Abstract
Mesenchymal stem cells (MSCs) have the potential to reduce healing time and treat nonunion in fracture patients. In this study, bone marrow MSCs-derived extracellular vesicles (B-EVs) were firstly extracted and identified. CD9−/− and normal mice were enrolled for the establishment of fracture models and then injected with B-EVs. Osteoblast differentiation and fracture recovery were estimated. The levels of osteoblast-related genes were detected, and differentially expressed microRNAs (miRs) in B-EVs-treated normal fracture mice were screened and verified. The downstream mechanisms of miR were predicted and assessed. The loss-of functions of miR-335 in B-EV and gain-of-functions of VapB were performed in animal and cell experiments to evaluate their roles in bone fracture. Collectively, B-EVs promoted bone fracture recovery and osteoblast differentiation by releasing miR-335. miR-335 downregulation in B-EVs impaired B-EV functions in fracture recovery and osteoblast differentiation. miR-335 could target VapB, and VapB overexpression reversed the effects of B-EVs on osteoblast differentiation. B-EV treatment activated the Wnt/β-catenin pathway in fracture mice and osteoblasts-like cells. Taken together, the study suggested that B-EVs carry miR-335 to promote bone fracture recovery via VapB and the Wnt/β-catenin pathway. This study may offer insights into bone fracture treatment.
Highlights
Fractures are the most common traumatic injuries to humans[1], and almost occur on subcondylar, parasymphyseal, joints, and mandibular, pelvis and third metacarpal bone[2,3]
Identification of bone marrow-derived MSCs (BMSCs) and BMSCs-derived EVs (B-EVs) Murine BMSCs were able to be induced toward osteogenic and lipogenic differentiation, and flow cytometry verified that the cells used in our experiment were in line with the definition of BMSCs (Supplementary Fig. 1A)
The results showed that EV size was about 120 nm, and EVs were spherical or ellipsoidal under the transmission electron microscope (TEM), and CD63 and CD9 were both positive; no secretion of EVs was found in the extraction after GW4869 intervention; at the same time, adding phosphatebuffered saline (PBS) to the medium of BMSC had no effect on EVs (Supplementary Fig. 1C–E)
Summary
Fractures are the most common traumatic injuries to humans[1], and almost occur on subcondylar, parasymphyseal, joints, and mandibular, pelvis and third metacarpal bone[2,3]. EVs are endocytic membrane-derived vesicles released by cells, which could mediate intercellular communication by carrying proteins, microRNAs (miRs), and RNAs between cells or remote organs[13]. In the bone microenvironment, EVs play an important role in the communication between bone cells, namely osteoblasts, osteoclasts, and BMSCs14. MSCs-derived EVs saved the delay of fracture healing in CD9−/− mice and wild-type (WT) mice[7]. It has been suggested that a large number of miRs participate in EV-mediated intercellular communication and play significant roles in morphogenesis, tissue repair, and organogenesis[16]. In this study, we firstly explored the dysregulated miRs in BMSCs-derived EVs (B-EVs), and studied the effects of B-EVs and the molecular mechanisms in fracture recovery
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