| BackgroundApical periodontitis is the inflammatory disease caused by microbial infection that occurring in periapical tissue. Many factors can lead to periapical infection, among which infection is the most important, followed by physical stimulation, chemical stimulation and immune factors. Clinically, if patients suffer from periapical periodontitis, they need for root canal therapy. What’s more, immaturate young permanent teeth tend to need further treatments including Ca (OH) 2 apexification and MTA apical barrier technique, which now is more commonly used in clinical treatment. But the treatment time and effect of Ca (OH) 2 apexification were uncertain. Although MTA apical barrier technique can improve patient compliance and increase the healing potential of treatment, it is difficult to obtain complete root development. Short and weak root wall can increase the possibility of subsequent root fracture, and bring greater risk for clinical treatment. The treatment for sick young permanent teeth periapical expected to achieve results including curing the periapical periodontitis, promoting root development and the restoration of the normal function of the pulp tissue (both histological and sensuous). However, the current therapeutic scheme is difficult to achieve this goal, these treatments can only provide guarantee for curing periapical inflammation. A series of clinical studies and case reports show that if young permanent teeth with apical periodontitis undergo rigorous root canal disinfection, inducing blood into root canal and the tightly filling of the crown access, the root can continue to develop and reborn tissue will grow into root canal. X-ray showed that the root length increased, root wall thickened, apical foramen closed and periapical lesion disappeared. This kind of treatment method is called pulp revascularization.The occurrence and development of apical periodontitis and the process of pulp revascularization is a multi-factor interaction process. The mechanism is very complex and has not yet formed a mature theory. There are many assumed theory existing and the exact mechanism need further study. Now many studies emphasize on the pathogenesis of periapical periodontitis, especially the role of various cytokines and transcription factors, has become a hot topic over the past decade.Nuclear factor-erythroid 2-related factor-2 (also known as NFE2L2 or Nrf2) is a key nuclear transcription factor that can mediate transcription of antioxidative and phase-2 detoxifying enzymes gene through Keapl-Nrf2-ARE signaling pathway, which is considered to be the most important mechanism of cellular antioxidant pathways so far discovered. The pathogenesis of many diseases is related to oxidative stress, and Keapl-Nrf2-ARE signaling pathway is widely antioxidant mechanism that can defend a variety of cells and tissues from injury. It has been confirmed that Nrf2 plays a key role in cancer, Alzheimer’s disease, Parkinson’s disease, diabetes and other diseases.Recent studies have demonstrated that Keapl-Nrf2-ARE signaling pathway is also involved in impairing inflammation-associated pathogenesis.. In addition, Nrf2 also has pro-angiogenic function. Studies have shown that Nrf2 related to the formation and reparation of vessel, the protection, proliferation, differentiation, migration of endothelial cells and the capacity of angiogenesis. There were not much studies about Nrf2 and oral diseases. In recent years, scholars showed that Keapl-Nrf2-ARE pathway activation might inhibit H2O2-induced human dental pulp cell necrosis, P.gingivalis LPS-stimulated macrophages inflammation in rats. Development of apical periodontitis is inflammation-related pathological process. Pulp revascularization is an process that newborn tissue grow into root canal after inflammation control, and revascularization is necessary in the survival of newborn tissue. It suggests that Nrf2 may play a role in the control of apical periodontitis and the process of pulp revascularization.In summary, Nrf2 is a transcription factor that plays an important role in cell protection, inflammation control and angiogenesis. But because of the limitations on clinical practice, it is difficult to detect the dynamic expression of Nrf2 in apical periodontitis and pulp revascularization procedure of the human body. In this study, we chosed mice as experimental subjects to measure the dynamic expression of Nrf2 in apical periodontitis and pulp revascularization procedure to provide theoretical basis for studing the mechanism of apical periodontitis and pulp revascularization.ObjectiveTo establish an animal model of experimental apical apical periodontitis and pulp revascularization in mice, and measure the dynamic expression of Nrf2 in apical periodontitis and pulp revascularization procedure to lay the foundation for the future study of the mechanism.Materials and methodsPart I Establishment of an animal model of experimental apical periodontitis in mice20 3-week-old C57BL/6 mice, randomly select 10 as the experimental group, the other 10 untreated normal mice served as blank controls. First mandibular molars of 10 mice were exposed to the oral environment for 3周 eeks to induce experimental periapical periodontitis. Animals were killed at 3 weeks after surgery for radiographical, histological and qRT-PCR analysis.5 mice of experimental group were killed, isolated mandible and fixed the mandible in neutral 4% paraformaldehyde for 24 h. Use Micro-CT imaging system to scan the specimen for subsequent observation and analysis. After scanning the specimen were made paraffin sections and HE staining. The other 5 mice of the experimental group were killed, isolated mandible and collected periapical and inner root cannel tissue. Extract tissue total RNA and use qRT-PCR to detect mRNA expression of inflammatory cytokines IL-1β, IL-6, TNF-a. Record the results for statistically analysis.Part II Establishment of an animal model of pulp revascularization in mice303-week-old C57BL/6 mice, randomly select 20 as the experimental group (10 filled with flowable resin composite and 10 with glass ionomer cement (GIC)), the other 10 untreated normal mice served as blank controls. First mandibular molars of 20 mice underwent pulp revascularization. Use 6#,8# K file to remove pulp tissues, and bleeding was induced in the canal space to the level of the cementoenamel junction. After the formation of the blood clot, cover its surface with mineral trioxide aggregate (MTA) to cover the entire surface of a blood clot. Than restored the crown access of 10 mice with GIC and other 10 mice with flowable resin composite. Check up the fillings of first mandibular molars in pulp revascularization group 3 weeks after surgery, and record the results for statistically analysis. Animals were killed at 3 weeks after surgery for radiographical, histological analysis and qRT-PCR to detect mRNA expression VEGF.Part III Nrf2 Expression in apical periodontitis and pulp revascularization procedure10 3-week-old C57BL/6 mice were killed, isolated mandible and fixed the mandible in neutral 4% paraformaldehyde for 24 h. Make paraffin sections for subsequent observation. Select sections similar to HE staining sections for immunohistochemical staining to detect the expression of Nrf2.20 3-week-old C57BL/6 mice, the left first mandibular molars were underwent pulp revascularization treatment and the right first mandibular molars were exposed to the oral environment for 3 weeks to induce experimental periapical periodontitis. Animals were killed at 1 and 4 weeks after surgery for radiographical and histological analysis. Extract tissue total RNA and use qRT-PCR to detect mRNA expression of Nrf2, IL-1β, IL-6, TNF-a of apical periodontitis group and mRNA expression of Nrf2 and VEGF of pulp revascularization group. Select sections similar to HE staining sections for immunohistochemical staining to detect the expression of Nrf2 atl and 4 week in each group. Untreated mice served as blank controls.Results1. The experimental animals were in good general condition during the experimental period, the experimental teeth had no fracture or odontoseisis.3 weeks after surgery, Micro-CT showed apical bone resorption in apical periodontitis group. The root canal was filled with necrotic tissues, apical lesion was present and periapical portion had inflammatory cells infiltration. IL-1p、IL-6 and TNF-a mRNA expression was higher than the control group (P<0.05).2.3 weeks after surgery, in pulp revascularization group, a portion of the fillings were off from the crown. Compared with flowable resin composite group, GIC group cost less surgery time-consuming and filling dropout rate was lower, the difference was statistically significant (P<0.05). The canal space was narrowed and hard tissue was observed in root canal in the pulp revascularization group under Micro-CT. Histologic evaluation showed the canal space was filled with connective tissue containing numerous blood vessels, and the apical portion of root was full of calcification tissue and bonelike tissues. VEGF mRNA expression was higher than the control group (P<0.05).3. In control group, Nrf2 expression was low both in the root canal and periapical tissue, and the expression site was mainly in cytoplasm. Compared with the control group, Nrf2-positive cells had increased at each time point of the experimental groups. In both Cytoplasm and nucleus there were Nrf2 expression and the nuclear Nrf2 expression is greatly increased. In apical periodontitis group, at 1 week Nrf2 positive cells were more than at 4 weeks. The Nrf2-positive cells were present in both root canal and periapical inflammation region, which were mainly lymphocytes, neutrophils and other inflammatory cells. The cells were round or oval, the cytoplasm or nucleus was stained brown, and fibroblasts inner root canal can also found Nrf2 expression. In pulp revascularization, group 4 weeks after surgery Nrf2-positive cells than 1 week, at 1 week Nrf2 positive cells were less than at 4 weeks. There were Nrf2 expression both in root canal and periapical inflammation region and positive cells were mainly irregular fibroblast-like cells.Conclusions1. In this study, the animal models of experimental apical periodontitis in mice was established successfully. Radiographical, histological and molecular biochemistry methods were used to evaluate the animal models, Results showed that periapical lesion was present in apical periodontitis group. The successful establishment of the animal model can contribute to the further research of the mechanism of apical periodontitis.2. In this study, the animal models of pulp revascularization in mice were established and evaluated successfully. It suggests that GIC may be more suitable for crown access filling after pulp revascularization in mice. Results showed that the root continued to grow in pulp revascularization group. The successful establishment of the animal model can contribute to the further research of the mechanism of pulp revascularization.3. During apical periodontitis and pulp revascularization procedure, Nrf2 expression was significantly increased, and the expression was time-dependent. It suggests that Nrf2 may has influence on the inflammation control of periapical periodontit and tissue repair and newborn during pulp revascularization, but the mechanism still needs further study. |
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