Dimethyloxaloylglycine-stimulated human bone marrow mesenchymal stem cell-derived exosomes enhance bone regeneration through angiogenesis by targeting the AKT/mTOR pathway

Abstract

Background: Mesenchymal stem cell (MSC)-derived exosomes have been recognized as new candidate agents for treating critical-sized bone defects; they promote angiogenesis and may be an alternative to cell therapy. In this study, we evaluated whether exosomes derived from bone marrow-derived MSCs (BMSCs) preconditioned with a low dose of dimethyloxaloylglycine (DMOG), DMOG-MSC-Exos, exert superior proangiogenic activity in bone regeneration and the underlying mechanisms involved.

Methods: To investigate the effects of these exosomes, scratch wound healing, cell proliferation, and tube formation assays were performed in human umbilical vein endothelial cells (HUVECs). To test the effects in vivo, a critical-sized calvarial defect rat model was established. Eight weeks after the procedure, histological/histomorphometrical analysis was performed to measure bone regeneration, and micro-computerized tomography was used to measure bone regeneration and neovascularization.

Results: DMOG-MSC-Exos activated the AKT/mTOR pathway to stimulate angiogenesis in HUVECs. This contributed to bone regeneration and angiogenesis in the critical-sized calvarial defect rat model in vivo.

Conclusions: Low doses of DMOG trigger exosomes to exert enhanced proangiogenic activity in cell-free therapeutic applications.

Keywords: Angiogenesis; Bone regeneration; Dimethyloxaloylglycine; Exosome; Mesenchymal stem cell; Tissue engineering.

Fig. 1

Internalization and proangiogenic effects of MSC-Exos in HUVECS. a Fluorescent microscopy showing internalization of DIO-labeled DMOG-MSC-Exos by HUVECS. Green-labeled exosomes are visible in the perinuclear region of HUVECs. b Proliferation of HUVECS treated with exosomes (50 μg/mL). c Wound healing assay of HUVECs treated with exosomes (50 μg/mL). d Quantitative analysis of wound closure. e Tube formation capacity of HUVECs treated with exosomes. f Quantitative analysis of total tube length (*P < 0.05, versus control; ^#P < 0.05, versus MSC-Exos)

Fig. 2

Micro-CT evaluation of repaired craniums and blood vessel formation at 8 weeks post-implantation. a Three-dimensional reconstruction and sagittal images showed different reparative effects of HA, MSC-Exos, and DMOG-MSC-Exos. b New blood vessels in calvarial defects are shown in three-dimensional reconstruction images. c CD31 immunohistochemistry of bone defect regions of all groups at 8 weeks post-surgery (green fluorescence indicates newly formed blood vessels). d Bone mineral density (BMD) of each group. e Bone volume-to-total volume ratio (BV/TV) in each group. f Morphometric analysis to determine local vessel area in bone defects (*P < 0.05, versus control; #P < 0.05, versus MSC-Exos)

Fig. 3

Histomorphometric analysis of new bone regeneration via fluorochrome labeling and histological analysis. a Staining with tetracycline (column 1, yellow) after 2 weeks, alizarin red (column 2, red) after 4 weeks, and calcein (column 3, green) after 6 weeks. Column 4 shows the merged images of the three fluorochromes for the same groups (scale bar = 100 μm, white arrows indicate round shapes, high intensity, and large mass of newly formed bone in HA pores; white asterisks indicate edges of bone defect). b Percentage of each fluorochrome area in each group. c Undecalcified craniums were sliced, and sections were stained with van Gieson’s picrofuchsin. New bone and HA are shown in red and black, respectively. d New bone area in each group (*P < 0.05, versus control; #P < 0.05, versus MSC-Exos)

Fig. 4

Mechanism of proangiogenic effects of DMOG-MSC-Exos. a HUVECs were treated with MSC-Exos with or without DMOG, treated with PBS as the control, mRNA levels of p53, p21, p65, Hippo-YAP, JAK-STAT, β-catenin, and PTEN were evaluated by qRT-PCR. b HUVECs were treated with MSC-Exos, DMOG-MSC-Exos, and PBS as a control; protein levels of members of the AKT/mTOR pathway were detected by western blotting. Actin was used as a loading control (*P < 0.05, versus control; #P < 0.05, versus MSC-Exos)

Fig. 5

Proangiogenic effects of DMOG-MSC-Exos in HUVECS after AKT/mTOR pathway was blocked. a DMOG-MSC-Exos were treated with MK2206 and blocking of AKT/mTOR signaling was detected by western blotting. b Wound healing assay in HUVECs treated with exosomes (50 μg/mL). c Tube-forming capacity of HUVECs treated with exosomes (50 μg/mL). d Quantitative analysis of wound closure. e Quantitative analysis of total tube length (*P < 0.05, versus control; #P < 0.05, versus MSC-Exos)