Effect Of Dual Delivery Of Exosome/Platelet-Rich Fibronectin MiR-26a On Osteogenic Differentiation Mechanism Of Osteoblasts

Objective:Purpose: Difficulty in restoring missing teeth due to insufficient bone volume in the alveolar bone is one of the major problems plaguing dental implant clinics.To circumvent the problems such as trauma caused by conventional bone augmentation,gene therapy tools are gradually becoming a hot research topic.The aim of this study was to construct a PRF/exosome/miR-26 a complex for dual and efficient delivery of miR-26 a into osteogenic-related cells to promote their osteogenic differentiation.Methods:1.1.Exosomes were extracted by ultra-high speed centrifugation and morphological features were observed by transmission electron microscopy.Exosomes were identified by particle size analysis and protein immunoblotting(Western blot,WB).2.Exosomes/miR-26 a complexes were constructed by loading fluorescently labeled miR-26 a into exosomes by electroporation.3.The exosomes carrying miR-26 a were co-cultured with mouse embryo osteoblast precursor cells(MC3T3-E1)to verify the feasibility and efficiency of cell transfection by exosomes.4.4-week-old rats were selected for blood collection via heart,and platelet rich fibrin(PRF)gel was prepared by centrifugation,and its ultrastructure was observed by electron microscopy.5.exosomes carrying miR-26 a were mixed with rat blood,and PRF/exosome/miR-26 a complexes were prepared by centrifugation.The miR-26 a loading and distribution in PRF/exosome/miR-26 a complexes were observed by fluorescence microscopy.6.The complex was co-cultured with MC3T3-E1 and the effect of the complex on cell proliferation was detected by CCK8 method.7.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the effects of alkaline phosphatase(ALP),Runt-related transcription factor 2(RUNX2),type I collagen(COL-1),osteopontin(OST),and osteopontin(OST)during the osteogenic differentiation of MC3T3-E1.phosphatase(ALP),Runt-related transcription factor 2(RUNX2),type I collagen(COL-1),osteopontin(OPN)and osteocalcin(OCN)during osteogenic differentiation of MC3T3-E1.RNA expression of osteocalcin(OCN)and thus the effect of the complex on osteogenic differentiation was observed.Results:1.Under transmission electron microscopy,the exosomes isolated and extracted in this study showed a characteristic "teato-like" structure.The results of particle size analysis showed that the particle size of exosomes was 90-120 nm;the particle size of exosomes loaded with miR-26 a after electroporation was 100-150 nm.WB results showed that the surface marker protein CD9 of exosomes was highly expressed;the specific protein Calnexin of endoplasmic reticulum was lowly expressed.2.Fluorescence microscopy showed scattered miR-26 a fluorescent markers in MC3T3-E1 cells co-cultured with exosomes carrying miR-26a;whereas no fluorescent markers were seen in MC3T3-E1 cells co-cultured with exosomes not carrying miR-26 a.This demonstrates the feasibility of transfecting cells by exosomes.3.Transmission electron microscopy showed that the fibrous structure of PRF crossed each other and showed a three-dimensional meshwork structure,which accommodated multiple cellular components.4.The PRF/exosome/miR-26 a complex was observed under fluorescence microscopy,and uniformly distributed fluorescently labeled spots were visible;while no fluorescent labeling was seen in the PRF/exosome complex.CCK8 results showed that cell proliferation was significantly accelerated in the PRF group,PRF/exosome group and PRF/exosome/miR-26 a complex group compared with the blank and exosome groups from day 3(p<0.05).Among them,the PRF/exosome/miR-26 a complex had the most significant promotion effect on cell proliferation.6.During osteogenic differentiation of MC3T3-E1,the osteogenic effect of blank group and exosome alone group was not significant;PRF group,PRF/exosome group and PRF/exosome/miR-26 a complex group all significantly upregulated the expression of osteogenic-related genes in MC3T3-E1(p<0.05).CONCLUSION: The dual gene delivery system constructed by PRF/exosome/miR-26 a complex can promote osteogenic differentiation of osteoblast-associated cells.It provides a new approach for gene therapy to repair bone defects.