Viability Of Human Gingival Fibroblasts Research Articles

Effect of Mineral Trioxide Aggregate Cements on Transforming Growth Factor β1 and Bone Morphogenetic Protein Production by Human Fibroblasts In Vitro

The aim of this study was to evaluate and compare the effects of two commercial mineral trioxide aggregate (MTA) cements (ProRoot MTA and MTA Angelus) on transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP)-2 levels produced by cultured human gingival fibroblasts (HGFs). Human gingival tissues were obtained from individuals with healthy periodontium. HGFs were grown at 37 degrees C in humidified atmosphere of 5% CO(2) in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, penicillin, and streptomycin. After 24 and 72 hours of exposure to the MTA products, HGF viability was determined by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay. TGF-beta1 and BMP-2 levels in cell-free culture media were determined by enzyme-linked immunosorbent assay. Cell viability of the test groups was significantly lower than that of control at 24 and 72 hours (p < 0.05) but showed an increase at 72 hours (p < 0.05). Both test groups showed increased TGF beta-1 levels at 72 hours (p < 0.05), whereas the MTA Angelus group displayed higher TGF beta-1 levels than control and ProRoot MTA groups at 24 and 72 hours (p < 0.05). At 24 hours, BMP-2 levels of the ProRoot group were significantly higher than that of MTA Angelus (p < 0.05). Both test materials increased the BMP-2 levels within time (p < 0.05) and displayed similar levels at 72 hours (p > 0.05). These results suggest that both MTA products are capable of stimulating HGF to produce BMP-2, whereas the stimulatory effect for TGF beta-1 is material dependent.

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 2O 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 2O 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 24 h, treated with various concentrations of TEGDMA (0.5–5.0 mM) for 24 h and subsequently for 90 min with 0.2 mM H 2O 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 ≥1.0 mM. 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 2O 2, compared to experiments without H 2O 2. Significance We conclude from our findings that TEGDMA and H 2O 2 have additive adverse effects on GSH metabolism and cell viability.

Cytotoxic effects of dental desensitizers on human gingival fibroblasts

The purpose of this study was to evaluate the effects of three different desensitizers on the cell viability and morphology of human gingival fibroblasts (HGF). Human gingival tissues were obtained from individuals who have clinically, healthy periodontium. HGF were grown at 37 degrees C in humidified atmosphere of 5% CO2 in Dulbecco's modified eagle's medium, supplemented with glutamine, penicillin, streptomycin, and 10% fetal bovine serum. The cells were treated with different concentrations (0.1, 0.3, and 0.5 microL/mL) of desensitizers (Gluma Desensitizer, Seal&Protect, and MicroPrime). After 24- and 48-h exposure to the desensitizer solutions, the viable cells were examined using a hemocytometer. To monitor HGF viability, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used and cell morphology was also observed at 48 h. Following exposure to concentrations of 0.1 microL/mL of test materials for 24 h, cell survival rates for Gluma Desensitizer (106%) and Micro Prime (62%) were not significantly different from the control, while it was significant for Seal&Protect (50%). Growing cells were significantly inhibited by all tested materials for 48 h (p < 0.05) in survival rates of 51, 47, and 31%, respectively. On the basis of the MTT assay, the cytotoxic effect of MicroPrime was more prominent, especially at high concentrations, than does Gluma Desensitizer and Seal&Protect. After exposure to Seal&Protect and MicroPrime, HGF became retracted, rounded in appearance and had loss of normal organization, leading to enlargement of intercellular space when compared with Gluma Desensitizer. As a conclusion, taking the limitations of an in vitro experiment into consideration, the cytotoxic effects were varied, depending on the chemical composition and exposure periods of the tested desensitizers.

A Functional Virulence Complex Composed of Gingipains, Adhesins, and Lipopolysaccharide Shows High Affinity to Host Cells and Matrix Proteins and Escapes Recognition by Host Immune Systems

Arg-gingipain (Rgp) and Lys-gingipain (Kgp) are Porphyromonas gingivalis cysteine proteinases implicated as major virulence factors in pathologies of periodontitis. We purified a 660-kDa cell-associated gingipain complex existing as a homodimer of two catalytically active monomers which comprises their catalytic and adhesin domains. Electron microscopy revealed that the complex was composed of a globular particle with a 10-nm external diameter possessing one or two electron-dense hole-like structures. Two-dimensional gel electrophoresis and immunoblot analyses revealed the association of lipopolysaccharide (LPS) with the catalytic domains and a hemagglutinin domain, Hgp44, of Rgp and Kgp in the complex. The complex significantly degraded human type I collagen and elastin and strongly disrupted viability of human gingival fibroblasts and umbilical vein endotherial cells with an efficiency which was higher than that of the monomeric gingipains. The native complex produced only a small amount of nitrogen dioxide, tumor necrosis factor alpha, and interleukin-6 by macrophages, whereas the heat-denatured complex resulted in increased production. Inhibition of the proteolytic activities of the gingipain complex did not up-regulate the cytokine production, indicating that the functional domains in LPS are structurally masked by the complex proteins. These results indicate the importance of the complex in evasion of host defense mechanisms as well as in host tissue breakdown.

Cytotoxic effects of dental resin liquids on primary gingival fibroblasts and periodontal ligament cells in vitro

Cytotoxic effects of resin liquids of three in situ relining dental polymers, Alike, Kooliner, and Tokuso Rebase, and their major components, methyl methacrylate (MMA), isobutyl methacrylate (IBMA), and 1,6-hexanediol dimethacrylate (1,6-HDMA) were investigated. The concentrations of major monomers in these resin liquids were determined by high-performance liquid chromatography. Cellular viability of human gingival fibroblasts (GF) and periodontal ligament (PDL) cells were evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay. Moreover, patterns of cell death were analysed using annexin V/propidium iodide staining with flow cytometry. The results indicated that Alike liquid contained 91.3% MMA, Kooliner liquid contained 94.5% IBMA, and Tokuso Rebase liquid contained 65.8% 1,6-HDMA. All materials examined had cytotoxic effects on GF and PDL cells in dose-dependent manners. Tokuso Rebase liquid appeared to be the most cytotoxic among the various resin liquids examined. The effects of Kooliner and Tokuso Rebase liquids may have resulted from IBMA and 1,6-HDMA, respectively. Furthermore, the majority of treated cells died from necrosis; whereas a small portion of cells died from apoptosis. In conclusion, the results demonstrated that these liquid forms of dental polymers and their major monomers cause cytotoxic reactions. The direct relining procedure that cures these materials in situ should be used cautiously.

The volatile fraction of cigarette smoke induces alterations in the human gingival fibroblast cytoskeleton.

Several in vitro investigations have indicated that the particulate phase of cigarette smoke, such as nicotine, affects many cell types, including gingival fibroblasts. However, few studies have been performed on the effects of the volatile fraction on the cellular structures that are involved in cell functions, such as adhesion and proliferation. Since the survival and reproduction of gingival fibroblasts are fundamental in maintaining the integrity of the oral connective tissue, as well as in wound healing, the effects on the cytoskeleton of acrolein and acetaldehyde, which are the volatile fractions of cigarette smoke, were examined in vitro for human gingival fibroblasts (HGFs). HGF strains that were taken from healthy subjects with non-inflamed-gingiva were utilized in this investigation. The cells were incubated in the presence of different concentrations of acrolein and acetaldehyde. Cell adhesion and viability were evaluated after incubation for 3 h and 5 days, respectively. The influence on cytoskeletal structures (tubulin, actin and vimentin intermediate filaments) was investigated with the indirect immunofluorescence technique. The results show that both substances produced similar effects, which resulted in a dose-dependent inhibition of HGF adhesion and viability. Disturbance of the HGF cytoskeleton consisted of disruption of the microtubules, actin filaments and vimentin microfilaments, which was accompanied by alterations to cell shape. Our experimental findings suggest that the volatile fractions of cigarette smoke, such as acrolein and acetaldehyde, have a cytotoxic effect on HGFs, with the result that they lose their capacity for adhesion and proliferation. The consequences of this could be impairment of the maintenance, integrity and remodelling of the oral connective tissue. According to our morphological evidence, these findings show that cigarette smoke can lead to the development and progression of periodontal disease, and indicate the need for appropriate therapy.

Ultrastructural changes in human gingival fibroblasts in vitro after exposure to vapour phase smoke components

Tobacco and some of its volatile and non-volatile components have been found to affect many types of cells including gingival fibroblasts. Because normal gingival fibroblast functioning is fundamental to the maintenance of the oral connective tissue as well as to wound healing, we examined the effect of two vapour phase smoke components (acrolein and acetaldehyde) on proliferation and ultrastructure of human gingival fibroblasts (HGFs) in culture. A human gingival fibroblast strain derived from healthy individuals was used in this study. The cells were incubated in the presence of different concentrations of acrolein and acetaldehyde and cell proliferation and fine morphology were evaluated. The results show that acrolein and acetaldehyde produced dose dependent inhibition of HGF viability and alteration of cytoplasmic organelles. The main ultrastructural finding for the HGF cytoplasm was the presence of vacuoles and lysosomal structures which became prominent with increasing concentration of acrolein and acetaldehyde. Our results suggest that the ultrastructural alterations we observed in HGFs may be due to the uptake and storage of acrolein and acetaldehyde by the cells.

Effect of glycyrrhetinic acid on the viability of human gingival fibroblasts

Some therapeutic agents and medicines such as phenytoin(PHT), nifedipine and cyclosporine may lead to adverse effects causing pathologic changes in tissue, especially on the gingivae1-8). The common histological finding is a marked gingival hyperplasia, with proliferation of gingival fibroblasts and an increase of collagen fibers in connective tissue. PHT-induced human gingival overgrowth may be genetically programmed for increased synthesis of collagen9). PHT-induced gingival overgrowth was preceded by the inflammation of gingivae10-11). Previous studies reported that PHT, nifedipine and cyclosporine increased the cell activity of human gingival fibroblasts12-17). Some differentiation factors such as glycyrrhetinic acid(GA), retinoic acid, ursolic acid and oleanolic acid inhibited18-25). Previous studies reported that GA inhibited intercellular communication between human fibroblasts26-27). Glycyrrhizin(GL), a major component of licorice (Glycyrrhiza glabra L.), is ingested orally as a sweetener or as an ingredient in oriental medicine. GL has a steroid-like action and anti-viral activity (Pompei et al., 1979). Indeed, use of GL to treat chronic viral hepatitis has shown that it can reduce hepatocellular injury by immunopathologic mechanisms and hepatotoxins, possibly through its ability to inhibit phospholipase A2 activity preventing liver lysosome labilization29-30). GL is metabolized to GA, and then 3-epi-18-GA via 3-keto-18-GA by human intestinal flora31). GA shows pronounced antiinflammatory activity and anti-estrogenic action via alteration of cortisol metabolism32-35). GA inhibits the growth and stimulates melanogenesis in cultured melanoma cells36). There are many reports about the use of differentiation factors for the resolution of uncontrolled growth such as in tumors, but there are few report about its use in the dental field including gingival hyperplasia. The purpose of this study was to investigate the 대한치주과학회지 : Vol. 28, No. 3, 1998

Corrosion and cell culture evaluations of nickel-chromium dental casting alloys.

In this study, the corrosion and surface properties of four commercially available nickel-chromium dental casting alloys, were evaluated using electrochemical corrision testing and Auger electron microscopy. The corrosion tests were conducted under cell culture conditions of 5% CO 2 humidified atmosphere at 37 degrees C in minimum essential medium (MEM) balanced salt solution, 95% MEM-5% FBS (fetal bovine serum) cell culture media, and in 95% MEM-5% FBS media after cold solution sterilization of test samples. The results of the surface and corrision analyses were correlated to cytotoxicity and metal ion release from the alloys using agarose overlay and direct contact cell culture tests. The surface and electrochemical corrision analyses demonstrated that the non-beryllium containing alloys were more resistant to accelerated corrosion processes as compared to the beryllium-containing alloys. All alloys demonstrated decreased corrision rates in cell culture solutions after cold solution sterilization treatment. The corrision products released from the nickel-based alloys failed to alter the cellular morphology and viability of human gingival fibroblasts, however they did cause reductions in cellular proliferation. The potential for accelerated corrision and the exposure of local and systemic tissues to elevated levels of corrision products raises concerns over the biocompatibility of these alloys.

Treponema denticola induces actin rearrangement and detachment of human gingival fibroblasts.

Spirochetes are associated with destructive periodontal diseases, and one cultivatable oral species, Treponema denticola, binds to mammalian cells and perturbs metabolism. To evaluate the cytoskeletal responses and attachment functions of human gingival fibroblasts (HGF) exposed to T. denticola, monolayers of HGF were incubated with T. denticola strains ATCC 35405, e, and e' in serum-free medium. HGF retracted pseudopods, rounded up, and ultimately detached from the substratum. Scanning electron microscopy showed spirochetes in close contact with HGF surfaces; occasionally, bacteria were partially submerged between folds in the HGF membrane. Blebbing and numerous microvilli formed on the cell surface as the HGF retracted. By confocal microscopy, spirochetes were detected in contact with the HGF surface but were never found on the ventral surface of fibroblasts between the substratum and cell. Morphological alterations were associated with and preceded by actin assembly, as measured by microscopic fluorimetry: there was a 263% increase in actin fluorescence over controls within 30 min. Detachment of fibroblasts from the substratum was related to incubation time and was dependent on the concentration of T. denticola. Detachment was observed for all strains tested and was also dependent on the viability of T. denticola: UV light, heat, and metronidazole treatment markedly reduced the HGF detachment response. Detachment was also significantly reduced by the protease inhibitor phenylmethylsulfonyl fluoride. HGF viability was not significantly affected by coincubation with spirochetes, as measured by lactate dehydrogenase release. Thus, T. denticola induces rapid cytoskeletal remodelling followed by cell detachment, which might be stimulated by a bacterially associated protease but is not likely directly mediated by proteolytic degradation at the cell-substratum adhesive contact points.

Cellular effects of human leukocyte hydrolases I. Human gingival fibroblasts

Experiments were carried out to determine whether lysosomal enzymes had the ability to adversely affect the viability of human gingival fibroblasts. These studies were accomplished in vitro using lysosomal enzymes derived from human polymorphonuclear leukocytes. The results strongly indicated that under the conditions of these experiments, lysosomal enzymes were incapable of mediating cell destruction. In fact, the only measurable effect was to release the cells from their growth surface in a manner similar to other known proteases. Even this capability was not expressed in the presence of naturally occurring inhibitors which are widely distributed in many body fluids, such as serum and inflammatory exudates. These results suggest that PMN leukocyte granule enzymes may not contribute significantly as mediators of the cellular destruction observed in inflammatory diseases in general, and inflammatory periodontal disease in particular.