In Vivo Observation Of Biological Effects Of IRoot BP Plus And Related Mechanism Study

Part one:Study on the application of i Root BP Plus in Beagle dog and humansin vivo Objective: Various factors,such as caries,pulp disease or trauma,have been implicated to influence the development of young permanent teeth.These factors may lead to pulp exposed,and delayed treatment will cause pulpitis,periapical inflammation and even pulp necrosis,thus affecting the completion of root development.To preserve the vitality of dental pulp and maintain the function of teeth,we designed the experiment of the application of i Root BP Plus in Beagle dog and humans.In this animal experiment,i Root BP Plus(experimental group)and Pro Root MTA(control group)were applied directly to the exposed dental pulp and the results of histological changes of dental pulp tissue were recorded;To observe the composition of new tissue in root canal after the application of i Root BP Plus in pulp revascularization;In clinical practice,to observe theprognosis of young permanent teeth treated with i Root BP Plusafterpartial pulpotomy.Methods:(1)After induction of general anesthesia in Beagle dog and oral local disinfection,maxilla and mandibular 12 anterior teeth wereanesthetized,isolated and accessed in lingual surface of anterior teeth with high-speed round burs.The diameter of cavity was 1 mm,and hemostasis was achieved by sterile saline cotton pellets.Then the pulp capping procedurewas conducted with i Root BP Plus or MTA,which was placed in the pulp perforation sites and packed using a sterile cotton pellet.Subsequently,the cavities were filled with glass-ionomer cement and light-cured resin.The changes of pulp tissues were analyzed by HE staining after 3 months.(2)12permanent teeth with open apices in beagle dog were selected.Periapical periodontitis was induced by pulp exposure.After root canal preparation anddisinfection,apical bleeding was stimulated to make blood fill the root canal to form blood clots.The i Root BP Pluswas placed on the blood clot.X-ray examination was performed to observe the root development after 3 months,and HE staining was performed to analyze the morphological characteristics of new tissues in the root canal.(3)27 cases of young permanent teeth with pulp exposure were selected with the informed consent of the patients and their families.Partial pulpotomy was performed and the exposed pulp was covered with i Root BP Plus.Teeth were filled with resin.The success rate was recored.Results: In the direct pulp capping model,no pulp necrosis and no periapical periodontitis occurred in experimental and control group,and some root canals contained inflammatory cells.Under the pulp capping agent,the formation of reparativedentin can be seen,and single layer cells with uniform arrangement can be seen on the surface.Under thereparativedentin,pulp-like tissueswere formed with abundant blood supply.The arrangement of collagen fibers is consistent with that of the normal pulp,and bone and periodontal ligament-like tissue structures are not found.In the pulp revascularization model of young permanent teeth,tissue regeneration was observed in root canals of the experimental group(7/10)and the control group(8/10),and some of which contained inflammatory cells.The new hard tissue was the dentin-like structure formed along the dentin wall,and different sizes of osteoid tissuecan be seen in the pulp cavity and root canal;the regenerating soft tissue wasthe pulp-like loose tissue containing a large number of small blood vessels,and periodontal ligament-like tissue can be seen in some samples.In the partial pulpotomyexperiment,the success rate of 27 patients was 100% after 1 year.Conclusion: the uninfected pulp of exposed teeth can be protected by direct pulp capping with i Root BP Plus or MTA,and there is no significant difference between the two groups.In dental pulp rescularization experiments,teeth with open apices and periapical periodontitiswere found pulp vascular regenerationaftertreated withi Root BP Plus or MTA.The regeneration of tissue wassimilarto normal dental pulp.No differences were found between the two groups.Samples had more inflammatory cells infiltrationin MTA group.The success rate suggested that i Root BP Plus and MTA might give similar effects in partial pulpotomy.This study provides a referencefor the application of i Root BP Plus in pulp reascularization.i Root BP Plus can be used in partial pulpotomy.Part Two:The effect of i Root BP Plus on the biological behavior of macrophagesin vitroObjective: i Root BP Plus is a kind of pre-mixed nano-bioceramicmaterials,whichis widely used as root canal repair or filling material.Root canal filling material is contact with inflammationperiapical tissuedirectlyduring root canal treatment.The interactionbetween filling material and periapical tissuewere studied,butthe effectof i Root BP Plus on macrophage function has not yet been reported.The purpose of this study was to evaluate the effect of i Root BP Plus on the biological behavior of macrophages.Methods: In the absence of LPS stimulation and LPS stimulation condition,RAW264.7 cellswere cultivated.Different concentration of i Root BP Plus material leaching solution was added into the culture medium.The samples wereincubatedat 37℃in a chamber.CCK-8 kit was used to detect the proliferation of macrophages.Intracellular ros accumulation was detected by DCFH-DA staining and flow cytometry.The changes of macrophage apoptosis and IRF8 expression were observed by immunofluorescence staining.Results: i Root BP Plus had no effect on the activity of macrophages in vitro.LPS significantly increased intracellular ros levels,while i Root BP Plus decreased intracellular ros levels in the normal control group and LPS stimulation group.Conclusion: The new nano-bioceramic materials i Root BP Plus can promote the proliferation of macrophages in vitro and reduce the level of reactive oxygen species in macrophages.Part Three: The Activation of Interferon regulatory factor 8(IRF8)in human Periapical lesionsIntroduction: Interferon regulatory factor 8(IRF8)is a critical transcription factor in innate immune responses that regulates the development and function of myeloid cells.Human periapical lesions are caused by endodontic microbial infections.However,the presence of IRF8 in human periapical lesions remains elusive.This study aims toexplore the expression of IRF8 in human periapical lesions,and the possible association of IRF8 with macrophages,nuclear factor(NF)-κB signaling,and autophagy process.Methods:Thirty-nine human periapical tissues,including healthy control tissues(n=15),radicular cysts(RCs,n =11),and periapical granulomas(PG,n = 13),were examined.Tissues were fixed in paraformaldehyde and analyzed.The inflammatory infiltrates of lesions were evaluated by hematoxylin–eosin,and the presence of IRF8 was analyzed by immunohistochemistry.Double-immunofluorescence assessment was performed to colocalize IRF8 with CD68,NF-κB p65 and LC3 B.Results: The expression of IRF8 was significantly higher in RCs and PGs than in the healthy control group,but nosignificant differences was found between RCs and PGs.IRF8-CD68 double-positive cells were significantly stronger in RCs and PGs than in the healthy control group,but no significant difference was observed between RCs and PGs.Double-labeling analysis of IRF8 with NF-κB and LC3 B indicated that IRF8 associate with NF-κB signaling and autophagy process during periapical lesions.Conclusions:IRF8 is activated in periapical lesions.IRF8 may be a critical regulator of macrophages in periapical lesions.The activation of IRF8 associates with NF-κBsignaling and autophagy process.