| Root canal therapy is often used to treat endodontic and periapical diseases. However, a variety of bacterial infections, the complication of root canal system and improper operation, make some teeth keep infected even if these teeth have been treated by root canal therapy repeatedly. That is a so-called refractory periapical disease. The main reason of forming refractory periapical diseases is the formation of apical multispecies biofilms.Apical multispecies biofilms are bacterial communities on the apical cementum, which are formed by various bacteria embedded in a matrix of proteins and polysaccharides. Gram-positive bacteria in anaerobic or facultative anaerobes are commonly seen in the apical multispecies biofilms, such as Enterococcus faecalis, Actinomyces israelii and Streptococcus sanguis and so on. Enterococcus faecalis is the most commonly detected bacteria in apical lesions, containing divrse virulence factors such as lipoteichoic acid(LTA), cytolysin and peptidoglycan, which can promote the formation of biofilms and have highly pathogenic. These features also make it difficult to be removed. Actinomyces israelii is often found in the cases of treatment failure, and is closely related to apical abscess. Streptococcus sanguis which can cause persistent infection of the root canal is often detected in the apical lesions too. The formation of biofilms can delay the diffusion of antibiotics and enhance the resistance to antimicrobial, making antibiotic drugs more difficult to kill bacteria. It also contributes to the aggregation of toxic products of bacteria and improving of bacterial virulence. So it is important to remove apical biofilm in the process of treating refractory periapical.Apicectomy is the most common method to treat refractory periapical. Apicectomy is a method of root canal surgery, which is used under the condition of root canal therapy can not cure the diseased teeth. It can remove infected tissue and the apical microorganism biofilm with the means of surgical, blocking inflammation sources, so as to eliminate the symptoms and save teeth. To achieve this goal, it is necessary to design a reasonable surgical incision. Scaling apical lesions thoroughly to ensure that all the bacteria, infected tissue and material are completely removed. 3mm of root-end is removed, and then root-end filling after prepares the apical. However, apicectomy must reduce the root length and make the support of teeth root decline, especially not suitable for patients with periodontitis and patients need to do prosthetic treatment. So other methods must be considered. We want to remove the biofilm in the apical and cure the diseased teeth without root-ending excision.Er: YAG laser has been used in area of dental treatment for its traits, including the small thermal injury, precision cutting, the capability of reduce bleeding and antibacterial etc. The study found that Er: YAG laser can kill most bacteria with Enterococcus faecalis included. Especially when it is combined with root canal irrigations, laser can produce air bubbles and fluid pressure pulse wave to develop bactericidal effect of laser and irrigations, and then remove bacteria in apical and dentinal tubules effectively. Meanwhile Er: YAG can also reduce the diffusion of bacterial toxins in cementum and inhibit biofilm regeneration. However, the effect of Er:YAG laser combined with root canal irrigants on apical multispecies biofilm is rare, no matter at home or at abroad. Therefore, we consider establishing multispecies biofilms in vitro, and dealing it with Er: YAG laser combined with some kinds of root canal irrigations. Observe the changes of bacteria morphology and the numbers of bacteria to explore the possibilities of Er: YAG laser used in apical biofilms.1、 Study the forming process of mutispecies biofilm on bovine cementum slicesChoose Enterococcus faecalis, Streptococcus sanguis and Actinomyces israelii as the study bacteria. Determine the sequences of bacteria inoculated on the cementum slices by drawing the bacterial-growth curve of these bacteria. Split bovine teeth roots and cut cementum down from root surface. After being disinfection, Actinomyces israelii were inoculated on the slices. 2 days later, other two kinds of bacteria were inoculated to be cultured together. During the period of co-culture, take one sample to observe by SEM every day to make sure the multispecies biofilms can form on bovine cementum slices. And understand the formation of biofilm and time required to a mature biofilm.2、 Observe the efficiency of Er: YAG laser combined with root canal irrigants to apical biofilms by scanning electron microscopyFinally multispecies biofilms established in vitro as the experimental model according to the result of experiment one. Divide samples into four experimental groups and two control groups randomly. Four experimental groups: A(5.25% Na Cl O / Er: YAG), B(5g / L Na Cl O / Er: YAG), C(20g / L CHX / Er: YAG) and D(SAEW / Er: YAG). Two control groups: E(normal saline) and F(5.25% Na Cl O). Group E was negative control group, and Group F was positive control group. Each group was divided into three subgroups by the function time. It was 30 s, 1min and 3min. After corresponding treatment, these samples were fixed with 4% paraformaldehyde overnight, then dehydrated with 30%, 50%, 70%, 80%, 90% ethanol solution for 15 min each and 100% twice. After drying, sputter coated with platinum, and observed by scanning electron microscopy. It was found that bacteria changed in all the groups no matter in morphological or in numbers except normal saline group, but the degree of change varies among different groups. It suggested Er: YAG laser combined with irrigation had effect on apical biofilm, but the effect depended on the kind of irrigations and experimental time.3、 A quantitative study of Er: YAG laser combined with root canal irrigants to apical biofilmsOn the basis of experiment two, this investigations were conducted on the number of bacteria changed in each group and analyzed the effect of bactericidal in each group quantitatively. Extracted the DNA of Enterococcus faecalis and performed PCR amplification, agarose gel electrophoresis, cut plastic, plastic recycling. Finally, we use the DNA fragment as a standard to perform real-time PCR and manufacture a standard curve. DNA of all the groups was extracted after treatment too. Performed real-time PCR and estimated the number of bacteria in each group according to the standard curve. The results showed 5.25% Na Cl O + Er: YAG had the best bactericidal effect, which showed the same antimicrobial activity as the positive group in 30 s and 3min and better availability in 1min. 0.5%Na Cl O + Er: YAG group and SAEW + Er: YAG group had slightly inferior bactericidal effects on 30 s comparing with 5.25% Na Cl O group, but they can be achieved with the 5.25% Na Cl O group level in 1min and 3min. CHX + Er: YAG group had the weakest bactericidal effect. It can’t achieve same effect with positive control group, but it still played a role in remove apical biofilms comparing with the negative control group. |
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