Adipose-derived stem cell exosomes promote critical-sized bone defect repair by enhancing the homing of bone marrow mesenchymal stem cells

Adipose-derived stem cell exosomes promote critical-sized bone defect repair by enhancing the homing of bone marrow mesenchymal stem cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Adipose-derived stem cell exosomes promote critical-sized bone defect repair by enhancing the homing of bone marrow mesenchymal stem cells Jingjie Yang, Jing Jing, Xuesha Tong, Songyang Ma, Ye Qiu, Yang Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8646512/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The repair of critical-sized bone defects remains a significant clinical challenge. As a cell-free alternative, exosomes derived from adipose-derived stem cells (ADSCs-Exos) hold promise, yet their precise mechanism in endogenous repair is unclear. This study investigated whether ADSCs-Exos enhance bone repair by promoting the homing and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). ADSCs-Exos were isolated and co-cultured with BMSCs to assess proliferation, migration, and osteogenic differentiation in vitro. Key gene expression (e.g., Cxcr4, Runx2) was analyzed. A highly elastic hydrogel was used for sustained exosome delivery in a rat calvarial defect model. mouse bone marrow-derived mesenchymal stem cells (mBMSCs) homing was monitored via live imaging, and bone regeneration was evaluated by micro-CT and histology. Results showed that ADSCs-Exos promoted BMSC migration and osteogenesis, rapidly upregulating homing-related genes (Ccr7, Cxcr4) and subsequently activating osteogenic genes (Runx2, OPN). In vivo, the ADSCs-Exo/hydrogel complex significantly enhanced BMSC recruitment to the defect site, leading to markedly improved new bone formation. This study elucidates a novel, cell-free strategy wherein ADSCs-Exos orchestrate endogenous bone repair by enhancing BMSC homing and differentiation, providing a potential therapeutic approach. Biological sciences/Cell biology Biological sciences/Stem cells Adipose-derived stem cells Exosomes Cell homing Bone marrow mesenchymal stem cells Endogenous repair Bone defect Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The regeneration of critical-sized bone defects remains a formidable clinical challenge in orthopedics 1 – 3 . Although autogenous bone grafts are considered the gold standard for bone defect repair 4 , 5 , they are associated with significant drawbacks, including insufficient graft supply, iatrogenic injury, and postoperative infection, which can lead to poor prognosis 5 – 7 . With recent breakthroughs in tissue engineering technology, stem cell transplantation has been gradually applied for bone tissue regeneration 8 – 10 . However, concerns regarding immune rejection, tumorigenicity, and low cell survival post-transplantation hinder its clinical translation 1 , 8 , 11 – 13 . Consequently, research attention has shifted toward the paracrine mechanisms of stem cells, particularly toward exosomes, as a potent cell-free alternative 1 , 14 – 16 . Exosomes, which are cell-secreted vesicles with a diameter of 30–150 nm, carry a variety of biological signal molecules, can mediate intercellular communication and replicate the therapeutic effects of their parent cells 17 – 19 . Exosomal components, such as miRNAs have been shown to be involved in various metabolic activities between cells, including those of adipose-derived stem cells (ADSCs), BMSCs, immune cells, and other cell types 20 , 21 , and are involved in regulating bone homeostasis 22 , 23 . ADSCs, which are isolated from adipose tissue, possess multi-lineage differentiation potential 24 – 26 . This capacity, combined with their abundant availability, accessible isolation, and robust proliferation, makes them a highly attractive source of therapeutic exosomes 26 – 28 . Although exosomes offer numerous advantages, their application is limited by rapid in vivo metabolism 20 , 29 , 30 . Therefore, there is a need for materials that enable the slow release of exosomes. Biomaterials such as hydrogels and titanium nano-scaffolds have been used for this purpose 1 , 15 , 29 – 31 . Although hydrogels and other materials have been explored for this purpose, the combined effect of ADSCs-Exos with a highly elastic hydrogel on specifically promoting the homing and osteogenic differentiation of BMSCs for endogenous repair of critical-sized defects remains insufficiently investigated. We hypothesize that ADSCs-Exos, when delivered via a highly elastic hydrogel, promote bone regeneration primarily by enhancing the homing and subsequent osteogenic differentiation of endogenous BMSCs. This mechanism aligns with the emerging paradigm of endogenous regeneration, which aims to recruit and activate the body's own reparative cells rather than relying on externally implanted ones. This study aims to: (1) investigate the in vitro effects of ADSCs-Exos on BMSCs migration and osteogenesis; (2) visualize and verify the enhanced homing of BMSCs in vivo using a luciferase-based imaging system; and (3) evaluate the efficacy of a sustained-release ADSCs-Exo/hydrogel complex in repairing critical-sized calvarial defects in a rat model. Our findings provide new mechanistic insights into ADSCs-Exos-mediated endogenous bone regeneration, supporting their development as a cell-free therapeutic strategy. 2. Materials and Methods 2.1 Cell culture Subcutaneous white adipose tissue was harvested from the inguinal region of 4-week-old male Sprague-Dawley (SD) rats. Fascia and blood vessels within the adipose tissue were meticulously removed, and the tissue was minced and digested with 3 mg/mL collagenase type I (Sigma-Aldrich, USA) for 1 hour at 37°C, and plated in 15 cm² culture dishes (Corning, USA) with growth medium (α-MEM supplemented with 10% FBS and 1% penicillin-streptomycin).Rat BMSCs were isolated from the femur and tibia by flushing the marrow cavity with serum-free α-MEM. The cell suspension was filtered, centrifuged, and cultured in the same growth medium as ADSCs. Both ADSCs and BMSCs at passages 3–5 were used for experiments and cultured at 37°C in a 5% CO₂ humidified incubator. 2.2 Stem cell multidirectional differentiation To assess differentiation potential, BMSCs underwent adipogenic and osteogenic induction. For adipogenic differentiation, cells were cultured in induction medium (α-MEM supplemented with 10% FBS, 1 µM dexamethasone, 10 µg/mL insulin, 200 µM indomethacin, and 0.5 mM IBMX) with medium replacement every 3 days. Lipid accumulation was evaluated after 14–21 days using Oil Red O staining (Solarbio, China). For osteogenic differentiation, cells were maintained in induction medium (α-MEM containing 10% FBS, 50µM ascorbic acid, 100nM dexamethasone, and 10mM β-glycerophosphate) with medium changes every 3 days. Calcium deposition was assessed by Alizarin Red S staining (Sigma-Aldrich, USA) after 21–28 days, while early osteogenic differentiation was evaluated through ALP staining. 2.3 Cell identification ADSCs and BMSCs were analyzed for mesenchymal stromal cell marker expression using a Rat Mesenchymal Stem Cell Characterization Kit (Cyagen Biosciences Inc, USA). Briefly, cell suspensions were prepared at a density of 3 × 10⁶ cells per 100 µL phosphate-buffered saline (PBS). Cells were pelleted by centrifugation (250 ×g for 5 min), washed once with PBS, and resuspended in 100 µL PBS. The cell suspension was then incubated with fluorochrome-conjugated primary antibodies (as specified in the kit) at 4°C for 30 minutes in the dark. Following incubation, cells were washed twice with PBS, resuspended in 400 µL PBS, and analyzed immediately using a FACS flow cytometer (BD Bioscience, USA). 2.4 Isolation and collection of exosomes Exosomes were isolated from the supernatant of ADSCs cultured in osteogenic induction medium for 14 days. The medium consisted of α-MEM supplemented with 10% exosome-depleted FBS, 10 mM β-glycerophosphate, 50 µM ascorbic acid, and 100 nM dexamethasone, and was replaced every 3 days. The collected supernatant was sequentially centrifuged at 300 × g for 10 min, 2,000 × g for 10 min, and 10,000 × g for 30 min to remove cells and debris. Exosomes were then pelleted by ultracentrifugation at 100,000 × g for 70 min, washed in PBS, and recentrifuged under the same conditions. The final pellet was resuspended in PBS and stored at − 80°C. Exosome protein concentration was determined using a BCA assay kit (Beyotime, China). 2.5 Identification of exosomes The exosomes suspension was prepared in PBS and applied dropwise onto a copper mesh. After air-drying, the sample was negatively stained with 1% phosphotungstic acid for 5 min and allowed to dry again. Morphological examination of exosomes was performed using transmission electron microscopy (TEM; Hitachi, Japan). Nanoparticle tracking analysis (NTA) determined the particle size and concentration of exosomes. Exosomes were diluted with PBS and analyzed using the ZetaViewPMX110 (Particle Matrix, Germany). Results were analyzed with NTA analysis software (ZetaView8.04.02SP2). icle Metrix, Germany) and corresponding ZetaView 8.04.02 software. Then, western blotting (WB) was conducted to verify the specific markers of exosomes. Exosomes were lysed in RIPA lysis buffer (Beyotime, China), and protein content was determined using the BCA protein analysis kit (Beyotime, China). Briefly, exosomes were loaded onto 10% gels, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore, USA). The membranes were closed with 5% distilled skim milk and then incubated overnight at 4°C with primary antibodies against anti-CD81, anti-CD9, anti-TSG101, and anti-GAPDH (Abcam, USA, 1:1000 dilution). Secondary antibodies (Abcam, USA, 1:2000 dilution) were incubated at room temperature for 1 h. After each incubation, membranes were washed 3 times with TBST, and protein bands were displayed with ECL kit (Beyotime, China). 2.6 Rheological measurements We tested the rheological properties of Thewell-Exos using a Discovery hybrid rheometer (Anton Paar, Austria). Rheological testing was performed at 25°C with a 1000 µm gap setting. (1) oscillation frequency scan (0.1–10 rad/s at 0.5% constant strain) to monitor the elasticity (storage modulus; G′) and viscosity (loss modulus; G″); (2) Steady-state flow measurements (0.1–100 s⁻¹ shear rates) to evaluate shear-thinning behavior; (3) The self-healing test consisting of high-strain deformation (1 Hz, 0.1–1000% strain, 200 s) followed by recovery phase (1 Hz, 0.5% strain, 200 s), monitoring G′/G″ evolution for self-healing capacity; (4) Amplitude sweeps (0.1–100% strain at 1 Hz) to determine yield stress, identified as the shear stress at the G'-G" crossover point. 2.7 Slow release behavior of exosomes To further determine the release efficiency of exosomes in the hydrogel, Hydrogels (TheWell Inc, USA) were loaded with exosomes by mixing equal volumes (500 µL) of the exosome suspension (0.4 mg/mL) and hydrogel, following the manufacturer's protocol. An equivalent volume mixture of hydrogel and PBS served as negative control. To evaluate exosome release efficiency, exosome-loaded hydrogels were incubated in 0.8 mL PBS at 4℃. At predetermined intervals from t 1 = 5 min to t 23 =168 h, 0.2 mL release medium was collected and replaced with fresh PBS. The collected samples were quantified using a bicinchoninic acid (BCA) protein assay kit. Absorbance at 562 nm was measured via microplate reader (Perkin Elmer, USA), with protein concentrations calculated against a standard curve. All experiments were performed in triplicate. 2.8 Exosome uptake assay. Purified exosomes were labelled with the lipophilic dye DiI (Beyotime, China) according to the manufacturer's instructions. The labeling reaction proceeded for 15 min at room temperature in light-protected conditions. The labelled exosomes were washed in PBS at 100,000 × g for 70 minutes. BMSCs were then incubated with labelled exosomes for 24 hours. After incubation, the cells were washed twice with PBS and fixed in 4% paraformaldehyde (Beyotime, China) for 15 min, permeabilized with 0.1% Triton X-100 (Beyotime, China) at room temperature, and stained with Alexa Fluor 488-phalloidin (Yeasen, China) for 30 min for F-actin visualization. The cells were washed three times with PBS, and the nuclei were stained with DAPI for 10 min. Images were obtained using a confocal laser scanning microscope (Nikon, Japan) and analyzed using NIS-Elements Viewer software. 2.9 BMSCs migration assay Cell migration capacity of BMSCs was assessed using scratch wound and Transwell assays. For the scratch assay, BMSCs in logarithmic growth phase were digested with trypsin to generate single-cell suspensions, seeded into 6-well plates at 6×10⁵ cells/well in 2 mL complete medium, and cultured to 100% confluence. Sterile pipette tips were used to create uniform scratches perpendicular to the plate surface. After three PBS washes to remove detached cells, the cells were treated with serum-free medium containing either ADSC-Exos (experimental group) or an equal volume of PBS (control group). Wound closure was documented at 0, 12, and 24 h post-scratching, with migration distances quantified using ImageJ software. For Transwell migration, 5×10⁵ cells were loaded into the upper chamber of the 24-well transwell plate (Corning, USA) with an 8-mm aperture filter. Chambers containing 0, 10, 20, or 30 µg/mL Exo-14d in complete medium were placed in the lower chamber. Chambers containing 0, 10, 20, or 30 µg/mL ADSC-Exos in complete medium were placed in the lower chamber. After co-culturing the cells for 24 h, cells were gently rinsed with PBS. and non-migrated cells on the upper membrane surface were removed by cotton swab abrasion. Membranes were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Beyotime, China) for 10 min. After three washes, positively stained cells were counted under an inverted microscope (Leica, Germany). Both assays were independently repeated in triplicate. 2.10 BMSCs proliferation assay The proliferation of BMSCs was measured using CCK-8 assay (EnoGene, China). Briefly, BMSCs (3×10³ cells/well in 100 µL medium) were seeded in the medium containing various concentrations of ADSCs-Exos (0, 10, 20, and 30 µg/mL) using 96-well plates. After 6, 12, or 24 h incubation, 10 µl of CCK-8 solution was added to each well and incubated for 3 h at 37°C. Absorbance was measured at 450 nm. 2.11 ALP activity assay staining BMSCs were plated in 24-well plates and cultured for 7 days in osteogenic differentiation medium under two conditions: control medium and medium supplemented with ADSCs-Exos. Following culture, cells were fixed, and ALP activity was assessed. ALP staining was performed using the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China), according to the manufacturer's instructions. ALP activity levels were quantified using an ALP assay kit (Beyotime, China). 2.12 Quantitative Real Time-Polymerase Chain Reaction (qRT‒PCR) To assess the effect of ADSCs-Exos on the expression of osteogenic differentiation and migration-related genes in BMSCs, qRT-PCR was performed under distinct culture conditions. For the assessment of migration-related gene expression, BMSCs were cultured in normal growth medium either supplemented with or without ADSCs-Exos. To evaluate osteogenic differentiation, BMSCs were divided into three groups: cultured in normal growth medium, normal growth medium supplemented with ADSCs-Exos, or standard osteoinductive medium (OM). After the treatment period, total RNA was isolated from the cells using RNAiso Plus reagent (TaKaRa, Japan) for mRNA analysis. Then, the first-strand cDNA was synthesized from equal amounts of total RNA using the PrimeScript RT Master Mix (Takara, Japan). For qRT-PCR analysis, 20µL of reaction mixture was used, including 10 µL of TBGreen Premix Ex Taq (Takara, Japan), 2.0 µL of cDNA, 0.8 µL each of the forward and reverse primers (10 µM), and 6.4 µL of RNase-free water. A dissociation curve analysis was subsequently performed to confirm amplification specificity. The expression of target genes, including osteogenic genes: runt-related transcription factor 2 (RUNX2), osteopontin (OPN), collagen type I alpha 1 chain (Col1a1), ALP, osteocalcin (OCN) and migration-related genes: C-X-C chemokine receptor type 4 (Cxcr4), C-C chemokine receptor 7 (Ccr7), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), Ras-related C3 botulinum toxin substrate 1 (Rac1) was analyzed, with GAPDH acting as the housekeeping gene. The relative gene expression levels were calculated using the 2 − ΔΔCt method. The sequences of all primers used are listed in Supplementary Table 1. 2.13 Lentivirus transfection Primary mBMSCs were purchased from Cyagen Biosciences (MUBMX-01001, China). 5×10 4 mBMSCs were seeded in 24-well plates, and transduced with PASLlenti-pA-Luc2-CMF-EF1-EGFP-P2A-Puro-WPRE lentivirus (OBiO Technology Corp, China) using 5 µg/ml polybrene (Beyotime, China). Following 18-hour incubation, the medium was replaced, and transduced cells were selected with puromycin (Beyotime, China). Then the transfection efficiency was detected by inverted fluorescence microscopy (Leica, Germany). 2.14 Bioluminescence Imaging of Luciferase-Labeled Cells In Vitro and In Vivo To verify the bioluminescence functionality of lentivirally transduced mBMSCs in vitro, the cells were cultured in a 96-well plate, incubated with luciferin substrate, and imaged using an IVIS Spectrum Imaging System (Revvity, USA). For in vivo imaging, C57BL/6 mice were placed in an induction chamber and anesthetized via inhalation of isoflurane (3–4% for induction, 1.5–2.5% for maintenance) delivered in 100% medical oxygen at a flow rate of 0.8–1.2 L/min using a calibrated vaporizer and a non-rebreathing system. Anesthesia depth was monitored by the absence of response to toe pinch. Once anesthetized, mice were administered luciferin potassium salt solution (Beyotime, China; 15 mg/mL) via intraperitoneal injection at a dose of 10 µL/g, and remained under anesthesia for the duration of imaging. No other anesthetic or analgesic agents were used during this procedure. The study included three experimental groups: a control group that had not undergone tail injection of lentivirus-labeled BMSC, a tail vein injection group, and a exosome-loaded hydrogel combined with tail vein injection group. Longitudinal bioluminescence imaging was performed using the IVIS Spectrum system under consistent exposure settings at 24 hours, 48 hours, 3 days, and 1 week post-treatment. 2.15 Evaluation of Bone Regeneration All experiments conducted in this study were approved by the Ethics Committee of the College of Stomatology at Chongqing Medical University (CQHS-REC-2025-078). Eight-week-old male SD rats were randomly divided into five groups (n = 6):(1) Control group: calvarial defect + intravenous PBS. (2) BMSCs group: calvarial defect + intravenous BMSCs. (3) Hydrogel group: calvarial defect + hydrogel implantation. (4) ADSCs-Exos group: calvarial defect + exosome-loaded hydrogel implantation.(5) BMSCs+ADSCs-Exos group: calvarial defect + exosome-loaded hydrogel implantation+ intravenous BMSCs. Bilateral critical-sized calvarial defects (5 mm diameter) were created under isoflurane anesthesia. Following hydrogel implantation according to group assignment and intravenous injection of BMSCs (3×10⁶) or PBS, animals were euthanized at 4 and 8 weeks. All animal euthanasia was conducted in compliance with institutional ethical guidelines. Specifically, rats were placed in a carbon dioxide euthanasia chamber, and carbon dioxide (CO₂) was delivered at a flow rate of 5–6 L/min until respiratory and cardiac arrest occurred. CO₂ flow was maintained for an additional 1 minute, after which the chamber was closed and kept under observation for 2 minutes to confirm irreversible death. No physical or additional chemical methods were employed. Calvarial specimens were analyzed by microcomputerized tomography (micro-CT) scan to quantify bone mineral density (BMD), Bone Volume / Tissue Volume (BV/TV), and trabecular parameters. After decalcification, sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome or immunostained for osteocalcin. All images were acquired using an Olympus VS200 microscope (Olympus, Japan). 2.16 Statistical analysis All experiments were repeated at least three times, and all data were expressed as mean ± standard deviation. Data were analyzed by t-test or one-way ANOVA, and GraphPadPrism 9.0 software was used for statistical processing. Statistical significance was considered when p < 0.05.(*P < 0.05; **P < 0.01; ***P < 0.001.) 3. Results 3.1 Isolation and Characterization of ADSCs and BMSCs As shown in Fig. 1 A, third-passage ADSCs exhibited classical fibroblast-like morphology with homogeneous size distribution, demonstrating characteristic plastic-adherent growth under light microscopy 32 – 34 . while BMSCs exhibited a spindle-like morphology. Both phenotypes characteristic of mesenchymal stem cells 35 . Osteogenic differentiation was confirmed by ALP staining for ADSCs and Alizarin Red S staining for BMSCs (Fig. 1 B). while adipogenic differentiation of both cell types was demonstrated through Oil Red O staining (Fig. 1 C).These results indicated that ADSCs and BMSCs were successfully isolated. The results of flow cytometry analysis demonstrated that ADSCs and BMSCs highly expressed the surface markers CD29, CD90, CD44 and CD73 but expressed low levels of CD11b/c, CD45 and CD34 (Fig. 1 D). 3.2 Characterization of exosomes of ADSCs origin Exosomes were isolated from ADSCs culture supernatants via ultracentrifugation (Fig. 1 E). The ultrastructure of cytosolic exosomes was observed by TEM (Fig. 1 F) with a typical spherical double-membrane structure. WB (Fig. 1 G; see Supplementary Information 2 for full-length blots) results showed that exosomes of ADSCs origin expressed exosomal surface markers (CD81,CD9, TSG101). In addition, NTA (Fig. 1 H) results showed that the particle size of most ADSCs-Exos was primarily distributed around 123.5nm, and their concentration was 3.14 × 10 10 particles/mL. These results attested that we successfully isolated exosomes of ADSCs origin. 3.3 Thewell-Exos hydrogel characterization Rheological testing was performed to characterize the viscoelastic properties of the Thewell-Exos hydrogel. In Fig. 2 A,storage modulus (G′ ≈240 Pa) approximately 5-fold greater than loss modulus (G″ ≈50 Pa) with a frequency range of 0.1–100 rad/s, exhibiting negligible strain dependence and indicating robust network formation conducive to controlled release kinetics;Figure 2 B showed that both G′ and G″ values were relatively stable in the strain range from 0.1% to 1%, which indicated the linear viscoelastic region of Thewell-Exos hydrogel. Additionally, Fig. 2 C shows the shear-thinning properties of the Thewell-Exos hydrogel. the Thewell-Exos hydrogel could sustain high shear forces before the sol/gel transition. The self-healing test (Fig. 2 D) show that the Thewell-Exos hydrogel was subjected to five cycles of time-sweep experiments with low (0.1%) and high (1000%) strain values.At 1000% strain, inversion of modulus values (G′ G″). This recovery behavior persisted through all cycles.confirming outstanding self healing behavior of the Thewell-Exos hydrogel. the Thewell-Exos hydrogel exhibited injectability through 26G needles and could be used as “ink” to draw pattern, This physical adaptability indicates potential suitability for complex bone defect geometries. (Fig. 2 E). According to BCA assay, the cumulative release of ADSCs-Exos reached 90.25 ± 3.65% at day 7. Moreover, the release profile (Fig. 2 F ) demonstrated excellent sustained-release capacity of Thewell hydrogel for ADSCs-Exos throughout the 7-day monitoring span. Taken the above results together, suggesting a successful preparation of the hydrogel capable of slow-releasing ADSCs-Exos was demonstrated. 3.4 Uptake of exosomes by Bone marrow mesenchymal stem cells Fluorescence microscopy showed that the cytoskeleton of BMSCs was labeled green by Alexa Fluor 488-phalloidin, the nucleus of BMSCs was labeled blue by DAPI, the ADSCs-Exos was labeled red by Dil, After coincubating Dil-labeled exosomes with BMSCs ,and there were a large number of exosomes (red dots) in the cytoplasm (green fluorescence) around the nucleus (blue fluorescence) (Fig. 3 A), which indicated that exosomes could be taken up by BMSCs. 3.5 ADSCs-Exos induce the migration, proliferation and osteogenesis of BMSCs. To assess the effect of ADSCs-Exos on BMSCs function, we performed a series of in vitro assays. Wound healing and Transwell migration assays revealed that ADSCs-Exos significantly promoted BMSCs migration in a concentration-dependent manner, with the most pronounced effect observed at 20 µg/mL (Fig. 3 B-E). CCK-8 assaysindicated that exosome treatment significantly enhanced BMSCs proliferation after 12 and 24 hours (Fig. 3 F). qRT-PCR analysis of migration-related genes revealed that ADSCs-Exos treatment significantly upregulated multiple genes associated with cell motility. After 12 hours, mRNA levels of Ccr7, MMP9, and Cxcr4 were markedly elevated compared to the control (Fig. 3 G). This upregulation persisted and expanded by 24 hours, with MMP2, Cxcr4, and Rac1 also showing significantly increased expression (Fig. 3 H). Furthermore, after 7 days of culture with ADSCs-Exos, BMSCs exhibited enhanced osteogenic differentiation, demonstrated by both more intense ALP staining (Fig. 3 I) and higher ALP activity (Fig. 3 J) compared to the control. qRT-PCR analysis further showed sustained upregulation of osteogenic genes across multiple time points: RUNX2, OCN, and COL1A1 were elevated at day 3 (Fig. 3 K); ALP, RUNX2, and OPN were increased at day 7 (Fig. 3 L); and ALP, OCN, and OPN remained significantly higher at day 14 (Fig. 3 M). The osteogenic induction medium group served as a positive control and consistently showed robust gene induction, confirming system responsiveness. Together, these results demonstrate that ADSCs-Exos effectively enhance the migration, proliferation, and osteogenic differentiation of BMSCs; these functional enhancements correlate with the upregulation of a specific set of migration-related and osteogenesis-related genes. 3.6 Cell transfection assay and Functional Bioluminescence Validation Lentivirus successfully transfected mBMSCs was confirmed by the presence of EGFP green fluorescence observed under microscopy (Fig. 4 A). After luciferin addition, the transduced cells emitted a strong and distinguishable bioluminescent signal, and can be detected by the IVIS Spectrum Imaging System (Fig. 4 B), demonstrating that the lentivirus-modified mBMSCs remained functional activity and suitability for in vivo tracking via bioluminescence imaging. 3.7 Live imaging Following intravenous injection of mBMSCs, in vivo cell tracking was conducted at 24 hours, 48 hours, 3 days, and 1 week (Fig. 4 C). No bioluminescent signal was detected at any time point in the blank control group that received no lentivirus-labeled mBMSCs. In contrast, both the tail vein injection group and the exosome-loaded hydrogel combined with tail vein injection group exhibited bioluminescent signals at 24 hours, 48 hours, 3 days, and 1 week after administration. Signals were observed in multiple regions including the skull, heart, lungs, limbs, and maxillofacial area, with initial accumulation primarily in the cardiopulmonary region. By 48 hours post-injection, while signals in the cardiopulmonary area began to diminish, distinct signals emerged in the calvarial defect region in both experimental groups. The combined treatment group demonstrated significantly stronger signals in the calvarial region compared to the injection-only group, indicating that the hydrogel-exosome conjugate enhanced BMSCs homing to the defect site. Due to substantially diminished signal intensity by one week, no further imaging was conducted beyond this time point. 3.8 Micro-CT Based on promising in vitro results, we further evaluated the bone regeneration efficacy of Thewell hydrogel loaded with ADSCs-Exos, alone or combined with intravenously administered BMSCs, in a rat critical-sized calvarial defect model (Fig. 5 A). Micro-CT and histological analyses were performed at 4 and 8 weeks post-surgery. Three-dimensional micro-CT reconstructions from both coronal (Fig. 5 B) and sagittal (Fig. 5 C) views revealed limited bone formation in the control and hydrogel-only groups at both time points. In contrast, the BMSCs, ADSC-Exos, and BMSCs+ADSC-Exos groups showed progressively increasing bone regeneration over time, with the BMSCs+ADSCs-Exos group exhibiting the most substantial bone regeneration, nearly bridging the defect site by week 8. Quantitative micro-CT analysis (Fig. 5 C) at 4 and 8 weeks showed that the BMSCs+ADSCs-Exos group, the BMSCs group, and the ADSCs-Exos group all exhibited significantly improved bone microarchitecture compared to the hydrogel and control groups. This was reflected in increased BV/TV, trabecular number (Tb.N), and trabecular thickness (Tb.Th), along with decreased trabecular separation (Tb.Sp) and bone surface/bone volume (BS/BV). Notably, the combined BMSCs and ADSCs-Exos treatment led to the most substantial enhancement in bone regeneration, outperforming all other groups at both time points. These results indicate that both systemic administration of BMSCs via tail vein injection and local delivery of exosome-loaded hydrogel effectively promote in vivo bone healing, with synergistic effects observed when combined. 3.9 Histological analysis Histological analysis (Fig. 6 A and 6 B) including H&E and Masson's trichrome staining revealed that the defects in both the control and hydrogel groups were primarily filled with fibrous connective tissue, characterized by fibroblast infiltration and hyperplastic fibers, which bridged the defect edges. Even at 8 weeks post-operation, no conspicuous new bone formation was observed within the defects. In contrast, the BMSCs, ADSC-Exos, and BMSCs+ADSC-Exos groups all exhibited robust collagen deposition and substantial new bone formation at both 4 and 8 weeks. The defect area was largely covered by bone collagen and newly formed bone tissue extending from the periphery to the central region. Compared to the other groups, BMSCs+ADSC-Exos groups showed bone bridges with greater continuity and thickness, as well as significantly more new bone formation. By 4 weeks, scarcely any residual hydrogel material was discernible in the hydrogel, ADSCs-Exos and BMSCs+ADSC-Exos groups, indicating extensive degradation and replacement by newly formed bone. By 8 weeks, the BMSCs+ADSC-Exos groups displayed well-organized and robust bone structures throughout the defect site. Immunohistochemical analysis (Fig. 6 C) further revealed strong expression of OCN in the three treatment groups, localized predominantly within the newly formed bone and osteoid regions. In contrast, only minimal staining was observed in the control and hydrogel-only groups. At the 8-week time point, the BMSCs+ADSC-Exos group showed markedly stronger immunoreactivity for both markers than the BMSCs and ADSCs-Exos groups, underscoring a synergistic and sustained osteogenic effect. In conclusion, the combined application of BMSCs and exosomes exerted a synergistic and sustained osteogenic effect, promoted osteogenic matrix deposition, mineralization, and structural maturation, likely through facilitating BMSCs homing and sustaining osteogenic activation. 4. Discussion In clinical periodontitis, inflammation caused by bacteria usually leads to alveolar bone destruction resulting in alveolar bone defects 36 , 37 , and there are still many problems for bone defect repair in clinical practice 38 , 39 . While stem cell-based therapies have shown promise, their clinical application is hampered by issues such as immune rejection and low cell survival 40 , 41 . Consequently, research has shifted towards the paracrine mechanisms of stem cells, particularly exosomes, as a cell-free therapeutic alternative 20 , 42 . ADSCs are a group of mesenchymal stem cells isolated from white adipose at the rat groin and are a type of stem cell of mesodermal origin. They can self-renewal and cellular differentiation, such as osteoblasts and adipocytes. Compared to BMSCs, ADSCs offer practical advantages, including broader tissue availability, less invasive harvesting, fewer ethical concerns, and lower cost. These advantages make them good seed cells for providing exosomes 27 , 43 , 44 . There are growing evidences that adipose stem cell exosomes can promote the regeneration of neural tissue 45 , 46 , muscle 47 ,skin tissue 32 , 34 , 48 , 49 , and other tissues 50 , 51 . Furthermore, Studies 52 , 53 have shown that changing cell culture conditions can cause alterations in stem cell-derived exosomal miRNAs, resulting in changes in the functions produced by exosomes acting on cells. However, the specific role of ADSCs-Exos in bone defect repair remains less explored. We therefore hypothesized that ADSCs-Exos facilitate the healing of critical-sized defects by orchestrating the homing and osteogenic differentiation of endogenous BMSCs. To test this, a series of in vitro experiments were conducted. Confocal microscopy confirmed the efficient cellular uptake of fluorescently labeled ADSCs-Exos by BMSCs. Functional assays demonstrated that ADSCs-Exos significantly promoted BMSCs migration, this pro-migratory effect is a critical prerequisite for the homing process in vivo. Furthermore, ADSCs-Exos enhanced the osteogenic differentiation of BMSCs, as evidenced by increased ALP activity. Critically, qRT-PCR analysis provided preliminary mechanistic insights underlying these phenotypic changes. The rapid upregulation of migration-related genes, including the key chemokine receptor Cxcr4 and matrix-remodeling enzymes Mmps, offers a molecular basis for the enhanced cell motility, suggesting ADSCs-Exos prime BMSCs for chemotaxis and tissue infiltration. Furthermore, the sustained and stage-specific upregulation of osteogenic markers (Runx2, OPN, OCN, Alp, Col1a1) delineates a clear activation of the osteogenic transcriptional program in BMSCs induced by ADSCs-Exos. These results align with previous studies 54 , 55 indicating that exosomes can act as potent messengers to direct cellular behavior. A pivotal challenge in harnessing exosomes for therapy is their rapid clearance in vivo 30 , 56 – 58 . To address this, we employed a highly elastic hydrogel as a delivery scaffold. Characterization confirmed that the hydrogel successfully encapsulated ADSCs-Exos, ensuring their sustained release and creating a conducive local microenvironment at the defect site, thereby preventing their rapid dissipation in the systemic circulation. Building on the in vitro evidence of enhanced migration potential, we directly investigated the central hypothesis of enhanced homing in vivo. Using small animal live imaging, we provided visual evidence that the ADSCs-Exos/hydrogel complex significantly augmented the recruitment of BMSCs to the calvarial defect site. The confluence of our in vitro and in vivo data supports a coherent “recruit-and-activate” mechanism. While previous studies, such as those by Kang, Y et al. 59 and Li, X et al. 60 have established the efficacy of exosome-biomaterial composites in bone repair, the involvement of endogenous BMSCs homing as a central mechanism has been less directly investigated. The hydrogel-mediated sustained release of ADSCs-Exos creates a bioactive niche at the defect site. These exosomes appear to systemically “prime” BMSCs, potentially via upregulation of genes like Cxcr4, Mmp9, enhancing their responsiveness to homing signals. Once recruited, the local exosome-rich microenvironment further “activates” the BMSCs, driving their commitment to the osteogenic lineage as evidenced by the sequential activation of osteogenic genes, ultimately leading to robust bone regeneration confirmed by micro-CT and histology. This strategy successfully leverages endogenous repair mechanisms, avoiding the pitfalls of direct cell transplantation. This study has several limitations. First, while we identified key gene expression changes, the specific bioactive cargoes within ADSCs-Exos (e.g., miRNAs, proteins) responsible for these effects remain unidentified. Future miRNA sequencing and functional validation are essential to pinpoint the definitive molecular drivers. Second, although in vivo tracking confirmed BMSCs homing to the defect site, the long-term survival, precise differentiation fate, and relative functional contribution of these recruited cells to the regenerated bone remain to be determined. Finally, the ADSCs-Exos were used as a heterogeneous mixture; the potential distinct functions of specific exosome subpopulations warrant future investigation. In summary, This study demonstrates that a sustained local delivery of ADSCs-Exos via an elastic hydrogel effectively promotes the repair of critical-sized bone defects. Mechanistically, ADSCs-Exos appear to prime BMSCs by upregulating homing-related genes like Cxcr4 and MMPs, thereby enhancing their recruitment to the defect site. subsequently activating their intrinsic osteogenic differentiation program. Thus, this work establishes a cell-free therapeutic paradigm that focuses on mobilizing the body's own regenerative capacity, holds significant promise for clinical translation in orthopedics and craniofacial reconstruction. Declarations Acknowledgments We are thankful to the staff of Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences for technical support. Authors’ contributions Conceptualization: J.Y., J.J. and Y.W.; methodology: J.Y., J.J. and X.T.; Validation: J.Y., S.M. and X.T.; Formal analysis: J.Y. and J.J.; Investigation: J.Y.; Resources: Y.W., Y.Q. and Y.L.; data curation: Y.L., S.M. and X.T.; project administration: J.Y., J.J. and X.T.; writing—original draft preparation: J.Y.; writing—review and editing: J.Y., Y.W. and Y.L.; supervision: Y.W., Y.Q. and Y.L.; funding acquisition: Y.W., Y.Q. and Y.L. All authors have read and agreed to the published version of the manuscript. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Funding This work was supported by the National Natural Science Foundation of China (82571133); Chongqing Science and Technology Commission (CSTB2025NSCQ-GPX1172); Science Health Joint Medical Scientific Research Project of Chongqing (2021MSXM114). Conflicts of Interest The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8646512","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":581815503,"identity":"613f39a7-edfb-49a8-a92b-b53c955a5288","order_by":0,"name":"Jingjie Yang","email":"","orcid":"","institution":"The Affiliated Stomatological Hospital of Chongqing Medical University, Chongqing Key Laboratory of Oral Diseases","correspondingAuthor":false,"prefix":"","firstName":"Jingjie","middleName":"","lastName":"Yang","suffix":""},{"id":581815504,"identity":"aa12e59f-ec19-476f-a973-3095a93a99a6","order_by":1,"name":"Jing Jing","email":"","orcid":"","institution":"The Affiliated Stomatological Hospital of Chongqing Medical University, Chongqing Key Laboratory of Oral Diseases","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Jing","suffix":""},{"id":581815505,"identity":"4ebbce84-d391-488b-9f75-a20bd8e96bc8","order_by":2,"name":"Xuesha Tong","email":"","orcid":"","institution":"The Affiliated Stomatological Hospital of Chongqing Medical University, Chongqing Key Laboratory of Oral Diseases","correspondingAuthor":false,"prefix":"","firstName":"Xuesha","middleName":"","lastName":"Tong","suffix":""},{"id":581815506,"identity":"08fae976-ad0f-4b06-ad41-19ea8345782d","order_by":3,"name":"Songyang Ma","email":"","orcid":"","institution":"The Affiliated Stomatological Hospital of Chongqing Medical University, Chongqing Key Laboratory of Oral Diseases","correspondingAuthor":false,"prefix":"","firstName":"Songyang","middleName":"","lastName":"Ma","suffix":""},{"id":581815507,"identity":"ee010093-370f-46d8-8c30-4a90b6d9fa82","order_by":4,"name":"Ye Qiu","email":"","orcid":"","institution":"The Affiliated Stomatological Hospital of Chongqing Medical University, Chongqing Key Laboratory of Oral Diseases","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Qiu","suffix":""},{"id":581815508,"identity":"eb4fba89-1c97-4d7e-abbd-6dd328468cba","order_by":5,"name":"Yang Liu","email":"","orcid":"","institution":"The Affiliated Stomatological Hospital of Chongqing Medical University, Chongqing Key Laboratory of Oral Diseases","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Liu","suffix":""},{"id":581815509,"identity":"25d5e7b3-797d-4ea2-ba19-0195a5cb4665","order_by":6,"name":"Yunji Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBAC+2bG5geS/2ygXDYitBiwNx8zsGBLI0ULz7EEiQq2wyRoMZfIMTC4wXNenu/aGQOGD2WHGfhnN+DXYjkjx+DhDInbhjNv5xgwzjh3mEHizgEC1tzIMTCWMLidYADUwszbdpjBQCKBsBbpPwnnIFr+EqPF4AzQ+xIHDkC0MBKjRbIdGMiSDclAv6QVHOw5l84jcYOAFn5mUFQ22Mnz3U7e+OBHmbUc/wxCfoGDA2DEwEOseoiWUTAKRsEoGAVYAQAQh0XVqywJXQAAAABJRU5ErkJggg==","orcid":"","institution":"The Affiliated Stomatological Hospital of Chongqing Medical University, Chongqing Key Laboratory of Oral Diseases","correspondingAuthor":true,"prefix":"","firstName":"Yunji","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-01-20 08:01:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8646512/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8646512/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101438785,"identity":"ba120084-4917-487a-97d2-ad40d4e64ece","added_by":"auto","created_at":"2026-01-29 16:41:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":895129,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of ADSCs, BMSCs and ADSCs-Exos. (\u003cstrong\u003eA\u003c/strong\u003e) Representative light microscopic images of ADSCs and BMSCs. Scale bars: 200 µm. (\u003cstrong\u003eB\u003c/strong\u003e) Osteogenic differentiation of ADSCs and BMSCs. Scale bar = 500 µm. (\u003cstrong\u003eC\u003c/strong\u003e) Adipogenic differentiation of ADSCs and BMSCs. Scale bar = 500 µm. (\u003cstrong\u003eD\u003c/strong\u003e) Flow cytometry analysis for ADSCs and BMSCs markers (CD11b/c, CD45, CD34, CD29, CD90, CD44 and CD73). \u003cstrong\u003e(E) \u003c/strong\u003eSchematic diagram of the isolation of the ADSCs-Exos. \u003cstrong\u003e(F)\u003c/strong\u003e TEM image of ADSCs-Exos.Scale bars: 200 nm. \u003cstrong\u003e(G)\u003c/strong\u003e Exosome-specific markers (CD81, CD9 and TSG101) detected by WB. n=3. \u003cstrong\u003e(H)\u003c/strong\u003e NTA for the diameters of ADSCs-Exos.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8646512/v1/d3ac82c3d133613d71669448.png"},{"id":101438826,"identity":"5614acec-bbf6-4e15-bc83-0b958ea572f0","added_by":"auto","created_at":"2026-01-29 16:41:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":206928,"visible":true,"origin":"","legend":"\u003cp\u003eRheological analyses of the Thewell-Exos hydrogel and release of Exosomes. (\u003cstrong\u003eA\u003c/strong\u003e) Oscillatory frequency sweep test showing the frequency dependence of storage and loss modulus of the Thewell-Exos hydrogel. (\u003cstrong\u003eB\u003c/strong\u003e) Rheology analysis of Thewell-Exos hydrogel in strain sweep experiment. (\u003cstrong\u003eC\u003c/strong\u003e) Shear-thinning property of the Thewell-Exos hydrogel. (\u003cstrong\u003eD\u003c/strong\u003e) Self-healing behavior of the Thewell-Exos hydrogel during destructive shearing and recovery. (\u003cstrong\u003eE\u003c/strong\u003e) Evaluation of the injectability of Thewell-Exos hydrogel (\u003cstrong\u003eF\u003c/strong\u003e) Exosome release profile of the Thewell hydrogel in vitro over 7d.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8646512/v1/bcedd900b49f7f4835aea882.png"},{"id":101438757,"identity":"d5d1259b-4110-4365-87d1-a80f8248959a","added_by":"auto","created_at":"2026-01-29 16:40:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1146152,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of different concentrations (0,10,20,30µg/mL) of ADSCs-Exos on the migration, proliferation and osteogenesis of BMSCs. (\u003cstrong\u003eA\u003c/strong\u003e) ADSCs-Exos were endocytosed by BMSCs. ADSCs-Exos were labeled by Dil (red); nuclei were labeled by DAPI (blue) ;cytoskeleton was labeled by phalloidin (green). Scale bar: 50 μm. (\u003cstrong\u003eB\u003c/strong\u003e) ADSCs-Exos significantly promoted the migration of BMSCs as determined by the transwell assay at 24 h. Scale bars 100 μm. (\u003cstrong\u003eC\u003c/strong\u003e) Quantitative analysis of the numbers of migrated BMSCs in the Transwell assay. (\u003cstrong\u003eD\u003c/strong\u003e) ADSCs-Exos significantly promoted the migration of BMSCs as determined by the scratch wound assay at 12 and 24 h. Scale bars 100 μm. (\u003cstrong\u003eE\u003c/strong\u003e) Quantitative analysis of the migration rates in the scratch wound assay. (\u003cstrong\u003eF\u003c/strong\u003e) ADSCs-Exos significantly promoted the proliferation of BMSCs, as demonstrated by CCK-8. Expression of migration-related genes at 12 hours (\u003cstrong\u003eG\u003c/strong\u003e) and 24 hours (\u003cstrong\u003eH\u003c/strong\u003e). (\u003cstrong\u003eI\u003c/strong\u003e) ALP staining revealed significantly greater alkaline phosphatase activity in the ADSCs-Exos group compared to the control group. (\u003cstrong\u003eJ\u003c/strong\u003e) Quantification of ALP staining after incubation of BMSCs with exosomes for 7 days. Expression of osteogenesis-related genes at 3 days (\u003cstrong\u003eK\u003c/strong\u003e) , 7 days (\u003cstrong\u003eL\u003c/strong\u003e) and 14 days (\u003cstrong\u003eM\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8646512/v1/b553a278633cb83590c0d3e0.png"},{"id":101438781,"identity":"a5422879-f9c9-47bc-86c8-67f4cee53e78","added_by":"auto","created_at":"2026-01-29 16:41:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1103887,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo homing and tracking of lentivirally transduced mBMSCs. (\u003cstrong\u003eA\u003c/strong\u003e) Observation of EGFP expression in lentivirus-transduced mBMSCs under a fluorescence microscope (scale bar, 300 µm). (\u003cstrong\u003eB\u003c/strong\u003e) Detection of bioluminescent signals from transduced mBMSCs following incubation with luciferin substrate. (\u003cstrong\u003eC\u003c/strong\u003e) Bioluminescent imaging of mice after mBMSCs injection and hydrogel-exosome treatment. Bioluminescent signal was detected on 24 hours, 48 hours, 3 days, and 1 week after mBMSCs injection and hydrogel-exosome treatment.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8646512/v1/bf6b35cb5bb24788950283fd.png"},{"id":101438766,"identity":"3632c0f4-d9b5-4f03-9be0-6797afb3439e","added_by":"auto","created_at":"2026-01-29 16:40:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":871740,"visible":true,"origin":"","legend":"\u003cp\u003eADSCs-Exos and ADSCs-Exos with intravenous BMSCs therapy promote bone formation in critical-sized rat calvarial defects. (\u003cstrong\u003eA\u003c/strong\u003e) Photo of the establishment of a rat calvarial defect model. Representative 3D reconstruction images of the defect area are presented in coronal (\u003cstrong\u003eB\u003c/strong\u003e) and sagittal (\u003cstrong\u003eC\u003c/strong\u003e) sectional views at 4 and 8 weeks post-surgery. Scale bar = 5 mm. (\u003cstrong\u003eD\u003c/strong\u003e) Quantitative analysis of bone volume(BV), bone volume/tissue volume(BV/TV), bone surface(BS), bone surface/tissue volume(BS/TV), trabecular number(Tb.N), trabecular separation(Tb. Sp) and trabecular thickness(Tb.Th) at 4 and 8 weeks.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8646512/v1/b759e4e7a84f2a869d72831f.png"},{"id":101438746,"identity":"376439d6-9e8a-4715-9521-e6c60e39f619","added_by":"auto","created_at":"2026-01-29 16:40:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1156668,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of newly formed bone in rat calvarial defects at 4 and 8 weeks post-surgery. (\u003cstrong\u003eA\u003c/strong\u003e) H\u0026amp;E staining, (\u003cstrong\u003eB\u003c/strong\u003e) Masson's trichrome staining, and (\u003cstrong\u003eC\u003c/strong\u003e) Immunohistochemical staining. The lower panels are the magnification of black frames; HB host bone; NB, new bone. Scale bar = 1 cm (top); scale bar = 200 µm (below). IHC staining against OCN in each group, and black arrows point to the positive staining area; scale bar = 200 µm.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8646512/v1/42be55f8d105131c2b4f4b37.png"},{"id":102994733,"identity":"48a7d469-3196-4e56-ad28-3db4bc9a131e","added_by":"auto","created_at":"2026-02-19 11:57:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6444098,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8646512/v1/98cbf91e-f180-43c8-9c00-15ef69e4b6b4.pdf"},{"id":101438756,"identity":"49b1361a-d950-4bf8-aab1-b89d93985356","added_by":"auto","created_at":"2026-01-29 16:40:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":504167,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8646512/v1/c3520e8bae65e0fa61cf4073.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Adipose-derived stem cell exosomes promote critical-sized bone defect repair by enhancing the homing of bone marrow mesenchymal stem cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe regeneration of critical-sized bone defects remains a formidable clinical challenge in orthopedics\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Although autogenous bone grafts are considered the gold standard for bone defect repair\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, they are associated with significant drawbacks, including insufficient graft supply, iatrogenic injury, and postoperative infection, which can lead to poor prognosis\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. With recent breakthroughs in tissue engineering technology, stem cell transplantation has been gradually applied for bone tissue regeneration\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, concerns regarding immune rejection, tumorigenicity, and low cell survival post-transplantation hinder its clinical translation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Consequently, research attention has shifted toward the paracrine mechanisms of stem cells, particularly toward exosomes, as a potent cell-free alternative\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Exosomes, which are cell-secreted vesicles with a diameter of 30\u0026ndash;150 nm, carry a variety of biological signal molecules, can mediate intercellular communication and replicate the therapeutic effects of their parent cells\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Exosomal components, such as miRNAs have been shown to be involved in various metabolic activities between cells, including those of adipose-derived stem cells (ADSCs), BMSCs, immune cells, and other cell types\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and are involved in regulating bone homeostasis\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. ADSCs, which are isolated from adipose tissue, possess multi-lineage differentiation potential\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This capacity, combined with their abundant availability, accessible isolation, and robust proliferation, makes them a highly attractive source of therapeutic exosomes\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Although exosomes offer numerous advantages, their application is limited by rapid in vivo metabolism\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Therefore, there is a need for materials that enable the slow release of exosomes. Biomaterials such as hydrogels and titanium nano-scaffolds have been used for this purpose\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Although hydrogels and other materials have been explored for this purpose, the combined effect of ADSCs-Exos with a highly elastic hydrogel on specifically promoting the homing and osteogenic differentiation of BMSCs for endogenous repair of critical-sized defects remains insufficiently investigated. We hypothesize that ADSCs-Exos, when delivered via a highly elastic hydrogel, promote bone regeneration primarily by enhancing the homing and subsequent osteogenic differentiation of endogenous BMSCs. This mechanism aligns with the emerging paradigm of endogenous regeneration, which aims to recruit and activate the body's own reparative cells rather than relying on externally implanted ones. This study aims to: (1) investigate the in vitro effects of ADSCs-Exos on BMSCs migration and osteogenesis; (2) visualize and verify the enhanced homing of BMSCs in vivo using a luciferase-based imaging system; and (3) evaluate the efficacy of a sustained-release ADSCs-Exo/hydrogel complex in repairing critical-sized calvarial defects in a rat model. Our findings provide new mechanistic insights into ADSCs-Exos-mediated endogenous bone regeneration, supporting their development as a cell-free therapeutic strategy.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture\u003c/h2\u003e \u003cp\u003eSubcutaneous white adipose tissue was harvested from the inguinal region of 4-week-old male Sprague-Dawley (SD) rats. Fascia and blood vessels within the adipose tissue were meticulously removed, and the tissue was minced and digested with 3 mg/mL collagenase type I (Sigma-Aldrich, USA) for 1 hour at 37\u0026deg;C, and plated in 15 cm\u0026sup2; culture dishes (Corning, USA) with growth medium (α-MEM supplemented with 10% FBS and 1% penicillin-streptomycin).Rat BMSCs were isolated from the femur and tibia by flushing the marrow cavity with serum-free α-MEM. The cell suspension was filtered, centrifuged, and cultured in the same growth medium as ADSCs. Both ADSCs and BMSCs at passages 3\u0026ndash;5 were used for experiments and cultured at 37\u0026deg;C in a 5% CO₂ humidified incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Stem cell multidirectional differentiation\u003c/h2\u003e \u003cp\u003eTo assess differentiation potential, BMSCs underwent adipogenic and osteogenic induction. For adipogenic differentiation, cells were cultured in induction medium (α-MEM supplemented with 10% FBS, 1 \u0026micro;M dexamethasone, 10 \u0026micro;g/mL insulin, 200 \u0026micro;M indomethacin, and 0.5 mM IBMX) with medium replacement every 3 days. Lipid accumulation was evaluated after 14\u0026ndash;21 days using Oil Red O staining (Solarbio, China). For osteogenic differentiation, cells were maintained in induction medium (α-MEM containing 10% FBS, 50\u0026micro;M ascorbic acid, 100nM dexamethasone, and 10mM β-glycerophosphate) with medium changes every 3 days. Calcium deposition was assessed by Alizarin Red S staining (Sigma-Aldrich, USA) after 21\u0026ndash;28 days, while early osteogenic differentiation was evaluated through ALP staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell identification\u003c/h2\u003e \u003cp\u003eADSCs and BMSCs were analyzed for mesenchymal stromal cell marker expression using a Rat Mesenchymal Stem Cell Characterization Kit (Cyagen Biosciences Inc, USA). Briefly, cell suspensions were prepared at a density of 3 \u0026times; 10⁶ cells per 100 \u0026micro;L phosphate-buffered saline (PBS). Cells were pelleted by centrifugation (250 \u0026times;g for 5 min), washed once with PBS, and resuspended in 100 \u0026micro;L PBS. The cell suspension was then incubated with fluorochrome-conjugated primary antibodies (as specified in the kit) at 4\u0026deg;C for 30 minutes in the dark. Following incubation, cells were washed twice with PBS, resuspended in 400 \u0026micro;L PBS, and analyzed immediately using a FACS flow cytometer (BD Bioscience, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Isolation and collection of exosomes\u003c/h2\u003e \u003cp\u003eExosomes were isolated from the supernatant of ADSCs cultured in osteogenic induction medium for 14 days. The medium consisted of α-MEM supplemented with 10% exosome-depleted FBS, 10 mM β-glycerophosphate, 50 \u0026micro;M ascorbic acid, and 100 nM dexamethasone, and was replaced every 3 days. The collected supernatant was sequentially centrifuged at 300 \u0026times; g for 10 min, 2,000 \u0026times; g for 10 min, and 10,000 \u0026times; g for 30 min to remove cells and debris. Exosomes were then pelleted by ultracentrifugation at 100,000 \u0026times; g for 70 min, washed in PBS, and recentrifuged under the same conditions. The final pellet was resuspended in PBS and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Exosome protein concentration was determined using a BCA assay kit (Beyotime, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Identification of exosomes\u003c/h2\u003e \u003cp\u003eThe exosomes suspension was prepared in PBS and applied dropwise onto a copper mesh. After air-drying, the sample was negatively stained with 1% phosphotungstic acid for 5 min and allowed to dry again. Morphological examination of exosomes was performed using transmission electron microscopy (TEM; Hitachi, Japan). Nanoparticle tracking analysis (NTA) determined the particle size and concentration of exosomes. Exosomes were diluted with PBS and analyzed using the ZetaViewPMX110 (Particle Matrix, Germany). Results were analyzed with NTA analysis software (ZetaView8.04.02SP2). icle Metrix, Germany) and corresponding\u003c/p\u003e \u003cp\u003eZetaView 8.04.02 software. Then, western blotting (WB) was conducted to verify the specific markers of exosomes. Exosomes were lysed in RIPA lysis buffer (Beyotime, China), and protein content was determined using the BCA protein analysis kit (Beyotime, China). Briefly, exosomes were loaded onto 10% gels, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore, USA). The membranes were closed with 5% distilled skim milk and then incubated overnight at 4\u0026deg;C with primary antibodies against anti-CD81, anti-CD9, anti-TSG101, and anti-GAPDH (Abcam, USA, 1:1000 dilution). Secondary antibodies (Abcam, USA, 1:2000 dilution) were incubated at room temperature for 1 h. After each incubation, membranes were washed 3 times with TBST, and protein bands were displayed with ECL kit (Beyotime, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Rheological measurements\u003c/h2\u003e \u003cp\u003eWe tested the rheological properties of Thewell-Exos using a Discovery hybrid rheometer (Anton Paar, Austria). Rheological testing was performed at 25\u0026deg;C with a 1000 \u0026micro;m gap setting. (1) oscillation frequency scan (0.1\u0026ndash;10 rad/s at 0.5% constant strain) to monitor the elasticity (storage modulus; G\u0026prime;) and viscosity (loss modulus; G\u0026Prime;); (2) Steady-state flow measurements (0.1\u0026ndash;100 s⁻\u0026sup1; shear rates) to evaluate shear-thinning behavior; (3) The self-healing test consisting of high-strain deformation (1 Hz, 0.1\u0026ndash;1000% strain, 200 s) followed by recovery phase (1 Hz, 0.5% strain, 200 s), monitoring G\u0026prime;/G\u0026Prime; evolution for self-healing capacity; (4) Amplitude sweeps (0.1\u0026ndash;100% strain at 1 Hz) to determine yield stress, identified as the shear stress at the G'-G\" crossover point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Slow release behavior of exosomes\u003c/h2\u003e \u003cp\u003eTo further determine the release efficiency of exosomes in the hydrogel, Hydrogels (TheWell Inc, USA) were loaded with exosomes by mixing equal volumes (500 \u0026micro;L) of the exosome suspension (0.4 mg/mL) and hydrogel, following the manufacturer's protocol. An equivalent volume mixture of hydrogel and PBS served as negative control. To evaluate exosome release efficiency, exosome-loaded hydrogels were incubated in 0.8 mL PBS at 4℃. At predetermined intervals from t\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5 min to t\u003csub\u003e23\u003c/sub\u003e=168 h, 0.2 mL release medium was collected and replaced with fresh PBS. The collected samples were quantified using a bicinchoninic acid (BCA) protein assay kit. Absorbance at 562 nm was measured via microplate reader (Perkin Elmer, USA), with protein concentrations calculated against a standard curve. All experiments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Exosome uptake assay.\u003c/h2\u003e \u003cp\u003ePurified exosomes were labelled with the lipophilic dye DiI (Beyotime, China) according to the manufacturer's instructions. The labeling reaction proceeded for 15 min at room temperature in light-protected conditions. The labelled exosomes were washed in PBS at 100,000 \u0026times; g for 70 minutes. BMSCs were then incubated with labelled exosomes for 24 hours. After incubation, the cells were washed twice with PBS and fixed in 4% paraformaldehyde (Beyotime, China) for 15 min, permeabilized with 0.1% Triton X-100 (Beyotime, China) at room temperature, and stained with Alexa Fluor 488-phalloidin (Yeasen, China) for 30 min for F-actin visualization. The cells were washed three times with PBS, and the nuclei were stained with DAPI for 10 min. Images were obtained using a confocal laser scanning microscope (Nikon, Japan) and analyzed using NIS-Elements Viewer software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 BMSCs migration assay\u003c/h2\u003e \u003cp\u003eCell migration capacity of BMSCs was assessed using scratch wound and Transwell assays. For the scratch assay, BMSCs in logarithmic growth phase were digested with trypsin to generate single-cell suspensions, seeded into 6-well plates at 6\u0026times;10⁵ cells/well in 2 mL complete medium, and cultured to 100% confluence. Sterile pipette tips were used to create uniform scratches perpendicular to the plate surface. After three PBS washes to remove detached cells, the cells were treated with serum-free medium containing either ADSC-Exos (experimental group) or an equal volume of PBS (control group). Wound closure was documented at 0, 12, and 24 h post-scratching, with migration distances quantified using ImageJ software. For Transwell migration, 5\u0026times;10⁵ cells were loaded into the upper chamber of the 24-well transwell plate (Corning, USA) with an 8-mm aperture filter. Chambers containing 0, 10, 20, or 30 \u0026micro;g/mL Exo-14d in complete medium were placed in the lower chamber. Chambers containing 0, 10, 20, or 30 \u0026micro;g/mL ADSC-Exos in complete medium were placed in the lower chamber. After co-culturing the cells for 24 h, cells were gently rinsed with PBS. and non-migrated cells on the upper membrane surface were removed by cotton swab abrasion. Membranes were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Beyotime, China) for 10 min. After three washes, positively stained cells were counted under an inverted microscope (Leica, Germany). Both assays were independently repeated in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 BMSCs proliferation assay\u003c/h2\u003e \u003cp\u003eThe proliferation of BMSCs was measured using CCK-8 assay (EnoGene, China). Briefly, BMSCs (3\u0026times;10\u0026sup3; cells/well in 100 \u0026micro;L medium) were seeded in the medium containing various concentrations of ADSCs-Exos (0, 10, 20, and 30 \u0026micro;g/mL) using 96-well plates. After 6, 12, or 24 h incubation, 10 \u0026micro;l of CCK-8 solution was added to each well and incubated for 3 h at 37\u0026deg;C. Absorbance was measured at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 ALP activity assay staining\u003c/h2\u003e \u003cp\u003eBMSCs were plated in 24-well plates and cultured for 7 days in osteogenic differentiation medium under two conditions: control medium and medium supplemented with ADSCs-Exos. Following culture, cells were fixed, and ALP activity was assessed. ALP staining was performed using the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China), according to the manufacturer's instructions. ALP activity levels were quantified using an ALP assay kit (Beyotime, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Quantitative Real Time-Polymerase Chain Reaction (qRT‒PCR)\u003c/h2\u003e \u003cp\u003eTo assess the effect of ADSCs-Exos on the expression of osteogenic differentiation and migration-related genes in BMSCs, qRT-PCR was performed under distinct culture conditions. For the assessment of migration-related gene expression, BMSCs were cultured in normal growth medium either supplemented with or without ADSCs-Exos. To evaluate osteogenic differentiation, BMSCs were divided into three groups: cultured in normal growth medium, normal growth medium supplemented with ADSCs-Exos, or standard osteoinductive medium (OM). After the treatment period, total RNA was isolated from the cells using RNAiso Plus reagent (TaKaRa, Japan) for mRNA analysis. Then, the first-strand cDNA was synthesized from equal amounts of total RNA using the PrimeScript RT Master Mix (Takara, Japan). For qRT-PCR analysis, 20\u0026micro;L of reaction mixture was used, including 10 \u0026micro;L of TBGreen Premix Ex Taq (Takara, Japan), 2.0 \u0026micro;L of cDNA, 0.8 \u0026micro;L each of the forward and reverse primers (10 \u0026micro;M), and 6.4 \u0026micro;L of RNase-free water. A dissociation curve analysis was subsequently performed to confirm amplification specificity. The expression of target genes, including osteogenic genes: runt-related transcription factor 2 (RUNX2), osteopontin (OPN), collagen type I alpha 1 chain (Col1a1), ALP, osteocalcin (OCN) and migration-related genes: C-X-C chemokine receptor type 4 (Cxcr4), C-C chemokine receptor 7 (Ccr7), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), Ras-related C3 botulinum toxin substrate 1 (Rac1) was analyzed, with GAPDH acting as the housekeeping gene. The relative gene expression levels were calculated using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method. The sequences of all primers used are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Lentivirus transfection\u003c/h2\u003e \u003cp\u003ePrimary mBMSCs were purchased from Cyagen Biosciences (MUBMX-01001, China). 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e mBMSCs were seeded in 24-well plates, and transduced with PASLlenti-pA-Luc2-CMF-EF1-EGFP-P2A-Puro-WPRE lentivirus (OBiO Technology Corp, China) using 5 \u0026micro;g/ml polybrene (Beyotime, China). Following 18-hour incubation, the medium was replaced, and transduced cells were selected with puromycin (Beyotime, China). Then the transfection efficiency was detected by inverted fluorescence microscopy (Leica, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Bioluminescence Imaging of Luciferase-Labeled Cells In Vitro and In Vivo\u003c/h2\u003e \u003cp\u003eTo verify the bioluminescence functionality of lentivirally transduced mBMSCs in vitro, the cells were cultured in a 96-well plate, incubated with luciferin substrate, and imaged using an IVIS Spectrum Imaging System (Revvity, USA). For in vivo imaging, C57BL/6 mice were placed in an induction chamber and anesthetized via inhalation of isoflurane (3\u0026ndash;4% for induction, 1.5\u0026ndash;2.5% for maintenance) delivered in 100% medical oxygen at a flow rate of 0.8\u0026ndash;1.2 L/min using a calibrated vaporizer and a non-rebreathing system. Anesthesia depth was monitored by the absence of response to toe pinch. Once anesthetized, mice were administered luciferin potassium salt solution (Beyotime, China; 15 mg/mL) via intraperitoneal injection at a dose of 10 \u0026micro;L/g, and remained under anesthesia for the duration of imaging. No other anesthetic or analgesic agents were used during this procedure. The study included three experimental groups: a control group that had not undergone tail injection of lentivirus-labeled BMSC, a tail vein injection group, and a exosome-loaded hydrogel combined with tail vein injection group. Longitudinal bioluminescence imaging was performed using the IVIS Spectrum system under consistent exposure settings at 24 hours, 48 hours, 3 days, and 1 week post-treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Evaluation of Bone Regeneration\u003c/h2\u003e \u003cp\u003e All experiments conducted in this study were approved by the Ethics Committee of the College of Stomatology at Chongqing Medical University (CQHS-REC-2025-078). Eight-week-old male SD rats were randomly divided into five groups (n\u0026thinsp;=\u0026thinsp;6):(1) Control group: calvarial defect\u0026thinsp;+\u0026thinsp;intravenous PBS. (2) BMSCs group: calvarial defect\u0026thinsp;+\u0026thinsp;intravenous BMSCs. (3) Hydrogel group: calvarial defect\u0026thinsp;+\u0026thinsp;hydrogel implantation. (4) ADSCs-Exos group: calvarial defect\u0026thinsp;+\u0026thinsp;exosome-loaded hydrogel implantation.(5) BMSCs+ADSCs-Exos group: calvarial defect\u0026thinsp;+\u0026thinsp;exosome-loaded hydrogel implantation+ intravenous BMSCs. Bilateral critical-sized calvarial defects (5 mm diameter) were created under isoflurane anesthesia. Following hydrogel implantation according to group assignment and intravenous injection of BMSCs (3\u0026times;10⁶) or PBS, animals were euthanized at 4 and 8 weeks. All animal euthanasia was conducted in compliance with institutional ethical guidelines. Specifically, rats were placed in a carbon dioxide euthanasia chamber, and carbon dioxide (CO₂) was delivered at a flow rate of 5\u0026ndash;6 L/min until respiratory and cardiac arrest occurred. CO₂ flow was maintained for an additional 1 minute, after which the chamber was closed and kept under observation for 2 minutes to confirm irreversible death. No physical or additional chemical methods were employed. Calvarial specimens were analyzed by microcomputerized tomography (micro-CT) scan to quantify bone mineral density (BMD), Bone Volume / Tissue Volume (BV/TV), and trabecular parameters. After decalcification, sections were stained with hematoxylin and eosin (H\u0026amp;E) and Masson's trichrome or immunostained for osteocalcin. All images were acquired using an Olympus VS200 microscope (Olympus, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were repeated at least three times, and all data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Data were analyzed by t-test or one-way ANOVA, and GraphPadPrism 9.0 software was used for statistical processing. Statistical significance was considered when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.(*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.)\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Isolation and Characterization of ADSCs and BMSCs\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, third-passage ADSCs exhibited classical fibroblast-like morphology with homogeneous size distribution, demonstrating characteristic plastic-adherent growth under light microscopy\u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. while BMSCs exhibited a spindle-like morphology. Both phenotypes characteristic of mesenchymal stem cells\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Osteogenic differentiation was confirmed by ALP staining for ADSCs and Alizarin Red S staining for BMSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). while adipogenic differentiation of both cell types was demonstrated through Oil Red O staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).These results indicated that ADSCs and BMSCs were successfully isolated. The results of flow cytometry analysis demonstrated that ADSCs and BMSCs highly expressed the surface markers CD29, CD90, CD44 and CD73 but expressed low levels of CD11b/c, CD45 and CD34 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Characterization of exosomes of ADSCs origin\u003c/h2\u003e \u003cp\u003eExosomes were isolated from ADSCs culture supernatants via ultracentrifugation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The ultrastructure of cytosolic exosomes was observed by TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) with a typical spherical double-membrane structure. WB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG; see Supplementary Information 2 for full-length blots) results showed that exosomes of ADSCs origin expressed exosomal surface markers (CD81,CD9, TSG101). In addition, NTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) results showed that the particle size of most ADSCs-Exos was primarily distributed around 123.5nm, and their concentration was 3.14 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e particles/mL. These results attested that we successfully isolated exosomes of ADSCs origin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Thewell-Exos hydrogel characterization\u003c/h2\u003e \u003cp\u003eRheological testing was performed to characterize the viscoelastic properties of the Thewell-Exos hydrogel. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,storage modulus (G\u0026prime; \u0026asymp;240 Pa) approximately 5-fold greater than loss modulus (G\u0026Prime; \u0026asymp;50 Pa) with a frequency range of 0.1\u0026ndash;100 rad/s, exhibiting negligible strain dependence and indicating robust network formation conducive to controlled release kinetics;Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB showed that both G\u0026prime; and G\u0026Prime; values were relatively stable in the strain range from 0.1% to 1%, which indicated the linear viscoelastic region of Thewell-Exos hydrogel. Additionally, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC shows the shear-thinning properties of the Thewell-Exos hydrogel. the Thewell-Exos hydrogel could sustain high shear forces before the sol/gel transition. The self-healing test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) show that the Thewell-Exos hydrogel was subjected to five cycles of time-sweep experiments with low (0.1%) and high (1000%) strain values.At 1000% strain, inversion of modulus values (G\u0026prime; \u0026lt; G\u0026Prime;) confirmed transition to sol state. When the strain was reduced to 0.1%, the hydrogel recovered (G\u0026prime;\u0026gt;G\u0026Prime;). This recovery behavior persisted through all cycles.confirming outstanding self healing behavior of the Thewell-Exos hydrogel. the Thewell-Exos hydrogel exhibited injectability through 26G needles and could be used as \u0026ldquo;ink\u0026rdquo; to draw pattern, This physical adaptability indicates potential suitability for complex bone defect geometries. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). According to BCA assay, the cumulative release of ADSCs-Exos reached 90.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.65% at day 7. Moreover, the release profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e demonstrated excellent sustained-release capacity of Thewell hydrogel for ADSCs-Exos throughout the 7-day monitoring span. Taken the above results together, suggesting a successful preparation of the hydrogel capable of slow-releasing ADSCs-Exos was demonstrated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Uptake of exosomes by Bone marrow mesenchymal stem cells\u003c/h2\u003e \u003cp\u003eFluorescence microscopy showed that the cytoskeleton of BMSCs was labeled green by Alexa Fluor 488-phalloidin, the nucleus of BMSCs was labeled blue by DAPI, the ADSCs-Exos was labeled red by Dil, After coincubating Dil-labeled exosomes with BMSCs ,and there were a large number of exosomes (red dots) in the cytoplasm (green fluorescence) around the nucleus (blue fluorescence) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), which indicated that exosomes could be taken up by BMSCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5 ADSCs-Exos induce the migration, proliferation and osteogenesis of BMSCs.\u003c/h2\u003e \u003cp\u003eTo assess the effect of ADSCs-Exos on BMSCs function, we performed a series of in vitro assays. Wound healing and Transwell migration assays revealed that ADSCs-Exos significantly promoted BMSCs migration in a concentration-dependent manner, with the most pronounced effect observed at 20 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-E). CCK-8 assaysindicated that exosome treatment significantly enhanced BMSCs proliferation after 12 and 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). qRT-PCR analysis of migration-related genes revealed that ADSCs-Exos treatment significantly upregulated multiple genes associated with cell motility. After 12 hours, mRNA levels of Ccr7, MMP9, and Cxcr4 were markedly elevated compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). This upregulation persisted and expanded by 24 hours, with MMP2, Cxcr4, and Rac1 also showing significantly increased expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Furthermore, after 7 days of culture with ADSCs-Exos, BMSCs exhibited enhanced osteogenic differentiation, demonstrated by both more intense ALP staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI) and higher ALP activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ) compared to the control. qRT-PCR analysis further showed sustained upregulation of osteogenic genes across multiple time points: RUNX2, OCN, and COL1A1 were elevated at day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK); ALP, RUNX2, and OPN were increased at day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL); and ALP, OCN, and OPN remained significantly higher at day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). The osteogenic induction medium group served as a positive control and consistently showed robust gene induction, confirming system responsiveness. Together, these results demonstrate that ADSCs-Exos effectively enhance the migration, proliferation, and osteogenic differentiation of BMSCs; these functional enhancements correlate with the upregulation of a specific set of migration-related and osteogenesis-related genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Cell transfection assay and Functional Bioluminescence Validation\u003c/h2\u003e \u003cp\u003eLentivirus successfully transfected mBMSCs was confirmed by the presence of EGFP green fluorescence observed under microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). After luciferin addition, the transduced cells emitted a strong and distinguishable bioluminescent signal, and can be detected by the IVIS Spectrum Imaging System (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), demonstrating that the lentivirus-modified mBMSCs remained functional activity and suitability for in vivo tracking via bioluminescence imaging.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Live imaging\u003c/h2\u003e \u003cp\u003eFollowing intravenous injection of mBMSCs, in vivo cell tracking was conducted at 24 hours, 48 hours, 3 days, and 1 week (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). No bioluminescent signal was detected at any time point in the blank control group that received no lentivirus-labeled mBMSCs. In contrast, both the tail vein injection group and the exosome-loaded hydrogel combined with tail vein injection group exhibited bioluminescent signals at 24 hours, 48 hours, 3 days, and 1 week after administration. Signals were observed in multiple regions including the skull, heart, lungs, limbs, and maxillofacial area, with initial accumulation primarily in the cardiopulmonary region. By 48 hours post-injection, while signals in the cardiopulmonary area began to diminish, distinct signals emerged in the calvarial defect region in both experimental groups. The combined treatment group demonstrated significantly stronger signals in the calvarial region compared to the injection-only group, indicating that the hydrogel-exosome conjugate enhanced BMSCs homing to the defect site. Due to substantially diminished signal intensity by one week, no further imaging was conducted beyond this time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Micro-CT\u003c/h2\u003e \u003cp\u003eBased on promising in vitro results, we further evaluated the bone regeneration efficacy of Thewell hydrogel loaded with ADSCs-Exos, alone or combined with intravenously administered BMSCs, in a rat critical-sized calvarial defect model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Micro-CT and histological analyses were performed at 4 and 8 weeks post-surgery. Three-dimensional micro-CT reconstructions from both coronal (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and sagittal (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) views revealed limited bone formation in the control and hydrogel-only groups at both time points. In contrast, the BMSCs, ADSC-Exos, and BMSCs+ADSC-Exos groups showed progressively increasing bone regeneration over time, with the BMSCs+ADSCs-Exos group exhibiting the most substantial bone regeneration, nearly bridging the defect site by week 8. Quantitative micro-CT analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) at 4 and 8 weeks showed that the BMSCs+ADSCs-Exos group, the BMSCs group, and the ADSCs-Exos group all exhibited significantly improved bone microarchitecture compared to the hydrogel and control groups. This was reflected in increased BV/TV, trabecular number (Tb.N), and trabecular thickness (Tb.Th), along with decreased trabecular separation (Tb.Sp) and bone surface/bone volume (BS/BV). Notably, the combined BMSCs and ADSCs-Exos treatment led to the most substantial enhancement in bone regeneration, outperforming all other groups at both time points. These results indicate that both systemic administration of BMSCs via tail vein injection and local delivery of exosome-loaded hydrogel effectively promote in vivo bone healing, with synergistic effects observed when combined.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Histological analysis\u003c/h2\u003e \u003cp\u003eHistological analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) including H\u0026amp;E and Masson's trichrome staining revealed that the defects in both the control and hydrogel groups were primarily filled with fibrous connective tissue, characterized by fibroblast infiltration and hyperplastic fibers, which bridged the defect edges. Even at 8 weeks post-operation, no conspicuous new bone formation was observed within the defects. In contrast, the BMSCs, ADSC-Exos, and BMSCs+ADSC-Exos groups all exhibited robust collagen deposition and substantial new bone formation at both 4 and 8 weeks. The defect area was largely covered by bone collagen and newly formed bone tissue extending from the periphery to the central region. Compared to the other groups, BMSCs+ADSC-Exos groups showed bone bridges with greater continuity and thickness, as well as significantly more new bone formation. By 4 weeks, scarcely any residual hydrogel material was discernible in the hydrogel, ADSCs-Exos and BMSCs+ADSC-Exos groups, indicating extensive degradation and replacement by newly formed bone. By 8 weeks, the BMSCs+ADSC-Exos groups displayed well-organized and robust bone structures throughout the defect site. Immunohistochemical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) further revealed strong expression of OCN in the three treatment groups, localized predominantly within the newly formed bone and osteoid regions. In contrast, only minimal staining was observed in the control and hydrogel-only groups. At the 8-week time point, the BMSCs+ADSC-Exos group showed markedly stronger immunoreactivity for both markers than the BMSCs and ADSCs-Exos groups, underscoring a synergistic and sustained osteogenic effect. In conclusion, the combined application of BMSCs and exosomes exerted a synergistic and sustained osteogenic effect, promoted osteogenic matrix deposition, mineralization, and structural maturation, likely through facilitating BMSCs homing and sustaining osteogenic activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn clinical periodontitis, inflammation caused by bacteria usually leads to alveolar bone destruction resulting in alveolar bone defects\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, and there are still many problems for bone defect repair in clinical practice\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. While stem cell-based therapies have shown promise, their clinical application is hampered by issues such as immune rejection and low cell survival\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Consequently, research has shifted towards the paracrine mechanisms of stem cells, particularly exosomes, as a cell-free therapeutic alternative\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eADSCs are a group of mesenchymal stem cells isolated from white adipose at the rat groin and are a type of stem cell of mesodermal origin. They can self-renewal and cellular differentiation, such as osteoblasts and adipocytes. Compared to BMSCs, ADSCs offer practical advantages, including broader tissue availability, less invasive harvesting, fewer ethical concerns, and lower cost. These advantages make them good seed cells for providing exosomes\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. There are growing evidences that adipose stem cell exosomes can promote the regeneration of neural tissue\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, muscle\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e ,skin tissue\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, and other tissues\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Furthermore, Studies\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e have shown that changing cell culture conditions can cause alterations in stem cell-derived exosomal miRNAs, resulting in changes in the functions produced by exosomes acting on cells.\u003c/p\u003e \u003cp\u003eHowever, the specific role of ADSCs-Exos in bone defect repair remains less explored. We therefore hypothesized that ADSCs-Exos facilitate the healing of critical-sized defects by orchestrating the homing and osteogenic differentiation of endogenous BMSCs. To test this, a series of in vitro experiments were conducted. Confocal microscopy confirmed the efficient cellular uptake of fluorescently labeled ADSCs-Exos by BMSCs. Functional assays demonstrated that ADSCs-Exos significantly promoted BMSCs migration, this pro-migratory effect is a critical prerequisite for the homing process in vivo. Furthermore, ADSCs-Exos enhanced the osteogenic differentiation of BMSCs, as evidenced by increased ALP activity.\u003c/p\u003e \u003cp\u003eCritically, qRT-PCR analysis provided preliminary mechanistic insights underlying these phenotypic changes. The rapid upregulation of migration-related genes, including the key chemokine receptor Cxcr4 and matrix-remodeling enzymes Mmps, offers a molecular basis for the enhanced cell motility, suggesting ADSCs-Exos prime BMSCs for chemotaxis and tissue infiltration. Furthermore, the sustained and stage-specific upregulation of osteogenic markers (Runx2, OPN, OCN, Alp, Col1a1) delineates a clear activation of the osteogenic transcriptional program in BMSCs induced by ADSCs-Exos. These results align with previous studies\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003eindicating that exosomes can act as potent messengers to direct cellular behavior.\u003c/p\u003e \u003cp\u003eA pivotal challenge in harnessing exosomes for therapy is their rapid clearance in vivo\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. To address this, we employed a highly elastic hydrogel as a delivery scaffold. Characterization confirmed that the hydrogel successfully encapsulated ADSCs-Exos, ensuring their sustained release and creating a conducive local microenvironment at the defect site, thereby preventing their rapid dissipation in the systemic circulation. Building on the in vitro evidence of enhanced migration potential, we directly investigated the central hypothesis of enhanced homing in vivo. Using small animal live imaging, we provided visual evidence that the ADSCs-Exos/hydrogel complex significantly augmented the recruitment of BMSCs to the calvarial defect site.\u003c/p\u003e \u003cp\u003eThe confluence of our in vitro and in vivo data supports a coherent \u0026ldquo;recruit-and-activate\u0026rdquo; mechanism. While previous studies, such as those by Kang, Y et al.\u003csup\u003e59\u003c/sup\u003e and Li, X et al.\u003csup\u003e60\u003c/sup\u003e have established the efficacy of exosome-biomaterial composites in bone repair, the involvement of endogenous BMSCs homing as a central mechanism has been less directly investigated. The hydrogel-mediated sustained release of ADSCs-Exos creates a bioactive niche at the defect site. These exosomes appear to systemically \u0026ldquo;prime\u0026rdquo; BMSCs, potentially via upregulation of genes like Cxcr4, Mmp9, enhancing their responsiveness to homing signals. Once recruited, the local exosome-rich microenvironment further \u0026ldquo;activates\u0026rdquo; the BMSCs, driving their commitment to the osteogenic lineage as evidenced by the sequential activation of osteogenic genes, ultimately leading to robust bone regeneration confirmed by micro-CT and histology. This strategy successfully leverages endogenous repair mechanisms, avoiding the pitfalls of direct cell transplantation.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, while we identified key gene expression changes, the specific bioactive cargoes within ADSCs-Exos (e.g., miRNAs, proteins) responsible for these effects remain unidentified. Future miRNA sequencing and functional validation are essential to pinpoint the definitive molecular drivers. Second, although in vivo tracking confirmed BMSCs homing to the defect site, the long-term survival, precise differentiation fate, and relative functional contribution of these recruited cells to the regenerated bone remain to be determined. Finally, the ADSCs-Exos were used as a heterogeneous mixture; the potential distinct functions of specific exosome subpopulations warrant future investigation.\u003c/p\u003e \u003cp\u003eIn summary, This study demonstrates that a sustained local delivery of ADSCs-Exos via an elastic hydrogel effectively promotes the repair of critical-sized bone defects. Mechanistically, ADSCs-Exos appear to prime BMSCs by upregulating homing-related genes like Cxcr4 and MMPs, thereby enhancing their recruitment to the defect site. subsequently activating their intrinsic osteogenic differentiation program. Thus, this work establishes a cell-free therapeutic paradigm that focuses on mobilizing the body's own regenerative capacity, holds significant promise for clinical translation in orthopedics and craniofacial reconstruction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are thankful to the staff of Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences for technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: J.Y., J.J. and Y.W.; methodology: J.Y., J.J. and X.T.; Validation: J.Y., S.M. and X.T.; Formal analysis: J.Y. and J.J.; Investigation: J.Y.; Resources: Y.W., Y.Q. and Y.L.; data curation: Y.L., S.M. and X.T.; project administration: J.Y., J.J. and X.T.; writing\u0026mdash;original draft preparation: J.Y.; writing\u0026mdash;review and editing: J.Y., Y.W. and Y.L.; supervision: Y.W., Y.Q. and Y.L.; funding acquisition: Y.W., Y.Q. and Y.L. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82571133); Chongqing Science and Technology Commission (CSTB2025NSCQ-GPX1172); Science Health Joint Medical Scientific Research Project of Chongqing (2021MSXM114).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll studies were approved by the Ethics Committee of the College of Stomatology at Chongqing Medical University (approval No. CQHS-REC-2025-078). 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Nanobiotechnol.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12951-024-02342-6\u003c/span\u003e\u003cspan address=\"10.1186/s12951-024-02342-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Adipose-derived stem cells, Exosomes, Cell homing, Bone marrow mesenchymal stem cells, Endogenous repair, Bone defect","lastPublishedDoi":"10.21203/rs.3.rs-8646512/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8646512/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe repair of critical-sized bone defects remains a significant clinical challenge. As a cell-free alternative, exosomes derived from adipose-derived stem cells (ADSCs-Exos) hold promise, yet their precise mechanism in endogenous repair is unclear. This study investigated whether ADSCs-Exos enhance bone repair by promoting the homing and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). ADSCs-Exos were isolated and co-cultured with BMSCs to assess proliferation, migration, and osteogenic differentiation in vitro. Key gene expression (e.g., Cxcr4, Runx2) was analyzed. A highly elastic hydrogel was used for sustained exosome delivery in a rat calvarial defect model. mouse bone marrow-derived mesenchymal stem cells (mBMSCs) homing was monitored via live imaging, and bone regeneration was evaluated by micro-CT and histology. Results showed that ADSCs-Exos promoted BMSC migration and osteogenesis, rapidly upregulating homing-related genes (Ccr7, Cxcr4) and subsequently activating osteogenic genes (Runx2, OPN). In vivo, the ADSCs-Exo/hydrogel complex significantly enhanced BMSC recruitment to the defect site, leading to markedly improved new bone formation. This study elucidates a novel, cell-free strategy wherein ADSCs-Exos orchestrate endogenous bone repair by enhancing BMSC homing and differentiation, providing a potential therapeutic approach.\u003c/p\u003e","manuscriptTitle":"Adipose-derived stem cell exosomes promote critical-sized bone defect repair by enhancing the homing of bone marrow mesenchymal stem cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 16:38:38","doi":"10.21203/rs.3.rs-8646512/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"70b36972-9cbc-4b84-97c9-79dbed643301","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61899438,"name":"Biological sciences/Cell biology"},{"id":61899439,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-04-11T16:23:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 16:38:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8646512","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8646512","identity":"rs-8646512","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}