Four beagle dogs were anaesthetized by intraperitoneal injection with 3% pentobarbital sodium (30 mg/kg body weight, Aikonchem, Jiangsu, China) and the pulp chambers of the teeth studied were opened. The canals were exposed to the oral environment for 4 weeks to induce a chronic AP. Cone beam computed tomography (CBCT) was taken to observe the periapical lesion. One of the dogs was sacrificed, and its canals were randomly divided into two groups, including 10 exposed canals and 10 non-infected canals (control group), for hematoxylin and eosin (H&E) stains. Periradicular tissue responses were histologically classified into four categories, according to the amount of inflammatory cells and the degree of apical bone and root destruction (Table S1 ).
Four weeks later, the canals of another 3 dogs were instrumented using K-files and finished at #25 by the step-back technique, with irrigation by 1.0% sodium hypochlorite (Longly, Wuhan, China). Then, the root canals were dried with sterile paper points and filled with calcium hydroxide (Ivoclar Vivadent, Schaan, Liechtenstein), followed by access cavity sealing with FiltekTMZ350XT adhesive resin (3M, St. Paul, MN, USA). CBCT was taken to observe the periapical lesion for all 3 dogs, and one of the dogs was sacrificed for histopathological examination.
After 2 weeks of disinfection, the canals of the remaining two dogs were divided into the control group, E. faecalis group and multi-bacteria group. Then, the canals were prepared to #30 and irrigated with 1.0% sodium hypochlorite (Longly, Wuhan, China), followed by inoculation of bacterial suspension. For the E.faecalis group, the bacteria concentration was 106 colony-forming units [CFUs]/mL. For the multi-bacteria group, bacterial suspensions were mixed to obtain an inoculum containing E.faecalis (106 CFUs/mL), L. acidophilus (106 CFUs/mL), A. neisseriae (106 CFUs/mL) and S. gordonii (106 CFUs/mL). For the control group, the root canals were injected with sterile normal saline. The cavity was sealed with adhesive resin (3M, St. Paul, MN, USA). Two weeks later, CBCT was taken to observe the periapical lesion for 2 dogs, and all of them were sacrificed for histopathological examination.
Four weeks later, the canals of another 3 dogs were instrumented using K-files and finished at #25 by the step-back technique, with irrigation by 1.0% sodium hypochlorite (Longly, Wuhan, China). Then, the root canals were dried with sterile paper points and filled with calcium hydroxide (Ivoclar Vivadent, Schaan, Liechtenstein), followed by access cavity sealing with FiltekTMZ350XT adhesive resin (3M, St. Paul, MN, USA). CBCT was taken to observe the periapical lesion for all 3 dogs, and one of the dogs was sacrificed for histopathological examination.
After 2 weeks of disinfection, the canals of the remaining two dogs were divided into the control group, E. faecalis group and multi-bacteria group. Then, the canals were prepared to #30 and irrigated with 1.0% sodium hypochlorite (Longly, Wuhan, China), followed by inoculation of bacterial suspension. For the E.faecalis group, the bacteria concentration was 106 colony-forming units [CFUs]/mL. For the multi-bacteria group, bacterial suspensions were mixed to obtain an inoculum containing E.faecalis (106 CFUs/mL), L. acidophilus (106 CFUs/mL), A. neisseriae (106 CFUs/mL) and S. gordonii (106 CFUs/mL). For the control group, the root canals were injected with sterile normal saline. The cavity was sealed with adhesive resin (3M, St. Paul, MN, USA). Two weeks later, CBCT was taken to observe the periapical lesion for 2 dogs, and all of them were sacrificed for histopathological examination.
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