INTRODUCTION
Osteoporosis is a systemic skeletal disorder characterized by reduced bone mass, deterioration of bone microarchitecture, and increased bone fragility[1,2]. It represents a major cause of fracture risk in middle-aged and elderly populations, particularly among postmenopausal women[3,4]. The underlying pathological basis of osteoporosis is the disruption of bone metabolic homeostasis, specifically impaired coupling between bone resorption and bone formation. In postmenopausal osteoporosis, the marked decline in estrogen levels leads to excessive osteoclast-mediated bone resorption, whereas osteogenic bone formation remains insufficient, resulting in progressive bone loss[5]. Current pharmacological treatments, including bisphosphonates and receptor activator of nuclear factor-kappa B ligand inhibitors, primarily suppress bone resorption but exert limited effects on bone formation[6]. Consequently, the development of therapeutic strategies that actively enhance osteogenesis and restore skeletal homeostasis has become a key research priority.
Mesenchymal stem cells (MSCs), particularly bone marrow-derived MSCs (BMSCs), are essential cellular contributors to osteogenesis. Their osteogenic differentiation capacity directly determines their effectiveness in bone repair and regeneration[7]. In recent years, cell-free therapeutic strategies based on the stem cell secretome, especially exosomes, have attracted increasing attention[8]. Exosomes are nanoscale extracellular vesicles released by cells and contain diverse bioactive components, including proteins, lipids, and messenger RNAs. They function as important mediators of intercellular communication by transferring molecular cargo that influences the biological state and fate of recipient cells[9,10]. Accumulating evidence indicates that exosomes derived from adipose-derived stem cells (ADSCs) promote angiogenesis, inhibit apoptosis, regulate immune responses, and exhibit pronounced osteoinductive activity during bone regeneration[11]. Enhancement of exosomal bioactivity through parent cell pretreatment has emerged as an effective strategy. Among these approaches, hypoxic preconditioning mimics the physiological microenvironment of the stem cell niche in vivo[12]. Hypoxia alters exosomal cargo composition, enriching factors associated with cell survival, angiogenesis, and osteogenic differentiation, which may improve therapeutic outcomes[13]. Despite these advances, the molecular mechanisms through which hypoxia-conditioned exosomes initiate and regulate osteogenic processes remain incompletely defined.
In addition to biochemical cues, mechanical stimulation plays a central role in bone development and homeostasis. Bone tissue exhibits pronounced mechanosensitivity, and mechanical loading is required to maintain structural integrity and function[14-16]. Piezo1, a mechanosensitive cation channel widely expressed across tissues, serves as a key molecular transducer that converts mechanical forces into intracellular biochemical signals[17]. Changes in membrane tension activate Piezo1 and induce calcium influx, positioning this channel as a core mediator of mechanotransduction. Within the osteoblast lineage, Piezo1 functions as a critical conduit through which mechanical stimulation promotes bone formation[18,19]. Mechanical activation of Piezo1 initiates calcium signaling, which engages downstream pathways, including calcineurin/NFAT and CaMKII/CREB, and culminates in the induction of osteogenesis-related genes[18,20,21]. Piezo1 further integrates mechanical inputs with paracrine regulation of bone remodeling by influencing sclerostin expression, Wnt/β-catenin signaling, and the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin axis that controls osteoclastogenesis[22,23]. Converging evidence shows that reduced Piezo1 signaling in osteoblasts or osteocytes impairs load responsiveness and suppresses bone formation, whereas Piezo1 activation enhances osteogenic differentiation and bone accrual[24-27]. These findings position the Piezo1/Ca2+ axis as a central node linking mechanical stimuli to osteoblast-lineage function and skeletal remodeling.
Based on this framework, hypoxia-conditioned ADSC-derived exosomes were hypothesized to activate Piezo1 signaling in BMSCs. Such activation may result from the delivery of bioactive components that modulate cellular mechanosensitivity, thereby inducing calcium influx and downstream osteogenic signaling. Transcriptomic analysis supported this hypothesis by revealing significant enrichment of calcium signaling and osteogenic differentiation pathways in cells treated with hypoxic exosomes. These observations motivated systematic in vitro and in vivo investigations to define the functional outcomes and molecular mechanisms by which hypoxic exosomes promote osteogenic differentiation of BMSCs and attenuate bone loss in ovariectomized rats through the Piezo1/Ca2+ axis. This work advances an exosome-based therapeutic concept that couples biochemical cues with mechanotransduction to address osteoporosis.
MATERIALS AND METHODS
Reagents and instruments
The following reagents and instruments were used: Sterile phosphate-buffered saline (PBS, Servicebio, G4202, China); fetal bovine serum (FBS, Cytiva, SVV30208.01, MA, United States); penicillin-streptomycin (NCM Biotech C100C5, China); low-glucose Dulbecco’s modified Eagle medium (DMEM; Jin Yibai, PM150312, China); trypsin solution (NCM Biotech C100C10, China); anti-CD81 (Abways, CY5337, China); anti-tumor susceptibility gene 101 (Abways, CY5985, China); anti-calnexin (Abways, CY5839, China); anti-runt-related transcription factor 2 (anti-Runx2, Affinity, AF5186, OH, United States); anti-osterix (Affinity, DF7731, OH, United States); GAPDH (Proteintech, 81640-5-RR, IL, United States); goat anti-rabbit IgG-HRP (Proteintech, SA00001-2, IL, United States); PKH67 staining kit (Solarbio, D0031, China); alizarin red S staining solution (Solarbio, G1450, China); BCIP/NBT alkaline phosphatase (ALP) staining kit (Solarbio, G1480, China); paraformaldehyde (Solarbio, P1112, China); and GsMTx4 TFA (TargetMol, TP1300, MA, United States).
Experimental animals
Forty 8-week-old female specific pathogen-free Sprague-Dawley rats were obtained from Jiangsu Qinglongshan Biotechnology Co., Ltd. (License No.: CXK (Su) 2024-0001). Animals were housed in a specific pathogen-free facility at the Experimental Animal Center of Nanjing University of Chinese Medicine under a 12 hours light/dark cycle, with ad libitum access to food and water. All experimental procedures followed animal welfare guidelines and were approved by the Experimental Animal Ethics Committee of Nanjing University of Chinese Medicine (Approval No. 202312A044). The study was reported in accordance with the ARRIVE guidelines.
Cell culture and hypoxia treatment
BMSCs were isolated from 1-week-old rats according to a previously established protocol[28]. Briefly, bone marrow cavities were flushed with low-glucose DMEM to obtain a cell suspension. After centrifugation, cells were cultured in complete L-DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, and maintained at 37 °C in a humidified atmosphere containing 5% CO2. At 80%-90% confluence, cells were passaged using 0.25% trypsin-EDTA. Cells at passages 3-5 were used for subsequent experiments.
ADSCs were isolated from the inguinal fat pads of Sprague-Dawley rats. Adipose tissue was finely minced and digested with 1 mg/mL type I collagenase for 1 hour at 37 °C. After centrifugation and resuspension, cells were cultured under the same conditions as BMSCs. At passage 3, cells were characterized by morphology and flow cytometry. Both cell types exhibited a spindle-shaped morphology. The cells were positive for standard MSC markers (CD29, CD90) and negative for hematopoietic markers (CD34, CD45). Representative flow cytometry histograms are presented in Supplementary material.
Osteogenic differentiation of BMSCs
For osteogenic induction, BMSCs were seeded in 12-well plates at a density of 1 × 105 cells per well. At approximately 70% confluence, the culture medium was replaced with osteogenic induction medium consisting of complete medium supplemented with 50 μmol/L ascorbic acid, 10 mmol/L β-glycerophosphate, and 100 nM dexamethasone. The induction medium was refreshed every three days, with the concurrent addition of 200 μL exosomes (200 μg/mL), hypoxia-preconditioned exosomes (HY-Exos) (200 μg/mL), or phosphate buffered saline.
Exosome isolation, electron microscopy, and size distribution analysis
ADSCs were cultured in medium supplemented with exosome-depleted FBS for 48 hours in an oxygen-controlled incubator at 37 °C under either normoxic (21% O2, 5% CO2) or hypoxic (1% O2, 5% CO2, 94% N2) conditions. Culture supernatants were collected separately. Exosomes were isolated by differential centrifugation as follows: 300 × g for 10 minutes to remove intact cells, 2000 × g for 20 minutes to eliminate dead cells, and 10000 × g for 30 minutes to remove cellular debris. The resulting supernatant was then subjected to ultracentrifugation at 110000 × g for 70 minutes. The final exosome-containing pellet was resuspended in PBS. For transmission electron microscopy, exosome suspensions were placed onto copper grids, negatively stained with 2% phosphotungstic acid, and examined using a transmission electron microscope to assess vesicle morphology. Nanoparticle tracking analysis was performed to determine exosome size distribution and concentration using a NanoSight NS300 system (Malvern, United Kingdom).
Exosome uptake
To evaluate exosome uptake by BMSCs, isolated exosomes were labeled with the green fluorescent dye PKH67. BMSCs were incubated with PKH67-labeled exosomes (200 μg/mL) or HY-Exos (200 μg/mL) for 12 hours. Cells were then fixed with 4% paraformaldehyde, and nuclei were counterstained with DAPI. Fluorescent signals were observed using a confocal laser scanning microscope.
Alizarin red S and ALP staining
After 7 days of osteogenic induction, cells were subjected to ALP staining using a BCIP/NBT ALP staining kit according to the manufacturer’s instructions to assess early osteogenic differentiation. For alizarin red S staining, cells were induced for 21 days, fixed with 4% paraformaldehyde, and stained with 2% alizarin red S solution for 10 minutes to visualize calcium nodule formation. For semi-quantitative analysis, calcium deposits were dissolved in 10% cetylpyridinium chloride, and absorbance was measured at 562 nm.
Ovariectomy model establishment
Forty female Sprague-Dawley rats were randomly divided into four groups (n = 10 per group): The sham-operated group (sham), the ovariectomy (OVX) model group (model), the OVX treated with exosomes group (Exos), and the OVX treated with HY-Exos group (HY-Exos). OVX was performed under general anesthesia induced by intraperitoneal injection of 1% sodium pentobarbital. Following shaving and disinfection of the dorsal region, bilateral longitudinal incisions were made along the spine, and both ovaries were excised. In the sham group, an equivalent amount of peri-ovarian adipose tissue was removed without ovary excision. Penicillin sodium was administered intramuscularly for three consecutive days after surgery to prevent infection. One week postoperatively, rats received weekly tail vein injections of 200 μg exosomes or HY-Exos suspended in 200 μL phosphate buffered saline, while control groups received PBS alone. After an 8-week treatment period, all animals were euthanized for subsequent analyses.
Micro-computed tomography analysis
After euthanasia, the right femur from three randomly selected rats in each group was harvested and fixed in 4% paraformaldehyde for 48 hours. The distal femoral metaphysis was scanned using a high-resolution micro-computed tomography system. Three-dimensional microstructural parameters, including bone volume/tissue volume, trabecular number, trabecular thickness, and trabecular separation, were quantified using the accompanying analysis software.
Hematoxylin and eosin staining
After decalcification, femoral tissues were embedded in paraffin and sectioned into 5 μm slices. Sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin. Bone tissue morphology was examined under a light microscope.
Western blot analysis
Total protein was extracted from cells or bone tissue using radioimmunoprecipitation assay lysis buffer. Protein concentration was determined using the bicinchoninic acid assay. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% skim milk and incubated overnight at 4 °C with primary antibodies against Piezo1 (1:2000), RUNX2 (1:2000), osterix (1:2000), and GAPDH (1:10000). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 hours at room temperature. Protein bands were detected using an enhanced chemiluminescence reagent and visualized with an imaging system.
Cellular immunofluorescence staining
Immunofluorescence staining was performed to assess protein expression and subcellular localization. BMSCs were cultured in confocal dishes, treated as indicated, and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Cells were permeabilized with 0.1% Triton X-100 for 10 minutes and blocked with 5% bovine serum albumin for 1 hour to reduce nonspecific binding. Samples were incubated overnight at 4 °C with anti-Piezo1 antibody (1:150). After washing with PBS, cells were incubated with fluorescence-labeled secondary antibodies for 1 hour at room temperature in the dark. Nuclei were counterstained using an antifade mounting medium containing DAPI. Images were acquired using a laser scanning confocal microscope.
Calcium oscillation assay
Real-time intracellular calcium oscillations in BMSCs were assessed using the calcium-sensitive fluorescent probe Fluo-4 AM. BMSCs were seeded in 96-well plates. After cell attachment, the culture medium was replaced with Hank’s balanced salt solution containing 5 μmol/L Fluo-4 AM, followed by incubation at 37 °C with 5% CO2 for 30 minutes. Cells were then washed with Hank’s balanced salt solution and equilibrated for 15 minutes. Fluorescence images were recorded every second for 400 seconds using a fluorescence microscope at excitation and emission wavelengths of 488 nm and 525 nm, respectively, with minimal light exposure.
Statistical analysis
Quantitative data are presented as mean ± SD from three biological replicates (n = 3). Statistical analyses were conducted using GraphPad Prism version 9.0 and SPSS version 25.0. Comparisons between two groups were performed using Student’s t-test, while one-way analysis of variance was applied for comparisons among multiple groups, followed by Bonferroni post hoc testing. A P value < 0.05 was considered statistically significant.
RESULTS
Characterization of exosomes and uptake by BMSCs
To evaluate the effects of hypoxic preconditioning on exosomal properties, ADSC-derived exosomes were isolated and characterized under normoxic and hypoxic conditions. Transmission electron microscopy and nanoparticle tracking analysis demonstrated that exosomes from both groups displayed a typical cup-shaped morphology, with comparable particle size distributions (Figure 1A and B). Western blot analysis of exosomal marker proteins showed that the expression levels of CD81 and tumor susceptibility gene 101 were markedly higher in hypoxia-conditioned exosomes than in normoxic exosomes. The endoplasmic reticulum marker calnexin was detected exclusively in whole-cell lysates and was absent from exosomal fractions in both groups, indicating high exosome purity and minimal cellular contamination (Figure 1C). Exosome uptake by BMSCs was subsequently examined using PKH67 fluorescent labeling. Confocal microscopy revealed efficient internalization of exosomes derived from both conditions. The fluorescence intensity was notably higher in cells treated with hypoxia-conditioned exosomes than in those treated with normoxic exosomes, suggesting enhanced uptake following hypoxic preconditioning (Figure 1D).
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.
Hypoxia-conditioned exosomes promote osteogenic differentiation of BMSCs
The osteogenic effects of exosomes derived under different conditions were assessed in BMSCs. ALP staining, an early marker of osteogenic differentiation, and alizarin red S staining, indicating late-stage mineral deposition, showed that normoxic exosomes moderately enhanced osteogenic differentiation compared with the blank control. Hypoxia-conditioned exosomes produced the strongest effect, as reflected by increased ALP activity and more extensive mineralized nodule formation (Figure 2A and B). These phenotypic observations were supported by molecular analyses. Western blot results demonstrated progressive increases in the expression of key osteogenic transcription factors, including RUNX2 and osterix, across the blank control, normoxic exosome, and hypoxia-conditioned exosome groups, with the highest expression observed following hypoxia-conditioned exosome treatment (Figure 2C and D). These findings indicate that hypoxia-conditioned exosomes exert a greater capacity to promote osteogenic differentiation in BMSCs.
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.
HY-Exos promote bone formation in OVX rats
To assess the in vivo therapeutic effects of hypoxia-conditioned exosomes, an OVX-induced osteoporosis model was established in rats. Micro-computed tomography and bone morphometric analyses showed that, relative to the sham group, the OVX model group exhibited pronounced trabecular bone loss and structural disruption in the distal femur. Treatment with normoxic exosomes partially attenuated bone deterioration. Hypoxia-conditioned exosomes produced the most pronounced improvement in bone microarchitecture, as evidenced by significant increases in bone mineral density, bone volume fraction, and trabecular number, along with a marked reduction in trabecular separation (Figure 3A and B). Histological evaluation using hematoxylin and eosin staining further demonstrated that trabecular bone in the hypoxia-conditioned exosome-treated group was denser and more continuous than in other groups (Figure 3C). Consistent with these structural findings, western blot analysis of bone tissue extracts showed that RUNX2 and osterix protein levels were significantly elevated in the hypoxia-conditioned exosome group compared with all other groups (Figure 3D), supporting an association between hypoxia-conditioned exosome treatment and enhanced osteogenic activity in vivo.
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.
HY-Exos induce calcium influx via Piezo1 upregulation
To explore the mechanism underlying the osteogenic effects of hypoxia-conditioned exosomes, Piezo1 expression and localization were examined in BMSCs. Immunofluorescence analysis showed a marked increase in Piezo1 fluorescence intensity in BMSCs treated with hypoxia-conditioned exosomes compared with normoxic exosome treatment. This increase was substantially attenuated by pretreatment with GsMTx4, a selective Piezo1 inhibitor (Figure 4A). Consistent with these observations, western blot analysis demonstrated that hypoxia-conditioned exosomes significantly increased Piezo1 protein expression, which was effectively suppressed by GsMTx4 (Figure 4B). Functional activation of Piezo1 was evaluated by monitoring intracellular calcium dynamics using the Fluo-4 AM probe. Normoxic exosomes elicited only a modest calcium response. In contrast, hypoxia-conditioned exosomes induced strong and sustained calcium oscillations. This calcium influx was almost completely abolished by GsMTx4 pretreatment (Figure 4C). These findings indicate that hypoxia-conditioned exosomes promote calcium influx in BMSCs through upregulation and activation of the Piezo1 channel.
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.
HY-Exos promote osteogenic differentiation of BMSCs through the Piezo1/Ca2+ signaling axis
After establishing that hypoxia-conditioned exosomes activate the Piezo1-calcium signaling pathway, the requirement of this pathway for osteogenic differentiation was examined. The hypoxia-conditioned exosome-induced increase in ALP activity was significantly reduced following inhibition of Piezo1 with GsMTx4 (Figure 5A). Consistently, during prolonged osteogenic induction, GsMTx4 markedly attenuated mineralized nodule formation promoted by hypoxia-conditioned exosomes (Figure 5A). Molecular analyses supported these phenotypic findings. Western blot results showed that hypoxia-conditioned exosomes significantly increased the protein expression of key osteogenic transcription factors, including RUNX2 and osterix. This upregulation was substantially suppressed by Piezo1 inhibition with GsMTx4 (Figure 5B). These results indicate that activation of the Piezo1/Ca2+ signaling pathway contributes to the pro-osteogenic effects of hypoxia-conditioned exosomes in BMSCs. Modulation of Piezo1 activity appears to be closely associated with hypoxia-conditioned exosome-induced osteogenic differentiation through regulation of RUNX2 and osterix expression.
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.
DISCUSSION
This study examines the enhanced osteogenic effects of hypoxia-preconditioned ADSC-derived exosomes and explores the associated molecular mechanisms. The main findings are as follows: Hypoxic preconditioning did not alter exosome particle size or morphology but increased the abundance of exosomal marker proteins and improved uptake by BMSCs. Both in vitro and in vivo experiments showed that hypoxia-conditioned exosomes exerted stronger pro-osteogenic effects than normoxic exosomes. Mechanistic analyses further suggested that these effects are associated with increased Piezo1 expression and activation of downstream calcium signaling. Together, these findings support hypoxic preconditioning as a means to enhance the biological activity of ADSC-derived exosomes and highlight a potential link between mechanosensitive signaling and osteogenic regulation.
At the level of exosome characteristics, hypoxic culture conditions did not affect vesicle morphology, consistent with previous reports on MSC-derived exosomes[29]. In contrast, hypoxia significantly increased exosome uptake efficiency and the expression of characteristic marker proteins. Hypoxia represents a key physiological feature of the native MSC niche and is known to enhance paracrine activity[30]. Li et al[31] reported that hypoxic stimulation modifies exosomal membrane adhesion proteins, thereby facilitating endocytosis by recipient cells. In addition, activation of hypoxia-inducible factor-1α signaling has been shown to promote exosome biogenesis and secretion[31]. The enhanced uptake observed in the present study may therefore reflect hypoxia-induced changes in exosomal membrane composition that improve recognition and internalization by BMSCs, increasing the efficiency of cargo delivery.
Functionally, HY-Exos exhibited pronounced osteogenic activity, as evidenced by increased ALP activity, enhanced mineralized nodule formation, and elevated expression of RUNX2 and osterix. RUNX2 governs early osteogenic lineage commitment, whereas osterix is required for osteoblast maturation and functional bone formation[32-34]. Current osteoporosis therapies predominantly suppress bone resorption, with limited emphasis on stimulating bone formation[35]. The coordinated upregulation of both RUNX2 and osterix observed here suggests that hypoxia-conditioned exosomes influence core regulatory pathways involved in osteogenic differentiation. These effects were further supported by in vivo findings in OVX rats, in which hypoxia-conditioned exosome treatment produced the most substantial improvements in bone mineral density and trabecular architecture. These results are consistent with previous reports using hypoxia-preconditioned bone marrow MSC-derived exosomes for bone repair[36] and extend these observations to ADSC-derived exosomes and a systemic osteoporosis model. Collectively, the data indicate that hypoxia-conditioned exosomes may represent a promising cell-free approach for enhancing osteogenesis and mitigating osteoporotic bone loss.
A key finding of this study is the involvement of the Piezo1/Ca2+ signaling axis in the osteogenic effects associated with hypoxia-conditioned exosomes. Transcriptomic analyses indicated marked enrichment of pathways related to mechanosensitivity and calcium signaling, offering insight into exosome-mediated regulation of intercellular communication. These results support the concept that exosomes can transmit biochemical cues while modulating cellular responsiveness to mechanical stimuli[37,38]. Piezo1 functions as a principal mechanosensitive ion channel in skeletal tissue and plays an essential role in bone homeostasis. A seminal study demonstrated that Piezo1 deficiency results in severe osteoporosis, closely resembling disuse-induced bone loss[39]. In the present study, hypoxia-conditioned exosomes significantly increased Piezo1 protein expression. Whereas previous work has largely emphasized direct mechanical activation of Piezo1[37,40,41], these findings suggest that exosome-derived biochemical signals may prime cellular mechanosensitivity at the transcriptional level. Under osteoporotic conditions, where mechanical loading is often reduced, hypoxia-conditioned exosomes may enhance osteogenic responsiveness by elevating basal Piezo1 expression and lowering the activation threshold for mechanotransduction. This proposed sensitization effect provides insight into adaptive regulation of mechanosensitive pathways in vivo.
Further mechanistic analyses demonstrated that Piezo1 upregulation was accompanied by functional changes in intracellular signaling. Hypoxia-conditioned exosomes induced pronounced calcium oscillations in BMSCs, an effect effectively abolished by the Piezo1 inhibitor GsMTx4. Calcium ions act as ubiquitous second messengers, and oscillatory signaling patterns encode distinct biological information[42]. Sustained or frequency-specific calcium influx has been reported to activate pathways such as calcineurin/NFAT and CaMKII/CREB, which converge on transcriptional programs governing osteogenic differentiation, including RUNX2 expression[43,44]. Consistent with these reports, Piezo1-mediated calcium signaling has been implicated in osteogenesis through the calcineurin/NFATc1 axis[45]. Loss-of-function experiments in this study showed that Piezo1 inhibition suppressed calcium signaling and largely reversed hypoxia-conditioned exosome-induced osteogenic differentiation. These findings strengthen the association between exosome uptake, Piezo1 activation, and downstream osteogenic signaling[46]. It remains possible that hypoxia-conditioned exosomes are enriched in specific regulatory molecules, such as hypoxia-responsive microRNAs or proteins, that influence Piezo1 transcription or post-transcriptional regulation. Identification of these components will require further investigation.
The discovery of Piezo ion channels (Piezo1 and Piezo2) was recognized by the 2021 Nobel Prize in Physiology or Medicine to David Julius and Ardem Patapoutian - closely related to translational medicine as below. Beyond osteogenic lineage effects, Piezo1 is increasingly recognized as a mechanosensory integrator within the bone marrow microenvironment, where calcium influx couples’ structural perturbation to reparative programs. In this context, Zhang et al[47] demonstrated that Piezo1 upregulation in bone marrow macrophages after irradiation injury promotes sinusoidal vascular niche regeneration via a calcineurin/NFAT/hypoxia-inducible factor-1 alpha axis and increased vascular endothelial growth factor-A production. This independent niche-regeneration framework provides a biologically coherent backdrop for our study’s observation that ADSC-exosome treatment is associated with activation of a Piezo1/Ca2+ signaling node during bone repair. Importantly, these bodies of work point toward Piezo1 as a shared “mechanotransduction bottleneck” linking extracellular microenvironmental cues to downstream regenerative responses across distinct marrow-resident cell types. Nevertheless, the current data do not establish cell-type-specific Piezo1 necessity in vivo, and future studies employing genetic or lineage-restricted perturbation will be required to define whether exosome-associated effects converge on macrophage-vascular niche remodeling, osteoprogenitor differentiation, or both.
Several limitations should be acknowledged. Although the necessity of the Piezo1/Ca2+ signaling pathway was supported, its sufficiency remains to be established. Direct evidence describing the biodistribution and retention of intravenously administered exosomes within bone tissue was not obtained, limiting precise interpretation of systemic delivery and localized effects. Future studies incorporating in vivo imaging approaches, such as near-infrared or fluorescent labeling, would address this issue. In addition, the specific molecular cargo within hypoxia-conditioned exosomes responsible for Piezo1 upregulation remains undefined. Hypoxia-associated molecules, including selected microRNAs or long non-coding RNAs, may contribute to this regulatory effect and warrant targeted analysis.
