Abstract
ObjectivesThe present study aimed to investigate whether exosomes derived from miR‐375‐overexpressing human adipose mesenchymal stem cells (hASCs) could enhance bone regeneration.Materials and MethodsExosomes enriched with miR‐375 (Exo [miR‐375]) were generated from hASCs stably overexpressing miR‐375 after lentiviral transfection and identified with transmission electron microscopy, nanosight and western blotting. The construction efficiency of Exo (miR‐375) was evaluated with qRT‐PCR and incubated with human bone marrow mesenchymal stem cells (hBMSCs) to optimize the effective dosage. Then, the osteogenic capability of Exo (miR‐375) was investigated with ALP and ARS assays. Furthermore, dual‐luciferase reporter assay and western blotting were conducted to reveal the underlying mechanism of miR‐375 in osteogenic regulation. Finally, Exo (miR‐375) were embedded with hydrogel and applied to a rat model of calvarial defect, and μ‐CT analysis and histological examination were conducted to evaluate the therapeutic effects of Exo (miR‐375) in bone regeneration.ResultsmiR‐375 could be enriched in exosomes by overexpressing in the parent cells. Administration of Exo (miR‐375) at 50 μg/mL improved the osteogenic differentiation of hBMSCs. With miR‐375 absorbed by hBMSCs, insulin‐like growth factor binding protein 3 (IGFBP3) was inhibited by binding to its 3′UTR, and recombinant IGFBP3 protein reduced the osteogenic effects triggered by Exo (miR‐375). After incorporated with hydrogel, Exo (miR‐375) displayed a slow and controlled release, and further in vivo analysis demonstrated that Exo (miR‐375) enhanced the bone regenerative capacity in a rat model of calvarial defect.ConclusionsTaken together, our study demonstrated that exosomes derived from miR‐375‐overexpressing hASCs promoted bone regeneration.
Highlights
Mesenchymal stem cells (MSCs) are broadly utilized in bone tissue engineering owing to their ability of multipotential differentiation
With the development of cell‐free transplantation strategy, we considered whether human adipose mesenchymal stem cells (hASCs)‐derived exo‐ somes could be applied as a carrier of osteogenic miRNA to achieve a combination of their functions and effects
Exosomal concentration was evaluated according to the protein level, and 25 μg/mL exosomes could be obtained from almost 100 mL hASC supernatants. qRT‐PCR analysis showed that Exo treatment led to a remarkable increase in miR‐375 in human bone marrow mesenchymal stem cells (hBMSCs) for 4 hours, and the effect was dependent on dosage, while no significant difference was detected between the 50 μg/mL and 100 μg/mL groups when the incubation time prolonged to 24 hours (Figure 2B)
Summary
Mesenchymal stem cells (MSCs) are broadly utilized in bone tissue engineering owing to their ability of multipotential differentiation. Encapsulated with a lipid bilayer, exosomes can protect its contents from degradation and transport a variety of small bio‐ molecules including mRNAs, miRNAs, non‐coding RNAs and pro‐ teins to surrounding cells.[3,4] As natural vesicles of gene delivery, MSC‐derived exosomes exhibit a broad range of therapeutic effects, which were previously attributed to MSCs, such as tissue repair, immunological regulation and inflammatory control.[5,6,7] recent studies have revealed MSC‐derived exosomes were able to regulate osteogenic differentiation, promote bone regeneration and ameliorate osteopenia in vivo.[8,9,10] Despite their great potential in therapeutic delivery, MSC‐de‐ rived exosomes have shown limited application in clinical studies be‐ cause of many problems, and the low yield poses a major challenge to further applications.[11,12] Several strategies have been developed to facilitate the release of exosomes, including raising intracellular calcium concentration and serum starvation. We aimed to investigate whether exosomes derived from miR‐375‐overexpress‐ ing hASCs could enhance the therapeutic effects of bone regener‐ ation and provide a basis for the application of exosomes as a gene delivery vehicle to transport therapeutic miRNAs for regenerative therapy
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