Cytotoxicity And Microbicidal Efficacy Of Ozonated Water In Root Canal Irrigation: An In Vitro Study

The root canal irrigation plays an important role on the disinfection andcleaning to the root canal system. The structural complexity of the root canalsystem, such as dentinal tubule,accessory canal,root tip bifurcation and so on,which makes it inaccessible to clean up thoroughly just by the pure instrumentalpreparation, so it is necessary to assist the root canal irrigating agents. Therefore,the root canal irrigation becomes the essential link during the root canalpreparation process. The ideal root canal irrigating agents should have: 1,Goodsterilization performance; 2,Proteolytic role which can dissolve organics insidethe root canal; 3,Dissolve inorganic tissues, remove the smear layer; 4,Goodbiocompatibility; 5,Easy to operate and obtain, low in price; 6,Nontoxic andharmless. At present time, the irrigating agents used in clinical varies a lot.Overall evaluation from microbicidal efficacy, cleaning function, biologicalcompatibility and so on, the irrigating agents have both the advantages and thedisadvantages. So, searching for a kind of new ideal irrigating agents becomes animportant task in the pulpitis and the periapical periodontitis clinical research.The ozonated water has stronger bacteriostasis and sterilization ability, lowtoxicity, hard to produce drug resistance, which makes it applied increasinglywidespread in Dentistry, but still at the basic research stage in the Root CanalTherapy as an irrigating agent. The ozone is unstable, we should usenew-produced ozonated water when in clinical practice. Until now, there is not akind of ozone therapeutic equipment to be able to produce the effectiveconcentration of ozonated water which is conforms completely to the clinicalinfectious root canal irrigation. In view of this question, this research chooses akind of small family type of ozone generator (Type JM-D3000B), using the iodine titration to determine the concentration of ozonated water produced byType JM-D3000B ozone generator, researching the variation rule of theconcentration with time, then select the effective concentration and measure theRIR(%) of the HGFCs, evaluate the cytotoxicity of ozonated water, byestablishing the E. faecalis infected model on extracted teeth in vitro andcalculate the CFUml-1 through the plate count test, evaluate the microbicidalefficacy of ozonated water, in order to provide the microbiological foundation forthe application of ozonated water as a root canal irrigating agent in clinical, andtheoretical basis for developing the equipment to be able to produce the effectiveconcentration of ozonated water which is conforms completely to the clinicalinfectious root canal irrigation in future.ResearcResearch Methods:Study 1:Concentration Measurement of Ozonated WaterIn our experiment condition (room temperature = water temperature =1℃±0.5℃), iodine titration is applied to measure the concentration of ozonatedwater. According to the formula C(O3) = 2400?M?V/V0 where, C(O3) is for theconcentration of ozonated water (mg/L); M is for the concentration of Na2S2O3standard solution (mol/L); V is for the dosage of Na2S2O3 standard solution intitration (ml); V0 is for the sampling volume of ozonated water (ml). Calculatethe concentration of ozonated water produced by Type JM-D3000B ozonegenerator within different time, then draw the time-concentration gradient curve.Researching the variation rule of the concentration with time, in order to selectthe effective concentration of ozonated water for the next experiment, andprovide the theoretical basis for developing the equipment to be able to producethe effective concentration of ozonated water which is conforms completely tothe clinical infectious root canal irrigation in future.Study 2:Cytotoxicity Test of Ozonated WaterThe 4th generation of the HGFCs in well grown condition are treated with 0.25 % trypsin then collected, adjusting the cell concentration at 1×104 /ml with10% fetal calf serum DMEM culture medium. Based on the time-concentrationgradient curve in the former experiment, we select the experimental ozoneconcentration (3.994 mg/L). Measure the 30-minute RIR of the ozonated wateron the HGFCs by the MTT colorimetric method. We gain the OD values at eachhole on the wavelength of 490 nm through enzyme-linked immunosorbent assaymeasuring instrument. Calculate the RIR of the HGFCs according to the formula,RIR(%)= (1- mean OD value of experimental group / mean OD value of controlgroup)×100 %, and then according to the reference, evaluates the cytotoxicity ofozonated water, in order to provide a reliable basis for ozonated water used inclinical irrigation.StudStudy 3:Microbicidal Efficacy Observation of Ozonated Water in vitroSelect thirty single-rooted human premolars, remove the crowns, unify theroots into a standard length of 10 mm. Eliminate the pulp, dredge the root canalswith the stainless steel K-Files, clean them for 2 minutes with 15% EDTAassistedultrasonic irrigation, then wash the root canals with distilled water for 1minute, dried, seal up the apical foramen with the Filtek P90. Afterstandardization, samples are sterilized through high temperature and highpressure. We use the UV spectrophotometer to adjust the monocloned bacteriaturbidity which the OD value equals 0.5 at 600 nm wavelength. Then pour 200μlinto each sample in the super clean bench. 37℃and anaerobic incubated for 21days to establish infection model. All specimens average are divided into threegroups stochastically, and then prepared by the step-down technique with sterileMtwo machine nickel-titanium instruments up to size 35. Once changing the filesize, do the irrigation. The irriganting volume is 3 ml, and application time is 1minute. During each irrigation, syringe needle is inserted as deep as possible. Thestandard for the syringe needle is no resistance, no impaction, reflux smoothly.The needles are moved around up and down from time to time. After the end ofeach irrigation, rinse immediately with 5ml sterile distilled water. Roots are then sampled using proper sterile paper points inserting into the root canals, beforeand after the treatment respectively on root canals, the paper tip reaches to theapical foramen and keeps in contact with the root canal wall for 1 minute. Usingplate count test the number of the CFU/ml at 24 hours and 7 days after theirrigation. Data are analyzed by Mann-Whitney tests.ExperimentaExperimental Results:Study 1:Type JM-D3000B ozone generator in our experimental conditions,after the 22-minute working time, the concentration of ozonated water reachesthe peak value, about 3.994 mg/L, then shows a downward trend.Study 2:It is showed from Table.3 and Fig.3, the RIR of 5.25 % NaOClsolution, 1.339 mg/L ozonated water, 3.994 mg/L ozonated water and 0.9 %sodium chloride, which acts respectively on the HGFCs after 30 minutes is37.57 %, 23.32 %, 19.21 % and 0.03 %. According to the reference, 5.25 %NaOCl solution is low inhibition to the HGFCs, the last three groups arenon-inhibitory action to the HGFCs. The concentration and the inhibitory degreeof ozonated water is positively correlated.Study 3:Before irrigation, there is no statistic differences of the CFUamong three groups (P > 0.05). After irrigation, the 24-hour killing ratio of thethree irrigating agents on E. faecalis in root canals are all 100 %. But after 7 days,a small amount of E. faecalis have been detected, no statistically significantdifference among the three groups (P > 0.05). It's suggested that the E. faecalis inroot canals can be killed instantaneously by the three irrigating agents, but cannot be removed off, only reduced in its quantity and inhibited during a period oftime; the concentration and the microbicidal efficacy of ozonated water arepositively correlated.Conclusions:1, The ozonated water shows instantaneous microbicidal efficacy on E.faecalis in root canals. 2, The inhibitory degree to the HGFCs and the microbicidal efficacy ofozonated water is positively correlated with its concentration.3, The ozonated water produced in our experimental conditions isnon-inhibition to the HGFCs.Besides, the ozonated water is hard to produce drug resistance, withoutsecondary pollution of its decomposition products, convenient to operate, low inprice and so on. It takes a certain significance in clinical as a root canal irrigatingagent.