| Background:At present,the incidence of pulpitis and dental pulp necrosis is increasing year by year Clinical treatment methods for severe dental pulp lesions—apexification and root canal therapy—could not restore the functions of teeth completely.With the development of stem cells and tissue engineering technology in recent years,dental pulp regeneration has become a new treatment method to cure severe pulp lesions.The use of stem cells from human exfoliated deciduous teeth(SHED)and cell aggregate technique improved the efficiency of pulp regeneration to a great extent.In the previous experiments,SHED aggregates was applied to dental pulp regeneration in minipig young permanent teeth and human young permanent teeth and permanent teeth with closed root apex.However,dental pulp regeneration was observed only in young permanent teeth,but not in permanent teeth with closed root apex.This is most likely related to insufficient blood and nutriment needed for tissue regeneration due to the stenosis in permanent teeth root apexExosomes secreted by cells are tiny vesicles which can be picked up and used by surrounding cells.Exosomes can transport proteins,target receptor cells specifically,exchange proteins and lipids and activate downstream pathways.They can also transport micro ribonucleic acid(miRNA)and many other nucleic acids,and promote the regeneration of blood vessels,nerves and other organs.According to the previous studies,exosomes derived from dental pulp stem cells promoted the expression of genes needed for odontogenic differentiation and pulp regeneration.There were differences in exosomes secreted by different cells and by the same cell in different states.There are still no reports on whether there are differences between cell-aggregate-derived exosomes and single-cell-derived exosomes.And if there are differences between cell-aggregate-derived exosomes and single-cell-derived exosomes,whether the differences make some sense in superiority regeneration effect of cell aggregates to single cells.Objective:The aim of this study is to explore the mechanism of the superiority regeneration effect of cell aggregates to single cells in dental pulp regeneration by comparing the differences between cell-aggregate-derived exosomes and single-cell-derived exosomes.In order to find out clinical treatment method for pulp regeneration in permanent teeth with closed root apex.Methods:1.Isolation,culturing and identification of SHED:In this study,collagenase digestion method and tissue adherent culturing method were used to separate and purify SHED.Flow cytometry identification was used to differentiate the surface markers of SHED.The ability of Colony Forming was detected by toluidine blue staining.Cell growth curve was detected by Methyl thiazolyl tetrazolium.The multidirectional differentiation potential was detected by osteogenesis induction and lipogenic induction.2.Extraction and identification of exosomes;data collection with information analysis of micro ribonucleic acid(miRNA):Exosomes were extracted by overspeed centrifugation.The particle morphology and size were examined by transmission electron microscope.The diameters of exosomes were measured and analyzed by particle size analysis.BGISEQ-500 sequencing technology was used for sequencing,and the data were compared with the 22nd version miRBase database3.Comparison of the effects of exosomes derived from different sources on bioactivity and vessel/nerve regeneration of SHED:Cell Counting kit-8 and flow cytometry were used to detect the effects of exosomes on proliferation and apoptosis of SHED.The influence of exosomes on multidirectional differentiation potential of SHED was studied by osteoblastic and lipid induction.Real-time Polymerase Chain Reaction was used to detect the effects of exosomes on secretion of vascular/nerve neurotrophic factors by SHED4.Heterotopicly subcutaneous transplantation in nude mice back for dental pulp regeneration.Permanent incisors collected from clinical patients were made to full-length roots.SHED aggregates,exosomes-inhibited SHED aggregates,and exosomes-added SHED aggregates were stored in full-length roots respectively,which were subcutaneously transplanted in the back of nude mice.Nude mice were sacrificed 3 months later.The full-length roots were stained after decalcification and section.The structure of regenerated dental pulp tissue was observed by hematoxylin and eosin stain.The regeneration of odontoblast layer(OL)was observed by immunohistochemical staining(IHC)of DSPP.The regeneration of vessel and nerve were observed by immunofluorescence staining(IF)of CD31 and S100 respectively5.Orthotopic transplantation in dental pulp cavity of minipig permanent teeth with closed root apex for dental pulp regeneration.The original dental pulps were taken out SHED aggregates,exosomes-inhibited SHED aggregates,and exosomes-added SHED aggregates were implantated into the pulp cavities of minipigs.The minipigs were sacrificed 6 months later.The pulp cavities were reconstructed by micro computed tomography scanning after tooth extraction.The structure of regenerated dental pulp tissues was observed by hematoxylin and eosin stain.The regeneration of OL was observed by IHC of DSPP.The regeneration of vessel and nerve were observed by IF of CD31 and S100 respectivelyResults:1.SHED grew in fusiform after separation and purification.The surface markers of MSCs were positive while those of hematopoietic stem cells were negative.SHED had good abilities of self-renewal and multidirectional differentiation2.According to transmission electron microscope observation results,SHED-aggregate-derived exosomes and SHED-derived exosomes were similar to saucers or concave hemispheroids with a diameter about 100 nm.The main peak of SHED-derived exosomes was smaller than that of SHED-aggregate-derived exosomes,but the average particle size of these two exosomes were consistent.Exosomes surface markers were all positive.The expression of miRNA which inhibiting cell apoptosis and promoting vascular/nerve regeneration was higher in SHED-aggregate-derived exosomes than in SHED-derived exosomes3.Exosomes made no effect on SHED proliferation,but inhibited SHED apoptosis Exosomes promoted SHED in multidirectional differentiation and secretion of vascular/nerve neurotrophic factors.SHED-aggregate-derived exosomes was more effective than SHED-derived exosomes4.Exosomes promoted SHED differentiatied into dentine cells,vascular endothelial cells and nerve cells in regenerated dental pulp in heterotopic transplantation.SHED-aggregate-derived exosomes was more effective than SHED-derived exosomes.Inhibition of exosomes resulted in necrosis in regenerated dental pulp5.Human SHED aggregate transplantated into minipig dental pulp cavities made no rejection.Exosomes promoted the regeneration of dental pulp tissue,OL,vessel,and nerve in regenerated dental pulp in orthotopic transplantation.SHED-aggregate-derived exosomes was more effective than SHED-derived exosomes.Regenerated vessels accompanied with regenerated nerves.Exosomes promoted the regeneration of continuous nerve tissue along the main vessel.Inhibition of exosomes resulted in calcification in regenerated dental pulpConclusions:1.Cell-aggregate-derived exosomes have more large-diameter particles than single-cell-derived exosomes.The expression of miRNA which inhibiting cell apoptosis and promoting vascular/nerve regeneration was higher in cell-aggregate-derived exosomes than in single-cell-derived exosomes2.Exosomes not only inhibit SHED apoptosis,but also promote SHED multi-directional differentiation and secretion of vessel/nerve neurotrophic factors.The effect of cell-aggregate-drived exosomes is better than that of cell-derived exosomes3.Exosomes promote dental pulp regeneration in minipig teeth with closed root apex and in nude mice back.The effect of cell-aggregate-drived exosomes is better than that of cell-derived exosomes.Cell-aggregate-derived exosomes significantly promote vessel and nerve regeneration.Cell aggregate combined with cell-aggregate-derived exosomes can be used in clinical treatment for dental pulp regeneration in permanent teeth with closed root apex. |
PDF Full Text Request