Bibliometric analysis of extracellular vesicles in osteoarthritis

Bibliometric analysis of extracellular vesicles in osteoarthritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bibliometric analysis of extracellular vesicles in osteoarthritis Ga Liao, Hongyu XIe, Lunwei Kang, Lin Zhao, Weikun Meng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5627430/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Osteoarthritis (OA) is a common degenerative joint disease with complex risk factors and an unclear pathogenesis. The onset is insidious, with mild early symptoms, and the disease progression is irreversible. Current treatment options cannot completely cure the disease. Therefore, the development of novel therapeutic approaches and early prevention strategies is crucial for alleviating the disease burden and enhancing the quality of life for patients with OA. Extracellular vesicles (EVs) naturally possess biocompatibility and low immunogenicity, showing significant potential in drug delivery and cell-free therapy. To comprehensively understand the research status and application prospects of EVs in OA, this review employs bibliometric methods to explore the development and collaboration patterns within this research field, current significant research statuses, and emerging directions. Additionally, it thoroughly reviews the mechanisms of EVs in OA, and the integration of EVs with engineering technologies and biomaterials to enhance their efficacy in OA treatment. The goal is to provide scientific evidence and references for the development of OA therapies and the clinical application of EVs. Health sciences/Pathogenesis Biological sciences/Physiology/Bone Osteoarthritis Extracellular vesicles Cell-free therapy Microbiota Cartilage repair Immunomodulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Osteoarthritis (OA) is a globally prevalent chronic degenerative joint disease characterized primarily by the wear and degradation of articular cartilage, accompanied by osteophyte formation, periarticular soft tissue inflammation, and chronic inflammatory responses 1 . Symptoms include joint pain and stiffness, which can lead to functional loss and even disability in severe cases, imposing a significant socioeconomic burden (Fig. 1 A). According to the Global Burden of Disease report published in The Lancet Rheumatology 2 , In 1990, about 256 million people (95% UI 232–282) globally had OA. By 2020, this number had risen to 595 million people (95% UI 535–656 million). Over the span of ten years, the prevalence of OA across all age groups has steadily increased. It is projected that by 2050, nearly 1 billion people will be affected by OA 3 . To date, the exact etiology of OA remains not fully understood. Major risk factors include age, gender, obesity, joint injuries, genetic predisposition, and occupational factors 4 . The incidence of this disease is higher among the elderly population, but there is a noticeable trend towards younger individuals being affected 5 . Compared to men, postmenopausal women have significantly lower estrogen levels and bone density, making them more susceptible to joint diseases (Fig. 1 B) 6 , 7 . As a multifactorial degenerative disease, OA is not a single entity but rather a collection of distinct clinical phenotypes driven by different and/or overlapping molecular subtypes 8 . Consequently, patients exhibit diverse combinations of pain, symptoms, and functional impairments, contributing to considerable heterogeneity in clinical features and responses to existing treatments. This variability not only complicates the comprehensive understanding of OA pathogenesis but also hinders the development of standardized therapeutic strategies. Current treatment approaches primarily focus on symptom management through medications, physical therapy, and injection therapies. However, these methods do not cure the disease. Pharmaceutical interventions, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroid injections, can provide short-term relief of pain and inflammation but are associated with significant long-term side effects 9 , 10 . For advanced-stage OA, joint replacement surgery is often the primary treatment option. Nevertheless, postoperative complications, such as infections, prosthesis loosening, and wear, may necessitate revision surgeries. Additionally, some patients experience suboptimal recovery, with persistent pain and limited mobility remaining a challenge after surgery 11 (Fig. 1 C). With a deeper understanding of the molecular mechanisms underlying OA, various emerging therapies are being actively explored to reverse or halt disease progression at the molecular and cellular levels. Against this backdrop, extracellular vesicles (EVs) have garnered significant attention as a critical mediator of intercellular communication and a promising therapeutic tool. Due to their unique biocompatibility and low immunogenicity, EVs are considered to hold great potential for OA treatment 12 , 13 . EVs are nanometer-scale particles encapsulated by a lipid bilayer, incapable of self-replication, and secreted by cells into the extracellular environment. They participate in various biological processes, including cell communication, immune regulation, and modulation of pathophysiological mechanisms. Based on their size and biogenesis, EVs can be categorized into three main types: exosomes (30–150 nm, formed within multivesicular bodies and released via exocytosis), microvesicles (100–1000 nm, generated through direct budding of the plasma membrane), and apoptotic bodies (500–2000 nm, formed during the fragmentation of apoptotic cells) 14 (Fig. 1 D-G). Nearly all cell types can secrete EVs, including mesenchymal stem cells, dendritic cells, and T cells. Extensive research has demonstrated that EVs carry bioactive molecules capable of promoting tissue repair and regeneration, inhibiting cartilage degradation, and regulating inflammatory responses in damaged joints. Furthermore, EVs serve as effective drug delivery vehicles, transporting therapeutic molecules to target sites 15 . However, the high biological heterogeneity of EVs, as well as the lack of standardized techniques for their isolation, purification, and quality control, remains a significant barrier to their clinical translation. To address these challenges, the international research community has been working to standardize EV-related studies. In 2011, scholars from multiple European countries established the International Society for Extracellular Vesicles (ISEV), which has since published three editions of the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. The latest edition, MISEV2023, further refines the standards for EV nomenclature, sample preparation, isolation, characterization, and functional analysis, providing essential guidance for experimental design and reporting 16 . Transparent and standardized workflows are expected to enhance the validity, accuracy, and reproducibility of EV research, laying a solid foundation for their clinical application. To advance understanding of the mechanisms underlying EV-mediated effects in OA treatment, explore their clinical potential, and identify gaps and unresolved issues in current research, this study employs bibliometric methods to review and evaluate the status of EV-related research in OA from January 1, 1998, to June 1, 2024. By summarizing the research landscape, identifying key areas of focus, and outlining future directions, this work aims to provide scientific evidence supporting the clinical translation of EVs in OA therapy. Results Development Trends and Research Field Distribution The annual publication volume of research on EVs related to OA can reflect the development trends in the mechanisms and applications of EVs in OA. From the line graph, it is evident that the annual publication volume has shown a significant upward trend. In 2017, the annual growth rate exceeded double digits for the first time, and in 2020, the field entered a period of rapid development, marked by a notable increase in annual growth (Fig. 2 A). This rapid development period may be attributed to the introduction of new technologies and methods, such as ultracentrifugation, size exclusion chromatography, and microfluidics-based techniques, which have made the isolation, purification, and characterization of EVs more accurate and efficient 17 – 19 . The potential of EVs in disease diagnosis, prognosis assessment, regenerative medicine, and cell therapy has gradually been recognized 20 . Numerous studies have identified microRNAs and proteins within EVs as important biomarkers, attracting widespread attention. Research areas on EVs in OA is multidisciplinary, encompassing basic research, applied research, and clinical studies. The primary research areas are distributed across Cell Biology (163), Research Experimental Medicine (81), Engineering (73), Biochemistry Molecular Biology (72), and Pharmacology Pharmacy (60) (Fig. 2 B). Since the mechanisms of EVs are not yet fully understood, current research focuses on understanding the molecular components, secretion mechanisms, and roles of extracellular vesicles in cell communication, as well as their potential applications in disease models. Additionally, efforts are being made to develop new biomaterials and scaffolds to enhance the applications of EVs in tissue engineering and regenerative medicine. Collaboration Among Institutions and Countries A total of 52 countries have contributed to the research on extracellular vesicles in osteoarthritis (Fig. 2 C). Among the top 10 countries by publication volume are three Asian countries, five European countries, one North American country, and one Oceanian country. China stands out with a significantly higher number of publications, reflecting the country’s strong focus on this field. This emphasis is likely driven by China’s large population and high number of elderly individuals, resulting in a higher prevalence of osteoarthritis compared to other countries. Consequently, China is investing heavily in research to mitigate the social and economic burdens associated with this condition. In terms of institutional contributions, 235 research institutions have been involved in studies related to extracellular vesicles in osteoarthritis 21 . Among the top six institutions by publication volume, four are based in China, one in Italy, and one in Singapore. The National University of Singapore, which began publishing related research as early as 2014, holds the highest total publication volume from 1998 to June 2024. Sichuan University has rapidly advanced, ranking second in publication volume in 2023 and becoming the leading institution in 2024 as of June 1 (Fig. 2 D). The collaboration network among these institutions reveals extensive cooperation, underscoring the widespread attention that research on extracellular vesicles in osteoarthritis has garnered across various research entities. Keyword Clustering to Interpret Research Frontiers Keyword co-occurrence refers to the phenomenon where two or more keywords appear simultaneously in the same document or group of documents 22 . By counting the frequency of these co-occurrences, one can understand the degree of association between the keywords. As shown in Fig. 3 A, the core keywords include “exosomes,” “mesenchymal stem cells,” and “osteoarthritis.” These keywords are more prominent in the figure and have a strong co-occurrence relationship with many other keywords, indicating that they are central themes in this field of research (Fig S1 a). The red cluster mainly involves exosomes, mesenchymal stem cells, osteoarthritis, and their related biological mechanisms and therapeutic potential. In contrast, the blue cluster is associated with stromal cells, articular cartilage, and themes related to their repair and regeneration. Using CiteSpace, a co-occurrence network analysis of keywords identified nine clusters: Gut Microbiota, Immunomodulation, Cartilage Regeneration, Activation, miRNA Sequencing, Knee Osteoarthritis, Mesenchymal Stromal Cells, Knee Osteoarthritis Progression, and Microvesicles (Fig. 3 B). Gut microbiota plays a crucial role in regulating the host’s immune system and inflammatory responses 23 . Dysbiosis of the gut microbiota can impair the gut barrier function, leading to increased intestinal permeability. Bacterial endotoxins, such as lipopolysaccharides (LPS), can enter the bloodstream, activating systemic immune responses and increasing systemic inflammation 24 . This inflammatory response can extend to the joints, promoting the development and exacerbation of osteoarthritis 25 . Recently, the concept of the “Gut-Joint Axis” has been proposed. Uzma Amin et al. 26 demonstrated that the concentrations of gamma-aminobutyric acid (GABA) in the serum and small intestine contents of treated mice increased, inhibiting cartilage catabolism and protecting mouse joints from degeneration. Furthermore, synthetic markers in mouse knee joints and chondrocytes were upregulated, while inflammatory markers decreased. Probiotics such as Streptococcus thermophilus and Lactobacillus pentosus, along with GABA, were shown to counteract osteoarthritis in vivo and mitigate IL-1β-induced changes in chondrocytes in vitro. These findings open new avenues for understanding the gut-joint axis and its therapeutic implications for osteoarthritis. Identification of Core Literature Each research article incorporates numerous references, and reference analysis can identify the frontiers and hotspots of current research, as well as the core literature that holds significant influence and plays a critical role. The co-citation relationship refers to the scenario when two documents (A and B) are simultaneously cited by a third document (C), establishing a co-citation link between A and B (Fig. 3 C, Fig S1 b) 27 . Key nodes in the co-citation network typically represent the core literature and significant authors within the field. Zhang et al. 28 demonstrated that MSC exosomes could induce higher infiltration of CD163-positive regenerative M2 macrophages rather than inflammatory CD86-positive M1 macrophages. They also reduced pro-inflammatory cytokines (IL-1β and TNF-α) in the synovial fluid and induced phosphorylation of AKT and ERK in chondrocytes, enhancing the survival and proliferation of chondrocytes. This reveals the potential of MSC exosomes to achieve cartilage repair and regeneration through the coordinated regulation of multiple cellular processes, including cell migration, proliferation, matrix synthesis, macrophage infiltration, and cytokine production. Tao et al. 29 were the first to use synovial mesenchymal stem cells (SMSC) as a source of exosomes. They transfected SMSCs with or without miR-140-5p, extracted and characterized the exosomes. Their findings indicated that Wnt5a and Wnt5b in the exosomes activated YAP through the alternative Wnt signaling pathway, thereby enhancing chondrocyte proliferation and migration but inhibiting the secretion of SOX9 and ECM components. Overexpression of miR-140-5p could restore SOX9 expression by inhibiting RalA, thus preventing the inhibition of ECM secretion. In a rat model of osteoarthritis, SMSC-140-Exos successfully prevented the onset and progression of osteoarthritis, showing significant protective effects. Compared to the control group, rats treated with SMSC-140-Exos exhibited significantly reduced joint cartilage damage, maintained levels of type II collagen and aggrecan expression, and reduced type I collagen expression. Zhang et al.’s study 30 was the first to demonstrate the effectiveness of human embryonic mesenchymal stem cell (hEMSC)-derived exosomes in repairing critical-sized osteochondral defects in an immunocompetent adult rat model. An osteochondral defect model was created on the femoral trochlear grooves of 12 adult rats. One side of the defect was treated with 100 micrograms of exosomes, while the other side was treated with phosphate-buffered saline (PBS) as a control. Age-matched animals that did not undergo surgery served as the control group. The defects were injected intra-articularly with exosomes or PBS weekly for 12 weeks. Exosome-treated defects showed significant tissue regeneration within 12 weeks, forming hyaline cartilage and subchondral bone with good surface regularity and complete integration with the adjacent cartilage. In contrast, PBS-treated defects were primarily fibrous tissue and failed to effectively repair cartilage and subchondral bone. This study provided a novel, cell-free therapeutic strategy with significant advantages over cell-based therapies. Clustering based on reference titles reveals that the therapeutic potential of EVs has garnered widespread attention, as evidenced by cluster labels such as #1 Potential Role, #7 Therapeutic Effect, #9 MSC Exosome, #11 MSC Therapy, and #13 Cartilage Degradation (Fig. 3 D). Combining these cluster results with keyword clustering indicates that current research hotspots and future directions focus on the potential application of EVs in cartilage repair, particularly those derived from MSCs. Overview of Extracellular Vesicle Isolation and Characterization Techniques The separation of EVs is the primary and critical step in EV research, while characterization serves as an essential means to assess the purity and yield of separation methods and to identify the features of EVs. Among the 296 included research articles, differential ultracentrifugation is the most widely described separation method (N = 177, 59.80%), with commercial kits being the second most common (N = 41, 13.85%) (Fig S1 c). Ultracentrifugation utilizes centrifugal force at high speeds (typically 100,000 g or higher) to separate different components in a sample based on density and size, effectively isolating EVs from complex samples. This method is suitable for various types of EVs and has been validated by numerous studies, demonstrating high credibility and reproducibility. However, it entails high equipment costs, requires multiple centrifugation steps, is time-consuming, and the high centrifugal forces may cause physical damage to EVs, potentially affecting their biological activity. Commercial kits available on the market primarily employ principles such as polymer precipitation, membrane affinity, antibody capture, and filtration for the “separation” of exosomes. These kits optimize operational workflows, reduce cumbersome steps, and decrease time consumption, enabling separation to be completed in a relatively short period. Nonetheless, compared to ultracentrifugation, commercial kits usually yield lower amounts of EVs, are challenging to process large-volume samples, and technical differences among kits from different manufacturers may lead to inconsistent separation outcomes. Additionally, they rely on specific reagent formulations, making it difficult to adjust protocols according to specific needs. The MISEV2023 guidelines recommend prioritizing commercial EV kits with publicly disclosed separation or concentration principles to avoid potential interference from unknown contaminants in experimental results. The characterization methods for EVs are more diverse than the separation methods. Single vesicle analysis is mainly performed using electron microscopy techniques, such as scanning or transmission electron microscopy, or confocal laser scanning microscopy. A total of 227 studies (76.69%) provided detailed descriptions of electron microscopy methods, while 4 studies (1.35%) used confocal laser scanning microscopy. Quantification of particle concentration was conducted using nanoparticle tracking analysis (NTA) (N = 188; 63.51%) or dynamic light scattering (DLS) (N = 34; 11.49%). Although many articles provided quantitative values, few studies reported ratios—such as particle number to protein and/or lipid content—to estimate product purity. In evaluating protein marker expression, 207 studies (69.93%) quantified total protein content using Western blotting (WB), 46 studies (15.54%) employed flow cytometry, and a few studies utilized other protein analysis methods like ELISA kits (N = 7; 2.36%) (Fig S1 d). The most commonly used specific markers are surface proteins of the tetraspanin family, including CD63 (N = 196; 66.22%), CD9 (N = 161; 54.39%), and CD81 (N = 145; 48.99%). Additionally, 98 studies (33.11%) and 39 studies (13.18%) chose to use TSG101 and Alix, which are protein components of the endosomal sorting complexes required for transport (ESCRT). Through a systematic analysis of the EV characterization and separation purification methods in the included studies, we gained an in-depth understanding of the characteristics and shortcomings of current research practices. These methodological choices not only affect the reliability of experimental results but also reflect researchers’ focal points and technical proficiency, highlighting both the diversity and commonality of technological applications in this field. To more comprehensively grasp the current state of development and future trends in EV research, exploring research hotspots and frontier directions is highly necessary. Multifaceted Applications of Extracellular Vesicles in Osteoarthritis Treatment EVs are nano-sized vesicles secreted by cells that contain proteins, mRNA, miRNA, and other biomolecules. These vesicles play a crucial role in intercellular communication, contributing to the regulation of inflammatory responses and promoting tissue repair and regeneration. In the treatment of osteoarthritis (OA), EVs have been shown to reduce inflammation, facilitate cartilage repair, and improve joint health through multiple molecular mechanisms. Based on an analysis of 296 studies on the therapeutic effects of EVs in OA, the results were categorized into five outcome groups: “significant improvement,” “partial improvement,” “no effect,” “unclear results,” and “reported adverse effects.” Of these studies, 248 (83.78%) reported significant improvements in OA treatment outcomes, and 32 studies (10.81%) reported partial improvement (i.e., at least one outcome was improved, but not all). Additionally, 2 study (0.68%) observed no therapeutic effect, 2 studies (0.68%) yielded unclear results, and 12 studies (4.05%) documented adverse effects. In a representative experimental study, Liu et al. 31 demonstrated that EVs derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) suppressed IL-1β-induced apoptosis in chondrocytes and reversed the upregulation of matrix metalloproteinase-13 (MMP13), thereby enhancing chondrocyte viability. Furthermore, bioengineered hUC-EVs were found to significantly promote chondrocyte proliferation and increase the expression of aggrecan and type II collagen, accelerating cartilage repair (Fig S1 e). Most studies focused on the regulation of inflammatory pathways (n = 152, 51.35%)), followed by apoptosis (n = 87, 29.39%)) and cell proliferation (n = 79, 26.69%) (Fig S1 f). These findings highlight the importance of understanding the underlying mechanisms through which EVs exert therapeutic effects in OA, especially their capacity to modulate the interactions between chondrocytes and the immune microenvironment. Such insights are crucial for elucidating how EVs slow disease progression and promote tissue repair in OA. Extracellular Vesicles in Regulating Inflammation and Promoting Cartilage Protection in OA The persistent high expression and activation of inflammatory factors in OA are core mechanisms driving its progression. Pro-inflammatory cytokines such as IL-1β and TNF-α induce the expression of matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS), accelerating cartilage matrix degradation 32 . Reactive oxygen species (ROS) generated during inflammation cause oxidative damage to chondrocytes, further exacerbating cartilage degeneration 33 . EVs carrying anti-inflammatory molecules target and regulate key modulators of the NF-κB signaling pathway, thereby reducing NF-κB activity and the expression of pro-inflammatory cytokines (Fig. 4 A) 34 . Marta Varela-Eirín et al. 35 demonstrated that extracellular vesicles from Cx43-overexpressing T/C-28a2 chondrocytes (sEVs-T/C-Cx43) promoted NF-κB nuclear translocation, significantly increasing the expression of pro-inflammatory genes in osteocytes and synoviocytes, leading to a pro-inflammatory phenotype and degenerative state. Therefore, downregulating Cx43 in human osteoarthritic chondrocytes can restore chondrocyte redifferentiation, reduce the accumulation of senescent cells, and inhibit NF-κB nuclear translocation. Using specific peptides targeting the C-terminal domain of Cx43 may help reduce inflammation and cellular senescence, promoting tissue regeneration. The polarization state of macrophages can significantly influence the nature and duration of inflammation 36 . Macrophages are primarily categorized into two phenotypes: M1 (pro-inflammatory) and M2 (anti-inflammatory and tissue repair). miRNAs in EVs, such as miR-223, and anti-inflammatory cytokines like TGF-β and IL-10, modulate multiple targets and receptor-related signaling pathways, such as the Smad pathway. These molecules downregulate the expression of M1-associated genes, including TNF-α and IL-1β, inhibiting the production of pro-inflammatory cytokines, reducing the number and activity of M1 macrophages, and inducing M2 macrophage polarization. This promotes the expression of anti-inflammatory genes and maintains the M2 phenotype. Qian et al. 37 found that exosomes from M2 macrophages contain miR-26b-5p, which can repolarize M1 macrophages into the anti-inflammatory M2 phenotype by targeting the TLR3 signaling pathway. In vivo experiments showed that injecting miR-26b-5p agomir improved gait abnormalities and mechanical allodynia in OA mice, alleviated synovitis and cartilage degeneration, and slowed OA progression. This provides a potential therapeutic approach for OA. EVs-Mediated Modulation of Inflammatory Bone Loss and Osteogenesis Inflammatory factors not only affect cartilage but also influence bone metabolism and remodeling. They inhibit osteoblast differentiation and function while promoting osteoclast activity, thereby slowing bone repair and reconstruction, leading to increased bone resorption and loss (Fig. 4 B) 38 . In an inflammatory state, pro-inflammatory cytokines such as IL-1β and TNF-α can upregulate the expression of RANKL. RANKL, by binding to its receptor RANK, activates the differentiation and maturation of osteoclast precursor cells 39 . Activated osteoclasts form resorption lacunae on the bone surface, releasing acids and proteases that degrade the bone matrix, resulting in bone loss. Concurrently, these pro-inflammatory factors inhibit the generation and maturation of osteoblasts by downregulating the expression of osteoblast differentiation markers such as Runx2 and Osterix, and suppress the synthesis and secretion of bone matrix components like type I collagen and osteocalcin, thereby impairing new bone formation 40 . EVs promote osteoblast differentiation, function, and survival through multiple pathways, regulate osteoclast activity, reduce bone resorption, and promote the balance between bone formation and resorption. For instance, miR-196a in EVs can target the HOXC8 gene to promote osteoblast differentiation and bone matrix synthesis. miR-503 inhibits osteoclast activity by targeting upstream regulators of RANKL 41 , while miR-218 promotes osteoblast differentiation and mineralization by regulating the Wnt/β-catenin signaling pathway 42 . Bone morphogenetic proteins (BMPs) carried by EVs promote osteoblast differentiation and function through the Smad signaling pathway 43 . Insulin-like growth factor (IGF-1) in EVs enhances osteoblast proliferation and survival via the PI3K/Akt signaling pathway 44 . Chen et al. 45 demonstrated that the combined application of BMP-2/macrophage-derived exosomes and titanium nanotubes significantly promoted the expression of osteogenesis-related genes (such as ALP, osteopontin, Runx2, BMP-2, and BMP-7) and activated autophagy in human bone marrow stromal cells (hBMSCs), indicating a substantial osteogenic effect. Targeted EV Therapies for Enhancing Cartilage Repair and Chondrocyte Survival Cartilage tissue is avascular, lacking direct blood supply, which means that nutrients and oxygen from the bloodstream cannot reach chondrocytes directly 46 . Instead, these must diffuse through the synovial fluid. Chondrocytes are sparsely distributed within the matrix and exhibit low metabolic activity. These characteristics make cartilage tissue difficult to repair quickly through cell proliferation and matrix synthesis, thus attracting significant attention to exogenous therapeutic interventions. EVs carrying miRNAs, anti-apoptotic molecules, and growth factors can promote chondrocyte proliferation and survival, as well as regulate the balance between synthesis and degradation of the extracellular matrix (ECM) 47 – 49 . Growth factors like TGF-β and IGF-1 promote chondrocyte proliferation and survival through receptor-mediated signaling pathways such as PI3K/Akt and Smad (Fig. 4 C) 50 , 51 . Liu et al. 31 indicate that dual-engineered cartilage-targeted extracellular vesicles (hUC-EVs) derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) significantly improve chondrocyte survival rates and reverse the IL-1β-induced downregulation of anabolic factors (Collagen II and SOX9), while inhibiting the upregulation of matrix metalloproteinase 13 (MMP13), thereby promoting cartilage anabolism. High-throughput miRNA sequencing revealed that miR-223 was significantly upregulated in the hUC-EVs treatment group, directly targeting NLRP3 mRNA and modulating the activation of the NLRP3 inflammasome, thereby exerting protective effects on cartilage. In summary, EVs exhibit significant potential in the treatment of OA by demonstrating anti-inflammatory properties, modulating immune responses, repairing cartilage, and regulating bone remodeling. This provides new approaches and methods for OA treatment. To further enhance the therapeutic effects of EVs, researchers have begun exploring the combination of EVs with engineering technologies and biomaterials. This paves the way for new directions in future research and clinical applications. Advancements in EV-Based Therapies through Engineering and Biomaterial Integration Natural EVs have a broad range of sources, as nearly all cell types can secrete them, and they are distributed throughout the body. Their composition is complex, containing various proteins, RNA, and lipids. Consequently, natural EVs are easily degraded in vivo, preventing them from exerting their effects for extended periods. They lack specific targeting abilities, which may result in inefficient delivery to diseased sites, reduced therapeutic efficacy, or increased side effects 52 . To overcome the limitations of natural EVs, chemical modifications or combinations with biomaterials can be employed to enhance their stability in vivo. Additionally, integrating EVs with different biomaterials can improve their effectiveness and duration of action. Hydrogel-Enhanced EVs Therapies for Osteoarthritis: Innovations in Delivery and Therapeutic Efficacy Hydrogels are a class of soft materials with unique physical and chemical properties 53 . They have a high-water-content, can gradually degrade into harmless small molecules in the body, and exhibit excellent biocompatibility 54 . Their softness and elasticity allow them to withstand certain mechanical stresses without breaking. Additionally, hydrogels typically possess high porosity and a three-dimensional network structure, which facilitates material exchange and cell migration 55 . These characteristics make hydrogels important materials in biomedical engineering and drug delivery fields. Sun et al. 56 utilized a mixture of gelatin, fibrinogen, hyaluronic acid (HA), and glycerol as a matrix to form a hydrogel through cross-linking. They embedded extracellular vesicles (T3-EV) derived from TGFβ3-preconditioned bone marrow mesenchymal stem cells (BMSCs) into the composite hydrogel. In a rat knee cartilage defect model, the T3-EV composite hydrogel treatment group showed better new cartilage formation, proteoglycan deposition, and tissue integrity after 24 weeks, significantly improving compared to the control group. Immunohistochemical staining results showed that in the T3-EV composite hydrogel treatment group, the expression of cartilage markers such as SOX9, ACAN, and COL2A1 was significantly enhanced, while the expression of the cartilage-degrading enzyme MMP13 was significantly reduced. Wan’s team 57 developed a spherical gelatin methacrylate (GelMA) hydrogel using photo-crosslinking technology, designed to encapsulate engineered exosomes modified with the WYRGRL peptide and loaded with the small molecule inhibitor LRRK2-IN-1 (W-Exo-L@GelMA). This encapsulation effectively delayed the release of exosomes and prolonged their retention time in the joint cavity. In vivo experiments showed that significant fluorescent signals of the encapsulated exosomes were detectable even 14 days after injection. Transcriptomic analysis revealed that W-Exo-L@GelMA significantly inhibited the upregulation of IL-1β-induced inflammation-related genes (such as Cxcl1, Cxcl5, Mmp3, and Mmp13) and restored the expression of anabolic genes suppressed by IL-1β. In a DMM-induced mouse OA model, the W-Exo-L@GelMA group demonstrated significant anti-inflammatory and cartilage repair effects, reducing joint cartilage degradation and subchondral bone loss, thereby exhibiting promising therapeutic effects. Due to their unique physicochemical properties, hydrogels exhibit significant advantages in EVs-based therapies for OA. The integration of microfluidic technology further enhances this approach by offering more refined control over EVs production and hydrogel encapsulation, thereby optimizing functionality (Fig. 5 A). Enhancing EV Therapy Precision for Osteoarthritis with Microfluidics Microfluidic technology is an advanced method for manipulating fluids on the micro- or nanoscale, allowing precise control over liquid, gas, and suspended particles. By fabricating microchannel structures in materials such as silicon, glass, or polymers through techniques like etching or 3D printing, microfluidic systems achieve high-precision fluid control within these small channels 58 . The rapid development of this technology in biomedicine has opened new pathways for enhancing the quality and functionality of EVs. For example, Neety et al. 59 used a polydimethylsiloxane (PDMS)-based non-planar microfluidic device to encapsulate mesenchymal stromal cells (MSCs) in sodium alginate hydrogel microbeads. These MSC microbeads were co-cultured with OA cartilage samples from patients, aiming to investigate the effects of MSC-secreted paracrine factors on cartilage regeneration. The results showed significant improvements in proteoglycan distribution and increased sulfated glycosaminoglycan (sGAG) content in the cartilage samples co-cultured with MSCs, compared to controls without MSCs, indicating enhanced chondrocyte proliferation and new sGAG synthesis. In a separate study, Yin et al. 60 employed microfluidic devices utilizing thiol-ene Michael addition to react thiolated hyaluronic acid (SH-HA) with hyperbranched polyethylene glycol diacrylate (HB-PEGDA), generating hydrogel microparticles (HMPs). In a mouse OA model, injection of HMPs@Exos allowed for gradual degradation and sustained exosome release, which inhibited the expression of ADAMTS4, a matrix metalloproteinase, and promoted cartilage matrix repair. Animal studies further demonstrated that encapsulating exosomes with high miR-99b-3p expression in HMPs (HMPs@ExosScAT-99b-3p) effectively slowed OA progression, preserved cartilage integrity, and achieved long-term therapeutic effects locally. Through precise production of EVs and targeted encapsulation in hydrogels, microfluidic technology enhances the stability and efficacy of EV-based therapies for OA, paving new directions for EV applications in regenerative medicine (Fig. 5 B). Advancements in Culture Systems for Enhanced EVs Yield and Bioactivity Optimizing the culture system is crucial for improving the yield, quality, and functionality of EVs 61 , 62 . By designing and optimizing culture conditions scientifically and reasonably, higher quality EVs can be produced to meet the diverse needs of basic research and clinical applications, thereby advancing the development of exosomes in the biomedical field. Consequently, researchers have conducted relevant studies on EV culture. Yan et al. 63 used a rotary cell culture system (RCCS) to culture MSCs in a mechanical environment to enhance the yield and biological function of exosomes. When Rab27a gene expression was knocked down using siRNA to reduce exosome secretion, the yield of exosomes decreased by approximately threefold under static conditions (0 rpm/min); however, under mechanical conditions (36 rpm/min), the yield decreased by only 1.5-fold. This indicates that mechanical stimulation can mitigate the reduction in exosome yield caused by siRNA interference. Additionally, exosomes produced in the mechanical environment significantly inhibited chondrocyte apoptosis. The expression level of LncRNA H19 in the exosomes increased nearly tenfold ( P < 0.01). Treatment with exosomes containing LncRNA H19 (S-Exos) significantly reduced chondrocyte apoptosis rate (11.1 ± 0.5%, P < 0.01), and the protein and mRNA levels of anti-apoptotic genes (Bcl-2 and Bax) consistently showed stronger anti-apoptotic activity. Zhou et al. 64 employed 3D culture techniques to simulate the three-dimensional structure and ECM of cells in vivo. The results showed that under 3D culture conditions, the number of exosomes produced by the cells significantly increased, and the expression of intracellular F-actin decreased. Compared to 2D culture, the composition of proteins and genetic material in exosomes also changed, resulting in stronger effects in promoting tissue regeneration and inhibiting inflammatory responses. The application of novel culture systems significantly enhances EVs yield and bioactivity, establishing a strong foundation for their widespread use in cell therapy and regenerative medicine (Fig. 5 C). Enhancing Tissue Regeneration with 3D-Printed Exosome-Releasing Scaffolds 3D printing technology, particularly desktop stereolithography (SLA), can create highly precise three-dimensional structures 65 . Using computer-aided design (CAD), scaffolds with specific shapes and internal structures can be printed. The 3D structure and internal channel design of the scaffolds, such as radially oriented channels, can significantly enhance cell migration ability, guiding cells to migrate to defect areas, thereby promoting tissue repair and regeneration. Chen et al. 66 designed a bio-scaffold capable of sustained MSC-derived exosome release, utilizing 3D printing technology to fabricate an ECM/GelMA/exosome scaffold with radial channels. Results demonstrated that the defect regions treated with the 3D-printed ECM/GelMA/exosome scaffold regenerated cartilage-like tissue, with significantly improved repair outcomes compared to other groups. Cartilage repair was assessed using the International Cartilage Repair Society (ICRS) macroscopic scoring system, showing significantly higher scores in the 3D-printed scaffold group at both 6 and 12 weeks. Exosomes within the scaffold restored mitochondrial function in degenerated chondrocytes, markedly reducing mitochondrial damage marker MDA levels and displaying antioxidative stress capacity. Additionally, the ECM/GelMA/exosome scaffold significantly promoted M2 macrophage polarization while reducing M1 infiltration, exhibiting strong immunomodulatory potential. Furthermore, the ECM/GelMA scaffold excelled in trabecular bone regeneration, facilitating osteogenic tissue formation in the defect area. The application of 3D-printed scaffolds highlights its substantial potential in precision tissue engineering, not only enhancing exosome delivery but also promoting cell migration and tissue regeneration, offering a novel solution for the repair of degenerated cartilage and bone defects (Fig. 5 D). The integration of engineering technologies with EVs has opened new avenues for applications in regenerative medicine. This interdisciplinary synergy not only enhances the stability, bioactivity, and delivery efficiency of EVs but also addresses challenges related to rapid degradation and low retention rates in vivo. As a result, EVs have demonstrated remarkable potential in cartilage and bone tissue repair. To maximize therapeutic efficacy, EV delivery strategies should be tailored to the specific stages of disease progression. In the early stage, precise targeting is critical; here, microfluidic technology and novel culture techniques can be utilized to deliver specific EV components at low doses and with high specificity, facilitating early intervention. During the mid-stage, sustained anti-inflammatory and reparative support becomes the focus. In this phase, hydrogels with controlled-release properties are particularly advantageous for gradual EV release, reducing the discomfort and compliance issues associated with frequent injections. In the late stage, the combination of EVs with 3D-printed scaffolds offers structural support to damaged tissues, aiding cartilage repair and preserving joint function, thereby slowing disease progression. Recently, research has increasingly explored the integration of multiple technologies. For instance, combining hydrogels with microfluidic systems enables precise EV release rates that can be adjusted according to disease progression, thereby enhancing therapeutic outcomes. Similarly, 3D-printed scaffolds used alongside hydrogels provide a stable delivery platform for EVs and facilitate tissue repair. This multi-technology approach compensates for the limitations of single technologies in terms of targeting specificity or stability, yielding improved therapeutic results. Looking forward, as these engineering technologies and biomaterials continue to advance and mature, EVs are expected to see broader and deeper applications in clinical therapies, tissue engineering, and regenerative medicine, with the potential to bring personalized precision medicine into routine clinical practice. Discussion and Future Outlook Numerous studies have demonstrated that EVs exhibit significant therapeutic potential in the treatment of OA. EVs can regulate immune cell functions and reduce the release of inflammatory factors, thereby significantly alleviating the inflammatory response in OA 67 , 68 . Additionally, they can inhibit apoptosis of chondrocytes and synovial cells through various signaling pathways, thus protecting joint tissues 69 , 70 . The engineering modification and combination with various biomaterials significantly enhance the efficacy and targeting capability of EVs 71 , 72 . Compared to traditional cell therapy, EVs have lower immunogenicity, avoiding immune rejection and the side effects of excessive cell proliferation. EVs are a heterogeneous population containing various bioactive molecules, with differences in size, composition, and function 73 , 74 . The isolation and purification of specific types of EVs while maintaining their activity and functionality present significant challenges. During EV preparation, contaminants such as cell debris, protein aggregates, and other impurities may be introduced. Different production batches of EVs may exhibit significant variability, affecting their therapeutic efficacy and safety. Although numerous international guidelines for the purification, isolation, and characterization of EVs have been issued, efficient and standardized methods for EV preparation and purification remain a bottleneck for clinical application. The difficulties in separating and purifying EVs arise from their inherent biological properties, and there are technical bottlenecks in achieving high-efficiency purification and high-resolution characterization. Existing EV separation and purification techniques are generally suitable for laboratory-scale production but face issues of low efficiency and high cost when scaled up. Ensuring the consistency, purity, and reproducibility of EVs while achieving large-scale production within a controllable cost range is crucial for their clinical application. Although preliminary studies have shown that EVs have good therapeutic effects and low side effects in OA, it is still necessary to verify these treatments’ long-term efficacy and safety in larger-scale and longer-term clinical trials. Currently, the most commonly used administration method is intra-articular injection, which can precisely target the affected area. However, this method requires high technical skill and carries a certain risk of infection, especially with repeated injections. Improper injection techniques may cause mechanical damage to joint structures, including cartilage, ligaments, and synovium 75 . Intravenous injection of EVs allows for systemic distribution through the bloodstream and is convenient to administer. However, due to the rapid degradation of EVs and the lack of specific targeting mechanisms, it is challenging to ensure that a sufficient quantity of EVs reaches the affected joint, resulting in suboptimal therapeutic effects. Therefore, optimizing the administration methods, such as using 3D-printed scaffolds, combining with hydrogels and other biomaterials, and engineering EVs to enhance their biological functions, is crucial to improving the precision and efficacy of treatments. These strategies are essential for realizing the large-scale clinical application of EVs. As a novel biological therapeutic agent, EVs primarily derived from stem cells necessitate the establishment of comprehensive legal and regulatory frameworks. These frameworks are crucial for strictly controlling potential hazards, ensuring the scientific integrity and reliability of clinical research, and protecting patient rights. A unified and clear regulatory framework can also guide research and development, as well as production, thereby promoting the global advancement of EV technologies and accelerating the clinical translation and market entry of products. In summary, future research on EVs will benefit from multidisciplinary collaboration across biology, medicine, and materials science to drive innovative applications in OA treatment. The development of relevant laws and regulations is also crucial. By combining these efforts, we can ensure the clinical efficacy and safety of EVs, paving the way for their extensive use in regenerative medicine and disease therapy, ultimately providing more effective treatment options for OA patients. Materials and Methods Search Strategy The data were sourced from the most commonly used scientific literature repository—Thomson Reuters’ Web of Science Core Collection (WoSCC). This includes the Science Citation Index Expanded (SCI-EXPANDED), the Emerging Sources Citation Index (ESCI), the Conference Proceedings Citation Index-Science (CPCI-S), and the Conference Proceedings Citation Index-Social Science and Humanities (CPCI-SSH). All electronic searches were conducted on June 1, 2024. The search strategy is as follows: (TS=(exosome* OR exosomes OR exosomal OR “extracellular vesicle” OR “extracellular vesicles” OR “extracellular particle” OR “extracellular particles” OR “microvesicle” OR “microvesicles” OR “Shedding Microvesicle” OR “Shedding Microvesicles” OR “Secretory Vesicle” OR “Secretory Vesicles” OR “Cell-Derived Microparticle” OR “Cell-Derived Microparticles” OR “Apoptotic Bodies” OR “Apoptotic Blebs” OR “Apoptotic Vesicles” OR “Cell Fragments” OR “Apoptotic Bodies” OR “ApoBDs”)) AND TS=(Osteoarthritides OR Osteoarthrosis OR Osteoarthroses OR Arthritis, Degenerative OR Arthritides, Degenerative OR Degenerative Arthritides OR Degenerative Arthritis OR Arthrosis OR Arthroses OR Osteoarthrosis Deformans OR osteoarthritis). The search was limited to articles and reviews published between January 1, 1998, and June 1, 2024. An initial retrieval yielded 754 articles. Based on predefined inclusion and exclusion criteria, titles and abstracts were further screened, resulting in the final inclusion of 533 articles for subsequent analysis (Fig S2 a). All data were stored in plain text format and imported into CiteSpace and Microsoft Excel 2016 for subsequent analysis. All data were downloaded from public databases and do not involve any medical ethics issues. All included studies meet the criteria for bibliometric analysis, and can be found in Appendix 1 (Table S1 , Fig S2 b). Data Analysis In this study, bibliometric methods were applied to analyze the historical evolution and development trends of research on extracellular vesicles (EVs) in osteoarthritis (OA). CiteSpace, a bibliometric visualization tool developed in Java by Dr. Chaomei Chen, was utilized to identify key literature and emerging trends through various parameters, including centrality, burstiness, and Sigma values. This approach effectively illustrates the developmental trajectory of the research field. Based on the research objectives, specific analytical functions, such as co-citation analysis and keyword co-occurrence analysis, were selected to detect influential core publications and identify research hotspots and key themes within the field. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Funding This work was supported by the Sichuan Provincial Science and Technology Department under the International Cooperation Project (Grant No. 25GJHZ0145). Conflict of Interests The authors report no declarations of interest. Author Declarations and Contributions: All authors gave their final approval and agree to be accountable for all aspects of the work. Xie Hongyu was responsible for article selection, bibliometric data analysis, data extraction and statistical evaluation, figure creation, and the drafting and revision of the manuscript. Zhao Lin and Kang Lunwei assisted with data extraction, statistical analysis, and data maintenance. Weikun Meng and Liao Ga contributed to the manuscript review. The corresponding author supervised the study, provided guidance throughout the research process, and contributed to the final revision and approval of the manuscript. Acknowledgements Not applicable References Hunter, D. J. & Bierma-Zeinstra, S. Osteoarthritis. The Lancet vol. 393 Preprint at https://doi.org/10.1016/S0140-6736(19)30417-9 (2019). Steinmetz, J. D. et al. Global, regional, and national burden of osteoarthritis, 1990–2020 and projections to 2050: a systematic analysis for the Global Burden of Disease Study 2021. Lancet Rheumatol 5, (2023). Katz, J. N., Arant, K. R. & Loeser, R. F. Diagnosis and Treatment of Hip and Knee Osteoarthritis: A Review. JAMA - Journal of the American Medical Association vol. 325 Preprint at https://doi.org/10.1001/jama.2020.22171 (2021). Allen, K. D., Thoma, L. M. & Golightly, Y. M. 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Cell and Bioscience vol. 12 Preprint at https://doi.org/10.1186/s13578-022-00786-7 (2022). Paganini, C. et al. Scalable Production and Isolation of Extracellular Vesicles: Available Sources and Lessons from Current Industrial Bioprocesses. Biotechnology Journal vol. 14 Preprint at https://doi.org/10.1002/biot.201800528 (2019). Kolasinski, S. L. et al. 2019 American College of Rheumatology/Arthritis Foundation Guideline for the Management of Osteoarthritis of the Hand, Hip, and Knee. Arthritis and Rheumatology 72, (2020). Additional Declarations There is no conflict of interest Supplementary Files FigueS12.0.pdf Figure S1 FigureS2.pdf Figure S2 Cite Share Download PDF Status: Posted Version 1 posted 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-5627430","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":394939008,"identity":"9a1b7cdc-3e3e-4227-8ba2-7beeb9295a71","order_by":0,"name":"Ga Liao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACPmYwZZMAphIKiNDCBtGSlsDABtJiQIwWCHUYooWBKC3szA8ffqk5n8cv35344YEBgzy/2AFCDmMzNpY5drtYso13swTQYYYzZycQ0sJgJi3BdjtxwzHeDSAtCQa3CWph/yYt8e8cSMvmH0Rq4TGT/Nh2AKRlG7G28BQbM/YlJ85sy91mkWAgQdgv/PzHNz788c0usZ/57OabPyps5PmlCWgBAWYeBFuCsHIQYPxBnLpRMApGwSgYqQAAIpk8muNA/FIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4327-0054","institution":"West China Hospital of Stomatology, Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Ga","middleName":"","lastName":"Liao","suffix":""},{"id":394939009,"identity":"8740fd0e-c46d-4df2-bc7b-6b076b4110ed","order_by":1,"name":"Hongyu XIe","email":"","orcid":"","institution":"West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"XIe","suffix":""},{"id":394939010,"identity":"1ca530e6-d998-4c3d-80b1-39801def2a4c","order_by":2,"name":"Lunwei Kang","email":"","orcid":"","institution":"West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Lunwei","middleName":"","lastName":"Kang","suffix":""},{"id":394939011,"identity":"93213d46-a621-420b-ab9f-f8c4d327a58c","order_by":3,"name":"Lin Zhao","email":"","orcid":"","institution":"West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Zhao","suffix":""},{"id":394939012,"identity":"15a37293-e5fd-4ccf-8a16-8e1b961b17d3","order_by":4,"name":"Weikun Meng","email":"","orcid":"","institution":"West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Weikun","middleName":"","lastName":"Meng","suffix":""}],"badges":[],"createdAt":"2024-12-12 01:35:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5627430/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5627430/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72643159,"identity":"47be642e-5afd-465a-a37d-e1b5d263d2d7","added_by":"auto","created_at":"2024-12-30 16:37:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":306599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathophysiology and Extracellular Vesicle Dynamics in Osteoarthritis Progression and Treatment\u003c/strong\u003e \u003cstrong\u003e(Created with BioRender.com). A. Affected Joints. \u003c/strong\u003eOsteoarthritis impacts multiple joints throughout the body. \u003cstrong\u003eB. Disease Progression. \u003c/strong\u003eMechanical stress or infection leads to cartilage damage, releasing alarmins (e.g., 14-3-3ε, heat shock proteins, S100A8/9, HMGB1, IL-33) that bind to PRRs like RAGE and TLR4/2, triggering inflammation, cartilage degradation, and bone remodeling. Pro-inflammatory mediators (e.g., TNF-α, IL-6, MCP1, MMP3) and cell polarization (fibroblasts, macrophages, chondrocytes) further drive progression. \u003cstrong\u003eC. Treatment. \u003c/strong\u003eEarly treatment involves NSAIDs (e.g., ibuprofen, aspirin) and steroids. Advanced stages may require knee replacement surgery. Commonly affected joints include hands, knees, hips, and spine. \u003cstrong\u003eD. Exosome Secretion. \u003c/strong\u003eExosomes (30–100 nm) are formed within early endosomes, maturing into multivesicular bodies (MVBs). Rab27A regulates MVB fusion with the cell membrane, releasing exosomes; unreleased MVBs are degraded in lysosomes. \u003cstrong\u003eE. Microvesicle Secretion. \u003c/strong\u003eMicrovesicles (100–1000 nm) are released by direct budding of the plasma membrane into the extracellular space. \u003cstrong\u003eF. Apoptotic Bodies. \u003c/strong\u003eThese EVs are released during apoptosis, containing cellular components from the dying cell. \u003cstrong\u003eG. Extracellular Vesicles (EVs) Structure. \u003c/strong\u003eEVs carry surface antigens and adhesion molecules, allowing target cell binding. Their membrane contains lipid rafts and tetraspanins, essential for vesicle formation and function, and various cargoes (nucleic acids, proteins, lipids) for intercellular communication.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5627430/v1/ebbb52b6dbb7eb80f6507027.jpg"},{"id":72643162,"identity":"3c99b6ea-2014-45fc-9cbb-26cc3572ecbc","added_by":"auto","created_at":"2024-12-30 16:37:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":307619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of Publication Trends, Research Focus, Methods, and International Collaboration in Extracellular Vesicle Research in Osteoarthritis. A. Annual Publication Growth.\u003c/strong\u003eThe line chart illustrates the annual growth in the number of publications from 1998 to 2024, showing a significant increase in recent years, particularly after 2020. \u003cstrong\u003eB. Distribution of Research Areas.\u003c/strong\u003e The pie chart displays the distribution of research areas within the EVs field, with the majority focused on cell biology, experimental medicine, and engineering, among other fields. \u003cstrong\u003eC. International Collaboration Network. \u003c/strong\u003eThe network map shows international collaborations, where each node represents a country, and the connections indicate partnerships between countries. Larger nodes indicate countries with a more central role in the collaboration network, with China and the USA being the most prominent. \u003cstrong\u003eD. Publication Trends of the Top 6 Institutions.\u003c/strong\u003e The line chart tracks the publication output over time for the top six institutions contributing to EV research, showing increasing productivity in recent years.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5627430/v1/acb6f281ddeab37d3f67ca47.jpg"},{"id":72643166,"identity":"5e8f13c7-00f1-4fea-8eff-fc603dd245fb","added_by":"auto","created_at":"2024-12-30 16:37:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":787245,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of Keywords and References in Osteoarthritis and Extracellular Vesicle Research. A. Keyword Co-occurrence Network.\u003c/strong\u003e The co-occurrence network of keywords displays the relationships between frequently appearing terms in osteoarthritis and extracellular vesicle research. Nodes represent keywords, and connections represent their co-occurrence in publications. Larger nodes indicate more frequently used keywords, such as “exosomes,” “mesenchymal stem cells,” and “osteoarthritis,” which are central in the research network. \u003cstrong\u003eB. Keyword Clustering.\u003c/strong\u003e The clustering analysis of keywords reveals distinct thematic groups in the field. Each cluster is color-coded and labeled according to its dominant research topic, including themes such as “microvesicles,” “knee osteoarthritis progression,” “cartilage regeneration,” and “gut microbiota.” \u003cstrong\u003eC. Reference Co-citation Network.\u003c/strong\u003e This co-citation network shows connections between frequently cited references, illustrating influential papers and their relationships within the research field. Larger nodes indicate references with higher citation frequencies, with key publications such as Zhang S (2016), Zhu Y (2017), and Wu JY (2019) prominently positioned within the network. \u003cstrong\u003eD. Reference Clustering.\u003c/strong\u003e The clustering of co-cited references highlights thematic groups within the cited literature, with each cluster labeled based on its primary focus. Clusters include areas such as “therapeutic application,” “mesenchymal stem cell therapy,” “cartilage degradation,” and “degenerative disease,” representing major research directions in the field.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5627430/v1/3b70947318badd14561c9cd8.jpg"},{"id":72645751,"identity":"b8a98dcc-fcf5-4a6e-8151-88e98640d32e","added_by":"auto","created_at":"2024-12-30 16:45:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":504462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanisms of Cartilage Damage, Bone Resorption, and Repair Processes in Osteoarthritis and the Role of EVs\u003c/strong\u003e \u003cstrong\u003e(Created with BioRender.com). A. Cartilage Damage and Inflammation Initiated by Mechanical Stress or Infection\u003c/strong\u003e. Mechanical stress or infection can lead to cartilage damage and inflammation, triggering the release of pro-inflammatory factors such as IL-1β and TNF-α. These factors induce the expression of matrix metalloproteinases (MMPs) and ADAMTS, accelerating the degradation of the cartilage matrix and causing oxidative damage to chondrocytes through reactive oxygen species (ROS). Extracellular vesicles (EVs) carrying anti-inflammatory factors like miR-21 and miR-146a can target TRAF6 and IRAK1, modulating the NF-κB signaling pathway to reduce pro-inflammatory cytokine expression. Additionally, they regulate macrophage polarization, downregulate NF-κB signaling, and decrease CXCL3 expression, thus preventing cartilage degeneration and suppressing inflammation. \u003cstrong\u003eB. Osteoclast Activation and Bone Resorption\u003c/strong\u003e. Inflammatory factors upregulate the expression of RANKL, which binds to RANK to activate osteoclasts and promotes the differentiation and maturation of osteoclast precursors. Activated osteoclasts form resorption pits on bone surfaces, releasing acids and proteolytic enzymes to break down the bone matrix, leading to bone loss. EVs carrying miR-196a, miR-503, and miR-218 promote osteoblast differentiation and function through various signaling pathways while inhibiting osteoclast activity. These miRNAs, delivered via EVs, enhance osteoblast differentiation and function, suppress osteoclast activity, and reduce bone resorption, thereby promoting a balance between bone formation and resorption. \u003cstrong\u003eC. Cartilage Repair and Extracellular Matrix Stabilization\u003c/strong\u003e. Cartilage tissue is avascular and has low metabolic activity, limiting its capacity for rapid repair. In osteoarthritis, joint inflammation leads to synovitis, cartilage degradation, and joint space narrowing. Anti-apoptotic molecules, such as miR-21 and Bcl-2 proteins, reduce chondrocyte apoptosis by inhibiting key factors in the apoptotic pathway, including Bax and caspases. miR-140 and TGF-β regulate the expression of genes associated with matrix synthesis and degradation, enhancing the synthesis of cartilage matrix components such as type II collagen and aggrecan, thus promoting remodeling and stabilization of the extracellular matrix (ECM).\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5627430/v1/f8482d4a661a5223ff9f7301.jpg"},{"id":72643165,"identity":"60d9fe50-e987-41d6-b636-da9f8a6e900d","added_by":"auto","created_at":"2024-12-30 16:37:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":533348,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdvanced Biomaterials and Engineering Technologies for Enhanced Extracellular Vesicle (EV) Delivery and Cartilage Regeneration\u003c/strong\u003e \u003cstrong\u003e(Created with BioRender.com)\u003c/strong\u003e. \u003cstrong\u003eA. Hydrogel-Based EV Delivery System\u003c/strong\u003e. The combination of hydrogels with EVs improves drug delivery efficiency, enabling sustained release and targeted delivery to cartilage. This approach enhances therapeutic outcomes by directly delivering EVs loaded with anti-inflammatory and cartilage-regenerative factors (such as SOX9 and ACAN) to damaged joint areas. \u003cstrong\u003eB. Microfluidics for Controlled EV Loading and Release\u003c/strong\u003e. Microfluidic technology allows precise control over EV loading and release, facilitating efficient EV delivery in cartilage repair. This technology creates a microenvironment that mimics physiological conditions, enhancing EV stability and ensuring their release upon injection into cartilage tissue. \u003cstrong\u003eC. Novel Culture Systems for Enhanced EV Production\u003c/strong\u003e. Novel cell culture systems simulate the in vivo extracellular matrix (ECM) environment, increasing EV yield and biological functionality. Compared to traditional 2D culture, these 3D ECM models provide cells with a concentrated gradient of soluble factors, enhanced cell-cell interactions, and structural support, significantly promoting tissue regeneration and anti-inflammatory effects. \u003cstrong\u003eD. 3D Printing for Scaffold Fabrication and Targeted Cell Migration\u003c/strong\u003e. 3D printing, particularly Stereolithography (SLA) technology, enables the creation of highly precise scaffolds through computer-aided design (CAD). These custom-designed scaffolds support enhanced cell migration and guide cells toward damaged regions, promoting tissue repair and regeneration. Exosome-laden 3D-printed scaffolds facilitate chondrocyte migration and increase mitochondrial biogenesis in chondrocytes, further improving cartilage repair outcomes.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5627430/v1/bc7b47a8724f24d1fe50cb95.jpg"},{"id":77697591,"identity":"6de0a691-9901-4d1b-99d7-14038e837148","added_by":"auto","created_at":"2025-03-04 10:43:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3521247,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5627430/v1/10835d08-194c-402b-8e6f-79a8e41a4ecb.pdf"},{"id":72643161,"identity":"4569047f-f531-443e-a9ee-80050a5996bc","added_by":"auto","created_at":"2024-12-30 16:37:22","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":321648,"visible":true,"origin":"","legend":"Figure S1","description":"","filename":"FigueS12.0.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5627430/v1/386c167a2843e4c48d8058d3.pdf"},{"id":72643169,"identity":"e72ed7b9-5258-4a12-be1c-d15b6799db6b","added_by":"auto","created_at":"2024-12-30 16:37:23","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":216726,"visible":true,"origin":"","legend":"Figure S2","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5627430/v1/2e69aca7dbac138ca5386d76.pdf"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Bibliometric analysis of extracellular vesicles in osteoarthritis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is a globally prevalent chronic degenerative joint disease characterized primarily by the wear and degradation of articular cartilage, accompanied by osteophyte formation, periarticular soft tissue inflammation, and chronic inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Symptoms include joint pain and stiffness, which can lead to functional loss and even disability in severe cases, imposing a significant socioeconomic burden (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). According to the Global Burden of Disease report published in The Lancet Rheumatology\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, In 1990, about 256\u0026nbsp;million people (95% UI 232\u0026ndash;282) globally had OA. By 2020, this number had risen to 595\u0026nbsp;million people (95% UI 535\u0026ndash;656\u0026nbsp;million). Over the span of ten years, the prevalence of OA across all age groups has steadily increased. It is projected that by 2050, nearly 1\u0026nbsp;billion people will be affected by OA\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. To date, the exact etiology of OA remains not fully understood. Major risk factors include age, gender, obesity, joint injuries, genetic predisposition, and occupational factors\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The incidence of this disease is higher among the elderly population, but there is a noticeable trend towards younger individuals being affected\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Compared to men, postmenopausal women have significantly lower estrogen levels and bone density, making them more susceptible to joint diseases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. As a multifactorial degenerative disease, OA is not a single entity but rather a collection of distinct clinical phenotypes driven by different and/or overlapping molecular subtypes\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Consequently, patients exhibit diverse combinations of pain, symptoms, and functional impairments, contributing to considerable heterogeneity in clinical features and responses to existing treatments. This variability not only complicates the comprehensive understanding of OA pathogenesis but also hinders the development of standardized therapeutic strategies. Current treatment approaches primarily focus on symptom management through medications, physical therapy, and injection therapies. However, these methods do not cure the disease. Pharmaceutical interventions, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroid injections, can provide short-term relief of pain and inflammation but are associated with significant long-term side effects\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. For advanced-stage OA, joint replacement surgery is often the primary treatment option. Nevertheless, postoperative complications, such as infections, prosthesis loosening, and wear, may necessitate revision surgeries. Additionally, some patients experience suboptimal recovery, with persistent pain and limited mobility remaining a challenge after surgery\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWith a deeper understanding of the molecular mechanisms underlying OA, various emerging therapies are being actively explored to reverse or halt disease progression at the molecular and cellular levels. Against this backdrop, extracellular vesicles (EVs) have garnered significant attention as a critical mediator of intercellular communication and a promising therapeutic tool. Due to their unique biocompatibility and low immunogenicity, EVs are considered to hold great potential for OA treatment\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. EVs are nanometer-scale particles encapsulated by a lipid bilayer, incapable of self-replication, and secreted by cells into the extracellular environment. They participate in various biological processes, including cell communication, immune regulation, and modulation of pathophysiological mechanisms. Based on their size and biogenesis, EVs can be categorized into three main types: exosomes (30\u0026ndash;150 nm, formed within multivesicular bodies and released via exocytosis), microvesicles (100\u0026ndash;1000 nm, generated through direct budding of the plasma membrane), and apoptotic bodies (500\u0026ndash;2000 nm, formed during the fragmentation of apoptotic cells)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-G). Nearly all cell types can secrete EVs, including mesenchymal stem cells, dendritic cells, and T cells. Extensive research has demonstrated that EVs carry bioactive molecules capable of promoting tissue repair and regeneration, inhibiting cartilage degradation, and regulating inflammatory responses in damaged joints. Furthermore, EVs serve as effective drug delivery vehicles, transporting therapeutic molecules to target sites\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, the high biological heterogeneity of EVs, as well as the lack of standardized techniques for their isolation, purification, and quality control, remains a significant barrier to their clinical translation. To address these challenges, the international research community has been working to standardize EV-related studies. In 2011, scholars from multiple European countries established the International Society for Extracellular Vesicles (ISEV), which has since published three editions of the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. The latest edition, MISEV2023, further refines the standards for EV nomenclature, sample preparation, isolation, characterization, and functional analysis, providing essential guidance for experimental design and reporting\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Transparent and standardized workflows are expected to enhance the validity, accuracy, and reproducibility of EV research, laying a solid foundation for their clinical application.\u003c/p\u003e \u003cp\u003eTo advance understanding of the mechanisms underlying EV-mediated effects in OA treatment, explore their clinical potential, and identify gaps and unresolved issues in current research, this study employs bibliometric methods to review and evaluate the status of EV-related research in OA from January 1, 1998, to June 1, 2024. By summarizing the research landscape, identifying key areas of focus, and outlining future directions, this work aims to provide scientific evidence supporting the clinical translation of EVs in OA therapy.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDevelopment Trends and Research Field Distribution\u003c/p\u003e \u003cp\u003eThe annual publication volume of research on EVs related to OA can reflect the development trends in the mechanisms and applications of EVs in OA. From the line graph, it is evident that the annual publication volume has shown a significant upward trend. In 2017, the annual growth rate exceeded double digits for the first time, and in 2020, the field entered a period of rapid development, marked by a notable increase in annual growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This rapid development period may be attributed to the introduction of new technologies and methods, such as ultracentrifugation, size exclusion chromatography, and microfluidics-based techniques, which have made the isolation, purification, and characterization of EVs more accurate and efficient\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The potential of EVs in disease diagnosis, prognosis assessment, regenerative medicine, and cell therapy has gradually been recognized\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Numerous studies have identified microRNAs and proteins within EVs as important biomarkers, attracting widespread attention.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResearch areas on EVs in OA is multidisciplinary, encompassing basic research, applied research, and clinical studies. The primary research areas are distributed across Cell Biology (163), Research Experimental Medicine (81), Engineering (73), Biochemistry Molecular Biology (72), and Pharmacology Pharmacy (60) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Since the mechanisms of EVs are not yet fully understood, current research focuses on understanding the molecular components, secretion mechanisms, and roles of extracellular vesicles in cell communication, as well as their potential applications in disease models. Additionally, efforts are being made to develop new biomaterials and scaffolds to enhance the applications of EVs in tissue engineering and regenerative medicine.\u003c/p\u003e \u003cp\u003eCollaboration Among Institutions and Countries\u003c/p\u003e \u003cp\u003eA total of 52 countries have contributed to the research on extracellular vesicles in osteoarthritis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Among the top 10 countries by publication volume are three Asian countries, five European countries, one North American country, and one Oceanian country. China stands out with a significantly higher number of publications, reflecting the country\u0026rsquo;s strong focus on this field. This emphasis is likely driven by China\u0026rsquo;s large population and high number of elderly individuals, resulting in a higher prevalence of osteoarthritis compared to other countries. Consequently, China is investing heavily in research to mitigate the social and economic burdens associated with this condition. In terms of institutional contributions, 235 research institutions have been involved in studies related to extracellular vesicles in osteoarthritis\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Among the top six institutions by publication volume, four are based in China, one in Italy, and one in Singapore. The National University of Singapore, which began publishing related research as early as 2014, holds the highest total publication volume from 1998 to June 2024. Sichuan University has rapidly advanced, ranking second in publication volume in 2023 and becoming the leading institution in 2024 as of June 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The collaboration network among these institutions reveals extensive cooperation, underscoring the widespread attention that research on extracellular vesicles in osteoarthritis has garnered across various research entities.\u003c/p\u003e \u003cp\u003eKeyword Clustering to Interpret Research Frontiers\u003c/p\u003e \u003cp\u003eKeyword co-occurrence refers to the phenomenon where two or more keywords appear simultaneously in the same document or group of documents\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. By counting the frequency of these co-occurrences, one can understand the degree of association between the keywords. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the core keywords include \u0026ldquo;exosomes,\u0026rdquo; \u0026ldquo;mesenchymal stem cells,\u0026rdquo; and \u0026ldquo;osteoarthritis.\u0026rdquo; These keywords are more prominent in the figure and have a strong co-occurrence relationship with many other keywords, indicating that they are central themes in this field of research (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). The red cluster mainly involves exosomes, mesenchymal stem cells, osteoarthritis, and their related biological mechanisms and therapeutic potential. In contrast, the blue cluster is associated with stromal cells, articular cartilage, and themes related to their repair and regeneration. Using CiteSpace, a co-occurrence network analysis of keywords identified nine clusters: Gut Microbiota, Immunomodulation, Cartilage Regeneration, Activation, miRNA Sequencing, Knee Osteoarthritis, Mesenchymal Stromal Cells, Knee Osteoarthritis Progression, and Microvesicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Gut microbiota plays a crucial role in regulating the host\u0026rsquo;s immune system and inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Dysbiosis of the gut microbiota can impair the gut barrier function, leading to increased intestinal permeability. Bacterial endotoxins, such as lipopolysaccharides (LPS), can enter the bloodstream, activating systemic immune responses and increasing systemic inflammation\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This inflammatory response can extend to the joints, promoting the development and exacerbation of osteoarthritis\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Recently, the concept of the \u0026ldquo;Gut-Joint Axis\u0026rdquo; has been proposed. Uzma Amin et al.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e demonstrated that the concentrations of gamma-aminobutyric acid (GABA) in the serum and small intestine contents of treated mice increased, inhibiting cartilage catabolism and protecting mouse joints from degeneration. Furthermore, synthetic markers in mouse knee joints and chondrocytes were upregulated, while inflammatory markers decreased. Probiotics such as Streptococcus thermophilus and Lactobacillus pentosus, along with GABA, were shown to counteract osteoarthritis in vivo and mitigate IL-1β-induced changes in chondrocytes in vitro. These findings open new avenues for understanding the gut-joint axis and its therapeutic implications for osteoarthritis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIdentification of Core Literature\u003c/p\u003e \u003cp\u003eEach research article incorporates numerous references, and reference analysis can identify the frontiers and hotspots of current research, as well as the core literature that holds significant influence and plays a critical role. The co-citation relationship refers to the scenario when two documents (A and B) are simultaneously cited by a third document (C), establishing a co-citation link between A and B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Key nodes in the co-citation network typically represent the core literature and significant authors within the field. Zhang et al.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e demonstrated that MSC exosomes could induce higher infiltration of CD163-positive regenerative M2 macrophages rather than inflammatory CD86-positive M1 macrophages. They also reduced pro-inflammatory cytokines (IL-1β and TNF-α) in the synovial fluid and induced phosphorylation of AKT and ERK in chondrocytes, enhancing the survival and proliferation of chondrocytes. This reveals the potential of MSC exosomes to achieve cartilage repair and regeneration through the coordinated regulation of multiple cellular processes, including cell migration, proliferation, matrix synthesis, macrophage infiltration, and cytokine production. Tao et al.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e were the first to use synovial mesenchymal stem cells (SMSC) as a source of exosomes. They transfected SMSCs with or without miR-140-5p, extracted and characterized the exosomes. Their findings indicated that Wnt5a and Wnt5b in the exosomes activated YAP through the alternative Wnt signaling pathway, thereby enhancing chondrocyte proliferation and migration but inhibiting the secretion of SOX9 and ECM components. Overexpression of miR-140-5p could restore SOX9 expression by inhibiting RalA, thus preventing the inhibition of ECM secretion. In a rat model of osteoarthritis, SMSC-140-Exos successfully prevented the onset and progression of osteoarthritis, showing significant protective effects. Compared to the control group, rats treated with SMSC-140-Exos exhibited significantly reduced joint cartilage damage, maintained levels of type II collagen and aggrecan expression, and reduced type I collagen expression. Zhang et al.\u0026rsquo;s study\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e was the first to demonstrate the effectiveness of human embryonic mesenchymal stem cell (hEMSC)-derived exosomes in repairing critical-sized osteochondral defects in an immunocompetent adult rat model. An osteochondral defect model was created on the femoral trochlear grooves of 12 adult rats. One side of the defect was treated with 100 micrograms of exosomes, while the other side was treated with phosphate-buffered saline (PBS) as a control. Age-matched animals that did not undergo surgery served as the control group. The defects were injected intra-articularly with exosomes or PBS weekly for 12 weeks. Exosome-treated defects showed significant tissue regeneration within 12 weeks, forming hyaline cartilage and subchondral bone with good surface regularity and complete integration with the adjacent cartilage. In contrast, PBS-treated defects were primarily fibrous tissue and failed to effectively repair cartilage and subchondral bone. This study provided a novel, cell-free therapeutic strategy with significant advantages over cell-based therapies. Clustering based on reference titles reveals that the therapeutic potential of EVs has garnered widespread attention, as evidenced by cluster labels such as #1 Potential Role, #7 Therapeutic Effect, #9 MSC Exosome, #11 MSC Therapy, and #13 Cartilage Degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Combining these cluster results with keyword clustering indicates that current research hotspots and future directions focus on the potential application of EVs in cartilage repair, particularly those derived from MSCs.\u003c/p\u003e \u003cp\u003eOverview of Extracellular Vesicle Isolation and Characterization Techniques\u003c/p\u003e \u003cp\u003eThe separation of EVs is the primary and critical step in EV research, while characterization serves as an essential means to assess the purity and yield of separation methods and to identify the features of EVs. Among the 296 included research articles, differential ultracentrifugation is the most widely described separation method (N\u0026thinsp;=\u0026thinsp;177, 59.80%), with commercial kits being the second most common (N\u0026thinsp;=\u0026thinsp;41, 13.85%) (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec). Ultracentrifugation utilizes centrifugal force at high speeds (typically 100,000 g or higher) to separate different components in a sample based on density and size, effectively isolating EVs from complex samples. This method is suitable for various types of EVs and has been validated by numerous studies, demonstrating high credibility and reproducibility. However, it entails high equipment costs, requires multiple centrifugation steps, is time-consuming, and the high centrifugal forces may cause physical damage to EVs, potentially affecting their biological activity. Commercial kits available on the market primarily employ principles such as polymer precipitation, membrane affinity, antibody capture, and filtration for the \u0026ldquo;separation\u0026rdquo; of exosomes. These kits optimize operational workflows, reduce cumbersome steps, and decrease time consumption, enabling separation to be completed in a relatively short period. Nonetheless, compared to ultracentrifugation, commercial kits usually yield lower amounts of EVs, are challenging to process large-volume samples, and technical differences among kits from different manufacturers may lead to inconsistent separation outcomes. Additionally, they rely on specific reagent formulations, making it difficult to adjust protocols according to specific needs. The MISEV2023 guidelines recommend prioritizing commercial EV kits with publicly disclosed separation or concentration principles to avoid potential interference from unknown contaminants in experimental results.\u003c/p\u003e \u003cp\u003eThe characterization methods for EVs are more diverse than the separation methods. Single vesicle analysis is mainly performed using electron microscopy techniques, such as scanning or transmission electron microscopy, or confocal laser scanning microscopy. A total of 227 studies (76.69%) provided detailed descriptions of electron microscopy methods, while 4 studies (1.35%) used confocal laser scanning microscopy. Quantification of particle concentration was conducted using nanoparticle tracking analysis (NTA) (N\u0026thinsp;=\u0026thinsp;188; 63.51%) or dynamic light scattering (DLS) (N\u0026thinsp;=\u0026thinsp;34; 11.49%). Although many articles provided quantitative values, few studies reported ratios\u0026mdash;such as particle number to protein and/or lipid content\u0026mdash;to estimate product purity. In evaluating protein marker expression, 207 studies (69.93%) quantified total protein content using Western blotting (WB), 46 studies (15.54%) employed flow cytometry, and a few studies utilized other protein analysis methods like ELISA kits (N\u0026thinsp;=\u0026thinsp;7; 2.36%) (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed). The most commonly used specific markers are surface proteins of the tetraspanin family, including CD63 (N\u0026thinsp;=\u0026thinsp;196; 66.22%), CD9 (N\u0026thinsp;=\u0026thinsp;161; 54.39%), and CD81 (N\u0026thinsp;=\u0026thinsp;145; 48.99%). Additionally, 98 studies (33.11%) and 39 studies (13.18%) chose to use TSG101 and Alix, which are protein components of the endosomal sorting complexes required for transport (ESCRT).\u003c/p\u003e \u003cp\u003eThrough a systematic analysis of the EV characterization and separation purification methods in the included studies, we gained an in-depth understanding of the characteristics and shortcomings of current research practices. These methodological choices not only affect the reliability of experimental results but also reflect researchers\u0026rsquo; focal points and technical proficiency, highlighting both the diversity and commonality of technological applications in this field. To more comprehensively grasp the current state of development and future trends in EV research, exploring research hotspots and frontier directions is highly necessary.\u003c/p\u003e \u003cp\u003eMultifaceted Applications of Extracellular Vesicles in Osteoarthritis Treatment\u003c/p\u003e \u003cp\u003eEVs are nano-sized vesicles secreted by cells that contain proteins, mRNA, miRNA, and other biomolecules. These vesicles play a crucial role in intercellular communication, contributing to the regulation of inflammatory responses and promoting tissue repair and regeneration. In the treatment of osteoarthritis (OA), EVs have been shown to reduce inflammation, facilitate cartilage repair, and improve joint health through multiple molecular mechanisms. Based on an analysis of 296 studies on the therapeutic effects of EVs in OA, the results were categorized into five outcome groups: \u0026ldquo;significant improvement,\u0026rdquo; \u0026ldquo;partial improvement,\u0026rdquo; \u0026ldquo;no effect,\u0026rdquo; \u0026ldquo;unclear results,\u0026rdquo; and \u0026ldquo;reported adverse effects.\u0026rdquo; Of these studies, 248 (83.78%) reported significant improvements in OA treatment outcomes, and 32 studies (10.81%) reported partial improvement (i.e., at least one outcome was improved, but not all). Additionally, 2 study (0.68%) observed no therapeutic effect, 2 studies (0.68%) yielded unclear results, and 12 studies (4.05%) documented adverse effects. In a representative experimental study, Liu et al.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e demonstrated that EVs derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) suppressed IL-1β-induced apoptosis in chondrocytes and reversed the upregulation of matrix metalloproteinase-13 (MMP13), thereby enhancing chondrocyte viability. Furthermore, bioengineered hUC-EVs were found to significantly promote chondrocyte proliferation and increase the expression of aggrecan and type II collagen, accelerating cartilage repair (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ee). Most studies focused on the regulation of inflammatory pathways (n\u0026thinsp;=\u0026thinsp;152, 51.35%)), followed by apoptosis (n\u0026thinsp;=\u0026thinsp;87, 29.39%)) and cell proliferation (n\u0026thinsp;=\u0026thinsp;79, 26.69%) (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ef). These findings highlight the importance of understanding the underlying mechanisms through which EVs exert therapeutic effects in OA, especially their capacity to modulate the interactions between chondrocytes and the immune microenvironment. Such insights are crucial for elucidating how EVs slow disease progression and promote tissue repair in OA.\u003c/p\u003e \u003cp\u003eExtracellular Vesicles in Regulating Inflammation and Promoting Cartilage Protection in OA\u003c/p\u003e \u003cp\u003eThe persistent high expression and activation of inflammatory factors in OA are core mechanisms driving its progression. Pro-inflammatory cytokines such as IL-1β and TNF-α induce the expression of matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS), accelerating cartilage matrix degradation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Reactive oxygen species (ROS) generated during inflammation cause oxidative damage to chondrocytes, further exacerbating cartilage degeneration\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. EVs carrying anti-inflammatory molecules target and regulate key modulators of the NF-κB signaling pathway, thereby reducing NF-κB activity and the expression of pro-inflammatory cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Marta Varela-Eir\u0026iacute;n et al.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e demonstrated that extracellular vesicles from Cx43-overexpressing T/C-28a2 chondrocytes (sEVs-T/C-Cx43) promoted NF-κB nuclear translocation, significantly increasing the expression of pro-inflammatory genes in osteocytes and synoviocytes, leading to a pro-inflammatory phenotype and degenerative state. Therefore, downregulating Cx43 in human osteoarthritic chondrocytes can restore chondrocyte redifferentiation, reduce the accumulation of senescent cells, and inhibit NF-κB nuclear translocation. Using specific peptides targeting the C-terminal domain of Cx43 may help reduce inflammation and cellular senescence, promoting tissue regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe polarization state of macrophages can significantly influence the nature and duration of inflammation\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Macrophages are primarily categorized into two phenotypes: M1 (pro-inflammatory) and M2 (anti-inflammatory and tissue repair). miRNAs in EVs, such as miR-223, and anti-inflammatory cytokines like TGF-β and IL-10, modulate multiple targets and receptor-related signaling pathways, such as the Smad pathway. These molecules downregulate the expression of M1-associated genes, including TNF-α and IL-1β, inhibiting the production of pro-inflammatory cytokines, reducing the number and activity of M1 macrophages, and inducing M2 macrophage polarization. This promotes the expression of anti-inflammatory genes and maintains the M2 phenotype. Qian et al.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e found that exosomes from M2 macrophages contain miR-26b-5p, which can repolarize M1 macrophages into the anti-inflammatory M2 phenotype by targeting the TLR3 signaling pathway. In vivo experiments showed that injecting miR-26b-5p agomir improved gait abnormalities and mechanical allodynia in OA mice, alleviated synovitis and cartilage degeneration, and slowed OA progression. This provides a potential therapeutic approach for OA.\u003c/p\u003e \u003cp\u003eEVs-Mediated Modulation of Inflammatory Bone Loss and Osteogenesis\u003c/p\u003e \u003cp\u003eInflammatory factors not only affect cartilage but also influence bone metabolism and remodeling. They inhibit osteoblast differentiation and function while promoting osteoclast activity, thereby slowing bone repair and reconstruction, leading to increased bone resorption and loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In an inflammatory state, pro-inflammatory cytokines such as IL-1β and TNF-α can upregulate the expression of RANKL. RANKL, by binding to its receptor RANK, activates the differentiation and maturation of osteoclast precursor cells\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Activated osteoclasts form resorption lacunae on the bone surface, releasing acids and proteases that degrade the bone matrix, resulting in bone loss. Concurrently, these pro-inflammatory factors inhibit the generation and maturation of osteoblasts by downregulating the expression of osteoblast differentiation markers such as Runx2 and Osterix, and suppress the synthesis and secretion of bone matrix components like type I collagen and osteocalcin, thereby impairing new bone formation\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. EVs promote osteoblast differentiation, function, and survival through multiple pathways, regulate osteoclast activity, reduce bone resorption, and promote the balance between bone formation and resorption. For instance, miR-196a in EVs can target the HOXC8 gene to promote osteoblast differentiation and bone matrix synthesis. miR-503 inhibits osteoclast activity by targeting upstream regulators of RANKL\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, while miR-218 promotes osteoblast differentiation and mineralization by regulating the Wnt/β-catenin signaling pathway\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Bone morphogenetic proteins (BMPs) carried by EVs promote osteoblast differentiation and function through the Smad signaling pathway\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Insulin-like growth factor (IGF-1) in EVs enhances osteoblast proliferation and survival via the PI3K/Akt signaling pathway\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Chen et al.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e demonstrated that the combined application of BMP-2/macrophage-derived exosomes and titanium nanotubes significantly promoted the expression of osteogenesis-related genes (such as ALP, osteopontin, Runx2, BMP-2, and BMP-7) and activated autophagy in human bone marrow stromal cells (hBMSCs), indicating a substantial osteogenic effect.\u003c/p\u003e \u003cp\u003eTargeted EV Therapies for Enhancing Cartilage Repair and Chondrocyte Survival\u003c/p\u003e \u003cp\u003eCartilage tissue is avascular, lacking direct blood supply, which means that nutrients and oxygen from the bloodstream cannot reach chondrocytes directly\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Instead, these must diffuse through the synovial fluid. Chondrocytes are sparsely distributed within the matrix and exhibit low metabolic activity. These characteristics make cartilage tissue difficult to repair quickly through cell proliferation and matrix synthesis, thus attracting significant attention to exogenous therapeutic interventions. EVs carrying miRNAs, anti-apoptotic molecules, and growth factors can promote chondrocyte proliferation and survival, as well as regulate the balance between synthesis and degradation of the extracellular matrix (ECM)\u003csup\u003e\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Growth factors like TGF-β and IGF-1 promote chondrocyte proliferation and survival through receptor-mediated signaling pathways such as PI3K/Akt and Smad (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC)\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Liu et al.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e indicate that dual-engineered cartilage-targeted extracellular vesicles (hUC-EVs) derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) significantly improve chondrocyte survival rates and reverse the IL-1β-induced downregulation of anabolic factors (Collagen II and SOX9), while inhibiting the upregulation of matrix metalloproteinase 13 (MMP13), thereby promoting cartilage anabolism. High-throughput miRNA sequencing revealed that miR-223 was significantly upregulated in the hUC-EVs treatment group, directly targeting NLRP3 mRNA and modulating the activation of the NLRP3 inflammasome, thereby exerting protective effects on cartilage.\u003c/p\u003e \u003cp\u003eIn summary, EVs exhibit significant potential in the treatment of OA by demonstrating anti-inflammatory properties, modulating immune responses, repairing cartilage, and regulating bone remodeling. This provides new approaches and methods for OA treatment. To further enhance the therapeutic effects of EVs, researchers have begun exploring the combination of EVs with engineering technologies and biomaterials. This paves the way for new directions in future research and clinical applications.\u003c/p\u003e \u003cp\u003eAdvancements in EV-Based Therapies through Engineering and Biomaterial Integration\u003c/p\u003e \u003cp\u003eNatural EVs have a broad range of sources, as nearly all cell types can secrete them, and they are distributed throughout the body. Their composition is complex, containing various proteins, RNA, and lipids. Consequently, natural EVs are easily degraded in vivo, preventing them from exerting their effects for extended periods. They lack specific targeting abilities, which may result in inefficient delivery to diseased sites, reduced therapeutic efficacy, or increased side effects\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. To overcome the limitations of natural EVs, chemical modifications or combinations with biomaterials can be employed to enhance their stability in vivo. Additionally, integrating EVs with different biomaterials can improve their effectiveness and duration of action.\u003c/p\u003e \u003cp\u003eHydrogel-Enhanced EVs Therapies for Osteoarthritis: Innovations in Delivery and Therapeutic Efficacy\u003c/p\u003e \u003cp\u003eHydrogels are a class of soft materials with unique physical and chemical properties\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. They have a high-water-content, can gradually degrade into harmless small molecules in the body, and exhibit excellent biocompatibility\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Their softness and elasticity allow them to withstand certain mechanical stresses without breaking. Additionally, hydrogels typically possess high porosity and a three-dimensional network structure, which facilitates material exchange and cell migration\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. These characteristics make hydrogels important materials in biomedical engineering and drug delivery fields. Sun et al.\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e utilized a mixture of gelatin, fibrinogen, hyaluronic acid (HA), and glycerol as a matrix to form a hydrogel through cross-linking. They embedded extracellular vesicles (T3-EV) derived from TGFβ3-preconditioned bone marrow mesenchymal stem cells (BMSCs) into the composite hydrogel. In a rat knee cartilage defect model, the T3-EV composite hydrogel treatment group showed better new cartilage formation, proteoglycan deposition, and tissue integrity after 24 weeks, significantly improving compared to the control group. Immunohistochemical staining results showed that in the T3-EV composite hydrogel treatment group, the expression of cartilage markers such as SOX9, ACAN, and COL2A1 was significantly enhanced, while the expression of the cartilage-degrading enzyme MMP13 was significantly reduced. Wan\u0026rsquo;s team\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e developed a spherical gelatin methacrylate (GelMA) hydrogel using photo-crosslinking technology, designed to encapsulate engineered exosomes modified with the WYRGRL peptide and loaded with the small molecule inhibitor LRRK2-IN-1 (W-Exo-L@GelMA). This encapsulation effectively delayed the release of exosomes and prolonged their retention time in the joint cavity. In vivo experiments showed that significant fluorescent signals of the encapsulated exosomes were detectable even 14 days after injection. Transcriptomic analysis revealed that W-Exo-L@GelMA significantly inhibited the upregulation of IL-1β-induced inflammation-related genes (such as Cxcl1, Cxcl5, Mmp3, and Mmp13) and restored the expression of anabolic genes suppressed by IL-1β. In a DMM-induced mouse OA model, the W-Exo-L@GelMA group demonstrated significant anti-inflammatory and cartilage repair effects, reducing joint cartilage degradation and subchondral bone loss, thereby exhibiting promising therapeutic effects. Due to their unique physicochemical properties, hydrogels exhibit significant advantages in EVs-based therapies for OA. The integration of microfluidic technology further enhances this approach by offering more refined control over EVs production and hydrogel encapsulation, thereby optimizing functionality (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEnhancing EV Therapy Precision for Osteoarthritis with Microfluidics\u003c/p\u003e \u003cp\u003eMicrofluidic technology is an advanced method for manipulating fluids on the micro- or nanoscale, allowing precise control over liquid, gas, and suspended particles. By fabricating microchannel structures in materials such as silicon, glass, or polymers through techniques like etching or 3D printing, microfluidic systems achieve high-precision fluid control within these small channels\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The rapid development of this technology in biomedicine has opened new pathways for enhancing the quality and functionality of EVs. For example, Neety et al.\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e used a polydimethylsiloxane (PDMS)-based non-planar microfluidic device to encapsulate mesenchymal stromal cells (MSCs) in sodium alginate hydrogel microbeads. These MSC microbeads were co-cultured with OA cartilage samples from patients, aiming to investigate the effects of MSC-secreted paracrine factors on cartilage regeneration. The results showed significant improvements in proteoglycan distribution and increased sulfated glycosaminoglycan (sGAG) content in the cartilage samples co-cultured with MSCs, compared to controls without MSCs, indicating enhanced chondrocyte proliferation and new sGAG synthesis. In a separate study, Yin et al.\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e employed microfluidic devices utilizing thiol-ene Michael addition to react thiolated hyaluronic acid (SH-HA) with hyperbranched polyethylene glycol diacrylate (HB-PEGDA), generating hydrogel microparticles (HMPs). In a mouse OA model, injection of HMPs@Exos allowed for gradual degradation and sustained exosome release, which inhibited the expression of ADAMTS4, a matrix metalloproteinase, and promoted cartilage matrix repair. Animal studies further demonstrated that encapsulating exosomes with high miR-99b-3p expression in HMPs (HMPs@ExosScAT-99b-3p) effectively slowed OA progression, preserved cartilage integrity, and achieved long-term therapeutic effects locally. Through precise production of EVs and targeted encapsulation in hydrogels, microfluidic technology enhances the stability and efficacy of EV-based therapies for OA, paving new directions for EV applications in regenerative medicine (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eAdvancements in Culture Systems for Enhanced EVs Yield and Bioactivity\u003c/p\u003e \u003cp\u003eOptimizing the culture system is crucial for improving the yield, quality, and functionality of EVs\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. By designing and optimizing culture conditions scientifically and reasonably, higher quality EVs can be produced to meet the diverse needs of basic research and clinical applications, thereby advancing the development of exosomes in the biomedical field. Consequently, researchers have conducted relevant studies on EV culture. Yan et al.\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e used a rotary cell culture system (RCCS) to culture MSCs in a mechanical environment to enhance the yield and biological function of exosomes. When Rab27a gene expression was knocked down using siRNA to reduce exosome secretion, the yield of exosomes decreased by approximately threefold under static conditions (0 rpm/min); however, under mechanical conditions (36 rpm/min), the yield decreased by only 1.5-fold. This indicates that mechanical stimulation can mitigate the reduction in exosome yield caused by siRNA interference. Additionally, exosomes produced in the mechanical environment significantly inhibited chondrocyte apoptosis. The expression level of LncRNA H19 in the exosomes increased nearly tenfold (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Treatment with exosomes containing LncRNA H19 (S-Exos) significantly reduced chondrocyte apoptosis rate (11.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the protein and mRNA levels of anti-apoptotic genes (Bcl-2 and Bax) consistently showed stronger anti-apoptotic activity. Zhou et al.\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e employed 3D culture techniques to simulate the three-dimensional structure and ECM of cells in vivo. The results showed that under 3D culture conditions, the number of exosomes produced by the cells significantly increased, and the expression of intracellular F-actin decreased. Compared to 2D culture, the composition of proteins and genetic material in exosomes also changed, resulting in stronger effects in promoting tissue regeneration and inhibiting inflammatory responses. The application of novel culture systems significantly enhances EVs yield and bioactivity, establishing a strong foundation for their widespread use in cell therapy and regenerative medicine (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eEnhancing Tissue Regeneration with 3D-Printed Exosome-Releasing Scaffolds\u003c/p\u003e \u003cp\u003e3D printing technology, particularly desktop stereolithography (SLA), can create highly precise three-dimensional structures\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Using computer-aided design (CAD), scaffolds with specific shapes and internal structures can be printed. The 3D structure and internal channel design of the scaffolds, such as radially oriented channels, can significantly enhance cell migration ability, guiding cells to migrate to defect areas, thereby promoting tissue repair and regeneration. Chen et al.\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e designed a bio-scaffold capable of sustained MSC-derived exosome release, utilizing 3D printing technology to fabricate an ECM/GelMA/exosome scaffold with radial channels. Results demonstrated that the defect regions treated with the 3D-printed ECM/GelMA/exosome scaffold regenerated cartilage-like tissue, with significantly improved repair outcomes compared to other groups. Cartilage repair was assessed using the International Cartilage Repair Society (ICRS) macroscopic scoring system, showing significantly higher scores in the 3D-printed scaffold group at both 6 and 12 weeks. Exosomes within the scaffold restored mitochondrial function in degenerated chondrocytes, markedly reducing mitochondrial damage marker MDA levels and displaying antioxidative stress capacity. Additionally, the ECM/GelMA/exosome scaffold significantly promoted M2 macrophage polarization while reducing M1 infiltration, exhibiting strong immunomodulatory potential. Furthermore, the ECM/GelMA scaffold excelled in trabecular bone regeneration, facilitating osteogenic tissue formation in the defect area. The application of 3D-printed scaffolds highlights its substantial potential in precision tissue engineering, not only enhancing exosome delivery but also promoting cell migration and tissue regeneration, offering a novel solution for the repair of degenerated cartilage and bone defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe integration of engineering technologies with EVs has opened new avenues for applications in regenerative medicine. This interdisciplinary synergy not only enhances the stability, bioactivity, and delivery efficiency of EVs but also addresses challenges related to rapid degradation and low retention rates in vivo. As a result, EVs have demonstrated remarkable potential in cartilage and bone tissue repair. To maximize therapeutic efficacy, EV delivery strategies should be tailored to the specific stages of disease progression. In the early stage, precise targeting is critical; here, microfluidic technology and novel culture techniques can be utilized to deliver specific EV components at low doses and with high specificity, facilitating early intervention. During the mid-stage, sustained anti-inflammatory and reparative support becomes the focus. In this phase, hydrogels with controlled-release properties are particularly advantageous for gradual EV release, reducing the discomfort and compliance issues associated with frequent injections. In the late stage, the combination of EVs with 3D-printed scaffolds offers structural support to damaged tissues, aiding cartilage repair and preserving joint function, thereby slowing disease progression. Recently, research has increasingly explored the integration of multiple technologies. For instance, combining hydrogels with microfluidic systems enables precise EV release rates that can be adjusted according to disease progression, thereby enhancing therapeutic outcomes. Similarly, 3D-printed scaffolds used alongside hydrogels provide a stable delivery platform for EVs and facilitate tissue repair. This multi-technology approach compensates for the limitations of single technologies in terms of targeting specificity or stability, yielding improved therapeutic results. Looking forward, as these engineering technologies and biomaterials continue to advance and mature, EVs are expected to see broader and deeper applications in clinical therapies, tissue engineering, and regenerative medicine, with the potential to bring personalized precision medicine into routine clinical practice.\u003c/p\u003e "},{"header":"Discussion and Future Outlook","content":"\u003cp\u003eNumerous studies have demonstrated that EVs exhibit significant therapeutic potential in the treatment of OA. EVs can regulate immune cell functions and reduce the release of inflammatory factors, thereby significantly alleviating the inflammatory response in OA\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Additionally, they can inhibit apoptosis of chondrocytes and synovial cells through various signaling pathways, thus protecting joint tissues\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. The engineering modification and combination with various biomaterials significantly enhance the efficacy and targeting capability of EVs\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Compared to traditional cell therapy, EVs have lower immunogenicity, avoiding immune rejection and the side effects of excessive cell proliferation. EVs are a heterogeneous population containing various bioactive molecules, with differences in size, composition, and function\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. The isolation and purification of specific types of EVs while maintaining their activity and functionality present significant challenges. During EV preparation, contaminants such as cell debris, protein aggregates, and other impurities may be introduced. Different production batches of EVs may exhibit significant variability, affecting their therapeutic efficacy and safety. Although numerous international guidelines for the purification, isolation, and characterization of EVs have been issued, efficient and standardized methods for EV preparation and purification remain a bottleneck for clinical application. The difficulties in separating and purifying EVs arise from their inherent biological properties, and there are technical bottlenecks in achieving high-efficiency purification and high-resolution characterization. Existing EV separation and purification techniques are generally suitable for laboratory-scale production but face issues of low efficiency and high cost when scaled up. Ensuring the consistency, purity, and reproducibility of EVs while achieving large-scale production within a controllable cost range is crucial for their clinical application.\u003c/p\u003e \u003cp\u003eAlthough preliminary studies have shown that EVs have good therapeutic effects and low side effects in OA, it is still necessary to verify these treatments\u0026rsquo; long-term efficacy and safety in larger-scale and longer-term clinical trials. Currently, the most commonly used administration method is intra-articular injection, which can precisely target the affected area. However, this method requires high technical skill and carries a certain risk of infection, especially with repeated injections. Improper injection techniques may cause mechanical damage to joint structures, including cartilage, ligaments, and synovium\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Intravenous injection of EVs allows for systemic distribution through the bloodstream and is convenient to administer. However, due to the rapid degradation of EVs and the lack of specific targeting mechanisms, it is challenging to ensure that a sufficient quantity of EVs reaches the affected joint, resulting in suboptimal therapeutic effects. Therefore, optimizing the administration methods, such as using 3D-printed scaffolds, combining with hydrogels and other biomaterials, and engineering EVs to enhance their biological functions, is crucial to improving the precision and efficacy of treatments. These strategies are essential for realizing the large-scale clinical application of EVs.\u003c/p\u003e \u003cp\u003eAs a novel biological therapeutic agent, EVs primarily derived from stem cells necessitate the establishment of comprehensive legal and regulatory frameworks. These frameworks are crucial for strictly controlling potential hazards, ensuring the scientific integrity and reliability of clinical research, and protecting patient rights. A unified and clear regulatory framework can also guide research and development, as well as production, thereby promoting the global advancement of EV technologies and accelerating the clinical translation and market entry of products.\u003c/p\u003e \u003cp\u003eIn summary, future research on EVs will benefit from multidisciplinary collaboration across biology, medicine, and materials science to drive innovative applications in OA treatment. The development of relevant laws and regulations is also crucial. By combining these efforts, we can ensure the clinical efficacy and safety of EVs, paving the way for their extensive use in regenerative medicine and disease therapy, ultimately providing more effective treatment options for OA patients.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eSearch Strategy\u003c/p\u003e \u003cp\u003eThe data were sourced from the most commonly used scientific literature repository\u0026mdash;Thomson Reuters\u0026rsquo; Web of Science Core Collection (WoSCC). This includes the Science Citation Index Expanded (SCI-EXPANDED), the Emerging Sources Citation Index (ESCI), the Conference Proceedings Citation Index-Science (CPCI-S), and the Conference Proceedings Citation Index-Social Science and Humanities (CPCI-SSH). All electronic searches were conducted on June 1, 2024. The search strategy is as follows: (TS=(exosome* OR exosomes OR exosomal OR \u0026ldquo;extracellular vesicle\u0026rdquo; OR \u0026ldquo;extracellular vesicles\u0026rdquo; OR \u0026ldquo;extracellular particle\u0026rdquo; OR \u0026ldquo;extracellular particles\u0026rdquo; OR \u0026ldquo;microvesicle\u0026rdquo; OR \u0026ldquo;microvesicles\u0026rdquo; OR \u0026ldquo;Shedding Microvesicle\u0026rdquo; OR \u0026ldquo;Shedding Microvesicles\u0026rdquo; OR \u0026ldquo;Secretory Vesicle\u0026rdquo; OR \u0026ldquo;Secretory Vesicles\u0026rdquo; OR \u0026ldquo;Cell-Derived Microparticle\u0026rdquo; OR \u0026ldquo;Cell-Derived Microparticles\u0026rdquo; OR \u0026ldquo;Apoptotic Bodies\u0026rdquo; OR \u0026ldquo;Apoptotic Blebs\u0026rdquo; OR \u0026ldquo;Apoptotic Vesicles\u0026rdquo; OR \u0026ldquo;Cell Fragments\u0026rdquo; OR \u0026ldquo;Apoptotic Bodies\u0026rdquo; OR \u0026ldquo;ApoBDs\u0026rdquo;)) AND TS=(Osteoarthritides OR Osteoarthrosis OR Osteoarthroses OR Arthritis, Degenerative OR Arthritides, Degenerative OR Degenerative Arthritides OR Degenerative Arthritis OR Arthrosis OR Arthroses OR Osteoarthrosis Deformans OR osteoarthritis). The search was limited to articles and reviews published between January 1, 1998, and June 1, 2024. An initial retrieval yielded 754 articles. Based on predefined inclusion and exclusion criteria, titles and abstracts were further screened, resulting in the final inclusion of 533 articles for subsequent analysis (Fig \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea). All data were stored in plain text format and imported into CiteSpace and Microsoft Excel 2016 for subsequent analysis. All data were downloaded from public databases and do not involve any medical ethics issues. All included studies meet the criteria for bibliometric analysis, and can be found in Appendix 1 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eIn this study, bibliometric methods were applied to analyze the historical evolution and development trends of research on extracellular vesicles (EVs) in osteoarthritis (OA). CiteSpace, a bibliometric visualization tool developed in Java by Dr. Chaomei Chen, was utilized to identify key literature and emerging trends through various parameters, including centrality, burstiness, and Sigma values. This approach effectively illustrates the developmental trajectory of the research field. Based on the research objectives, specific analytical functions, such as co-citation analysis and keyword co-occurrence analysis, were selected to detect influential core publications and identify research hotspots and key themes within the field.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Sichuan Provincial Science and Technology Department under the International Cooperation Project (Grant No. 25GJHZ0145).\u003c/p\u003e\n\u003cp\u003eConflict of Interests\u003c/p\u003e\n\u003cp\u003eThe authors report no declarations of interest.\u003c/p\u003e\n\u003cp\u003eAuthor Declarations and Contributions:\u003c/p\u003e\n\u003cp\u003eAll authors gave their final approval and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003eXie Hongyu was responsible for article selection, bibliometric data analysis, data extraction and statistical evaluation, figure creation, and the drafting and revision of the manuscript. Zhao Lin and Kang Lunwei assisted with data extraction, statistical analysis, and data maintenance. Weikun Meng and Liao Ga contributed to the manuscript review. The corresponding author supervised the study, provided guidance throughout the research process, and contributed to the final revision and approval of the manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHunter, D. J. \u0026amp; Bierma-Zeinstra, S. Osteoarthritis. \u003cem\u003eThe Lancet\u003c/em\u003e vol. 393 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0140-6736(19)30417-9\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(19)30417-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteinmetz, J. 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L. \u003cem\u003eet al.\u003c/em\u003e 2019 American College of Rheumatology/Arthritis Foundation Guideline for the Management of Osteoarthritis of the Hand, Hip, and Knee. \u003cem\u003eArthritis and Rheumatology\u003c/em\u003e 72, (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteoarthritis, Extracellular vesicles, Cell-free therapy, Microbiota, Cartilage repair, Immunomodulation","lastPublishedDoi":"10.21203/rs.3.rs-5627430/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5627430/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteoarthritis (OA) is a common degenerative joint disease with complex risk factors and an unclear pathogenesis. The onset is insidious, with mild early symptoms, and the disease progression is irreversible. Current treatment options cannot completely cure the disease. Therefore, the development of novel therapeutic approaches and early prevention strategies is crucial for alleviating the disease burden and enhancing the quality of life for patients with OA. Extracellular vesicles (EVs) naturally possess biocompatibility and low immunogenicity, showing significant potential in drug delivery and cell-free therapy. To comprehensively understand the research status and application prospects of EVs in OA, this review employs bibliometric methods to explore the development and collaboration patterns within this research field, current significant research statuses, and emerging directions. Additionally, it thoroughly reviews the mechanisms of EVs in OA, and the integration of EVs with engineering technologies and biomaterials to enhance their efficacy in OA treatment. The goal is to provide scientific evidence and references for the development of OA therapies and the clinical application of EVs.\u003c/p\u003e","manuscriptTitle":"Bibliometric analysis of extracellular vesicles in osteoarthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-30 16:37:18","doi":"10.21203/rs.3.rs-5627430/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6d1d9fb3-b9f9-4d2a-8405-d12c90e3b5a3","owner":[],"postedDate":"December 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42084717,"name":"Health sciences/Pathogenesis"},{"id":42084718,"name":"Biological sciences/Physiology/Bone"}],"tags":[],"updatedAt":"2025-03-04T10:35:49+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-30 16:37:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5627430","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5627430","identity":"rs-5627430","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}