Exosomes from Human Synovial-Derived Mesenchymal Stem Cells Prevent Glucocorticoid-Induced Osteonecrosis of the Femoral Head in the Rat

Abstract

Osteonecrosis of the femoral head (ONFH) represents a debilitating complication following glucocorticoid (GC)-based therapy. Synovial-derived mesenchymal stem cells (SMSCs) can exert protective effect in the animal model of GC-induced ONFH by inducing cell proliferation and preventing cell apoptosis. Recent studies indicate the transplanted cells exert therapeutic effects primarily via a paracrine mechanism and exosomes are an important paracrine factor that can be directly used as therapeutic agents for tissue engineering. Herein, we provided the first demonstration that the early treatment of exosomes secreted by human synovial-derived mesenchymal stem cells (SMSC-Exos) could prevent GC-induced ONFH in the rat model. Using a series of in vitro functional assays, we found that SMSC-Exos could be internalized into bone marrow derived stromal cells (BMSCs) and enhance their proliferation and have anti-apoptotic abilities. Finally, SMSC-Exos may be promising for preventing GC-induced ONFH.

Keywords: apoptosis.; glucocorticoid; osteonecrosis of the femoral head; synovial-derived mesenchymal stem cells.

Figure 1

Isolation of human synovial-derived mesenchymal stem cells (SMSCs) from human synovial membrane. (A) SMSCs exhibited a typical spindle-shaped morphology. Scale bar: 100μm. (B) Flow cytometry analysis of the cell surface markers on SMSCs. The isotype controls were illustrated as blank curves and the test samples were illustrated as solid gray curves. (C) SMSCs displayed ability of adipogenic differentiation (Yellow scale bar: 20 μm), osteogenic differentiation (Green scale bar: 100 μm) and chondrogenic differentiation ((white scale bar: 100 μm).

Figure 2

Characterization of exosomes released by human synovial-derived mesenchymal stem cells (SMSC-Exos). (A) Particle size distribution of SMSC-Exos measured by DLS. (B) Morphology of SMSC-Exos under a transmission electron microscopy. Scale bar: 100nm. (C) Western blotting analysis of exosomal surface markers (including CD9, CD63, CD81, and TSG101). Exosomes from SMSCs of six patients were used. The ultrafiltration liquid of MesenGro hMSC Medium was used as negative control (NC).

Figure 3

SMSC-Exos transplantation induces bone tissue-protective effects in GC-treated rats. (A) Reconstruction of coronal, transverse, and sagittal images of bones within the femoral heads in normal, methylprednisolone (MPS)-treated, and MPS+SMSC-Exos co-treated rats. (B) Quantitative analyses of the trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), bone volume per tissue volume (BV/TV), and trabecular number (Tb.N) in different treatment groups. (C) H&E staining of the femoral heads in rats receiving different treatments. Scale bar: 100μm. (D) Immunohistochemical staining of OCN of the specimen in different treatment groups. Scale bar: 100μm.

Figure 4

SMSC-Exos transplantation induces bone marrow cell-protective effects in GC-treated rats. (A) Cellular apoptosis in different treatment groups was analyzed by TUNEL assay. Scale bar: 50μm. Quantitative analysis of the number of total apoptotic cells in (A). (B) The proliferation of cells in different treatment groups was analyzed ki67 immunostaining. Scale bar: 50μm. Quantitative analysis of the number of total proliferative cells in (B). (*P < 0.05 compared with the normal rats (control), ^#P < 0.05 compared with the MPS-treated rats (model group).)

Figure 5

Uptake of SMSC-Exos by BMSCs and their proliferative and anti-apoptotic effects on endothelial cells. (A) Fluorescent microscopy analysis of DiL-labeled SMSC-Exos uptake by BMSCs. The red-labeled exosomes were visible in the perinuclear region of BMSCs. Scale bar: 50μm. (B) The proliferation of BMSCs was analyzed by Cell Counting Kit-8 assay. (C) The apoptosis of BMSCs was assessed by flow cytometry with annexin V-FITC/propidium iodide (PI) double staining. Cells only stained with annexin V-FITC represent the early apoptotic cells. (D) Quantitative analysis of the early apoptosis rate of BMSCs. (*P < 0.05 compared with the control group, ^#P < 0.05 compared with the dexamethasone (DEX)-treated group).