Microdroplet-mediated exosome delivery system for the therapeutic management of radiation-induced bone damage

Abstract

Radiation-induced skeletal deterioration and bone loss remain major clinical challenges. Although exosomes derived from human bone marrow-derived mesenchymal stromal cells (hBMSCs) (MSC-Exos) are promising cell-free therapeutics, their limited stability and low delivery efficiency hinder translational application. Here, we developed an innovative microdroplet-based exosome delivery platform (MD/Exos) and systematically evaluated its protective effects under irradiation-induced stress. MSC-Exos were successfully isolated and characterized using transmission electron microscopy and western blot analysis. Fluorescence imaging and confocal microscopy confirmed efficient uptake of MSC-Exos by both macrophages and hBMSCs. MD/Exos were fabricated by encapsulating exosomes within bovine serum albumin-stabilized microdroplets. Cytocompatibility assays showed that MD/Exos exhibited no detectable cytotoxicity toward macrophages. Functionally, MD/Exos significantly enhanced the viability, metabolic activity, and cytoskeletal integrity of irradiated hBMSCs. Notably, MD/Exos markedly promoted osteogenic differentiation after irradiation, as evidenced by increased alkaline phosphatase activity, enhanced mineralized matrix deposition, and upregulated osteogenesis-related gene expression. In parallel, MD/Exos modulated macrophage responses to radiation by suppressing aberrant multinucleated giant-cell formation and reducing pro-inflammatory cytokine expression. In vivo, administration of MD/Exos in an irradiation-induced bone injury model significantly attenuated bone loss and promoted bone regeneration, as evidenced by preserved bone microstructure, reduced osteoclast activity, and downregulated tumor necrosis factor-alpha (TNF-α) expression. Further in vivo cytokine analysis indicated that MD/Exos treatment significantly reduced pro-inflammatory cytokines compared to irradiated controls. Collectively, these findings demonstrate that MD/Exos represent a biocompatible and efficient exosome delivery platform capable of mitigating irradiation-induced cellular dysfunction, enhancing osteogenesis, and modulating immune-bone crosstalk. This study highlights the therapeutic potential of MD/Exos as a novel strategy for the prevention and treatment of radiation-induced bone loss.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13036-026-00748-4.

Introduction

Cancer remains one of the leading causes of mortality worldwide and continues to impose a substantial burden on patients, healthcare systems, and society [1]. Among the currently available treatment options, radiation therapy or radiotherapy remains one of the most commonly utilized and clinically effective approaches for cancer management [2]. Approximately 50% of all cancer patients receive radiotherapy during the course of their treatment [3]. Radiotherapy functions by delivering ionizing radiation (IR) or subatomic particles to induce irreversible DNA damage in cancer cells, thereby suppressing tumor growth and promoting malignant cell death [4]. However, despite its therapeutic effectiveness against tumors, surrounding healthy tissues are frequently affected during the irradiation process, leading to a variety of treatment-related complications [5]. Among these complications, radiation-induced musculoskeletal complications have emerged as a significant clinical concern, particularly in cancer survivors undergoing radiotherapy near skeletal tissues [6]. Bone remodeling cells, including osteoblasts, osteoclasts, and mesenchymal stem cells, are highly susceptible to radiation exposure due to their sensitivity to oxidative stress, DNA damage, and mitochondrial dysfunction induced by ionizing radiation [7]. In addition, the skeletal system absorbs relatively higher levels of radiation compared with many other tissues because of its mineral composition and dense structural characteristics, thereby further increasing its vulnerability to radiation-induced damage [8]. Consequently, irradiation can severely disrupt bone homeostasis and skeletal integrity through multiple pathological mechanisms. The detrimental effects of radiotherapy on bone tissue include suppression of osteoblast activity, enhanced osteoclast-mediated bone resorption, impaired bone remodeling, reduced bone mineral density, increased marrow adipogenesis, delayed fracture healing, and elevated risks of osteonecrosis and pathological fractures within irradiated regions [8–12]. Despite the growing clinical significance of radiation-associated skeletal complications, currently available therapeutic approaches remain limited and primarily focus on symptomatic management rather than active tissue regeneration. Therefore, there is an urgent need to develop innovative therapeutic strategies capable of simultaneously reducing radiation-induced inflammation, preserving bone homeostasis, and promoting functional skeletal regeneration.

Exosomes are a subset of extracellular vesicles ranging from approximately 30 to 150 nm in diameter that play critical roles in numerous physiological and pathological conditions, including intercellular communication, cellular signaling, immune regulation, and the exchange of biomolecules between cells [13, 14]. Encapsulated within a lipid bilayer membrane, exosomes function as highly specialized biological carriers capable of transporting a broad spectrum of bioactive cargos, including proteins, lipids, metabolites, and nucleic acids such as DNA, messenger RNA, microRNA, and circular RNA [14]. Through delivery of these functional biomolecules to recipient cells, exosomes can regulate diverse cellular activities including proliferation, differentiation, migration, angiogenesis, inflammatory signaling, and tissue repair [15]. Owing to their intrinsic biocompatibility, low immunogenicity, and cell-interactive properties, exosomes have attracted considerable attention as promising therapeutic agents and drug delivery platforms in regenerative medicine. Among the various cellular sources of exosomes, human bone marrow mesenchymal stem cells (MSC)-derived exosomes (MSC-Exos) have demonstrated particularly strong regenerative and immunomodulatory potential. Compared with direct stem cell therapy, MSC-Exos offer several advantages, including lower risk of immune rejection, reduced carcinogenic potential, improved biosafety, and easier storage and transportation [16]. Previous studies have demonstrated that MSC-Exos can promote skeletal muscle regeneration, enhance cartilage repair, accelerate fracture healing, and improve bone regeneration in various disease models [17, 18]. These findings highlight the considerable therapeutic potential of MSC-Exos as a safer and more controllable alternative to direct stem cell transplantation.

Despite the remarkable regenerative and immunomodulatory potential of MSC-Exos, the therapeutic application of unmodified or freely administered exosomes still faces numerous biological, pharmacological, and translational limitations that substantially hinder their clinical effectiveness, particularly in complex pathological conditions such as radiation-induced bone injury [19]. One of the most critical disadvantages of free exosome therapy is their short biological half-life and possible poor stability within hostile pathological microenvironments [20]. For example, Yuki Takahashi and colleagues demonstrated the extremely short biological half-life of systemically administered exosomes in vivo. Using Gaussia luciferase-labeled exosomes derived from B16-BL6 murine melanoma cells, their pharmacokinetic analysis revealed that exosomes were rapidly eliminated from blood circulation with a half-life of approximately 2 min following intravenous injection. Moreover, exosomal signals were barely detectable in serum after 4 h, indicating rapid systemic clearance [21]. These findings strongly highlight the intrinsic limitation of free exosome therapy, including their poor circulation stability and extremely short in vivo persistence, which significantly restrict sustained therapeutic efficacy at target injury sites. In addition, the therapeutic efficacy of free exosomes is often dependent on both dosage and cell passage number; however, achieving effective therapeutic concentrations remains difficult due to their poor retention and rapid clearance [22]. To compensate for low local bioavailability, many studies rely on either exceptionally high exosome doses or repeated administrations to achieve measurable therapeutic effects [23]. However, large-scale exosome production remains technically challenging because MSC cultures produce relatively low exosome yields, purification processes are labor-intensive, and maintaining batch-to-batch consistency remains difficult. These limitations often lead to prolonged MSC expansion, repeated ultracentrifugation steps, or expensive purification systems, all of which substantially increase production time and cost. These limitations strongly support the development of advanced biomaterial-assisted delivery systems and engineered exosome platforms to enhance exosome stability and delivery, ultimately improving long-term regenerative outcomes in radiation-induced bone repair.

Ultrasound refers to mechanical pressure waves with frequencies above the upper limit of human hearing (> 20 kHz). Among external stimuli for controlled therapeutic release, ultrasound has attracted considerable attention as a non-invasive, clinically safe, and highly controllable triggering method. Its unique capability for deep tissue penetration, combined with precise spatial and temporal focusing, makes it particularly suitable for site-specific activation of therapeutics within complex biological environments [24]. In clinical and biomedical applications, low-frequency ultrasound is widely utilized because it can effectively induce acoustic cavitation, generating localized mechanical forces, which in turn facilitate controlled release of therapeutic payloads from responsive carriers [25]. Among the various ultrasound-responsive systems, phase-change nanodroplets and microdroplet (MD)-based platforms have emerged as particularly versatile delivery vehicles. These emulsions typically consist of a liquid perfluorocarbon core stabilized by a shell composed of lipids, proteins, or polymers, with particle sizes generally ranging from the nanoscale to several hundred nanometers depending on formulation design [25]. Importantly, their unique structure allows for the encapsulation of diverse therapeutic cargos, including small-molecule drugs, proteins, nucleic acids, and can be engineered to enable both sustained release and ultrasound-triggered payload activation [26]. Therefore, the present study aims to develop and evaluate an ultrasound-responsive MD-based exosome delivery platform (MD/Exos) designed to enhance the therapeutic efficacy of MSC-Exos for the treatment of radiation-induced bone injury. Mechanistically, the MD/Exos system is designed to respond to ultrasound stimulation to enable temporally controlled release of exosomes, thereby improving targeting precision and therapeutic accumulation at the injury site. Through this approach, the delivered exosomes are expected to modulate radiation-induced cellular dysfunction, attenuate inflammatory responses, and restore impaired osteogenic signaling pathways, ultimately mitigating bone degradation and promoting structural and functional regeneration. Collectively, this ultrasound-responsive MD-based exosome platform represents a promising and adaptable strategy for precision-oriented treatment of radiation-induced skeletal damage.

Materials and methods

Cell culture and exosome purification

Human bone marrow-derived mesenchymal stromal cells (hBMSCs, ATCC^® PCS-500-012™) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies Pty Ltd., China) supplemented with 10% fetal bovine serum (FBS; Biological Industries, LTD, Beit Haemek, Israel), and 1% (v/v) penicillin/streptomycin (Solarbio, Beijing, China) in a 5% CO₂ atmosphere at 37 °C. The murine-derived macrophage cell line RAW264.7 cells were obtained from American Type Culture Collection (TIB-71™) and cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin in a humidified incubator containing 5% CO₂ at 37 °C. hBMSCs were used for exosome isolation. Briefly, the cells were cultured in DMEM supplemented with 10% FBS, and 1% (v/v) penicillin/streptomycin (Solarbio, Beijing, China) in a 5% CO₂ atmosphere at 37 °C. To collect cell culture medium for exosome isolation, passage 3 hBMSCs were grown in T75 flasks until reaching 90% confluence at 37 °C. Following three washes with phosphate-buffered saline (PBS), the cells were cultured with 10 mL DMEM supplemented with 10% exosome-depleted FBS at 37 °C for 24 h to obtain exosome-containing conditioned medium (CM). Exosomes were isolated by filtering the CM through 0.22 μm filters to remove live cells and other large membranous structures. Subsequently, the CM were centrifugated at 300×g at 4 °C for 10 min, transferred to new tubes, and centrifugated at 2,000×g at 4 °C for 20 min. The CM was then centrifuged in a 45Ti rotor (Beckman) at 10,000×g at 4 °C for 40 min. The supernatant underwent centrifugation at 100,000×g at 4 °C for 90 min to pellet the exosomes. The resulting exosome pellets were resuspended in PBS, aliquoted, and stored at -80 °C for future usage.

Transmission electron microscopy (TEM)

A total of 5 µL of exosomes was carefully applied onto 200 mesh TEM copper grids coated with formvar carbon film and allowed to adhere for 5 min at room temperature. After this incubation period, the grids were stained with a 1% uranyl acetate solution for 15–20 s. Following staining, excess uranyl acetate was gently washed off by rinsing the grids three times with deionized water. Subsequently, the samples were dried using Whatman filter paper. Images were captured using a JEOL transmission electron microscope (JEM-1400) at an 80 kV voltage.

Western blot

To identify the exosome marker, samples were resuspended in RIPA lysis buffer and analyzed by Western blot. Briefly, proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with Odyssey^® Blocking Buffer (LI-COR Biotechnology, USA) for 1 h. Membranes were incubated with the following primary antibody: CD81 Antibody (B-11) (1:100, sc-166029, Santa Cruz). The following secondary antibody was used: IRDye^® 680RD goat anti-mouse IgG (H + L) (1:10,000; LI-COR Biotechnology, USA). The protein signal intensity was detected using Odyssey infrared Imaging System (LI-COR Biotechnology, USA).

Internalization of exosomes into target cells

Exosome labeling was performed using the PKH26 red fluorescent cell linker kit for general cell membrane labeling (PKH26GL-1KT, Sigma, China) based on our previous study [27]. Briefly, purified exosomes were incubated with PKH26 dye and subsequently pelleted by ultracentrifugation using a Beckman Coulter Optima ultracentrifuge equipped with an SW41 Ti rotor at 100,000 × g for 70 min at 4 °C. The supernatant was carefully removed, and the exosome pellet was washed three times with PBS to remove unbound dye before final resuspension in PBS. As a dye-only control, PBS without exosomes was processed in parallel following the same labeling and ultracentrifugation procedures to evaluate background fluorescence. Following exosome internalization, the cells were washed three times with PBS and were fixed using 4% paraformaldehyde. After rinsing, the samples were permeabilized with 0.25% Triton and stained with Alexa Fluor 488-labeled phalloidin for 1 h. The samples were then mounted with ProLong™ Gold Antifade Mountant with DAPI (P36935, Thermo Fisher Scientific, China), and images were captured using a confocal laser scanning microscope (Leica DM IRB; Leica, Wetzlar, Germany). For exosome uptake assays, PKH26-labeled exosomes were incubated with target cells at 37 °C for the indicated time periods. After incubation, cells were washed three times with PBS and examined using a Xenogen IVIS Imaging System.

Fabrication and characterization of MD/Exos

The MD-Exos was fabricated according to the method in our previous study with slight modification [28]. Briefly, MD/Exos were fabricated through an emulsification-based process using exosomes, perfluorocarbon, and bovine serum albumin (BSA). In brief, purified exosomes (10 µg total protein) were first dispersed in 4 mL of PBS to ensure uniform suspension. Subsequently, 300 µL of perfluorocarbon was added, and 40 mg of bovine serum albumin (BSA; Sigma-Aldrich, China) was introduced as a stabilizing emulsifier to facilitate the formation of a stable microdroplet interface. The mixture was then subjected to an emulsification process in which exosomes were effectively encapsulated within the microdroplet structure. Following emulsification, the resulting suspension was collected and subjected to ultracentrifugation (Beckman Coulter Optima XPN-100, USA) to collect the microdroplet-encapsulated exosomes (MD/Exos). In addition, the MD-only group without the addition of exosomes was included as an essential control to clearly distinguish the biological effects of the encapsulated exosomes from any potential influence of the MD delivery vehicle itself. To validate the successful internalization of exosomes into MDs, exosomes were fluorescently labeled using the PKH26 Red Fluorescent Cell Linker Kit. A dye-only sample processed under identical conditions without exosomes was used as a negative control. The incorporation of PKH26-labeled exosomes into MDs was assessed by detecting fluorescence signals using a Xenogen IVIS Imaging System. To further validate the successful internalization of exosomes into MDs, PKH26-labeled exosomes were used for MD-Exos fabrication. MD/Exos were exposed to ultrasound stimulation using a portable therapeutic ultrasound device (MYCHWAY, China) operating at an acoustic frequency of 1 MHz for 5 min to trigger the ultrasound-responsive behavior of the system. Then hBMSCs were incubated with MD/Exos at 37 °C. Subsequently, the cells were washed three times with PBS, followed by fixation using 4% paraformaldehyde. After rinsing, the samples were permeabilized with 0.25% Triton and stained with Alexa Fluor 488-labeled phalloidin for 1 h. Subsequently, the samples were mounted with ProLong™ Gold Antifade Mountant with DAPI (P36935, Thermo Fisher Scientific, China). Images were acquired using a confocal laser scanning microscope (Leica DM IRB; Leica, Wetzlar, Germany). Fluorescein isothiocyanate (FITC)-labeled MD/Exos was fabricated using FITC-labelled BSA (A9771, Sigma, China) with the same formulation mentioned above. To validate FITC-labeled MD/Exos internalization into target cells, the cellular uptake of MD/Exos into target cells was further evaluated using Xenogen IVIS Imaging System and confocal laser scanning microscopy (Leica DM IRB; Leica, Wetzlar, Germany).

Live and dead staining

Live/Dead staining assay was conducted using the Calcein/PI live/dead viability assay kit according to the manufacturer’s instructions (Beyotime, China). Briefly, the cells were stained with staining solution containing Calcein-AM and propidium iodide (PI) at room temperature. Calcein-AM marked viable cells with green fluorescence, while PI-stained non-viable cells red. After staining, cells were gently rinsed with PBS to remove excess dye. Confocal laser scanning microscopy (Leica, Wetzlar, Germany) was used to visualize stained cells at ×10 magnification.

Morphological characterization

Cells were washed three times with PBS, fixed with 4% paraformaldehyde and permeabilized using 0.25% Triton X-100. Subsequently, the cells were stained with Alexa Fluor 594-labeled phalloidin for 1 h at room temperature. Samples were mounted with ProLong™ Gold Antifade Mountant with DAPI (P36935, Thermo Fisher Scientific, China), and images were acquired using a confocal laser scanning microscope (Leica DM IRB; Leica, Wetzlar, Germany).

X-ray exposure

The cells were exposed to a single dose of X-ray irradiation at 8 Gy using an X-ray irradiation system (160 kV, 25 mA; Rad Source Technologies, USA) according to our previous studies [28]. Mock-irradiated cells were placed into X-ray machine without radiation exposure, hereby defined as mock radiation exposure.

Internalization assay

The cellular uptake dynamics of MD/Exos into target cells under both non-irradiated and irradiated conditions was evaluated using confocal laser scanning microscopy. Briefly, cells were treated with FITC-labeled MD/Exos for 2 h, 6 h, 12 h, and 24 h. Following treatment, the cells were thoroughly rinsed with PBS to remove unbound MD/Exos. Subsequently, cells were fixed in 4% paraformaldehyde for 10 min at room temperature. To visualize the actin cytoskeleton, the fixed cells were stained with Alexa Fluor 594-labeled phalloidin followed by nuclei staining. The stained samples were subsequently examined using a confocal laser scanning microscope (Leica DM IRB; Leica, Wetzlar, Germany).

Cell metabolic activity

Cell metabolic activity was evaluated using the MTT assay. On day 1 and day 3 after treatment, 5 mg/mL 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT, Sigma, China) was added to the wells and incubated for an additional 4 h at 37 °C. 100 µL dimethyl sulfoxide (DMSO) was used to dissolve formazan generated during the incubation. The absorbance of the sample was measured using a microplate reader at 570 nm. For morphological analysis, cells were fixed and stained with Alexa Fluor 594-labeled phalloidin, followed by imaging using a confocal laser scanning microscope (Leica DM IRB; Leica, Wetzlar, Germany).

Alkaline phosphatase (ALP) staining

To induce osteogenic differentiation, hBMSCs were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and osteogenic-inducing factors, including 10 mM β-glycerophosphate, 50 mM L-ascorbic acid 2-phosphate, and 100 nM dexamethasone. To evaluate osteogenic differentiation in hBMSCs, ALP staining was conducted using the BCIP/NBT Alkaline Phosphatase Color Development Kit according to the manufacturer’s protocol (Beyotime, Shanghai, China). Images were captured using an inverted light microscope (Leica, Wetzlar, Germany).

Immunofluorescence staining

Immunofluorescence staining was performed to evaluate the expression of ALP. Briefly, cells were washed three times with PBS, fixed with 4% paraformaldehyde, and permeabilized using 0.25% Triton X-100. To reduce nonspecific binding, the samples were blocked with 4% BSA for 1 h at room temperature. Subsequently, the cells were incubated overnight at 4 °C with a rabbit polyclonal antibody against ALP (1:100, ab224335, Abcam). After washing with PBS, a FITC-conjugated goat anti-rabbit IgG (H + L) secondary antibody was applied. F-actin was stained with Alexa Fluor 594-labeled phalloidin for 1 h at room temperature. Subsequently, samples were mounted with ProLong™ Gold Antifade Mountant with DAPI (P36935, Thermo Fisher Scientific, China), and images were captured using a confocal laser scanning microscope (Leica DM IRB; Leica, Wetzlar, Germany).

Mineralization assay

The mineralization capacity of hBMSCs was evaluated using Alizarin Red S staining. Briefly, cells were fixed with 4% paraformaldehyde and washed three times with deionized water. The samples were then incubated with 2% Alizarin Red S solution (pH = 4.1) for 20 min at room temperature. Subsequently, the cells were washed thoroughly with deionized water and air-dried overnight. Images were captured using an inverted light microscope (Leica, Wetzlar, Germany).

Osteogenic-related gene expression analysis by reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

To investigate changes in the expression of osteogenic-related genes, total RNA was extracted from hBMSCs cultured for 14 days using TRIzol reagent (15596026; Thermo Fisher Scientific, China) following the manufacturer’s instructions. The purity and concentration of the isolated RNA were assessed using a NanoDrop spectrophotometer (NanoDrop Technologies). Subsequently, cDNA was synthesized from 500 ng of total RNA using a RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific, China). Quantitative analysis of osteogenic-related gene expression was carried out via reverse transcription-polymerase chain reaction (RT-qPCR) with SYBR Green qPCR Master Mix (Life Technologies, China) on the QuantStudio Real-Time PCR system. Relative gene expression levels were calculated using the comparative Ct (2^−ΔΔCT) method [29].

Human TGF beta array analysis

Human TGF beta array analysis was performed following the protocols provided by the manufacturer (Human TGF beta Array C2, Ray Biotech, GA, USA). Briefly, cells were collected and centrifuged to remove cellular debris. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, China). Membranes were placed in incubation tray wells and blocked with 2 mL of blocking buffer for 30 min at RT, followed by overnight incubation with equal amounts of protein lysates at 4 °C. After washing, membranes were incubated overnight at 4 °C with the Biotinylated Antibody Cocktail, then treated with 1× HRP-Streptavidin solution for an additional overnight incubation. Protein spots were visualized using the Li‑Cor Odyssey M Imaging System and semi‑quantified with ImageJ software.

Macrophage analysis

The response of macrophages following MD/Exos treatment was examined in vitro. Briefly, cells were fixed with 4% paraformaldehyde at room temperature, rinsed with PBS, and stained with Alexa Fluor 488-labeled phalloidin for 1 h. Samples were then mounted with ProLong™ Gold Antifade Mountant containing DAPI (P36935, Thermo Fisher Scientific, China). All samples were imaged using a confocal laser scanning microscope with a ×63 objective (Leica DM IRB; Leica, Wetzlar, Germany). CM was collected from macrophages in the irradiated (IR), MDs-treated, Exos-treated, and MD/Exos-treated groups, respectively. To further evaluate the effects of macrophage modulation on osteogenesis, hBMSCs were cultured under osteogenic induction conditions in the presence of CM (1:1 ratio) obtained from irradiated macrophages. Live/dead staining and phalloidin/DAPI staining were performed to assess hBMSCs viability and morphological changes during osteogenic differentiation. ALP staining and Alizarin Red S staining were performed as previously described.

Mouse antibody array analysis

The cytokine profiles changes in the macrophages following MD/Exos treatment were examined using a Mouse Inflammation Array C1 (Ray Biotech, GA, USA), following the protocols provided by the manufacturer. Briefly, cell lysates were harvested, and protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, China). The protein spots on the membranes were visualized using Li-Cor Odyssey M Imaging System. ImageJ software was utilized for semi-quantitative analysis of cytokine expression levels.

Animal study

Female Sprague-Dawley (SD, 8 weeks old) rats were used for this study. All animal experiments were in accord with institutional animal use and care regulations approved by the Zunyi Medical University Committee (ZMU21-2203-009). The rats were divided into four groups (1. Mock-irradiated control (control); 2. Radiation control (IR); 3. IR + MDs (20 mg/kg in saline); 4. IR + MD/Exos (20 mg/kg in saline), with each group consisting of six animals. The hind limbs of the experimental subjects were irradiated on Days 0 and 3, with each session delivering a dose of 8 Gy. Intraperitoneal injection (I.P.) was performed twice a week, on Days 1 and 4. At 14 days post-radiation, all animals were euthanized in accordance with approved protocols. Femurs were carefully harvested, cleaned, and prepared for subsequent histological and immunohistochemical analyses.

Histological assessment

Bones were carefully extracted and immediately fixed in 10% neutral buffered formalin. Following fixation, samples were decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH 7.4) at room temperature, with the solution refreshed regularly until complete decalcification was achieved. Then, the samples were embedded in paraffin wax and sectioned into slices with a thickness of 5 μm using a microtome. Hematoxylin-Eosin (H&E) Staining was performed using the Hematoxylin-Eosin (H&E) Staining Kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. Images were captured using an inverted light microscope (Leica, Wetzlar, Germany).

Tartrate-resistant acid phosphatase (TRAP) staining

Osteoclast activity was assessed through tartrate-resistant acid phosphatase staining (TRAP staining), following established protocols as detailed in our previous study [26]. TRAP-positive multinucleated cells were identified as osteoclasts and assessed qualitatively. Images of the stained samples were acquired using an inverted light microscope (Leica, Wetzlar, Germany).

Immunohistochemical staining

For immunohistochemical analysis, paraffin-embedded sections were first deparaffinized in 100% xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed using proteinase K (Thermo Fisher Scientific, China). After washing with PBS, the tissue sections were blocked with ReadyProbes 2.5% Normal Goat Serum (1X) (Thermo Fisher Scientific, China) and incubated overnight at 4 °C with the following primary antibodies: anti-TNF alpha antibody (Thermo Fisher Scientific, China). Hematoxylin was used as counterstaining for cell nuclei. Stained sections were visualized and imaged using an inverted light microscope (Leica, Wetzlar, Germany).

Rat cytokine array

The cytokine profiles changes in the sera were examined using a rat cytokine antibody array C1 (Ray Biotech, GA, USA), following the protocols provided by the manufacturer. Briefly, each membrane was placed in an incubation tray well and was subjected to a 30-min incubation with 2 mL of blocking buffer at room temperature (RT). Subsequently, the membranes were incubated with the diluted sera (1:2 dilution) overnight at 4 °C. After the washing steps with wash buffer I and II, the membranes were subjected to an overnight incubation at 4 °C with a prepared Biotinylated Antibody Cocktail. Subsequently, 2 mL of 1X HRP-Streptavidin solution was added into each well and incubated overnight at 4 °C. After this incubation, the membranes underwent a final wash with wash buffer I and II to ensure thorough removal of any unbound streptavidin. The protein spots on the membranes were visualized using Li-Cor Odyssey M Imaging System. Images of the arrays were semi-quantified using ImageJ software.

Statistical analysis

The data were presented as mean ± standard deviations (SD, n = 3). Statistical analysis was conducted using GraphPad Prism 7 (Version 7.02) for Windows (GraphPad Software Inc., USA). Differences among groups were assessed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test. A p-value of less than 0.05 was considered statistically significant.

Results

Isolation, characterization, and cellular uptake of hBMSCs-derived exosomes

We firstly isolated the exosomes derived from hBMSCs (Fig. 1A). Through transmission electron microscopy (TEM), the morphology of the isolated exosomes derived from hBMSCs was firstly examined. The TEM image revealed that the isolated vesicles exhibited a spherical or cup-shaped morphology characteristic of exosomes (Fig. 1B). To further confirm the exosomal identity of the isolated vesicles, western blot analysis was performed to detect canonical exosomal surface markers. As shown in Fig. 1C, the isolated vesicles were enriched for CD81, a well-established exosome marker, confirming the successful isolation of hBMSC-derived exosomes. The full, uncropped Western blot image is shown in Supplementary Figure S1. The cellular uptake of hBMSC-derived exosomes was next evaluated using PKH26 fluorescent labeling. IVIS imaging demonstrated robust uptake of labeled exosomes by RAW264.7 macrophages compared with control groups (Fig. 1D). Quantitative analysis of fluorescence intensity revealed a significant increase in signal in the exosome-treated group (Fig. 1E). Subsequently, confocal laser scanning microscopy was used to investigate the internalization kinetics of the isolated exosomes. Exosomes were pre-labeled with the red fluorescent marker PKH26 before co-incubation with recipient cells. RAW264.7 macrophages exhibited minimal background fluorescence under mock conditions (Fig. 1F), whereas strong intracellular PKH26 fluorescence was observed following exosome treatment, with a clear time-dependent increase in signal intensity (Fig. 1G). In parallel, the uptake of exosomes by hBMSCs was also investigated. Dye-only -treated hBMSCs showed no detectable fluorescence (Fig. 1H), while PKH26-labeled exosomes were progressively internalized over time, as demonstrated by confocal microscopy (Fig. 1I). These results indicate that hBMSCs-derived exosomes can be efficiently internalized by both macrophages and mesenchymal stem cells.

Fig. 1.

Isolation, characterization, and cellular uptake of exosomes derived from hBMSCs. (A) Schematic diagram of the isolation procedure for exosomes derived from hBMSCs, including sequential centrifugation steps to remove cells, debris, and larger vesicles, followed by ultracentrifugation to obtain purified exosomes. (B) Transmission electron microscopy (TEM) images showing the typical cup-shaped morphology of hBMSC-derived exosomes. Scale bar = 100 nm. (C) Western blot analysis confirming the expression of the exosomal surface marker CD81 in isolated exosomes. (D) Uptake of labeled PKH26-labeled MSC-Exos by RAW264.7 macrophages visualized using the IVIS imaging system. (E) Quantitative analysis of fluorescence intensity. Significant difference: ****p < 0.0001. (F) Representative confocal laser scanning microscopy images of RAW264.7 macrophages following dye-only treatment. Cell nuclei were stained with DAPI (blue), and F-actin filaments were stained with phalloidin (green). (G) Confocal laser scanning microscopy images showing the time-dependent internalization of PKH26-labeled exosomes by RAW264.7 macrophages. Scale bar = 50 μm (low magnification) and 20 μm (high magnification). (H) Representative confocal microscopy images of hBMSCs following dye-only treatment. (I) Confocal microscopy images demonstrating the time-dependent uptake of PKH26-labeled exosomes by hBMSCs. Scale bar = 50 μm (low magnification) and 20 μm (high magnification)

Physical characterization and cytocompatibility of MD/Exos

To fabricate MD/Exos, MSC-derived exosomes were first fluorescently labeled with PKH26 to enable tracking during fabrication and cellular uptake (Fig. 2A). Fluorescence imaging using the Xenogen IVIS Imaging System confirmed strong PKH26 signals in labeled exosomes, whereas negligible fluorescence was detected in the dye-only control, indicating effective labeling and minimal free dye contamination (Fig. 2B). The fabrication process of MD/Exos is schematically illustrated in Fig. 2C. Representative IVIS fluorescence images demonstrated a pronounced PKH26 signals within the MD/Exos (Fig. 2D). To evaluate whether MD/Exos could efficiently deliver exosomes into target cells, hBMSCs were incubated with MD/Exos and analyzed using confocal laser scanning microscopy. As shown in Fig. 2E, robust intracellular PKH26 red fluorescence was observed within hBMSCs, indicating that exosomes delivered via MD/Exos were effectively internalized throughout the cytoplasm. To further validate the cellular uptake of MD/Exos, FITC-labeled MD/Exos were fabricated using FITC-conjugated BSA. IVIS imaging revealed strong fluorescence signals associated with FITC-labeled MD/Exos (Fig. 2F), and quantitative analysis demonstrated a significant increase in fluorescence intensity compared with controls (Fig. 2G). A schematic diagram illustrating the internalization of FITC-labeled MD/Exos by target cells is presented in Fig. 2H. IVIS imaging further demonstrated efficient uptake of FITC-labeled MD/Exos by macrophages (Fig. 2I). Quantitative analysis confirmed a significant increase in fluorescence intensity compared to control group (Fig. 2J). As shown in Fig. 2K, FITC-labeled MD/Exos were readily internalized by hBMSCs, with fluorescence predominantly localized within the cytoplasm, supporting effective delivery of the MD/Exos. The cytocompatibility of MD/Exos was subsequently evaluated in RAW264.7 macrophages. Live/dead staining demonstrated a predominance of viable cells following MD/Exos treatment (Fig. 2L). These findings indicate that MD/Exos do not induce significant cytotoxicity under the tested conditions. In addition, cytoskeletal staining revealed well-organized F-actin filaments and intact nuclear morphology in macrophages treated with MD/Exos (Fig. 2M).

Fig. 2.

Synthesis and characterization of MDs/Exos. (A) Schematic illustration of the MSC-Exo labeling process. (B) Representative fluorescence images acquired using the Xenogen IVIS Imaging System showing PKH26-labeled exosomes. A dye-only sample was included as a negative control. (C) Schematic diagram illustrating the fabrication of MD/Exos. (D) Representative fluorescence images acquired using the Xenogen IVIS Imaging System showing fabrication of MD/Exos. (E) Confocal microscopy images showing the cellular uptake of MSC-Exo delivered via MD/Exos (red) in hBMSCs. (F) Representative fluorescence images acquired using the Xenogen IVIS Imaging System showing FITC-labeled MD/Exos. (G) Quantitative analysis of fluorescence intensity of FITC-labeled MD/Exos. Significant difference: ****p < 0.0001. (H) Schematic diagram illustrating the cellular uptake of FITC-labeled MD/Exos. (I) Representative fluorescence images acquired using the Xenogen IVIS Imaging System showing FITC-labeled MD/Exos internalization into macrophages. (J) Quantitative analysis of fluorescence intensity. Significant difference: ****p < 0.0001. (K) Representative confocal laser scanning microscopy images confirming the uptake of FITC-labeled MD/Exos by hBMSCs. (L) Representative confocal laser microscopy images of RAW264.7 macrophages treated with or without MD-Exos following live and dead staining. Viable cells are indicated by green fluorescence, representing calcein-AM staining, while dead cells are labeled with red fluorescence (PI). Scale bar = 500 μm. (M) Representative confocal laser microscopy images of RAW264.7 macrophages treated with or without MD-Exo following DAPI/phalloidin staining. Cell cytoskeleton and nuclei were visualized in red and blue, respectively

MD/Exos exhibit efficient cellular internalization under both physiological and irradiated conditions

To investigate the cellular internalization process of MD/Exos, we evaluated the internalization behavior of MD/Exos in macrophages under both non-irradiated and irradiated conditions (Fig. 3A). Under non-irradiated conditions, MD/Exos were readily internalized by macrophages within 2 h of incubation (Fig. 3B). Fluorescent signals corresponding to MD/Exos were predominantly localized within the cytoplasm, appearing as punctate vesicular structures distributed around the perinuclear region. Given that radiation exposure alters membrane permeability, endocytic activity, and inflammatory status of immune cells, we next examined MD/Exos internalization following irradiation. Confocal imaging revealed similar time-dependent uptake dynamics in irradiated macrophages (Fig. 3C). By 6 h, a marked increase in cytoplasmic puncta was evident, suggesting active MD/Exos internalization. The intracellular accumulation further intensified at 12 h, with fluorescent signals distributed throughout the cytoplasm and enriched in perinuclear regions. At 24 h, robust intracellular fluorescence was observed, reflecting sustained uptake and retention of MD/Exos within irradiated macrophages. Collectively, these results demonstrate that MD/Exos are efficiently internalized by macrophages under both physiological and irradiated conditions.

Fig. 3.

Uptake dynamics of MDs-Exo under non-irradiated and irradiated conditions. (A) Schematic diagram for the in vitro study. Macrophages were cultured under non-irradiated or irradiated conditions and treated with fluorescently labeled MD/Exos. Cellular uptake was evaluated at indicated time points using confocal laser scanning microscopy. (B) Confocal microscopy images showing internalization of MD/Exos into macrophages under non-irradiated conditions. Nuclei were stained purple, while F-actin filaments were labeled with phalloidin (red). (C) The internalization process of MD/Exos into macrophages after irradiation at 2 h, 6 h, 12 h, and 24 h was visualized using confocal laser scanning microscopy

The effect of MD/Exos on the metabolic activity of hBMSCs after irradiation

To investigate whether MD/Exos could mitigate irradiation-induced damage and support the metabolic activity of hBMSCs, we examined the effects of MD/Exos treatment on hBMSCs (Fig. 4A). We first validated the internalization of MD/Exos into hBMSCs following irradiation. Confocal laser scanning microscopy revealed efficient internalization of MD/Exos by hBMSCs after X-ray exposure, with fluorescent signals predominantly localized within the cytoplasm, indicating successful cellular uptake (Fig. 4B). Cell metabolic activity was subsequently assessed using the MTT assay on days 1 and 3 after treatment. As shown in Fig. 4C, MD/Exos treatment significantly increased the metabolic activity of irradiated hBMSCs compared with untreated controls on day 3. Consistent with these quantitative findings, confocal microscopy analysis at day 3 revealed a marked increase in cell density in the MD/Exos-treated group when compared to irradiated controls (Fig. 4D). In addition, cytoskeletal staining showed that MD/Exos treatment preserved F-actin organization and promoted the formation of well-defined actin filaments, whereas irradiated control cells displayed disrupted cytoskeletal architecture (Fig. 4E). These results indicate that MD/Exos not only enhance the metabolic activity and viability of irradiated hBMSCs but also maintain cytoskeletal integrity, which is essential for cell adhesion and migration.

Fig. 4.

MD/Exos promote the metabolic activity of hBMSCs following irradiation-induced injury. (A) Schematic diagram for the in vitro study. (B) The internalization process of MD/Exos into hBMSCs subsequent to X-ray exposure was visualized using confocal laser scanning microscopy. Nuclei were counterstained with DAPI (blue), and F-actin filaments were stained with phalloidin (red). Images were acquired at 63× magnification. Scale bar = 20 μm. Top panel (mock-treated); bottom panel (MD/Exos-treated). (C) Quantitative analysis of hBMSC metabolic activity following MD/Exos treatment using the MTT assay on days 1 and 3. After MTT incubation, the formazan crystals were solubilized by DMSO, and the absorbance of the resulting solution was measured spectrophotometrically. (D) Representative confocal microscopy images showing cell morphology and density of hBMSCs after MD/Exos treatment. (E) Representative confocal microscopy images illustrating cytoskeletal organization of hBMSCs following MD/Exos treatment. Nuclei were stained purple, and F-actin filaments were stained with phalloidin (red). Images were acquired at 63× magnification

MD/Exos promote osteogenic differentiation of irradiated hBMSCs in vitro

To evaluate the potential of MD/Exos in promoting osteogenic differentiation and mitigating the adverse effects of radiation exposure on hBMSCs, in vitro osteogenesis assay was performed (Fig. 5A). ALP activity, an early marker of osteogenic differentiation, was first examined. As shown in Fig. 5B, non-irradiated control hBMSCs exhibited strong ALP staining, indicating normal osteogenic differentiation capacity. However, irradiated hBMSCs exhibited relatively weak ALP staining, indicating impaired osteogenic differentiation. In contrast, treatment with MD/Exos resulted in a marked increase in ALP-positive staining areas. Quantitative analysis of ALP-stained areas confirmed a significant enhancement in ALP activity in the MD/Exos-treated group compared with untreated irradiated controls (Fig. 5C). To determine whether differences in cell density contributed to the observed enhancement in osteogenic differentiation, nuclei staining and quantitative analysis were performed after 7 days of osteogenic induction. Representative DAPI-stained fluorescence images and nuclei quantification showed no significant differences in nuclei number among the tested groups at this time point (Supplementary Figure S2). These results indicate that the enhanced osteogenic differentiation observed following MD/Exos treatment was not associated with differences in cell density at day 7. Consistent with the ALP staining results, immunofluorescence staining demonstrated substantially stronger ALP intensity in the non-irradiated control and MD/Exos-treated groups compared with irradiated control cells (Fig. 5D&E). Late-stage osteogenic differentiation was next assessed by Alizarin Red S staining to evaluate extracellular matrix mineralization. As shown in Fig. 5F, irradiated hBMSCs without treatment formed limited mineralized nodules. In contrast, MD/Exos-treated cells exhibited a pronounced increase in the formation of mineralized nodules, indicating enhanced calcium deposition. Quantitative analysis further confirmed a significant elevation in mineralized matrix formation in the MD/Exos-treated group compared with radiated control cells (Fig. 5G). Additionally, the expression levels of key osteogenic marker genes were analyzed using RT-qPCR. As shown in Fig. 5H, MD/Exos treatment significantly upregulated the mRNA expression of key osteogenic markers, including ALP, Colla1, OCN, BSP, and OPN, relative to untreated irradiated hBMSCs. Collectively, these results demonstrate that MD/Exos effectively enhance osteogenic differentiation and mineralization of hBMSCs following X-ray irradiation.

Fig. 5.

Regulation of osteogenic differentiation of hBMSCs in vitro. (A) Schematic diagram for the in vitro study. (B) ALP staining images of hBMSCs treated with or without MD/Exos. Following treatment with MD/Exos, hBMSCs exposed to X-ray exhibited a notable increase in ALP activity. Scale bar = 500 μm. (C) Quantification of ALP staining areas. Significant difference: ****p < 0.0001. (D-E) Immunofluorescence staining images showing ALP expression in hBMSCs. Scale bar = 50 μm. (F) Alizarin red S staining images showing mineralized nodules. The increased formation of mineralized nodules following MD/Exos treatment suggests the therapeutic potential of MD/Exos in mitigating the adverse effects of radiation exposure on hBMSCs. Scale bar = 500 μm. (G) Quantification of alizarin red S staining. Treatment with MD/Exos demonstrated a significant increase in the formation of mineralized nodules compared to untreated control cells. Significant difference: ****p < 0.0001. (H) The expression levels of genes related to osteogenesis in hBMSCs were evaluated at 14 days. GAPDH was used as a housekeeping gene. Significant difference: **p < 0.01, ***p < 0.001, ****p < 0.0001

MD/Exos restores and amplifies TGF-β-related signaling in irradiated hBMSCs

To determine whether MD/Exos could rescue this radiation-induced signaling impairment, we profiled cytokine expression using the Human TGF-β Array C2 platform under irradiated conditions (Fig. 6A). Representative cytokine array membranes demonstrated distinct and intensified cytokine expression patterns in the MD/Exos-treated group compared with MDs alone following irradiation (Fig. 6B). Quantitative densitometric analysis revealed significant upregulation of key osteoinductive mediators, including Activin A, beta-IG-H3, BMP2, BMP7, BMP15, BMPRII, follistatin, GDF15, and TGFbRII (Fig. 6C).

Fig. 6.

MD/Exos restores TGF-β-related signaling during osteogenic differentiation following irradiation. (A) Experimental schematic illustrating cytokine profiling of irradiated hBMSCs cultured under osteogenic induction conditions and treated with MDs or MD/Exos. Cell lysates were collected and analyzed using the Human TGF-β Array C2 platform. (B) Representative membrane images from the Human TGF-β cytokine antibody array showing differential expression patterns between MDs-only and MD/Exos groups under irradiated conditions. (C) Quantitative densitometric analysis of cytokine array. Significant difference: *p < 0.05

The effect of MD/Exos on macrophages after irradiation

To investigate the regulatory effects of MD/Exos on macrophage behavior following irradiation and the subsequent influence on osteogenic differentiation of hBMSCs, an in vitro macrophage-CM-based study was established (Fig. 7A). Following X-ray irradiation, RAW264.7 macrophages exhibited pronounced morphological changes, characterized by increased cell spreading and the formation of multinucleated giant cells (Fig. 7B). In contrast, treatment with MD/Exos markedly attenuated irradiation-induced multinucleation. In addition, cells treated with or without MD/Exos under non-irradiated conditions exhibited similar morphology and cellular organization, indicating that MD/Exos treatment did not induce noticeable morphological alterations under physiological conditions (Fig. 7B). To further evaluate the functional consequences of macrophage modulation on osteogenesis, hBMSCs were cultured under osteogenic induction conditions in the presence of CM collected from irradiated macrophages with or without MD/Exos treatment. Live/dead staining demonstrated that hBMSCs cultured with CM from MD/Exos-treated macrophages showed no significant difference at both 7 and 14 days compared with cells exposed to CM from irradiated macrophages alone (Fig. 7C). In addition, confocal laser scanning microscopy revealed similar cell morphology and cytoskeletal organization at day 14 of osteogenic differentiation (Fig. 7D). Early osteogenic differentiation was further assessed by ALP staining. hBMSCs treated with CM from MD/Exos-treated macrophages displayed markedly stronger ALP staining intensity compared with those cultured with CM from irradiated macrophages (Fig. 7E&F). In addition, extracellular matrix mineralization was examined using Alizarin Red S staining. As shown in Fig. 7G&H, hBMSCs cultured with CM from MD/Exos-treated macrophages formed abundant mineralized nodules, whereas limited calcium deposition was observed in the control CM group.

Fig. 7.

MD/Exos modulate macrophage responses following irradiation and indirectly enhance osteogenic differentiation of hBMSCs. (A) Schematic diagram for the in vitro study. (B) Representative confocal laser scanning microscopy images showing multinucleated macrophage formation after irradiation (IR) with or without MD/Exos treatment. MD/Exos treatment markedly reduced irradiation-induced multinuclear cell formation. Cells not subjected to radiation exposure were used as the non-irradiated control group. Scale bar = 50 μm (low magnification) and 10 μm (high magnification). (C) Representative live/dead staining images of hBMSCs cultured under osteogenic conditions and treated with CM from macrophages at 7 and 14 days. Live cells are indicated by calcein-AM (green), and dead cells by propidium iodide (red). (D) Representative confocal laser scanning microscopy images of hBMSCs cultured with CM from macrophages at 14 days. Cell nuclei were stained with DAPI (blue), and F-actin filaments were stained with phalloidin (red). Scale bar = 50 μm (low magnification) and 10 μm (high magnification). (E) ALP staining images of hBMSCs treated with or without different CM. (F) Quantification of ALP staining areas. Significant difference: ****p < 0.0001. (G) Alizarin Red S staining images showing mineralized nodule formation in hBMSCs cultured with different CM. (H) Quantification of alizarin red S staining. Significant difference: ****p < 0.0001. (I) MD/Exos modulates irradiation-induced macrophage cytokine profiles in vitro. (J) Quantitative analysis of cytokine profiles following MD/Exos treatment. Significant difference: *p < 0.05, **p < 0.01

To determine whether MD/Exos could modulate macrophage responses under irradiation stress, cytokine antibody array analysis was performed from irradiated macrophages treated with MDs or MD/Exos (Fig. 7I). Representative cytokine array membranes demonstrated pronounced differences in cytokine profiles following MD/Exos treatment. Irradiated macrophages in the MDs-only group exhibited high levels of pro-inflammatory mediators. In contrast, treatment with MD/Exos markedly reshaped the cytokine landscape. Quantitative analysis revealed significant downregulation of key pro-inflammatory cytokines, including IL-6, TNFRI, and TNF-alpha (Fig. 7J). In addition, GCSF, GM-CSF, IL-2, IL-12 p70, and IL-13 were also downregulated following MD/Exos treatment. Collectively, these findings demonstrate that MD/Exos effectively modulate macrophage responses after X-ray exposure, suppressing aberrant multinucleation and creating a pro-osteogenic environment.

The impact of MD/Exos on preventing irradiation-induced bone loss

To evaluate the therapeutic efficacy of MD/Exos in mitigating irradiation (IR)-induced bone loss in vivo, a post-irradiation bone injury model was established (Fig. 8A). Representative hematoxylin and eosin-stained femoral sections revealed that IR exposure resulted in pronounced disruption of bone microarchitecture, characterized by trabecular thinning, increased marrow cavity space, and loss of structural connectivity (Fig. 8B). In contrast, treatment with MD/Exos markedly preserved bone morphology, with improved trabecular organization and overall tissue integrity compared with the IR-only and IR + MDs control groups. Osteoclast activity was next assessed using TRAP staining. As shown in Fig. 8C&D, IR exposure significantly increased the number of TRAP-positive multinucleated osteoclasts along the bone surface, indicative of enhanced bone resorption. Notably, MD/Exos treatment substantially reduced the number of TRAP-positive cells, suggesting effective suppression of osteoclastogenesis and osteoclast-mediated bone resorption following irradiation. To investigate the inflammatory response associated with radiation-induced bone loss, tumor necrosis factor-α (TNF-α) expression was evaluated in vivo. IR exposure resulted in pronounced TNF-α staining throughout the bone tissue, indicating a sustained and heightened pro-inflammatory microenvironment. In contrast, MD/Exos treatment significantly decreased the number of TNF-positive cells (Fig. 8E&F), indicating effective attenuation of radiation-induced inflammatory signaling. To further investigate the systemic effect of MD/Exos on irradiation-induced damage, sera were collected from MDs-only and MD/Exo-treated groups and subjected to cytokine antibody array analysis (Fig. 8G). The cytokine antibody array analysis revealed distinguishable expression patterns between the MDs-only and MD/Exo-treated groups (Fig. 8H). Quantitative analysis further demonstrated that administration of MD/Exos significantly reduced the expression of LIX, MCP-1, MIP-3α, TIMP-1, and TNF-α (Fig. 8I). Collectively, these results demonstrate that MD/Exos effectively protect against irradiation-induced bone loss in vivo by preserving bone microarchitecture, suppressing osteoclast formation and activity, and mitigating inflammatory responses.

Fig. 8.

The effects of MD/Exos on preventing irradiation-induced bone loss. (A) Schematic illustration for the in vivo study. (B) Representative hematoxylin and eosin (H&E) staining of bone sections revealed marked differences in bone microarchitecture among the groups. In the IR-only and IR + MDs groups, severe structural deterioration was observed. In contrast, treatment with MD/Exos substantially preserved bone architecture and maintained trabecular integrity, suggesting a protective effect against irradiation-induced degeneration. (C) Representative tartrate-resistant acid phosphatase (TRAP) staining results. (D) Quantitative analysis of TRAP-positive cells demonstrated a significant increase in osteoclast numbers in the IR-only and IR + MDs groups, confirming enhanced bone resorption following irradiation. Notably, MD/Exos treatment significantly reduced the number of TRAP-positive cells. (E) Immunohistochemical (IHC) staining for TNF-α further revealed elevated expression of this pro-inflammatory cytokine in irradiated bone tissues. (F) Quantification of TNF-α expression. (G) Schematic diagram illustrating cytokine profiling using sera collected from MDs-only and MD/Exos-treated groups. (H) Rat cytokine antibody array results demonstrating distinct expression patterns between the MDs-only and MD/Exos-treated groups under irradiated conditions. (I) Quantitative densitometric analysis of cytokine expression levels. Significant difference: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Discussion

Radiation-induced bone loss represents a significant clinical challenge for patients undergoing radiotherapy, as well as for individuals exposed to space radiation or accidental irradiation. Ionizing radiation disrupts skeletal homeostasis through complex and multifactorial mechanisms, including impaired osteoblast function, excessive generation of reactive oxygen species, and heightened osteoclast activity [7]. Collectively, these effects result in accelerated bone resorption, reduced bone formation, structural deterioration, and an increased risk of fragility fractures. Despite advances in radiation biology, effective countermeasures to prevent or reverse radiation-induced bone loss remain limited. MSC-Exos have recently emerged as promising acellular regenerative therapy due to their potent paracrine, immunomodulatory, and pro-osteogenic effects [30]. By delivering bioactive cargos such as microRNAs, proteins, and lipids, MSC-Exos can modulate multiple pathways involved in osteoblast survival, osteogenic differentiation, angiogenesis, and inflammation-related processes that are severely compromised following radiation exposure. However, the clinical translation of exosome-based therapy is greatly hindered by several intrinsic limitations, including structural fragility, rapid clearance, and lack of robust, site-specific delivery. To overcome these challenges, the present study focuses on the development of a novel microdroplet-based exosome delivery platform specifically designed to counteract radiation-induced skeletal injury. By integrating the regenerative capacity of MSC-derived exosomes with the precision of ultrasound-responsive microdroplets, this engineered platform represents a highly promising, multifunctional strategy to restore skeletal integrity in patients exposed to ionizing radiation.

Ionizing radiation is well known to compromise mesenchymal stem cell viability and proliferative capacity by inducing DNA damage, excessive oxidative stress, and apoptosis [8, 31, 32]. Consistent with these reports, our study demonstrated a marked reduction in hBMSC metabolic activity following X-ray irradiation. Importantly, treatment with MD/Exos effectively counteracted these deleterious effects and significantly restored the metabolic activity of irradiated hBMSCs, highlighting the radioprotective and regenerative potential of this strategy. Mechanistically, MSC-derived exosomes have been reported to carry a broad array of pro-survival and pro-proliferative factors, including miRNAs, proteins, and lipids, that collectively promote cell survival and proliferation. Previous studies have shown that exosomal miRNAs can suppress apoptosis-related signaling pathways [33], enhance DNA damage repair mechanisms [34], and attenuate radiation-induced oxidative stress [35]. These multifaceted protective effects are particularly relevant in the irradiated microenvironment, where persistent DNA lesions and ROS accumulation severely limit stem cell regenerative function. In our study, the delivery of MSC-derived exosomes via microdroplets may further enhance their therapeutic efficacy by protecting exosomes from premature degradation and enabling localized release, thereby prolonging their bioactivity and amplifying their pro-proliferative effects. Beyond improving cell metabolic activity, MD/Exos treatment markedly preserved cytoskeletal integrity in irradiated hBMSCs, as evidenced by the maintenance of organized F-actin structures. Radiation-induced cytoskeletal disruption is closely associated with impaired cellular functions [36]. Moreover, excessive ROS generated after irradiation has been implicated in promoting actin depolymerization and inducing oxidative damage to the plasma membrane, leading to altered cell morphology and reduced motility [37]. Notably, previous proteomic analyses of MSC-derived exosomes have consistently revealed enrichment of proteins involved in cell adhesion, surface receptor signaling, and cytoskeletal regulation [38]. For example, exosomes derived from nasal olfactory mucosa MSCs were shown to promote proliferation and migration of human brain microvascular endothelial cells, with functional proteins highly enriched in cell adhesion molecules and signaling pathways [39]. By maintaining organized F-actin architecture, MD/Exos likely preserve focal adhesion signaling and cytoskeletal tension, which are essential for sustained proliferative capacity in hBMSCs. These findings demonstrate that MD/Exos not only improve hBMSC survival after irradiation but also maintain the structural and functional integrity required for effective cell proliferation, thereby reinforcing their potential as a regenerative therapeutic approach for radiation-induced skeletal damage.

Beyond impairing cellular proliferation, ionizing radiation profoundly suppresses the osteogenic differentiation potential of hBMSCs by disrupting lineage commitment, inducing mitochondrial dysfunction [40], inhibiting osteogenic signaling cascades, and altering gene expression profiles [41–43]. Consistent with these reports, radiation exposure in our study markedly attenuated osteogenic differentiation, as evidenced by the significant reduction in ALP expression and activity-an established early marker of osteoblast commitment [44]. Importantly, our results demonstrate that MD/Exos effectively restored the osteogenic differentiation capacity of irradiated hBMSCs at both early and late stages. Treatment with MD/Exos significantly enhanced ALP activity during early osteogenic induction and substantially promoted extracellular matrix mineralization at later stages, indicating a robust restoration of osteoblast lineage progression following X-ray-induced cellular injury. These findings suggest that MD/Exos not only prevent radiation-induced osteogenic suppression but actively reprogram irradiated hBMSCs toward an osteogenic lineage. At the molecular level, these functional improvements were supported by a pronounced upregulation of key osteogenic marker genes following MD/Exos treatment, including genes critical for extracellular matrix deposition and mineralization. This transcriptional activation suggests that MD/Exos may either directly deliver osteoinductive bioactive cargos-such as microRNAs, growth factors, or regulatory proteins-or indirectly stimulate autocrine and paracrine signaling pathways that re-establish osteogenic transcriptional networks disrupted by irradiation [45, 46].

Ionizing radiation is well known to impair osteogenic differentiation by disrupting key signaling pathways, particularly those associated with the TGF-β signaling pathway superfamily. Notably, MSC-derived exosomes have been shown to modulate major osteogenic signaling pathways, including Wnt/β-catenin, BMP/Smad, and PI3K/Akt, all of which are highly susceptible to radiation-induced inhibition [47, 48]. In the present study, we demonstrated that treatment with MD/Exos effectively restores and enhances key proteins related to TGF-β signaling network. The cytokine array results provide compelling evidence that MD/Exos can rescue radiation-induced dysfunction by reactivating multiple growth factors and receptors essential for bone formation. Among the upregulated proteins, BMP2 plays a central and well-established role in osteogenic commitment [49]. As a potent inducer of mesenchymal stem cell differentiation, BMP2 activates downstream SMAD1/5/8 signaling, leading to the expression of key osteogenic genes such as Runx2 [50]. Bone morphogenetic protein 7 (BMP-7), also known as OP-1, is a member of the TGF-β signaling pathway superfamily and has been widely recognized for its potent osteoinductive properties [51]. Clinically, recombinant human BMP-7 (rhBMP-7) has been successfully applied in the treatment of challenging conditions such as revision posterolateral spinal fusion and long bone non-unions, demonstrating both safety and efficacy as a bone-stimulating agent [52]. Preclinical studies further support its therapeutic potential in various animal models. For example, in rabbits and non-human primates, rhBMP-7 has been shown to significantly enhance bone regeneration in critical-sized and segmental defects [51, 53]. For example, in non-human primate models, defects treated with BMP-7 exhibited clear evidence of bone regeneration, whereas untreated defects or defects treated with collagen-only implants showed no signs of healing or bone formation [54]. Therefore, the significant upregulation of BMP2 and BMP7 following MD/Exos treatment indicates that MD/Exos can rescue radiation-induced defects in osteogenic commitment by reactivating key regulators of mesenchymal stem cell differentiation, thereby promoting the re-initiation of osteoblast lineage differentiation and enhancing the overall bone-forming capacity. In addition, the expression of BMPRII (bone morphogenetic protein receptor type II), a key receptor mediating BMP signaling, was significantly upregulated following MD/Exos treatment. BMPRII is a type II TGF-β serine threonine kinase receptor and is essential for the subsequent activation of downstream SMAD1/5/8 signaling cascades that drive osteogenic differentiation [55]. By improving receptor-mediated signal transduction, MD/Exos treatment likely amplifies downstream osteogenic pathways, promoting the activation of key transcription factors and ultimately facilitating more efficient osteoblast differentiation. Beta-IG-H3 (also known as TGFBI), is an extracellular matrix-associated protein induced by TGF-β signaling [56]. Recent study suggests that TGFBI functions as an MSC-derived chondroprotective factor that promotes anabolic activity and helps maintain cartilage homeostasis, highlighting its potential role in supporting tissue regeneration [57]. Activin A, a member of the TGF-β signaling pathway superfamily, signals through activin type II receptors (ActRIIA/IIB) to activate type I receptors, leading to downstream phosphorylation of Smad2/3 and regulation of gene transcription [58]. Previous study indicated that it plays a critical role in maintaining human embryonic stem cells (hESCs) self-renewal and pluripotency by upregulating key factors such as Oct4 and Nanog [59]. Although an inhibitory effect of activin A on osteogenic differentiation in vitro was reported [60], several studies also demonstrated that local administration of activin A promotes bone formation and fracture healing in vivo [61, 62]. Collectively, these findings suggest that MD/Exos exert a multifaceted regulatory effect on osteogenic signaling pathways in irradiated hBMSCs, restoring impaired signaling networks and promoting osteogenic differentiation.

Macrophages are among the most functionally plastic immune cell populations within bone tissue and play a central role in regulating tissue homeostasis, inflammation, and regeneration [63]. Following exposure to ionizing radiation, macrophages are rapidly recruited to irradiated sites, where they persist and actively shape the local microenvironment through cytokine secretion, cell-cell interactions, and modulation of stromal cell behavior [64]. For example, evidence from radiation-induced lung fibrosis (RIF) demonstrates extensive infiltration of both interstitial and alveolar macrophages in clinical specimens and preclinical models, highlighting the remarkable capacity of macrophages to survive irradiation and contribute to chronic tissue remodeling and fibrosis [65]. As a result, macrophages are not passive responders to radiation injury, but rather key drivers of long-term pathological outcomes. Radiation exposure further exacerbates macrophage dysfunction by promoting pathological activation states, including aberrant macrophage fusion. In chronically inflamed, irradiated tissues, macrophages are exposed to sustained damage-associated molecular patterns and inflammatory cues that drive their fusion into multinucleated giant cells (MNGCs), a hallmark of radiation-induced tissue injury [66]. Consistent with these mechanisms, our study demonstrated that irradiation markedly increased the formation of multinucleated macrophages. Notably, treatment with MD/Exos significantly attenuated this irradiation-induced multinucleation phenotype. This finding suggests that MD/Exos actively modulate macrophage activation states under irradiated conditions, suppressing aberrant fusion and possibly dampening sustained inflammatory signaling. In addition, the observed suppression of osteogenic differentiation in hBMSCs cultured with irradiation-educated conditioned medium highlights the detrimental paracrine influence of radiation-activated macrophages on bone-forming cells. In contrast, the restoration of osteogenic differentiation following treatment with MD/Exos-conditioned medium indicates that MD/Exos reprogram macrophage behavior toward a more regulated and reparative phenotype within the irradiated microenvironment. By attenuating pathological immune activation and reshaping macrophage-derived signaling cues, MD/Exos create a paracrine milieu that favors osteogenic commitment and matrix mineralization, thereby establishing a microenvironment more permissive for bone regeneration after irradiation.

Ionizing radiation is known to induce persistent macrophage activation characterized by excessive secretion of pro-inflammatory cytokines, which contributes to impaired tissue regeneration and delayed bone healing [67]. Our cytokine array analysis further revealed that MD/Exos profoundly altered the inflammatory landscape of irradiated macrophages. In particular, the levels of several key pro-inflammatory cytokines, including IL-6, TNF-α, and TNFRI, were significantly reduced following MD/Exos treatment. These cytokines are well-recognized mediators of radiation-induced inflammation and have been shown to inhibit osteogenic differentiation while promoting osteoclastogenesis and bone resorption [68–70]. This shift indicates that MD/Exos delivery counteracts irradiation-induced inflammatory activation and suggests partial reprogramming of macrophage responses. In addition to classical pro-inflammatory mediators, several immune regulatory cytokines, including IL-2, IL-12p70, and IL-13, were also significantly reduced following MD/Exos treatment. These cytokines play important roles in radiation-induced immune activation and inflammatory tissue injury. Previous studies have identified IL-13 as a critical regulator of radiation-induced tissue damage. For example, IL-13 levels were markedly elevated in irradiated lung tissue as early as two weeks after exposure and were predominantly localized within fibrotic regions [71]. Similarly, IL-12p70 is a bioactive heterodimeric cytokine primarily produced by antigen-presenting cells such as macrophages and dendritic cells, where it functions as a key mediator linking innate and adaptive immune responses. Radiation exposure has been reported to stimulate IL-12 secretion by macrophages across both low- and high-dose irradiation conditions [72]. Moreover, total body irradiation has been shown to enhance macrophage activation characterized by increased inducible nitric oxide synthase (iNOS) expression and elevated production of M1-associated pro-inflammatory cytokines, including TNF-α, IL-12p70, and IFN-γ [73]. The observed downregulation of IL-13 and IL-12p70 following MD/Exos treatment therefore suggests that MD/Exos may suppress radiation-induced macrophage inflammatory activation. Given that sustained inflammation after irradiation further disrupts osteogenic signaling and impairs bone regeneration, the ability of MD/Exos to rebalance macrophage-derived cytokine secretion suggests a critical immunomodulatory mechanism underlying its therapeutic efficacy. Collectively, these results indicate that MD/Exos broadly reshape irradiation-triggered immune responses, contributing to the establishment of a more favorable microenvironment for tissue repair and osteogenic regeneration.

In the present study, we demonstrate that MD/Exos exert robust protective effects against IR-induced bone loss in vivo by simultaneously targeting inflammatory signaling, osteoclastogenesis, and bone microarchitectural integrity. In addition, our cytokine array results also demonstrate that MD/Exos treatment significantly attenuates the expression of multiple key inflammatory and regulatory mediators, including LIX (CXCL5), MCP-1 (CCL2), MIP-3α, TIMP-1, and TNF-α, indicating an overall shift toward a less pro-inflammatory post-irradiation microenvironment. Collectively, these changes suggest that MD/Exos can effectively attenuate radiation-induced inflammatory signaling and contribute to the restoration of a more pro-regenerative bone niche. Consistent with our in vitro findings, TNF-α emerges as a key mediator of radiation-induced bone loss. As a potent pro-inflammatory cytokine, TNF-α is rapidly upregulated following irradiation and plays a pivotal role in initiating and sustaining the inflammatory cascade within the bone microenvironment [74]. Mechanistically, TNF-α has been shown to synergize with RANKL to significantly enhance osteoclast differentiation and activation, leading to increased bone resorptive activity [75]. The significant reduction of TNF-α following MD/Exos treatment suggests effective suppression of this important inflammatory mediator, which likely contributes to both decreased osteoclast activity and improved osteogenic potential. Similarly, MCP-1 and LIX were significantly decreased following MD/Exos treatment. MCP-1 is a chemokine involved in monocyte and macrophage recruitment to sites of tissue injury [76], and its upregulation has been associated with radiation-induced inflammatory responses and tissue damage [77]. LIX is primarily involved in neutrophil recruitment and activation and is known to be induced under inflammatory and irradiation conditions, potentially contributing to acute inflammatory infiltration [78]. In this study, the observed reduction of MCP-1 and CXCL5 may suggest a general attenuation of chemokine-driven inflammatory cell recruitment. However, further mechanistic studies will be required to decipher the precise signaling pathways and cell-specific contributions underlying the observed changes in MCP-1 and LIX expression. Taken together, our in vivo results demonstrate that MD/Exos are associated with reduced inflammatory mediator expression, decreased osteoclastogenic activity, and preservation of bone structure following irradiation.

Conclusion and limitations

In conclusion, this study highlights the therapeutic potential of MD/Exos as a multifunctional strategy for the prevention and treatment of radiation-associated bone loss. However, there are several limitations of this study. While our findings support a direct pro-osteogenic effect of MD/Exos, the potential interplay between cell proliferation and osteogenic differentiation warrants further investigation in future studies. We nevertheless acknowledge that the inclusion of a free exosome treatment group in vivo would have further strengthened the translational evaluation and mechanistic interpretation of the MD/Exos platform. However, the exclusion of this group from the in vivo experiments was primarily based on several technical and biological considerations. Due to their rapid clearance and poor retention, a substantially higher quantity of exosomes would be required for repeated in vivo administration to achieve comparable effective dosing, which remained technically challenging under current experimental conditions. Future studies incorporating direct in vivo comparisons with free exosomes, along with long-term safety and biodistribution analyses, will further clarify the translational potential of this platform for clinical applications in regenerative medicine. Another limitation of the present study is that, although the in vitro findings clearly demonstrated the pro-osteogenic effects of MD/Exos, the in vivo evaluation primarily focused on the inhibition of osteoclast activity and inflammatory responses following irradiation. While preservation of bone microarchitecture and reduction of TRAP-positive osteoclasts indirectly support the therapeutic efficacy of MD/Exos, direct assessment of osteogenic activity in vivo was not extensively investigated. Therefore, future studies incorporating detailed analyses of osteoblast activity and bone formation-related signaling pathways will be important to further elucidate the regenerative mechanisms and translational potential of the MD/Exos platform.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This work was financially supported by the National Natural Science Foundation of China (Grant No. 82560425, 82560664, 82460429), Guizhou Science and Technology Program Project (Grant No. Qiankehe Foundation Basic MS [2025] 370, Basic MS [2025] 410, and Basic - ZK[2024] General 296), Zunyi Science and Technology Program Project (Grant No. Zunshikehe HZ zi [2025] 385), Zunyi Municipal Key Laboratory of Medicinal Biotechnology (Grant No. ZSKH-SYS-[2025] 03), and Zunyi Science and Technology Innovation Team for Nutrition and Health (Grant No. Zun KCTD [2025] 89).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The study was approved by the Ethics Committee of the Zunyi Medical University, China (NO. ZMU21-2203-009). This study did not involve human participants, clinical specimens, or patient-derived samples; therefore, informed consent was not applicable.

Competing interests

The authors declare no competing interests.