| ObjectiveBone tissue defects are facing huge clinical repair needs.Bone tissue engineering technology,especially the strategy of using exosomes to promote tissue repair,has outstanding advantages.Exosomes can be encapsulated in microspheres,and the microspheres are then adhered to the porous scaffold to form a cell-free tissue engineering scaffold.Promoting cells to efficiently release exosomes can increase the amount of exosomes obtained,which will be beneficial to the tissue engineering application of exosomes.MethodMC3T3 cells were transfected with recombinant plasmid to overexpress the Rab27a gene.After transfection,the expression level of protein and mRNA was detected by RT-PCR and Western Blot experiment.The release level of exosomes is detected by BCA protein concentration,and exosomes are identified by Western Blot,NTA,TEM,internalization experiments.The exosomes secreted by MC3T3-WT and MC3T3-Rab27a under different culture conditions and culture time were added to BMSCs to promote osteogenic differentiation,and detected with alkaline phosphatase(ALP)staining,ALP content analysis,alizarin red staining,and osteogenic-related gene expression levels,in order to determine optimal time-point and concentration of exosomes.In addition,perform protein profiling of exosomes to study the protein composition of exosomes.Signal pathways related to differentially expressed genes are analyzed by mRNA sequencing.Then,mitochondrial biogenesis,mitophagy,glucose metabolism indicators,mitochondrial membrane potential are detected.Mitochondrial proteins are extracted for co-immunoprecipitation to detect proteins that interact with Rab27a.Furthermore,VDAC1 phosphorylation level,and mitochondrial MPTP opening degree is detected.In addition,BMSCs were also transfected with Rab27a to verify the effect of Rab27a on mitochondrial-related indexes.5%PEGDA/GelMA/Exo microspheres(PGExo)are produced by a microfluidic chip,and the sustained release effect of exosomes is determined by fluorescence intensity analysis and BCA.The PLA porous scaffold is prepared by phase separation combined with low-temperature 3D printing.After the scaffold is coated with a polydopamine(pDA)film,PGExo is added dropwise to bond the microspheres and the scaffold.ResultMC3T3-Rab27a have a 43.26%increase in exosoma secretion level compared with wild-type cells.Osteogenic induction cultured MC3T3-Rab27a secreted exosomes at a concentration of 100 μg/mL,which is beneficial to promote osteogenic differentiation of BMSCs.The exosomes contain paracrine factors and Rab27a protein that promote osteogenic differentiation.The mRNA sequencing of MC3T3-Rab27a showed that metabolism and mitophagy-related genes were differentially expressed compared with wild-type cells.The test results showed that overexpression of Rab27a promoted the aerobic oxidation of glucose in the early stage of osteogenic differentiation,and enhanced the mitochondrial biogenesis and mitophagy of the MC3T3-Rab27a.Furthermore,Rab27a promotes the opening of MPTP by promoting the phosphorylation of VDAC1,thereby promoting the formation of calcium phosphate in the mitochondria,and accelerating the process of osteogenic differentiation of MC3T3-Rab27a.Similarly,the overexpression of Rab27a in BMSCs also leads to the enhancement of mitochondrial related indicators and osteogenic differentiation.ConclusionThe overexpression of Rab27a gene promotes the efficient secretion of exosomes of MC3T3,and the exosomes secreted by MC3T3-Rab27a after osteoblastic induction,can promote osteogenic differentiation of BMSCs.Rab27a promotes the phosphorylation of VDAC1,accelerates the process of osteogenic differentiation of MC3T3,and makes its exosomes rich in paracrine factors.The PGExo microspheres encapsulating exosomes can release exosomes with a long-term effect.The PGExo microspheres and the PLA porous are bonded together through the pDA film to form a PGExo/pDA/PLA exosome sustained-release scaffold,which can be used as a cell-free tissue engineering scaffold for bone defect repair. |
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