Research On The Feasibility Of Inhibiting CTSK To Regulate Inflammatory Granulation Transformation To Treat Alveolar Bone Inflammatory Lesions

A variety of odontogenic inflammatory lesions,such as chronic inflammation around the crown of wisdom teeth,periodontitis,chronic periapical periodontitis,and peri-implant periodontitis,can lead to the resorption of supporting structure of teeth or implants.It is the main cause of dentition defect,which seriously affects human health.The common feature of these lesions is that the local bone tissue is eroded and replaced by inflammatory granulation tissue(IGT),so IGT is generally considered to be pathological tissue.In the clinical treatment strategy,while cleaning up the pathogenic factors such as dental pulp or periodontium,it is also required to remove local IGT,even if the teeth needed to be removed,the residual IGT in the alveolar socket should be scraped so as not to affect bone regeneration.In fact,the restorative granulation tissue(RGT)which is formed in the process of alveolar bone healing,is in common with IGT in capillaries and fibroblasts.The difference is that there are more inflammatory cells infiltrating in IGT,while RGT is dominated by collagen fibers secreted by mature fibroblasts.At present,studies have confirmed that there are mesenchymal stem cell with osteogenic differentiation potential in IGT.Therefore,if properly handled,IGT can be transformed into RGT,and repair bone defects in theory.As a key regulator of bone metabolism,it has been confirmed that inhibition of CTSK can effectively prevent bone loss by affecting osteoclasts and immune system during the occurrence of periodontitis.It can effectively prevent the occurrence of periodontitis and apical periodontitis,but whether it can treat alveolar bone inflammatory lesions has not been reported.In recent years,other studies have shown that mesenchymal stem cell also expresses CTSK,and their endogenous CTSK may be related to the osteogenic activity.This makes CTSK a potential target for the treatment of a variety of inflammatory bone diseases.Therefore,it is proposed for the first time that promoting the transformation of IGT to RGT may be a new strategy to treat alveolar bone inflammatory lesions and promote alveolar bone regeneration through inhibition of CTSK.To confirm this hypothesis,this topic is mainly carried out from the following five aspects.Part one: Expression and distribution of CTSK in human alveolar inflammatory granulation tissue Objective: To clarify the expression and distribution of CTSK in different types of human alveolar IGT.Methods: IGT caused by periodontitis,periapical periodontitis and pericoronitis of wisdom teeth were collected,and the histological characteristics of different types of IGT were identified by H&E staining.The expression of CTSK was detected by RT-q PCR and Western Blot.The distribution of CTSK was revealed by immunohistochemical(IHC)staining and TRAP staining.The existence of mesenchymal stem cell and the colocalization of CTSK were detected by immunofluorescence staining.Results: There is no significant difference is observed in the histological characteristics of different types of IGT,which are composed of a large number of inflammatory cells,capillaries and fibrous tissue.Osteoclasts were not detected in IGT,but the expression of CTSK could be detected,which was widely distributed in lymphocytes,plasma cells,vascular endothelial cells and extracellular fibrous tissues.The results of RT-q PCR and Western Blot showed that the expression of CTSK in IGT was significantly higher than that in healthy alveolar bone or periodontal ligament.Conclusion: The expression of CTSK is significantly increased in human alveolar bone IGT but the role of CTSK in the development and outcome of alveolar bone IGT need further study.Part two: Comparation of the difference between IGT and RGT in histology Objective: To investigate the histological differences between IGT and RGT,and the expression of CTSK and Osterix in them.Methods: IGT and RGT were collected with the informed consent of patients undergoing delayed autologous dental transplantation.Hematoxylin-eosin staining(H&E staining)were used to determine the differences of histological morphological between IGT and RGT;IHC staining was used to clarify the difference in types of vessel between IGT and RGT;IHC staining and Masson staining were used to clarify the difference of ECM between IGT and RGT;IHC staining was used to detect the expression of Osterix and CTSK in these two kinds of granulation tissues.Results: The most striking histological property of RGT was found to be ECM deposition,which significantly decreased inflammatory cells,prominently increased fibroblasts as well as triggered changes of vascular types.CD31 positive vessel was found both in IGT and RGT,but EMCN positive vessel was only found in RGT.The expression level of CTSK in RGT was down-regulated to 76.97%(P<0.01),but the number of Osterix positive cells was significantly higher than that of IGT.Conclusion: There are both some similarities and some differences between IGT and RGT in histology,which lays a foundation for the study of granulation tissue transformation,but further study is needed.Part three: Comparation of the difference between IGT and RGT in proteomic analysis Objective: To determine the difference in proteomic analysis between IGT and RGT.Methods: IGT and RGT were collected with the informed consent of patients undergoing delayed autologous dental transplantation.Proteins from these two granulation tissues were extracted for proteomic analysis.The KEGG(http://www.genome.jp/kegg/)was used to classify the differentially expressed proteins(DEP),the two-tailed Fisher’s exact test was employed to verify the enrichment of the differentially abundant proteins versus all identified proteins.Protein-protein interaction(PPI)and co-expression were analyzed on Dr.Tom platform(http://biosys.bgi.com/#/report/login)provided by Beijing Genomics Institution(BGI,Shenzhen,China).Results: Combined with histological findings and proteomic analysis,49 DEP were involved in five KEGG pathways associating with ECM,inflammation and angiogenesis.COL1A1 was highly up-regulated,and played a key role in protein-protein interaction regulatory network.The expression of specific protease CTSK in RGT was downregulated to 69.10%-76.97%(P<0.05).The expression of COL1A1,COL1A2,FN1 and TGFB1 was significantly up-regulated,which were associated with focal adhesion,PI3 KAkt signaling pathways and angiogenesis.Conclusion: CTSK might be a target to regulate transformation from IGT to RGT in alveolar bone,and the regulating effect might depend on ECM mechanisms.However,further research is also clearly required.Part four: The Study of CTSK regulate the biological characteristics of IGT-derived cell Objective: To clarify the biological differences between IGT-derived cell(IGTC)and RGT-derived cell(RGTC),and then explore the regulatory effect of ODN on IGTC.Methods: Flow cytometry was used to detect the surface markers of cells both in IGTC and RGTC.Western Blot,ALP staining and Alizarin red staining were used to detect the osteogenic differentiation ability.The corresponding biological characteristics of IGTC were observed after ODN stimulation.Results: The cell surface markers of IGTC and RGTC are consistent with the characteristics of stem cells.The expression of CTSK in IGTC was significantly higher than that in RGTC,which was mainly distributed in lysosome.CCK-8 results showed that the proliferation ability of IGTC was stronger,and ODN had no significant effect on the proliferation of IGTC.Scratch test showed that the migration ability of IGTC was stronger,and ODN could significantly reduce the migration ability of IGTC.Compared with RGTC,the expression levels of type I collagen(COL1),Runt-related transcription factor 2(RUNX2)and alkaline phosphatase(ALP)in IGTC were significantly lower in IGTC and the ability to form mineralized nodules was also relatively weakened.However,ODN stimulation could significantly increase the expression of them and the ability to form mineralized nodules in vitro.Conclusion: The stem cells can be isolated from IGT and RGT,but the ability of osteogenic differentiation of IGTC is obviously weakened,but ODN can partially restore the osteogenic differentiation ability of IGTC.Part five: Feasibility study of ODN in the treatment of rat periodontitis model Objective: To clarify the therapeutic effect of ODN on rat periodontitis model,and provide a new idea for the further treatment of alveolar inflammatory lesions.Methods: The periodontitis model was established by local injection of LPS,and ODN was administered orally for 4 weeks as a treatment.The therapeutic effect of ODN on rat periodontitis model was detected by Micro-CT and histological examination.Results: The rat model of periodontitis was successfully established,and the local alveolar bone resorption was obvious.After oral administration of ODN,the results of Micro-CT and H&E staining showed that local alveolar bone was partly recovered.The results of IHC staining showed that the expression of osteogenesis-associated proteins Osterix and COL I increased significantly.Conclusion: The feasibility of ODN in the treatment of periodontitis in rats was verified in animal model,which lays a foundation for the study of minimally invasive treatment of alveolar bone inflammatory disease.