The Effect Of Exosome Of Human Periodontal Ligament Fibroblasts On The Biological Behavior Of Osteoblasts

As people pay more and more attention to oral health,the prevention and treatment of oral diseases has become a research hotspot in recent years.In the course of periodontitis,alveolar bone remodeling disorder caused by inflammation will cause alveolar bone loss and even tooth loss,which is one of the main causes of tooth loss in adults.As the most important pathogenic factor,lipopolysaccharide(Lipopolysaccharide LPS)also plays an important role in bone resorption caused by periodontitis.How to control the progress of periodontal disease,reduce bone destruction,promote periodontal tissue repair and regeneration,and restore the normal biological width has always been the focus of periodontal disease research.Human periodontal ligament fibroblasts(Human periodontal ligament fibroblast HPDLFs)are the main components of periodontal ligament tissue and play an important role in periodontal tissue remodeling.Early studies have shown that the maintenance of bone homeostasis depends on direct intercellular interaction and intercellular substance secretion.Some studies have shown that HPDLFs can express ephrin/Eph family related factors and bind to the corresponding receptors on the surface of osteoblasts to complete bone remodeling.Some researchers believe that fibroblast growth factor acts on osteocytes,regulates calcium ions inside and outside the cells,and has an effect on bone mineralization and calcium deposition.Exosome(Exosome)is a kind of extracellular vesicle.In recent years,the research on the involvement of exosome in bone tissue remodeling has been gradually deepened.Repairing bone defects through the interaction and regulation between various cells and osteocytes is of great significance for the maintenance of bone homeostasis and intraosseous environmental balance.These studies suggest that exosome may affect the biological function of osteoblasts in the process of HPDLFs involved in alveolar bone remodeling.Therefore,this study will explore the effect of HPDLFs exosome on the biological behavior of osteoblasts,and provide new ideas and theoretical reference for alveolar bone repair.Objective:The exosome was extracted from primary cultured HPDLFs and acted on osteoblasts to detect the proliferation of osteoblasts,alkaline phosphatase activity and the expression of osteogenesis-related gene BGP,so as to provide a new theoretical reference for alveolar bone loss caused by periodontitis.Methods:Part One Primary culture and identification of human periodontal ligament fibroblastsIn the Department of Stomatology of Affiliated Hospital of North Sichuan Medical College,the healthy teeth of 14-year-old patients who needed to be extracted due to orthodontics or impaction were collected.The periodontal ligament was isolated under aseptic condition.Human periodontal ligament fibroblasts were cultured by tissue mass culture method.The morphology of the cells was observed under microscope and identified by immunohistochemical method.Part Two Extraction and identification of exosome from human periodontal ligament fibroblastsThe periodontitis model was established.HPDLFs was stimulated with 10 ?g/ml LPS and the culture supernatants of HPDLFs and LPS-HPDLFs were collected.The exosome was extracted by ultracentrifugation.The concentration of exosome was determined.After 100 ?1 HPDLFs-EXO,was diluted with PBS for 2 times,the morphology of exosome was observed by electron microscope,and the expression of HPDLFs-EXO surface marker protein CD63 and TSG101 was detected by Westernblot.Part Three Effect of exosome of human periodontal ligament fibroblasts on osteoblastsHPDLFs-EXO and LPS-HPDLFs-EXO acted on hFOB1.19 human osteoblasts,CCK-8 detected cell proliferation and alkaline phosphatase(ALP)activity,QPCR detected the expression of osteogenesis-related gene BGP,and observed the fluorescence uptake of HPDLFs-EXO by hFOB1.19 human osteoblasts under confocal microscope.Result:1 Primary human periodontal ligament fibroblasts were successfully cultured by tissue mass culture method,and then subcultured,expanded and cryopreserved.After passage,HPDLFs grew faster,vimentin was positive and keratin was negative by immunohistochemical method.2 HPDLFs-EXO and LPS-HPDLFs-EXO,were observed to be round or oval under electron microscope,and the expression of surface marker proteins CD63 and TSG101 were detected by Westernblot.3 CCK-8 detection showed that exosome could promote the proliferation of hFOB1.19 human osteoblasts,and the higher the concentration was,the more obvious the proliferation of hFOB1.19 human osteoblasts was,the difference was statistically significant,and the proliferation of hFOB1.19 human osteoblasts stimulated by the same concentration of HPDLFs-EXO and LPS-HPDLFs-EXO was more obvious in HPDLFs-EXO group.After alkaline phosphatase activity detection and osteogenesis-related gene BGP detection,it was found that the expression in the exosome group was higher than that in the blank control group.The uptake of HPDLFs-EXO by hFOB 1.19 human osteoblasts was observed after fluorescence staining.Conclusion:1 The exosome of human periodontal ligament fibroblasts can promote the proliferation of osteoblasts and participate in alveolar bone remodeling.2 The exosome of human periodontal ligament fibroblasts still plays a role in inflammatory environment,but the effect is lower than that in normal environment.3 The effect of exosome of human periodontal ligament fibroblasts on osteoblasts increased with the increase of concentration.