Exosomal miRNA-128-3p from mesenchymal stem cells of aged rats regulates osteogenesis and bone fracture healing by targeting Smad5

Abstract

Transplantation of mesenchymal stem cells (MSCs) has been considered an effective therapeutic treatment for a variety of diseases including bone fracture. However, there are associated complications along with MSCs transplantation. There is evidence to show that exosomes (Exos) derived from MSCs exert a similar paracrine function. In addition, repair capabilities of MSCs decline with age. Hence, this study aims to confirm whether the Exos protective function on osteogenic differentiation and fracture healing from aged MSCs was attenuated. This information was used in order to investigate the underlying mechanism. MSCs were successfully isolated and identified from young and aged rats, and Exos were then obtained. Aged-Exos exhibited significantly attenuated effects on MSCs osteogenic differentiation in vitro and facture healing in vivo. Using miRNA array analysis, it was shown that miR-128-3p was markedly upregulated in Aged-Exos. In vitro experiments confirmed that Smad5 is a direct downstream target of miR-128-3p, and was inhibited by overexpressed miR-128-3p. A series gain- and loss- function experiment indicated that miR-128-3P serves a suppressor role in the process of fracture healing. Furthermore, effects caused by miR-128-3P mimic/inhibitor were reversed by the application of Smad5/siSmad5. Taken together, these results suggest that the therapeutic effects of MSCs-derived Exos may vary according to differential expression of miRNAs. Exosomal miR-128-3P antagomir may act as a promising therapeutic strategy for bone fracture healing, especially for the elderly.

Keywords: Bone fracture; Exosomes; Mesenchymal stem cells; Smad5; miR-128-3P.

Fig. 1

a MSCs adopted a spindle-like morphology, whereas aged MSCs demonstrated a flattened morphology compared to young MSCs (scale: 100 μm). b Toluidine Blue, Oli Red O and ALP staining of MSCs (scale: 100 μm). c, d MSCs were positive for CD44 and CD90 but negative for CD34 or CD45

Fig. 2

a The morphology of Young-Exos and Aged-Exos (scale: 100 nm). b The diameter of Young- and Aged-Exos, ranging from 50 to 150 nm. c Specific surface markers of exosomes, including CD63 and CD81

Fig. 3

a, b The mRNA expression levels of Runx2, ALP and Col I (n = 5, PBS vs Age-Exos, P < 0.05, Age-Exos vs Young-Exos, P < 0.05). c, d ALP staining and Alizarin Red staining after 7 or 14 days of incubation with Exos

Fig. 4

a, b Micro-CT images and Callus volumes (scale: 1 mm). c BV/TV on day 28. d The mRNA levels of Runx2, ALP and Collagen I in calluses (n = 4, PBS vs Age-Exos, P < 0.05, Age-Exos vs Young-Exos, P < 0.05)

Fig. 5

a The heat map showed that miR-128-3p was upregulated in Aged-Exos. b The schematic diagram of microRNA screening and selection. c qRT-PCR showed that miR-128-3p was the most upregulated miRNAs in the Aged-Exos among the top 10 miRNAs

Fig. 6

a. The relative luciferase activity was lessened when miR-128-3p mimics were co-transfected with Wt-Smad5 luciferase construct (n = 6, NC-mimics vs miR-128-3p mimics, P < 0.05), but not with the Mut-Smad5. An enhanced luciferase activity was observed when the miR-128-3p inhibitor was co-transfected with Wt-Smad5 (NC-inhibitor vs the miR-128-3p inhibitor, P < 0.05). b, c After 14 days of administration of the miR-128-3P inhibitor, the protein levels of Smad5 in MSCs were markedly increased (n = 4, P < 0.05). In contrast, decreased Smad5 expression was found in MSCs incubated with the miR-128-3P mimics (n = 4, P < 0.05). d, e Western blot analysis of Runx2, ALP and Col I protein product levels. Upregulating Smad5 rescued the negative effects of MSCs osteogenic differentiation caused by the miR-128-3P mimics (n = 4, P < 0.05). f, g Osteogenic differentiation was accelerated when aged MSCs were incubated with the miR-128-3P inhibitor and these positive effects could also be reversed by silencing Smad5 (n = 4, P < 0.05)

Fig. 7

a The expression of miR-128-3P in fractured callus at day 14 (NC-antagomir vs miR-128-3P antagomir, P < 0.05). b, c The post-operative micro-CT reconstruction images at 2, 3 and 4 weeks. The CV and the BV/TV were increased by the miR-128-3P antagomir treatment (n = 4, NC-antagomir vs miR-128-3P antagomir, P < 0.05, scale: 1 mm). d The mRNA level of ALP, Runx2, Collagen I and Smad5 in the callus at day 28(NC-antagomir vs miR-128-3P antagomir, P < 0.05). e The results of immunohistochemistry (NC-antagomir vs miR-128-3P antagomir, P < 0.05, scale: 100 μm)