Experiment Of Different Drugs For The Local Treatment Of Periodontitis And Dry Socket In Rats

Objectives The study aimed to compare the effects of a self-made MetronidazoleCarboxymethyl Chitosan(C/CMCS) topical gel and other drugs for the treatment of two common alveolar diseases, i.e. periodontitis and dry socket. The involvement of inflammation and cell apoptosis aspects were the main points, and some histopathological and molecular biological techniques were adopted to analyze a number of specific cell factor in the pathological process of the two diseases, then we hope to provide a preliminary pharmacological theory for C/CMCS clinical applications.Methods Experimental Periodontitis: total 72 of 6-week-old male SD rats model were established for periodontitis animals by ligation wires under gingival margin, then these animals were divided into four different groups, i.e. control group(A group),metronidazole carboxymethyl chitosan gel group(group B), Palio group(group C),metronidazole gel group(group D), respectively. All the rats except for the control group were treated gingival partial coating different drugs in the periodontal pockets twice a day in the morning and evening. The rat’s gingival crevicular fluid were collected during the treatment in 1,3,5 weeks, and the PGE2 level in the collected rat gingival sulcus fluid were tested by ELISA assay. Rats were sacrificed to take the lesion tissue for paraffin sections after treatment of 1 week, 3 weeks, 5 weeks, respectively. Some of the prepared paraffin sections were stained by HE to observe the pathological morphology changes under a microscope and some sections were analyzed the change in alveolar partial osteoclasts by TRAP method. Dry socket experiment: total 90 of healthy male SD rats aged 6 weeks were established dry socket model through dental extraction wounds and blood clots evisceration and pollution. These animals were divided into model group(A),metronidazole carboxymethyl chitosan gel group(group B), Palio group(group C),aluminum chlorate solution group(group D), metronidazole gel group(E), and the aluminum chlorate solution was diluted to wash the local wound with a syringe in drops,the other three preparations are to be applied twice a day till to 3 weeks. Some animals were killed after 1 week, 2 weeks, 4 weeks for making maxillary lesion paraffin sections respectively. Some sections were chosen for HE staining to observe the pathological morphological changes; Some sections were stained by TUNEL method to calculate the apoptosis rate of the local gingival tissue of the wound sockets; another sections were performed for the IL-10 immunohistochemistry experiments to analysis of the expression of IL-10 in the gum tissue surrounding the tooth socket.Results Experiments models of periodontitis and dry socket model were created successfully and respectively. Changes of rat gingival crevicular fluid PGE2 expression correlated with the severity of periodontal inflammation, i.e. the PGE2 increased when rats suffered from severe inflammation but PGE2 decreased after the treatment with the local drugs which suppressed the inflammation.The most obvious decline of PGE2 after three drugs intervention was in metronidazole carboxymethyl chitosan gel group,and there are significant differences compared with metronidazole gel alone(P < 0.05).histological morphology observation of the HE staining on untreated model group sections,inflammatory cells were easy visible from the periodontal connective tissue to the root side diffused infiltration with junctional epithelial attachment site extends to the root side; deep periodontal pockets were clearly and tooth ligament fibers were disordered,and bone ridge height reduced significantly because of the resorption of the alveolar, while the alveolar bone crest shape was extremely irregular. After the treatment of metronidazole carboxymethyl chitosan gel for 3 weeks, periodontal ligaments repaired partially, bone resorption was inhibited as the repair had occurred, the depth of periodontal pocket became shallow. After the treatment of metronidazole carboxymethyl chitosan gel for 5 weeks, the phenomenon of bone reparation increased and the bone density was more pronounced, and the damaged periodontal collagen fibers were repaired, both the the fracture of collagen fibers and the severity of inflammation reduced significantly.TRAP staining showed that the osteoclast activities were high in rat periodontitis models and the damage increased in pace with the extended duration of the pathological process.Osteoclast activities decreased obviously after drug intervention, both metronidazole carboxymethyl chitosan gel and Palio were significantly reduced the number of osteoclasts among the three local medications and there were difference between these two drugss with the simple metronidazole Gel(P<0.05), and the differences are more significant(P<0.01)if the two compared with the untreated control group.The presence of apoptosis was observed in the dry socket disease process by TUNEL staining, and the apoptosis rate showed a significantly lower trend with statistical significance(P < 0.05) after treatment of four drugs, which the apoptosis index caused by metronidazole carboxymethyl chitosan gel decreased the most obvious among them.Immunohistochemistry results showed that there were no obvious statistically difference about the expression of IL-10 on the dry socket gum tissue in the absence of drug intervention at the beginning of the two weeks. IL-10-positive cell counts obviously increased after drugs intervention and IL-10-positive cell counts increase significantly after the local administration of metronidazole carboxymethylcellulose chitosan gel and Palio ointment in the first week and continued to the second week till the fourth week. There were statistically significant(P <0.05) compared these two drugs with the model group and simple metronidazole gel group.The IL-10 expression in gingival tissue cells was difference compared with the model group After the presence of the aluminum perchlorate treatment the first week, the sample was more obvious in the second week and the fourth week, but with no significant difference compared to metronidazole gel. On the other hand, IL-10 expression treated with metronidazole topical gel was not very different with the model group in the first week, but there was significant difference in the second week and the fourth week.Conclusions Tronidazole carboxymethyl chitosan gel reagent can relieve inflammation,reduce bone resorption and promote healing in periodontitis rat models; the above pharmacological effects may be involved in the inhibition of expression of PGE2 and reduce osteoclast activities due to the local drug treatment. The pharmacological effects of metronidazole carboxymethyl chitosan gel suppressed PGE2 expression and reduced activity of osteoclasts are the strongest among the three test drugs in the rat periodontitis model, the efficacy of the complex gel is nearly equivalent to Palio ointment but is better than the simple metronidazole gel. The dry socket shows the presence of an inflammatory process of apoptosis and drug intervention will produce anti-apoptotic effects; but different drug administration will bring different effects. Apoptotic index decreases the most obviously with the metronidazole carboxymethyl chitosan gel treatment in rat dry socket models among these drugs in this test.The local drug intervention will promote the expression of IL-10 in the tooth extraction wound surrounding gingival and bone tissue,and the effects of metronidazole carboxymethyl chitosan gels and ointments Palio are superior to simple metronidazole gel in dry socket inflammatory rats.