Cytotoxicity and apoptosis induction by e-cigarette fluids in human gingival fibroblasts

Objectives

Electronic cigarettes (e-cigarettes) are generally acknowledged as a safer alternative to the use of combusted tobacco products. Nevertheless, there are increasing conflicting claims concerning the effect of these novel industrial products on the health of e-cigarettes users. The aim of this work was to investigate the effects of the liquids of e-cigarettes on human gingival fibroblasts (HGFs) and to compare the effects of nicotine-containing fluid to the fluid itself.

Materials and methods

HGFs were treated with different concentrations (0–5 mg/mL) of fluids of e-cigarettes for different times (0–72 h) and cytotoxicity was analyzed by MTT assay. Fluids were administered also after being vaped (e.g., warmed into the cartomizer). Apoptosis occurrence and Bax expression were evaluated by flow cytometry; ROS production was analyzed by fluorescence optical microscopy.

Results

Both nicotine-containing and nicotine-free fluids induced an increased ROS production after 24 h, along with an increased Bax expression, followed by apoptosis occurrence after 48 h of exposure.

Conclusions

The cytotoxicity exerted on HGFs by e-cigarettes fluids is not entirely ascribable to nicotine. Since the e-cigarettes are advertised as a safer alternative to traditional ones, especially for the possibility of “smoking” nicotine-free fluids, further studies are necessary to clarify the mechanism involved in the occurrence of cytotoxicity exerted by such compounds.

Clinical relevance

Our results suggest a role for e-cigarette fluids in the pathogenesis of oral diseases, such as periodontitis.

     

Cytotoxicity of resin monomers on human gingival fibroblasts and HaCaT keratinocytes.

OBJECTIVES: The aim of this study was to evaluate and compare the biological effects of three resin monomers on three human gingival fibroblast (HGF) cell lines and immortalised human keratinocytes. METHODS: Primary HGFs and HaCaT keratinocytes were cultured for 24h and grown to sub-confluent monolayers. Resin monomers were dissolved in dimethyl sulphoxide (DMSO) and diluted with culture medium. Cultures were exposed to different concentrations of monomers (10(-2) to 10mM) for 24h. Cell viability measured by Alamar Blue assay, and cell culture supernatant was examined for the presence of human interlukin-1beta (IL-1beta) using sandwich enzyme-linked immunosorbant assay (ELISA). TC50 values were calculated from fitted dose-response curves. RESULTS: All monomers showed toxic effects on the HGFs and HaCaT cells and inhibited chemical reduction of Alamar Blue in high concentrations. Statistical analysis of TC50 values by one-way ANOVA followed by Tukey's analysis showed that there is a significant difference in TC50 values between the cell lines (p<0.05), although the rank order of monomer toxicity remained the same for different cell lines. None of these monomers-induced IL-1beta release from HGFs and HaCaT cells. SIGNIFICANCE: Dental resin monomers are toxic to human gingival fibroblasts and HaCaT keratinocytes. However, they cannot induce IL-1beta release from these cells by themselves. Alamar Blue assay is a sensitive method for the evaluation of cytotoxicity and it can detect different sensitivities of different cell lines to the resin monomers.     

Cytotoxicity of Porphyromonas gingivalis toward cultured human gingival fibroblasts

Direct Cytotoxicity of black-pigmented anaerobic rods was studied on the confluent monolayer of human gingival fibroblasts in vitro. Only strains of Porphyromonas gingivalis caused morphological alteration (cell-rounding) and notable depression of viability of fibroblasts. To determine the location of the Cytotoxicity, bacterial surface components, i.e., outer membrane, lipopolysaccharide, fimbriae and outer membrane vesicles were prepared from P. gingivalis and their cytotoxicity was assessed. Among these preparations, only outer membrane vesicles are supposed to have high affinity to human gingival fibroblasts, and the cytotoxicity of outer membrane vesicles was found to be much stronger than that of the other constituents. This cytotoxic factor seemed to consist largely of protein and to be associated with the enzyme activity of outer membrane vesicles. The effects of some protease inhibitors and L-cysteine on the cytotoxicity of outer membrane vesicles suggest that the mechanism of cell-rounding is different from that of cell death.     

Cytotoxicity and arecoline mechanisms in human gingival fibroblasts in vitro

Betel nut chewing, like cigarette smoking, is a popular oral habit which impinges on the daily lives of a population of approximately 200 million. People who chew betel nuts have a higher prevalence of periodontal diseases than those who do not. Many of the undesirable effects of betel nuts have been attributed to arecoline, a major component of the particular alkaloid in betel nuts. In this in vitro study, we have focused on the effects of arecoline and the role it could play in periodontal breakdown via its direct effects on human gingival fibroblasts. Human gingival fibroblasts were derived from three healthy individuals undergoing crown-lengthening procedures. We found that arecoline is cytotoxic to human gingival fibroblasts at a concentration higher than 50 μg/ml by depleting intracellular thiols and inhibiting mitochondrial activity (P<0.05). In addition, the cells displayed a marked arrest at G₂/M phase in a dose-dependent manner. Repeated and long-term exposure to arecoline could impair the gingival fibroblast functions. As they are cytotoxic, the use of betel nut products in conjunction with periodontal therapy may interfere with optimal healing and/or lead to further periodontal breakdown. Received: 16 February 2000 / Accepted: 25 July 2000     

Cytotoxicity of a silorane-based dental composite on human gingival fibroblasts

AIM: To evaluate the direct and indirect biocompatibility of Filtek Silorane on human gingival fibroblastic cells. METHODS: Sixty-three standardized cylindrical specimens (8 mm diameter and 2 mm thickness) of restorative material were prepared using a light emitting diode-curing unit. The sample were built up in one increment and divided in 2 groups. In the first group, 21 samples (unpolished samples) were left without a specific polishing procedure; in the second one, 42 samples (polished samples) were polished with 4 different grains of discs. Fibroblast cultures, obtained from gingiva of 2 subjects without systemic and oral disease, were used to assess the direct and indirect biocompatibility. Cells cultured for 48 h in normal culture medium were used as a control. RESULTS: The scanning electron microscope observations of fibroblasts cultured on the silorane samples, either polished or unpolished, confirmed the good biocompatibility of the material, favouring the cellular spreading. 3-dimethylthiazol-2, 5-diphenyltetrazolium bromide tests showed a significant reduction (P < 0.01) of gingival fibroblasts viability cultured both in polished samples (90.05% ± 19.00%) and unpolished samples (78.15% ± 11.00%) compared with the control. Cells growth in medium conditioned with the samples for 1 wk showed a significant viability reduction (P < 0.01) compared to the control. A reduction of cell viability was observed even in the groups containing the material for 3 wk (polished: 89.45% ± 10.00%; unpolished: 65.97% ± 10.00%), even if the cytotoxicity was reduced after this long time exposure. CONCLUSION: Although the poor chromatic availability of this material remains a big limit that restricts its use to posterior sectors, the silorane-based material can be considered an option to perform restorations when aesthetic demands are not the priority, such as the class II restorations     

Cytotoxicity of the bacterium Actinobacillus actinomycetemcomitans extracts in human gingival fibroblasts

Filter-sterilized sonic extracts (SE) of strains of Actinobacillus actinomycetemcomitans were shown to inhibit the proliferation of human gingival fibroblasts in vitro. The inhibition was dose-dependent: a 50 per cent inhibitory dose of 2 micrograms protein/ml was found for A. actinomycetemcomitans strain Y4. The inhibitory activity could be neutralized by homologous antiserum and was heat inactivated by temperatures of 80 degrees C or greater. The fibroblast-inhibitory activity was present in SEs of both leukotoxic-producing and non-leukotoxic strains of A. actinomycetemcomitans, suggesting that a separate agent is responsible for leukotoxicity and fibroblast inhibition. A short (10 min) exposure of the fibroblasts to the A. actinomycetemcomitans SE was sufficient to inhibit irreversibly cell proliferation, provided that serum was present at the time that the cells were exposed to the SE. SE-challenged fibroblasts exhibited a marked decrease in the rate of DNA synthesis, but no inhibition of RNA or protein synthesis. Although the SE-treated cells did not proliferate, they appeared to remain intact and viable; and displayed no gross morphological alterations.     

Cytotoxicity and DNA double-strand breaks in human gingival fibroblasts exposed to eluates of dental composites

Objective

Previously, single composite components were used to study cytotoxicity and induction of DNA double-strand breaks (DNA-DSBs) of dental composite resins. In the present study, cytotoxicity and induction of DNA-DSBs in human gingival fibroblasts (HGFs) were investigated with dental composite eluates consisting of multiple components. The eluates were qualified and quantified.

Cytotoxicity of a new root canal filling material on human gingival fibroblasts

This study evaluated the cytotoxicity of the root canal sealing materials Resilon and Epiphany versus gutta-percha, Grossman's sealer, Thermaseal, and Sealapex. Using human gingival fibroblasts the fibroblasts cultures were incubated for either 1 or 24 h to test the cytotoxicity after freshly mixing or after 24 h of setting. Fibroblasts were then stained with trypan blue, to determine number of dead cells. Data were analyzed using ANOVA and t tests. Resilon was similar to gutta-percha and the control. Epiphany was less cytotoxic than Grossman's sealer at both the 1 and 24 h time periods. Epiphany was more cytotoxic than Sealapex at the 1-h time period but less cytotoxic at the 24 h time period. These results indicated that Resilon had a lower cytotoxicity and that Epiphany was more cytotoxic than conventional materials.     

Efficient gene transfer to periodontal ligament cells and human gingival fibroblasts by adeno-associated virus vectors

Objectives

We explored for the first time the possibility to deliver a reporter gene (Green Fluorescence Protein) to human primary periodontal ligament (PDL) cells and human gingival fibroblasts (HGF) using shuttle vectors derived from adeno-associated virus (AAV). Since AAV transduction rates on other human primary fibroblasts have been previously shown to depend on the particular cell lineage and on the employed viral serotype, we determined the most effective AAV variant for periodontal cells comparing different vector types.

Methods

AAV serotypes 1–5 encoding GFP in single stranded (ss) and self-complementary (sc) vector genome conformations were used to infect primary HGF and PDL cells. Two days post-infection, the percentage of GFP expressing cells was determined by flow cytometry.

Results

Highest transduction rates for both cell types were achieved with self-complementary vectors derived from AAV-2, resulting in GFP expression in up to 86% of PDL cells and 50% of HGF. Transgene expression could be observed by optical microscopy for 2 months after infection. Lower but detectable rates were obtained with serotypes 1, 3 and 5.

Conclusions

The efficacy demonstrated here and the safety and versatility of AAV technology indicated in previous studies clearly suggest the potential of AAV vectors as tools for gene transfer to periodontal tissues.     

Response of human gingival fibroblasts to prostaglandins

The effects of various prostaglandins (PGs) on the functions of human gingival fibroblasts (Gin-1 cells; ATCC CRL 1292) were examined by phase-contrast microscopy, cell-counting and radioautographic experiments. Tested PGs were PGA₁, PGA₂, PGB₁, PGB₂, PGD₂, PGE₁, PGE₂, PGF₁α, PGF₂α, PGI₂, 6-keto-PGF₁α, 9α-11α-methanoepoxy-PGF₂α, and thromboxane (TX) B₂. PGA₁, and PGD₂ at 30 μM caused morphological deformation of Gin-1 cells. All the PGs tested at 30 μM suppressed the proliferation of Gin-1 cells in the logarithmic growth phase. Furthermore, all the PGs tested at 10 μM suppressed DNA synthesis, collagen synthesis, and noncollagenous protein synthesis in confluent Gin-1 cells, while exerting no effect on GAG synthesis. The concentrations of PGs used are beyond those found in healthy gingiva. However, in periodontitis the local concentrations of some PGs within the gingiva are expected to be extremely elevated beyond the physiological level. These results suggest that PGs may play an important role as a negative regulator in metabolism and some pathologic gingival conditions by suppressing the functions of gingival fibroblasts.     

Phagocytosis of collagen by human gingival fibroblasts in vitro.

Human gingival fibroblasts cultivated on collagen-coated cover slips had collagen fibrils in deep folds of the cell membranes after one hour and fully interiorized fibrils by 24 hours. In 5-day sections, fibrils were within dense bodies, some containing multiple dense granules (40 to 50 nm) aligned along the surface of the interiorized collagen fibrils.     

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

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 6 h. 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.     

Clarithromycin transport by gingival fibroblasts and epithelial cells

Macrolide antibiotics penetrate cells, but the mechanism by which this occurs is unclear. The objective of this study was to characterize the mechanisms of clarithromycin uptake by gingival fibroblasts and oral epithelium. Cultured human gingival fibroblasts and SCC-25 cells were incubated with [(3)H]-clarithromycin. We assayed clarithromycin transport by measuring cell-associated radioactivity over time. Fibroblasts and epithelial cells rapidly accumulated clarithromycin, attaining steady-state intracellular concentrations within 15 minutes. Incubation in medium containing 2 mug/mL clarithromycin yielded steady-state intracellular concentrations of 75.8 mug/mL in fibroblasts and 6.6 mug/mL in SCC-25 cells. Clarithromycin transport exhibited Michaelis-Menten kinetics and was inhibited below 37 degrees C. The Michaelis constants for fibro-blasts and SCC-25 cells were 78.4 and 227 mug/mL, respectively, while the maximum transport velocities were 264 and 381 ng/min/10(6) cells, respectively. Thus, both types of cells take up clarithromycin via a concentrative active transport system. By increasing intracellular clarithromycin levels, this system may enhance the effectiveness of clarithromycin against invasive periodontal pathogens.     

人牙周膜细胞和牙龈成纤维细胞生物学活性的研究

目的:体外原代培养人牙周膜细胞和牙龈成纤维细胞,并对其生物学活性作初步探讨.方法:采用组织块法原代培养牙周膜细胞和牙龈成纤维细胞,绘制生长曲线,测定二者碱性磷酸酶活性;流式细胞术和免疫组化染色法测定Ⅰ、Ⅲ型胶原、骨形成蛋白的表达情况,观察对比两种细胞的生物学特性的异同.结果:牙龈成纤维细胞原代培养成功率及细胞增殖活性明显高于牙周膜细胞.在牙周膜细胞中,Ⅰ、Ⅲ型胶原均为阳性表达,棕黄色颗粒克满整个胞浆内.在牙龈成纤维细胞中Ⅰ型胶原为弱阳性表达,Ⅲ型胶原阳性表达更弱.牙周膜细胞的ALP水平明显高于牙龈成纤维细胞.牙周膜细胞BMP2表达为强阳性,而牙龈成纤维细胞表达弱阳性.结论:牙周膜细胞具有较强的成骨能力,是理想的牙周组织工程的种子细胞.牙龈成纤维细胞易于培养成活,增殖力强,具有牙周膜细胞的一些特点,组织取材方便,也可作为牙周组织工程的种子细胞.     

Mitogenic activity of cementum components to gingival fibroblasts

Cementum forms the interface between root dentin and periodontal ligament through which periodontal connective tissue is attached to root surfaces. We have examined how cementum components influence the biological activities of gingival fibroblasts. Cementum was harvested from freshly extracted human teeth and extracted sequentially with 0.5 mol/L acetic acid, 4 mol/L guanidine-0.5 mol/L EDTA, and bacterial collagenase. The extracts were concentrated and analyzed for mitogenic activity to human gingival fibroblasts. DNA synthesis was assayed by measurement of [3H]thymidine incorporation by quiescent fibroblasts activated to divide, and cell growth was determined by the counting of cells over a 10-day period. Results showed that extracts of cementum stimulated quiescent gingival fibroblasts to synthesize DNA and grow. The stimulation was dose-dependent, and most of the stimulatory activity was extracted by acid. Addition of small quantities of serum potentiated the mitogenic activity to levels greater than those of control cultures containing 10% fetal calf serum. The mitogenic activity was heat-stable, but it was destroyed by trypsin. Neither platelet-derived growth factor (PDGF) nor epidermal growth factor (EGF) was detectable in the cementum extract, and extracts of human dentin and skin contained very little mitogenic activity. We conclude that cementum contains substances capable of regulating the growth of gingival fibroblasts, and that these substances may play an important role in gingival connective tissue formation and regeneration.     

Scleraxis在牙周韧带细胞、牙龈成纤维细胞中的表达

目的研究碱性螺旋-环-螺旋转录因子 Scleraxis 能否在人牙周韧带细胞、牙龈成纤维细胞、骨髓基质细胞中表达,及其与牙周韧带细胞分化能力的关系。方法体外培养人牙周韧带细胞、牙龈成纤维细胞和骨髓基质细胞,RT-PCR 法检测牙龈成纤维细胞、骨髓基质细胞及不同代次牙周韧带细胞中 Scleraxis 的表达。结果 Scleraxis 在牙周韧带细胞、骨髓基质细胞及牙龈成纤维细胞中的表达差异有统计学意义(P<0.05)。其 Scleraxis 与β-肌动蛋白吸光度比值分别为0.877±0.024,0.438±0.031,0.313±0.083。Scleraxis 在牙周韧带细胞中的表达最强,在牙龈成纤维细胞中表达最弱。牙周韧带细胞中 Scleraxis 的表达随培养代次的增加而减弱。结论 Scleraxis 可表达于体外培养的牙周韧带细胞、牙龈成纤维细胞和骨髓基质细胞,Scleraxis 可能在牙周韧带细胞的分化过程中起重要作用。     

Collagen degradation by human gingival fibroblasts. I. In vivo phagocytosis

An electron microscopic survey and stereological analysis were made of the lamina propria of human gingival tissues from 8 patients with periodontitis and 6 patients clinically free of pathology (normal). The connective tissue was classified and divided into Zones A, B, and C, according to their collagen content, fibroblastic status (active or inactive), and numbers of inflammatory cells. Fibroblastic phagocytosis of collagen was most prominent in relatively stable areas (Zone A) where the collagen content was high, fibroblasts were classified as inactive (resting), and there were no inflammatory cells. Active (synthesizing) fibroblasts were conspicuous in Zone B, where intercollagenous spaces were wide and mononuclear cells occurred. Phagocytosis of collagen in the human gingiva was found to be a minor function of fibroblasts in Zone B and the highly inflamed Zone C.     

Resin composite monomers alter MTT and LDH activity of human gingival fibroblasts in vitro.

OBJECTIVES: Substances such as monomers may be released from composite resin systems and may induce adverse effects in biological tissues. The aim of this study is to investigate the cytotoxic concentrations of resin composite monomers on cultures of human gingival fibroblasts. METHODS: A range of dilutions of five resin composite monomers (HEMA, HPMA, DMAEMA, TEGDMA, and Bis-GMA) were added to the culture medium of human gingival fibroblasts for 24 h. Their cytotoxic effects were measured by using two colorimetric functional assays, mitochondrial dehydrogenase activity (MTT) and lactate dehydrogenase activity (LDH) assay. The logP values (water/octanol partition) of test monomers were also calculated computationally. RESULTS: Mitochondrial reducing activity assessed with the MTT test was inhibited by all monomers and all the monomers increased the LDH release in a reproducible dose dependent manner. A wide range of TC 50 values (concentrations altering MTT and LDH activity by 50%) (0.32-5.8 mM by MTT assay and 0.36-6.7 mM by LDH assay) was observed. Ranking of composite resin monomer cytotoxicities (TC 50) were similar for both the MTT and LDH assays, (Bis-GMA>TEGDMA>DMAEMA>HPMA >HEMA). However, the MTT assay was found to be more sensitive than the LDH assay, particularly when lower doses of the tested monomers were determined. The ranking of TC 50 concentrations correlated with the calculated logP values. SIGNIFICANCE: Monomers used in dental restorative materials show a variety of toxic effects on gingival fibroblasts. A combination approach using MTT and LDH assays provides valuable information about their toxic effects.     

Strong cytotoxicity to human gingival fibroblasts by Porphyromonas gingivalis ATCC 33277

The aim of the present study was to analyze the cytotoxicity of some bacterial species associated with periodontal diseases. The specificity of cytotoxicity was estimated against cells of various origin and from different individuals. The reference bacteria were Actinobacillus actinomycetemcoinitans, Porphyromonas gingivalis and Fusobacterium nucleatum . These bacteria were cultured for 24 h in liquid media and the supernatants were used in cytotoxicity assays. The target cells used were human gingival fibroblasts (GF). dermal fibroblasts (K4), gingival epithelial cells (E) and HeLa-cells (HeLa). These cells were exposed at 4 h or 24 h, respectively, to various concentrations of culture supernatants from the selected bacteria. The influence on the viability and metabolism of the cells were estimated quantitatively as increase in neutral red uptake and lactic acid production. Growth medium supernatants of P. gingivalis 33277 were strongly cytotoxic to gingival fibroblasts after 24 h incubation, compared to supernatants of P. gingivalis 381 or W 50, A. actinomycetemcoinitans or F. nucleatum cultures. The toxic effect of P. gingivalis 33277 decreased drastically after heat inactivation, which indicates effects of proteins. By adding anti-sera the cytotoxicity of P. gingivalis 33277 could be dose dependently neutralized, which was not the case when supernatants of A. actinomycetemcoinitans was tested. Target cells of epithelial origin did not show increased cytotoxicity to P. gingivalis 33277 . The results of the present study strengthen the hypothesis that P. gingivalis remains as a suspect causative key component in periodontal diseases.