| Endodontic and periapical lesions are common diseases in stomatology.They are often manifested as irreversible inflammation of pulp and destruction of periapical tissue,sometimes accompanied by severe pain.At present,the main way to solve this kind of disease is root canal therapy.However,after the preparation of large taper instruments,the teeth lose the nutritional and sensory function of the dental nerve,and may have a series of complications such as root fracture and reinfection.With the development of tissue engineering,pulp regeneration is a new research direction.Exosomes are small functional vesicles secreted by living cells,which contain signal molecules from a variety of sources,including m RNA,protein,etc.target cells complete information transmission and signal exchange by ingesting these vesicles,so as to play similar biological functions of their source cells.As a signal molecule of tissue engineering,exosomes need a certain carrier,so that they can be slowly released locally and play a role.Studies have shown that gelatin and fibrin cross-linking modification as tissue engineering scaffolds can effectively promote the migration,proliferation,matrix synthesis and tissue fusion of chondrocytes after articular cartilage fragmentation.Therefore,we speculate that fibrin /gelatin scaffolds loaded with DPSCs-Exo can also play a similar role in pulp tissue engineering.Objective: Use fibrin/gelatin composite hydrogel scaffold to load dental pulp stem cells derived exosomes,and explore the possibility of promoting cell migration and growth,as well as dental pulp tissue regeneration through experiments in vivo and in vitro,and provide references for the development of dental pulp tissue engineering.Methods: 1.The dental pulp stem cells were isolated and cultured from human teeth,and the surface antigens were identified by flow cytometry.The exosome was isolated from the supernatant of pulp stem cells by ultracentrifugation.The extracted exosome was identified by Western blot,NTA particle size analysis and transmission electron microscope.2.Preparation of fibrin/gelatin composite hydrogel,recording the gelation time at different thrombin concentrations,selecting the best concentration for subsequent experiments;drying the hydrogel through critical point,observe the three-dimensional structure and the state of loading dental pulp cells and exosome respectively by scanning electron microscope;cells loaded on fibrin/gelatin hydrogel were stained with live/dead test kit on the third day of culture to explore the cytocompatibility of the scaffold.3.The exosomes stained with PKH26 were cocultured with dental pulp cells,and the uptake of exosomes in vivo was observed by confocal laser scanning;the exosomes and dental pulp cells were loaded on fibrin/gelatin scaffolds,and the difference of cell viability was explored by live/dead staining;transwell assay was used to detect the difference of cell migration ability of fibrin/gelatin scaffolds loaded with or without exosomes;q PCR detected the differences of DMP1,VEGF,SDF1,CXCR4 expression in fibrin/gelatin scaffolds with or without loading exosomes;injected hydrogel containing or without exosomes into the root canal of root segment and transplanted it to the back of the immunodeficient nude mice.After 8 weeks,the specimens were taken out,and the difference of tissue composition was observed by HE staining.Results: 1.Dental pulp stem cells were successfully isolated and cultured from human teeth in vitro.Mesenchymal stem cell markers CD73 and CD90 were positive,while CD31,CD45 and NESTIN were negative,which accorded with the characteristics of dental pulp stem cells.Exosomes from human dental pulp cells were successfully isolated by ultracentrifugation.Western blot was used to detect the exosome marker protein.Compared with PBS control group,CD9 and CD63 were positive;NTA particle size analysis showed that the particles were distributed in30-150 nm,with an average particle size of 84.06nm;transmission electron microscope showed that the exosome was typical bilateral membranous vesicles,which was consistent with the characteristics of exosome.2.Preparation of fibrin/gelatin composite hydrogel,when the fibrin concentration is 5mg/ml,gelatin mass fraction is 4%,thrombin concentration is 0.5U/ml,the composite hydrogel will gel for about 6minutes,which meets the experimental requirements.Scanning electron microscope results show that the three-dimensional structure of the hydrogel is porous,and can successfully load cells and exosomes,and the cells extend on the scaffolds.The live/dead assay results showed that the fibrin/gelatin hydrogel had good cytocompatibility and low cytotoxicity,and was a good choice for tissue engineering scaffold materials.3.The exosomal staining tracer experiment showed that exosomes could be absorbed by dental pulp cells within 30 minutes and entered the cytoplasm around the nucleus.Live/dead assay showed that the DPSCs-Exo loaded on the fibrin/gelatin hydrogel scaffolds had a positive effect on cell growth(P < 0.001).Transwell assay showed that fibrin/gelatin hydrogel loaded exosomes could effectively promote cell migration(P<0.0001).q PCR results showed that the expression levels of VEGF,DMP1,CXCR4 in pulp cells on fibrin/gelatin scaffold loaded with exosomes were significantly higher than those in the control group(P<0.05)and SDF1 was lower expression,indicating that DPSCs-Exo could promote the expression of genes related to angiogenesis,odontogenesis and migration of pulp cells in fibrin/gelatin three-dimensional environment.The root segments of fibrin/gelatin hydrogel containing or without exosomes were subcutaneously implanted on the back of nude mice.After8 weeks,the slices were observed.Two groups of pulpal-like soft tissues were inserted into the root canal,and the soft tissues were removed for HE staining.The exosome group showed more small vessels rich in abundant red blood cells.Conclusion: Fibrin/gelatin composite hydrogel as scaffold for dental pulp tissue engineering,DPSCs-Exo as signal factor,both in vivo and in vitro have shown the promotion effect on cell growth and tissue regeneration,which showed promissing application prospects in dental pulp tissue engineering. |
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