Exosomes from human umbilical cord mesenchymal stem cells enhance fracture healing through HIF‐1α‐mediated promotion of angiogenesis in a rat model of stabilized fracture

Abstract

Objectives: Exosomes, as important players in intercellular communication due to their ability to transfer certain molecules to target cells, are believed to take similar effects in promoting bone regeneration with their derived stem cells. Studies have suggested that umbilical cord mesenchymal stem cells (uMSCs) could promote angiogenesis. This study investigated whether exosomes derived from uMSCs (uMSC-Exos) could enhance fracture healing as primary factors by promoting angiogenesis.Materials and Methods: uMSCs were obtained to isolate uMSC-Exos by ultrafiltration, with exosomes from human embryonic kidney 293 cells (HEK293) and phosphate-buffered saline (PBS) being used as control groups. NanoSight, laser light scattering spectrometer, transmission electron microscopy and Western blotting were used to identify exosomes. Next, uMSC-Exos combined with hydrogel were transplanted into the fracture site in a rat model of femoral fracture. Bone healing processes were monitored and evaluated by radiographic methods on days 7, 14, 21 and 31 after surgery; angiogenesis of the fracture sites was assessed by radiographic and histological strategies on post-operative day 14. In vitro, the expression levels of osteogenesis-or angiogenesis-related genes after being cultured with uMSC-Exos were identified by qRT-PCR. The internalization ability of exosomes was determined using the PKH67 assay. Cell cycle analysis, EdU incorporation and immunofluorescence staining, scratch wound assay and tube formation analysis were also used to determine the altered abilities of human umbilical vein endothelial cells (HUVECs) administered with uMSC-Exos in proliferation, migration and angiogenesis. Finally, to further explore the underlying molecular mechanisms, specific RNA inhibitors or siRNAs were used, and the subsequent effects were observed.Results: uMSC-Exos had a diameter of approximately 100 nm, were spherical, meanwhile expressing CD9, CD63 and CD81. Transplantation of uMSC-Exos markedly enhanced angiogenesis and bone healing processes in a rat model of femoral fracture.In vitro, other than enhancing osteogenic differentiation, uMSC-Exos increased the expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α). uMSC-Exos were taken up by HUVECs and enhanced theirThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Biomaterials Functionalized with MSC Secreted Extracellular Vesicles and Soluble Factors for Tissue Regeneration

“…[ 222 ] A hyaluronan/heparin hydrogel loaded with MSC‐EVs enhanced angiogenesis and promoted significant bone healing in a femoral fracture model, with EV‐induced angiogenesis shown to be mediated by upregulation of HIF‐1α and VEGF expression in endothelial cells. [ 223 ] In another study, MSC‐EV laden hydrogels significantly accelerated bone healing in critical‐sized calvaria defects in rats, with EV‐shuttled miR‐196a identified as having the most potent osteogenic effects in vitro. [ 224 ] EVs from osteogenically primed MSCs immobilized on a poly(lactic‐ co ‐glycolic acid)/polydopamine (PLGA/pDA) scaffold promoted the recruitment of endogenous MSCs and increased bone healing in a critical‐sized mouse calvaria defect.…”

Angiogenic Effects of Human Dental Pulp and Bone Marrow-Derived Mesenchymal Stromal Cells and their Extracellular Vesicles

“…For example, human umbilical cord MSC-derived EVs could induce angiogenesis by Wnt4/β-catenin signalling [39], while human BM-MSC EVs stimulated in vitro tube formation via the NF-κB pathway [40]. In addition, VEGF and VEGF receptor expression could be enhanced in endothelial cells by HIF-1α activation [21,41]. By using an antibody array, we confirmed the presence of both pro-and anti-angiogenic proteins in EVs and EV-depleted CM of DPSCs and BM-MSCs.…”

Perspectives for Future Use of Extracellular Vesicles from Umbilical Cord- and Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells in Regenerative Therapies—Synthetic Review

“…Our synthetic review points to the most important mechanisms of extracellular vesicles activity, which has been identified following their administration in several neurological, cardiovascular, liver, kidney, and skin injuries. Moreover, there is currently growing evidence supporting important role of extracellular vesicles derived from ADSCs and UC-MSCs in other conditions including in bone regeneration [96][97][98] and cancers [99][100][101], indicating similar mechanisms to be involved (summarized in Figure 1). MSC-derived EVs may play their role by following mechanisms of action after transplantation: (i) regulation of inflammatory response (increase in level of anti-inflammatory cytokines and decrease in pro-inflammatory ones), (ii) increasing endogenous cells proliferation, differentiation and viability in place of injury (e.g., by autophagy regulation), (iii) reducing oxidative stress, and iv) enhancing angiogenesis in place of injury, which together may contribute to tissue repair and prevent organs and tissues from severe anatomical and functional damages (Figure 1).…”