Experimental Study Of Hyperoxia Solution On The Inhibition Of Common Pathogenic Bacteria In Root Canal

Root canal therapy(RCT)is the most perfect method of clinical treatment of pulpitis and periapical periodontitis.The main bacteria in the infected root canal are anaerobic bacteria,especially obligate anaerobes,including Bacteroides,fusitis,genuine bacteria,Propionibacterium,Streptococcus and radiation bacteria,all of which are closely related to the occurrence and development of periapical diseases.The effective control of the bacteria in the infected root canal determines whether the root canal therapy is successful,and the root canal washing procedure is critical.At present,0.5% sodium hypochlorite fluid,1% to 3% hydrogen peroxide solution and physiological saline are used to carry out root canal irrigation.But there are many disadvantages,such as irritation and tissue toxicity,smelling nose,easy to cause subcutaneous emphysema and so on.Therefore,in clinical work,it is very important to find a highly efficient,safe and low cost irrigant for improving the success rate of oral infection.In our previous work,the group used the technology of fast gas displacement and dissolved oxygen,invented the hyperoxia medical liquid therapeutic instrument,and prepared the hyperoxia solution with oxygen partial pressure of 106.6 + 7.8kPa,and theoxygen concentration reached 5.3 times that of the conventional liquid.According to the characteristics of the anaerobes mainly in the root canal,we use the high oxygen liquid as the washing agent in the process of root canal preparation to explore its bacteriostasis effect.According to the mechanism of oxygen toxicity,anaerobic microorganisms and gaseous oxygen is not kill,but because cannot remove some oxygen metabolites toxicity and death,superoxide dismutase(SOD)as a free radical scavenger,and the function of SOD is to protect the aerobic bacteria from the superoxide anion free radical toxicity,so we detected bacterial SOD gene expression,to explore mechanism of high oxygen liquid antibacterial effect.In addition,Caspase3 is known as the executor of apoptosis,and its complex structure,function and regulation determine the fate of the cell.We used Caspase3 as an indicator of bacterial apoptosis.We observed the bacterial apoptosis by detecting the expression of Caspase3,and further clarified the mechanism of bacteriostasis in hyperoxia solution.In the end,we apply the hyperoxia solution to clinical root canal irrigation to investigate whether it can inhibit the pathogenic bacteria in the infected root canal.It is an attempt to provide a new method for clinical prevention of oral infection by simple operation,low cost and non-toxic side effects.The first part of the experiment: The effects of hyperoxia solution on common pathogenic bacteria in root canals.Purpose: With three kinds of anaerobic bacteria infection in root canals in common(Streptococcus mutans,Actinomyces viscosus and Prevotella intermedia)as the research object,through bacteriological experiments,to explore the hyperoxia liquid on the infected root canal of three common pathogenic bacteria antibacterial effect,and high oxygen liquid and conventional washing liquid antibacterial difference effect.Methods:The high oxygen solution was prepared and the Streptococcus mutans,sticky actinomycetes and prusia intermediate were recovered,and the bacteria were cultured to the exponential growth period.The bacteriostasis experiment of hyperoxia solution was carried out to count the number of surviving colony on the medium,and the diameter of bacteriostasis was measured by vernier caliper.Bacteriostasis effect of comparative hyperoxia solution on three common root canal infection bacteria.SPSS19.0 software was used to analyze the data.The Student 's t-test method was used totest the diameter of the Bacteriostasis of the common bacteria in the same root canal.Single factor variance analysis(ANOVA)and Tukey HSD test were used to compare the diameter of bacteriostasis of bacteria in different common root canals.P < 0.05 had statistical difference,and the experiment was repeated three times.Results: This experiment successfully prepared the standard hyperoxia solution and successfully cultured Streptococcus mutans,mucilage actinomycetes and prilacella intermediate.Three kinds of bacteria were treated with hyperoxia,0.5% sodium hypochlorite solution and0.9% Sodium Chloride Solution respectively.The number of surviving colonies on the medium was counted.The results showed that: compared with 0.9% Sodium Chloride Solution group(1197.2±153.4),the colony counts of Streptococcus mutans in the hyperoxia solution group(818.7±121.4)and 0.5% sodium hypochlorite solution group(403.1±96.2)were significantly reduced(P < 0.05).Compared with 0.9% Sodium Chloride Solution group(914.9±132.1),the number of Actinomyces in the hyperoxia solution group(451.2±125.3)and 0.5% sodium hypochlorite solution group(301.4±92.2)decreased significantly(P < 0.05).Compared with 0.9% Sodium Chloride Solution group(710.8±131.2),the colony number of P.intermedia in high oxygen treatment group(422.8±105.0)and 0.5% sodium hypochlorite solution group(126.5±90.6)decreased significantly(P < 0.05).After three kinds of bacteria were treated with hyperoxia,0.5%sodium hypochlorite solution and 0.9% Sodium Chloride Solution respectively,vernier caliper was used to measure the diameter of bacteriostasis ring.The experimental results show that: Compared with the 0.9% Sodium Chloride Solution group(0.00mm),the diameter of the Streptococcus mutans in the hyperoxia solution group(5.02 + 0.12mm)and 0.5% sodium hypochlorite solution group(6.52 + 0.24M)increased significantly(P <0.05).Compared with the 0.9% Sodium Chloride Solution group(0.00mm),the number of Actinomyces in the hyperoxia solution group(3.14 + 0.26mm)and 0.5% sodium hypochlorite solution group(4.12 + 0.34mm)increased significantly(P < 0.05).Compared with 0.9% Sodium Chloride Solution group(0.00mm),the colony number of P.intermedia in high oxygen liquid treatment group(7.02 + 0.24mm)and 0.5% sodium hypochlorite solution group(7.92 + 0.36mm)decreased significantly(P < 0.05).Conclusion:hyperoxia solution has bacteriostatic effect on three common pathogenic bacteria in root canal,and the inhibition effect of hyperoxia solution to P.intermedia is higher than that of Streptococcus mutans and mucus actinomycetes.The second part of the experiment: The study of the mechanism of hyperoxia solution on three pathogenic bacteria in root canals.Purpose : After applying the hyperoxia solution,the expression of the SOD and Caspase-3 genes of three bacteria was detected,and the mechanism of the Bacteriostasis of hyperoxia solution was determined.Methods: three bacteria,including Streptococcus mutans,actinomyces,and P.intermedia,were resuscitation in the corresponding plates.Streptococcus mutans incubated 24 h,48h was incubated with Actinomyces actinomycetes and P.intermedia,and colony count was counted under light microscope.The total RNA from Streptococcus mutans,Streptococcus actinomycetes and B.pneumophila was extracted from hyperoxia solution and 0.9%Sodium Chloride Solution.The total RNA of bacteria was extracted and the total bacterial cDNA was transcribed to cDNA to achieve real-time quantitative PCR reaction.SPSS19.0software was used to analyze the data.Results:Compared with the 0.9% Sodium Chloride Solution group(1097.5±144.2),the number of Streptococcus mutans in the hyperoxic solution group(752.8±127.2)increased significantly(P < 0.05).Compared with the 0.9%Sodium Chloride Solution group(826.3±107.6),the number of colonies of actinomycetes in the hyperoxic solution group(425.4±108.9)decreased significantly(P < 0.05).Compared with the 0.9% Sodium Chloride Solution group(692.4±117.1),the number of colonies in the high oxygen solution group(415.4±113.2)decreased significantly(P <0.05).Real time quantitative PCR results showed that compared with Staphylococcus aureus(aerobic bacteria),the SOD gene expression of Streptococcus mutans,P.intermedia and mucus actinomycetes was low(P < 0.05).Compared with the 0.9% Sodium Chloride Solution group,the expression of Caspase3 gene in Streptococcus mutans,mucus Actinomyces and P.intermedia in the hyperoxia solution group was significantly higher than that in the Sodium Chloride Solution group(P < 0.05).Conclusion: The Bacteriostasis of hyperoxia solution may be achieved by increasing the apoptosis of bacteria through the toxic effect of oxygen.The third part of the experiment: The inhibitory effects of hyperoxia solution on common pathogenic bacteria in infected root canals.Purpose : To further explore whether hyperoxia solution can be used in clinical root canal irrigation and can inhibit the pathogenic bacteria in the infected root canal.Methods: 84 patients with chronic periapical periodontitis were selected,including 100 teeth and periodontitis without dental periodontitis.There was no systemic disease in the patient and no antibiotic treatment was accepted in the last 1 months.Among them,there were 39 men,43 women,and 17~69years old.According to the patient's order,the affected teeth were randomly divided into three groups: hyperoxia group,0.9% sodium chloride group and sodium hypochlorite group.Each washing with 5mL root canal irrigation,#30 disinfection fidibus 1/3 maintain1 min remove insert tip.The anaerobic bacteria were transferred into the laboratory in the constant temperature anaerobic incubator during 30 min culture,then transferred to blood agar Petri dishes by scribing method,and 48 h was cultured to observe colony formation.The SPSS 19 software was used to analyze the bacteriostasis effect of different irrigant on common bacteria in root canal with variance analysis and Tukey HSD test.The experiment was repeated 3 times.SPSS19.0 software was used to analyze the data.Results: colony forming experiment results showed that the number of bacterial colony in high oxygen liquid group was higher than that in sodium hypochlorite group(P<0.05),but it was significantly lower than that in 0.9% NaCl group(P<0.05).Conclusion: the application of hyperoxia solution to root canal irrigation may increase the apoptosis of bacteria in root canal by oxygen toxicity,thus inhibiting the growth of bacteria in the root canal.Although the antibacterial effect of hyperoxia solution is lower than that of sodium hypochlorite solution,because of its high safety advantages,it also has high practical value in clinical practice.