Study On The Biological Characteristics Of Mesenchymal Stem Cell-derived Microvesicles And Their Application Potential In Tissue Engineering

Biological characteristics of bone marrow-derived MSC-MVsAims:To investigate the biological characteristics of bone marrow-derived MSC-MVs, including their morphology, size and immunophenotype, and to study the expression of miRNAs enclosed in MSC-MVs, providing insight into the investigation of the biological function and potential clinical application of bone marrow-derived MSC-MVs in future.Methods:Rat bone marrow-derived MSCs were cultured and characterized by their immune-phenotype and tri-lineage differentiation capacity. MSC-MVs were isolated by differential centrifugation from the conditioned medium of MSCs and observed by a scanning electron microscope and a laser confocal microscope. The immunophenotype of MSC-MVs was determined by flow cytometry. The protein content of MVs was measured to compare the difference between the quantity of MVs released by cells under normal and stress conditions. The expression of miRNAs enclosed in MSC-MVs was determined by microarray analysis, and gene ontology analysis was performed to predict the biological processes in which the potential target genes of miRNAs may participate.Results:The immunophenotype of rat bone marrow-derived MSCs was in accordance with the classical immune-phenotype of MSCs, and the tri-lineage differentiation capacity of MSCs was confirmed in vitro. When observed by scanning electron microscopy and laser confocal microscopy, MSC-MVs showed a spheroidal morphology, and their sizes were heterogeneous with diameters in the range 100-1000 nm. The immunophenotype of MSC-MVs was similar to their parent cells, as demonstrated by flow cytometry, which suggests that they are miniature versions of their parent cells. By measuring the protein content of MVs released by MSCs under normal and stress condition, we found that stress can induce cells to release a larger quantity of MVs. Additionally, by using microarray analysis, we discovered that MSC-MVs contained 274 miRNAs, and these miRNAs were predicted to participate in various biological processes, including regulation of transcription, intracellular signaling cascade and regulation of cell proliferation, etc. We also found that many miRNAs that were reported to be involved in regulation of differentiation and fate of stem cells, angiogenesis, hematopoiesis and immune responses were expressed in MSC-MVs.Conclusions:Rat bone marrow-derived MSCs could secret a large quantity of MVs with a diameter between 100-1000 nm during their in vitro cultivation. These MVs share similar membrane marker expression pattern with their parent cells, and contain miRNAs that are involved in various biological processes.The in vitro biological function of bone marrow-derived MSC-MVsAims:In bone tissue engineering applications, the seed cells in scaffolds often suffer from hypoxia and poor nutrient supply immediately after in vivo transplantation due to the lack of vascularization. Based on the reported biological functions of MVs released by MSCs derived from other species and tissues, we investigated whether rat bone marrow-derived MSC-MVs possess proangiogenic effects on epithelial cells, exert anti-apoptotic effects on apoptotic seed cells and influence osteogenic differentiation capacity of the seed cells.Methods:The effects of MSC-MVs on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) were investigated by CCK-8 test, wound healing assay and matrigel-based tube formation assay. In addition, MSCs were cultured in a hypoxic and serum-deprivated condition to mimic their in vivo environment. TUNEL assay and qRT-PCR analysis were performed to investigate the effects of MSC-MVs on the hypoxia and serum-deprivation induced apoptotic MSCs. Meanwhile osteogenically induced MSCs and non-induced MSCs were incubated with MSC-MVs, alizarin red staining and qRT-PCR analysis were performed to investigate the effect of MSC-MVs on the osteogenic differentiation capacity of MSCs.Results:MSC-MVs could promote the proliferation, migration and tube formation of HUVECs in a dose-dependent manner. We also demonstrated that hypoxia and serum-deprivation could induce apoptosis of MSCs, and MSC-MVs could not decrease the number of TUNEL positive effect and had no significant effect on the expression of apoptosis-related genes in MSCs. Additionally, we proved that MSC-MVs could not promote osteogenic differentiation of their parent cells, and had no significant effect on the expression of osteogenesis-related genes in MSCs.Conclusions:Our findings indicate that MSC-MVs possess potent pro-angiogenic properties, and could not exert anti-apoptotic effect and pro-osteogenic effect on MSCs. These findings may provide insight into the potential application of MSC-MVs in bone tissue engineering.The application potential of bone marrow-derived MSC-MV in bone tissue engineeringAims:Since MSC-MVs possess potent pro-angiogenic properties, we speculate that MSC-MVs could promote bone regeneration through accelerating vascularization. Seed cells, construction method and biomaterials are the three pillars in bone tissue engineering applications. We investigated the potential application of MSC-MVs in the fabrication of cell-MV-biomaterial constructs and MV-modified biomaterials.Methods:From the standpoint of construction method, osteogenically induced MSCs and MSC-MVs were incorporated into alginate and seeded on 3D printed PCL scaffold to obtain cell-MV-biomaterial constructs. From the standpoint of biomaterial, MSC-MVs were coated onto DBM scaffold to fabricate MV-modified biomaterial. Cell-MV-biomaterial constructs and MV-modified biomaterial were characterized by phase contrast microscope, scanning electron microscope and laser confocal microscope. We implanted cell-MV-biomaterial constructs and MV-modified biomaterial and various controls into an ectopic bone formation model in nude mice. Micro CT, hematoxylin-eosin staining and CD31 immunohistochemical staining were carried out to investigate whether MSC-MVs could promote bone regeneration and vascularization in vivo.Results:The seed cells were homogeneously distributed in the cell-MV-biomaterial construct, and MSC-MVs were homogeneously distributed on the micro-porous surface of DBM scaffolds. When implanted in vivo, the cell-MV-biomaterial constructs and MV-modified biomaterials could enhance bone regeneration and vascularization in an ectopic bone formation model in nude mice, as demonstrated by micro CT, hematoxylin-eosin staining and CD31 immunohistochemical staining.Conclusions:To the best of our knowledge, it is the firet time to utilize MSC-MVs to fabricate cell-MV-biomaterial composite constructs and MV-modified biomaterials. Through an ectopic bone formation model, we proved that MSC-MVs promoted bone regeneration and vascularization in vivo. Our findings suggest that MSC-MVs hold great potential in bone tissue engineering applications.