Mesenchymal Stem Cell-Derived Exosomes Promote Fracture Healing in a Mouse Model

Abstract

: Paracrine signaling by bone-marrow-derived mesenchymal stem cells (MSCs) plays a major role in tissue repair. Although the production of regulatory cytokines by MSC transplantation is a critical modulator of tissue regeneration, we focused on exosomes, which are extracellular vesicles that contain proteins and nucleic acids, as a novel additional modulator of cell-to-cell communication and tissue regeneration. To address this, we used radiologic imaging, histological examination, and immunohistochemical analysis to evaluate the role of exosomes isolated from MSC-conditioned medium (CM) in the healing process in a femur fracture model of CD9^-/- mice, a strain that is known to produce reduced levels of exosomes. We found that the bone union rate in CD9^-/- mice was significantly lower than wild-type mice because of the retardation of callus formation. The retardation of fracture healing in CD9^-/- mice was rescued by the injection of exosomes, but this was not the case after the injection of exosomes-free conditioned medium (CM-Exo). The levels of the bone repair-related cytokines, monocyte chemotactic protein-1 (MCP-1), MCP-3, and stromal cell-derived factor-1 in exosomes were low compared with levels in CM and CM-Exo, suggesting that bone repair may be in part mediated by other exosome components, such as microRNAs. These results suggest that exosomes in CM facilitate the acceleration of fracture healing, and we conclude that exosomes are a novel factor of MSC paracrine signaling with an important role in the tissue repair process.

Significance: This work focuses on exosomes, which are extracellular vesicles, as a novel additional modulator of cell-to-cell communication. This study evaluated the role of exosomes isolated from mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) in the fracture-healing process of CD9^-/- mice, a strain that is known to produce reduced levels of exosomes. Retardation of fracture healing in CD9^-/- mice was rescued by the injection of MSC exosomes, but this was not the case after the injection of exosome-free CM. This study finds that MSC exosomes are a novel factor of MSC paracrine signaling, with an important role in the tissue repair process.

Keywords: Cytokine; Endochondral ossification; Exosomes; Fracture healing; Mesenchymal stem cells; microRNA.

Figure 1.

Body weight, bone density, and the entire width of the tibial growth plate in CD9^−/− mice. (A, B): The body weight and bone density were measured in WT (n = 10) and CD9^−/− (n = 10) mice at 17 weeks old, just before fracture. (C): The tibial growth plate length was measured in WT and CD9^−/− mice at 1 month old. Growth plate lengths in CD9^−/− mice were significantly shorter than those in WT. Scale bars = 200 μm. Values were expressed as means ± SE. Statistical analysis was performed by Mann-Whitney U test. ∗, p < .05. Abbreviation: WT, wild-type mice.

Figure 2.

Impaired fracture healing in CD9^−/− mice. (A, B): A transverse femoral shaft fracture was produced by using a C-shaped instrument applying three-point bending in WT (n = 15) and CD9^−/− (n = 15) mice. Radiographic imaging by x-ray and bone union rate were performed at 0, 1, 2, 4, and 6 weeks after the fracture. Statistical analysis was performed by Pearson's chi-square test. ∗, p < .05. (C): The femurs of WT and CD9^−/− mice were harvested at 2 and 3 weeks after fracture. After radiographic imaging by x-ray and µCT was performed, the femurs were stained with Toluidine blue, and H&E. Scale bars = 100 μm. Abbreviations: μCT, microcomputed tomography; PO, postoperative; 0W, 0 weeks; WT, wild-type mice.

Figure 3.

Isolation of exosomes from MSC-conditioned medium. (A): MSCs were seeded at 1.0 × 10⁵ cells per well in a six-well plate with MSC growth medium. One day later, the cells were washed with serum-free DMEM and cultured with 2 ml per well serum-free DMEM for 48 hours. To isolate the exosomes, 2 ml of conditioned medium (CM) was collected and centrifuged for 15 minutes at 2,380g and then further ultracentrifuged for 70 minutes at 180,000g. The supernatants were collected as exosome-depleted conditioned medium. The pellets were resuspended in PBS for use as exosomes. (B): Immunoblotting for exosome markers, CD9, CD81, and flotillin-1, in Exo, CM and CM-Exo. (C): Images of the untreated Exo and CM-Exo in solution by FTE system. Original magnification ×20,000. Scale bars = 1 μm. Abbreviations: CM, conditioned medium; CM-Exo, exosome-depleted conditioned medium; DMEM, Dulbecco’s modified Eagle’s medium; Exo, exosomes; MSC, mesenchymal stem cell; PBS, phosphate-buffered saline.

Figure 4.

MSC exosomes rescued delayed fracture healing in CD9^−/− mice. (A): Representative radiologic images of control (n = 15), CM (n = 9), CM-Exo (n = 9), and Exo (n = 9) treated CD9^−/− mice at 0, 1, 2, 4, and 6 weeks after fracture. (B): The average period for bone union. Statistical analysis was performed by Steel-Dwass test. #, p < .05 versus control; ∗, p < .05 versus CM-Exo. (C): Representative histological evaluation of callus formation after Exo injection in CD9^−/− mice. The femurs of PBS control and Exo-treated CD9^−/− mice were assayed 10 days after fracture. The femurs were stained with Toluidine blue (PBS, n = 6; Exo, n = 5), TRAP (PBS, n = 4; Exo, n = 4), and αSM antibody (PBS, n = 4; Exo, n = 4) for histologic analysis of callus. Scale bars = 200 μm (right column) and 100 μm (left column). (D): Quantification of fracture healing, osteoclasts (TRAP) and angiogenesis (αSM) in calluses. Values were expressed as mean ± SE. Statistical analysis was performed by Mann-Whitney U test, ∗, p < .05. (E): Representative radiologic images and µCT images of femurs. The femurs of PBS-injected control CD9^−/− mice, MSC-Exo-treated CD9^−/− mice, and HOS-Exo-treated CD9^−/− mice were harvested at 10 days after fracture. The femurs were stained with Toluidine blue (PBS, n = 6; MSC-Exo, n = 5; HOS-Exo, n = 5) for histologic analysis. Scale bars = 100 μm. Fracture healing scores were generated after histological evaluation. Statistical analysis was performed by Steel-Dwass test. ∗, p < .05. Control, no injection group. Abbreviations: CM, conditioned medium; CM-Exo, exosome-depleted conditioned medium; µCT, microcomputed tomography; Exo, exosomes; HOS, human osteosarcoma cells; MSC, mesenchymal stem cell; PBS, phosphate-buffered saline; αSM, α-smooth muscle actin; TB, Toluidine blue; TRAP, tartrate-resistant acid phosphatase; 0W, 0 weeks; WT, wild type.

Figure 5.

MSC exosomes promote fracture healing in wild-type mice. (A): Representative radiologic images of the femurs of control (n = 15), CM (n = 9), CM-Exo (n = 9), and Exo (n = 9) treated WT mice. (B): The average period for bone union. Statistical analysis was performed by Steel-Dwass test. ∗, p < .05 versus control. Control, no injection group. (C): The concentrations of the bone-repair-related cytokines, MCP-1, -3, and SDF-1 were measured in CM, exosomes, and CM-Exo from MSCs and HOS cells by using Bio-Plex assays. Some values in CM and CM-Exo from MSCs were used with 29,575.6 pg/ml as the maximum concentration within range, because the values of MCP-1 in CM and CM-Exo from MSCs were out of range of the standard curve. Values were expressed as mean ± SE. Statistical analysis was performed by Steel-Dwass test. ∗, p < .05 versus MSC-Exo; ∗∗, p < .01 versus MSC-Exo. hMSC was from nine donors (n = 17). Abbreviations: CM, conditioned medium; CM-Exo, exosome-depleted conditioned medium; Exo, exosomes; HOS, human osteosarcoma cells; MCP-1, monocyte chemotactic protein-1; MSC, mesenchymal stem cell; SDF-1, stromal cell-derived factor-1; 0w, 0 weeks.