The influence of exosomal miRNAs in OA has garnered attention due to their potential roles in mediating intercellular communication and gene regulation. Extensive research has been conducted to understand how exosomal miRNAs contribute to the progression of OA and their therapeutic implications (Table I). The dysregulation of these miRNAs may lead to an imbalance in matrix synthesis and degradation, promoting the pathological processes of OA (21). Additionally, exosomal miRNAs can influence neighboring cell types, such as synovial cells and macrophages, contributing to the chronic inflammatory environment characteristic of OA (21). By modulating gene expression in target cells, exosomal miRNAs coordinate a wide range of cellular responses, thereby serving an integral role in the progression of OA (Fig. 1) (31).

Several studies have highlighted exosomal miRNAs that facilitate the progression of OA by targeting key regulatory pathways (32-34). Kong et al (32) illustrated that exosomes secreted from osteoarthritic fibroblast-like synoviocytes contain miR-19b-3p, which promotes cartilage ferroptosis by targeting solute carrier family 7 member 11, thereby exacerbating cartilage damage. This finding underscores the contribution of specific miRNAs derived from pathologically altered tissue to the overall OA pathology. In another pivotal study, Mao et al (33) demonstrated that miR-92a-3p derived from exosomes of human mesenchymal stem cells (MSCs) enhanced chondrogenesis while simultaneously suppressing cartilage degradation through targeted modulation of WNT5A. This research indicates that certain exosomal miRNAs can serve a dual role, depending on the cellular context, complicating their impact on cartilage health. Furthermore, Xia et al (35) reported that miR-125a-5p-rich exosomes from MSCs suppress chondrocyte degeneration in traumatic OA by targeting E2F2, presenting evidence that even dysregulated miRNAs can have protective roles under certain conditions. Overall, these studies emphasize the critical role that exosomal miRNAs serve in propagating OA through the modulation of signaling pathways involved in cartilage homeostasis. Understanding these mechanisms is crucial for identifying potential therapeutic targets as well as biomarkers for monitoring disease progression.

A growing body of research has demonstrated the ability of certain exosomal miRNAs to inhibit the progression of OA, providing avenues for potential therapeutic interventions (36-38). Tao et al (36) revealed that exosomal miR-361-5p derived from human bone MSCs alleviates OA by downregulating DEAD-box helicase 20 and inactivating the NF-κB signaling pathway, consequently reducing inflammation and promoting chondrocyte viability. The study highlights the impact of MSC-derived exosomes in mitigating OA pathology through specific miRNA-mediated mechanisms. Further exploring protective roles, Li et al (37) observed that exosomal miR-100-5p derived from human umbilical cord MSCs alleviated cyclic strain-induced chondrocyte damage in OA by inhibiting reactive oxygen species production and apoptosis through direct targeting of NADPH oxidase 4 (NOX4). This finding not only provides insights into how exosomal miRNAs can counteract oxidative stress but also establishes potential therapeutic uses for MSC-derived exosomal products in OA treatment. Furthermore, Ye et al (38) demonstrated that exosomal miR-3960 can target the syndecan 1/Wnt/β-catenin axis, leading to the alleviation of chondrocyte injury in OA, further supporting the protective potential of exosomal miRNAs against degeneration in joint tissues. This dichotomy of miRNAs, both promoting and inhibiting OA progression, points towards a complex interplay where the recruitment, secretion and action of specific exosomes can be leveraged for therapeutic purposes. The choice of exosomal miRNA delivery systems may thus prove pivotal in therapeutic strategies aimed at OA management.

The diagnostic and therapeutic potential of exosomal miRNAs extends beyond their role in OA progression. As highlighted by a study, alterations in the levels of specific exosomal miRNAs, including miR-140-5p, miR-92a-3p, miR-100-5p and miR-155-5p, may serve as biomarkers for OA severity and response to treatment (39). These miRNAs can elucidate the molecular landscape of joint degeneration, providing insights into disease mechanisms and guiding therapeutic decisions (31). The therapeutic application of exosomal miRNAs offers promising strategies to combat OA. Utilizing exosomes enriched with protective miRNAs can be a novel approach to modulate inflammatory responses and enhance chondrocyte function. For instance, Wang et al (40) demonstrated that exosomal miR-155-5p derived from synovial MSCs enhanced proliferation, migration and extracellular matrix (ECM) secretion in chondrocytes, suggesting their potential for tissue engineering and regenerative medicine applications in OA. Furthermore, Qian et al (41) illustrated that M2 macrophage-derived exosomal miR-26b-5p modulates macrophage polarization to alleviate OA, underscoring the therapeutic potential of targeting inflammation in OA treatment using miRNA-based strategies. Overall, the ongoing exploration of exosomal miRNAs holds the potential to revolutionize the understanding and treatment of OA. The ability of miRNAs to act as diagnostic markers and therapeutic agents signals a pivotal shift in how OA may be managed in clinical settings, emphasizing the importance of further research to harness their full therapeutic potential.

Exosomes have emerged as key mediators of intercellular communication in OA (14). These vesicles carry a cargo rich in proteins that can influence the pathophysiological processes of OA (22). The role of exosomal proteins in OA is multifaceted and involves various signaling pathways essential for maintaining cartilage integrity, regulating inflammation and promoting chondrocyte survival (1,42). Through their ability to mediate complex cellular communication and modulate critical signaling pathways such as the TGF-β, Wnt/β-catenin, PI3K/Akt, MAPK/ERK and NF-κB signaling pathways, exosomes offer a novel avenue for therapeutic intervention in OA (43-47) (Fig. 2). Focusing on specific exosomal cargoes and their respective signaling pathways may reveal novel therapeutic targets for OA, paving the way for innovative treatment strategies that address the underlying mechanisms of the disease.

TGF-β is a well-characterized protein present in exosomes, with implications in the regulation of chondrocyte homeostasis and cartilage repair mechanisms (42). Studies have demonstrated that exosomal TGF-β promotes chondrocyte proliferation and matrix synthesis while inhibiting apoptosis (48,49). The activation of the TGF-β signaling pathway leads to the phosphorylation of SMAD proteins, which subsequently regulate the expression of ECM components, such as collagen and aggrecan (43). Furthermore, TGF-β signaling has been implicated in the modulation of inflammation within the OA joint, helping to counteract pro-inflammatory cytokine effects, including those mediated by TNF-α and IL-1β (50).

Exosomal proteins can also influence the Wnt/β-catenin signaling pathway, which serves a role in maintaining cartilage integrity and regulating osteophyte formation (44). For example, Wnt-5a has been shown to be present in MSC-derived exosomes and to activate β-catenin signaling in chondrocytes, promoting their differentiation and proliferation (44). This pathway may also enhance the synthesis of cartilage-specific markers while suppressing the expression of MMPs that contribute to cartilage degradation (1). In OA, the dysregulation of Wnt/β-catenin signaling is evident, and thus, exosomal proteins modulating this pathway may offer therapeutic benefits.

The PI3K/Akt signaling pathway is another crucial pathway influenced by exosomal proteins in OA (51). Exosomal delivery of growth factors such as VEGF can activate the PI3K/Akt pathway in target cells, leading to enhanced cell survival and proliferation (45). In the context of OA, activation of this pathway in chondrocytes can promote not only cell survival but also the production of anti-apoptotic factors such as Bcl-2 (45). Additionally, the PI3K/Akt pathway has been shown to inhibit the expression of inflammatory mediators, thus providing a protective effect against the inflammatory milieu associated with OA (52).

The MAPK/ERK pathway is another signaling pathway activated by exosomal proteins (53). Proteins such as fibroblast growth factor 2 carried by exosomes can activate this pathway in chondrocytes, leading to cellular responses that support cartilage anabolism and integrity (54). Activation of MAPK/ERK signaling leads to increased expression of chondrocyte-specific markers and ECM components, while downregulating inflammatory responses (46). Dysregulation of the MAPK pathway has been associated with OA progression (55), thus showcasing the therapeutic potential of exosomal proteins that can modulate this pathway effectively.

The NF-κB signaling pathway is central to the inflammatory response observed in OA (56). Exosomal proteins can modulate the activity of NF-κB, thereby influencing the expression of pro-inflammatory cytokines. For instance, exosomal delivery of cytokines such as IL-10 can inhibit the activation of NF-κB, leading to suppressed expression of inflammatory mediators such as IL-1β and TNF-α (47). This inhibitory effect not only protects chondrocytes from inflammation-induced apoptosis but also promotes a favorable microenvironment for cartilage regeneration (47).

In addition to direct signaling effects, exosomal proteins also interact with miRNAs that can post-transcriptionally regulate gene expression (57). For example, the exosomal protein cargo may deliver specific miRNAs, such as miR-140, which have been shown to serve a regulatory role in maintaining cartilage integrity and modulating inflammatory responses in OA (58). Similarly, miR-100-5p regulates NOX4 levels, reducing oxidative stress and apoptosis in response to cyclic strain (37). These findings illustrate how exosomal miRNAs can modulate protein activity, influencing OA pathology and presenting potential therapeutic targets for treatment strategies.

The lipid composition of exosomes also serves a pivotal role in the inflammatory response associated with OA (59). In OA, aberrant lipid profiles in exosomes can influence inflammation by activating pro-inflammatory pathways (59). For instance, the presence of specific sphingolipids can modulate inflammatory cytokine production in synovial cells, contributing to synovitis (60). These lipids can also affect the stability and functionality of exosomes, subsequently influencing their interaction with target cells (60). By altering the lipid composition in exosomes, researchers may be able to design therapeutic strategies to attenuate inflammation and promote cartilage health (Fig. 3). Further research into the lipid profiles of exosomes in OA could reveal novel insights into their role in inflammation and promote the development of lipid-based therapies targeting inflammatory mechanisms to improve joint health.

Exosomes are lipid bilayer vesicles that originate from MVBs and facilitate the transfer of bioactive molecules (molecules that are able to interact with molecules in living organisms to produce specific biological effects) between cells (61). The lipid composition of exosomes, which includes phospholipids, cholesterol and sphingolipids, is pivotal for their biogenesis, stability and cargo selection (61). For instance, certain lipids, including phosphoinositides and ether lipids, contribute to the fluidity of the exosomal membrane and influence the loading of proteins and RNA molecules (61). Mori et al (59) highlighted that exosomes can serve as nanocarriers for the delivery of therapeutic agents in inflammatory diseases, indicating that manipulating lipid composition could be an effective novel strategy for drug delivery.

In OA, the lipid metabolism is altered, which affects the properties and functionality of synovial fluid and cartilage (62). Sphingosine-1-phosphate (S1P) is a sphingolipid that has been implicated in OA pathogenesis (63). El Jamal et al (60) demonstrated that S1P signaling contributes to cartilage degradation and promotes inflammation in joint tissues, revealing that lipid alterations may directly impact disease mechanisms. Furthermore, alterations in the lipid composition of exosomes derived from MSCs may modulate the progression of OA, as shown by Toh et al (64), suggesting that therapeutic MSC-derived exosomes could improve cartilage repair by restoring normal lipid profiles.

Exosomal lipids serve crucial roles in immunomodulation, particularly in the context of OA, where inflammation is a central feature (22). Lipid components, including phospholipids and sphingolipids, are known to influence immune cell activation and cytokine release (22). Cosenza et al (65) emphasized the importance of MSCs-derived exosomes in modulating the inflammatory microenvironment. The bioactive lipids within these exosomes can reduce pro-inflammatory cytokines and promote an anti-inflammatory milieu (65). This effect suggests that lipid-carrying exosomes might be harnessed to mitigate OA-related inflammation and pain.

The therapeutic potential of exosomes, particularly those derived from MSCs, is underscored by their ability to deliver lipids and other bioactive molecules to affected joint tissues (66). Yang et al (67) provided evidence that exosomal treatments can enhance cartilage regeneration and joint function in OA models. Exosomes derived from MSCs can promote chondrogenesis and inhibit cartilage degradation by delivering specific lipids (59). Furthermore, Fan et al (15) indicated the possibility of utilizing exosomes as novel drug delivery systems, where lipid manipulation could optimize their therapeutic effects.

Exosomes derived from various sources can exert effects on chondrocyte proliferation and apoptosis (68,69) (Figs. 1 and 2). For example, exosomes from stem cells have been shown to enhance chondrocyte proliferation and survival, providing a potential avenue for therapeutic application in OA (28). These exosomes contain factors, such as TGF-β and MMPs, that can stimulate anabolism in chondrocytes and promote the synthesis of ECM components (42,70). Conversely, exosomes from degenerative cartilage can contain catabolic signals that trigger chondrocyte apoptosis and inhibit their repair capabilities (25,71). This dichotomy underscores the importance of identifying the origin and content of circulating exosomes in the context of OA, as their influence can determine the fate of chondrocytes and the overall joint health.

Chondrocytes are intrinsically sensitive to the inflammatory signals present in the OA milieu (8). Exosomes serve a crucial role in modulating this response by delivering pro-inflammatory or anti-inflammatory signals. For example, exosomes from inflamed joints may carry TNF-α or IL-1β, perpetuating the inflammatory feedback loop and resulting in further cartilage degradation (72). On the other hand, certain exosomal cargoes may have the potential to mitigate inflammation. MSCs-derived exosomes can reduce the infiltration of inflammatory cells and inhibit the production of specific inflammatory mediators by providing anti-inflammatory factors such as TGF-β and prostaglandin E2 (73). Furthermore, exosomes harboring anti-inflammatory miRNAs can promote resistance to apoptosis and enhance chondrocyte function (74). This balance between pro-inflammatory and anti-inflammatory exosome signaling is pivotal for maintaining cartilage homeostasis in OA (74).

The accumulation of exosomes in the synovial fluid during OA contributes to synovial inflammation (synovitis), a hallmark of the disease (75). Synovial cells, including fibroblasts and macrophages, respond to exosomal signals, leading to increased production of inflammatory mediators (32). This culminates in a self-perpetuating cycle of inflammation that contributes to joint pain and dysfunction. Exosomes from osteoarthritic synovial cells can influence local immunity and inflammation, promoting the recruitment of immune cells to the joint (32). This heightened inflammatory response can exacerbate tissue damage and further propagate the cycle of joint deterioration (76).

Exosomes also hold therapeutic potential for immune modulation within the context of OA (77). For instance, exosomes derived from MSCs can induce an anti-inflammatory response in synovial cells, downregulating the production of pro-inflammatory cytokines. This property makes them an attractive candidate for cell-based therapies aimed at reducing synovitis and promoting cartilage regeneration (77). Understanding the interaction between exosomes and synovial cells can open up novel avenues for targeting OA inflammation. Therapeutic approaches leveraging exosomes as delivery vehicles for anti-inflammatory agents or regenerative factors may ultimately enhance joint healing and function.

Exosomes have emerged as crucial mediators in the pathophysiology of OA, influencing key signaling pathways and serving as potential biomarkers (78). In OA, exosomes derived from chondrocytes, synovial cells and cartilage have been shown to contain various biomolecules, including proteins, lipids, mRNA and miRNA, which can modulate inflammatory responses and cartilage homeostasis (78).

One signaling pathway associated with exosome activity in OA is the Wnt/β-catenin pathway (79). Research has indicated that exosomes from OA chondrocytes can transfer Wnt ligands, activating this pathway, which contributes to chondrocyte hypertrophy and cartilage degradation (79). Additionally, exosomal miRNAs, particularly miR-21 and miR-140, have been implicated in the regulation of this pathway, further promoting catabolic processes in cartilage (80,81).

Exosomes serve a vital role in mediating the inflammatory response in OA. They facilitate communication between synovial cells and chondrocytes, promoting the release of pro-inflammatory cytokines such as TNF-α and IL-1β (82). Exosomal miRNAs, including miR-146a, can modulate the NF-κB pathway, enhancing inflammatory signaling and exacerbating joint damage (83).

Previous studies have also identified specific exosomal biomarkers that could help in OA diagnosis and prognosis. For instance, increased levels of exosomal proteins such as TGF-β and cyclooxygenase-2 have been reported in patients with OA, and are associated with disease severity (42,72). Furthermore, circulating exosomal miRNAs are being investigated as non-invasive biomarkers for OA progression and response to treatment (84).

In conclusion, exosomes are pivotal in the signaling cascades and molecular mechanisms underlying OA. The interactions between exosomal cargo and various signaling pathways reveal their dual role as mediators of cartilage degeneration and potential biomarkers for disease monitoring (85). Future research targeting exosomal contents could pave the way for novel therapeutic strategies aimed at modulating exosomal signaling and mitigating OA progression. This highlights the necessity for continued exploration of the role of exosomes in OA and their therapeutic prospects.

Exosomes derived from embryonic MSCs (eMSCs) have been shown to alleviate OA by balancing the synthesis and degradation of the cartilage ECM (90). This study demonstrated that eMSC-derived exosomes promote chondrocyte proliferation and enhanced matrix synthesis, while inhibiting catabolic pathways. Similarly, He et al (92) reported that bone marrow MSC-derived exosomes possess protective effects against cartilage damage in a rat OA model. The authors found that these exosomes reduce knee pain and enhanced joint function, suggesting a therapeutic potential for OA management. Furthermore, Xu et al (93) provided insights into the anti-inflammatory potential of bone marrow MSC-derived exosomes by demonstrating their ability to inhibit pyroptosis in chondrocytes through the specific delivery of miR-326, targeting the signaling pathways involved in inflammatory processes. This indicates a critical role of exosomal miRNAs in mediating the therapeutic effects of MSC-derived exosomes.

Adipose-derived stem cells (ADSCs) have also emerged as a source of therapeutic exosomes in OA (100). Wang et al (101) demonstrated that ADSCs-derived exosomes modulate chondrocyte metabolism, particularly glutamine metabolism, thus alleviating the progression of OA. Hypoxia-treated ADSCs-derived exosomes have been found to attenuate lumbar facet joint OA by delivering pro-survival signals (102). These findings suggest that the microenvironment and hypoxic preconditioning may enhance the exosomal therapeutic potential in joint diseases. Notably, Fu et al (103) identified that dental pulp stem cell-derived exosomes alleviate OA by inhibiting transient receptor potential cation channel subfamily V member 4-mediated osteoclast activation, underscoring the immunomodulatory capabilities of exosomal therapies in joint disease. Additionally, Meng et al (104) showcased that exosomal miR-429 from ADSCs can ameliorate chondral injury by targeting fasciculation and elongation protein ζ2 and promoting autophagy, highlighting the role of miRNAs in cartilage repair mechanisms.

Various novel sources of exosomes are emerging, including exosomes derived from human umbilical cord MSCs (105) and placenta-derived exosomes (106). The latter study characterized placenta-derived exosomes as a potential disease-modifying therapy for OA, emphasizing their unique biochemical composition and low immunogenicity, which make them suitable candidates for therapeutic applications.

Advancements in bioengineering have enhanced the therapeutic efficacy of exosomes. Zhao et al (107) reported that engineering exosomes from subcutaneous fat MSCs specifically promotes cartilage repair through targeted delivery of miR-199a-3p, showcasing an innovative approach to enhance the specificity and efficacy of exosomal therapy. Similarly, Yang et al (108) developed magnetic polysaccharide-based microcarriers loaded with MSC-derived exosomes, thereby enhancing targeted delivery and synergistic effects for OA treatment. Furthermore, Ma et al (109) explored the use of engineered exosomes with activating transcription factor 5-modified mRNA loaded into thermosensitive hydrogels, demonstrating that this novel delivery system can alleviate OA through mitochondrial signaling pathways. Such advancements indicate a shift towards more tailored exosomal therapies that could optimize treatment outcomes in OA.

In previous studies, the preconditioning of donor cells (hypoxia preconditioning or biochemical treatment) has been shown to enhance the therapeutic efficacy of exosomes (102), as highlighted by the findings of Zhao et al (102), where hypoxia enhanced the protective effect of ADSC-derived exosomes on lumbar facet joint OA. The application of exosomes derived from various stem cells represents a promising strategy for OA treatment, with preclinical studies revealing their potential to modulate inflammatory responses, promote cartilage repair and improve joint function (92,101,105,106). As research continues to unveil the complexities of exosomal biology and engineering, future studies should focus on optimizing exosomal therapies and translating these findings into clinical settings to address the considerable burden of OA.

Recent advancements in regenerative medicine have highlighted the potential of EVs, particularly those derived from umbilical cord tissues, as innovative therapeutic agents in the treatment of OA (110). The present review summarizes the current clinical research on the application of exosomes in OA therapy, focusing on findings and implications, while also highlighting the need for further studies.

Clinical trials have begun to explore the safety and efficacy of umbilical cord-derived Wharton's jelly (UCWJ) as a novel treatment for knee OA (95,96). In a study, UCWJ has been compared with traditional therapies, such as hyaluronic acid and saline, with the study design being a randomized, controlled, single-blind, multi-center trial (95). This approach is significant as it aims to evaluate not only the safety but also the potential benefits of UCWJ in improving knee function and alleviating pain in patients with OA. The anticipated results of the study are expected to shed light on the therapeutic benefits of UCWJ exosomes. Preliminary findings from earlier research have indicated that exosomes derived from Wharton's jelly may possess anti-inflammatory properties and the ability to enhance cartilage regeneration, providing a biological advantage over conventional treatments (95). These exosomes may stimulate cellular processes responsible for cartilage repair and modulate inflammatory pathways in the joint environment. Furthermore, Gupta et al (96) also conducted a non-randomized, open-label, multi-center trial to further assess the therapeutic potential of UCWJ in OA treatment. The focus was on the functional outcomes of patients as well as the severity of symptoms post-treatment. Collectively, these studies have highlighted the promising role of UCWJ in reducing pain and improving joint function in patients with OA. The findings underscore the potential for UCWJ to revolutionize current treatment options and enhance the quality of life of patients.

Currently, the research landscape regarding exosomal therapies in OA is expanding, with various studies underway (NCT06466850, NCT06431152 and NCT05060107). The emphasis is shifting towards understanding the underlying mechanisms by which exosomes influence cartilage health, inflammatory responses and joint function (73,111). In addition to UCWJ, studies are also exploring exosomes from other sources, such as ADSCs and bone marrow-derived stem cells (98,99). However, the current body of research remains limited, focusing primarily on specific types of exosomes rather than a broader spectrum of availed cellular sources. A number of ongoing studies (NCT06466850, NCT06431152 and NCT06463132) are expected to assess the impact of these therapeutic agents on various patient populations, different OA stages and their combinatory effects with other treatment modalities.

While the current clinical research indicates a promising direction for exosome-based therapies in OA (98,99), there is only one randomized controlled study and only preliminary efficacy has been achieved (95). Future studies should adopt more rigorous methodologies, including larger sample sizes and randomized controlled designs, to validate the efficacy and safety of exosome treatments. Additionally, research should focus on identifying predictive biomarkers that can help select the most suitable patients for exosome therapy, enhancing personalized treatment approaches. Continued clinical research and refinement of methodologies will be crucial for translating these findings into safe and effective therapies for patients, ultimately improving the management of OA.

A technical challenge in the field of exosome therapy lies in the isolation and characterization of exosomes (113), which can impact research and clinical applications. Exosomes are nanoscale EVs that vary in size, composition and functional roles, making standardization of isolation methods crucial (114). Currently, a variety of isolation techniques exist, including ultracentrifugation, ultrafiltration and commercial isolation kits, each with its advantages and disadvantages (114). Ultracentrifugation is highly effective for isolating pure exosomes but is time-consuming and requires specialized equipment. Ultrafiltration offers faster processing times and can concentrate exosomes but may lead to some loss of sample quality due to filter retention. Commercial separation kits are user-friendly and convenient but can be expensive and may result in lower yields. Overall, the choice of method depends on the specific research needs, sample type and available resources (114). One major issue is the lack of standardized protocols, leading to variability in yield and purity (115). As noted by Yadav et al (115), standardized reporting is crucial for ensuring rigor and reproducibility in exosome research. Furthermore, existing purification methods, such as ultracentrifugation and affinity chromatography, have limitations in specificity and efficiency (116). These challenges must be addressed to enhance the reliability of exosome studies and facilitate the eventual application of exosomes in OA.

The isolation process can lead to contamination with other EVs or proteins, compromising the purity and functional efficacy of the exosome preparation (117). Furthermore, variations in yield between and within different biological sources such as stem cells, platelets or other tissues can introduce inconsistencies in exosomal properties (118). As a result, the difficulty in achieving consistent and reproducible isolation hampers the ability to conduct comparative studies and can complicate the validation of treatment protocols.

In addition to isolation, there is a pressing need for reliable characterization techniques to assess the size, concentration and content of exosomes. Characterization methodologies typically involve nanoparticle tracking analysis, dynamic light scattering and electron microscopy, as well as profiling bioactive molecules through proteomic and genomic analyses (113). Fluctuations in these parameters can markedly influence the anticipated efficacy of the therapy (113), underscoring the need for robust quality control measures to ensure that only high-quality exosome preparations are utilized in clinical applications.

Another crucial technical hurdle is the development of effective delivery mechanisms that can ensure the successful targeting of exosomes to the desired tissues in patients with OA (119). While exosomes possess inherent targeting capabilities due to their lipid bilayer and surface markers that can facilitate cellular uptake, these natural targeting mechanisms alone are often insufficient for achieving optimal therapeutic outcomes (120). One major concern is the bioavailability of exosomes following administration. Traditional delivery routes such as intravenous or intra-articular injections may result in rapid systemic distribution and clearance, thereby limiting the local therapeutic concentration of exosomes at the target site, specifically the affected joints (121). Researchers are exploring innovative delivery strategies, including the use of hydrogels (110), nanoparticles (122) or other biomaterials as carriers for exosomes, which could enhance the retention and controlled release of exosomes at the joint site.

Optimizing the surface modification of exosomes to improve targeting specificity is critical. Genetic engineering or chemical modifications may enhance receptor-mediated targeting, increasing the likelihood of exosomes interacting with chondrocytes and other relevant cell types in the joint (123,124). Nonetheless, these adaptations may introduce complex regulatory and safety considerations, necessitating further research and validation before clinical use.

Biological variability in exosome content poses a challenge to the standardization and predictability of exosome-based therapies (125). The molecular makeup of exosomes, including proteins, lipids, mRNAs and non-coding RNAs, can differ based on their cellular source, culture conditions and even the physiological state of the donor organism (126). This variability can lead to inconsistent therapeutic effects when using exosomes derived from different sources or batches (127). For instance, exosomes derived from MSCs exhibit similar characteristic; however, their regenerative potential may differ based on the tissue source such as the bone marrow or adipose tissue. This inconsistency can make it challenging to predict treatment responses in patients with OA, as different exosomal preparations may yield varying degrees of anti-inflammatory effects or regenerative outcomes (127). Additionally, factors such as age, sex and the severity of OA can lead to alterations in exosome composition, further contributing to variability in experimental outcomes (127).

Variability in exosome isolation techniques can introduce discrepancies in the assessments of exosomal content (128). Methods such as ultrafiltration, precipitation and affinity-based techniques can yield exosomes of different purity and yield, complicating comparisons across studies (129). This variability poses challenges for the reproducibility and reliability of therapeutic results, as different exosomal profiles may yield divergent biological effects when applied in therapeutic contexts (129).

To tackle the challenges of exosome variability, it is crucial to standardize isolation and characterization protocols. Strict quality control measures, including the use of reference materials and calibration standards, alongside the establishment of consensus guidelines for exosome research, can enhance reproducibility (125). Additionally, integrating advanced techniques such as single-exosome characterization and multi-omics approaches will yield deeper insights into exosomal heterogeneity and its relevance to OA treatment (130). Coupling machine learning algorithms with high-throughput screening could further identify specific exosomal signatures associated with OA (131). Implementing standardized exosome handling and storage protocols will ensure consistency, ultimately aiding in the validation of exosomes as reliable biomarkers or therapeutic agents in OA management (132).

While the short-term effects of exosome therapies are gaining positive preliminary results, understanding the long-term outcomes and safety is crucial for establishing their clinical relevance (133). At present, to the best of our knowledge, there are no clinical data on long-term administration of exosomes for OA, especially in terms of immune response, potential immunogenicity and tissue side effects (78).

One concern is the possibility of unknown long-term effects on local tissues or systemic immunity (134). The administration of foreign exosomal material, even from autologous sources, may elicit immune responses in some patients, particularly in those with underlying inflammatory conditions (135). Long-term follow-up studies are essential to monitor potential adverse events associated with prolonged use of exosome-based therapies. Additionally, the regenerative effects of exosome therapies need thorough investigation in terms of sustainability. It remains unclear whether the benefits observed after treatment will persist over time or if repeat administrations will be required for sustained therapeutic effects. Ongoing research must focus on defining the optimal dosing strategies, frequency of administration and long-term monitoring to mitigate any adverse effects while maximizing regenerative benefits.

One of the most significant hurdles faced by exosome-based therapies is navigating the complex regulatory landscape. As biologics derived from living cells, exosomes fall under stringent regulations which vary across different regions and countries (136). Regulatory agencies require comprehensive data on the safety, efficacy, manufacturing and quality control of exosome-based products, which can be daunting for developers (137). The Food and Drug Administration categorizes exosomes as biological products (138); however, their specific classification can be ambiguous, leading to uncertainties in regulatory pathways. Unlike traditional pharmaceutical products, exosomes are derived from living cells, which introduces variability in their composition, function and manufacturing process, complicating the establishment of standardized protocols for their development and approval (139).

Regulations related to cell therapies impact exosome research. Current guidelines primarily focus on the source, manufacturing and clinical application of cells, often neglecting the unique aspects of exosome therapeutics. For example, compliance with Good Manufacturing Practices in the production of exosomes requires rigorous documentation and control of starting materials, processing, storage and quality testing (140). This can be particularly challenging given the inherent variability in exosome isolation techniques and characterization methodologies.

Safety assessments for exosome-based therapeutics must address risks associated with immune reactions or unintended biological effects, which are not yet fully understood (141). This necessitates extensive preclinical studies and clinical trials, aligning with a risk-benefit analysis approach typically reserved for more conventional therapies.

To navigate these regulatory hurdles, researchers must advocate for the development of specific guidelines tailored to exosome-based therapies. By engaging with regulatory agencies early in the research process, scientists can better clarify regulatory expectations and streamline the pathway for exosome applications in the treatment of OA. Establishing collaborative frameworks among researchers, clinicians and regulators will be crucial for advancing this field and ensuring that innovative exosome-based therapies can be safely and effectively translated into clinical practice.

The ethical considerations surrounding the sources of exosome isolation present another challenge in the development of exosome-based therapies. Exosomes can be derived from various sources, including stem cells, body fluids and tissues. However, ethical concerns may arise, especially when it comes to the need to obtain tissue samples or cells from human donors (142).

The use of embryonic stem cells for exosome production raises complex ethical dilemmas and concerns related to consent, donor rights and the implications of using embryonic tissue (143). Additionally, the collection of exosomes from human adipose or bone marrow sources necessitates ensuring that such procedures are performed ethically, with informed consent from donors and robust transparency regarding the implications of their contributions (144). Furthermore, as exosome therapies move forward, establishing clear guidelines and ethical frameworks for the use of both animal and human-derived materials will be crucial. This ensures that researchers and clinicians prioritize patient rights and societal norms while advancing the scientific understanding and therapeutic applications (145).