Osteoarthritis (OA), the most prevalent chronic degenerative joint disease, is characterized by articular cartilage deterioration, subchondral bone remodeling, osteophyte formation, and synovial inflammation. With an aging global population, OA's prevalence is increasing, affecting over 300 million people worldwide, with over 40% being over 70 years old. Articular cartilage, composed of chondrocytes and extracellular matrix (ECM), forms a functional unit with the subchondral bone, influencing OA development. Chondrocytes, the main cartilage cells, synthesize and maintain ECM components like lubricin, glycoproteins, and type II collagen (COL2). Physiological loading benefits chondrocytes, maintaining cartilage integrity, while abnormal loading leads to degeneration. Inflammatory mediators like interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) negatively affect chondrocyte function, accelerating cartilage degradation. In OA, homeostasis is disrupted; chondrocytes undergo hypertrophy, secreting matrix-degrading enzymes (ADAMTS5, MMP-3, MMP-13), leading to cartilage degeneration. Cartilage's limited regenerative capacity stems from its avascular nature and low cell turnover. Subchondral bone provides mechanical support and undergoes dynamic remodeling. In early OA, accelerated bone resorption precedes cartilage degeneration, with subsequent destruction occurring in areas of decreased subchondral bone plate thickness. As OA progresses, subchondral bone resorption slows, resulting in abnormal thickening of the growth plates. Current OA treatments include early medication (NSAIDs, glucosamine, hyaluronic acid, chondroitin sulfate) for symptom relief and surgery (microfracture, chondrocyte implantation, arthroplasty) when conservative methods fail. These treatments are costly and may have adverse effects, highlighting the need for early intervention and improved cartilage reconstruction. Cell-based therapies, particularly using mesenchymal stem cells (MSCs), show promise in cartilage repair; however, challenges like potential tumorigenicity and ethical considerations exist. MSC-derived extracellular vesicles (MSC-EVs), particularly exosomes, offer an advantageous alternative due to their low immunogenicity, stability, ease of storage, and ability to fuse with target cells.

Numerous preclinical studies demonstrate that injecting MSCs into the joint cavity enhances cartilage regeneration and reduces synovial inflammation, alleviating OA progression. While a systematic review showed MSC-based therapy significantly reduces pain and improves joint function, clinical implementation faces challenges. Recently, evidence suggests MSC-derived extracellular vesicles (MSC-EVs), including exosomes, play a crucial role in intercellular communication, retaining parental cell properties. Exosomes, nanoscale membrane-bound vesicles, are found in various bodily fluids and are derived from diverse cell types. They deliver bioactive molecules, influencing physiological and pathological processes like cellular homeostasis, apoptosis regulation, and inflammation modulation. Studies show that MSC-Exos are effective for tissue repair and immunomodulation and can attenuate OA progression by reducing cartilage degradation and enhancing chondrocyte phenotype. A completed clinical trial demonstrated that ExoFlo (BM-MSC-Exos product) significantly reduced pain and improved joint function after six months, showing safety and efficacy.

This study is a review article. The authors systematically reviewed existing literature on the application of various MSC-derived exosomes (MSC-Exos) for the treatment of osteoarthritis (OA). They explored the potential mechanisms of action of MSC-Exos, including their effects on cartilage repair, anti-inflammatory responses, immunomodulation, and extracellular matrix (ECM) balance. The review also analyzed current engineering strategies for improving the therapeutic efficacy of MSC-Exos, including cargo loading strategies (pre-loading and post-loading), surface modification techniques to enhance targeting, strategies to optimize the production environment (3D vs 2D culture, hypoxia, mechanical stimulation), and the use of biomaterials (hydrogels, ECM-derived scaffolds) for improved retention and delivery. The authors examined various sources of MSCs and the impact of these sources on the therapeutic efficacy of the derived exosomes. Specific studies cited in the review are extensively detailed, including methods employed, outcomes observed, and implicated mechanisms. The review considered in vitro and in vivo studies, noting the specific techniques used such as cell culture, animal models, and relevant molecular assays to assess the effects of MSC-Exos on various parameters. The study integrated multiple sources to provide a comprehensive overview of current knowledge, challenges, and future directions in this field. Figures and tables were utilized to visually represent the key findings and mechanisms of MSC-Exos in OA treatment. A thorough exploration of the literature, focusing on both preclinical and clinical trials (including a completed clinical trial on ExoFlo), provides a solid basis for the review's conclusions and future outlook on MSC-Exos-based therapy.

MSC-Exos demonstrate potential in mitigating OA progression through several mechanisms: **Effect on Cartilage Repair:** MSC-Exos enhance chondrocyte proliferation and migration, promoting cartilage restoration. Studies using exosomes from various MSC sources (induced pluripotent stem cell-derived MSCs, synovial MSCs, bone marrow-derived MSCs, umbilical cord-derived MSCs, Wharton's jelly MSCs, embryonic stem cell-derived MSCs) showed improved chondrocyte viability, proliferation, and migration. Specific mechanisms involved include improved mitochondrial activity, interaction with microRNAs (e.g., circHIPK3, miR-124-3p, miR-100-5p, miR-26a-5p), and long non-coding RNAs (e.g., LYRM4-AS1). MSC-Exos also inhibit chondrocyte apoptosis by influencing signaling pathways involving phosphorylation, affecting the Bcl-2/Bax ratio, and targeting proteins like NOX4. **Anti-inflammatory Response and Immunomodulation:** MSC-Exos regulate inflammatory responses by reducing pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and promoting anti-inflammatory cytokines (IL-4, IL-10, TGF-β). They modulate macrophage polarization towards the anti-inflammatory M2 phenotype, impacting the intra-articular microenvironment. Specific microRNAs within MSC-Exos (e.g., miR-24-3p, miR-222-3p, miR-146a-5p, miR-34a-5p, miR-181a-5p, miR-100-5p, miR-let-7a-5p, miR-122-5p, miR-486-5p, miR-148a-3p, miR-9-5p, miR-145, miR-221, miR-129-5p) contribute to these effects. MSC-Exos can prevent macrophage ferroptosis. **Maintain ECM Balance:** MSC-Exos regulate ECM balance by downregulating matrix-degrading enzymes (ADAMTS-5, MMP-3, MMP-13) and upregulating tissue inhibitors of metalloproteinases (TIMPs), COL2, glycosaminoglycans (GAGs), and SOX9. Specific exosomal RNAs (e.g., miR-320c, miR-3960, miR-125a-5p, miR-155-5p, miR-1208) play crucial roles. The study highlighted the importance of maintaining the balance of ECM synthesis and degradation for the preservation of cartilage integrity. **Engineering Strategies:** The review explored engineering strategies to overcome the limitations of natural exosomes, such as low yield and inadequate targeting. Strategies include cargo loading (overexpression of non-coding RNAs, encapsulation of small molecule drugs, and proteins), surface modification (ligand incorporation to enhance targeting, e.g., chondrocyte-binding peptide (CAP), MSC-binding peptide E7, chitosan oligosaccharides (COS)), and optimization of the production environment (3D culture, hypoxia, mechanical stimulation). Biomaterials, such as hydrogels (GelMA) and ECM-derived scaffolds, are used for improved retention and delivery.

This comprehensive review highlights the significant potential of MSC-Exos as a cell-free therapeutic strategy for knee OA. The multifaceted mechanisms of action, encompassing cartilage repair, immunomodulation, and ECM balance, underscore their therapeutic promise. The findings address the need for effective and less invasive treatment options for OA, particularly considering the limitations of existing therapies. The engineering strategies discussed provide avenues for enhancing the efficacy and clinical translation of MSC-Exo therapy. The diverse roles of specific microRNAs and non-coding RNAs within MSC-Exos suggest potential targets for future therapeutic development. The discussion emphasizes the need for further investigation and standardization in MSC-Exo production and characterization. The effectiveness of MSC-Exos across different MSC sources highlights the importance of carefully considering the origin of MSCs for optimal therapeutic outcomes.

MSC-Exos represent a promising cell-free therapy for knee OA. Their diverse mechanisms of action, combined with ongoing advancements in engineering strategies, support their potential as an effective alternative to joint replacement surgery. Future research should focus on standardization of production methods, large-scale production, and rigorous clinical trials to confirm their efficacy and safety profiles. Addressing challenges related to MSC source selection and ensuring sufficient delivery of bioactive factors are crucial for successful clinical translation.

While the review provides a comprehensive overview, limitations exist. The majority of studies reviewed are preclinical, primarily using small animal models. The translation to larger animal models and subsequent clinical trials is crucial before widespread clinical application. Variations in MSC-Exo preparation across studies may influence results. Furthermore, the complex composition of exosomes necessitates thorough characterization to understand their precise functions and improve therapeutic outcomes. The need for large-scale, standardized production methods remains a significant challenge for clinical translation. Finally, the potential for immune responses to engineered exosomes needs further investigation.