Fig 2: EV-NID1 regulates the formation and assembly of FAs by targeting myosin-10. (A)Adenovirus was used to overexpress NID1 in RAECs, and the overexpression efficiency was detected by WB. (B) The effect of endogenous overexpression of NID1 (adenovirus infection for 48h) on the wound healing ability of RAECs. (C) The effect of NID1 overexpression on the migration of RAECs. (D) Overexpression of NID1 affected the tubule formation of RAECs. (E) The effects of NID1 overexpression on the expression of Rac1/cdc42 and vinculin. (F)The binding of NID1 and myosin-10 was verified in Co-RAECs. Co-immunoprecipitation of myosin-10 by NID1 antibody. (G) Reverse Co-immunoprecipitation of NID1 by myosin-10 antibody. (H) The intracellular location and distribution of NID1 and myosin-10. (I) The expression of Rac1/cdc42, focal adhesion complex (vinculin/paxillin/FAK) and myosin-10 were measured by WB. Co-RAECs were used as positive control. (J) Relative mRNA expression of vinculin, paxillin, BCL6, MMP10 and MMP13 in RAECs. Data were represented as the mean ± SD. *p < 0.05; **p < 0.01; ns, not significant from Student's t-test.

Fig 3: Hydrogel loaded with EV-NID1 significantly promotes angiogenesis at bone defects sites. (A) Program diagram of the construction and application of EVs-hydrogel. (B) The microstructure and EVs distribution inside hydrogels were characterized by SEM. (C) At 4 weeks post operation, HE staining was used to observe the cell infiltration and the hydrogel degradation at the defect site. The green dashed box represents the integration of the interface between the regenerative tissue and the host bone, and the blue dashed box represents the status of tissue filling and material degradation in the defect center. (D) Sirius red staining showed collagen coverage in the defect area. Under polarized light, red/orange represents type?collagen, and green represents type ? collagen. Scale bar,200 µm. (E) The relative coverage of type ? collagen in the defect area, and the proportion of type ? collagen in total type?and ? collagen. (F) Immunofluorescence staining of CD31 and NID1, the white arrow represents the double-positive tubular structure of CD31 and NID1. Scale bar,100 µm. (G) Comparison of the relative number of CD31+ vessels, and count the CD31/NID1 double positive cells. Data were represented as the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant from Student's t-test.

Fig 4: Nidogen1-enriched EV (EV-NID1) derived from BMSCs may be a key candidate mediator. (A) Schematic diagram of the cytology experiment. RAECs were incubated with concentrated conditioned medium (CM) for 48 h, the EVs were analyzed by TMT proteomics. (B) Wound healing of RAECs incubated with CM or EVs-free-CM (PBS as the Control). (C) Relative migration capacity. (D) Comparison of tubule formation ability and total branching points. (E) The adhesion rate of RAECs in each group. (F) The expression of specific markers (CD63, TSG101 and HSP90) in BMSC-EVs, RAEC-EVs and Co-culture-EVs. (G) Nanoparticle tracking analysis (NTA) showed the particle size distribution of BMSC-EVs. (H) Transmission electron microscopy (TEM) imaging of BMSC-EVs, Scale bar, 50 nm. (I) Heat map of TMT proteomics analysis. The green box indicates that NID1 enriched in BMSC-EVs and Co-culture-EVs, but absented in RAEC-EVs, and was proposed as a candidate effector molecule. (J) The abundance of NID1 in EVs was verified by Western blot. Data were represented as the mean ± SD. *p < 0.05; **p < 0.01; ns, not significant from Student's t-test.

Fig 5: EV-NID1 loaded hydrogel accelerates bone repair. (A) The bone regeneration rate of the Hydrogel, EVs/Hydrogel, EVsshNID1/Hydrogel and Blank groups were evaluated by calcein (green)/alizarin red (red) fluorescence double labeling. (B) VG staining was performed on the samples without decalcification to observe the distribution of new trabecular bone. (C)Micro-CT analysis image, the red dashed ellipse represents the ROI of the defect area, and the orange column is the 3D reconstruction image of new bone. (D) Bone volume fraction (BV/TV%) and trabecular number (Tb.N) in the reconstruction area of Micro-CT. Data were represented as the mean ± SD. *p < 0.05; **p < 0.01. (E) The schematic diagram showed the role and mechanism of EV-NID1.