Mechanical Stimulation Activates Postn-Mediated Wnt/β-catenin Pathway to Enhance BMSCs Paracrine Function and Promote Wound Healing in Diabetic Rats

Mechanical Stimulation Activates Postn-Mediated Wnt/β-catenin Pathway to Enhance BMSCs Paracrine Function and Promote Wound Healing in Diabetic Rats | 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 Research Article Mechanical Stimulation Activates Postn-Mediated Wnt/β-catenin Pathway to Enhance BMSCs Paracrine Function and Promote Wound Healing in Diabetic Rats Dong Zhang, Haowei Zhou, Congying Zhao, Zhanjun Lei, Zhe Liu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8064472/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background Diabetic wounds exhibit impaired healing due to angiogenic deficiency and dysregulated extracellular matrix homeostasis. Although bone marrow mesenchymal stem cells (BMSCs) promote wound repair through paracrine signaling, their therapeutic efficacy is compromised in diabetic microenvironments. Crucially, emerging evidence implicates Periostin (Postn) as a mechanoresponsive matricellular protein that directly activates Wnt/β-catenin signaling - a pathway governing stem cell paracrine function and tissue regeneration. However, whether mechanical stimulation leverages this Postn-Wnt/β-catenin axis to optimize BMSCs secretory capacity remains unexplored. This study specifically interrogates this mechanotransduction mechanism to develop enhanced therapies for diabetic wounds. Methods BMSCs underwent cyclic stretching (15% strain, 0.5 Hz, 10 h), and paracrine factors (VEGF/TGF-β/bFGF) and pathway proteins were assessed via qRT-PCR, Western blot, and ELISA. Then, Postn knockdown (siRNA) or Wnt/β-catenin inhibition (XAV-939) was implemented to the stretched BMSCs, and paracrine factors were assessed again. Conditioned medium (CM) of stretched BMSCs functionality was evaluated using scratch assay and tube formation assays with Rat umbilical vein endothelial cells (RUVECs) and fibroblasts (Fbs). Finally, a full-thickness diabetic rat wound model was established to validate in vivo efficacy of the CM through wound closure rate, histochemistry, and immunofluorescence. Results Mechanical stimulation significantly increased Postn expression and Wnt/β-catenin signaling, boosting the secretion of VEGF, TGF-β1, and bFGF. Postn knockdown or Wnt/β-catenin pathway inhibition attenuated these effects, while exogenous Postn partially restored function. CM from stretched BMSCs promoted endothelial migration, tube formation, fibroblast migration in vitro, and accelerated wound healing, angiogenesis, and collagen deposition in vivo . Conclusion Mechanical stretch enhances BMSCs’ paracrine function through a Postn-Wnt/β-catenin axis, offering a mechanobiology-based, cell-free approach to improve diabetic wound repair. Mechanical stretch Bone marrow mesenchymal stem cells Periostin Wnt/β-catenin pathway Diabetic wound healing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Chronic refractory wounds (CRW), particularly those associated with diabetes mellitus, remain a significant clinical challenge due to impaired angiogenesis, persistent inflammation, and dysregulated extracellular matrix remodeling [ 1 , 2 ]. In recent years, bone marrow-derived mesenchymal stem cells (BMSCs) have emerged as a promising therapeutic approach for diabetic wound repair because of their multilineage differentiation potential and potent paracrine activity[ 3 – 5 ]. However, the regenerative efficacy of transplanted BMSCs is often limited by their poor survival, suboptimal secretory activity, and inadequate responsiveness due to the pathological wound microenvironment [ 5 – 7 ]. Thus, strategies to optimize BMSCs function and enhance their paracrine effects have become critical for improving therapeutic outcomes. Mechanical stimulation, as an essential biophysical cue, has received increasing attention in stem cell biology [ 8 , 9 ]. It has been shown that mechanical forces can profoundly influence stem cell proliferation, migration, differentiation, and secretory functions by inducing cytoskeletal remodeling and activating downstream signaling pathways, thereby improving cell-mediated tissue repair [ 10 – 13 ]. For example, in tissue expansion, bone regeneration, and angiogenesis models, mechanical stress has been reported to enhance the secretion of pro-angiogenic factors and improve the regenerative microenvironment [ 14 – 16 ]. However, the precise molecular mechanisms by which mechanical stimulation enhances BMSCs paracrine function remain largely unclear. Our previous studies revealed that cyclic mechanical stretching significantly enhanced the paracrine function of BMSCs, leading to elevated secretion of pro-angiogenic and pro-regenerative cytokines [ 13 ]. Transcriptomic profiling further showed that Periostin (Postn) expression was markedly upregulated in stretched BMSCs. Postn is a matricellular protein enriched in mechanically dynamic tissues such as periosteum and tendons and is recognized as a key mediator linking extracellular mechanical cues to intracellular signaling events [ 17 – 19 ]. Previous studies have demonstrated that Postn can directly bind to Wnt ligands and potentiate the canonical Wnt/β-catenin signaling pathway, which plays a crucial role in regulating BMSCs migration, secretion, and tissue regeneration [ 20 – 22 ]. Based on these observations, we hypothesized that mechanical stimulation enhances BMSCs paracrine activity through Postn-mediated activation of the Wnt/β-catenin pathway. Therefore, the present study aimed to validate this hypothesis by investigating whether cyclic tensile strain enhances BMSCs paracrine function via Postn-mediated activation of the canonical Wnt/β-catenin signaling pathway. We further evaluated the functional impact of this enhanced paracrine activity on endothelial cells, fibroblasts, and diabetic wound healing in vivo . This study not only provides new mechanistic insights into how BMSCs respond to mechanical stimulation but also offers a potential strategy to optimize stem cell-based therapies for chronic diabetic wounds. Materials and Methods Isolation and Culture of BMSCs One week - old Sprague–Dawley (SD) rats were obtained from the Animal Center of the Medical University. Bone marrow was extracted from both femurs of the rats with the approval of the Institutional Animal Care and Use Committee of the Fourth Military Medical University (No. 20240252), and conducted in accordance with the National Institutes of Health guidelines. One-week-old SD rats were anesthetized with inhaled isoflurane until loss of the pedal withdrawal reflex. Animals were then euthanized by cervical dislocation under deep isoflurane anesthesia, and femurs were harvested immediately for bone marrow isolation. BMSCs were isolated via density gradient centrifugation and then suspended in cell culture dishes with Minimum Essential Medium-α (MEM α, Procell, Wuhan, China) containing 0.272 g/L of L-glutamine, 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin. The cell cultures were incubated at 37°C under 5% carbon dioxide for 24 h. After 24 h, nonadherent cells were washed with phosphate-buffered saline and adherent cells were passaged at 80% − 90% confluence. Cells from passage two were used in subsequent experiments and maintained by replacing the entire medium every two days. Application of Mechanical Stimulation A spherical automatic cell - stretching device was applied for the mechanical stretch loading of cells and cell sheets. The device comprised mainly two components: a mechanical stretch loading machine and a control system [ 23 ]. BMSCs were seeded on 6-well Flexcell Bioflex® culture plates (TTCF 5001C, Flexcell® International Corporation, USA) and stretched at 15% elongation, 0.5 Hz, for 10 hours to simulate physiological mechanical loading. Unstretched cells served as the control. Postn and Wnt/β-catenin Pathway Interventions Postn and Wnt/β-catenin Pathway Interventions For gene silencing, BMSCs were transfected with siRNA targeting Postn (si-Postn) or scrambled control siRNA using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's protocol. For overexpression, Postn-expressing plasmids were transfected into BMSCs. To inhibit Wnt/β-catenin signaling, cells were treated with 10 µM XAV-939 (a tankyrase inhibitor) 1h prior to mechanical stimulation. Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted using TRIzol reagent, and cDNA was synthesized using a reverse transcription kit (Takara). qRT-PCR was performed using SYBR Green Master Mix on a QuantStudio 5 Real-Time PCR system. Gene expression levels were normalized to GAPDH and analyzed using the 2^−ΔΔCt method. Western Blotting Proteins were extracted using RIPA lysis buffer with protease and phosphatase inhibitors. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were probed with antibodies against VEGF, TGF-β, bFGF, Postn, β-catenin, Wnt1, AXIN2 and GAPDH, followed by HRP-conjugated secondary antibodies. Bands were visualized using ECL reagent and analyzed with ImageJ. Collection of BMSC-Conditioned Medium (CM) BMSCs at passages 3–5 were expanded in T75 flasks using α-MEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C/5% CO₂. At 70%–80% confluency, cells were washed 3× with PBS to remove serum residues. Serum-free α-MEM was added for a 24-h adaptation period. After discarding the adaptation medium, fresh serum-free α-MEM (10 mL/flask) was added and incubated for 48 h. The supernatant was collected, centrifuged at 500 × g for 10 min (4°C) to remove debris, and sterilized through a 0.22-µm filter. Aliquots were stored at − 80°C until functional assays. Enzyme-Linked Immunosorbent Assay (ELISA) CM from BMSCs under various treatments were collected and analyzed for paracrine factors (VEGF, TGF-β, bFGF, Postn, β-catenin, Wnt1, AXIN2) using ELISA kits (R&D Systems) according to the manufacturer’s instructions. Functional Assays Using Conditioned Media Rat umbilical vein endothelial cells (RUVECs, CP-R232, Pricella, China) and Fibroblast (Fbs, CP-R086, Pricella, China) were treated with different groups of BMSCs-CM. RUVECs migration was assessed via scratch assay: confluent monolayers were scratched with a pipette tip, and gap closure (%) was quantified at 0 and 12 hours using ImageJ. RUVECs tube formation was evaluated on growth factor-reduced Matrigel, with total tube length measured after 12h. HDF migration was analyzed using Transwell inserts (8-µm pores); cells migrating through membranes after 12 h were stained with crystal violet and counted. All experiments included ≥ 3 biological replicates, and data were analyzed by ANOVA ( P < 0.05 ). Establishment of Diabetic Wound Model The work has been reported in line with the ARRIVE guidelines 2.0. All animal experiments were approved by the Experimental Animal Center of the Fourth Military Medical University (No. 20240252) and conducted in accordance with the National Institutes of Health guidelines. 8–10-week-old SD rats were selected in this study. After 2 weeks of adaptive feeding, the diabetic rat model was induced by a single intraperitoneal injection of 70 mg/kg of streptozotocin (STZ) on an empty stomach. One week after injection, the random blood glucose levels were measured from the tail vein. Rats with blood glucose levels of > 16.7 mmol/L that maintained this level were confirmed to have successfully developed the diabetic rat model. After the blood glucose levels had stabilized for 2 weeks, 30 rats were selected and randomly allocated into 5 groups: (1) Control group, (2) Unstretched group, (3) Stretched group, (4) Stretched + si-Postn group and (5) Stretched + XAV-939 group (n = 6). All the rats were anesthetized with isoflurane (3–4% for induction and 1.5–2% for maintenance) in oxygen using an inhalation anesthesia system. Adequate anesthesia was confirmed by the absence of pedal reflex. The dorsal hair was then shaved and the skin disinfected before surgery. A full-thickness skin defect with a diameter of 1.5 cm was created using tissue scissors. A silicone ring with an inner diameter of 1.6 cm was attached to the surrounding skin using skin adhesive, and the outer edge of the silicone ring was further secured to the skin with 6 − 0 sutures to prevent skin contraction. (1) Control group: The wound was treated with an equal volume of serum-free medium (vehicle control); (2) Unstretched CM group: The wound received an intradermal injection of conditioned medium collected from BMSCs cultured under static (unstretched) conditions; (3) Stretched CM group: The wound received an intradermal injection of conditioned medium collected from BMSCs subjected to cyclic mechanical stretching; (4) Stretched + si-Postn CM group: The wound received an injection of conditioned medium derived from Postn-silenced BMSCs under mechanical stretching; (5) Stretched + XAV-939 CM group: The wound was injected with conditioned medium collected from mechanically stimulated BMSCs pretreated with the Wnt/β-catenin inhibitor XAV-939. All injections were administered around the wound margins at four equidistant points immediately after wound creation. The wounds were subsequently covered with sterile oil gauze and wrapped with a self-adhesive bandage to maintain a moist environment. Photographs of the wound area were taken on days 0, 5, 7, 10, 13, 15, 17, 19 and 21. At the experimental endpoint (day 21), rats were anesthetized with inhaled isoflurane and euthanized by cervical dislocation under deep anesthesia prior to tissue harvesting. Histological and Immunohistochemical Analysis Excised tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) staining was used for general morphology, and Masson’s trichrome staining for collagen deposition. Immunohistochemical and immunofluorescent staining were performed for markers including CD31, vWF, Collagen-Ⅰ (COL-Ⅰ) and Collagen-Ⅲ (COL-Ⅲ). Stained sections were imaged and quantified using ImageJ. Statistical Analysis Data were presented as mean ± standard deviation. One-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons. P < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism 10.0. Results Mechanical stretch enhances the paracrine function of BMSCs and activates the Wnt/β-catenin signaling pathway Our previous study demonstrated that cyclic mechanical stretching (15% strain, 0.5 Hz, 10 h) significantly enhanced the secretion of VEGF, bFGF, and TGF-β1 by BMSCs, thereby promoting angiogenesis and wound healing in diabetic rats[ 13 ]. Transcriptomic profiling further revealed that mechanical stretching markedly upregulated the mechanosensitive matricellular protein Postn, suggesting its potential role as a key mediator in the cellular response to mechanical cues. Building on these findings, the present study sought to elucidate the molecular mechanisms through which mechanical stimulation enhances BMSC paracrine activity. KEGG pathway enrichment analysis indicated that the canonical Wnt/β-catenin signaling pathway was among the most significantly enriched pathways (Fig. 1 E). These transcriptomic data provide direct evidence that cyclic mechanical stretch activates Wnt/β-catenin signaling in BMSCs. To further validate these findings at the protein level, qRT-PCR, Western blot, and ELISA analyses were performed. Consistently, mechanical stretching significantly increased the expression of Postn, β-catenin, Wnt1, and AXIN2 at both mRNA and protein levels compared with the unstretched controls (Fig. 1 A–D). Collectively, these results indicate that cyclic mechanical stimulation enhances the paracrine function of BMSCs through the activation of the Postn-associated Wnt/β-catenin signaling axis. Mechanical stretch induces cytoskeletal remodeling, Postn upregulation, and nuclear translocation of β-catenin in BMSCs To obtain direct morphological evidence of Wnt/β-catenin pathway activation, immunofluorescence staining was performed to visualize cytoskeletal organization as well as the subcellular localization of Postn and β-catenin in BMSCs after mechanical stretching. F-actin staining revealed pronounced cytoskeletal remodeling in stretched BMSCs, characterized by thickened stress fibers, elongated pseudopodia, and increased cell spreading area (Fig. 2 A, B), indicating enhanced cellular mechanosensitivity. Furthermore, immunofluorescence staining for Postn and β-catenin demonstrated that, compared with unstretched controls, mechanical stretching markedly increased Postn expression and promoted nuclear accumulation of β-catenin (Fig. 2 C–E). Quantitative fluorescence analysis confirmed that both Postn fluorescence intensity and the nuclear-to-cytoplasmic ratio of β-catenin were significantly elevated in the stretched group (P < 0.05) . Collectively, these findings indicate that mechanical stimulation activates the Wnt/β-catenin pathway through Postn-mediated mechanotransduction, establishing a direct structural link between cytoskeletal remodeling and canonical Wnt signaling activation in BMSCs. Postn is required for Wnt/β-catenin activation and paracrine enhancement induced by mechanical stimulation To deeply investigate whether Postn mediates the mechanoactivation of the Wnt/β-catenin signaling pathway, BMSCs were transfected with Postn-targeting siRNA (si-Postn) prior to mechanical stimulation. WB and ELISA analyses revealed that Postn silence significantly reduced the protein levels of VEGF, TGF-β, and bFGF compared to the stretched control group without silence (Fig. 3 A, B). Furthermore, expression of β-catenin, Wnt1, and AXIN2 was downregulated in the si-Postn group (Fig. 3 C, D), indicating that Postn silencing markedly attenuated Wnt/β-catenin signaling. To confirm the involvement of Wnt/β-catenin signaling, BMSCs were treated with the pathway inhibitor XAV-939 before mechanical stimulation. Pharmacological inhibition of Wnt/β-catenin signaling significantly suppressed the secretion of paracrine factors in stimulated BMSCs (Fig. 3 E, F). These findings substantiate the hypothesis that the Wnt/β-catenin pathway acts as a critical downstream effector of Postn-mediated mechanotransduction in BMSCs. For definitive validation of Postn’s role in mechanoactivating Wnt/β-catenin signaling, a rescue experiment was performed: Following Postn silence, cells were supplemented with exogenous recombinant Postn protein prior to re-applying mechanical stimulation. Subsequent analysis of Wnt/β-catenin pathway activation and paracrine factor expression demonstrated that Postn add-back significantly upregulated both the paracrine factors and Wnt/β-catenin-related proteins compared to the si-Postn group (Fig. 3 G, H). Notably, however, their expression levels did not fully restore to those observed in the no-silence control group. Conditioned medium from stimulated BMSCs promotes endothelial and fibroblast cell functions To functionally characterize the enhanced paracrine activity of mechanically stimulated BMSCs, the biological effects of CM from various treatment groups on RUVECs and fibroblast function were examined. Comparative analysis revealed that CM from stretched BMSCs markedly promoted endothelial cell migration, capillary-like tube formation, and fibroblast migration relative to control, Postn-silenced, or Wnt/β-catenin pathway-inhibited groups (Fig. 4 A-F). Partial abolition of these effects by either Postn silence or XAV-939-mediated Wnt/β-catenin inhibition established the Postn-Wnt/β-catenin axis as the critical mechanistic link. In rescue experiments, Postn-reconstituted BMSCs restored approximately of the paracrine activity, as evidenced by significant improvements in RUVECs migration (Fig. 4 H, I) and tube formation (Fig. 4 J, K) and fibroblast migration (Fig. 4 G, I) after 12 hours of CM treatment compared to the knockdown group (Fig. 4 H-J). These findings collectively demonstrate that mechanical stimulation enhances BMSC paracrine function primarily through Postn-mediated Wnt/β-catenin activation. Stretched BMSCs-CM Promotes Diabetic Wound Repair in vivo To evaluate the in vivo therapeutic potential of mechanically stimulated BMSCs, conditioned media from different BMSCs groups were injected around full-thickness wounds in diabetic rats. Macroscopic observations revealed that wounds treated with CM from stretched BMSCs exhibited significantly accelerated healing compared to other groups, particularly when contrasted with the si-Postn group and the XAV-939 (Wnt/β-catenin inhibitor) group (Fig. 5 A, B). This difference became more pronounced by day 21. Histological analysis via H&E and Masson staining demonstrated that, in comparison to the control group, the stretched BMSCs group showed more robust epithelial regeneration, increased granulation tissue formation, and enhanced collagen deposition (Fig. 5 C). Co-localization fluorescence staining of CD31-vWF-DAPI in day-21 tissue samples revealed an increased number of blood vessels in wounds treated with mechanically stretched CM, indicating enhanced angiogenesis (Fig. 5 C). Meanwhile, COL-Ⅰ and COL-Ⅲ-DAPI staining indicated that CM from mechanical stretching BMSCs promoted collagen deposition in the wound (Fig. 5 C). Conversely, both the Postn silence group and the XAV-939 inhibition group showed significantly reduced levels of angiogenesis and collagen neogenesis. These in vivo findings are consistent with the in vitro results, further confirming the role of the Postn-mediated Wnt/β-catenin pathway in enhancing BMSCs function and facilitating diabetic wound healing. Discussion In this study, we demonstrated that cyclic mechanical stretching profoundly enhances the paracrine activity of BMSCs through activation of a Postn-mediated Wnt/β-catenin signaling axis, ultimately improving angiogenesis and tissue repair in diabetic wounds. Transcriptomic profiling provided the first indication that mechanical stimulation activates canonical Wnt signaling, which was further confirmed by the upregulation of Postn, β-catenin, Wnt1, and AXIN2 at both mRNA and protein levels. Immunofluorescence staining offered direct morphological evidence that mechanical strain induced Postn overexpression and nuclear translocation of β-catenin in BMSCs, accompanied by pronounced cytoskeletal remodeling. Together, these findings identify a Postn-dependent mechanotransduction mechanism linking external tensile strain to intracellular Wnt pathway activation and enhanced paracrine signaling. This aligns with previous work showing mechanical stretch enhances MSCs angiogenic and anti-apoptotic capacities [ 24 , 25 ]. Moreover, mechanical stretch is known to induce endothelial markers and pro-angiogenic behavior in MSCs, enhancing their regenerative efficacy [ 26 , 27 ]. Our results expand on these findings by demonstrating that even without direct differentiation into RUVECs or Fbs, BMSCs under mechanical loading potentiate paracrine signaling - supported by enriched secretion of FGF family factors, consistent with canonical paracrine signaling mechanisms. Mechanotransduction is mediated by integrins, cytoskeletal remodeling, and focal adhesion complexes such as YAP/TAZ, MAPK-ERK, and Wnt/β-catenin pathways [ 28 – 30 ]. Our observation that mechanical stretch reorganizes F-actin architecture - with thickened stress fibers and pseudopodia formation - reveals dynamic cytoskeletal responses enabling nucleus-transduced signaling. Concurrently, mechanical strain sharply elevated Postn at both mRNA and protein levels intra- and extracellularly, matched by increased expression and nuclear translocation of β-catenin, and upregulation of Wnt1 and AXIN2, corroborating activation of the canonical Wnt/β-catenin pathway. Postn, a matricellular protein enriched in mechanically active tissues such as periosteum and tendon, is known to interact with integrins and modulate the Akt-GSK3β axis [ 31 – 33 ]. The present study shows that Postn expression is markedly induced by tensile strain, and that its suppression attenuates Wnt/β-catenin activation and growth factor secretion. This finding aligns with reports that Postn enhances osteogenic and angiogenic signaling by stabilizing β-catenin and facilitating ligand-receptor binding [ 34 , 35 ]. Moreover, our results demonstrate that exogenous Postn partially rescues the inhibitory effects of si-Postn on paracrine function, indicating that Postn is necessary but not solely sufficient for the full mechanotransductive response. These parallels support our conclusion that mechanical stretch induces Postn expression, which acts as an ECM mechano-transducer that enhances Wnt/β-catenin signaling. To test causality, we silenced Postn via siRNA before mechanical stretch. The result was marked attenuation of VEGF, TGF-β, and bFGF secretion and reduced levels of β-catenin, Wnt1, and AXIN2, both in cell lysates and conditioned medium. This underscores Postn as an essential driver linking mechano-sensing to Wnt pathway activation. The addition of the Wnt inhibitor XAV-939 similarly abolished stretch-enhanced paracrine secretion, reinforcing that canonical Wnt/β-catenin acts downstream of Postn. Rescue of mechanoactivated BMSCs with recombinant Postn restored paracrine factor levels and Wnt signaling activity partially, further confirming that Postn is necessary - but perhaps not solely sufficient - for full mechanotransductive effect. These results align with studies showing that Postn binds integrins and Wnt ligands to activate downstream signaling. For example, Postn enhances osteogenic Wnt/β-catenin signaling in BMSCs and dentinogenesis systems [ 34 ]. Taken together, our data demonstrate that mechanical stretch triggers Postn-dependent Wnt/β-catenin activation, which in turn elevates transcription and secretion of angiogenic and regenerative cytokines in BMSCs. Functionally, CM from mechanically stimulated BMSCs significantly enhanced RUVECs migration, capillary-like tube formation, and fibroblast migration in vitro . These effects were lost when Postn was silenced or Wnt/β-catenin signaling inhibited and partially restored after Postn rescue - demonstrating the functional relevance of mechanical - Postn - Wnt/β-catenin axis on paracrine-mediated cell biology. These results build on foundational concepts of paracrine signaling, notably that bFGF, VEGF, and TGF-β superfamilies regulate adjacent cell behavior via local secretion pathways [ 13 ]. Moreover, in MSCs and endothelial co-culture systems, mechanical stretch augments paracrine VEGF to promote osteogenesis and neo-angiogenesis [ 36 ]. Here, our findings affirm that mechanical conditioning of BMSCs boosts their regenerative secretome, which in turn enhances the migration, tube formation, and activity of key repair-associated cell types. In diabetic rat full-thickness wound models, injections of CM from mechanically stretched BMSCs significantly accelerated wound closure, as shown macroscopically by day 21. Histologically, treated wounds displayed increased epithelial regeneration, granulation tissue formation, and mature collagen deposition. Immunofluorescence demonstrated increased CD31-vWF co-localization, confirming angiogenesis. COL-I/III staining also showed enhanced matrix deposition. In contrast, CM from Postn-silenced or Wnt/β-catenin-inhibited cells failed to produce these enhancements, highlighting necessity of the Postn - Wnt/β-catenin axis for in vivo efficacy. These findings corroborate clinical observations where mechanical stretch in skin or tissue expansion models induces angiogenesis and paracrine-driven repair, often involving Wnt/β-catenin pathway activation. Our work is also consistent with large bone regeneration studies demonstrating Postn expression in myeloid and stromal cells during healing, reinforcing that Postn-mediated signaling is a conserved mechanism in regenerative microenvironments [ 37 ]. The molecular sequence we describe - mechanical stretch to Postn upregulation to Wnt/β-catenin activation to enhanced paracrine secretion to accelerated wound healing - is supported by multiple external studies: In skin tissue expansion, transcriptomic analyses have shown mechanical stretch activates Wnt/β-catenin, MAPK/ERK, and other pathways to drive regeneration [ 38 – 40 ]. In bone and periodontal ligament MSCs, Postn modulates integrin-Akt-GSK3β signaling upstream of β-catenin, promoting osteogenesis [ 41 ]. In cementoblasts, mechanical forces regulate autophagy and Wnt/β-catenin through Postn, affecting mineralization and differentiation. More generally, paracrine secretion of VEGF and bFGF by BMSCs under mechanical forces aligns with canonical mechanics of growth factor-mediated microenvironmental modulation [ 42 ]. Collectively, previous research aligns with our mechanistic model, while our study advances it by experimentally validating Postn's central role in mechanically induced paracrine enhancement and demonstrating downstream repair efficacy. Our data suggest that mechanical preconditioning of BMSCs could be deployed as a non-cellular therapy - via delivering optimized CM - for chronic wounds like diabetic ulcers. This approach avoids issues related to cell survival, delivery, and potential immunogenicity or oncogenic risks. Conclusion In sum, our study offers a cohesive mechanistic model of mechanotransduction in BMSCs, centered on Postn-mediated Wnt/β-catenin activation, leading to elevated secretion of therapeutic paracrine factors and functional enhancement of endothelial and fibroblast repair processes. By translating in vitro findings to in vivo diabetic wound repair, we demonstrate clinical relevance and pave the way for a next-generation, cell-free regenerative therapy founded on mechanobiology. This enriches current understanding of MSCs mechanoregulation and suggests innovative, safer alternatives to conventional cell-based therapies. Abbreviations bFGF Fibroblast Growth Factor-basic BMSCs Bone marrow mesenchymal stem cells CM Conditioned medium COL Collagen CRW Chronic refractory wounds DAPI 4',6-diamidino-2-phenylindole DNA Deoxyribonucleic acid DNase Deoxyribonuclease ELISA Enzyme-linked Immunosorbent Assay ECM Extracellular matrix EGF Epidermal growth factor Fbs Fibroblast FBS Fetal bovine serum H&E Hematoxylin and Eosin MEM Minimum essential medium MSCs Mesenchymal stem cells PBS Phosphate buffer saline PDGF Platelet derived growth factor Postn Periostin RNA Ribonucleic acid RUVECs Rat umbilical vein endothelial cells SD Sprague-Dawley STZ Streptozotocin SWE Shear wave elastography TGF-β Transforming growth factor-β TNF-α Tumor necrosis factor-α VEGF Vascular endothelial growth factor VWF Von willebrand factor WB Western blot. Declarations Ethics approval and consent to participate All animal procedures were reviewed and approved by the Ethics Committee of the Experimental Animal Center, Fourth Military Medical University (Approval No. 20240252; approved on 29 February 2024) under the project titled “Study on Mechanical Reprogramming Modulating Paracrine Signaling of BMSCs to Promote Personalized Repair of ECM in Chronic Wounds.” All experiments were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and are reported in compliance with the ARRIVE 2.0 guidelines. Consent to participate is not applicable as this study involved only animals. Consent for publication All authors confirm their consent for publication. Competing interests The authors declare that they have no competing interests. Funding This work was financially supported by the Clinical Project at the Fourth Military Medical University (No. 2024LC2420). Author Contribution DZ, HZ and CZ contributed equally to this work and share the first authorship. DZ conducted all the experiments. HZ, and CZ assisted in the experiments and contributed to writing the manuscript. JL and ZL performed data analysis and interpretation. JS and CG formatted all the figures. ZZ reviewed the manuscript and refined the language. ZL and JL conceived and designed the research, revised the manuscript, and offered financial support. All authors reviewed and approved the manuscript. Acknowledgements The authors declare that they have not use AI-generated work in this manuscrip. Data Availability Raw RNAseq data is available in the Sequence Read Archive (SRA) at the National Center for Biotechnology Information (NCBI) under BioProject Accession: PRJNA1333633. The data used to support the findings of this study are available from the corresponding author upon request. References Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. 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12:16:31","extension":"xml","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133379,"visible":true,"origin":"","legend":"","description":"","filename":"bf05b9d3ab7a41b48530d1fd197443ed1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8064472/v1/c45596db1ffde61ce01bf73f.xml"},{"id":100587643,"identity":"1612f48e-d306-4368-88d4-e39021cba0a3","added_by":"auto","created_at":"2026-01-19 12:16:44","extension":"html","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146117,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8064472/v1/665d2a6ae3009a07673f9a0e.html"},{"id":100587615,"identity":"e561a543-e6b8-42b2-911a-4b02081ad06b","added_by":"auto","created_at":"2026-01-19 12:16:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3332937,"visible":true,"origin":"","legend":"\u003cp\u003eMechanical stretching enhances BMSC paracrine function and activates the Wnt/β-catenin signaling pathway. (A) qRT-PCR analysis showing increased mRNA expression of Postn, β-catenin, Wnt1, and AXIN2 in BMSCs subjected to cyclic mechanical stretch compared with unstretched controls. (B) ELISA quantification of secreted Wnt/β-catenin-related proteins in conditioned medium from stretched and unstretched BMSCs. (C) Western blot analysis of Postn, β-catenin, Wnt1, and AXIN2 protein expression, with representative WB bands displayed. (D) Densitometric quantification of Western blot results. (E) KEGG pathway enrichment analysis of RNA-sequencing data comparing stretched and unstretched BMSCs, showing significant enrichment of the canonical Wnt/β-catenin signaling pathway. \u003cem\u003e**P \u0026lt; 0.01 and ****P \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8064472/v1/1167fea745454f3f9382b5a7.png"},{"id":100587567,"identity":"af8ac670-c6ad-46ed-a80d-1ad0c448032e","added_by":"auto","created_at":"2026-01-19 12:15:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12655040,"visible":true,"origin":"","legend":"\u003cp\u003eMechanical stretching induces cytoskeletal remodeling, Postn upregulation, and β-catenin nuclear translocation in BMSCs. (A) The distribution of cytoskeletal proteins in BMSCs shows significant sensitivity in response to stretching stress and (B) quantitative analysis of cell spreading area and F-actin length in two groups. (C-F) Immunofluorescence of Postn and β-catenin in stretched and unstretched groups: (C) cell populations (low magnification); (D) individual cells (high magnification). (E, F) Quantitative analysis. *\u003cem\u003e*P \u0026lt; 0.01, ***P \u0026lt; 0.001 and ****P \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8064472/v1/f799be227454389629adad40.png"},{"id":100587509,"identity":"99b7fbca-e394-45c6-8ec2-c826af3c85b6","added_by":"auto","created_at":"2026-01-19 12:15:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5588825,"visible":true,"origin":"","legend":"\u003cp\u003ePostn is required for Wnt/β-catenin activation and mechanically induced paracrine enhancement in BMSCs. (A, B) Western blot (A) and ELISA (B) analysis of paracrine factors (VEGF, TGF-β, bFGF) in BMSCs after Postn silence. (C, D) Western blot (C) and ELISA (D) of Wnt pathway-related proteins in Postn-silent BMSCs. (E, F) Western blot (E) and ELISA (F) of paracrine factors after Wnt pathway inhibition in BMSCs. (G, H) Western blot (G) and ELISA (H) showing restored paracrine factor secretion upon exogenous Postn supplementation. (I, J) Western blot (I) and ELISA (J) of Wnt pathway proteins after exogenous Postn treatment. \u003csup\u003e\u003cem\u003e*P\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u0026lt; 0.05\u003c/em\u003e;\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003e**P\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u0026lt; 0.01\u003c/em\u003e;\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.001\u003c/em\u003e; and \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8064472/v1/321e25ff25e0a08ad17101d7.png"},{"id":100587547,"identity":"0a94dd9a-b81e-4866-9507-11d471bfaedc","added_by":"auto","created_at":"2026-01-19 12:15:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13725625,"visible":true,"origin":"","legend":"\u003cp\u003eConditioned medium from mechanically stimulated BMSCs enhances RUVECs and fibroblast functions. (A, B) Fibroblast migration (A: representative images; B: quantitative analysis) treated with conditioned medium (CM) from different BMSC groups. (C, D) RUVECs migration (C: representative images; D: quantitative analysis) under CM treatment. (E, F) RUVECs tube formation (E: representative images; F: quantification of tube length and nodes). (G–I) Rescue effects of exogenous Postn on fibroblast (G: images; I: migration rate) and RUVECs migration (H: images; I: migration rate). (J, K) Exogenous POSTN restores RUVECs tube formation (J: images; K: tube/node quantification). \u003cem\u003e*P \u0026lt; 0.05\u003c/em\u003e;\u003cem\u003e **P \u0026lt; 0.01\u003c/em\u003e;\u003cem\u003e ***P \u0026lt; 0.001\u003c/em\u003e; and \u003cem\u003e****P \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8064472/v1/975417e3ce13e2bf2e65290d.png"},{"id":100587633,"identity":"e74cd982-a4a4-4036-a253-e6730345629a","added_by":"auto","created_at":"2026-01-19 12:16:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14186972,"visible":true,"origin":"","legend":"\u003cp\u003eStretched BMSCs-CM Promotes Diabetic Wound Repair \u003cem\u003ein vivo\u003c/em\u003e. (A) Macroscopic overview of wound healing across various treatment groups, and (B) a quantitative analysis of healing rates. (C) Hematoxylin-eosin (HE) as well as Masson staining of skin wound tissues for each group at 21 days were performed. Immunofluorescent colocalization staining of CD31 (green)–vWF (red)–DAPI and collagen types I (red)–collagen types III (green) –DAPI on skin wound tissue were shown. (D) Quantitative analysis of the ratio of COL- I to COL- III and (E) collagen volume fractions in the five groups. (F) Quantitative analysis of vascular counts and lumen diameters for the five groups. \u003cem\u003e*P \u0026lt; 0.05\u003c/em\u003e;\u003cem\u003e **P \u0026lt; 0.01\u003c/em\u003e;\u003cem\u003e ***P \u0026lt; 0.001\u003c/em\u003e; and \u003cem\u003e****P \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8064472/v1/a64af33ba8e9c83118dc74c9.png"},{"id":100588073,"identity":"01cd43d9-6ab9-4191-a7e6-8e1d620e8edb","added_by":"auto","created_at":"2026-01-19 12:23:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":46764822,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8064472/v1/ce40b34a-6bd2-4bcf-b489-5da4c60ce279.pdf"},{"id":100587515,"identity":"4d7d3beb-11ce-4bb0-9b30-847420c90b43","added_by":"auto","created_at":"2026-01-19 12:15:35","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14996562,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialTheoriginaluncroppedimagesofwesternblot.docx","url":"https://assets-eu.researchsquare.com/files/rs-8064472/v1/91099e60ad40715595de4ef1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanical Stimulation Activates Postn-Mediated Wnt/β-catenin Pathway to Enhance BMSCs Paracrine Function and Promote Wound Healing in Diabetic Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic refractory wounds (CRW), particularly those associated with diabetes mellitus, remain a significant clinical challenge due to impaired angiogenesis, persistent inflammation, and dysregulated extracellular matrix remodeling [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In recent years, bone marrow-derived mesenchymal stem cells (BMSCs) have emerged as a promising therapeutic approach for diabetic wound repair because of their multilineage differentiation potential and potent paracrine activity[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the regenerative efficacy of transplanted BMSCs is often limited by their poor survival, suboptimal secretory activity, and inadequate responsiveness due to the pathological wound microenvironment [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Thus, strategies to optimize BMSCs function and enhance their paracrine effects have become critical for improving therapeutic outcomes.\u003c/p\u003e \u003cp\u003eMechanical stimulation, as an essential biophysical cue, has received increasing attention in stem cell biology [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It has been shown that mechanical forces can profoundly influence stem cell proliferation, migration, differentiation, and secretory functions by inducing cytoskeletal remodeling and activating downstream signaling pathways, thereby improving cell-mediated tissue repair [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. For example, in tissue expansion, bone regeneration, and angiogenesis models, mechanical stress has been reported to enhance the secretion of pro-angiogenic factors and improve the regenerative microenvironment [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the precise molecular mechanisms by which mechanical stimulation enhances BMSCs paracrine function remain largely unclear.\u003c/p\u003e \u003cp\u003eOur previous studies revealed that cyclic mechanical stretching significantly enhanced the paracrine function of BMSCs, leading to elevated secretion of pro-angiogenic and pro-regenerative cytokines [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Transcriptomic profiling further showed that Periostin (Postn) expression was markedly upregulated in stretched BMSCs. Postn is a matricellular protein enriched in mechanically dynamic tissues such as periosteum and tendons and is recognized as a key mediator linking extracellular mechanical cues to intracellular signaling events [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Previous studies have demonstrated that Postn can directly bind to Wnt ligands and potentiate the canonical Wnt/β-catenin signaling pathway, which plays a crucial role in regulating BMSCs migration, secretion, and tissue regeneration [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Based on these observations, we hypothesized that mechanical stimulation enhances BMSCs paracrine activity through Postn-mediated activation of the Wnt/β-catenin pathway.\u003c/p\u003e \u003cp\u003eTherefore, the present study aimed to validate this hypothesis by investigating whether cyclic tensile strain enhances BMSCs paracrine function via Postn-mediated activation of the canonical Wnt/β-catenin signaling pathway. We further evaluated the functional impact of this enhanced paracrine activity on endothelial cells, fibroblasts, and diabetic wound healing \u003cem\u003ein vivo\u003c/em\u003e. This study not only provides new mechanistic insights into how BMSCs respond to mechanical stimulation but also offers a potential strategy to optimize stem cell-based therapies for chronic diabetic wounds.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and Culture of BMSCs\u003c/h2\u003e \u003cp\u003eOne week - old Sprague\u0026ndash;Dawley (SD) rats were obtained from the Animal Center of the Medical University. Bone marrow was extracted from both femurs of the rats with the approval of the Institutional Animal Care and Use Committee of the Fourth Military Medical University (No. 20240252), and conducted in accordance with the National Institutes of Health guidelines. One-week-old SD rats were anesthetized with inhaled isoflurane until loss of the pedal withdrawal reflex. Animals were then euthanized by cervical dislocation under deep isoflurane anesthesia, and femurs were harvested immediately for bone marrow isolation. BMSCs were isolated via density gradient centrifugation and then suspended in cell culture dishes with Minimum Essential Medium-α (MEM α, Procell, Wuhan, China) containing 0.272 g/L of L-glutamine, 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin. The cell cultures were incubated at 37\u0026deg;C under 5% carbon dioxide for 24 h. After 24 h, nonadherent cells were washed with phosphate-buffered saline and adherent cells were passaged at 80% \u0026minus;\u0026thinsp;90% confluence. Cells from passage two were used in subsequent experiments and maintained by replacing the entire medium every two days.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eApplication of Mechanical Stimulation\u003c/h3\u003e\n\u003cp\u003eA spherical automatic cell - stretching device was applied for the mechanical stretch loading of cells and cell sheets. The device comprised mainly two components: a mechanical stretch loading machine and a control system [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. BMSCs were seeded on 6-well Flexcell Bioflex\u0026reg; culture plates (TTCF 5001C, Flexcell\u0026reg; International Corporation, USA) and stretched at 15% elongation, 0.5 Hz, for 10 hours to simulate physiological mechanical loading. Unstretched cells served as the control.\u003c/p\u003e\n\u003ch3\u003ePostn and Wnt/β-catenin Pathway Interventions\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003ePostn and Wnt/β-catenin Pathway Interventions\u003c/div\u003e \u003cp\u003eFor gene silencing, BMSCs were transfected with siRNA targeting Postn (si-Postn) or scrambled control siRNA using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's protocol. For overexpression, Postn-expressing plasmids were transfected into BMSCs. To inhibit Wnt/β-catenin signaling, cells were treated with 10 \u0026micro;M XAV-939 (a tankyrase inhibitor) 1h prior to mechanical stimulation.\u003c/p\u003e\n\u003ch3\u003eQuantitative Real-Time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol reagent, and cDNA was synthesized using a reverse transcription kit (Takara). qRT-PCR was performed using SYBR Green Master Mix on a QuantStudio 5 Real-Time PCR system. Gene expression levels were normalized to GAPDH and analyzed using the 2^\u0026minus;ΔΔCt method.\u003c/p\u003e\n\u003ch3\u003eWestern Blotting\u003c/h3\u003e\n\u003cp\u003eProteins were extracted using RIPA lysis buffer with protease and phosphatase inhibitors. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were probed with antibodies against VEGF, TGF-β, bFGF, Postn, β-catenin, Wnt1, AXIN2 and GAPDH, followed by HRP-conjugated secondary antibodies. Bands were visualized using ECL reagent and analyzed with ImageJ.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCollection of BMSC-Conditioned Medium (CM)\u003c/h2\u003e \u003cp\u003eBMSCs at passages 3\u0026ndash;5 were expanded in T75 flasks using α-MEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37\u0026deg;C/5% CO₂. At 70%\u0026ndash;80% confluency, cells were washed 3\u0026times; with PBS to remove serum residues. Serum-free α-MEM was added for a 24-h adaptation period. After discarding the adaptation medium, fresh serum-free α-MEM (10 mL/flask) was added and incubated for 48 h. The supernatant was collected, centrifuged at 500 \u0026times; g for 10 min (4\u0026deg;C) to remove debris, and sterilized through a 0.22-\u0026micro;m filter. Aliquots were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until functional assays.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/h3\u003e\n\u003cp\u003eCM from BMSCs under various treatments were collected and analyzed for paracrine factors (VEGF, TGF-β, bFGF, Postn, β-catenin, Wnt1, AXIN2) using ELISA kits (R\u0026amp;D Systems) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eFunctional Assays Using Conditioned Media\u003c/h3\u003e\n\u003cp\u003eRat umbilical vein endothelial cells (RUVECs, CP-R232, Pricella, China) and Fibroblast (Fbs, CP-R086, Pricella, China) were treated with different groups of BMSCs-CM. RUVECs migration was assessed via scratch assay: confluent monolayers were scratched with a pipette tip, and gap closure (%) was quantified at 0 and 12 hours using ImageJ. RUVECs tube formation was evaluated on growth factor-reduced Matrigel, with total tube length measured after 12h. HDF migration was analyzed using Transwell inserts (8-\u0026micro;m pores); cells migrating through membranes after 12 h were stained with crystal violet and counted. All experiments included\u0026thinsp;\u0026ge;\u0026thinsp;3 biological replicates, and data were analyzed by ANOVA (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of Diabetic Wound Model\u003c/h2\u003e \u003cp\u003eThe work has been reported in line with the ARRIVE guidelines 2.0. All animal experiments were approved by the Experimental Animal Center of the Fourth Military Medical University (No. 20240252) and conducted in accordance with the National Institutes of Health guidelines. 8\u0026ndash;10-week-old SD rats were selected in this study. After 2 weeks of adaptive feeding, the diabetic rat model was induced by a single intraperitoneal injection of 70 mg/kg of streptozotocin (STZ) on an empty stomach. One week after injection, the random blood glucose levels were measured from the tail vein. Rats with blood glucose levels of \u0026gt;\u0026thinsp;16.7 mmol/L that maintained this level were confirmed to have successfully developed the diabetic rat model.\u003c/p\u003e \u003cp\u003eAfter the blood glucose levels had stabilized for 2 weeks, 30 rats were selected and randomly allocated into 5 groups: (1) Control group, (2) Unstretched group, (3) Stretched group, (4) Stretched\u0026thinsp;+\u0026thinsp;si-Postn group and (5) Stretched\u0026thinsp;+\u0026thinsp;XAV-939 group (n\u0026thinsp;=\u0026thinsp;6). All the rats were anesthetized with isoflurane (3\u0026ndash;4% for induction and 1.5\u0026ndash;2% for maintenance) in oxygen using an inhalation anesthesia system. Adequate anesthesia was confirmed by the absence of pedal reflex. The dorsal hair was then shaved and the skin disinfected before surgery. A full-thickness skin defect with a diameter of 1.5 cm was created using tissue scissors. A silicone ring with an inner diameter of 1.6 cm was attached to the surrounding skin using skin adhesive, and the outer edge of the silicone ring was further secured to the skin with 6\u0026thinsp;\u0026minus;\u0026thinsp;0 sutures to prevent skin contraction. (1) Control group: The wound was treated with an equal volume of serum-free medium (vehicle control); (2) Unstretched CM group: The wound received an intradermal injection of conditioned medium collected from BMSCs cultured under static (unstretched) conditions; (3) Stretched CM group: The wound received an intradermal injection of conditioned medium collected from BMSCs subjected to cyclic mechanical stretching; (4) Stretched\u0026thinsp;+\u0026thinsp;si-Postn CM group: The wound received an injection of conditioned medium derived from Postn-silenced BMSCs under mechanical stretching; (5) Stretched\u0026thinsp;+\u0026thinsp;XAV-939 CM group: The wound was injected with conditioned medium collected from mechanically stimulated BMSCs pretreated with the Wnt/β-catenin inhibitor XAV-939. All injections were administered around the wound margins at four equidistant points immediately after wound creation. The wounds were subsequently covered with sterile oil gauze and wrapped with a self-adhesive bandage to maintain a moist environment. Photographs of the wound area were taken on days 0, 5, 7, 10, 13, 15, 17, 19 and 21. At the experimental endpoint (day 21), rats were anesthetized with inhaled isoflurane and euthanized by cervical dislocation under deep anesthesia prior to tissue harvesting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistological and Immunohistochemical Analysis\u003c/h2\u003e \u003cp\u003eExcised tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin and eosin (H\u0026amp;E) staining was used for general morphology, and Masson\u0026rsquo;s trichrome staining for collagen deposition. Immunohistochemical and immunofluorescent staining were performed for markers including CD31, vWF, Collagen-Ⅰ (COL-Ⅰ) and Collagen-Ⅲ (COL-Ⅲ). Stained sections were imaged and quantified using ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. One-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was used for multiple group comparisons. \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e was considered statistically significant. All analyses were performed using GraphPad Prism 10.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMechanical stretch enhances the paracrine function of BMSCs and activates the Wnt/β-catenin signaling pathway\u003c/h2\u003e \u003cp\u003eOur previous study demonstrated that cyclic mechanical stretching (15% strain, 0.5 Hz, 10 h) significantly enhanced the secretion of VEGF, bFGF, and TGF-β1 by BMSCs, thereby promoting angiogenesis and wound healing in diabetic rats[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Transcriptomic profiling further revealed that mechanical stretching markedly upregulated the mechanosensitive matricellular protein Postn, suggesting its potential role as a key mediator in the cellular response to mechanical cues. Building on these findings, the present study sought to elucidate the molecular mechanisms through which mechanical stimulation enhances BMSC paracrine activity.\u003c/p\u003e \u003cp\u003eKEGG pathway enrichment analysis indicated that the canonical Wnt/β-catenin signaling pathway was among the most significantly enriched pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These transcriptomic data provide direct evidence that cyclic mechanical stretch activates Wnt/β-catenin signaling in BMSCs.\u003c/p\u003e \u003cp\u003eTo further validate these findings at the protein level, qRT-PCR, Western blot, and ELISA analyses were performed. Consistently, mechanical stretching significantly increased the expression of Postn, β-catenin, Wnt1, and AXIN2 at both mRNA and protein levels compared with the unstretched controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;D). Collectively, these results indicate that cyclic mechanical stimulation enhances the paracrine function of BMSCs through the activation of the Postn-associated Wnt/β-catenin signaling axis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMechanical stretch induces cytoskeletal remodeling, Postn upregulation, and nuclear translocation of β-catenin in BMSCs\u003c/h2\u003e \u003cp\u003eTo obtain direct morphological evidence of Wnt/β-catenin pathway activation, immunofluorescence staining was performed to visualize cytoskeletal organization as well as the subcellular localization of Postn and β-catenin in BMSCs after mechanical stretching. F-actin staining revealed pronounced cytoskeletal remodeling in stretched BMSCs, characterized by thickened stress fibers, elongated pseudopodia, and increased cell spreading area (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B), indicating enhanced cellular mechanosensitivity.\u003c/p\u003e \u003cp\u003eFurthermore, immunofluorescence staining for Postn and β-catenin demonstrated that, compared with unstretched controls, mechanical stretching markedly increased Postn expression and promoted nuclear accumulation of β-catenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u0026ndash;E). Quantitative fluorescence analysis confirmed that both Postn fluorescence intensity and the nuclear-to-cytoplasmic ratio of β-catenin were significantly elevated in the stretched group \u003cem\u003e(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eCollectively, these findings indicate that mechanical stimulation activates the Wnt/β-catenin pathway through Postn-mediated mechanotransduction, establishing a direct structural link between cytoskeletal remodeling and canonical Wnt signaling activation in BMSCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePostn is required for Wnt/β-catenin activation and paracrine enhancement induced by mechanical stimulation\u003c/h2\u003e \u003cp\u003eTo deeply investigate whether Postn mediates the mechanoactivation of the Wnt/β-catenin signaling pathway, BMSCs were transfected with Postn-targeting siRNA (si-Postn) prior to mechanical stimulation. WB and ELISA analyses revealed that Postn silence significantly reduced the protein levels of VEGF, TGF-β, and bFGF compared to the stretched control group without silence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Furthermore, expression of β-catenin, Wnt1, and AXIN2 was downregulated in the si-Postn group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D), indicating that Postn silencing markedly attenuated Wnt/β-catenin signaling.\u003c/p\u003e \u003cp\u003eTo confirm the involvement of Wnt/β-catenin signaling, BMSCs were treated with the pathway inhibitor XAV-939 before mechanical stimulation. Pharmacological inhibition of Wnt/β-catenin signaling significantly suppressed the secretion of paracrine factors in stimulated BMSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). These findings substantiate the hypothesis that the Wnt/β-catenin pathway acts as a critical downstream effector of Postn-mediated mechanotransduction in BMSCs.\u003c/p\u003e \u003cp\u003eFor definitive validation of Postn\u0026rsquo;s role in mechanoactivating Wnt/β-catenin signaling, a rescue experiment was performed: Following Postn silence, cells were supplemented with exogenous recombinant Postn protein prior to re-applying mechanical stimulation. Subsequent analysis of Wnt/β-catenin pathway activation and paracrine factor expression demonstrated that Postn add-back significantly upregulated both the paracrine factors and Wnt/β-catenin-related proteins compared to the si-Postn group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). Notably, however, their expression levels did not fully restore to those observed in the no-silence control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eConditioned medium from stimulated BMSCs promotes endothelial and fibroblast cell functions\u003c/h2\u003e \u003cp\u003eTo functionally characterize the enhanced paracrine activity of mechanically stimulated BMSCs, the biological effects of CM from various treatment groups on RUVECs and fibroblast function were examined. Comparative analysis revealed that CM from stretched BMSCs markedly promoted endothelial cell migration, capillary-like tube formation, and fibroblast migration relative to control, Postn-silenced, or Wnt/β-catenin pathway-inhibited groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-F). Partial abolition of these effects by either Postn silence or XAV-939-mediated Wnt/β-catenin inhibition established the Postn-Wnt/β-catenin axis as the critical mechanistic link.\u003c/p\u003e \u003cp\u003eIn rescue experiments, Postn-reconstituted BMSCs restored approximately of the paracrine activity, as evidenced by significant improvements in RUVECs migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, I) and tube formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, K) and fibroblast migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, I) after 12 hours of CM treatment compared to the knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-J). These findings collectively demonstrate that mechanical stimulation enhances BMSC paracrine function primarily through Postn-mediated Wnt/β-catenin activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eStretched BMSCs-CM Promotes Diabetic Wound Repair\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the \u003cem\u003ein vivo\u003c/em\u003e therapeutic potential of mechanically stimulated BMSCs, conditioned media from different BMSCs groups were injected around full-thickness wounds in diabetic rats. Macroscopic observations revealed that wounds treated with CM from stretched BMSCs exhibited significantly accelerated healing compared to other groups, particularly when contrasted with the si-Postn group and the XAV-939 (Wnt/β-catenin inhibitor) group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). This difference became more pronounced by day 21.\u003c/p\u003e \u003cp\u003eHistological analysis via H\u0026amp;E and Masson staining demonstrated that, in comparison to the control group, the stretched BMSCs group showed more robust epithelial regeneration, increased granulation tissue formation, and enhanced collagen deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Co-localization fluorescence staining of CD31-vWF-DAPI in day-21 tissue samples revealed an increased number of blood vessels in wounds treated with mechanically stretched CM, indicating enhanced angiogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Meanwhile, COL-Ⅰ and COL-Ⅲ-DAPI staining indicated that CM from mechanical stretching BMSCs promoted collagen deposition in the wound (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Conversely, both the Postn silence group and the XAV-939 inhibition group showed significantly reduced levels of angiogenesis and collagen neogenesis. These \u003cem\u003ein vivo\u003c/em\u003e findings are consistent with the \u003cem\u003ein vitro\u003c/em\u003e results, further confirming the role of the Postn-mediated Wnt/β-catenin pathway in enhancing BMSCs function and facilitating diabetic wound healing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that cyclic mechanical stretching profoundly enhances the paracrine activity of BMSCs through activation of a Postn-mediated Wnt/β-catenin signaling axis, ultimately improving angiogenesis and tissue repair in diabetic wounds. Transcriptomic profiling provided the first indication that mechanical stimulation activates canonical Wnt signaling, which was further confirmed by the upregulation of Postn, β-catenin, Wnt1, and AXIN2 at both mRNA and protein levels. Immunofluorescence staining offered direct morphological evidence that mechanical strain induced Postn overexpression and nuclear translocation of β-catenin in BMSCs, accompanied by pronounced cytoskeletal remodeling. Together, these findings identify a Postn-dependent mechanotransduction mechanism linking external tensile strain to intracellular Wnt pathway activation and enhanced paracrine signaling.\u003c/p\u003e \u003cp\u003eThis aligns with previous work showing mechanical stretch enhances MSCs angiogenic and anti-apoptotic capacities [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, mechanical stretch is known to induce endothelial markers and pro-angiogenic behavior in MSCs, enhancing their regenerative efficacy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our results expand on these findings by demonstrating that even without direct differentiation into RUVECs or Fbs, BMSCs under mechanical loading potentiate paracrine signaling - supported by enriched secretion of FGF family factors, consistent with canonical paracrine signaling mechanisms.\u003c/p\u003e \u003cp\u003eMechanotransduction is mediated by integrins, cytoskeletal remodeling, and focal adhesion complexes such as YAP/TAZ, MAPK-ERK, and Wnt/β-catenin pathways [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Our observation that mechanical stretch reorganizes F-actin architecture - with thickened stress fibers and pseudopodia formation - reveals dynamic cytoskeletal responses enabling nucleus-transduced signaling. Concurrently, mechanical strain sharply elevated Postn at both mRNA and protein levels intra- and extracellularly, matched by increased expression and nuclear translocation of β-catenin, and upregulation of Wnt1 and AXIN2, corroborating activation of the canonical Wnt/β-catenin pathway.\u003c/p\u003e \u003cp\u003ePostn, a matricellular protein enriched in mechanically active tissues such as periosteum and tendon, is known to interact with integrins and modulate the Akt-GSK3β axis [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The present study shows that Postn expression is markedly induced by tensile strain, and that its suppression attenuates Wnt/β-catenin activation and growth factor secretion. This finding aligns with reports that Postn enhances osteogenic and angiogenic signaling by stabilizing β-catenin and facilitating ligand-receptor binding [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Moreover, our results demonstrate that exogenous Postn partially rescues the inhibitory effects of si-Postn on paracrine function, indicating that Postn is necessary but not solely sufficient for the full mechanotransductive response. These parallels support our conclusion that mechanical stretch induces Postn expression, which acts as an ECM mechano-transducer that enhances Wnt/β-catenin signaling.\u003c/p\u003e \u003cp\u003eTo test causality, we silenced Postn via siRNA before mechanical stretch. The result was marked attenuation of VEGF, TGF-β, and bFGF secretion and reduced levels of β-catenin, Wnt1, and AXIN2, both in cell lysates and conditioned medium. This underscores Postn as an essential driver linking mechano-sensing to Wnt pathway activation.\u003c/p\u003e \u003cp\u003eThe addition of the Wnt inhibitor XAV-939 similarly abolished stretch-enhanced paracrine secretion, reinforcing that canonical Wnt/β-catenin acts downstream of Postn. Rescue of mechanoactivated BMSCs with recombinant Postn restored paracrine factor levels and Wnt signaling activity partially, further confirming that Postn is necessary - but perhaps not solely sufficient - for full mechanotransductive effect.\u003c/p\u003e \u003cp\u003eThese results align with studies showing that Postn binds integrins and Wnt ligands to activate downstream signaling. For example, Postn enhances osteogenic Wnt/β-catenin signaling in BMSCs and dentinogenesis systems [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Taken together, our data demonstrate that mechanical stretch triggers Postn-dependent Wnt/β-catenin activation, which in turn elevates transcription and secretion of angiogenic and regenerative cytokines in BMSCs.\u003c/p\u003e \u003cp\u003eFunctionally, CM from mechanically stimulated BMSCs significantly enhanced RUVECs migration, capillary-like tube formation, and fibroblast migration \u003cem\u003ein vitro\u003c/em\u003e. These effects were lost when Postn was silenced or Wnt/β-catenin signaling inhibited and partially restored after Postn rescue - demonstrating the functional relevance of mechanical - Postn - Wnt/β-catenin axis on paracrine-mediated cell biology.\u003c/p\u003e \u003cp\u003eThese results build on foundational concepts of paracrine signaling, notably that bFGF, VEGF, and TGF-β superfamilies regulate adjacent cell behavior via local secretion pathways [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Moreover, in MSCs and endothelial co-culture systems, mechanical stretch augments paracrine VEGF to promote osteogenesis and neo-angiogenesis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Here, our findings affirm that mechanical conditioning of BMSCs boosts their regenerative secretome, which in turn enhances the migration, tube formation, and activity of key repair-associated cell types.\u003c/p\u003e \u003cp\u003eIn diabetic rat full-thickness wound models, injections of CM from mechanically stretched BMSCs significantly accelerated wound closure, as shown macroscopically by day 21. Histologically, treated wounds displayed increased epithelial regeneration, granulation tissue formation, and mature collagen deposition. Immunofluorescence demonstrated increased CD31-vWF co-localization, confirming angiogenesis. COL-I/III staining also showed enhanced matrix deposition. In contrast, CM from Postn-silenced or Wnt/β-catenin-inhibited cells failed to produce these enhancements, highlighting necessity of the Postn - Wnt/β-catenin axis for \u003cem\u003ein vivo\u003c/em\u003e efficacy.\u003c/p\u003e \u003cp\u003eThese findings corroborate clinical observations where mechanical stretch in skin or tissue expansion models induces angiogenesis and paracrine-driven repair, often involving Wnt/β-catenin pathway activation. Our work is also consistent with large bone regeneration studies demonstrating Postn expression in myeloid and stromal cells during healing, reinforcing that Postn-mediated signaling is a conserved mechanism in regenerative microenvironments [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe molecular sequence we describe - mechanical stretch to Postn upregulation to Wnt/β-catenin activation to enhanced paracrine secretion to accelerated wound healing - is supported by multiple external studies: In skin tissue expansion, transcriptomic analyses have shown mechanical stretch activates Wnt/β-catenin, MAPK/ERK, and other pathways to drive regeneration [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In bone and periodontal ligament MSCs, Postn modulates integrin-Akt-GSK3β signaling upstream of β-catenin, promoting osteogenesis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In cementoblasts, mechanical forces regulate autophagy and Wnt/β-catenin through Postn, affecting mineralization and differentiation. More generally, paracrine secretion of VEGF and bFGF by BMSCs under mechanical forces aligns with canonical mechanics of growth factor-mediated microenvironmental modulation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Collectively, previous research aligns with our mechanistic model, while our study advances it by experimentally validating Postn's central role in mechanically induced paracrine enhancement and demonstrating downstream repair efficacy.\u003c/p\u003e \u003cp\u003eOur data suggest that mechanical preconditioning of BMSCs could be deployed as a non-cellular therapy - via delivering optimized CM - for chronic wounds like diabetic ulcers. This approach avoids issues related to cell survival, delivery, and potential immunogenicity or oncogenic risks.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn sum, our study offers a cohesive mechanistic model of mechanotransduction in BMSCs, centered on Postn-mediated Wnt/β-catenin activation, leading to elevated secretion of therapeutic paracrine factors and functional enhancement of endothelial and fibroblast repair processes. By translating in vitro findings to in vivo diabetic wound repair, we demonstrate clinical relevance and pave the way for a next-generation, cell-free regenerative therapy founded on mechanobiology. This enriches current understanding of MSCs mechanoregulation and suggests innovative, safer alternatives to conventional cell-based therapies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ebFGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFibroblast Growth Factor-basic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBMSCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBone marrow mesenchymal stem cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eConditioned medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCollagen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChronic refractory wounds\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e4',6-diamidino-2-phenylindole\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDeoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNase\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDeoxyribonuclease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-linked Immunosorbent Assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpidermal growth factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFbs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFibroblast\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin and Eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimum essential medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMesenchymal stem cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate buffer saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlatelet derived growth factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePostn\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeriostin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRibonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRUVECs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRat umbilical vein endothelial cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSprague-Dawley\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTZ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStreptozotocin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSWE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eShear wave elastography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTGF-β\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransforming growth factor-β\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF-α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factor-α\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVEGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVascular endothelial growth factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVWF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVon willebrand factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWestern blot.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e All animal procedures were reviewed and approved by the Ethics Committee of the Experimental Animal Center, Fourth Military Medical University (Approval No. 20240252; approved on 29 February 2024) under the project titled \u0026ldquo;Study on Mechanical Reprogramming Modulating Paracrine Signaling of BMSCs to Promote Personalized Repair of ECM in Chronic Wounds.\u0026rdquo; All experiments were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and are reported in compliance with the ARRIVE 2.0 guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to participate\u003c/strong\u003e \u003cp\u003eis not applicable as this study involved only animals.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors confirm their consent for publication.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the Clinical Project at the Fourth Military Medical University (No. 2024LC2420).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDZ, HZ and CZ contributed equally to this work and share the first authorship. DZ conducted all the experiments. HZ, and CZ assisted in the experiments and contributed to writing the manuscript. JL and ZL performed data analysis and interpretation. JS and CG formatted all the figures. ZZ reviewed the manuscript and refined the language. ZL and JL conceived and designed the research, revised the manuscript, and offered financial support. All authors reviewed and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors declare that they have not use AI-generated work in this manuscrip.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eRaw RNAseq data is available in the Sequence Read Archive (SRA) at the National Center for Biotechnology Information (NCBI) under BioProject Accession: PRJNA1333633. The data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature. 2008;453(7193):314\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature07039\u003c/span\u003e\u003cspan address=\"10.1038/nature07039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmstrong DG, Boulton AJM, Bus SA. Diabetic Foot Ulcers and Their Recurrence. 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J Cell Physiol. 2023;238(9):2147\u0026ndash;60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jcp.31075\u003c/span\u003e\u003cspan address=\"10.1002/jcp.31075\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mechanical stretch, Bone marrow mesenchymal stem cells, Periostin, Wnt/β-catenin pathway, Diabetic wound healing","lastPublishedDoi":"10.21203/rs.3.rs-8064472/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8064472/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDiabetic wounds exhibit impaired healing due to angiogenic deficiency and dysregulated extracellular matrix homeostasis. Although bone marrow mesenchymal stem cells (BMSCs) promote wound repair through paracrine signaling, their therapeutic efficacy is compromised in diabetic microenvironments. Crucially, emerging evidence implicates Periostin (Postn) as a mechanoresponsive matricellular protein that directly activates Wnt/β-catenin signaling - a pathway governing stem cell paracrine function and tissue regeneration. However, whether mechanical stimulation leverages this Postn-Wnt/β-catenin axis to optimize BMSCs secretory capacity remains unexplored. This study specifically interrogates this mechanotransduction mechanism to develop enhanced therapies for diabetic wounds.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eBMSCs underwent cyclic stretching (15% strain, 0.5 Hz, 10 h), and paracrine factors (VEGF/TGF-β/bFGF) and pathway proteins were assessed via qRT-PCR, Western blot, and ELISA. Then, Postn knockdown (siRNA) or Wnt/β-catenin inhibition (XAV-939) was implemented to the stretched BMSCs, and paracrine factors were assessed again. Conditioned medium (CM) of stretched BMSCs functionality was evaluated using scratch assay and tube formation assays with Rat umbilical vein endothelial cells (RUVECs) and fibroblasts (Fbs). Finally, a full-thickness diabetic rat wound model was established to validate \u003cem\u003ein vivo\u003c/em\u003e efficacy of the CM through wound closure rate, histochemistry, and immunofluorescence.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMechanical stimulation significantly increased Postn expression and Wnt/β-catenin signaling, boosting the secretion of VEGF, TGF-β1, and bFGF. Postn knockdown or Wnt/β-catenin pathway inhibition attenuated these effects, while exogenous Postn partially restored function. CM from stretched BMSCs promoted endothelial migration, tube formation, fibroblast migration in vitro, and accelerated wound healing, angiogenesis, and collagen deposition \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eMechanical stretch enhances BMSCs\u0026rsquo; paracrine function through a Postn-Wnt/β-catenin axis, offering a mechanobiology-based, cell-free approach to improve diabetic wound repair.\u003c/p\u003e","manuscriptTitle":"Mechanical Stimulation Activates Postn-Mediated Wnt/β-catenin Pathway to Enhance BMSCs Paracrine Function and Promote Wound Healing in Diabetic Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 12:07:03","doi":"10.21203/rs.3.rs-8064472/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"165486606977422348222609111544565547975","date":"2026-05-06T13:43:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-05T04:18:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180795320578858143347173180271580585536","date":"2026-03-11T03:19:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-14T17:03:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-05T21:03:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-11T23:41:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2025-12-11T02:02:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cb91d2e6-5e13-468d-b563-41cd58371e41","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"165486606977422348222609111544565547975","date":"2026-05-06T13:43:38+00:00","index":52,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T12:07:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 12:07:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8064472","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8064472","identity":"rs-8064472","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}