Worldwide Research Trends on Cell-Free Treatments Using Extracellular Vesicles for Osteoarthritis

Cell-free therapy based on EVs has surfaced as a groundbreaking approach in regenerative medicine, leveraging the curative capacity of cell-secreted bioactive substances, as opposed to the cellular components themselves, to promote tissue restoration and regeneration [48, 49], and offering an alternative to traditional cell-based therapies that require direct transplantation of living cells into the target tissue [28]. These vesicles, including exosomes and MVs [50], made up of various functional substances, such as proteins, lipids, and nucleic acids [20], which transport to affected cells within injured joint structures, subsequently altering their responses and capabilities [51, 52]. Among these alternatives, vesicles released by MSCs have display favorable medicinal impacts in multiple test scenarios, such as heart-related illnesses, sudden kidney failure, hepatic impairment, and lung tissue damage [52 –56]. These vesicles influence immune system activity by decreasing the release of inflammation-related proteins, such as IL-1β and TNF-α, and by fostering an environment conducive to tissue regeneration [16]. Meanwhile, gene-modifying activities driven by miRNAs are essential for the medicinal properties of cell-free approaches, with EVs being enriched in miRNAs that can modulate the expression of genes involved in inflammation, autophagy, and other relevant pathways. Studies indicate these vesicles promote macrophage conversion from inflammatory M1 state to regenerative M2 form, supporting decreased joint inflammation [23, 57]. Additionally, studies reveal that EVs enhance cartilage matrix restoration in osteoarthritic tissue by reducing levels of proteins that break down matrix components, including MMP13 and ADAMTS5, thereby maintaining cartilage structure and delaying OA development [32, 46, 58, 59]. Moreover, EVs modify chondrocyte metabolic processes by boosting cellular energy production and ATP generation, thus strengthening their performance and stress resistance [60]. The cell homing properties of certain EVs, mediated by chemokines and other signaling molecules, allow for the recruitment of endogenous stem cells or progenitor cells to the site of injury, where they can participate in the tissue repair and regeneration process [61]. Some studies have also revealed that microparticles can upregulate the expression of type II collagen, suppress the expression of inflammatory markers and macrophage activation, protect chondrocytes from apoptosis, and improve cartilage parameters [30]. These microparticles exhibit therapeutic effects similar to those of exosomes, thus playing a positive role in the treatment of OA [62]. Beyond the direct actions of EVs, this treatment also enhances tissue regeneration through paracrine effects, with therapeutic cells secreting various forms of cytokines, growth factors, and other bioactive molecules that modulate the microenvironment of the damaged joint tissues, suppressing inflammation, promoting blood vessel formation, and modulating immune responses [63, 64]. Cell-free approaches target various OA mechanisms, offering an integrated solution for joint disease while advancing innovative regenerative treatments in orthopedic medicine [65, 66].

OA, a common joint disorder, causes progressive degeneration that significantly diminishes patient well-being and daily functioning. As researchers seek to better understand the pathogenesis of this debilitating condition and develop more effective treatments, they have turned their attention to the role of EVs in the disease process. Exosomes and MVs, subclasses of EVs, have garnered significant attention in the pathogenesis and treatment of OA. The mechanisms underlying their roles have emerged as a focal point of research in this field. Cosenza et al. discovered that EVs derived from MSCs possess a chondroprotective effect, capable of mitigating IL-1β-induced chondrocyte apoptosis and matrix degradation [30]. Tao et al. identified key miRNAs, including miR-140-5p, EVs isolated from synovial fluid MSCs of individuals with OA. They found that these miRNAs promote chondrocyte proliferation and migration, suppress the expression of MMP-13 and ADAMTS-5, and are essential for EV-facilitated cartilage restoration [32]. Wu et al. further elucidated that MSC-derived EVs transport miR-100-5p and other miRNAs to inhibit the mTOR pathway in macrophages, thereby attenuating synovial inflammation and exerting an immunomodulatory mechanism [23]. Liu et al. further revealed that the lncRNA KLF3-AS1 in MSCs-exos could adsorb miR-206 by sponging, thus alleviating the suppressive impact of miR-206 on GIT1. Therefore, EVs mediate the restraining influence of the lncRNA-KLF3-AS1/miR-206/GIT1 axis [67]. Taken together, EVs, as essential intercellular communication mediators, regulate the functions of chondrocytes and immunomodulatory cells through the transfer of functional molecules such as non-coding RNAs. They are essential to the development and treatment of OA, emerging as a frontier and hotspot in precision medicine and regenerative medicine research.

Exosomes, a type of vesicles secreted by cells, have developed into a novel drug delivery vehicle owing to their distinctive advantages in various aspects, such as biocompatibility, stability, and ability to cross biological barriers [68, 69]. Exosomes possess a multitude of unique properties, for example capability to cross biological boundaries, strong blood circulation persistence, and precise targeting ability, rendering them outstanding drug delivery vehicles. These distinctive features can be attributed to their biological composition and tiny dimensions, typically ranging from 40 to 100 nm [68, 70]. Engineered exosomes, which have undergone surface modifications including genetic engineering and chemical alterations, demonstrate improved targeting abilities. These adjustments elevate regional concentrations, reduced immunogenicity, minimal toxicity, and strong engineerability, thereby enhance their potential for treating numerous medical conditions [71]. Beyond serving as drug delivery vehicles, exosomes have natural properties that support osteoarthritic cartilage restoration. These inherent capabilities include the mitigation of local inflammation, the promotion of anabolic metabolism, and the suppression of catabolic processes [27, 72]. Several genes within chondrocytes have been identified as closely related to the progression mechanism of OA. Consequently, genome editing is considered a possible therapeutic approach that could offer long-term benefits for patients suffering from this debilitating condition [73, 74]. Liang et al. genetically engineered the exosomal surface protein Lamp2b to express a chondrocyte-affinity peptide on the exosome exterior, allowing vesicles to locate cartilage cells and be retained within the joint after intra-articular injection. Moreover, he combination of exosomes and lipid vesicles allowed the resulting hybrid exosomes to encapsulate CRISPR/Cas9 plasmids meanwhile maintaining the chondrocyte-homing properties, thereby delaying cartilage degradation in vivo and accelerating bone tissue repair [75].

Chen et al. researched the relationship between FGF18 silencing and the short of assorted cartilage-specific genes with in vivo and in vitro studies of OA progression. They s effectively created a innovative in vivo approach founded on CRISPR/Cas9 technology to activate the FGF18 gene in OA chondrocytes on a genetic basis. This method endowed chondrocytes with targeting capabilities, thereby enhancing in vivo binding efficiency and biosafety [76].

Zhao et al. utilized engineered exosomes derived from subcutaneous (SC) adipose tissue-derived MSCs-exos to achieve targeted transport of miR-199a-3p into chondrocytes. This approach was feasible due to the relative ease of obtaining healthy donor subcutaneous adipose tissue. In an OA mouse model, this targeted delivery system displayed considerable cartilage regeneration [77].

Wan et al. designed a novel engineered exosome-based drug delivery system to alleviate OA symptoms. They chemically modified exosomes by acylating methacrylic anhydride with the amino groups at the exosomal membrane, forming olefin double bond-modified exosomes. Through acylation with the peptides' amino groups, targeting peptides were conjugated to the modified exosomes, resulting in peptide-exosome binding. The engineered exosomes, modified with the cartilage-affinity peptide WYRGRL, were encapsulated in a new light-crosslinked spherical gelatin methacryloyl hydrogel (GelMA) for OA treatment. The performance of the engineered exosomes abundant in the small inhibitor LRRK2-IN-1 (W-Exo-L@GelMA) was surveyed both in cell cultures and animal models. W-Exo@GelMA evidenced effective chondrocyte targeting and markedly suppressed the expression of genes related to inflammation and immunity in OA [78]. These innovative, in-depth and extensive studies have laid an extremely solid foundation for the cell-free treatment of OA.

Looking ahead, several challenges remain. To advance exosome-centered OA therapy, standardization of exosome isolation and characterization protocols must be achieved, effective regulatory processes must be implemented, and therapeutic efficacy must be validated through clinical trials [79].

Recently, the development of novel delivery systems has become a focal point of investigation in OA therapy. The treatment approaches for OA can be divided into two primary groups: pharmacological and non-pharmacological interventions. Pharmacological therapies can be further subdivided based on the route of administration, which includes oral, injectable, and topical modalities [80]. An ideal drug delivery platform should efficiently cross biological barriers and precisely release the necessary quantity of active compounds at the designated time and location. Utilizing naturally derived materials with excellent biocompatibility for targeted drug delivery to specific cells or molecules ensures the safety of the drug delivery vehicle [81 –83].

The microenvironment of osteoarthritic joints is characterized by distinct pathological changes. These changes are relevant to factors such as pH levels, reactive oxygen species (ROS), and enzyme concentrations. Consequently, endogenous stimuli-responsive mechanisms that are sensitive to these conditions are currently under development. Nanoplatform-based drug delivery systems present innovative solutions for precise drug administration. These platforms enable enhanced drug accumulation and release kinetics at targeted disease locations, with the ability to respond to both endogenous and exogenous triggers for controlled therapeutic delivery. This strategy improves therapeutic uptake while reducing adverse effects throughout the body [81, 84]. To date, multiple delivery vehicles engineered for cartilage specificity have been created that facilitate the penetration of the compact ECM and improve therapeutic transport. Notably, nanoparticles incorporating chondrocyte-affinity peptides exhibit prolonged retention within the cartilage, causing them to become viable alternatives for the treatment of OA. In this burgeoning area of drug delivery study, the surface of exosomes is altered to boost their capabilities, facilitating site-specific drug delivery, along with in vivo visualization and tracing. Notably, nanoparticles incorporating chondrocyte-affinity peptides exhibit prolonged retention within the cartilage, making them promising candidates for the treatment of OA. Surface modification of exosomes in this innovative area of drug delivery research aims to augment their capabilities, facilitating targeted drug delivery, in vivo imaging, and monitoring [85].

Some researchers have attempted to establish an effective delivery strategy to enable exosomes to achieve continuous release in the superficial defect area [86]. According to a study carried out by Zhang et al., an injectable hydrogel with robust bonding properties to the moist chondral tissue was prepared through the enzymatic cross-linking of alginate-dopamine, chondroitin sulfate, and regenerated silk fibroin (AD/CS/RSF). The interfacial shear strength of the hydrogel reached 120 kPa. By encapsulating exosomes within the hydrogel, endogenous BMSCs in a rat model were recruited to the lesion location through chemoattractant transduction cascades and induced to differentiate into chondrocytes, assisting in the restoration of chondral lesions in the trochlear groove. This research has yielded an encouraging bioengineered substance for individuals with shallow chondral lesions, potentially deliverable via minimally invasive arthroscopic techniques [87]. Chen et al. designed a bioscaffold for the delivery of MSCs-exos and fabricated a 3D-printed scaffold composed of chondrocyte ECM, gelatin methacrylate (GelMA), and exosomes with radial channels utilizing desktop stereolithography technology. In a rabbit osteochondral lesion model, the ECM/GelMA/exosome scaffold productively rehabilitated chondrocyte mitochondrial dysfunction, increased chondrocyte motility, as well as polarized the response of synovial macrophages towards the M2 phenotype. The 3D printed scaffold remarkably promoted cartilage restoration in the in vivo study. The approach may serve as an encouraging approach for the therapy of OA [88]. Zeng et al. designed a multifunctional hydrogel system inspired by mussels, which exhibited thermosensitivity, self-healing properties, and adhesive characteristics. This system enabled the co-delivery and complementary actions of MSCs-exos and icariin (ICA). The intra-articular injection system combining ICA and MSCs-exos is expected to be retained within the joint cavity, alleviate cartilage atrophy, and ensure proper cartilage thickness [27].

Future research will focus on developing novel EVs-biomaterial composite carriers, optimizing 3D printing processes, and improving their loading capacity and bioavailability. Furthermore, EVs modification strategies, such as lipid insertion metabolic labeling, and peptide CAQK-modified, are important means to enhance the pharmacokinetic properties of EVs [89 –92]. Continued efforts to strengthen the fundamental research and industrialization exploration of exosome engineering, along with the establishment of standardized technical guidelines and translational pathways, will undoubtedly promote the widespread application of engineered exosomes in the precise diagnosis and treatment of OA. With the progress in nanotechnology, tissue engineering, and other fields, novel EVs delivery systems are anticipated to assume a more significant function in the identification and management of degenerative joint disease.