Hypoxia-preconditioned adipose-derived stem cell exosomes promote osteogenic differentiation and bone formation in bone marrow mesenchymal stem cells through the Piezo1/Ca2+ signaling axis

Figure 1 Hypoxia promotes exosomal release from adipose-derived stem cells and uptake by bone marrow mesenchymal stem cells. A: Representative transmission electron microscopy images showing exosome morphology (scale bar: 200 nm); B: Nanoparticle tracking analysis showing size distribution of adipose-derived stem cells-derived exosomes; C: Western blot analysis and quantification of exosomal markers CD81 and tumor susceptibility gene 101, with calnexin as a negative control (n = 3, one-way ANOVA); D: Fluorescence microscopy images showing colocalization of exosomes with bone marrow mesenchymal stem cells. Nuclei are stained with DAPI (blue), PKH67-labeled exosomes are shown in green, and merged images are presented (scale bar: 100 μm). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. Exos: Exosomes; HY-Exos: Hypoxia-preconditioned exosomes; ADSCs: Adipose-derived stem cells; TSG101: Tumor susceptibility gene 101.

Figure 2 Hypoxia-preconditioned exosomes regulate osteogenic differentiation of bone marrow mesenchymal stem cells. A and B: Alkaline phosphatase and alizarin red S staining with corresponding quantitative analyses after osteogenic induction (scale bar: 100 μm); C and D: Western blot analysis and quantification of runt-related transcription factor 2 and osterix expression in bone marrow mesenchymal stem cells treated with normoxic and hypoxia-conditioned exosomes (n = 3, one-way ANOVA). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. ALP: Alkaline phosphatase; ARS: Alizarin red S; PBS: Phosphate-buffered saline; Exos: Exosomes; HY-Exos: Hypoxia-preconditioned exosomes; Runx2: Runt-related transcription factor 2.

Figure 3 Hypoxia-preconditioned exosomes regulate bone microstructure in ovariectomy rats. A: Quantitative analysis of bone mineral density, bone volume/tissue volume, trabecular number, and trabecular separation (Tb.Sp) (n = 3, one-way ANOVA); B: Representative micro-computed tomography images of the distal femur; C: Hematoxylin and eosin-stained sections showing trabecular bone morphology (scale bar: 100 μm); D: Western blot analysis and quantification of runt-related transcription factor 2 and osterix expression in bone tissue from different groups (n = 3, one-way ANOVA). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. BMD: Bone mineral density; BV/TV: Bone volume/tissue volume; Tb.N: Trabecular number; Tb.Sp: Trabecular separation; OVX: Ovariectomy; Exos: Exosomes; HY-Exos: Hypoxia-preconditioned exosomes; Runx2: Runt-related transcription factor 2.

Figure 4 Hypoxia-preconditioned exosomes mediate calcium influx by upregulating Piezo1. A: Immunofluorescence staining showing Piezo1 expression in bone marrow mesenchymal stem cells (scale bar: 100 μm); B: Western blot analysis and quantification of Piezo1 expression in bone marrow mesenchymal stem cells (n = 3, one-way ANOVA); C: Representative real-time fluorescence images of intracellular calcium dynamics in the same field of view (scale bar: 100 μm). ^cP < 0.001. Exos: Exosomes; HY-Exos: Hypoxia-preconditioned exosomes.

Figure 5 Hypoxia-preconditioned exosomes promote osteogenic differentiation of bone marrow mesenchymal stem cells through the Piezo1/Ca²⁺ signaling axis. A: Alkaline phosphatase and alizarin red S staining with corresponding quantitative analyses after osteogenic induction (scale bar: 100 μm); B: Western blot analysis and quantification of runt-related transcription factor 2 and osterix expression in bone marrow mesenchymal stem cells (n = 3, one-way ANOVA). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. ALP: Alkaline phosphatase; ARS: Alizarin red S; PBS: Phosphate-buffered saline; Exos: Exosomes; HY-Exos: Hypoxia-preconditioned exosomes; Runx2: Runt-related transcription factor 2.