Platelet-rich plasma-derived exosomes accelerate the healing of diabetic foot ulcers by promoting macrophage polarization to the M2 phenotype

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Although platelet-rich plasma (PRP) and platelet-rich plasma-derived exosomes (PRP-Exos) have emerged as promising therapeutic agents in tissue regeneration, the mechanisms underlying the immunomodulatory effects of PRP and PRP-Exos—particularly their role in macrophage polarization—remain poorly understood. In this study, we isolated and characterized PRP-Exos and systematically evaluated their therapeutic potential in diabetic wound healing via comprehensive in vivo and in vitro experiments. Our results revealed that both PRP-gel and PRP-Exos significantly enhanced diabetic wound healing by promoting macrophage polarization toward the anti-inflammatory M2 phenotype. These findings suggest that PRP-Exos represent a novel and effective therapeutic strategy for DFUs, providing a robust rationale for future clinical translation. Diabetic foot ulcers Platelet-rich plasma Platelet-rich plasma-derived exosomes Macrophage polarization M2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Diabetic foot ulcers (DFUs) affect 10 to 15% of the diabetic population and are among the most formidable chronic complications, conveying profound clinical risks that demand urgent attention. This debilitating condition not only significantly extends hospital stays and sustains a persistently high disability rate, but also carries a grave risk of amputation, profoundly impacting patient quality of life[ 1 ]. Epidemiological studies suggest that between 10 and 15% of individuals with diabetes develop foot ulcers that are highly susceptible to infection. Patients with DFUs have a five-year mortality rate of 50%, increasing to 80% after amputation[ 2 ]. The development of DFUs is intricately linked to a complex interplay of mechanisms—including impaired angiogenesis, chronic inflammatory responses, and compromised tissue-repair functions—that collectively contribute to the progression and persistence of this condition[ 3 ]. In current clinical practice, wound management primarily relies on standard therapeutic approaches such as surgical debridement, reducing weight-bearing pressure, treating lower-extremity ischemia, antimicrobial treatment, and glycemic control[ 1 , 4 ]. Although these conventional methods offer partial symptomatic improvement, they are frequently limited by prolonged healing durations and significant recurrence rates, and fail to meet the increasing demands of modern wound care. For many patients, the efficacy of traditional treatments remains suboptimal, and this is largely attributed to impaired cellular functions and insufficient bioactive components within the wound microenvironment[ 5 ]. This clinical dilemma not only severely impacts the quality of life of patients but also imposes a significant financial burden on their families[ 6 , 7 ]. Therefore, searching for more effective treatment strategies has become an urgent need in the field of DFU therapy. In recent years, the rapid advancements in regenerative medicine and biotherapy have led to the emergence of innovative treatment modalities[ 8 , 9 ], and, of these, platelet-rich plasma (PRP) therapy, an autologous biotherapy approach, has garnered significant attention due to its unique advantages. Sourced directly from the patient’s bloodstream, PRP offers the unique advantage of being immunogen-free while containing a concentrated array of growth factors, thus emerging as a particularly promising therapeutic approach in clinical practice. PRP has also demonstrated superior efficacy in wound healing compared to traditional therapies[ 2 ]. PRP is a bioactive complex of substances extracted through a meticulous process of centrifuging and concentrating a patient's whole blood, and the method yields plasma with a platelet concentration that is a remarkable 3–7 times higher than that found in normal blood. These platelets, rich in α-granules, harbor an array of growth factors, adhesion molecules, and cytokines[ 10 ]. Upon activation, these granular contents are released and play an indispensable role in stimulating cellular proliferation, differentiation, and tissue regeneration; this, in turn, significantly expedites the wound-healing process. Recent research has revealed that PRP markedly reduces both the healing time and wound area in patients suffering from DFUs [ 11 ]. Moreover, a systematic review, a meta-analysis, and several randomized controlled trials (RCTs) have shown that an autologous platelet-gel surpasses topical antibacterial dressings in healing rates and infection prevention with respect to clean DFU wounds[ 12 – 14 ]. While these outcomes are encouraging, the existing evidence remains inadequate for a definitive determination of the efficacy of PRP therapy in treating chronic DFU wounds. Consequently, additional rigorous studies are required to establish PRP’s clinical utility and to provide a solid foundation of experimental evidence. Macrophages, crucial actors in the healing process, are classified into two distinct phenotypes based on their functional characteristics and cytokine profiles: the classically activated macrophages (M1 type), which predominantly secrete pro-inflammatory cytokines potentially hindering the maturation of granulation tissue[ 15 ]; and the alternatively activated macrophages (M2 type), which suppress inflammatory responses and facilitate wound healing. The literature is replete with evidence confirming the pivotal role of macrophages in managing the inflammatory phase of wound repair, with their dynamic phenotypic plasticity mediating their involvement in tissue injury and regeneration[ 16 , 17 ]. The pathogenesis of DFUs is multifaceted, and recent research suggests that an impaired transition from the M1 to the M2 macrophage phenotype is a critical factor in the development of chronic non-healing ulcers in DFUs. The therapeutic potential of PRP-derived exosomes (PRP-Exos) in facilitating the healing of DFUs has emerged as a compelling area of regenerative medicine that has garnered sustained interest from the scientific community [ 18 , 19 ]. However, further investigation is needed to understand the impact of PRP-gel and PRP-Exos on DFU treatments, particularly their role in modulating macrophage polarization. In this study, we thoroughly investigated the mechanisms underlying the actions of PRP-gel and PRP-Exos in modulating macrophage polarization during diabetic wound healing, with a focus on their influence on DFU treatment outcomes. By merging clinical observations with experimental data, we have attempted to establish a robust framework for the clinical implementation of these therapies, thereby enhancing the efficacy of DFU management and advancing patient care. Materials and methods Patients This study was sanctioned by the Ethics Committee of the Wannan Medical College. Written informed consent was obtained from all participants. These patients had been diagnosed with stage II–IV DFU’s based on the Wagner classification system. Clinical information regarding the patients is presented in Supplemental Table S1 . The wound of the patient with diabetic foot was treated using only autologous PRP-gel derived from the patient themselves. The ulcer area was measured every 3 days using a digital camera, and wound closure rates were calculated. We analyzed wound-area measurements from images using ImageJ (NIH, USA) software. Data were not identifiable to any patient. Diabetic skin-wound animal model and treatment Adult male BALB/c mice (6–8 weeks of age and weighing 26–28 g; grade, clean; license, SCXK 2023-005) were purchased from the Experimental Animal Center of Qinglongshan (Nanjing, China) and raised in a specific-pathogen-free (SPF) mouse colony. Mice were housed in a controlled environment with a 12-h light/dark cycle, maintained at 22 ± 2°C, and provided with standard rodent chow and water ad libitum. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 50 mg/kg) for 5 days consecutively. After 4 weeks, blood glucose levels were monitored, and mice with sustained blood glucose levels above 16.8 mmol/L were considered successful diabetic models. An electronic blood glucose meter (Roche, China) was used to measure mouse serum glucose levels. Subsequently, full-thickness skin ulcers with a diameter of 6 mm were created on the dorsal side of the mice to serve as a DFU model. Chronic wound-healing in mice A model of DFUs was created in mice anesthetized with isoflurane inhalation. A single round full-thickness skin wound was then created with a 6-mm disposable skin biopsy punch on the dorsum of the diabetic mice. Digital images of the wound area were captured on the day of surgery and on days 5, 7, 9, and 11 after operation. A ruler reference was placed nearby to confirm the distance between the animals and the camera. The wound area was quantified by three blinded observers using ImageJ (NIH, USA) software and expressed as a percentage of the original area. The wound was covered with gauze and a bandage, and the dressings were changed every 3 days according to the standard of care; and wound tissues were sampled and harvested at 5, 7, 9, and 11 days. Harvested wound tissues were fixed in 10% formalin solution, and adopted for further immunofluorescence (IF) investigations. Preparation of PRP and PRP-gel Ten healthy adult volunteers, aged between 18 and 45 years and with no history of hematological or infectious diseases were selected. PRP was isolated utilizing a fully automatic blood separator (COM.TEC; Fresenius Kabi, German), and PRP-gel was produced by activating PRP with a prepared mixture of thrombin and calcium gluconate at a ratio of 10:1. PRP-gel derived from volunteers was used for the diabetic wound experiment in mice. Morphological staining A graded series of ethanol and formaldehyde were applied to the wound tissues from the mice after they had been sampled at 5, 7, 9, and 11 days; and paraffin was applied after the tissues were fixed in 4% formaldehyde overnight. The sections were cut at a thickness of 5 mm, and then treated with hematoxylin and eosin (Servicebio, China). We performed Masson trichrome staining using a staining kit (Servicebio, China) in accordance with the manufacturer’s guidelines. Immunofluorescence staining The tissues were fixed with 4% paraformaldehyde for 16–24 h at 4°C, followed by embedding in paraffin and sectioning at 4 µm. After deparaffinization with a dewaxing solution and anhydrous ethanol, the sections were rinsed in PBS and then blocked with 5% of BSA blocking solution for 30 min. The sections were subsequently incubated overnight at 4°C with primary antibodies targeting iNOS (Servicebio, China), Arg-1 (Servicebio, China), and F4/80 (Servicebio, China). For immunofluorescence staining, the sections were incubated with Alexa Fluor 488, Alexa Fluor 555, and conjugated secondary antibodies (Servicebio, China). To evaluate the angiogenesis of PRP-gel and PRP-Exos in the treatment of diabetic wounds, the sections were incubated with antibodies to α-SMA (Servicebio, China) and CD31 (Servicebio, China) at 4°C overnight. After washing several times with PBS, DAPI (Servicebio, China) was used to stain the nuclei for 10 min. Fluoroshield mounting medium (G1401, Servicebio, China) was applied to preserve fluorescence when imaging tissues. Isolation and purification of PRP-Exos The PRP samples were centrifuged at 3000× g for 15 min at 4°C to remove cell debris, and the supernatant was then centrifuged at 10,000× g for 30 min at 4°C to remove large particles; this was followed by ultracentrifugation of the supernatant at 100,000× g for 70 min at 4°C to pellet the exosomes. The exosomal pellets were ultracentrifuged in PBS and centrifuged again at 100,000× g for 70 min to wash the exosomes. Identification of PRP-Exos A transmission electron microscope (TEM) was used to identify the morphology of the exosomes. Briefly, PRP-Exos were fixed in 3% glutaraldehyde for 2 h and washed twice with PBS. The exosomes were negatively stained with 2% uranyl acetate for 30 s and applied to a continuous carbon grid. We ultimately visualized the exosomes on a FEI Tecnai G2 12 TEM (FEI, America). The size distribution of PRP-derived exosomes (PRP-Exos) was determined using nanoparticle tracking analysis (NTA) with a ZetaView PMX-120 system (Particle Metrix, Germany). Prior to sample analysis, the instrument was calibrated using 100-nm polystyrene nanoparticles as reference standards. The exosome pellet, resuspended in phosphate-buffered saline (PBS), was subsequently introduced into the NTA system for particle-size characterization. It should be noted that while polystyrene beads serve as reliable calibration standards, the inherent differences in material composition between synthetic beads and biological exosomes necessitate cautious interpretation of size-distribution data. Therefore, the NTA results were complemented and validated by TEM for direct visualization of exosomal morphology and western-blot (WB) analysis for specific exosomal marker identification, ensuring comprehensive characterization of the isolated exosomes. The marker proteins of the exosomes were detected by western blotting. A QubitTM Protein Assay Kit (Thermo Fisher Scientific, USA) was used to measure the protein concentration of the isolated PRP-Exos. PRP-Exos (10 µg protein) were lysed in loading buffer (Solarbio, China), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a 0.22-µm nitrocellulose (NC) filter membrane (Millipore, USA). The NC filter membranes were incubated with the following primary antibodies: Alix (SantaCruz, USA), CD81 (SantaCruz, USA), CD63 (Abcam, USA), and flotillin-1 (Abcam, USA). Imaging was performed using a chemiluminescence imaging system (P&Q Science & Technology, USA). In Vitro experiments of bone marrow-derived macrophages BMDMs were separated from the mouse femurs and tibias under sterile conditions. BMDMs were seeded in six-well plates and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco,, USA) containing 20% fetal bovine serum (FBS, Gibco, USA) and 20% L929 cell supernatant at 37°C in a 5% CO 2 incubator (M0). Seven days later, the cells were transferred in fresh RPMI-1640 medium containing 10% FBS for 24 h. BMDMs were cultured with DMEM medium (10% FBS) supplemented with 100 ng/ml lipopolysaccharide (LPS, Sigma, USA) plus 20 ng/ml interferon-γ (IFN-γ, Sigma, USA) for 24 h to activate M1 polarization or 20 ng/ml interleukin-4 (IL-4, Sigma, USA) for 48 h to activate M2 polarization. Statistical analyses Results are expressed as the mean ± standard deviation (SD). We analyzed the data using GraphPad Prism software (version10.4), with Student’s t test used to compare the differences between the two groups. A P value < 0.05 was considered statistically significant. Results Therapeutic effects of PRP-gel in enhancing wound healing in DFUs We demonstrated that the application of PRP-gel to DFUs significantly enhanced the healing process. As illustrated in Fig. 1 A, the transformative effects of PRP-gel were vividly captured through typical images of five patients before and after treatment. Figure 1 B further elucidates the patients' wound-healing trajectory, providing a clear visual representation of the progressive improvement. Additionally, Fig. 1 C outlines the wound-healing rate and the reduction in wound area, offering a comprehensive overview of the therapeutic outcomes. These findings underscore the efficacy of PRP-gel in accelerating the recovery of DFUs, and represent a promising avenue for clinical intervention. PRP-gel promotes wound healing in diabetic mice We subsequently investigated the potential of PRP-gel to modulate wound healing by creating full-thickness cutaneous wounds on the dorsal surfaces of diabetic mice (DM), followed by the application of PRP-gel to the wounds (the experimental design for evaluating diabetic wound healing is illustrated in Fig. 2 A). Blood glucose levels were monitored at the time of tissue collection (D0/D5 D7/D9 post-wounding) to ensure the diabetic status of the mice. As shown in Fig. 2 B, the blood glucose levels remained consistently elevated in the STZ-induced diabetic mice throughout the experimental period, in contrast to the non-diabetic BALB/c mice that maintained normal blood glucose levels. Mice exhibiting sustained levels of hyperglycemia (> 16.7 mmol/L), indicative of diabetes, were chosen for subsequent experiments in this study. To evaluate the therapeutic potential of PRP-gel, full-thickness wounds in diabetic mice were treated with either 0.9% physiological saline (DM-Non-PRP, DM-NPRP group) or PRP-gel, and digital photographs were taken at 0, 5, 7, 9, and 11 days post-wounding to monitor the healing process. PRP-gel-treated groups exhibited accelerated wound closure compared to the DM-NPRP group (Fig. 2 C). At 5 days post-treatment, the PRP-gel group demonstrated a significantly greater reduction in wound area compared to the DM-NPRP group (Fig. 2 D). On days 5, 7, and 9, the wound area in the PRP-gel treatment group demonstrated a significant reduction compared to the control group. By day 11, the wounds in the PRP-gel treatment group were nearly completely healed, showcasing the remarkable efficacy of this treatment (Fig. 2 E). For histological analysis, wound samples were stained with hematoxylin and eosin (H&E) to assess re-epithelialization at 14 days post-wounding. The PRP-gel treatment groups showed significantly better epithelia than DM-NPRP group (Fig. 2 F). Masson’s trichrome staining revealed significantly increased collagen deposition in the PRP-gel group at 11 days, characterized by densely packed and well-organized collagen fibers compared to the DM-NPRP group. This observation underscores the potential of PRP-gel in promoting collagen synthesis and tissue regeneration. PRP-gel modulates neutrophil activity to regulate local immune-inflammatory response in DM To investigate the immunomodulatory effects of PRP-gel on diabetic ulcer healing, we collected wound-tissue samples on day 11 post-treatment for immune cell profiling, and our flow-cytometric analysis revealed a significant reduction in neutrophil infiltration in PRP-gel-treated wounds compared to those of the DM-NPRP group ( P < 0.05). However, we noted no difference in the proportions of CD3 + T cells, CD49b + NK cells, CD19 + B cells, or F4/80 + CD11b + macrophages between the two groups (Fig. 3 B). Notably, we found that the proportion of F4/80 + CD206 + M2 macrophages within the total macrophage population was significantly increased in the PRP-gel-treated group than in the DM-NPRP group. Figure 3 C presents the results of our statistical analysis of these immune cell populations, revealing the differences between different groups. These data suggest that PRP-gel exerts its immunomodulatory effects in diabetic wound healing predominantly through neutrophil regulation, while maintaining homeostasis of other immune cell populations. PRP-gel promotes macrophage polarization toward the M2 phenotype in the late stage of ulcer wound healing in diabetic mice In our investigation of ulcer healing in diabetic mice, IF staining showed the profound influence of PRP-gel treatment on macrophage polarization. On day 5 (Fig. 4 A), quantitative analysis revealed a substantial decrease in the co-localization of F4/80 (green fluorescence) and iNOS (red fluorescence) in the PRP-gel treated group compared to the DM-NPRP group. This observation suggested that PRP-gel treatment predominantly inhibited macrophage polarization toward the M1 phenotype in the initial phase of wound healing. As the healing progressed, by day 11 (Fig. 4 B), quantitative analysis showed a significant increase in the co-localization of F4/80 (green) and Arg-1 (red) in the PRP-gel treated group, with statistical analyses corroborating the imaging results. This sequence of findings demonstrates the efficacy of PRP-gel treatment in facilitating macrophage polarization toward the M2 phenotype during the advanced stages of wound healing. Characterization of PRP-Exos Purified nanoparticles derived from PRP were identified using TEM, NanoSight NTA and western blotting. The exosomes appeared as cup-shaped or sphere-shaped vesicles with a diameter of approximately 100 nm as observed under TEM (Fig. 5 A), and NTA revealed that the peak particle size of the nanoparticles was 105.1 nm (Fig. 5 B). WB results confirmed that PRP-Exos exhibited high levels of expression for exosomal markers, including Alix, CD63, CD81, and Flot-1 (Fig. 5 C). These results collectively confirmed the presence of PRP-Exos. PRP-Exos promote the diabetic wound healing process To evaluate the in vivo efficacy of PRP-Exos, full-thickness excisional wound models mimicking human conditions were established in STZ-induced diabetic mouse models. This study comprised two treatment groups: PRP-gel and PRP-Exos. Digital photographs documenting the healing process were obtained at 0, 5, 7, 9, and 11 days post-treatment, and wound areas were quantified using ImageJ software. We noted that the PRP-Exo group exhibited significantly accelerated wound healing compared to the control group (Fig. 5 D); and by day 11, wounds in the PRP-Exo group showed near-complete re-epithelialization, demonstrating the significant pro-healing effects of this treatment (Figs. 5 E, F). For histological assessment, wound samples were stained with hematoxylin and eosin (H&E) on day 7 to assess neoepithelium formation, and we observed that the PRP-Exo group demonstrated significantly enhanced epithelial regeneration compared to the control group (Fig. 5 G). In addition, collagen deposition and organization were evaluated using Masson’s trichrome staining on day 14. The PRP-Exo group exhibited a greater degree of collagen deposition marked by large, wavy collagen fibers and a well-organized extracellular matrix, as compared to the control group (Fig. 5 H). These findings collectively underscore the significant efficacy of PRP-Exos in promoting epithelial regeneration and collagen remodeling, suggesting its therapeutic potential for diabetic wound healing. PRP-Exos boost angiogenesis and M2 macrophage polarization to expedite diabetic foot ulcer healing To assess the impact of PRP-Exos on angiogenesis on day 14, IF staining was employed. CD31, a marker specifically expressed on endothelial cell surfaces, and alpha-smooth muscle actin (α-SMA), a protein localized in vascular smooth muscle cells, are both well-established indicators of vascular networks. In the IF analysis, the number of CD31-positive and α-SMA-positive vessels within each wound was quantified to evaluate the formation of new blood vessels. The quantitative assessment of neovascularization demonstrated that the PRP-Exo group exhibited significantly higher angiogenic activity relative to the PRP-gel group, highlighting its superior efficacy in vascular regeneration (Fig. 6 A). To elucidate the regulatory mechanism underlying PRP-Exo action in healing ulcer wounds in diabetic mice, we conducted IF analysis on ulcer tissues collected on day 11 of the experiment. Our results revealed a marked increase in the number of F4/80 (a marker for macrophages) and Arg-1 (a marker for M2 macrophages) co-positive cells in the PRP-Exo group compared to the PRP-gel group. These findings indicate that PRP-Exos effectively promotes the polarization of macrophages toward the anti-inflammatory M2 phenotype, thereby significantly enhancing the healing process of ulcer wounds in diabetic mice (Fig. 6 B). To further explicate the regulatory mechanisms underlying PRP-Exo action on macrophage polarization, we initiated our investigation by isolating BMDMs and establishing a comprehensive in vitro culture system (Fig. 6 C). Through systematic experimental analyses, we ascertained that PRP-Exos modulated macrophage polarization by suppressing the transition of M0 macrophages into the pro-inflammatory M1 phenotype while promoting their differentiation into the anti-inflammatory M2 phenotype (Fig. 6 D). Discussion DFUs represent a critical complication that poses a significant threat to the lower limbs of individuals with diabetes, potentially causing systemic harm and even resulting in death[ 1 ]. The management of DFUs is particularly challenging, as these injuries tend to heal slowly or inadequately in diabetic patients. Due to its marked prevalence—affecting approximately 15–25% of diabetic patients during their lifetime—and associated high healthcare expenditures, DFUs have emerged as a pressing global health concern[ 2 ]. Therefore, it is essential to conduct thorough research into the pathophysiological processes involved in diabetic wound healing and to investigate potential interventions so as to expedite the healing process. PRP has been previously demonstrated to enhance the healing and regeneration of diabetic skin wounds[ 13 , 20 – 22 ], and we herein also demonstrated that PRP-gel effectively promoted wound healing in patients with DFUs (Fig. 1 ). We observed similar results in the diabetic mouse model, as the application of PRP-gel significantly enhanced wound healing in diabetic mice. Furthermore, PRP-gel reduced inflammation within the wound tissue and promoted collagen accumulation, which in turn accelerated the healing process (Fig. 2 ). Neutrophils are key cells in the early phase of an inflammatory response, and are capable of clearing infected and damaged tissues by releasing inflammatory mediators and reactive oxygen species. However, excessive neutrophil activity can lead to tissue damage[ 23 , 24 ]. In the present study, we provided evidence that PRP-gel can regulate the local inflammatory microenvironment by reducing neutrophil infiltration that is crucial for tissue repair and regeneration (Fig. 3 ). Emerging evidence highlights the crucial role of macrophages in the inflammatory response stage of wound repair[ 16 , 17 ], with their dynamic plasticity mediating their involvement in both tissue damage and repair processes[ 9 ]. The etiology of DFUs is multifactorial, involving a combination of vascular, neurological, and immunological dysfunctions. Recent studies in macrophage biology suggest that impaired transformation of M1 to M2 macrophages constitutes a critical factor that contributes to poor wound healing and the development of chronic open ulcers in DFUs[ 25 ]. Our experimental data indicated that PRP-gel exhibited a significant dual-regulatory effect in the wound healing process. In the early stages, PRP-gel reduced inflammation by inhibiting M1-type macrophages, while in the later stages it promoted macrophage polarization toward the M2 phenotype—thereby accelerating tissue repair and creating a favorable microenvironment for regeneration (Fig. 4 ). PRP-Exos have been shown by other investigators to exhibit significantly enhanced efficacy in promoting diabetic wound healing compared to PRP alone[ 18 – 20 , 26 ]. In our study, we successfully isolated and characterized PRP-Exos, and demonstrated their potential as an accessible bioactive substance that can promote DFU healing (Fig. 5 ). Blood vessels are characterized as essential for tissue regeneration, primarily due to their roles in supplying nutrients and oxygen to cells surrounding wounds[ 27 ]. We thus assessed the levels of α-SMA and CD31 to evaluate newly formed vessels within the regenerated tissue, and observed that PRP-Exo-treated diabetic wounds exhibited much higher expression of α-SMA and CD31 than the PRP-gel groups. We thus hypothesized that PRP-Exos exerted their functions in DFU progression by regulating macrophage polarization. To verify this, we conducted both in vitro and in vivo studies, and we found that PRP-Exos inhibited macrophage polarization toward the M1 phenotype, thereby promoting a shift to the M2 phenotype (Fig. 6 ). These data indicated that PRP-Exos successfully promoted angiogenesis and blood vessel formation in the diabetic wounds, accelerating the healing process. Our findings signify that the precise regulation of macrophage polarization toward the M2 phenotype—combined with PRP-Exo application—can significantly enhance DFU wound healing. Despite the promising therapeutic effects of PRP-gel and PRP-Exos on wound healing in DFUs that we demonstrated herein, there were limitations to the present study, including limited sample size and lack of diversity in the cohort that would have enhanced the generalizability of our findings. In addition, although we explored the immune-inflammatory responses and macrophage polarization facilitated by PRP-gel and PRP-Exos, the precise underlying molecular mechanisms involved remain incompletely understood. We recommend that investigators in the future explore the synergistic potential of PRP-gel and PRP-Exos when combined with other therapeutic modalities such as stem cell therapy or advanced wound dressings, which we expect would improve healing outcomes in DFUs. Conclusions In this study, we provided evidence that PRP-gel and PRP-Exos enhanced the healing of DFUs by modulating macrophage polarization and promoting the transition from the pro-inflammatory M1 phenotype to the anti-inflammatory and pro-healing M2 phenotype. These findings provided robust experimental evidence for the clinical application of PRP-gel and PRP-Exos in the treatment of DFUs. Additionally, novel insights were offered into the development of biological therapies that target DFUs. Abbreviations DFUs，Diabetic foot ulcers；PRP，Platelet-rich plasma； PRP-Exos，plasma-derived exosomes; RCTs, Randomized Controlled Trials; SPF, specific-pathogen-free; STZ, streptozotocin; BMDM, Bone Marrow-Derived Macrophages; DMEM, Dulbecco’s Modified Eagle’s medium; FBS, fetal bovine serum; LPS, Lipopolysaccharide; IFN-γ, Interferon-γ; IL-4, Interleukin-4; SD, standard deviation; DM, diabetic mice; DM-NPRP, DM-NPRP: Non-PRP treatment of ulcers in diabetic mice ; DM-PRP-gel, DM-PRP-gel: PRP-gel treatment of ulcers in diabetic mice ; IF, immunofluorescence; WB, Western blotting; Neu , neutrophils ; MΦ , macrophages . Declarations Funding This work was supported by grants from Key projects of Natural Science in Colleges and Universities of Anhui Province [grant numbers: 2023AH051754]. The funders had no role in study design; in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to submit the article for publication. Ethics approval and consent to participate This study was sanctioned by Ethics Committee of Wannan Medical College (202375). The protocols used in this study were approved by the First Affiliated Hospital of Wannan Medical College, and written informed consent was provided from all the patients. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory and approved by the Animal Research Ethics Committee of Wannan Medical College (WNMC-AWE-2023102). Consent for publication All subjects have written informed consent. Data availability statement All data relevant to the study are included in the article. Primary data are available upon request. Conflicts of interest We declare that we have no conflicts of interest relevant to the content of this paper. Author Contributions Ling He:wrote the main manuscript text; Nan Zhao: acquisition of data;Xiaoling Chen and Wenjie Zhang: ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Kun LV:made substantial contributions to the conception or design of the work；Yuanhong Xu: approved the version to be published. References Armstrong DG, Tan TW, Boulton AJM, Bus SA. Diabetic Foot Ulcers: A Review. JAMA. 2023;330(1):62–75. Dixon D, Edmonds M. Managing Diabetic Foot Ulcers: Pharmacotherapy for Wound Healing. Drugs. 2021;81(1):29–56. Gao W, Jin W, Li Y, et al. A highly bioactive bone extracellular matrix-biomimetic nanofibrous system with rapid angiogenesis promotes diabetic wound healing. J Mater Chem B. 2017;5(35):7285–7296. Qian Z, Wang H, Bai Y, et al. Improving Chronic Diabetic Wound Healing through an Injectable and Self-Healing Hydrogel with Platelet-Rich Plasma Release. ACS Appl Mater Interfaces. 2020;12(50):55659–55674. Han G, Ceilley R. Chronic Wound Healing: A Review of Current Management and Treatments. Adv Ther. 2017;34(3):599–610. Xiao J, Zhu Y, Huddleston S, et al. Copper Metal-Organic Framework Nanoparticles Stabilized with Folic Acid Improve Wound Healing in Diabetes. ACS Nano. 2018;12(2):1023–1032. Marti-Carvajal AJ, Gluud C, Nicola S, et al. Growth factors for treating diabetic foot ulcers. Cochrane Database Syst Rev. 2015;2015(10):CD008548. Nie X, Liu Y, Yuan T, et al. Platelet-rich plasma-derived exosomes promote blood-spinal cord barrier repair and attenuate neuroinflammation after spinal cord injury. J Nanobiotechnology. 2024;22(1):456. Jiang G, Li S, Yu K, et al. A 3D-printed PRP-GelMA hydrogel promotes osteochondral regeneration through M2 macrophage polarization in a rabbit model. Acta Biomater. 2021;128:150–162. Everts P, Onishi K, Jayaram P, Lana JF, Mautner K. Platelet-Rich Plasma: New Performance Understandings and Therapeutic Considerations in 2020. Int J Mol Sci. 2020;21(20). Qu W, Wang Z, Hunt C, et al. The Effectiveness and Safety of Platelet-Rich Plasma for Chronic Wounds: A Systematic Review and Meta-analysis. Mayo Clin Proc. 2021;96(9):2407–2417. Martinez-Zapata MJ, Marti-Carvajal AJ, Sola I, et al. Autologous platelet-rich plasma for treating chronic wounds. Cochrane Database Syst Rev. 2016;2016(5):CD006899. Hirase T, Ruff E, Surani S, Ratnani I. Topical application of platelet-rich plasma for diabetic foot ulcers: A systematic review. World J Diabetes. 2018;9(10):172–179. Del Pino-Sedeno T, Trujillo-Martin MM, Andia I, et al. Platelet-rich plasma for the treatment of diabetic foot ulcers: A meta-analysis. Wound Repair Regen. 2019;27(2):170–182. Louiselle AE, Niemiec SM, Zgheib C, Liechty KW. Macrophage polarization and diabetic wound healing. Transl Res. 2021;236:109–116. Willenborg S, Sanin DE, Jais A, et al. Mitochondrial metabolism coordinates stage-specific repair processes in macrophages during wound healing. Cell Metab. 2021;33(12):2398–2414 e2399. Boniakowski AE, Kimball AS, Jacobs BN, Kunkel SL, Gallagher KA. Macrophage-Mediated Inflammation in Normal and Diabetic Wound Healing. J Immunol. 2017;199(1):17–24. Cao W, Meng X, Cao F, Wang J, Yang M. Exosomes derived from platelet-rich plasma promote diabetic wound healing via the JAK2/STAT3 pathway. iScience. 2023;26(11):108236. Rui S, Dai L, Zhang X, et al. Exosomal miRNA-26b-5p from PRP suppresses NETs by targeting MMP-8 to promote diabetic wound healing. J Control Release. 2024;372:221–233. He M, Chen T, Lv Y, et al. The role of allogeneic platelet-rich plasma in patients with diabetic foot ulcer: Current perspectives and future challenges. Front Bioeng Biotechnol. 2022;10:993436. Nolan GS, Smith OJ, Heavey S, Jell G, Mosahebi A. Histological analysis of fat grafting with platelet-rich plasma for diabetic foot ulcers-A randomised controlled trial. Int Wound J. 2022;19(2):389–398. Hesseler MJ, Shyam N. Platelet-rich plasma and its utility in medical dermatology: A systematic review. J Am Acad Dermatol. 2019;81(3):834–846. Takagi N, Kawakami K, Kanno E, et al. IL-17A promotes neutrophilic inflammation and disturbs acute wound healing in skin. Exp Dermatol. 2017;26(2):137–144. Phillipson M, Kubes P. The Healing Power of Neutrophils. Trends Immunol. 2019;40(7):635–647. Geng K, Ma X, Jiang Z, et al. WDR74 facilitates TGF-beta/Smad pathway activation to promote M2 macrophage polarization and diabetic foot ulcer wound healing in mice. Cell Biol Toxicol. 2023;39(4):1577–1591. Chen C, Wang Q, Li D, Qi Z, Chen Y, Wang S. MALAT1 participates in the role of platelet-rich plasma exosomes in promoting wound healing of diabetic foot ulcer. Int J Biol Macromol. 2023;238:124170. Wang C, Wang M, Xu T, et al. Engineering Bioactive Self-Healing Antibacterial Exosomes Hydrogel for Promoting Chronic Diabetic Wound Healing and Complete Skin Regeneration. Theranostics. 2019;9(1):65–76. Additional Declarations No competing interests reported. Supplementary Files Supplement20250220.docx Cite Share Download PDF Status: Published Journal Publication published 15 May, 2025 Read the published version in Clinical and Experimental Medicine → Version 1 posted Editorial decision: Accepted 25 Mar, 2025 Reviews received at journal 24 Mar, 2025 Reviewers agreed at journal 20 Mar, 2025 Reviewers invited by journal 20 Mar, 2025 Submission checks completed at journal 19 Mar, 2025 First submitted to journal 19 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5945926","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431623525,"identity":"41fc8796-1c74-4193-a791-6bd8eb5a9f5a","order_by":0,"name":"Ling He","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"He","suffix":""},{"id":431623526,"identity":"f42d7cf6-4808-4311-95d0-d0add90b985b","order_by":1,"name":"Nan Zhao","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Zhao","suffix":""},{"id":431623529,"identity":"e4c14b98-5f3a-42a8-81b3-9712f6972707","order_by":2,"name":"Xiaoling Chen","email":"","orcid":"","institution":"The First Affiliated Hospital of Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Chen","suffix":""},{"id":431623530,"identity":"358c8e8e-511b-4213-8a04-4d39f6a23dd1","order_by":3,"name":"Wenjie Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Wenjie","middleName":"","lastName":"Zhang","suffix":""},{"id":431623532,"identity":"7f88f9d2-6bcd-47a8-a349-fca05ddf3bd9","order_by":4,"name":"Kun Lv","email":"","orcid":"","institution":"The First Affiliated Hospital of Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Lv","suffix":""},{"id":431623534,"identity":"29f1e853-944a-4dc2-bee4-5a9895b9ade1","order_by":5,"name":"Yuanhong Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYFACHhAhIcfPcPjAgQ8/iNdiYSzZeCzx4Mwe4rVUJG5oPmN8mIONCA0GN3IPPi74JZG4ge3Mh8NA/fL8Ygfwa5GckZdsPLNPwng7z9kNhwssGAxnzk7Ar4VfIsdMmrdHQnbnDKCWGTwMCQa3CWhhg2ph3HD/zYPDPGxEaAHbwvNDQnHDgTMMxGmR7HljbMzbIGEs2XDMABjIEoT9YnA8x/Axz586UFQ+/vDhh408vzQBLQwCQAWMbXCuBAHlIMB/AEj8IULhKBgFo2AUjFwAACiCSRpoeyBPAAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yuanhong","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2025-02-02 13:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5945926/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5945926/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10238-025-01651-w","type":"published","date":"2025-05-15T15:57:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79320926,"identity":"ff83e873-006c-4b3a-83b3-c70b066df64f","added_by":"auto","created_at":"2025-03-27 04:27:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27009319,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlatelet-rich plasma gel(PRP-gel) promotes wound healing in patients with diabetic foot ulcers\u003c/strong\u003e (\u003cstrong\u003eDFUs)\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003eA. \u003c/strong\u003eRepresentative images of DFU wounds before and after PRP-gel treatment. \u003cstrong\u003eB. \u003c/strong\u003eWound healing trajectory of DFU wounds before and after PRP-gel treatment. \u003cstrong\u003eC. \u003c/strong\u003eWound healing rate and area changes in patients with DFU after PRP-gel treatment. \u003cstrong\u003e**\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01, ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.0001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5945926/v1/a837e7b4eb016efec63eecef.png"},{"id":79319652,"identity":"bcb0ed1d-add5-49f1-aed6-17a53e4a5e9a","added_by":"auto","created_at":"2025-03-27 04:19:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":32507813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRP-gel enhances ulcer healing in diabetic mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. Schematic of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e wound-healing assay. BALB/c mice were intraperitoneally injected with streptozotocin (STZ, 50 mg/kg) for 5 days consecutively, and blood glucose levels were measured after 4 weeks. Our model was successfully established when blood glucose levels were greater than 16.8 mmol/L. Experiments were conducted on days 0, 5, 7, 9, and 11. B. The plasma glucose concentration in STZ-induced diabetic mice was measured on days 0, 5, 7, 9, and 11 post-wound and compared with that of BALB/c control mice. C. Representative diagram of PRP-gel on the ulcers of diabetic mice on days 0, 5, 7, 9, and 11. DM-NPRP, non-PRP treatment of ulcers in diabetic mice; DM-PRP-gel, PRP-gel treatment of ulcers in diabetic mice. D. Healing progression of ulcer wounds in diabetic mice treated with non-PRP treatment control and PRP-gel. E. Changes in the ulcer area in diabetic mice treated with non-PRP treatment control and PRP-gel. F. Hematoxylin and eosin \u003c/strong\u003e(\u003cstrong\u003eH\u0026amp;E\u003c/strong\u003e) \u003cstrong\u003estaining of ulcer tissues in diabetic mice treated with non-PRP treatment control and PRP-gel. G. Masson's trichrome staining of ulcer tissues in diabetic mice treated with the non-PRP control treatment and PRP-gel. **\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01, ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.0001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5945926/v1/c34270da0ab07baaf94c5e44.png"},{"id":79319651,"identity":"8413ef40-aea4-4fc4-a0e4-647857b503f9","added_by":"auto","created_at":"2025-03-27 04:19:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34827980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow-cytometric analysis of immune cell populations in diabetic ulcer tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. Representative density dot plots of CD3+ T cells, CD49b+ NK cells, and CD19+ B cells in ulcers of diabetic mice treated with non-PRP and PRP-gel using flow cytometry. B. Representative density plots of neutrophils (Neu), macrophages (MΦ), and M2 macrophages in ulcer of diabetic mice treated with non-PRP and PRP-gel using flow cytometry. C. Quantitative analysis of immune cell populations (CD3+ T cells, CD49b+ NK cells, CD19+ B cells, neutrophils, Mφ, and M2 macrophages) in ulcers of diabetic mice treated with non-PRP and PRP-gel. *\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05, ***\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.001; NS, no statistical difference.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5945926/v1/df5070c2e61a050c34d5818c.png"},{"id":79319636,"identity":"d0512a9a-fd86-43dd-b4e2-78b44fb432af","added_by":"auto","created_at":"2025-03-27 04:19:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2487838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRP-gel promotes M2 polarization of macrophages in ulcer tissue of diabetic mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. \u003cstrong\u003eRepresentative immunofluorescence images of F4/80 (green) and iNOS (red) co-localized cells in groups treated with non-PRP treatment control or PRP-gel. F4/80 and iNOS fluorescence area were quantified by adopting five high-magnification fields using ImageJ software. \u003c/strong\u003eB. \u003cstrong\u003eRepresentative immunofluorescence images of F4/80 (green) and Arg-1 (red) co-localized cells in groups treated with non-PRP treatment control or PRP-gel. F4/80 and Arg-1 fluorescence area were quantified under five high-magnification fields using ImageJ software. *\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05, **\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01; NS, no statistical difference.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5945926/v1/ec8cabe93cabb0bc61570ad5.png"},{"id":79319659,"identity":"5cdb859f-6d06-4ea7-a455-b3450c00d9b7","added_by":"auto","created_at":"2025-03-27 04:19:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":34830850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasma-derived exosomes\u003c/strong\u003e (\u003cstrong\u003ePRP-Exos) promote ulcer healing in diabetic mice via enhanced tissue regeneration and reduced inflammation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. \u003cstrong\u003eMorphology of exosomes observed under a \u003c/strong\u003etransmission electron microscope (\u003cstrong\u003eTEM) showing typical cup-shaped structures with a diameter range of 50–150 nm. B. Size distribution and concentration of exosomes were evaluated by nanoparticle tracking analysis (NTA). C. Western-blot analysis confirmed the presence of exosomal surface biomarkers, including Alix, CD63, CD81, and Flot-1 in PRP-Exos.\u003cbr\u003e\nD. Representative diagram illustrating the application of PRP-gel and PRP-Exos on ulcer wounds in diabetic mice. E. Healing progression of ulcer wounds in diabetic mice treated with PRP-gel and PRP-Exos. F. Changes in ulcer area in diabetic mice treated with PRP-gel and PRP-Exos showing a significant reduction in wound size compared to PRP-gel groups \u003c/strong\u003eG. \u003cstrong\u003eH\u0026amp;E staining of ulcer tissue in diabetic mice treated with PRP-gel and PRP-Exos, revealing enhanced re-epithelialization and reduced inflammatory cell infiltration. H. Masson's trichrome staining of ulcer tissue in diabetic mice treated with PRP-gel and PRP-Exos demonstrating increased collagen deposition and improved tissue remodeling. *\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05, **\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5945926/v1/0876c5687546b315fa847f66.png"},{"id":79319629,"identity":"e95cac7a-9361-4f91-a34a-2710afe9cd58","added_by":"auto","created_at":"2025-03-27 04:19:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3879693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRP-Exos enhance M2 polarization of macrophages in ulcer tissue of diabetic mice.\u003cbr\u003e\n A. Representative immunofluorescence images and quantitative analysis of α-SMA (red) and CD31 (green) co-localized cells in groups treated with PRP-gel and PRP-Exos. α-SMA and CD31 fluorescence areas were quantified under high-magnification fields using ImageJ, and statistical analysis was performed with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003et \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etests. B. Representative immunofluorescence images of F4/80 (green) and Arg-1 (red) co-localized cells in groups treated with PRP-gel or PRP-Exos. F4/80 and Arg-1 fluorescence areas were quantified under five high-magnification fields using ImageJ, and statistical analysis was performed with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003et\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e tests. C. Schematic diagram illustrating the induction of M0, M1, and M2 macrophages from BMDM under specific conditions (LPS/IFN-γ for M1, IL-4 for M2) and their detection by immunofluorescence after treatment with PRP-Exos (diagram was constructed using Figdraw 2.0). D. Representative immunofluorescence images of iNOS+ in LPS/IFN-γ-activated BMDM and Arg-1+ in IL-4-activated BMDM.\u003c/strong\u003e \u003cstrong\u003e*\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05, ***\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.001, ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.0001; NS, no statistical difference.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5945926/v1/de0ff00b3c314d1afeb2f99f.png"},{"id":83067781,"identity":"ca1bbe66-a8f6-4b72-bf3b-59ee8e4a8ebe","added_by":"auto","created_at":"2025-05-19 16:05:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28693362,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5945926/v1/4e0737c5-a9db-4a76-b869-590d7e107aad.pdf"},{"id":79319635,"identity":"c9303912-edcf-4b0b-abbf-8642e4213585","added_by":"auto","created_at":"2025-03-27 04:19:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15577,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement20250220.docx","url":"https://assets-eu.researchsquare.com/files/rs-5945926/v1/318deebc31ca202cd86e3564.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Platelet-rich plasma-derived exosomes accelerate the healing of diabetic foot ulcers by promoting macrophage polarization to the M2 phenotype","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic foot ulcers (DFUs) affect 10 to 15% of the diabetic population and are among the most formidable chronic complications, conveying profound clinical risks that demand urgent attention. This debilitating condition not only significantly extends hospital stays and sustains a persistently high disability rate, but also carries a grave risk of amputation, profoundly impacting patient quality of life[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Epidemiological studies suggest that between 10 and 15% of individuals with diabetes develop foot ulcers that are highly susceptible to infection. Patients with DFUs have a five-year mortality rate of 50%, increasing to 80% after amputation[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe development of DFUs is intricately linked to a complex interplay of mechanisms\u0026mdash;including impaired angiogenesis, chronic inflammatory responses, and compromised tissue-repair functions\u0026mdash;that collectively contribute to the progression and persistence of this condition[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In current clinical practice, wound management primarily relies on standard therapeutic approaches such as surgical debridement, reducing weight-bearing pressure, treating lower-extremity ischemia, antimicrobial treatment, and glycemic control[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although these conventional methods offer partial symptomatic improvement, they are frequently limited by prolonged healing durations and significant recurrence rates, and fail to meet the increasing demands of modern wound care. For many patients, the efficacy of traditional treatments remains suboptimal, and this is largely attributed to impaired cellular functions and insufficient bioactive components within the wound microenvironment[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This clinical dilemma not only severely impacts the quality of life of patients but also imposes a significant financial burden on their families[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, searching for more effective treatment strategies has become an urgent need in the field of DFU therapy.\u003c/p\u003e \u003cp\u003eIn recent years, the rapid advancements in regenerative medicine and biotherapy have led to the emergence of innovative treatment modalities[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and, of these, platelet-rich plasma (PRP) therapy, an autologous biotherapy approach, has garnered significant attention due to its unique advantages. Sourced directly from the patient\u0026rsquo;s bloodstream, PRP offers the unique advantage of being immunogen-free while containing a concentrated array of growth factors, thus emerging as a particularly promising therapeutic approach in clinical practice. PRP has also demonstrated superior efficacy in wound healing compared to traditional therapies[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePRP is a bioactive complex of substances extracted through a meticulous process of centrifuging and concentrating a patient's whole blood, and the method yields plasma with a platelet concentration that is a remarkable 3\u0026ndash;7 times higher than that found in normal blood. These platelets, rich in α-granules, harbor an array of growth factors, adhesion molecules, and cytokines[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Upon activation, these granular contents are released and play an indispensable role in stimulating cellular proliferation, differentiation, and tissue regeneration; this, in turn, significantly expedites the wound-healing process.\u003c/p\u003e \u003cp\u003eRecent research has revealed that PRP markedly reduces both the healing time and wound area in patients suffering from DFUs [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, a systematic review, a meta-analysis, and several randomized controlled trials (RCTs) have shown that an autologous platelet-gel surpasses topical antibacterial dressings in healing rates and infection prevention with respect to clean DFU wounds[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. While these outcomes are encouraging, the existing evidence remains inadequate for a definitive determination of the efficacy of PRP therapy in treating chronic DFU wounds. Consequently, additional rigorous studies are required to establish PRP\u0026rsquo;s clinical utility and to provide a solid foundation of experimental evidence.\u003c/p\u003e \u003cp\u003eMacrophages, crucial actors in the healing process, are classified into two distinct phenotypes based on their functional characteristics and cytokine profiles: the classically activated macrophages (M1 type), which predominantly secrete pro-inflammatory cytokines potentially hindering the maturation of granulation tissue[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]; and the alternatively activated macrophages (M2 type), which suppress inflammatory responses and facilitate wound healing. The literature is replete with evidence confirming the pivotal role of macrophages in managing the inflammatory phase of wound repair, with their dynamic phenotypic plasticity mediating their involvement in tissue injury and regeneration[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The pathogenesis of DFUs is multifaceted, and recent research suggests that an impaired transition from the M1 to the M2 macrophage phenotype is a critical factor in the development of chronic non-healing ulcers in DFUs.\u003c/p\u003e \u003cp\u003eThe therapeutic potential of PRP-derived exosomes (PRP-Exos) in facilitating the healing of DFUs has emerged as a compelling area of regenerative medicine that has garnered sustained interest from the scientific community [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, further investigation is needed to understand the impact of PRP-gel and PRP-Exos on DFU treatments, particularly their role in modulating macrophage polarization. In this study, we thoroughly investigated the mechanisms underlying the actions of PRP-gel and PRP-Exos in modulating macrophage polarization during diabetic wound healing, with a focus on their influence on DFU treatment outcomes. By merging clinical observations with experimental data, we have attempted to establish a robust framework for the clinical implementation of these therapies, thereby enhancing the efficacy of DFU management and advancing patient care.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eThis study was sanctioned by the Ethics Committee of the Wannan Medical College. Written informed consent was obtained from all participants. These patients had been diagnosed with stage II\u0026ndash;IV DFU\u0026rsquo;s based on the Wagner classification system. Clinical information regarding the patients is presented in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The wound of the patient with diabetic foot was treated using only autologous PRP-gel derived from the patient themselves. The ulcer area was measured every 3 days using a digital camera, and wound closure rates were calculated. We analyzed wound-area measurements from images using ImageJ (NIH, USA) software. Data were not identifiable to any patient.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDiabetic skin-wound animal model and treatment\u003c/h3\u003e\n\u003cp\u003eAdult male BALB/c mice (6\u0026ndash;8 weeks of age and weighing 26\u0026ndash;28 g; grade, clean; license, SCXK 2023-005) were purchased from the Experimental Animal Center of Qinglongshan (Nanjing, China) and raised in a specific-pathogen-free (SPF) mouse colony. Mice were housed in a controlled environment with a 12-h light/dark cycle, maintained at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and provided with standard rodent chow and water ad libitum. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 50 mg/kg) for 5 days consecutively. After 4 weeks, blood glucose levels were monitored, and mice with sustained blood glucose levels above 16.8 mmol/L were considered successful diabetic models. An electronic blood glucose meter (Roche, China) was used to measure mouse serum glucose levels. Subsequently, full-thickness skin ulcers with a diameter of 6 mm were created on the dorsal side of the mice to serve as a DFU model.\u003c/p\u003e\n\u003ch3\u003eChronic wound-healing in mice\u003c/h3\u003e\n\u003cp\u003eA model of DFUs was created in mice anesthetized with isoflurane inhalation. A single round full-thickness skin wound was then created with a 6-mm disposable skin biopsy punch on the dorsum of the diabetic mice. Digital images of the wound area were captured on the day of surgery and on days 5, 7, 9, and 11 after operation. A ruler reference was placed nearby to confirm the distance between the animals and the camera. The wound area was quantified by three blinded observers using ImageJ (NIH, USA) software and expressed as a percentage of the original area. The wound was covered with gauze and a bandage, and the dressings were changed every 3 days according to the standard of care; and wound tissues were sampled and harvested at 5, 7, 9, and 11 days. Harvested wound tissues were fixed in 10% formalin solution, and adopted for further immunofluorescence (IF) investigations.\u003c/p\u003e\n\u003ch3\u003ePreparation of PRP and PRP-gel\u003c/h3\u003e\n\u003cp\u003eTen healthy adult volunteers, aged between 18 and 45 years and with no history of hematological or infectious diseases were selected. PRP was isolated utilizing a fully automatic blood separator (COM.TEC; Fresenius Kabi, German), and PRP-gel was produced by activating PRP with a prepared mixture of thrombin and calcium gluconate at a ratio of 10:1. PRP-gel derived from volunteers was used for the diabetic wound experiment in mice.\u003c/p\u003e\n\u003ch3\u003eMorphological staining\u003c/h3\u003e\n\u003cp\u003eA graded series of ethanol and formaldehyde were applied to the wound tissues from the mice after they had been sampled at 5, 7, 9, and 11 days; and paraffin was applied after the tissues were fixed in 4% formaldehyde overnight. The sections were cut at a thickness of 5 mm, and then treated with hematoxylin and eosin (Servicebio, China). We performed Masson trichrome staining using a staining kit (Servicebio, China) in accordance with the manufacturer\u0026rsquo;s guidelines.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eThe tissues were fixed with 4% paraformaldehyde for 16\u0026ndash;24 h at 4\u0026deg;C, followed by embedding in paraffin and sectioning at 4 \u0026micro;m. After deparaffinization with a dewaxing solution and anhydrous ethanol, the sections were rinsed in PBS and then blocked with 5% of BSA blocking solution for 30 min. The sections were subsequently incubated overnight at 4\u0026deg;C with primary antibodies targeting iNOS (Servicebio, China), Arg-1 (Servicebio, China), and F4/80 (Servicebio, China). For immunofluorescence staining, the sections were incubated with Alexa Fluor 488, Alexa Fluor 555, and conjugated secondary antibodies (Servicebio, China). To evaluate the angiogenesis of PRP-gel and PRP-Exos in the treatment of diabetic wounds, the sections were incubated with antibodies to α-SMA (Servicebio, China) and CD31 (Servicebio, China) at 4\u0026deg;C overnight. After washing several times with PBS, DAPI (Servicebio, China) was used to stain the nuclei for 10 min. Fluoroshield mounting medium (G1401, Servicebio, China) was applied to preserve fluorescence when imaging tissues.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation and purification of PRP-Exos\u003c/h3\u003e\n\u003cp\u003eThe PRP samples were centrifuged at 3000\u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min at 4\u0026deg;C to remove cell debris, and the supernatant was then centrifuged at 10,000\u0026times; \u003cem\u003eg\u003c/em\u003e for 30 min at 4\u0026deg;C to remove large particles; this was followed by ultracentrifugation of the supernatant at 100,000\u0026times; \u003cem\u003eg\u003c/em\u003e for 70 min at 4\u0026deg;C to pellet the exosomes. The exosomal pellets were ultracentrifuged in PBS and centrifuged again at 100,000\u0026times; \u003cem\u003eg\u003c/em\u003e for 70 min to wash the exosomes.\u003c/p\u003e\n\u003ch3\u003eIdentification of PRP-Exos\u003c/h3\u003e\n\u003cp\u003eA transmission electron microscope (TEM) was used to identify the morphology of the exosomes. Briefly, PRP-Exos were fixed in 3% glutaraldehyde for 2 h and washed twice with PBS. The exosomes were negatively stained with 2% uranyl acetate for 30 s and applied to a continuous carbon grid. We ultimately visualized the exosomes on a FEI Tecnai G2 12 TEM (FEI, America).\u003c/p\u003e \u003cp\u003eThe size distribution of PRP-derived exosomes (PRP-Exos) was determined using nanoparticle tracking analysis (NTA) with a ZetaView PMX-120 system (Particle Metrix, Germany). Prior to sample analysis, the instrument was calibrated using 100-nm polystyrene nanoparticles as reference standards. The exosome pellet, resuspended in phosphate-buffered saline (PBS), was subsequently introduced into the NTA system for particle-size characterization. It should be noted that while polystyrene beads serve as reliable calibration standards, the inherent differences in material composition between synthetic beads and biological exosomes necessitate cautious interpretation of size-distribution data. Therefore, the NTA results were complemented and validated by TEM for direct visualization of exosomal morphology and western-blot (WB) analysis for specific exosomal marker identification, ensuring comprehensive characterization of the isolated exosomes.\u003c/p\u003e \u003cp\u003eThe marker proteins of the exosomes were detected by western blotting. A QubitTM Protein Assay Kit (Thermo Fisher Scientific, USA) was used to measure the protein concentration of the isolated PRP-Exos. PRP-Exos (10 \u0026micro;g protein) were lysed in loading buffer (Solarbio, China), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a 0.22-\u0026micro;m nitrocellulose (NC) filter membrane (Millipore, USA). The NC filter membranes were incubated with the following primary antibodies: Alix (SantaCruz, USA), CD81 (SantaCruz, USA), CD63 (Abcam, USA), and flotillin-1 (Abcam, USA). Imaging was performed using a chemiluminescence imaging system (P\u0026amp;Q Science \u0026amp; Technology, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eexperiments of bone marrow-derived macrophages\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBMDMs were separated from the mouse femurs and tibias under sterile conditions. BMDMs were seeded in six-well plates and cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM, Gibco,, USA) containing 20% fetal bovine serum (FBS, Gibco, USA) and 20% L929 cell supernatant at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator (M0). Seven days later, the cells were transferred in fresh RPMI-1640 medium containing 10% FBS for 24 h. BMDMs were cultured with DMEM medium (10% FBS) supplemented with 100 ng/ml lipopolysaccharide (LPS, Sigma, USA) plus 20 ng/ml interferon-γ (IFN-γ, Sigma, USA) for 24 h to activate M1 polarization or 20 ng/ml interleukin-4 (IL-4, Sigma, USA) for 48 h to activate M2 polarization.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eResults are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). We analyzed the data using GraphPad Prism software (version10.4), with Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test used to compare the differences between the two groups. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTherapeutic effects of PRP-gel in enhancing wound healing in DFUs\u003c/h2\u003e \u003cp\u003eWe demonstrated that the application of PRP-gel to DFUs significantly enhanced the healing process. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the transformative effects of PRP-gel were vividly captured through typical images of five patients before and after treatment. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB further elucidates the patients' wound-healing trajectory, providing a clear visual representation of the progressive improvement. Additionally, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC outlines the wound-healing rate and the reduction in wound area, offering a comprehensive overview of the therapeutic outcomes. These findings underscore the efficacy of PRP-gel in accelerating the recovery of DFUs, and represent a promising avenue for clinical intervention.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePRP-gel promotes wound healing in diabetic mice\u003c/h2\u003e \u003cp\u003eWe subsequently investigated the potential of PRP-gel to modulate wound healing by creating full-thickness cutaneous wounds on the dorsal surfaces of diabetic mice (DM), followed by the application of PRP-gel to the wounds (the experimental design for evaluating diabetic wound healing is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Blood glucose levels were monitored at the time of tissue collection (D0/D5 D7/D9 post-wounding) to ensure the diabetic status of the mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the blood glucose levels remained consistently elevated in the STZ-induced diabetic mice throughout the experimental period, in contrast to the non-diabetic BALB/c mice that maintained normal blood glucose levels. Mice exhibiting sustained levels of hyperglycemia (\u0026gt;\u0026thinsp;16.7 mmol/L), indicative of diabetes, were chosen for subsequent experiments in this study. To evaluate the therapeutic potential of PRP-gel, full-thickness wounds in diabetic mice were treated with either 0.9% physiological saline (DM-Non-PRP, DM-NPRP group) or PRP-gel, and digital photographs were taken at 0, 5, 7, 9, and 11 days post-wounding to monitor the healing process. PRP-gel-treated groups exhibited accelerated wound closure compared to the DM-NPRP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). At 5 days post-treatment, the PRP-gel group demonstrated a significantly greater reduction in wound area compared to the DM-NPRP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). On days 5, 7, and 9, the wound area in the PRP-gel treatment group demonstrated a significant reduction compared to the control group. By day 11, the wounds in the PRP-gel treatment group were nearly completely healed, showcasing the remarkable efficacy of this treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). For histological analysis, wound samples were stained with hematoxylin and eosin (H\u0026amp;E) to assess re-epithelialization at 14 days post-wounding. The PRP-gel treatment groups showed significantly better epithelia than DM-NPRP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Masson\u0026rsquo;s trichrome staining revealed significantly increased collagen deposition in the PRP-gel group at 11 days, characterized by densely packed and well-organized collagen fibers compared to the DM-NPRP group. This observation underscores the potential of PRP-gel in promoting collagen synthesis and tissue regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePRP-gel modulates neutrophil activity to regulate local immune-inflammatory response in DM\u003c/h2\u003e \u003cp\u003eTo investigate the immunomodulatory effects of PRP-gel on diabetic ulcer healing, we collected wound-tissue samples on day 11 post-treatment for immune cell profiling, and our flow-cytometric analysis revealed a significant reduction in neutrophil infiltration in PRP-gel-treated wounds compared to those of the DM-NPRP group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, we noted no difference in the proportions of CD3\u0026thinsp;+\u0026thinsp;T cells, CD49b\u0026thinsp;+\u0026thinsp;NK cells, CD19\u0026thinsp;+\u0026thinsp;B cells, or F4/80\u0026thinsp;+\u0026thinsp;CD11b\u0026thinsp;+\u0026thinsp;macrophages between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Notably, we found that the proportion of F4/80\u0026thinsp;+\u0026thinsp;CD206\u0026thinsp;+\u0026thinsp;M2 macrophages within the total macrophage population was significantly increased in the PRP-gel-treated group than in the DM-NPRP group. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC presents the results of our statistical analysis of these immune cell populations, revealing the differences between different groups. These data suggest that PRP-gel exerts its immunomodulatory effects in diabetic wound healing predominantly through neutrophil regulation, while maintaining homeostasis of other immune cell populations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePRP-gel promotes macrophage polarization toward the M2 phenotype in the late stage of ulcer wound healing in diabetic mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn our investigation of ulcer healing in diabetic mice, IF staining showed the profound influence of PRP-gel treatment on macrophage polarization. On day 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), quantitative analysis revealed a substantial decrease in the co-localization of F4/80 (green fluorescence) and iNOS (red fluorescence) in the PRP-gel treated group compared to the DM-NPRP group. This observation suggested that PRP-gel treatment predominantly inhibited macrophage polarization toward the M1 phenotype in the initial phase of wound healing. As the healing progressed, by day 11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), quantitative analysis showed a significant increase in the co-localization of F4/80 (green) and Arg-1 (red) in the PRP-gel treated group, with statistical analyses corroborating the imaging results. This sequence of findings demonstrates the efficacy of PRP-gel treatment in facilitating macrophage polarization toward the M2 phenotype during the advanced stages of wound healing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of PRP-Exos\u003c/h2\u003e \u003cp\u003ePurified nanoparticles derived from PRP were identified using TEM, NanoSight NTA and western blotting. The exosomes appeared as cup-shaped or sphere-shaped vesicles with a diameter of approximately 100 nm as observed under TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and NTA revealed that the peak particle size of the nanoparticles was 105.1 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). WB results confirmed that PRP-Exos exhibited high levels of expression for exosomal markers, including Alix, CD63, CD81, and Flot-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results collectively confirmed the presence of PRP-Exos.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePRP-Exos promote the diabetic wound healing process\u003c/h2\u003e \u003cp\u003eTo evaluate the \u003cem\u003ein vivo\u003c/em\u003e efficacy of PRP-Exos, full-thickness excisional wound models mimicking human conditions were established in STZ-induced diabetic mouse models. This study comprised two treatment groups: PRP-gel and PRP-Exos. Digital photographs documenting the healing process were obtained at 0, 5, 7, 9, and 11 days post-treatment, and wound areas were quantified using ImageJ software. We noted that the PRP-Exo group exhibited significantly accelerated wound healing compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD); and by day 11, wounds in the PRP-Exo group showed near-complete re-epithelialization, demonstrating the significant pro-healing effects of this treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003eFor histological assessment, wound samples were stained with hematoxylin and eosin (H\u0026amp;E) on day 7 to assess neoepithelium formation, and we observed that the PRP-Exo group demonstrated significantly enhanced epithelial regeneration compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). In addition, collagen deposition and organization were evaluated using Masson\u0026rsquo;s trichrome staining on day 14. The PRP-Exo group exhibited a greater degree of collagen deposition marked by large, wavy collagen fibers and a well-organized extracellular matrix, as compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). These findings collectively underscore the significant efficacy of PRP-Exos in promoting epithelial regeneration and collagen remodeling, suggesting its therapeutic potential for diabetic wound healing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePRP-Exos boost angiogenesis and M2 macrophage polarization to expedite diabetic foot ulcer healing\u003c/h2\u003e \u003cp\u003eTo assess the impact of PRP-Exos on angiogenesis on day 14, IF staining was employed. CD31, a marker specifically expressed on endothelial cell surfaces, and alpha-smooth muscle actin (α-SMA), a protein localized in vascular smooth muscle cells, are both well-established indicators of vascular networks. In the IF analysis, the number of CD31-positive and α-SMA-positive vessels within each wound was quantified to evaluate the formation of new blood vessels. The quantitative assessment of neovascularization demonstrated that the PRP-Exo group exhibited significantly higher angiogenic activity relative to the PRP-gel group, highlighting its superior efficacy in vascular regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the regulatory mechanism underlying PRP-Exo action in healing ulcer wounds in diabetic mice, we conducted IF analysis on ulcer tissues collected on day 11 of the experiment. Our results revealed a marked increase in the number of F4/80 (a marker for macrophages) and Arg-1 (a marker for M2 macrophages) co-positive cells in the PRP-Exo group compared to the PRP-gel group. These findings indicate that PRP-Exos effectively promotes the polarization of macrophages toward the anti-inflammatory M2 phenotype, thereby significantly enhancing the healing process of ulcer wounds in diabetic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo further explicate the regulatory mechanisms underlying PRP-Exo action on macrophage polarization, we initiated our investigation by isolating BMDMs and establishing a comprehensive \u003cem\u003ein vitro\u003c/em\u003e culture system (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Through systematic experimental analyses, we ascertained that PRP-Exos modulated macrophage polarization by suppressing the transition of M0 macrophages into the pro-inflammatory M1 phenotype while promoting their differentiation into the anti-inflammatory M2 phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDFUs represent a critical complication that poses a significant threat to the lower limbs of individuals with diabetes, potentially causing systemic harm and even resulting in death[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The management of DFUs is particularly challenging, as these injuries tend to heal slowly or inadequately in diabetic patients. Due to its marked prevalence\u0026mdash;affecting approximately 15\u0026ndash;25% of diabetic patients during their lifetime\u0026mdash;and associated high healthcare expenditures, DFUs have emerged as a pressing global health concern[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, it is essential to conduct thorough research into the pathophysiological processes involved in diabetic wound healing and to investigate potential interventions so as to expedite the healing process.\u003c/p\u003e \u003cp\u003ePRP has been previously demonstrated to enhance the healing and regeneration of diabetic skin wounds[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and we herein also demonstrated that PRP-gel effectively promoted wound healing in patients with DFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We observed similar results in the diabetic mouse model, as the application of PRP-gel significantly enhanced wound healing in diabetic mice. Furthermore, PRP-gel reduced inflammation within the wound tissue and promoted collagen accumulation, which in turn accelerated the healing process (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNeutrophils are key cells in the early phase of an inflammatory response, and are capable of clearing infected and damaged tissues by releasing inflammatory mediators and reactive oxygen species. However, excessive neutrophil activity can lead to tissue damage[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In the present study, we provided evidence that PRP-gel can regulate the local inflammatory microenvironment by reducing neutrophil infiltration that is crucial for tissue repair and regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEmerging evidence highlights the crucial role of macrophages in the inflammatory response stage of wound repair[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], with their dynamic plasticity mediating their involvement in both tissue damage and repair processes[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The etiology of DFUs is multifactorial, involving a combination of vascular, neurological, and immunological dysfunctions. Recent studies in macrophage biology suggest that impaired transformation of M1 to M2 macrophages constitutes a critical factor that contributes to poor wound healing and the development of chronic open ulcers in DFUs[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our experimental data indicated that PRP-gel exhibited a significant dual-regulatory effect in the wound healing process. In the early stages, PRP-gel reduced inflammation by inhibiting M1-type macrophages, while in the later stages it promoted macrophage polarization toward the M2 phenotype\u0026mdash;thereby accelerating tissue repair and creating a favorable microenvironment for regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePRP-Exos have been shown by other investigators to exhibit significantly enhanced efficacy in promoting diabetic wound healing compared to PRP alone[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our study, we successfully isolated and characterized PRP-Exos, and demonstrated their potential as an accessible bioactive substance that can promote DFU healing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Blood vessels are characterized as essential for tissue regeneration, primarily due to their roles in supplying nutrients and oxygen to cells surrounding wounds[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We thus assessed the levels of α-SMA and CD31 to evaluate newly formed vessels within the regenerated tissue, and observed that PRP-Exo-treated diabetic wounds exhibited much higher expression of α-SMA and CD31 than the PRP-gel groups. We thus hypothesized that PRP-Exos exerted their functions in DFU progression by regulating macrophage polarization. To verify this, we conducted both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies, and we found that PRP-Exos inhibited macrophage polarization toward the M1 phenotype, thereby promoting a shift to the M2 phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These data indicated that PRP-Exos successfully promoted angiogenesis and blood vessel formation in the diabetic wounds, accelerating the healing process. Our findings signify that the precise regulation of macrophage polarization toward the M2 phenotype\u0026mdash;combined with PRP-Exo application\u0026mdash;can significantly enhance DFU wound healing.\u003c/p\u003e \u003cp\u003eDespite the promising therapeutic effects of PRP-gel and PRP-Exos on wound healing in DFUs that we demonstrated herein, there were limitations to the present study, including limited sample size and lack of diversity in the cohort that would have enhanced the generalizability of our findings. In addition, although we explored the immune-inflammatory responses and macrophage polarization facilitated by PRP-gel and PRP-Exos, the precise underlying molecular mechanisms involved remain incompletely understood. We recommend that investigators in the future explore the synergistic potential of PRP-gel and PRP-Exos when combined with other therapeutic modalities such as stem cell therapy or advanced wound dressings, which we expect would improve healing outcomes in DFUs.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we provided evidence that PRP-gel and PRP-Exos enhanced the healing of DFUs by modulating macrophage polarization and promoting the transition from the pro-inflammatory M1 phenotype to the anti-inflammatory and pro-healing M2 phenotype. These findings provided robust experimental evidence for the clinical application of PRP-gel and PRP-Exos in the treatment of DFUs. Additionally, novel insights were offered into the development of biological therapies that target DFUs.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDFUs，Diabetic foot ulcers；PRP，Platelet-rich plasma；\u0026nbsp;PRP-Exos，plasma-derived exosomes; RCTs, Randomized Controlled Trials; SPF, specific-pathogen-free; STZ, streptozotocin; BMDM, Bone Marrow-Derived Macrophages; DMEM, Dulbecco’s Modified Eagle’s medium; FBS, fetal bovine serum; LPS, Lipopolysaccharide; IFN-γ, Interferon-γ; IL-4, Interleukin-4; SD, standard deviation; DM, diabetic mice; DM-NPRP,\u0026nbsp;\u003cstrong\u003eDM-NPRP: Non-PRP treatment of ulcers in diabetic mice\u003c/strong\u003e; DM-PRP-gel,\u0026nbsp;\u003cstrong\u003eDM-PRP-gel: PRP-gel treatment of ulcers in diabetic mice\u003c/strong\u003e; IF, immunofluorescence; WB, Western blotting;\u0026nbsp;\u003cstrong\u003eNeu\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eneutrophils\u003c/strong\u003e\u003cstrong\u003e;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMΦ\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cstrong\u003emacrophages\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from Key projects of Natural Science in Colleges and Universities of Anhui Province [grant numbers: 2023AH051754]. The funders had no role in study design; in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to submit the article for publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was sanctioned by Ethics Committee of Wannan Medical College (202375). The protocols used in this study were approved by the First Affiliated Hospital of Wannan Medical College, and written informed consent was provided from all the patients. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory and approved by the Animal Research Ethics Committee of Wannan Medical College (WNMC-AWE-2023102).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll subjects have written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data relevant to the study are included in the article. Primary data are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that we have no conflicts of interest relevant to the content of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLing He:wrote the main manuscript text; Nan Zhao: acquisition of data;Xiaoling Chen and Wenjie Zhang: ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Kun LV:made substantial contributions to the conception or design of the work；Yuanhong Xu: approved the version to be published.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArmstrong DG, Tan TW, Boulton AJM, Bus SA. Diabetic Foot Ulcers: A Review. JAMA. 2023;330(1):62\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDixon D, Edmonds M. Managing Diabetic Foot Ulcers: Pharmacotherapy for Wound Healing. Drugs. 2021;81(1):29\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao W, Jin W, Li Y, et al. A highly bioactive bone extracellular matrix-biomimetic nanofibrous system with rapid angiogenesis promotes diabetic wound healing. J Mater Chem B. 2017;5(35):7285\u0026ndash;7296.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian Z, Wang H, Bai Y, et al. Improving Chronic Diabetic Wound Healing through an Injectable and Self-Healing Hydrogel with Platelet-Rich Plasma Release. ACS Appl Mater Interfaces. 2020;12(50):55659\u0026ndash;55674.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan G, Ceilley R. Chronic Wound Healing: A Review of Current Management and Treatments. Adv Ther. 2017;34(3):599\u0026ndash;610.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao J, Zhu Y, Huddleston S, et al. Copper Metal-Organic Framework Nanoparticles Stabilized with Folic Acid Improve Wound Healing in Diabetes. ACS Nano. 2018;12(2):1023\u0026ndash;1032.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarti-Carvajal AJ, Gluud C, Nicola S, et al. Growth factors for treating diabetic foot ulcers. Cochrane Database Syst Rev. 2015;2015(10):CD008548.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNie X, Liu Y, Yuan T, et al. Platelet-rich plasma-derived exosomes promote blood-spinal cord barrier repair and attenuate neuroinflammation after spinal cord injury. J Nanobiotechnology. 2024;22(1):456.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang G, Li S, Yu K, et al. A 3D-printed PRP-GelMA hydrogel promotes osteochondral regeneration through M2 macrophage polarization in a rabbit model. Acta Biomater. 2021;128:150\u0026ndash;162.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEverts P, Onishi K, Jayaram P, Lana JF, Mautner K. Platelet-Rich Plasma: New Performance Understandings and Therapeutic Considerations in 2020. Int J Mol Sci. 2020;21(20).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQu W, Wang Z, Hunt C, et al. The Effectiveness and Safety of Platelet-Rich Plasma for Chronic Wounds: A Systematic Review and Meta-analysis. Mayo Clin Proc. 2021;96(9):2407\u0026ndash;2417.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinez-Zapata MJ, Marti-Carvajal AJ, Sola I, et al. Autologous platelet-rich plasma for treating chronic wounds. Cochrane Database Syst Rev. 2016;2016(5):CD006899.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirase T, Ruff E, Surani S, Ratnani I. Topical application of platelet-rich plasma for diabetic foot ulcers: A systematic review. World J Diabetes. 2018;9(10):172\u0026ndash;179.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDel Pino-Sedeno T, Trujillo-Martin MM, Andia I, et al. Platelet-rich plasma for the treatment of diabetic foot ulcers: A meta-analysis. Wound Repair Regen. 2019;27(2):170\u0026ndash;182.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLouiselle AE, Niemiec SM, Zgheib C, Liechty KW. Macrophage polarization and diabetic wound healing. Transl Res. 2021;236:109\u0026ndash;116.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWillenborg S, Sanin DE, Jais A, et al. Mitochondrial metabolism coordinates stage-specific repair processes in macrophages during wound healing. Cell Metab. 2021;33(12):2398\u0026ndash;2414 e2399.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoniakowski AE, Kimball AS, Jacobs BN, Kunkel SL, Gallagher KA. Macrophage-Mediated Inflammation in Normal and Diabetic Wound Healing. J Immunol. 2017;199(1):17\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao W, Meng X, Cao F, Wang J, Yang M. Exosomes derived from platelet-rich plasma promote diabetic wound healing via the JAK2/STAT3 pathway. iScience. 2023;26(11):108236.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRui S, Dai L, Zhang X, et al. Exosomal miRNA-26b-5p from PRP suppresses NETs by targeting MMP-8 to promote diabetic wound healing. J Control Release. 2024;372:221\u0026ndash;233.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe M, Chen T, Lv Y, et al. The role of allogeneic platelet-rich plasma in patients with diabetic foot ulcer: Current perspectives and future challenges. Front Bioeng Biotechnol. 2022;10:993436.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNolan GS, Smith OJ, Heavey S, Jell G, Mosahebi A. Histological analysis of fat grafting with platelet-rich plasma for diabetic foot ulcers-A randomised controlled trial. Int Wound J. 2022;19(2):389\u0026ndash;398.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHesseler MJ, Shyam N. Platelet-rich plasma and its utility in medical dermatology: A systematic review. J Am Acad Dermatol. 2019;81(3):834\u0026ndash;846.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakagi N, Kawakami K, Kanno E, et al. IL-17A promotes neutrophilic inflammation and disturbs acute wound healing in skin. Exp Dermatol. 2017;26(2):137\u0026ndash;144.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhillipson M, Kubes P. The Healing Power of Neutrophils. Trends Immunol. 2019;40(7):635\u0026ndash;647.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeng K, Ma X, Jiang Z, et al. WDR74 facilitates TGF-beta/Smad pathway activation to promote M2 macrophage polarization and diabetic foot ulcer wound healing in mice. Cell Biol Toxicol. 2023;39(4):1577\u0026ndash;1591.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen C, Wang Q, Li D, Qi Z, Chen Y, Wang S. MALAT1 participates in the role of platelet-rich plasma exosomes in promoting wound healing of diabetic foot ulcer. Int J Biol Macromol. 2023;238:124170.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C, Wang M, Xu T, et al. Engineering Bioactive Self-Healing Antibacterial Exosomes Hydrogel for Promoting Chronic Diabetic Wound Healing and Complete Skin Regeneration. Theranostics. 2019;9(1):65\u0026ndash;76.\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Diabetic foot ulcers, Platelet-rich plasma, Platelet-rich plasma-derived exosomes, Macrophage polarization, M2","lastPublishedDoi":"10.21203/rs.3.rs-5945926/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5945926/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiabetic foot ulcers (DFUs) impose a significant clinical and socioeconomic burden on patients and healthcare systems. Although platelet-rich plasma (PRP) and platelet-rich plasma-derived exosomes (PRP-Exos) have emerged as promising therapeutic agents in tissue regeneration, the mechanisms underlying the immunomodulatory effects of PRP and PRP-Exos\u0026mdash;particularly their role in macrophage polarization\u0026mdash;remain poorly understood. In this study, we isolated and characterized PRP-Exos and systematically evaluated their therapeutic potential in diabetic wound healing via comprehensive \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments. Our results revealed that both PRP-gel and PRP-Exos significantly enhanced diabetic wound healing by promoting macrophage polarization toward the anti-inflammatory M2 phenotype. These findings suggest that PRP-Exos represent a novel and effective therapeutic strategy for DFUs, providing a robust rationale for future clinical translation.\u003c/p\u003e","manuscriptTitle":"Platelet-rich plasma-derived exosomes accelerate the healing of diabetic foot ulcers by promoting macrophage polarization to the M2 phenotype","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 04:19:35","doi":"10.21203/rs.3.rs-5945926/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-03-25T17:42:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-24T19:15:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124687580277668544717978562188259282521","date":"2025-03-20T13:48:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-20T09:38:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-20T01:40:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical and Experimental Medicine","date":"2025-03-19T07:07:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6402bae5-3ede-421a-a883-9b75317bf5b5","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T16:00:22+00:00","versionOfRecord":{"articleIdentity":"rs-5945926","link":"https://doi.org/10.1007/s10238-025-01651-w","journal":{"identity":"clinical-and-experimental-medicine","isVorOnly":false,"title":"Clinical and Experimental Medicine"},"publishedOn":"2025-05-15 15:57:26","publishedOnDateReadable":"May 15th, 2025"},"versionCreatedAt":"2025-03-27 04:19:35","video":"","vorDoi":"10.1007/s10238-025-01651-w","vorDoiUrl":"https://doi.org/10.1007/s10238-025-01651-w","workflowStages":[]},"version":"v1","identity":"rs-5945926","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5945926","identity":"rs-5945926","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}