribution and stability of CQ within 3D-CCMs.

Methods: 3D-CCMs consist of confluent keratinocytes (OKF6/TERT2) on cell culture inserts on top of human gingival fibroblasts (HGFs) in collagen. The treatment was carried out by adding CQ to the cell culture inserts at two time points with declining concentrations. Mass spectrometry was used to analyze the CQ concentration above and underneath the OKF6/TERT2-layer. The expression of antioxidant genes was analyzed by qRT-PCR and western blot. The regulation of extracellular proteases from different families was analyzed by qRT-PCR and Proteome Profiler arrays.

Results: GC/MS analysis showed that CQ was evenly distributed within the model. Heme oxygenase-1, NAD(P)H quinone dehydrogenase 1 (NQO1), and superoxide dismutase 1 were induced on the mRNA and protein level in OKF6/TERT2 cells. In HGFs, only the transcription of NQO1 was induced. The transcription of extracellular proteases was increased mainly in OKF6/TERT2 cells 72 h after the initial treatment. The quantity of ten out of 25 analyzed extracellular proteases in the cell culture supernatant above and six underneath the keratinocyte-layer were modulated by CQ.

Significance: Despite its high reactivity, CQ is able to penetrate a dense keratinocyte-layer, presumably across plasma membranes. CQ initially induced the cellular defense machinery against oxidative stress and altered the expression of extracellular proteases. We assume a relationship between both processes.
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http://dx.doi.org/10.1016/j.dental.2020.11.005DOI Listing
February 2021

Root coverage using a connective tissue graft with epithelial striation in combination with enamel matrix derivatives - a long-term retrospective clinical interventional study.

BMC Oral Health 2019 07 15;19(1):148. Epub 2019 Jul 15.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.

Background: The application of a connective tissue graft with epithelial striation (CTG-ES) has been shown to improve the outcome of root coverage (RC) using the coronally advanced flap (CAF) and adjunctive administration of enamel matrix derivatives (EMD). Aim of the present study was to evaluate the long-term (mean: 16.19 ± 1.80 years, range: 13 to 18 years) stability of this treatment method with special focus on the location of the gingival margin and the width of keratinized tissue (WKT).

Methods: 16 patients (10 female, 6 male, aged 35.36 ± 14.70 years at surgery) with 25 Miller class I or II gingival recession (GR) defects were treated using the CAF combined with the CTG-ES and EMD. The clinical measurements recorded at baseline (t0), 6 months (t1), and 13 to 18 years (t2) after surgery included recession depth (RED), probing pocket depth (PPD), clinical attachment level (CAL), and WKT. In addition, the number of sites with complete RC (CRC) and the mean RC (MRC) were documented at t1 and t2. The statistical analysis was performed using a linear mixed model.

Results: The RED (t0: 4.52 ± 1.56 mm; t1: 0.36 ± 0.76 mm; t2: 0.30 ± 0.60 mm) and CAL (t0: 6.16 ± 1.62 mm; t1: 1.86 ± 0.87 mm; t2: 1.54 ± 0.92 mm) were significantly reduced at t1 and t2 compared to t0 (p <  0.001). The PPD was significantly reduced at t2 compared to t0 (p = 0.016). The WKT (t0: 1.18 ± 1.28 mm; t1: 3.26 ± 0.98 mm; t2: 4.26 ± 1.83 mm) significantly increased from t0 to t1, from t0 to t2 (p <  0.001) and from t1 to t2 (p = 0.007). A CRC was recorded at 19 sites (76.0%) at t1 and t2. The MRC was 93.6 ± 12.8% at t1 and 93.3 ± 13.3% at t2.

Conclusions: The use of the CAF combined with CTG-ES and EMD leads to stable long-term outcomes on teeth with Miller Class I or II GR defects. The CTG-ES represents a hybrid graft with increased position stability and advantageous properties for the healing process. We assume that the ES is responsible for the increase of the WKT.
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http://dx.doi.org/10.1186/s12903-019-0849-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631897PMC
July 2019

Effects of HEMA on Nrf2-related gene expression using a newly developed 3D co-culture model of the oral mucosa.

Dent Mater 2019 09 28;35(9):1214-1226. Epub 2019 May 28.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, D-30625 Hannover, Germany. Electronic address:

Objective: 2-Hydroxyethyl methacrylate (HEMA) is a component of many resin-modified materials and elutes from dental restorations into the oral cavity. Objective of our investigation was to determine the impact of HEMA on oral keratinocytes (OKF6/TERT2) and gingival fibroblasts (HGFs) in a newly established 3D co-culture model (3D-CCM) and to analyze the permeability of OKF6/TERT2 cells for HEMA.

Methods: Well-characterized 3D-CCMs, consisting of confluent OKF6/TERT2 cells on cell culture inserts above HGF-containing collagen gels, were treated supra-epithelial with HEMA. Mass spectrometry was used to measure the supra- and sub-epithelial distribution of HEMA after 24 h. The impact of HEMA on nuclear factor erythroid 2-related factor 2 (Nrf2) target genes was measured by qRT-PCR and western blot analysis.

Results: Mass spectrometry showed that HEMA was evenly distributed above and below the keratinocyte layer after 24 h. Analyzed target genes of Nrf2 were induced in both cell types on the mRNA-level but less pronounced in HGFs. On the protein-level, both cell types showed similar effects: At 5 mM HEMA, heme oxygenase-1 was induced 5.1-fold in OKF6/TERT2 cells and 4.1-fold in HGFs. NAD(P)H quinone dehydrogenase-1 was approximately induced 1.85-fold in both cell types.

Significance: Our 3D-CCM is suitable to analyze the biocompatibility of dental materials due to an improved simulation of the oral mucosa compared to monolayer cultures. Our results indicate that HEMA is able to penetrate a dense layer of keratinocytes and to activate the cellular oxidative defense response. This may be due to the activation of the Nrf2-pathway in both cell types.
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http://dx.doi.org/10.1016/j.dental.2019.05.006DOI Listing
September 2019

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

Dent Mater 2019 03 25;35(3):501-510. Epub 2019 Jan 25.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, D-30625 Hannover, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.dental.2019.01.011DOI Listing
March 2019

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

Dent Mater 2019 01 28;35(1):144-155. Epub 2018 Nov 28.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, D-30625 Hannover, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.dental.2018.11.006DOI Listing
January 2019

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

Dent Mater 2018 12 17;34(12):1783-1796. Epub 2018 Oct 17.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

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 DCFH 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.
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http://dx.doi.org/10.1016/j.dental.2018.09.015DOI Listing
December 2018

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

Stem Cell Res Ther 2017 Nov 2;8(1):247. Epub 2017 Nov 2.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School (MHH), Hannover, Germany.

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 (β-galactosidase (SA-β-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.
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http://dx.doi.org/10.1186/s13287-017-0705-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667471PMC
November 2017

Advances and New Technologies towards Clinical Application of Oral Stem Cells and Their Secretome.

Stem Cells Int 2017 24;2017:6367375. Epub 2017 Jan 24.

Aix-Marseille University, CNRS, Institute of Movement Science (ISM), Marseille, France.

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http://dx.doi.org/10.1155/2017/6367375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5294372PMC
January 2017

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

Dent Mater 2016 08 11;32(8):e159-75. Epub 2016 Jun 11.

Department of Fixed Prosthesis & Implant Prosthodontics, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki GR-54124, Greece. Electronic address:

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 TGFβ-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.
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http://dx.doi.org/10.1016/j.dental.2016.05.013DOI Listing
August 2016

A biomimetic approach to ameliorate dental hypersensitivity by amorphous polyphosphate microparticles.

Dent Mater 2016 06 6;32(6):775-83. Epub 2016 Apr 6.

ERC Advanced Investigator Grant Research Group at the Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Duesbergweg 6, D-55128 Mainz, Germany. Electronic address:

Objective: Dental hypersensitivity has become one of the most common and most costly diseases in the world, even though those maladies are very rarely life threatening. Using amorphous microparticles, fabricated from the natural polymer (polyphosphate), we intend to reseal the dentinal tubules exposed and reduce by that the hypersensitivity.

Methods: Amorphous microparticles (termed aCa-polyP-MP) were prepared from Na-polyphosphate (polyP) and CaCl2, then incubated with human teeth. The potential of the microparticles to plug the dentinal tubules was determined by microscopic and spectroscopic techniques.

Results: We demonstrate that, in contrast to polyP, the aCa-polyP-MP efficiently reseal dentinal tubules exposed at the tooth surface. Scanning electron microscopical (SEM) and energy dispersive X-ray spectroscopic (EDX) studies showed that the tooth cement and dentin surfaces, incubated with aCa-polyP-MP, form a nearly homogenous, approximately 50-μm thick solid polyP layer on the tooth cement and dentin surfaces, while no coating on the tooth surface, incubated with Na-polyP [Ca(2+)], was observed. Determination of the mechanical properties of the polyP coating revealed a Martens hardness of 3.85±0.64GPa and a reduced elastic modulus of 94.72±8.54GPa already after a 3h exposure to the aCa-polyP-MP, which become close to those of the natural enamel (4.33±0.69GPa and 101.61±8.52GPa, respectively) after prolonged incubation periods. In addition, aCa-polyP-MP turned out to display morphogenetic activity. Incubation of precursor odontoblasts cultures in the presence of aCa-polyP-MP resulted in a 7-fold increase of the steady-state-expression level of the gene encoding for the alkaline phosphatase (ALP) during a 7 d incubation period.

Significance: Ca-polyP microparticles, consisting of the biocompatible natural polymer polyP, provide a potential sealing material for dentinal tubules on the tooth surface.
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http://dx.doi.org/10.1016/j.dental.2016.03.027DOI Listing
June 2016

Curcumin in Combination with Piperine Suppresses Osteoclastogenesis In Vitro.

J Endod 2015 Oct 20;41(10):1638-45. Epub 2015 Aug 20.

Department of Conservative/Preventive Dentistry and Periodontology, Hannover Medical School, Hannover, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.joen.2015.05.009DOI Listing
October 2015

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

Stem Cells Dev 2015 Nov 2;24(21):2496-512. Epub 2015 Sep 2.

5 Laboratory of Pharmacology, School of Pharmaceutical Sciences, Aristotle University of Thessaloniki (A.U.TH.) , Thessaloniki, Greece .

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.
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http://dx.doi.org/10.1089/scd.2015.0197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620528PMC
November 2015

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

Dent Mater 2015 Oct 15;31(10):1159-68. Epub 2015 Jul 15.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

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.
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http://dx.doi.org/10.1016/j.dental.2015.06.007DOI Listing
October 2015

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

Dent Mater 2015 May 28;31(5):542-55. Epub 2015 Feb 28.

Department of Conservative Dentistry, Periodontology & Preventive Dentistry, School of Dentistry, Hannover Medical School, Hannover D-30625, Germany. Electronic address:

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/β-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 GSK3β inhibitor Lithium (L:1-10mM), used both as activators of Wnt/β-catenin signaling. Cell viability was evaluated by MTT assay, cell cycle profiles by flow cytometry and expression of key molecules of Wnt/β-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 β-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.
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http://dx.doi.org/10.1016/j.dental.2015.02.004DOI Listing
May 2015

Release and protein binding of components from resin based composites in native saliva and other extraction media.

Dent Mater 2015 May 24;31(5):496-504. Epub 2015 Feb 24.

Department of Operative/Restorative Dentistry, Periodontology and Pedodontics, Ludwig-Maximilians-University of Munich, Goethestr. 70, 80336 Munich, Germany; Walther-Straub-Institute of Pharmacology and Toxicology, Nussbaumstr. 26, 80336 Munich, Germany.

Objectives: Unpolymerized (co)monomers and additives can be released from resin based composites (RBCs) and can enter the human organism. In this study, the binding of ingredients from composites to salivary proteins and plasma proteins was investigated.

Methods: The composites investigated were Admira(®) flow, Venus(®) Diamond flow, Filtek™ Supreme XTE flow, Tetric EvoCeram(®), Tetric EvoFlow(®). The samples (n=4) were polymerized according to the instructions of the manufacturer of RBCs. The samples were immersed into native saliva, protein-free saliva (artificial saliva), water and ethyl acetate, and incubated at 37°C for 24h or 72h. The eluates were analyzed by gas chromatography/mass spectrometry. To determine the binding to salivary proteins, the concentration of (co)monomers and additives detected in native saliva was compared to the concentration of (co)monomers and additives detected in protein-free saliva, water and ethyl acetate respectively. To assess the affinity of TEGDMA, EGDMA, DEGDMA, PMGDMA, BPA, and DCHP to human serum albumin (HSA) and human α1-acid glycoprotein (AGP), a plasma protein binding assay (ABNOVA, Transil XL PPB Prediction Kit TMP-0212-2096) was performed. The statistical significance (p<0.05) of the difference between the experimental groups was tested using the one-way-analysis of variance (ANOVA), followed by Tukey's analysis.

Results: The concentration of TEGDMA, GMA and CyHEMA released in native saliva was significantly lower than the concentration released in protein-free saliva or water (Admira(®) flow: concentration of TEGDMA after 72h: 0.08 mmol/L (native saliva), 0.34 mmol/L (protein-free saliva), 0.39 mmol/L (water)). The concentrations of HEMA, EGDMA, DDDMA and CQ released in native saliva remained even below the detection limit, compared to the other extraction media. Protein binding of the tested methacrylates to HSA+AGP was 82-85%, the binding of DCHP was 96.6%, and the binding of BPA was 95.2%.

Significance: Artificial saliva or water as extraction medium does not reflect the real physiological situation in the body. Salivary and plasma proteins may bind (co)monomers and additives and may thereby contribute to a lower bioavailability of leachables from RBCs in vivo than previously thought.
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http://dx.doi.org/10.1016/j.dental.2015.01.016DOI Listing
May 2015

Effects of alendronate on osteoclast formation and activity in vitro.

J Endod 2015 Jan 14;41(1):45-9. Epub 2014 Oct 14.

Department of Conservative/Preventive Dentistry and Periodontology, Hannover Medical School, Hannover, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.joen.2014.07.010DOI Listing
January 2015

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

Dent Mater 2014 Dec 18;30(12):e405-18. Epub 2014 Sep 18.

Department of Fixed Prosthesis & Implant Prosthodontics, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki GR-54124, Greece. Electronic address:

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-β1 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-β1 and a TEGDMA concentration-dependent increase of both TGF-β1 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.
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http://dx.doi.org/10.1016/j.dental.2014.08.377DOI Listing
December 2014

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

Clin Oral Investig 2014 Jul 16;18(6):1705-10. Epub 2014 Jan 16.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, 30625, Hannover, Germany,

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.
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http://dx.doi.org/10.1007/s00784-013-1178-xDOI Listing
July 2014

Curing mode affects bond strength of adhesively luted composite CAD/CAM restorations to dentin.

Dent Mater 2014 Mar 11;30(3):281-91. Epub 2014 Jan 11.

KU Leuven - BIOMAT, Department of Oral Health Sciences, KU Leuven (University of Leuven) & Dentistry, University Hospitals Leuven, Belgium.

Objectives: To determine the effect of curing mode and restoration-surface pre-treatment on the micro-tensile bond strength (μTBS) to dentin.

Methods: Sandblasted CAD/CAM composite blocks (LAVA Ultimate, 3M ESPE) were cemented to bur-cut dentin using either the etch & rinse composite cement Nexus 3 ('NX3', Kerr) with Optibond XTR ('XTR', Kerr), or the self-etch composite cement RelyX Ultimate ('RXU', 3M ESPE) with Scotchbond Universal ('SBU', 3M ESPE). All experimental groups included different 'curing modes' (light-curing of adhesive and cement ('LL'), light-curing of adhesive and auto-cure of cement ('LA'), co-cure of adhesive through light-curing of cement ('AL'), or complete auto-cure ('AA')) and different 'restoration-surface pre-treatments' of the composite block (NX3: either a silane primer (Kerr), or the XTR adhesive; RXU: either silane primer (RelyX Ceramic Primer, 3M ESPE) and SBU, or solely SBU). After water-storage (7 days, 37°C), the μTBS was measured. Additionally, the degree of conversion (DC) of both cements was measured after 10min and after 1 week, either auto-cured (21°C/37°C) or light-cured (directly/through 3-mm CAD/CAM composite).

Results: The linear mixed-effects model (α=0.05) revealed a significant influence of the factors 'curing mode' and 'composite cement', and a less significant effect of the factor 'restoration-surface pre-treatment'. Light-curing 'LL' revealed the highest μTBS, which decreased significantly for all other curing modes. For curing modes 'AA' and 'AL', the lowest μTBS and a high percentage of pre-testing failures were reported. Overall, DC increased with light-curing and incubation time.

Significance: The curing mode is decisive for the bonding effectiveness of adhesively luted composite CAD/CAM restorations to dentin.
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http://dx.doi.org/10.1016/j.dental.2013.11.016DOI Listing
March 2014

Composite cements benefit from light-curing.

Dent Mater 2014 Mar 7;30(3):292-301. Epub 2014 Jan 7.

KU Leuven - BIOMAT, Department of Oral Health Sciences, KU Leuven (University of Leuven) & Dentistry, University Hospitals Leuven, Belgium.

Objective: To investigate the effect of curing of composite cements and a new ceramic silanization pre-treatment on the micro-tensile bond strength (μTBS).

Methods: Feldspathic ceramic blocks were luted onto dentin using either Optibond XTR/Nexus 3 (XTR/NX3; Kerr), the silane-incorporated 'universal' adhesive Scotchbond Universal/RelyX Ultimate (SBU/RXU; 3M ESPE), or ED Primer II/Panavia F2.0 (ED/PAF; Kuraray Noritake). Besides 'composite cement', experimental variables were 'curing mode' ('AA': complete auto-cure at 21°C; 'AA*': complete auto-cure at 37°C; 'LA': light-curing of adhesive and auto-cure of cement; 'LL': complete light-curing) and 'ceramic surface pre-treatment' ('HF/S/HB': hydrofluoric acid ('HF': IPS Ceramic Etching Gel, Ivoclar-Vivadent), silanization ('S': Monobond Plus, Ivoclar-Vivadent) and application of an adhesive resin ('HB': Heliobond, Ivoclar-Vivadent); 'HF/SBU': 'HF' and application of the 'universal' adhesive Scotchbond Universal ('SBU'; 3M ESPE, only for SBU/RXU)). After water storage (7 days at 37°C), ceramic-dentin sticks were subjected to μTBS testing.

Results: Regarding the 'composite cement', the significantly lowest μTBSs were measured for ED/PAF. Regarding 'curing mode', the significantly highest μTBS was recorded when at least the adhesive was light-cured ('LA' and 'LL'). Complete auto-cure ('AA') revealed the significantly lowest μTBS. The higher auto-curing temperature ('AA*') increased the μTBS only for ED/PAF. Regarding 'ceramic surface pre-treatment', only for 'LA' the μTBS was significantly higher for 'HF/S/HB' than for 'HF/SBU'.

Significance: Complete auto-cure led to inferior μTBS than when either the adhesive (on dentin) or both adhesive and composite cement were light-cured. The use of a silane-incorporated adhesive did not decrease luting effectiveness when also the composite cement was light-cured.
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http://dx.doi.org/10.1016/j.dental.2013.11.012DOI Listing
March 2014

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

Dent Mater 2014 Feb 17;30(2):215-26. Epub 2013 Dec 17.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, Hannover 30625, Germany.

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.
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http://dx.doi.org/10.1016/j.dental.2013.11.008DOI Listing
February 2014

The enzyme carbonic anhydrase as an integral component of biogenic Ca-carbonate formation in sponge spicules.

FEBS Open Bio 2013 16;3:357-62. Epub 2013 Aug 16.

ERC Advanced Investigator Grant Research Group at Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Duesbergweg 6, Mainz D-55128, Germany.

The inorganic scaffold of the spicules, the skeletal elements of the calcareous sponges, is formed of calcium carbonate (CaCO3). The growth of the approximately 300-μm large spicules, such as those of the calcareous sponge Sycon raphanus used in the present study, is a rapid process with a rate of about 65 μm/h. The formation of CaCO3 is predominantly carried out by the enzyme carbonic anhydrase (CA). The enzyme from the sponge S. raphanus was isolated and prepared by recombination. The CA-driven deposition of CaCO3 crystallites is dependent on temperature (optimal at 52 °C), the pH value of the reaction assay (7.5/8.0), and the substrate concentration (CO2 and Ca(2+)). During the initial phase of crystallite formation, ≈40 μm large round-shaped deposits are formed that remodel to larger prisms. These crystal-like prisms associate to each other and form either rope-/bundle-like aggregates or arrange perfectly with their smaller planes along opposing surfaces of the sponge spicule rays. The CA-dependent CaCO3 deposition can be inhibited by the CA-specific inhibitor acetazolamide. The Michaelis-Menten constant for the CA-driven mineralization has been determined to be around 8 mM with respect to CaCO3. The deposits formed have a Martens hardness of ≈5 GPa. The data presented here highlights for the first time that calcite deposition in the sponge system is decisively controlled enzymatically. This data will contribute to the development of new strategies applicable for the fabrication of novel biomaterials.
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http://dx.doi.org/10.1016/j.fob.2013.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821024PMC
November 2013

Periodontal conditions in patients with Marfan syndrome - a multicenter case control study.

BMC Oral Health 2013 Oct 28;13:59. Epub 2013 Oct 28.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany.

Background: Marfan syndrome (MFS) is a disorder of the connective tissues. Alterations of the elastic fibers may manifest in different tissues especially in the skeletal, cardiovascular and ocular system. Oral manifestations like orthodontic or skeletal anomalies and fragility of the temporomandibular joint have been well described by various authors. However, no data are available regarding a possible periodontal involvement of MFS. Hence, the aim of the present study was to investigate for the first time if MFS may increase the susceptibility to periodontitis.

Methods: A comprehensive periodontal examination including documentation of probing pocket depth, gingival recession, clinical attachment level, and bleeding on probing was conducted in all patients. In addition, dental conditions were assessed by determining the Index for Decayed, Missing and Filled Teeth (DMFT) and a self-administered questionnaire was filled out by patients. For statistical analysis, the unpaired t-Test was applied (level of significance: p < 0.05). Both groups were matched concerning well known periodontal risk factors like age, gender and smoking habits.

Results: 82 participants, 51 patients with MFS (30 female and 21 male, mean age: 40.20 ± 15.35 years) and 31 sound controls (17 female and 14 male, mean age: 40.29 ± 13.94 years), were examined. All assessed periodontal and dental parameters were not significantly different between groups.

Conclusions: Based on our data, patients with MFS did not reveal a higher prevalence of periodontitis compared to the control group. However, Marfan patients showed a tendency to more inflammation signs, which can be explained by the crowded teeth. Therefore, a regular professional cleaning of the teeth is recommendable (i.e., 6 months intervals) in order to reduce the bacterial biofilm in the oral cavity and thus resulting in a decreased risk of systemic diseases, specifically endocarditis.
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http://dx.doi.org/10.1186/1472-6831-13-59DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816571PMC
October 2013

Cytotoxicity and induction of DNA double-strand breaks by components leached from dental composites in primary human gingival fibroblasts.

Dent Mater 2013 Sep 31;29(9):971-9. Epub 2013 Jul 31.

Department of Operative/Restorative Dentistry, Periodontology and Pedodontics, Ludwig-Maximilians-University of Munich, Goethestr. 70, 80336 Munich, Germany.

Introduction: The public interest steadily increases in the biological adverse effects caused by components released from resin-based dental restorations.

Objective: In this study, the cytotoxicity and the genotoxicity were investigated of following released components from dental resin restorations in human gingival fibroblasts (HGF): tetraethyleneglycol dimethacrylate (TEEGDMA), neopentylglycol dimethacrylate (Neopen), diphenyliodoniumchloride (DPIC), triphenyl-stibane (TPSB) and triphenylphosphane (TPP).

Methods: XTT based cell viability assay was used for cytotoxicity screening of substances. γ-H2AX assay was used for genotoxicity screening. In the γ-H2AX assay, HGFs were exposed to the substances for 6h. Induced foci represent double DNA strand breaks (DSBs), which can induce ATM-dependent phosphorylation of the histone H2AX. Cell death effects (apoptosis and necrosis), induced by the substances were visually tested by the same investigator using the fluorescent microscope.

Results: All tested substances induced a dose-dependent loss of viability in HGFs. Following toxicity ranking among the substances at EC50-concentration were found in the XTT assay (mM, mean±SEM; n=5): DPIC>Neopen>TPSB>TPP>TEEGDMA. DSB-foci per HGF-cell were obtained, when HGFs were exposed to the EC50-concentration of each substance in the following order (mean±SEM; n=3): DPIC>Neopen>TPSB>TPP>TEEGDMA. Multi-foci cells (cells that contain more than 40 foci each) in 80 HGF-cells at EC50-concentration of each substance were found as follow (mean±SEM; n=3): DPIC>Neopen>TPP>TPSB>TEEGDMA. Cell apoptosis contained in each substance at EC50-concentration in the following order (mean±SEM; n=3): DPIC>Neopen>TPSB>TPP >TEEGDMA. Cell necrosis contained in each substance at EC50-concentration in the following order (mean±SEM; n=3): DPIC>Neopen>TPSB>TPP>TEEGDMA.

Conclusion: Leached components from dental resin restorations can induce DNA DSBs and cell death effects in HGFs.
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http://dx.doi.org/10.1016/j.dental.2013.07.007DOI Listing
September 2013

An early oral health care program starting during pregnancy--a long-term study--phase V.

Clin Oral Investig 2014 Apr 28;18(3):863-72. Epub 2013 Jul 28.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany,

Objectives: Objective was to analyze the effects of a long-term prevention program on dental and oral health of adolescents.

Materials And Methods: The entire study was subdivided into five phases. Phase I comprised an individual preventive care during pregnancy, phase II assessed mothers and their children until the age of 3, and in phase III until the age of 6. In phase IV, 13- to 14-year-old teenagers were investigated. In phase V, 18-19-year-old adolescents were examined (18.4 ± 0.4 years, n = 26). All phases consisted of an examination, education, and treatment based on the concept of an "early oral health care promotion." The control group consisted of randomly selected adolescents of the same age (n = 35). The following clinical parameters were assessed: DMF-T/DMF-S, HI, PBI, PSI, and Streptococcus mutans/lactobacilli concentration in saliva.

Results: The adolescents of the prevention group revealed a share of 92.3 % caries-free dentition. Mean DMF-T was 1.4 ± 2.6. The control group showed a significantly higher mean DMF-T of 3.8 ± 3.2 (p < 0.05) and revealed 71.4 % of caries-free dentition. The prevention group showed a significant lower PSI of 1.2 ± 0.8 compared to the control group (2.1 ± 0.4) (p < 0.05).

Conclusion: An "early oral health care promotion" starting during pregnancy may cause a sustained and long-term improvement of the oral health of young adults.

Clinical Relevance: Prevention programs starting during pregnancy may establish an improved health behavior. Caries, periodontitis, and dietary complications in mother and child can be avoided by improving maternal oral health and by a tooth-friendly diet.
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http://dx.doi.org/10.1007/s00784-013-1059-3DOI Listing
April 2014

Level of information about the relationship between diabetes mellitus and periodontitis--results from a nationwide diabetes information program.

Eur J Med Res 2013 Mar 11;18. Epub 2013 Mar 11.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover, 30625, Germany.

Background: A comprehensive knowledge about the mutual influence between diabetes and periodontitis is decisive for the successful treatment of both diseases. The present investigation aimed at assessing the diabetic and periodontal conditions and, in particular, the degree of knowledge about the relationship between diabetes and periodontitis.

Methods: During a diabetes information program, 111 nondiabetics (ND), 101 type 1 diabetics (T1D), and 236 type 2 diabetics (T2D) were subject to a medical and dental examination and completed a self-administered questionnaire. Medical examination included measurements of glycated hemoglobin (HbA1c), blood glucose (BG), and body mass index (BMI). Full-mouth examination consisted of the assessment of the decayed, missing, filled teeth index (DMFT) and the periodontal screening index (PSI). Chi-square test, ANOVA, t test of independent samples, univariate and multivariate logistic regression models with variable selection strategies were used for statistical analyses. Due to the exploratory character of the investigation a value of P≤0.05 was considered to be statistically substantial.

Results: T2D had a significantly higher PSI when compared to T1D and ND (t test: P<0.001; P=0.005). Approximately 90% of T2D suffered from periodontitis. In addition, diabetics with periodontitis showed a significantly higher BMI when compared to diabetics without periodontitis (multivariate logistic regression: P=0.002). Almost 60% of all investigated subjects were not informed about the mutual influence between diabetes and periodontitis. T2D had almost as little information about the increased risk for periodontitis as ND.

Conclusions: The data of the present investigation suggest that there is a strong association between type 2 diabetes and chronic periodontitis. The lack of awareness of the mutual influence between diabetes and periodontitis, especially in T2D, demonstrates that this topic is still neglected in dental and diabetic treatment.
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http://dx.doi.org/10.1186/2047-783X-18-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605295PMC
March 2013

Does inhibition of proteolytic activity improve adhesive luting?

Eur J Oral Sci 2013 Apr 22;121(2):121-31. Epub 2013 Feb 22.

KU Leuven-BIOMAT, Department of Oral Health Sciences, KU Leuven (University of Leuven), & Dentistry, University Hospitals Leuven, Leuven, Belgium.

Endogenous enzymes may be involved in the biodegradation of adhesive restoration-tooth interfaces. Inhibitors of matrix metalloproteinases (MMPs) have been suggested to retard the bond-degradation process. Limited data are available on whether composite cements may also benefit from MMP inhibitors. Therefore, the aim of this study was to determine the effect of two MMP inhibitors--chlorhexidine digluconate (CHX) and galardin--on the microtensile bond strength (μTBS) of two self-adhesive composite cements to dentin. Ceramic specimens were cemented to bur-cut dentin surfaces using the self-adhesive composite cements RelyX Unicem 2 (3M ESPE) or Clearfil SA (Kuraray), or the etch-and-rinse composite cement Nexus 3 (Kerr) that served as the control. The surfaces were left untreated or were pretreated with MMP inhibitors (2% CHX or 0.2 mM galardin). The μTBS was determined 'immediately' and upon ageing (water storage for 6 months). Statistical analysis revealed a significant effect of the factors 'composite cement' and 'storage', as well as all interactions, but no effect of the MMP inhibitors. After 6 months of ageing, the μTBS decreased for all cements, except for the multistep etch-and-rinse luting composite when it was applied without MMP inhibitors. The MMP inhibitors could not prevent the decrease in μTBS upon ageing and therefore do not improve the luting durability of the composite cements tested.
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http://dx.doi.org/10.1111/eos.12018DOI Listing
April 2013

Periodontal status of HIV-infected patients undergoing antiretroviral therapy compared to HIV-therapy naive patients: a case control study.

Eur J Med Res 2012 Jan 30;17. Epub 2012 Jan 30.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany.

Background: Although severe oral opportunistic infections decreased with the implementation of highly active antiretroviral therapy, periodontitis is still a commonly described problem in patients infected with human immunodeficiency virus (HIV). The objective of the present investigation was to determine possible differences in periodontal parameters between antiretroviral treated and untreated patients.

Methods: The study population comprised 80 patients infected with HIV divided into two groups. The first group was receiving antiretroviral therapy while the second group was therapy naive. The following parameters were examined: probing pocket depth, gingival recession, clinical attachment level, papilla bleeding score, periodontal screening index and the index for decayed, missed and filled teeth. A questionnaire concerning oral hygiene, dental care and smoking habits was filled out by the patients.

Results: There were no significant differences regarding the periodontal parameters between the groups except in the clinical marker for inflammation, the papilla bleeding score, which was twice as high (P < 0.0001) in the antiretroviral untreated group (0.58 ± 0.40 versus 1.02 ± 0.59). The participants of this investigation generally showed a prevalence of periodontitis comparable to that in healthy subjects. The results of the questionnaire were comparable between the two groups.

Conclusion: There is no indication for advanced periodontal damage in HIV-infected versus non-infected patients in comparable age groups. Due to their immunodeficiency, HIV-infected patients should be monitored closely to prevent irreversible periodontal damage. Periodontal monitoring and early therapy is recommended independent of an indication for highly active antiretroviral therapy.
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http://dx.doi.org/10.1186/2047-783X-17-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3337541PMC
January 2012
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