Exosomes Derived from Cyclic Mechanical Stretch-exposed Bone Marrow Mesenchymal Stem Cells Inhibit RANKL-induced Osteoclastogenesis through the NF-κB Signaling Pathway

Abstract

Background: Skeletal unloading usually induces severe disuse osteoporosis (DOP), which often occurs in patients subjected to prolonged immobility or in spaceflight astronauts. Increasing evidence suggests that exosomes are important mediators in maintaining the balance between bone formation and bone resorption. We hypothesized that exosomes play a pivotal role in the maintenance of bone homeostasis through intercellular communication between bone marrow mesenchymal stem cells (BMSCs) and osteoclasts under mechanical loading.Methods: Cells were divided into cyclic mechanical stretch (CMS)-treated BMSCs and normal static-cultured BMSCs, and exosomes were extracted by ultracentrifugation. After incubation with CMS-treated BMSC-derived exosomes (CMS_Exos) or static-cultured BMSC-derived exosomes (static_Exos), the apoptosis rates of bone marrow macrophages (BMMs) were determined by flow cytometry, and cell viability was detected with a CCK-8 assay. Osteoclast differentiation was determined with an in vitro osteoclastogenesis assay. Signaling pathway activation was evaluated by Western blotting and immunofluorescence staining. Hindlimb unloading (HU)-induced DOP mouse models were prepared to evaluate the function of exosomes in DOP.Results: Both CMS_Exos and static_Exos could be internalized by BMMs, and CMS_Exos did not affect BMM viability or increase apoptosis. CMS_Exos effectively suppressed receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclast differentiation and F-actin ring formation. Further molecular investigation demonstrated that CMS_Exos impaired osteoclast differentiation via inhibition of the RANKL-induced NF-κB signaling pathway. Both CMS_Exos and static_Exos partly rescued the osteoporosis caused by mechanical unloading; however, the CMS_Exo group showed more obvious rescue. CMS_Exo treatment significantly decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts.Conclusions: Exosomes derived from CMS-treated BMSCs strongly inhibited osteoclast differentiation by attenuating the NF-κB signaling pathway in vitro and rescued osteoporosis caused by mechanical unloading in an HU mouse model in vivo.

Emerging biogenesis technologies of extracellular vesicles for tissue regenerative therapeutics

“…In another similar study with MSCs, the cyclically stretched cells synthesized EVs that could inhibit osteoclast differentiation. 36 The event was demonstrated to relate with the attenuated RANKL-induced NF-κB signaling pathway, and the EVs could effectively rescue the in vivo osteoporosis caused by the unloading of the hindlimb, suggesting that the anti-osteoclastic role of endogenous MSCs in vivo might be due to the generation of stretch-induced therapeutic EVs. This study envisions mechanical conditioning as a potential strategy to collect therapeutic EVs from MSCs targeting diseases in skeletal systems.…”