Chinese Hamster Lung Fibroblast Cells Research Articles

50-Hz magnetic field induces EGF-receptor clustering and activates RAS

Purpose: In a previous study, we found that exposure to a 50 Hz magnetic field (MF) could activate stress-activated protein kinase (SAPK) and P38 mitogen-activated protein (MAP) kinase (P38 MAPK) in Chinese hamster lung (CHL) fibroblast cells, and simultaneous exposure to a ‘noise’ MF of the same intensity inhibited these effects. In order to explore the possible target sites and upstream signal transduction molecules of SAPK and P38 MAPK, and further validate the interference effects of ‘noise’ MF on 50 Hz MF, the effects of MF exposure on clustering of epidermal growth factor (EGF) receptors and Ras protein activation were investigated.Materials and methods: CHL cells were exposed to a 50 Hz sinusoidal MF at 0.4 mT for different durations, and clustering of EGF receptors on cellular membrane and Ras protein activation were analyzed using immunofluorescence confocal microscopy and co-precipitation technology. EGF treatment served as the positive control.Results: The results showed that, compared with sham-exposed cells, exposure to a 50 Hz MF at 0.4 mT for 5 min slightly induced EGF receptor clustering, whereas exposure for 15 min enhanced receptor clustering significantly. Corresponding to receptor clustering, Ras protein was also activated after exposure to the 50 Hz MF. Exposure to a ‘noise’ MF (with frequency ranges from 30 – 90 Hz) at the same intensity and durations, did not significantly affect EGF receptor clustering and Ras protein. However, by superimposing the ‘noise’ MF, receptor clustering and Ras activation induced by 50 Hz MF were inhibited.Conclusion: The results suggested that membrane receptors could be one of the most important targets where extremely low frequency (ELF) MF interacts with cells, and Ras may participate in the signal transduction process of 50 Hz MF. Furthermore, a ‘noise’ MF could inhibit these effects caused by ELF-MF.

Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.

Evaluation of the Mutagenic and Genotoxic Potential of Ubiquinol

Ubiquinol (the reduced form of coenzyme Q(10)) is the two-electron reduction product of ubiquinone (the oxidized form of coenzyme Q(10)), and has been shown to be an integral part of living cells, where it functions as an antioxidant in both mitochondria and lipid membranes. To provide information to enable a Generally Regarded as Safe (GRAS) evaluation for the use of ubiquinol in selected foods, a series of Organisation of Economic Cooperation and Development (OECD) and good laboratory practice (GLP) toxicological studies was conducted to evaluate the mutagenic and genotoxic potential of Kaneka QH brand of ubiquinol. Ubiquinol did not induce reverse mutations in Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA at concentrations up to 5000 mu g/plate, in either the absence and presence of exogenous metabolic activation by rat liver S9. Likewise, ubiquinol did not induce chromosome aberrations in Chinese hamster lung fibroblast (CHL/IU) cells in short-term (6-h) tests with or without rat liver S9 at concentrations up to 5000 mu g/ml or in a continuous (24-h) treatment test at concentrations up to 1201 mu g/ml. Finally, no mortalities, no abnormal clinical signs, and no significant increase in chromosome damage were observed in an in vivo micronucleus test when administered orally at doses up to 2000 mg/kg/day. Thus, ubiquinol was evaluated as negative in the bacterial reverse mutation, chromosomal aberration, and rat bone marrow micronucleus tests under the conditions of these assays.

Protective effect of irisolidone, a metabolite of kakkalide, against hydrogen peroxide induced cell damage via antioxidant effect

The protective properties of irisolidone (a metabolite of kakkalide by intestinal bacteria) against hydrogen peroxide (H 2O 2) induced cell damage were investigated. Irisolidone was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and the intracellular reactive oxygen species (ROS), thereby preventing lipid peroxidation and DNA damage. Irisolidone inhibited apoptosis in Chinese hamster lung fibroblast (V79-4) cells induced by H 2O 2 via radical scavenging activity. This was achieved by the activation of the extracellular signal regulated kinase (ERK) and DNA binding activity of activator protein-1 (AP-1) (a downstream transcription factor of ERK) by irisolidone.

Ficus racemosa Stem Bark Extract: A Potent Antioxidant and a Probable Natural Radioprotector

Ethanol extract (FRE) and water extract (FRW) of Ficus racemosa (family: Moraceae) were subjected to free radical scavenging both by steady state and time resolved methods such as nanosecond pulse radiolysis and stopped-flow spectrophotometric analyses. FRE exhibited significantly higher steady state antioxidant activity than FRW. FRE exhibited concentration dependent DPPH, ABTS•−, hydroxyl radical and superoxide radical scavenging and inhibition of lipid peroxidation with IC50 comparable with tested standard compounds. In vitro radioprotective potential of FRE was studied using micronucleus assay in irradiated Chinese hamster lung fibroblast cells (V79). Pretreatment with different doses of FRE 1h prior to 2 Gy γ-radiation resulted in a significant (P < 0.001) decrease in the percentage of micronucleated binuclear V79 cells. Maximum radioprotection was observed at 20 μg/ml of FRE. The radioprotection was found to be significant (P < 0.01) when cells were treated with optimum dose of FRE (20 μg/ml) 1 h prior to 0.5, 1, 2, 3 and 4 Gy γ-irradiation compared to the respective radiation controls. The cytokinesis-block proliferative index indicated that FRE does not alter radiation induced cell cycle delay. Based on all these results we conclude that the ethanol extract of F. racemosa acts as a potent antioxidant and a probable radioprotector.

Measurement of genotoxicity in wastewater samples with the in vitro micronucleus test—Results of a round-robin study in the context of standardisation according to ISO

In the course of standardisation of the in vitro micronucleus test for analysis of effluents according to ISO, a national round-robin study was organised by the German Federal Institute of Hydrology ( BfG), involving 10 laboratories of private companies, universities and public authorities. The micronucleus assay was performed with the permanently growing Chinese hamster lung fibroblast cell line V79. All participants tested four encoded samples from one municipal and one industrial wastewater treatment plant with and without metabolic activation by S9-mix. Two of these samples were spiked in advance with defined concentrations of the clastogenic substances cyclophosphamide and mitomycin C, respectively. Cyclophosphamide and ethyl methanesulfonate were used as positive controls. The defined assessment criterion for genotoxicity was the lowest dilution of a sample that does not show any significant induction of micronuclei. Cytotoxicity was judged by determining the cell-survival index, i.e. the percentage growth rate of the cells compared with the corresponding negative controls. As supplementary qualitative criteria, the mitotic index and the proliferation index were assessed. All participants successfully established the method within a few weeks and generated viable test results in time. The two non-genotoxic samples were detected as negative by 90% (with S9-mix) and 95% (without S9-mix) of the participants. The mitomycin C-spiked wastewater sample (expected to be positive without S9-mix supplementation) was correctly judged as positive by all laboratories. The cyclophosphamide-spiked sample (expected to be positive with S9-mix addition) was evaluated correctly as genotoxic by 80% of the laboratories. A post-test analysis found evidence that the false negative results were due to technical failure, but not of a methodological nature. In 94% of all tests the sample LID values (lowest ineffective dilution = dilution stage of the sample in the test at which a statistically significant increase in the micronucleus rate was not detectable any more) varied by no more than one dilution step around the median LID value. The survival index was proven to be a robust measure for estimation of toxicity. This round-robin study is the first inter-laboratory comparison of the in vitro micronucleus test using wastewater samples. The test system is intended to complement the already DIN- and ISO-standardised bacterial tests, i.e. the umu-test and the Ames plate-incorporation assay. The data provide evidence that the robust and practicable in vitro micronucleus test is suitable as a routine method for wastewater testing.

Relative antioxidant and cytoprotective activities of common herbs

Many studies have been carried out on bioactivities of individual herbs, however, no collective study on their comparative antioxidant and cytoprotective activities against oxidative damage has been reported. We selected 17 common commercial herbs and studied their relative phenolic contents, antioxidant activities, and cytoprotective activities on gap–junction intercellular communication and antioxidative enzymes in vitro under the same conditions. Total polyphenol content ranged from 464 to 870 gallic acid equivalents (GAE) mg/100 g and total flavonoid content from 212 to 494 catechin equivalents (CE) mg/100 g. Among the samples, chamomile, rosehip, hawthorn, lemon verbena, and green tea contained relatively high total phenolics (769–844 mg GAE/100 g) and flavonoids (400–4 mg CE/100 g). Chamomile also showed the highest antioxidant activity with 960 mg/100 g of vitamin C equivalent (VCE), followed by hawthorn (929 mg VCE/100 g) and black tea (916 mg VCE/100 g). Total phenolic and total flavonoids showed a higher correlation with antioxidant activity. Most of herbs enhanced cell viability and showed protective effects against oxidative stress induced by hydrogen peroxide in Chinese hamster lung fibroblast (V79-4) cells. Furthermore, herbs used in this study showed higher protective effect on gap–junction intercellular communication (GJIC) as compared to gallic acid and catechin, and also enhanced activity of the antioxidative enzymes such as superoxide dismutase (SOD) and catalase (CAT) in a dose-dependent manner.

Rhapontigenin from Rheum undulatum Protects Against Oxidative-Stress-Induced Cell Damage Through Antioxidant Activity

The antioxidant properties of rhapontigenin and rhaponticin isolated from Rheum undulatum were investigated. Rhapontigenin was found to scavenge intracellular reactive oxygen species (ROS), the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and hydrogen peroxide (H2O2). The radical scavenging effect of rhapontigenin was more effective than rhaponticin. Rhapontigenin protected against H2O2-induced membrane lipid peroxidation and cellular DNA damage, which are the main targets of oxidative stress-induced cellular damage. The radical scavenging activity of rhapontigenin protected Chinese hamster lung fibroblast (V79-4) cells exposed to H2O2 by inhibiting apoptosis. Rhapontigenin inhibited cell damage induced by serum starvation and was also found to increase the activity of catalase and its protein expression. Further, rhapontigenin increased phosphorylation of extracellular signal-regulated kinase (ERK) and inhibited the activity of activator protein 1 (AP-1), a redox-sensitive transcription factor. In summary, these results suggest that rhapontigenin protects V79-4 cells against oxidative damage by enhancing the cellular antioxidant activity and modulating cellular signal pathways.

Geonotoxicity study of illegal drug MDMA and its nitroso derivative N-MDMA by micronucleus and chromosomal aberration tests using Chinese hamsger lung fibroblast cell line.

An increase in incidence of the illegal use of tablets containing 3,4-methylenedioxymethamphetamine hydrochloride (MDMA) has recently become a widespread social problem. MDMA ingested orally reacts with nitrite in the stomach and is synthesized intoN-nitroso-3,4-methylenedioxymethamphetamine (N-MDMA). The aim of this study is to investigate the genotoxic effects of MDMA and N-MDMA on the basis of the results of an in vitro micronucleus (MN) test and an in vitro chromosomal aberration (CA) test using a Chinese hamster lung fibroblast cell line (CHL/IU). Tablets containing MDMA obtained from the Regional Bureau of the Ministry of Health, Labor and Welfare were purified, and N-MDMA was synthesized from MDMA in our laboratory. To evaluate the effects of MDMA and N-MDMA, the MN test established by our laboratory and the CA test in accordance with the guidelines for toxicity studies of drugs recommended by the Ministry of Health, Labor and Welfare were performed. In the MN test, no increased frequency of MNs was not found for MDMA. On the other hand, an apparently increased frequency of MNs was observed for N-MDMA. In the CA test, no CA was found for MDMA, but CA was observed for N-MDMA apparently. N-MDMA genotoxicity was observed in the MN and CA tests. However, no MDMA genotoxicity was observed.

1P1-G03 IPMCの生体適合性(ソフトロボティクス)

Ionic polymer-metal composite (IPMC) is promising material in biomedical applications, since IPMC is soft and flexible, leading to the safety of the device itself. The purpose of this study is to investigate the biocompatibility of IPMC by in vitro experiment, in order to evaluate the applicability to biomedical field. Embryo cell of cynops pyrrhogaster could be cultured around IPMC films. Colony-forming test using Chinese hamster lung fibroblast cell shows high cytotoxicity of IPMC. Further tests are necessary to investigate the cause of cytotoxicity of IPMC. IPMC actuators immersed in medium for 7 to 13 weeks could show bending response driven by a rectangular wave and a sin wave.

Investigation of the specific α1‐adrenergic receptor pathway link to phosphorylation of sodium‐hydrogen exchanger by RhoA kinase

Previous work in our lab has shown that phenylephrine stimulation of chinese hamster lung fibroblast cells has resulted in activation of the well-characterized growth factor pathway. However, current evidence points to a second RhoA dependent pathway involved in NHE1 activation. Stimulation of RhoA kinase (ROCK) has been demonstrated to result in phosphorylation of the carboxy-terminus of NHE but neither the direct phosphorylation nor the phosphorylation site of NHE1 have yet to be shown. We have sought to characterize the specific links between the α1-adrenergic pathway and the EGF pathway. Our goal is to generate and purify an affinity, histadine-tagged C-term NHE1 (CT-NHE1) peptide and use this to identify the adrenergic mediated phosphorylation as well as the direct phosphorylation of the recombinant peptide. Three different strains of bacteria were used to screen the expression of CT-NHE1. This truncated protein product, purified to >80%, was then used as a target for phenylephrine-dependent phosphorylation. In vitro kinase studies are presented using recombinant ROCK (both active and dominant negative forms of the kinase) or lysates from cells treated with or without kinase inhibitors. These studies should help to identify novel phosphorylation sites for ROCK and indicate that α1-adrenergic receptors mediate a non-growth factor pathway activation of NHE1. This work is funded by NIH 1 R15 HL074924-01A1.

Genotoxicity Study of Illegal Drug MDMA and Its Nitroso Derivative N-MDMA by Micronucleus and Chromosomal Aberration Tests using Chinese Hamster Lung Fibroblast Cell Line

Genotoxicity Study of Illegal Drug MDMA and Its Nitroso Derivative N-MDMA by Micronucleus and Chromosomal Aberration Tests using Chinese Hamster Lung Fibroblast Cell Line

Increased expression of genes in a radioresistant cell strain: modulation of hnRNP E2, Hsp90 and SSBP2 genes in γ-irradiated Chinese hamster V79 cells

The methotrexate-resistant cell strain M5, derived from Chinese hamster lung fibroblast cell line V79, provides a good model to study differential gene expression in response to stress. Genes coding for human homologues of myosin phosphatase (AF458589), filamin-like protein (AAA92644), phosphate transporter 1 (AAH199441) and hnRNP E2 (NP_035172.1) were upregulated in M5 as determined by RAP-PCR. The Gene Expression Array (GEArray) analysis showed increased expression of caspase-6 and Hsp90 in M5 cells compared to that observed in V79 cells. Genomic amplification of SSBP2 as well as its transcriptional upregulation was also detected in M5 cells. Following γ-irradiation, expressions of SSBP2, Hsp90 and hnRNP E2 increased steadily in V79 cells. However, expression of hnRNP K and hnRNP A2 genes decreased at first and then increased, while hnRNP C1 gene expression remained unaffected up to the time studied. Thus, SSBP2, Hsp90 and hnRNP E2 are involved in the radiation response of V79 cells.

Inhibitory effects of glycitein on hydrogen peroxide induced cell damage by scavenging reactive oxygen species and inhibiting c-Jun N-terminal kinase

The present study investigated the cytoprotective properties of glycitein, a metabolite formed by the transformation of glycitin by intestinal microflora, against oxidative stress. Glycitein was found to scavenge intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and thereby preventing lipid peroxidation and DNA damage. Glycitein inhibited apoptosis of Chinese hamster lung fibroblast (V79-4) cells exposed to hydrogen peroxide (H2O2) via radical scavenging activity. Glycitein abrogated the activation of c-Jun N-terminal kinase (JNK) induced by H2O2 treatment and inhibited DNA binding activity of activator protein-1 (AP-1), a downstream transcription factor of JNK. Taken together, these findings suggest that glycitein protected H2O2 induced cell death in V79-4 cells by inhibiting ROS generation and JNK activation.

GSM 1,800 MHz radiofrequency electromagnetic fields induced clustering of membrane surface receptors and interference by noise magnetic fields

To investigate the possible effect of exposure to GSM 1,800 MHz radiofrequency electromagnetic fields (RF EMF) on epidermal growth factor (EGF) receptor and its possible interference by noise magnetic fields (MF). Chinese hamster lung fibroblasts (CHL) were exposed to 1,800 MHz RF EMF (modulated by 217 Hz or 50 Hz, or unmodulated), 2 microT noise MF, and RF EMF combined with 2 microT noise MF for 15 min, respectively. The specific absorption rates (SARs) of RF EMF were 0.1, 0.5, 1.0, 2.0 and 4.0 W/kg. Commercial EGF (1 ng/ml) treatment was used as positive control. EGF receptors on the cell membrane were observed under a laser scanning confocal microscope after indirect immunofluorescence staining. EGF receptor clustering was induced after exposure to GSM 1,800 MHz RF EMF modulated by 217 Hz or 50 Hz MF at SARs of 0.5, 1.0, 2.0, 4.0 W/kg for 15 min as induced by 1 ng/ml EGF, but not at SAR of 0.1 W/kg. And no EGF receptor clustering was found in cells after exposure to unmodulated RF EMF or 2 microT noise MF. In addition, superposition of 2 microT noise MF could inhibit the EGF receptor clustering induced by GSM 1,800 MHz RF EMF. EGF receptor clustering in CHL cells can be induced by GSM 1,800 MHz RF EMF at the lowest SAR of 0.5 W/kg and inhibited by noise MF. The modulation of wave may play an important role in the inducement of receptor clustering after RF exposure.

Protective effect of Ecklonia cava enzymatic extracts on hydrogen peroxide-induced cell damage

In this study, Ecklonia cava was enzymatically hydrolyzed to prepare water-soluble extracts, using five carbohydrases (Viscozyme, Celluclast, AMG, Termamyl, and Ultaraflo) and five proteases (Protamex, Kojizyme, Neutase, Flavourzyme, and Alcalase), and the potential antioxidant activity of each was assessed. The Celluclast and Viscozyme extracts of E. cava evidenced good hydrogen peroxide (H 2O 2) scavenging activities (73.25% and 72.92%, respectively) as compared to those of other enzymatic extracts. Therefore, the Celluclast enzymatic extract was selected for use in further experiments, and separated into four different molecular weight fractions (<1, 1–10, 10–30 and >30 kDa). Among these fractions, the >30 kDa fraction manifested the most profound H 2O 2 scavenging activity, with a measured IC 50 of 13 μg/ml. The >30 kDa fraction also strongly enhanced cell viability against H 2O 2-induced oxidative damage, and evidenced relatively good lipid peroxidation inhibitory activity in a Chinese hamster lung fibroblast (V79-4) cell line. This fraction also effected a reduction in the proportion of cells undergoing H 2O 2-induced apoptosis, as was demonstrated by a decreased quantity of sub-G 1 hypodiploid cells and decreased apoptotic body formation on the flow cytometry assay. These results clearly indicate that the >30 kDa fraction of E. cava possesses good antioxidant activity against H 2O 2 mediated cell damage in vitro.

Genotoxic effects of N-nitrosoketamine and ketamine as assessed by in vitro micronucleus test in Chinese hamster lung fibroblast cell line

Ketamine hydrochloride (KT) is a secondary amine that has been safely used as an injectable anesthetic and analgesic to avoid the production of nitroso compounds in the stomach. However, ketamine in the tablet form has recently become an abused, recreational drug. The aim of this study was to investigate the genotoxic effects of N-nitrosoketamine (NKT) and KT on the basis of an in vitro micronucleus (MN) test using a Chinese hamster lung fibroblast cell line (CHL/IU). NKT was synthesized from KT in our laboratory. In the MN tests, CHL/IU cells were continuously treated with either NKT or KT for 24, 48, or 72 hours without the S9 mix. The cells were also treated with NKT or KT with or without the S9 mix for 6 hours, followed by a recovery period of 18, 42, or 66 hours (short-term treatment). The results were considered to be statistically significant when the p-values of both Fisher's exact test and the trend test were less than 0.05. After the short-term treatment with either NKT or KT with and without the S9 mix, the frequency of micronuclei significantly increased. However, the frequency of micronuclei did not significantly increase after the continuous treatment with either NKT or KT. Both NKT and KT were determined to be genotoxic in the short-term treatment with or without the S9 mix, but they were determined to be nongenotoxic in continuous treatment. Our findings suggest that NKT has a stronger genotoxic effect than KT.

Evaluation of cytotoxic effect of photodynamic therapy in combination with electroporation in vitro

Under the influence of electric pulses cells undergo membrane electroporation (EP), which results in increased permeability of the membrane to exogenous compounds. EP is applied in oncology as a method to enhance delivery of anticancer drugs. For that reason it was essential to combine photodynamic tumor therapy (PDT)—the cancer treatment method based on the use of photosensitizers that localize selectively in malignant tumors and become cytotoxic when exposed to light, and EP, with the aim to enhance the delivery of photosensitizers into the tumor and therefore to increase the efficacy of PDT. Thus, the aim of study was to evaluate the cytotoxic effect of PDT in combination with EP. A Chinese hamster lung fibroblast cell line (DC-3F) was used. The cells were affected by photosensitizers chlorin e 6 (C e 6) at the dose of 10 μg/ml and aluminium phthalocyanine tetrasulfonate (AlPcS4) at the dose of 50 μg/ml. Immediately after adding of photosensitizers the cells were electroporated with 8 electric pulses at 1200 V/cm intensity, 0.1 ms duration, 1 Hz frequency. Then, after 20 min of incubation the cells were irradiated using a light source—a visible light passing through a filter (KC 14, emitted light from 660 nm). The fluence rate at the level of the cells was 3 mW/m 2. Cytotoxic effect on cells viability was evaluated using MTT assay. Our in vitro data showed that the cytotoxicity of PDT in combination with EP increases fourfold on the average. Based on the results we suggest that EP could enhance the effect of PDT.

The Attachment of V79 and Human Periodontal Ligament Fibroblasts on Periodontally Involved Root Surfaces Following Treatment with EDTA, Citric Acid, or Tetracycline HCL: An SEM in vitro Study

The present in vitro study has been designed to establish and compare the effects of citric acid, EDTA, and tetracycline HCl on human periodontally diseased roots on the structure, attachment, and orientation of V79 (primary Chinese hamster lung fibroblasts) cells and human periodontal ligament fibroblasts (HPDL). Commercially available V79 cells and HPDL derived from healthy human third molars were used in this study. These fibroblasts were left in solution for seven days in order to attain confluence. Forty single-rooted teeth were obtained from patients diagnosed with periodontitis. The crown part was removed under constant irrigation and the root was split vertically into two equal halves, thus, yielding 80 specimens. Following scaling and root planing, the specimens were washed with phosphate buffered saline (PBS) and kept in 50 microg/ml gentamycin sulphate solution for 24 hours. The root pieces were then treated as follows: citric acid at pH 1, 24% EDTA, or with a 10% solution of tetracycline HCl and were then placed in V79 fibroblast cultures and HPDL cultures. The specimens were harvested after four weeks and were fixed in 2.5% glutaraldehyde in PBS before preparation for scanning electron microscopy (SEM). The behavior of V79 cells was similar to that of human periodontal ligament cells on root conditioned surfaces. V79 and HPDL showed a healthy morphology on root surfaces treated with citric acid and EDTA and a relatively unhealthy appearance on root surfaces treated with tetracycline HCl and distilled water (control group). The results suggest the use of citric acid and EDTA as root conditioning agents favorably affects the migration, attachment, and morphology of fibroblasts on human root surfaces, which may play a significant role in periodontal healing and regeneration.

The Attachment of V79 and Human Periodontal Ligament Fibroblasts on Periodontally Involved Root Surfaces Following Treatment with EDTA, Citric Acid, or Tetracycline HCL: An SEM in vitro Study

Abstract Objective The present in vitro study has been designed to establish and compare the effects of citric acid, EDTA, and tetracycline HCl on human periodontally diseased roots on the structure, attachment, and orientation of V79 (primary Chinese hamster lung fibroblasts) cells and human periodontal ligament fibroblasts (HPDL). Materials and Methods Commercially availableV79 cells and HPDL derived from healthy human third molars were used in this study. These fibroblasts were left in solution for seven days in order to attain confluence. Forty single-rooted teeth were obtained from patients diagnosed with periodontitis. The crown part was removed under constant irrigation and the root was split vertically into two equal halves, thus, yielding 80 specimens. Following scaling and root planing, the specimens were washed with phosphate buffered saline (PBS) and kept in 50 μg/ml gentamycin sulphate solution for 24 hours. The root pieces were then treated as follows: citric acid at pH 1, 24% EDTA, or with a 10% solution of tetracycline HCl and were then placed in V79 fibroblast cultures and HPDL cultures. The specimens were harvested after four weeks and were fixed in 2.5% glutaraldehyde in PBS before preparation for scanning electron microscopy (SEM). Results The behavior of V79 cells was similar to that of human periodontal ligament cells on root conditioned surfaces. V79 and HPDL showed a healthy morphology on root surfaces treated with citric acid and EDTA and a relatively unhealthy appearance on root surfaces treated with tetracycline HCl and distilled water (control group). Conclusion The results suggest the use of citric acid and EDTA as root conditioning agents favorably affects the migration, attachment, and morphology of fibroblasts on human root surfaces, which may play a significant role in periodontal healing and regeneration. Citation Chandra RV, Jagetia GC, Bhat KM. The Attachment of V79 and Human Periodontal Ligament Fibroblasts on Periodontally Involved Root Surfaces Following Treatment with EDTA, Citric Acid, or Tetracycline HCL: An SEM in vitro Study. J Contemp Dent Pract 2006 February;(7)1:044-059.