Mechanism Of Angiogenesis Promotion By SHED Aggregate Exosomes In Pulp Regeneration

Periapical pulp disease caused by caries and trauma is one of the most common diseases in clinical.At present,traditional root canal therapy is still the main treatment,and young permanent teeth are often treated with apexification.After treatment,due to the loss of dental pulp nutrition supply,immune defense and regulatory protective function,complications such as tooth fracture caused by brittle tooth tissue and reinfection caused by incomplete filling material are easy to occur in the affected teeth.With the development of tissue engineering and regenerative medicine,pulp regeneration has become possible.Since there are no reports of successful pulp regeneration using stem cell homing and cell-free therapy,exogenous stem cell transplantation is still required to reconstruct the natural pulp with highly organized three-dimensional morphology and realize neurovascularized pulp regeneration with full-function.Dental pulp stem cells(DPSCs)are undifferentiated mesenchymal cells that exist in the pulp tissue with the properties of stem cells.The pulp stem cells from deciduous teeth are called SHED.These odontogenic stem cells have the ability to differentiate into various dental tissues.Our research group carried out a clinical trial which using autologous SHED aggregate to treat pulp necrosis in young permanent teeth,and achieved the dental pulp tissue regeneration with total length(full-length pulp regeneration).The regenerated tissue not only had normal dental pulp and dentine complex morphological structure,but also the recovery of physiological function as dentine formation,root development,revascularization,and pulp feeling.But the underlying mechanism is still unclear.At the same time,the previous study found that the pulp regeneration rate and structural stability of mature permanent teeth of miniature pigs were worse than those of young permanent teeth,and the difference in the number of new blood vessels was particularly significant.Due to the narrow and long pulp cavity,closed apical foramen,poor local blood supply,and poor survival of the implanted aggregate,it is very important to clarify the function and mechanism of SHED aggregate in the angiogenesis of pulp regeneration to further improve the regeneration efficiency of dental pulp in permanent teeth.Cell-to-cell communication via direct cell-cell contact or paracrine mediated communication is essential for maintaining cellular function and tissue homeostasis.Exosomes derived from the endosome and plasma membrane serve as delivery carriers for cytoplasmic proteins,lipids,mi RNAs,and RNA,influencing the fate of target cells in a variety of ways.It has been shown that odontogenic stem cell-derived exosomes can induce the regeneration of tooth tissues such as pulp and dentin,and regulate the epithelial-mesenchymal transition during tooth germ development.Whether or not SHED aggregate exosomes can promote angiogenesis through two modes in dental pulp vascular regeneration(stem cell differentiation;induction of endothelial cell migration)has not been reported.In this study,firstly,the vascular formation pattern was observed by constructing pulp regeneration model,and the influence of SHED aggregate exosomes on pulp tissue regeneration was clarified.Secondly,the effect of SHED aggregate exosomes on inducing angiogenic differentiation and promoting cell migration was examined.Then the molecular mechanism of mi RNA-induced vascular formation and pulp regeneration induced by exosomes was explored.Finally,the efficacy of SHED aggregate exosomes in pulp regeneration was verified to provide a research basis and experimental basis for clinical transformation and application of pulp regenerationPart one: Study on the effect of SHED derived exosomes in promoting the vascular formation of pulp regenerationObjective: To establish an ectopic pulp regeneration model and examine the role of SHED derived exosomes in the vascular formation of pulp regeneration.Methods: Stem cells from human exfoliated deciduous teeth were isolated and cultured to be induced into aggregate.Then the pulp regeneration model of subcutaneous transplantation in nude mice was constructed by composite dental matrix with SHED aggregate.The relationship between vascular formation and pulp tissue regeneration was determined by HE staining.Exosomes were extracted from the supernatant of the SHED aggregates.SHED aggregates(Normal),SHED aggregates treated with GW4869 to inhibit exosome secretion(GW4869),SHED aggregates treated with added exosome(Exosomes),SHED aggregates treated with GW4869 and then added supplemented exosomes(GW4869+Exo).Four groups of SHED aggregates prepared in different ways composited with dental matrixes were implanted subcutaneous into nude mice to establish the dental pulp regeneration model.HE and immunofluorescence staining were used to observe and compare the effects of dental pulp and vascular regeneration in each group.Results: He staining showed that the tissue structure of SHED aggregates remained undifferentiated one week after implantation.Two weeks after implantation,the cells of aggregates were scattered and a small number of necrotic apoptotic cells were observed.Three weeks after implantation,a cord-like structure of vascular endothelial cells could be observed in the center of the aggregates.Microvessels contained with rich red cells were observed in the regenerated tissue 4 weeks after implantation.Three months after implantation,there were typical layers of odontoblasts and abundant vascular structures in the regenerated tissue.HE staining results of Normal group showed that the tissue structure of the regenerated pulp was continuous and complete,and the odontoblast cell layer and new blood vessel structure were visible.Immunofluorescence staining results showed that cells positive for CD31 were clustered around the tube-like structure in the regenerated tissue,while a large number of cells positive for CD31 also expressed Mitochondria.The results of HE staining in the exosome secretion inhibition group(GW4869)showed that the SHED aggregate was not fully differentiated as cords,the regenerated tissue structure was loose and irregular,and there was no neovascularization or odontoblast cell layer structure in the dental matrix.Immunofluorescence staining showed that there were almost no CD31 positive cells or tissues in GW4869 group.The results of HE staining in the exosomes added group(Exosomes)showed that the regenerated pulp tissues were arranged regularly,containing abundant neovascularization tissues and typical odontoblast cell layer structure,and the regeneration effect was significantly improved compared with the normal group.The results of HE and immunofluorescence staining in the last group showed that after the supplementation of exosomes,the regeneration effect of dental pulp tissue was restored in some degree compared with the GW4869 group,but the number of regenerated blood vessels was significantly reduced compared with the normal group.Conclusion:Microvascular system reconstruction plays an important role in pulp regeneration.SHED aggregate-derived exosomes can affect pulp regeneration by regulating the angiogenic differentiation between implant and host cells.Part two: Study on the function of SHED aggregate exosomes in promoting angiogenic differentiationObjective: To investigate the promoting function of SHED aggregate exosomes in angiogenic differentiation.Methods: The phagocytosis of SHED aggregate exosomes(SA-Exo)was detected by immunofluorescence staining and fluorescence tracer.Tube formation assay and cell migration assay were used to evaluate the effect of SA-Exo on the angiopoiesis ability of SHED and HUVEC.The regulation effect of SA-Exo on the expression of angiogenic genes and proteins of SHED and HUVEC was verified by q-PCR and Western blot.Results: Immunofluorescence staining results showed that after3 h co-culture of SHED or HUVEC with SA-Exo,a large number of exosomes with positive expression of PKH67 were observed in cells and dotted distributed in cytoplasm.The results of the tube formation experiment showed that the number of points,the connections,branches and the total length of branches were significantly increased after SA-Exo co-culture with SHED and HUVEC.Transwell results showed that the number of HUVEC cells migrated from the upper chamber to the lower chamber was significantly increased after added SA-Exo.q-PCR results showed that the expression levels of vascular endothelial growth factor(VEGF),angiogenin and platelet-derived growth factor(PDGF)in SHED and HUVEC cells were significantly up-regulated after SA-Exo treatment.GW4869 inhibited the secretion of SHED exosomes,and the expression levels of related genes were significantly decreased.Adding exogenous SA-Exo after GW4869 treatment could partially restore the expression of angiogenesis related genes.Western blot results showed the same trend as q-PCR results.Conclusion: SA-Exo can promote the differentiation of SHED cells to vascular endothelium by endocytosis,and also promote the angiogenic differentiation and migration of HUVEC.These results further reveal the mechanism of SHED aggregate-mediated pulp regeneration.Part three: Study on the molecular mechanism of SHED aggregate exosomes in regulating angiogenesisObjective: To investigate the molecular regulation mechanism of SA-Exo in promoting angiogenesis.Methods: The expression differences of mi RNAs in SHED exosomes(S-Exo)and SA-Exo were detected by transcriptome sequencing technology,and the enrichment of differentially expressed mi RNAs in diverse signaling pathways were determined.The target mi RNAs in SHED and HUVEC were treated with inhibitor and mimics by cell transfection method.The influence of target mi RNAs on the angiogenic function of SHED and HUVEC was determined by the tube formation experiment and cell migration experiment,then the regulatory effect of target mi RNAs on angiogenic related gene and proteins was verified by q-PCR and Western blot.KEGG pathway enrichment analysis was applied to the sequencing results to clarify the enrichment of differentially expressed mi RNAs in different signaling pathways,and major related pathways were selected.Western blot was used to clarify the regulatory effects of target mi RNA on pathway-related proteins.Results: Gene sequencing results showed that mi R-23 a,mi R-199 a,let-7i,mi R-23 a,mi R-21 a,mi R-23 a and other mi RNAs were highly expressed in SA-Exo,and the expression level of mi R-26 a was significantly increased,which was consistent with the q-PCR results.KEGG pathway enrichment analysis results showed that the differentially expressed mi RNA mainly enriched in TGF-beta,PI3K-Akt,VEGF,MAPK,Ras,and other signal pathways.Mi R-26 a regulates the expression of angiogenic related gene and proteins(VEGF,Angiogenin/ angiopoietin-2,and PDGF)in SHED and HUVEC.Further it plays an important role in the regulation of HUVEC migration and the tube formation ability of SHED and HUVEC through TGF-β/Smad2/3pathway.Conclusion: mi R-26 a transported by SA-Exo can promote the angiogenic differentiation ability of SHED and activating HUVEC through TGF-β/Smad2/3 signaling pathwayPart four: Study on the function of mi R-26 a in SA-Exo to promoting vascular formation in pulp regeneration of Animal experimentalObjective: To evaluate the effect of mi R-26 a overexpressed SA-Exo on promoting angiogenesis in dental pulp ectopical regeneration model with subcutaneous transplantation on nude mice.Methods: SA-EXO with overexpression of mi R-26a(Mimic group)and normal SA-EXO(Control group)were prepared respectively.SHED aggregates of the two groups composited with dental matrix were implanted for pulp regeneration.HE and immunofluorescence staining were used to observe and compare the effects of pulp regeneration and vascular reconstruction between the two groups.Results:The results of HE staining in Mimic group showed that the tissue structure of the regenerated pulp was similar to that of the normal pulp with continuous and complete odontoblast cell layer.A large number of tube or streak-like vascular structures could be seen in the interstitium of the pulp.Immunofluorescence staining results showed that a large number of cells with positive expression of CD31 could be seen gathered around the tube.The results of HE and immunofluorescence staining in the control group showed that the regenerated tissue had similar structure to the normal pulp,but the tissue maturity and the number of regenerated blood vessels in the control group were lower than those in the Mimic group.Conclusion: SA-Exo overexpression of mi R-26 a can improve the regeneration ability of SHED aggregate,especially angiogenesis.Its application can promote pulp tissue regeneration and is an effective way to optimize pulp regeneration.