Fig. 2: Plasma sEVs from TBI rats contribute to TBI-stimulated osteogenesis.

a, b In vitro, freshly isolated rat BMSCs and MC3T3-E1 cells (2 × 10⁵/well) were treated with plasma of rats (20 μL). Total proteins were extracted and subjected to Western blot analysis showing the expression of RUNX2, β-actin, and ALP staining of BMSCs and MC3T3-E1 cells treated with plasma isolated from sham and TBI groups (n = 3; Student’s two-sided unpaired t test). P_{(BMSC TBI vs. sham)} = 0.029, P_{(MC3T3-E1 TBI vs. sham)} = 0.0444. c, d sEVs and non-sEV component were isolated from the plasma of sham or TBI rats. BMSCs and MC3T3-E1 cells (2 × 10⁵/well) were treated with the sEVs or the non-sEV component isolated from equal amount of plasma. Western blot analysis showing the expression of RUNX2, β-actin, and ALP staining of BMSCs and MC3T3-E1 cells treated with sham sEV, TBI sEV, sham non-sEV, or TBI-non-sEV (n = 3; one-way analysis of variance with Turkey’s multiple comparisons test was performed). P_{(BMSC sham sEV vs. TBI sEV)} = 0.0028, P_{(BMSC TBI sEV vs. sham non-sEV)} < 0.0001, P_{(BMSC TBI sEV vs. TBI non-sEV)} < 0.0001, P_{(MC3T3-E1 sham sEV vs. TBI sEV)} = 0.0008, P_{(MC3T3-E1 TBI sEV vs. sham non-sEV)} = 0.0002, P_{MC3T3-E1 TBI sEV vs. TBI non-sEV)} = 0.0002. e Concentration of sEVs isolated from the plasma of rats (n = 5, Student’s two-sided unpaired t test). P_{(TBI-sEV vs. sham-sEV)} = 0.0076. f Micro-CT 3D structure image of proximal tibial defect healing at 7 and 14 days postsurgery with intraperitoneal injection of GW4869 or DMSO saline. BV/TV (g) and Tb.N (h) of region with proximal tibial defect were obtained from micro-CT analysis (n = 5; Student’s two-sided unpaired t test). P_{(7d BV/TV PTD+TBI+GW4869 vs. PTD+TBI)} = 0.0015, P_{(14d BV/TV PTD+TBI+GW4869 vs. PTD+TBI)} = 0.011, P_{(7d Tb.N PTD+TBI+GW4869 vs. PTD+TBI)} = 0.001, P_{(14d Tb.N PTD+TBI+GW4869 vs. PTD+TBI)} = 0.0392. i Representative H&E staining images of proximal tibial defect region at 7 days and 14 days post-impact. Scale bars, 200 μm, (n = 5 animals per group per time point). j, k Representative immunohistochemical staining and quantitation of expression of OCN in rat PTD sites (n = 5; Student’s two-sided unpaired t test). P_{(7d PTD+TBI+GW4869 vs. PTD+TBI)} = 0.0004, P_{(14d PTD+TBI+GW4869 vs. PTD+TBI)} = 0.0012. Scale bars, 40 μm. The quantitation result were plotted as dot plots, showing the mean ± SEM of three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.