Werner Geurtsen

Comparison of the number and diameter of dentinal tubules in human and bovine dentine by scanning electron microscopic investigation

Detailed information on dentine structure is essential for interpreting data from investigations on dentine-adhesive materials. The purpose here was to compare the number and diameter of dentinal tubules at similarly prepared surfaces of bovine permanent central incisors and human deciduous and third molars. In bovine teeth, crowns and roots were used; in human samples only the crowns were investigated. Tubule density in the middle layer was higher in bovine root (BR) dentine (number of tubules per mm(2)+/-SD: 23, 760+/-2453) than in human deciduous (HD) (18,243+/-3845), human permanent (HP) (18,781+/-5855), and bovine coronal (BC) (17, 310+/-2140) dentine. The corresponding values for the deep layer were 23,738+/-4457 (BR), 24,162+/-5338 (HD), 21,343+/-7290 (HP), and 20,980+/-4198 (BC). No significant differences were found for the number of dentinal tubules in bovine coronal dentine compared to the dentine of human deciduous and permanent molars. The mean diameter of bovine dentinal tubules was slightly, but not significantly, higher than in human dentine (middle layer/deep layer+/-SD): BC, 2. 85 microm+/-0.18/3.50 microm+/-0.33; BR, 3.10 microm+/-0.33/3.23 microm+/-0.30; HD, 2.55 microm+/-0.16/2.82 microm+/-0.28; HP, 2.65 microm+/-0.19/2.90 microm+/-0.22. These findings demonstrate that corresponding coronal dentine layers of human deciduous and permanent molars, and of bovine central incisors, are not significantly different in their number of tubules per mm(2) and their tubule diameter, whereas tubule density in bovine root dentine is significantly higher. These results suggest that provided standardized preparations are used, bovine incisor crown dentine is a suitable substitute for human molar dentine in adhesion studies.

Biocompatibility of Dental Casting Alloys

Most cast dental restorations are made from alloys or commercially pure titanium (cpTi). Many orthodontic appliances are also fabricated from metallic materials. It has been documented in vitro and in vivo that metallic dental devices release metal ions, mainly due to corrosion. Those metallic components may be locally and systemically distributed and could play a role in the etiology of oral and systemic pathological conditions. The quality and quantity of the released cations depend upon the type of alloy and various corrosion parameters. No general correlation has been observed between alloy nobility and corrosion. However, it has been documented that some Ni-based alloys, such as beryllium-containing Ni alloys, exhibit increased corrosion, specifically at low pH. Further, microparticles are abraded from metallic restorations due to wear. In sufficient quantities, released metal ions-particularly Cu, Ni, Be, and abraded microparticles-can also induce inflammation of the adjacent periodontal tissues and the oral mucosa. While there is also some in vitro evidence that the immune response can be altered by various metal ions, the role of these ions in oral inflammatory diseases such as gingivitis and periodontitis is unknown. Allergic reactions due to metallic dental restorations have been documented. Ni has especially been identified as being highly allergenic. Interestingly, from 34% to 65.5% of the patients who are allergic to Ni are also allergic to Pd. Further, Pd allergy always occurrs with Ni sensitivity. In contrast, no study has been published which supports the hypothesis that dental metallic materials are mutagenic/genotoxic or might be a carcinogenic hazard to man. Taken together, very contradictory data have been documented regarding the local and systemic effects of dental casting alloys and metallic ions released from them. Therefore, it is of critical importance to elucidate the release of cations from metallic dental restorations in the oral environment and to determine the biological interactions of released metal components with oral and systemic tissues.

Long-term cytocompatibility of various endodontic sealers using a new root canal model.

It was the purpose of our study to determine the cytotoxicity of several types of root canal sealers in vitro over the period of 1 yr by using a new test model. Roots of extracted human teeth were filled with N2, Apexit, Roekoseal, AH Plus, Ketac Endo, Endomethasone, and one gutta-percha point. In addition, roots filled with laterally condensed gutta-percha/N2. Teeth filled with one gutta-percha point only were controls. All specimens were consecutively extracted with distilled water for a total period of 1 yr. Extracts were investigated for cytotoxicity by using immortalized 3T3 fibroblasts and primary human periodontal ligament fibroblasts. Results were statistically analyzed with Dunnetts t tests (p < 0.05). Pronounced cytotoxic effects were only caused by N2-extracts in both cell cultures (p < 0.05). Furthermore, statistically significant cytotoxic alterations were also induced by 10-week eluates of Endomethasone (p < 0.05). All other investigated materials did not significantly alter cell metabolism.

Genotoxicity and cytotoxicity of the epoxy resin-based root canal sealer AH plus

Previous studies with four rapid in vitro and in vivo test systems have shown that the epoxy resin-based root canal sealer AH26 may be genotoxic and cytotoxic (9). The purpose of this study was to determine the cytotoxic and genotoxic effects of the new resinous root canal sealer AH Plus by means of the growth inhibition test with primary human periodontal ligament fibroblasts and permanent 3T3 monolayers, the procaryotic umu test, the eucaryotic DNA synthesis inhibition test, and the in vivo alkaline filter elution test. In addition, Ames tests were performed with extracts from AH Plus. AH Plus caused only slight or no cellular injuries. Furthermore, no genotoxicity and mutagenicity were revealed by AH Plus. These data should be taken into consideration when deciding about a root canal sealer.

The Cellular Compatibility of Five Endodontic Sealers during the Setting Period

The purpose of this study was to evaluate the cellular compatibility of five endodontic sealers in the first 24 h after mixing. Specimens of N2, Endomethasone, Apexit, AH Plus, and Ketac Endo were extracted with cell culture medium 0, 1, 5, and 24 h after mixing. Eluates were tested for cytotoxicity with immortal 3T3 cells and primary human periodontal ligament fibroblasts using XTT-assays. Data were analyzed for statistically significant differences by means of Dunnetts t tests (p < 0.05). All extracts of N2 completely inhibited cell metabolism (p < 0.05). Similar effects were provoked by the first three eluates of Endomethasone, but the 24-h extract irritated cells significantly less (p < 0.05). Severe cytotoxicity was also observed with all Ketac Endo extracts (p < 0.05). A significant inhibition of mitochondrial activity was induced by the first (3T3) or the first and second eluate (periodontal ligament fibroblasts) of AH Plus (p < 0.05). The subsequent eluates of this sealer and all extracts of Apexit did not reveal any cytotoxic potency.

Biocompatibility of various light-curing and one conventional glass-ionomer cement

The purpose of this study was to determine and to compare the cellular compatibility of modern light-curing (lc) glass-ionomer cements (GICs) to one conventional (co) GIC. The following materials were investigated: Ionoseal (IS, lc) (VOCO, Germany), Vitrebond (VB, lc) (3M, USA), Compoglass (CG, lc) (Vivadent, FL) and Ketac Fil Applicap (KF, co) (ESPE, Germany). From all GICs, equally sized specimens (height 2 mm, diameter 5 mm) were polymerized or set according to the instructions of the manufacturers. Various extracts of all specimens were obtained by subsequent elutions. Human primary fibroblasts of the attached gingiva (HGF) and permanent mouse fibroblasts (3T3) were used for the experiments. HGF and 3T3 cells were exposed to the extracts of all materials for 48 h. Growth inhibition due to cytotoxic effects was determined by staining the cultures with Hoechst 33342 (determination of DNA and cell vitality). It was found that the material CG induced no growth inhibition in any of the assays. Proliferation of HGF was not, or only slightly, inhibited by the extracts of the materials IS and KF, whereas severe alterations were caused by the extracts of the material VB. Growth of 3T3 cells was only moderately or slightly reduced by the extracts of materials IS and KF respectively, but was severely or totally inhibited by all extracts of VB. From our results we conclude that the GIC VB is very cytotoxic and therefore may also induce alterations in vivo. All other investigated GICs revealed excellent (CG), or good (IS, KF) cellular compatibility.

Nicotine-induced alterations in human primary periodontal ligament and gingiva fibroblast cultures

Abstract Various in vivo and in vitro investigations have indicated that tobacco smoking as well as the use of smokeless tobacco products may be important risk factors for the development and severity of inflammatory periodontal disease. The purpose of this study was to determine the cytotoxicity of nicotine by means of human primary oral fibroblast cultures and a permanent cell line. The cytotoxicity of nicotine was evaluated by determination of cell growth, cell membrane integrity, protein content, and alterations of the cytoskeleton. Furthermore, recovery following nicotine exposure was assessed by vital staining (trypan blue). Dose-dependent toxic effects of nicotine were measured within a range of 0.48 mM to 62 mM. Growth of fibroblasts was decreased by nicotine concentrations higher than 7.8 mM. Additionally, the protein content was significantly decreased and cell membranes were damaged. Morphological alterations of microtubules and vimentin filaments were observed at concentrations higher than 3.9 mM. Nicotine-exposed cells revealed atypical shapes and vacuoles. The toxic effects of nicotine became irreversible in the range between 10.5 and 15.5 mM, whereas at lower concentrations cells recovered after the withdrawal of nicotine. Our results confirm clinical oberservations regarding the important role of nicotine as a risk factor in the etiology and progression of periodontal disease.

Compatibility of resorbable and nonresorbable guided tissue regeneration membranes in cultures of primary human periodontal ligament fibroblasts and human osteoblast-like cells

Abstract The purpose of this study was (a) to evaluate the cytocompatibility of three resorbable and nonresorbable membranes in fibroblast and osteoblast-like cell cultures and (b) to observe the growth of those cells on the various barriers by scanning electron microscopy (SEM). Primary human periodontal ligament fibroblasts (HPLF) and human osteoblast-like cells (SAOS-2) were incubated with nonresorbable polytetrafluoroethylene (ePTFE) barriers and resorbable polylactic acid as well as collagen membranes. Cytotoxic effects were determined by XTT (mitochondrial metabolic activity) and sulforhodamine B assays (cellular protein content). In addition, HPLF and SAOS-2 grown for 21 days on the investigated barriers were evaluated by SEM. Data were analyzed statistically by ANOVA using the Wilcoxon-Mann- Whitney test (P<0.05). No changes were established in the periodontal ligament fibroblasts and human osteoblast-like cells after incubation with the collagen membrane. Cytotoxic effects, however, were induced by the polylactic acid barrier which slightly inhibited cell metabolism of the periodontal fibroblasts (XTT: 90.1%±3.6 of control value). Moderate cytotoxic reactions were caused by the nonresorbable ePTFE membrane in HPLF-cultures (XTT: 82.7%±3.5) and osteoblast-like cell monolayers (XTT: 80.0%±0.6%). Mitochondrial activity in both cell cultures was significantly reduced by ePTFE barriers in comparison to nonincubated control cells (P = 0.028). SEM analysis of cell behavior on barriers demonstrated the differences between these materials: collagen barriers were densely populated with HPLF and SAOS-2, whereas only few or no cells were seen to adhere to the ePTFE and polylactic acid membranes. Our findings indicate that the collagen barrier investigated is very cytocompatible and may be integrated into connective tissue well. On the contrary, the ePTFE and polylactic acid membranes induced slight to moderate cytotoxic reactions which may reduce cellular adhesion. Thus, gap formation between the barrier surface and the connective tissue may be promoted which may facilitate epithelial downgrowth and microbial accumulation. Consequently, these effects may reduce the potential gain in periodontal attachment.

Effects of HEMA and TEDGMA on the in vitro odontogenic differentiation potential of human pulp stem/progenitor cells derived from deciduous teeth

OBJECTIVES The aim of this study was to investigate the effects of HEMA and TEGDMA on the odontogenic differentiation potential of dental pulp stem/progenitor cells. METHODS Dental stem/progenitor cell cultures were established from pulp biopsies of human deciduous teeth of 1-3 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures were characterized for stem cell markers, including STRO-1, CD146, CD34, CD45 using flow cytometry. Cytotoxicity was evaluated with the MTT assay. DTSCs were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4),β-glycerophosphate and L-ascorbic acid phosphate in the presence of nontoxic concentrations of HEMA (0.05-0.5mM) and TEGDMA (0.05-0.25mM) for 3 weeks. Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2mM) and TEGDMA (1mM) were also evaluated. RESULTS DTSCs cultures were positive for STRO-1 (7.53±2.5%), CD146 (91.79±5.41%), CD34 (11.87±3.02%) and negative for CD45. In the absence of monomers cell migration, differentiation and production of mineralized dentin-like structures could be observed. Cells also progressively expressed differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA significantly delayed the differentiation and mineralization processes of DTSCs, whereas, one time exposure to higher concentrations of these monomers almost completed inhibited mineral nodule formation. BSP, OCN, ALP and DSPP expression were also significantly down-regulated. SIGNIFICANCE These findings suggest that HEMA and TEGDMA can severely disturb the odontogenic differentiation potential of pulp stem/progenitor cells, which might have significant consequences for pulp tissue homeostasis and repair.

Cytotoxicity of four root canal sealers in permanent 3T3 cells and primary human periodontal ligament fibroblast cultures

OBJECTIVES The purposes of this study were to determine the cytocompatibility of four endodontic sealers and gutta-percha using various extracts and to compare the cellular injuries resulting from these materials in permanent 3T3 monolayers and primary human periodontal ligament fibroblasts. STUDY DESIGN Set specimens from four sealers (AH26, Apexit, Sealapex, N2) as well as gutta-percha were eluted with cell culture medium for 24 hours, 5 days, 5 days, and 24 hours, respectively. Cytotoxic effects due to these extracts were evaluated by determining proliferation of permanent 3T3 cells and primary human periodontal ligament fibroblasts by means of the fluorochrome propidiumiodide. RESULTS No statistically significant cellular alterations were induced by gutta-percha and Apexit, whereas various extracts of AH26 and Sealapex caused significant moderate or severe growth inhibition. Severe cellular damage was documented for all extracts of N2, which was statistically significantly different from the other materials in both cell lines (p < 0.05). CONCLUSIONS Our results confirm that various root canal sealers constantly segregate substances after being exposed to an aqueous environment for extended periods, possibly causing moderate or severe cytotoxic reactions and possibly contributing to failure.