| Objective:The aim of this research is observing the Enterococcus faecalis adhesion to the dentin treated with 17%EDTA irrigation,uncovering the effect of 17%EDTA irrigation on the adhesion of E.faecalis,so as to provide the experimental reference for the rational use of EDTA irrigation in clinic.Methods:The standard strain(ATCC19433)of E.faecalis was identified by morphological analysis as the pure strain.Then the OD value of E.faecalis in different growth periods was measured and its standard curve was plotted.The bacteria in the logarithmic growth period were used in the experiment.38 mandibular premolars extracted due to orthodontic treatment were cut away the crown,splited into two halves after root canal preparation..60 samples with intact morphology were selected,and 12 of them were cut into 5mm x 5mm x 1mm dentin slices.48 half split samples and 12 dentin slices were randomly divided into 3 experimental groups and 1 control group.Each group consisted of 12 half split samples and 3 dentin slices.Group A:soaked 1 minute with 17%EDTA;Group B:soaked 3 minutes with 17%EDTA;Group C:soaked 5 minutes with 17%EDTA;Control group:soaked 5 minutes with 0.9%normal saline.1 sample was randomly selected from each group to observe the dentinal tubules by SEM.The E.faecalis was inoculated into 24 well plates with teeth samples and incubated for 7 days.The morphology and adhesion of E.faecalis biofilm on dentin surface were observed by SEM.The thickness of E.faecalis biofilm on dentin surface was measured by CLSM.And the depth of E.faecalis intruded in dentine tubules was measured by histological gram staining and CLSM.And the CFU counts were used to calculate the colony number of E.faecalis.Results:1.The standard strain(ATCC19433)of E..faecalis was identified as a pure strain.The standard curve showed that the logarithmic growth period of E.faecalis was during the 4-12 hours.2.The opening situation of dentinal tubules was observed by SEM.In experimental groups,the dentinal tubules opened in varying degrees after soaked with 17%EDTA.In 1 minute group,the least number of opened dentinal tubules were observed.In 5 minutes group,the most number of opened dentinal tubules were observed,almost all dentinal tubules were clearly exposed.Yet the dentinal tubules of the control group were not opened.3.The situation of E.faecalis adhesion on dentin surface was observed by SEM.E.faecalis attached to the dentin surface of each group.The E.faecalis that adhered to the 1 minute group and control group were less attached,bacteria scattered in the distribution without intruding into dentine tubules.In 3 minutes group.The samples in 5 minutes group showed a large number of bacterial adhesion,formed a multilevel biofilm and colonized in the dentinal tubule.4.After histological gram staining,there were different degrees of blue dye particles in each group under the optical microscope.The invasive depth of bacteria in each group was compared with each other.The invasive depths of bacteria in 3 minutes group and 5 minutes group were deeper than control group,and the differences were statistically significant(P<0.05).The depth difference between 1 minute group and control group was not statistically significant(P>0.05).Among the experimental groups,the invasive depth of bacteria in 5 minutes group was deeper than 1 minute group and 3 minutes group,and the differences were statistically significant(P<0.05).The depth difference between 1 minute group and 3 minutes group was not statistically significant(P>0.05).5.The thickness of E.faecalis biofilm on dentin surface was measured by CLSM.The fluorescence staining showed that the red and green fluorescence of bacterial biofilm on the sample surface was mixed with each other,the bacteria showed dispersion distribution.The bacterial biofilm thickness in 1 minute group,3 minutes group and 5 minutes group were greater than control group,and the differences were statistically significant(P<0.05).Among the experimental groups,the bacterial biofilm thickness in 1 minute group,3 minutes group and 5 minutes group were gradually increased,and the differences were statistically significant(P<0.05).6.The depth of E.faecalis intruded in dentine tubules was measured by CLSM.The invasive depths of bacteria in 1 minute group,3 minutes group and 5 minutes group were greater than control group,and the differences were statistically significant(P<0.05).Among the experimental groups,the invasive depths of bacteria in 1 minute group,3 minutes group and 5 minutes group were gradually increased,and the differences were statistically significant(P<0.05).7.The colonies on the BHI plates were count by CFU analyse.The colony numbers of E.faecalis in 1 minute group,3 minutes group and 5 minutes group were greater than control group,and the differences were statistically significant(P<0.05).Among the experimental groups,the colony numbers in 1 minute group,3 minutes group and 5 minutes group were gradually increased,and the differences were statistically significant(P<0.05).Conclusion:17%EDTA root canal irrigation can promote the E.faecalis adhesing to dentin surface and intruding in dentine tubules.The adhesion and the invasive depth of E.faecalis to dentin would increase with the prolongation of EDTA treat time which was increasing from 1 minute to 5 minutes. |
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