The conventional method of culturing mesenchymal stem cells (MSCs) involves a two-dimensional (2D) monolayer setup. However, this approach has limitations, particularly in terms of proliferation efficiency, falling short of meeting the clinical demand for high doses of MSCs. Furthermore, the development and functionality of MSCs are significantly influenced by molecular interactions within their environment. Traditional 2D cultures fail to replicate the spatial distribution, nutrient transfer, cell migration, and mechanical stimulation characteristics of a three-dimensional (3D) extracellular matrix (ECM) microenvironment. This discrepancy can lead to variations in gene expression, signaling, and morphology of MSCs from their natural state (Li et al., 2015), potentially impacting the biological function of exosomes.

Recognizing these limitations, the 3D culture and amplification of MSCs using 3D printed scaffolds have gained widespread acceptance. Microcarriers (MCs), consisting of hydrogel microparticles made from materials like gelatin, hyaluronic acid, collagen, or natural biological macromolecules such as chitosan, sodium alginate, and their derivatives combined with polyethylene glycol, offer a promising solution (Figure 2) (Tavassoli et al., 2018). These MCs present several advantages, including facilitating large-scale cell culture and expansion, mimicking in vivo 3D microenvironments, establishing cell-cell and cell-ECM interactions, and promoting gene expression and secretion activities akin to in vivo conditions (; Tseng et al., 2016; Shekaran, et al., 2016).

The extraction of exosomes typically involves techniques such as overspeed centrifugation, ultrafiltration, immunoaffinity capture, charge-neutralization-based polymer precipitation, size exclusion chromatography, and microfluidic separation (Wu J. et al., 2019). These methods yield exosomes with varying levels of purity, yield, size, surface potential, and inclusion composition, and currently, there is no standardized extraction protocol (Yang et al., 2019). The identification of exosomes is predominantly based on their morphological characteristics under electron microscope, particle size specific protein markers (Zhao X. et al., 2021). However, the efficacy of these identification methods can be influenced by the quality of the kits used and other external factors (Momen-Heravi F. 2017).

To enhance tissue regeneration efficiency, further enrichment of functional components in exosomes is crucial (Sun et al., 2021). The enrichment of exosomes with functional components is primarily achieved through either the overexpression of these components in parental cells or direct delivery to the exosomes (Pegtel and Gould, 2019). The former approach, involving genetic expression of functional components by parental cells and their subsequent secretion into exosomes, faces challenges in specifically regulating functional components and often results in low yields of exosomes enriched with targeted components (Sun et al., 2021). In contrast, physical and chemical methods such as incubation, electroporation, heat shock, ultrasound, freeze-thaw cycles, extrusion, gradient pH regulation, and transient penetration of phospholipid bilayers have proven more efficient and feasible (Colombo et al., 2014; Kalluri and LeBleu, 2020; Mondal et al., 2023). These methods enhance the interaction of target functional components with the exosomes’ surface.

The lipid bilayers of exosomes play a crucial role in shielding internal functional components from external environmental damage. However, their thermodynamic instability during storage makes them susceptible to alterations in the medium, temperature, time, and physical and chemical properties. Such changes can result in the loss of internal functional components, formation of multilayer vesicle structures, and aggregation. Repeated freeze-thaw cycles can also alter the lipid bilayer’s marker characteristics and composition (Maroto et al., 2017).

When administered subcutaneously, intravenously, or intraperitoneally, exosomes are known to accumulate in organs like the liver, spleen, lungs, and gastrointestinal tract within 2 h and are rapidly cleared by macrophages (Takahashi et al., 2013). The incorporation of exosomes into hydrogels for sustained release can mitigate the low tissue retention observed with direct injections. However, this passive diffusion release mechanism is difficult to control due to the influence of hydrogel porosity and degradation rate. Moreover, the negative charge on both exosome and cell membranes creates electrostatic barriers that reduce the bio-utilization efficiency of exosomes (Feng et al., 2021).

Surface modification of exosomes can enhance their physicochemical properties. Sushil et al. utilized cholesterol-modified DNA strands for complementary pairing and embedding a polymer onto the exosome membrane surface, improving stability and therapeutic efficacy without altering in vivo distribution or specific targeting. However, this method’s chemical synthesis complexity and the temperature-sensitive annealing of DNA chain segments may impact exosome activity. In contrast, Feng et al. (2021) demonstrated the incubation of exosomes with polylysine (ε PL)-PEG-distearoyl phosphatidylethanolamine (DSPE) (PPD), integrating the DSPE segment into the exosome membrane while reversing the ε PL segment from electronegative to electropositive. This modification increased exosome uptake, penetration, and retention in cartilage, thereby enhancing OA treatment effects (Lathwal et al., 2021). While this approach is simpler and does not affect the structure and content of exosomes, it results in increased exosome size and reduced cartilage absorption rate.

Exosomes derived from bone marrow MSCs play a crucial role in mitigating chondrocyte injury in OA induced by IL-1β, with miR-3960 emerging as a principal effector. In chondrocytes, the increase in PHLDA2 expression activates the Wnt/β-catenin pathway through the upregulation of SDC1 expression, leading to the degradation of the cartilage extracellular matrix due to elevated levels of MMP-13 and ADAMTS5, alongside reduced levels of collagen II and aggrecan. However, miR-3960, enriched in the exosomes secreted by MSCs, can be delivered to chondrocyte cells, subsequently degrading PHLDA2 mRNA. Moreover, the expression profiles of miR-3960, PHLDA2, SDC1, and β-catenin in cartilage tissues from OA patients align with findings from their prospective study (Ye et al., 2022).Xia Qingqing et al. identified a negative correlation between miR-125a-5p and E2F2 in the cartilage of traumatic osteoarthritis patients and in a corresponding mouse model. The exosomes derived from bone marrow MSCs, which are taken up by chondrocytes in mice, contain miR-125a-5p that suppresses E2F2 expression. This downregulation leads to decreased MMP-13 levels and increased levels of collagen II, aggrecan, and SOX9, thereby facilitating chondrocyte migration and alleviating extracellular matrix degradation (Xia et al., 2021).Through miRNA microarray analysis, Mao Guping et al. found that miR-92a-3p levels in MSC exosomes were upregulated following chondrogenic induction in bone marrow MSCs, but downregulated in OA chondrocyte-secreted exosomes compared to normal cartilage. MiR-92a-3p suppresses MMP-13 and RUNX2 expression while enhancing aggrecan, COL2A1, and SOX9 expression, thereby inhibiting cartilage degradation—a process regulated by reduced WNT5A expression in chondrocytes (Mao et al., 2018).Sun Hao et al. demonstrated that miR-320c, enriched in exosomes secreted by bone marrow MSCs undergoing chondrogenic differentiation, promotes osteoarthritis chondrocyte proliferation, enhances SOX9 expression, and inhibits MMP-13 expression. This contributes to chondrogenesis induction and inflammation suppression in chondrocytes (Sun et al., 2019).Chen Xue et al. observed higher ELF3 levels and lower miR-136-5p levels in traumatic OA cartilage tissues compared to healthy cartilage. Exosomes secreted by bone marrow MSCs, rich in miR-136-5p, are internalized by chondrocytes, leading to downregulated ELF3 expression, which in turn alleviates cartilage degeneration and enhances chondrocyte migration through the upregulation of collagen II, aggrecan, and SOX9, and the downregulation of MMP-13 expression (Chen et al., 2020).DDX20, identified as a key regulator in chondrocyte damage induced by IL-1β, activates the NF-κB signaling pathway, promoting chondrocyte damage through increased expression of MMP-3 and MMP-13. Exosomes derived from bone marrow MSCs can counteract this effect by downregulating DDX20 levels, thanks to the enrichment of miR-361-5p within them (Tao et al., 2021).Liu Yubao et al. demonstrated that in a mouse OA model, MSC-exosomes promoted chondrocyte proliferation, inhibited apoptosis, and maintained cartilage homeostasis. This was achieved through the upregulation of COL2A1 and aggrecan and the downregulation of MMP-13 and RUNX2, mediated by the lncRNA-KLF3-AS1/miR-206/GIT1 axis (Liu et al., 2018).

Exosomes derived from synovial MSCs have demonstrated a capacity to foster chondrocyte regeneration, although they did not show benefits for ECM synthesis. These exosomes aid in OA prevention by encouraging chondrocyte proliferation and migration and by reducing apoptosis, yet they do not affect ECM secretion. However, exosomes overexpressing miR-155-5p were found to enhance ECM secretion in OA chondrocytes through the degradation of Runx2 (Wang et al., 2021). In a similar vein, Tao Shicong et al. reported that exosomes from synovial MSCs could activate YAP through the delivery of wnt5a and wnt5b, key activators of the Wnt signaling pathway. This activation leads to increased chondrocyte proliferation and migration, albeit at the expense of ECM secretion. Nonetheless, this limitation could be addressed by miR-140-5p, which elevates the levels of SOX9, aggrecan, and collagen II by inhibiting RalA (Tao et al., 2017). Further, Qiu Min et al. observed a reduced expression of miR-129-5p in the cartilage tissues of OA patients and in chondrocytes induced by IL-1β, while HMGB1 was more highly expressed compared to healthy tissue. Exosomes from human synovial MSCs, rich in miR-129-5p, were able to elevate its levels in chondrocytes, subsequently downregulating HMGB1. Consequently, there was a reduction in the inflammatory response and apoptosis of chondrocytes (Qiu et al., 2021).

Yan Litao et al. demonstrated that mechanical stimulation during cell culture could amplify the secretion of exosomes by umbilical cord mesenchymal stem cells. These exosomes proved advantageous for repairing cartilage defects, a benefit attributed to the encapsulation of LncRNA H19 (Yan et al., 2020). Zhou Hao et al. further revealed that exosomes derived from umbilical cord mesenchymal stem cells exerted a protective effect on OA, as evidenced in a mouse OA model created by surgical destabilization of the medial meniscus. The protective mechanism was attributed to exosomal miR-1208, which reduced the m6A modification level of NLRP3 mRNA by targeting METTL3 mRNA, leading to a decrease in pro-inflammatory factor levels. Consequently, chondrocyte regeneration was enhanced due to increased proliferation and migration and reduced apoptosis. Additionally, ECM degradation was curtailed through the upregulation of COL2A1 and aggrecan and the downregulation of ADAMTS5 and MMP-13 (Zhou et al., 2022).

Exosomes secreted by urine-derived stem cells cultured under hypoxic conditions were found to be enriched in miR-26a-5p, which effectively inhibits PTEN expression in chondrocytes, thereby promoting their proliferation and migration (Wan et al., 2022). Moreover, exosomes from human urine-derived stem cells were shown to enhance the proliferation and migration of chondrocytes induced by IL-1β, while concurrently inhibiting apoptosis, albeit with a reduction in ECM secretion. Intriguingly, when overexpressed with miR-140-5p, these exosomes facilitated the upregulation of collagen II and aggrecan. Mechanistically, miR-140-5p was found to decrease VEGFA levels in cartilage and synovial fluid samples obtained from patients with knee osteoarthritis or from a rat model of the disease (Liu et al., 2022).

In vitro studies reveal that exosomes derived from adipose stem cells effectively downregulate the expression of prostaglandin E2, IL-6, IL-1β, and TNF-α in the murine chondroprogenitor cell line ADTC5. The encapsulated miR-338-3p within these exosomes is delivered to interleukin-1β-induced ADTC5 cells, promoting the degradation of RUNX2 mRNA. Consequently, this results in the inhibition of cell degradation and apoptosis, alongside the promotion of cell proliferation (Li et al., 2022).Wu Jiangyi et al. demonstrated that exosomes secreted by infrapatellar fat pad MSCs can maintain cartilage homeostasis by enhancing autophagy levels, attributed to the inhibition of mTOR. The encapsulated miR-100-5p within these exosomes can bind to the 3′-untranslated region (3′UTR) of mTOR, promoting its degradation. The exosomes ameliorated OA severity in vivo and inhibited cell apoptosis, enhanced matrix synthesis, and reduced the expression of catabolic factors in vitro. Given the relative ease of harvesting infrapatellar fat pad MSCs from OA patients, this study may guide future clinical therapies for OA (Wu X. et al., 2019).