Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo

Abstract

Emerging evidence suggests that extracellular vesicles (EVs) are secreted by diverse tissues and play important roles in cell-cell communication, organ interactions and tissue homeostasis. Studies have reported the use of EVs to stimulate tissue regeneration, such as hepatic cell regeneration, and to treat diseases, such as pulmonary hypertension. However, little is known about the osteogenic effect of EVs. In this study, we explore the role of bone marrow stromal cell-derived EVs in the regulation of osteoblast activity and bone regeneration. We isolated bone marrow stromal/stem cell (BMSC)-derived EVs through gradient ultracentrifugation and ultrafiltration, and tested the influence of the EVs on osteogenesis both in vivo and in vitro. The results indicated that EVs positively regulated osteogenic genes and osteoblastic differentiation but did not inhibit proliferation in vitro. Furthermore, we constructed an EVs delivery system to stimulate bone formation in Sprague Dawley (SD) rats with calvarial defects. We found that BMSC-derived EVs led to more bone formation in the critical-size calvarial bone defects. Moreover, we found that miR-196a plays an essential role in the regulation of osteoblastic differentiation and the expression of osteogenic genes. We anticipate that our assay using bone marrow stromal cell-derived EVs will become a valuable tool for promoting bone regeneration.

Figure 1

(a) The macroscopic Alizarin Red staining for the OM-group, the BM-group and the FM-group from Day 1 to Day 14 at each time point. (b) The OD ratio of Alizarin Red staining indicated that there was no statistically significant difference between the OM-group (0.84 ± 0.04) and the BM-group (0.82 ± 0.01) at day 14 (P = 0.97, P > 0.05), whereas there was a statistically significant differences between the BM-group and the FM-group (0.57 ± 0.03) (P = 0.001, P < 0.05). (c) The ALP expression increased by 2.2-fold and 1.6-fold in the OM-group and the BM-group compared to the FM-group, respectively. The OCN expression increased by 6.1-fold and 4.0-fold in the OM-group and the BM-group compared to the FM-group, respectively. The OPN expression increased by 4.1-fold and 2.9-fold in the OM-group and the BM-group compared to the FM-group, respectively. The RUNX2 expression increased by 2.8-fold and 2.1-fold in the OM-group and the BM-group compared to the FM-group, respectively.

Figure 2

(a) A representative electron microscopic image of EVs derived from BMSCs (White arrow). Scale bar, 200 nm. The western blot of EV-depleted medium and EVs pallet also demonstrated that BMSC-derived EVs were isolated. For the FACS analysis, EVs (open trace) and negative control (filled trace) are shown. (b) Confocal fluorescence analysis was performed 4 h after incubation. The EVs were PKH67-labeled according to the manufacturer’s protocol (green fluorescence). The endoplasmic reticulum, Golgi apparatus and lysosomes were stained by ER-Tracker Red, Golgi-RFP and Lyso-Tracker Red. DAPI was used to stain the cell nuclei (blue fluorescence). Red arrows showed shallow green round-like staining indicating that the foreign EVs were degraded. The white arrow showed the round Golgi apparatus lumen.

Figure 3

(a) The macroscopic Alizarin Red staining for the OM-group, the BM-group, the EV-group and the FM-group from Day 1 to Day 14 at each time point. (b) The OD ratio of Alizarin Red staining indicated that there was no statistically significant difference between the OM-group (0.83 ± 0.02) and the EV-group (0.78 ± 0.01) at day 14 (P = 0.09, P > 0.05), whereas there was a statistically significant difference between the EV-group and the FM-group (0.53 ± 0.03) (P = 0.001, P < 0.05). (c) The expression of ALP, OCN, OPN and RUNX2 increased by 1.9-, 3.8-, 3.2- and 2.4-fold, respectively, in the EV-group compared to the FM-group. (d) The western blot of ALP, OCN, OPN, and RUNX2 at day 14.

Figure 4

(a,b) FACS cell cycle analysis indicated that there was a statistically significant difference between the OM-group (S phase: 49.53 ± 0.68%) and the FM-group (S phase: 55.40 ± 0.51%), P = 0.001, P < 0.05. The EV-group (S phase: 52.16% ± 0.71%) showed statistical significance when compared to the FM-group (P = 0.001, P < 0.05). There was a statistically significant difference between the EV-group and the OM-group, P = 0.04, P < 0.05. There was no statistically significant difference between the EV-group and the BM-group (S phase: 53.76 ± 0.50%), P = 0.073, P > 0.05), *P < 0.05, **P < 0.001. (c) The OD ratio of the FM-group is 1.12 ± 0.02, the BM-group (0.89 ± 0.05) and the EV-group (0.87 ± 0.04) were higher than that of the OM-group (0.80 ± 0.05) but lower than that of the FM-group. There was a statistically significant difference between the FM-group and the EV-group (P = 0.028, P < 0.05). There was a statistically significant difference between the OM-group and the FM-group (P = 0.025, P < 0.05). There was no significant difference between the EV-group and the OM group (P = 0.121, P > 0.05). There was no significance difference between the EV-group and the BM-group (P = 1.000, P > 0.05).

Figure 5

(a) The RNA sequencing of BMSC and EVs indicated that miR-196a, miR27a and miR-206 were highly enriched in EVs. (b) Alizarin Red staining of osteoblasts treated with miR-196a, miR-27a and miR-206 at 3 days. (c) The OD ratio of Alizarin Red staining indicated a statistically significant difference compare miR-196a Mimic-group (0.42 ± 0.01) to the miR-27a Mimic-group (0.36 ± 0.01, P = 0.001, P < 0.05), or to the miR-206 Mimic group (0.35 ± 0.01, P = 0.001, P < 0.05). (d) The expression of ALP increased 1.4-fold in the Mimic-group, 1.9-fold in the EV-group, and 1.2-fold in the IH-group compared to the NC-group. The expression of OCN increased 3.3-fold in the Mimic-group, 4.0-fold in the EV-group, and 2.0-fold in the IH-group compared to the NC-group. The expression of OPN increased 2.4-fold in the Mimic-group, 2.6-fold in the EV-group, and 1.3-fold in the IH-group compared to the NC-group. The expression of RUNX2 increased 1.7-fold in the Mimic-group, 2.2-fold in the EV-group, and 1.4-fold in the IH-group compared to the NC-group.

Figure 6

(a) The EV delivery system improved bone repair. Micro-CT of live mice from 0 to 8 weeks after surgery. The EV-group is the right defect and the Gel-group is the left defect (b) Bone surface (8 weeks, EV-group 32.8 ± 16.9 mm², Gel-group 11.3 ± 4.0 mm², P = 0.041, P < 0.05, n = 6). Bone volume (8 weeks, EV-group 4.0 ± 1.9 mm³, Gel-group, 1.3 ± 0.7 mm³, P = 0.042, P < 0.05, n = 6). Bone Volume/Tissue Volume (8 weeks, EV-group 32 ± 17.4%, Gel-group 9 ± 4.4%, P = 0.043, P < 0.05). BMD (8 weeks, EV-group 0.53 ± 0.06 g/cm³, Gel-group 0.49 ± 0.19 g/cm³, P = 0.627, P > 0.05). (c) HE and Masson staining revealed that the EV-groups were characterized by complete repair and the Gel-groups were characterized by moderate repair.