Abstract:Objective: To explore the repairing effect of exosomes derived from human umbilical cord mesenchymal stem cells (HucMSCs) on the skull defect of osteoporosis rats. Methods: Isolation the exosomes from HucMSCs, the morphology and size of the exosomes were identified by transmission electron microscopy and nanoparticle tracking analysis, and the surface markers were identified by Western blot. Rat bone marrow mesenchymal stem cells (BMSCs) were isolated and their surface markers were identified by flow cytometry. Exosomes (control, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL) were co-cultured with BMSCs, and the effects of different concentra-tions of exosomes on proliferation and early osteogenic differentiation of BMSCs were detected. Osteoporosis model was established by ovariectomy (OVX) in SD rats. Exosomes suspension was injected at the same time as the skull defect model was estab-lished. Eight weeks after operation, the rats were euthanized. micro-CT scanning and bone mass analysis was performed on the tissue of the operation area, and the healing of bone defect was observed under microscope after HE staining, so as to evaluate the repairing potential of exosomes from HucMSCs on the skull defect of osteoporosis rats. Results: The results of transmission electron microscope and nanoparticle tracking analysis showed that the shape and size of exosomes derived from HucMSCs met the requirements, and the expression of surface protein markers CD63 and CD81 were positive. The surface markers CD29, CD90 and CD45 of rats BMSCs were expressed as 99.86%, 99.50% and 4.12% respectively, which accorded with the characteristics of mesenchymal stem cells. After co-culturing 60 μg/mL exosomes with BMSCs for 5 days, the cell viability was (127.782±7.184) % of the control group (P=0.001), the expression of alkaline phosphatase (ALP) was (2.715±0.095) times of the control group (P=0.003), and the expression of Runx family transcription factor 2 (Runx2) was (2.175±0.731) times of the control group (P=0.140). In the repair of an OVX-induced osteoporosis SD rat skull defect model, micro-CT showed that the exosome group had a significant increase in bone healing compared with the control group. The results of bone mass analysis showed that in the exosome group, the bone volume/tissue volume was (11.000±1.708) % (P=0.008), the trabecular number was (0.742±0.112) mm-1 (P=0.014), the trabecular thickness was (0.175±0.008) mm (P=0.005), and the trabecular separation was (1.402±0.507) mm (P=0.022). Histological results showed that the new bone had a complete structure, with clear layers, as dense lamellar bone, and uniform bone matrix and bone lacunae. Conclusion: The exosomes derived from HucMSCs can promote the proliferation and osteogenic differentiation of BMSCs, and have the ability to repair osteoporotic bone defects.
