GelMA Injectable Microspheres Laden With Exosomes Derived From Epithelial Root Sheath Cells For Periodontal Regeneration

Periodontal disease is a common and frequently-occurring disease in oral diseases.Periodontal defect caused by severe periodontal disease is the main cause of tooth loss in adults.Current treatment methods still have some limitations,they fail to predictably achieve physiological regeneration of periodontal tissue.During tooth development,Hertwig’s Epithelial Root Sheath(HERS)plays a vital role in the development of periodontal tissue and tooth root.It recruited dental mesenchymal stem cells through epithelial-mesenchymal interaction(EMI)and induced mesenchymal stem cells to differentiate into pulp tissue and periodontal tissue.Exosomes carry a variety of proteins,lipids,and nucleic acid molecules,which are important mediators of intercellular communication and have become an important tool for cell-free therapy.Accordingly,based on the role of HERS in tooth development,this study isolated exosomes derived from HERS,studied its role and regulatory mechanism in periodontal tissue regeneration,and provide a new idea for periodontal regeneration.Methods:1.HERS-Exos was isolated from HERS-H1,an immortalized HERS cell line established by our research group,and characterized by TEM,NTA,WB and other methods;cultured periodontal ligament stem cells(PDLSCs),bone marrow mesenchymal stem cells(BMSCs),and human umbilical vein endothelial Cells(HUVECs),studied the effects of the HERS-Exos on cell proliferation,migration,osteogenic differentiation and angiogenesis after different concentrations of HERS-Exos intervention.2.HERS-Exos-loaded GelMA microspheres were prepared by microfluidic technology,and observed the morphology of the microspheres,the release function and the degradability of microspheres in vitro.3.The rat periodontal defect model was constructed and HERS-Exos-loaded GelMA microspheres were implanted into the periodontal defect of rats for in vivo experiments,evaluated periodontal tissue regeneration by Micro-CT,HE staining and immunohistochemical staining.4.The mechanism of HERS-Exos on endogenous stem cells around periodontal tissue was explored through RNA sequencing and bioinformatics analysis after intervention of PDLSCs,BMSCs.Results:1.HERS-Exos has a typical extracellular vesicle-like structure with a particle size of about 100 nm,expressing CD63,HSP70 and other marker proteins;it can promote the proliferation and migration and osteogenic differentiation of PDLSCs,BMSCs;and promote the proliferation,migration and angiogenesis of HUVECs.2.Through microfluidic technology,we successfully prepared HERS-Exos-loaded GelMA microspheres with an average diameter of 225μm,were porous,sustainable sustained release HERS-Exos up to 8 days,and their sustained release HERS-Exos could be intaked by PDLScs,BMSCs,HUVECs.3.The HERS-Exos-loaded GelMA microspheres were transplanted in vivo,which could significantly promote periodontal tissue regeneration in the defect area compared with the control group.Immunohistochemical staining showed that it can increase the proportion of Ki67 positive cells and the expression CXCR4 of cells.4.RNA sequencing and bioinformatics analysis showed that ITGA2,ITGA10,ITGB2,WNT5A,WNT7B,WNT10B,CXCL1,CXCL2,CXCL6,CXCL8,CXCR12 and other gene expression were up-regulated and Focal adhesion,PI3K-AKT,MAPK signaling pathways were significantly activated of BMSCs,PDLSCs after HERS-Exos intervention.Conclusion:HERS-Exos can promote proliferation,migration and differentiation of endogenous stem cells and realize periodontal endogenous regeneration.The mechanism may be closely related to Focal adhesion,PI3K-AKT,MAPK and other signaling pathways.This study showed that HERS-Exos could activate periodontal endogenous stem cells to achieve periodontal tissue regeneration and provide a new HERS-Exos-based cell-free treatment strategy for periodontal defects.