Exosomes from bone marrow mesenchymal stem cells enhance fracture healing through the promotion of osteogenesis and angiogenesis in a rat model of nonunion

Abstract

Background: As important players in cell-to-cell communication, exosomes (exo) are believed to play a similar role in promoting fracture healing. This study investigated whether exosomes derived from bone marrow mesenchymal stem cells (BMMSC-Exos) could improve fracture healing of nonunion. Methods: BMMSC-Exos were isolated and transplanted into the fracture site in a rat model of femoral nonunion (Exo group) every week. Moreover, equal volumes of phosphate-buffered saline (PBS) and exosome-depleted conditioned medium (CM-Exo) were injected into the femoral fracture sites of the rats in the control and CM-Exo groups. Bone healing processes were recorded and evaluated by radiographic methods on weeks 8, 14 and 20 after surgery. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative week 20. The expression levels of osteogenesis-or angiogenesis-related genes were evaluated in vitro by western blotting and immunohistochemistry. The ability to internalize exosomes was assessed using the PKH26 assay. Altered proliferation and migration of human umbilical vein endothelial cells (HUVECs) and mouse embryo osteoblast precursor cells (MC3TE-E1s) treated with BMMSC-Exos were determined by utilizing EdU incorporation, immunofluorescence staining, and scratch wound assay. The angiogenesis ability of HUVECs was evaluated through tube formation assays. Finally, to explore the effect of exosomes in osteogenesis via the BMP-2/ Smad1/RUNX2 signalling pathway, the BMP-2 inhibitors noggin and LDN193189 were utilized, and their subsequent effects were observed. Results: BMMSC-Exos were observed to be spherical with a diameter of approximately 122 nm. CD9, CD63 and CD81 were expressed. Transplantation of BMMSC-Exos obviously enhanced osteogenesis, angiogenesis and bone healing processes in a rat model of femoral nonunion. BMMSC-Exos were taken up by HUVECs and MC3T3-E1 in vitro, and their proliferation and migration were also improved. Finally, experiments with BMP2 inhibitors confirmed that the BMP-2/Smad1/RUNX2 signalling pathway played an important role in the pro-osteogenesis induced by BMMSC-Exos and enhanced fracture healing of nonunion.

Exosomes: roles and therapeutic potential in osteoarthritis

“…93 Similarly, BMSC-derived exosomes can enhance osteogenesis and angiogenesis partially through the BMP-2/Smad1/RUNX2 and HIF-1alpha/VEGF pathways at fracture sites to accelerate fracture repair in a femoral nonunion rat model. 94 Moreover, umbilical cord MSC-derived exosomes also promote fracture healing in a rat model of stabilized fracture, which is involved in the HIF-1alpha-mediated regulation of angiogenesis. 95 In addition to osteoporosis and fracture, an increasing number of studies have revealed that exosomes are closely involved in OA pathology and have strong therapeutic potential for this disease.…”

“…171,172 Moreover, BMSCderived exosomes can regulate a variety of physiological and pathological processes, including the immune response, osteogenesis, fibrosis, and angiogenesis. 94,[173][174][175] Several studies have reported that BMSC-derived exosomes significantly promote the regeneration and repair of injured tissues, including cartilage and subchondral bone. 174,[176][177][178][179][180][181] The exosomes as well as MVs/ microparticles (MPs) from TGFβ3-pretreated BMSCs significantly increased the expression of anabolic markers and decreased the levels of catabolic marker genes in osteoarthritic chondrocytes.…”

Mesenchymal stem cell exosomes in bone regenerative strategies—a systematic review of preclinical studies

“…Increasingly, MSC exosomes have replicated the wide-ranging therapeutic efficacies of MSCs in several injury and disease indications such as myocardial ischemia reperfusion injury [ 29 ], drug-induced liver injury [ 30 ], retinal laser injury [ 31 ], pulmonary hypertension [ 32 ], and graft-versus-host-disease [ 33 , 34 ]. MSC exosomes were also reported to be efficacious in enhancing skeletal muscle regeneration [ 35 , 36 ] ​and more recently cartilage [ 23 , [37] , [38] , [39] , [40] ] and bone regeneration [ [41] , [42] , [43] , [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] , [56] , [57] , [58] , [59] , [60] , [61] , [62] , [63] ].…”

Exosomes Enhance Adhesion and Osteogenic Differentiation of Initial Bone Marrow Stem Cells on Titanium Surfaces

“…Moreover, many studies have shown that multiple regulatory factors and complex signaling pathways involved in the process of osteogenesis differentiation are regulated by MSC-exosomes. Specific pathways including Wnt, BMP, PI3K/Akt, insulin, TGFβ, and calcium signaling pathways may be affected by MSC-exosomes ( Cooper et al, 2019 ; Wei et al, 2019 ; Zhang et al, 2020 ). In aggregate, these researches demonstrate that MSC-exosomes carry much information that impacts key gene activation for osteogenesis including SATB2, Runx2, Dlx5, and Osterix (Osx; Fang et al, 2015 ; Huang et al, 2017 ).…”