Strategies for Cartilage Repair in Osteoarthritis Based on Diverse Mesenchymal Stem Cells‐Derived Extracellular Vesicles

Abstract

Osteoarthritis (OA) causes disability and significant economic and social burden. Cartilage injury is one of the main pathological features of OA, and is often manifested by excessive chondrocyte death, inflammatory response, abnormal bone metabolism, imbalance of extracellular matrix (ECM) metabolism, and abnormal vascular or nerve growth. Regrettably, due to the avascular nature of cartilage, its capacity to repair is notably limited. Mesenchymal stem cells‐derived extracellular vesicles (MSCs‐EVs) play a pivotal role in intercellular communication, presenting promising potential not only as early diagnostic biomarkers in OA but also as efficacious therapeutic strategy. MSCs‐EVs were confirmed to play a therapeutic role in the pathological process of cartilage injury mentioned above. This paper comprehensively provides the functions and mechanisms of MSCs‐EVs in cartilage repair.

We summarized the functions and mechanisms of extracellular vesicles in cartilage repair (including protecting excessive chondrocyte death, regulating the generation of osteoblasts and osteoclasts, balancing extracellular matrix metabolism, alleviating inflammatory response, regulating angiogenesis, etc.) and presented our views, which confirmed that exosomes are potential strategies for cartilage repair.

Introduction

Osteoarthritis (OA) often occurs in the joints subjected to high movements, including the knees, shoulders, spine, hips, elbows, wrists, ankles, and even the temporomandibular joint. ¹ The articular surface, joint capsule, and joint cavity form the joint, allowing human body to perform complex movements. The articular surface is the contact surface of all relevant bones involved in the composition of the joint, which is covered by articular cartilage that makes the rough and uneven articular surface smooth, reduces friction, and cushions the impact on the articular surface during intensive physical activities. ² OA progressions are often presented by progressive degeneration of articular cartilage, aseptic inflammation, restricted joint movement, and symptomatic pain, with the consequences of severe disability, substantial economic burden, and deteriorated life quality. ³ Cartilage damage is often considered one of the most important progression of OA and is often accompanied by the destruction of cartilage structures. Mainstay treatments for OA include conservative management (physiotherapy and pharmacotherapy) and surgical intervention. However, both conservative and surgical treatment inevitably have negative effects in cartilage repair. ⁴ , ⁵ , ⁶ To make matters worse, articular cartilage, similar to the intervertebral disc, is avascular, relying almost entirely on its intrinsic nutrient supply, resulting in a severely limited self‐repair ability once damaged. ⁷ Therefore, neither conservative nor surgical treatments aimed at symptom relief have been successful to repair the damaged cartilage or delay the degeneration process. As a result, extensive studies aim to repair the damaged cartilage are ongoing. Early studies validated that platelet‐rich plasma (PRP) can effectively mitigate OA symptoms, though the clinical effectiveness of PRP remains controversial, with randomized controlled trial results indicating no improvement in OA. ⁸ Cartilage damage is a principal pathological feature of OA, typically presenting as chondrocyte loss, activated inflammatory response, and imbalanced cartilage matrix metabolism (including excessive collagen, proteoglycans, and water consumption) induced by various detrimental conditions. ⁹

A great deal of research has been devoted to developing new strategies for cartilage repair. Although mesenchymal stem cells (MSCs) have shown considerable promise in the field of tissue engineering, however, their use in cell therapy is hindered by potential immune rejection and tumorigenicity. Interestingly, in 2005, extracellular vesicles (EVs) containing IL‐10 and EVs derived from IL‐10‐treated dendritic cells showed strong anti‐inflammatory properties when injected around the joint. ¹⁰ While in 2015, EVs derived from MSCs (MSCs‐EVs) demonstrated superior efficacy in treating OA compared to MSCs, as evidenced by histological improvements. Consequently, some experts posit that EVs could serve as a viable alternative to cell therapy for OA, although more studies are required to substantiate this notion. ¹¹ , ¹² This also lay the foundation for the subsequent treatment of cartilage injury with EVs. Gradually, EVs are considered to be the latest candidates for cartilage injury, and study has demonstrated that EVs show excellent potential in cartilage repair. ¹³ A mass of studies have confirmed the safety and effectiveness of EVs for the treatment of cartilage defects in small animals, although their utility in large animal models is less well established In a study based on OA in equine chondrocytes, MSCs‐EVs (less than 200 nm in diameter) were observed to be more effective than MSCs in increasing chondrocyte proliferation and migration to maintain the equine chondrocytes phenotype, suggesting that MSCs‐EVs promoted cartilage repair and delayed the progression of OA. ¹⁴ Furthermore, a study demonstrated superior outcomes using EVs combined with hyaluronic acid over hyaluronic acid alone in treating cartilage defects in micropigs, with improvements noted in MRI scores, histology, biomechanics, and bone mass. Importantly, no adverse reactions were reported in any of the micropigs. ¹⁵ The promising results provide a strong basis for the safety and efficacy of EVs in clinical trials for the treatment of cartilage defects. In the laboratory, the researchers demonstrated some exciting results of EVs in cartilage repair, including maintaining chondrocyte phenotype, regulating cartilage matrix metabolism, alleviating inflammation, and regulating neovascularization. In this review, we comprehensively summarize the established functions and mechanisms of different MSCs‐derived EVs in OA treatment, particularly focusing on cartilage repair, which provides us with a deeper understanding of cartilage damage and repair. Moreover, it may assist in identifying reliable OA treatment strategies in the future.

Search Strategy

A comprehensive literature search was conducted on the PubMed (MEDLINE) database spanning the period from January 2015 to August 2022 by keywords (Cartilage damage) OR (Cartilage repair) AND (Extracellular vesicles) OR (EVs) OR (Exosomes). A total of 145 publications were retrieved. Thirteen articles not related to (Cartilage damage) OR (Cartilage repair) AND (Extracellular vesicles) OR (EVs) OR (Exosomes) and forty‐seven reviews/case report articles were excluded. Finally, sixty‐eight articles were included for review (Figure 1). Our imperfect search strategy may have inadvertently led to the omission of some relevant articles from this study.

FIGURE 1.

Shows the search flow chart for this review, including the inclusion and exclusion criteria.

Extracellular Vesicles

EVs manifest in an array of forms, including microvesicles, exosomes, and apoptotic bodies. The Minimal information for studies of extracellular vesicles 2018(MISEV2018) guidelines suggested that the terminology pertaining to EV subtypes should be appropriately formulated to define the object of research in EV studies. In this paper, we define EVs as extracellular particles with a diameter of 40–200 nm. ¹⁶ All included studies were meticulously vetted to meet the definition and criteria for EVs, such as size, purity, source, and biomarkers. ¹⁷ Some of the included research articles also referred to these particles as exosomes.

EVs can be secreted by a wide range of cell types under both normal and pathological conditions. ¹⁸ Secretion of EVs has been proven as a multifactorial involved process, including endosomal sorting complex required for transport (ESCRT), recombinant human programmed cell death 6 interacting protein, phospholipase, vacuolar sorting protein 4 (Vps4), Rab GTPase activating protein, sphingomyelinase, and ceramide. ¹⁹ , ²⁰ , ²¹ , ²² Of these, the ESCRT is the central mechanism governing the production and release of EVs. This complex is further divided into ESCRT‐0, ESCRT‐I, ESCRT‐II, and ESCRT‐III, with the first three proteins tasked with recognizing ubiquitinated membrane proteins on endosomes, and the last involved in the budding and detachment of EVs. ²³ , ²⁴ Artificial blockage of this complex has been shown to negatively impact EVs production and release. Almost all cell types are capable of producing EVs via paracrine secretion. The biological function of EVs tends to be dictated by their cell of origin. In other words, the originating cell type typically determines the functionalities of the derived EVs. EVs contain cytokines, proteins, lipids, and non‐coding RNAs that participate in and regulate many physiological and pathological processes. ²⁵ Numerous studies have disclosed the ability of EVs to modulate immune responses, inflammation, cellular senescence, apoptosis, migration, and differentiation across various diseases. Meanwhile, EVs from different sources have been shown to reverse these pathological processes. Among the many biological functions attributed to EVs, their roles in mediating intercellular communication by ferrying substances and signals, and as novel drug delivery vehicles, are considered paramount. These functions of EVs have been shown to have implications in disease development, diagnosis, treatment, and prognosis. ²⁶ , ²⁷ Over recent years, research into the role of EVs in OA is still in its nascent stages, however, it has already yielded promising outcomes, particularly in relation to cartilage injuries.

Maintain Chondrocytes Density

Articular cartilage, alternatively known as hyaline cartilage or fibrocartilage, is constituted of sparsely distributed chondrocytes and surrounding various macromolecules. In the progression of OA, the reduced chondrocytes in articular cartilage struggle to typically regenerate and remodel the cartilage matrix, characterized by a failure to furnish the necessary nutritional support for extracellular matrix (ECM) remodeling. However, excessive matrix degrading enzymes induces the degradation of ECM, which cannot provide a stable internal environment for chondrocytes. ²⁸ The extent of chondrocyte loss is directly associated with the degree of ECM degradation and the progression of OA. The avascular physiological structure also limits the exogenous nutrient supply. In short, excessive chondrocyte apoptosis and matrix loss make them fail to support each other, ultimately leading to a vicious cycle of continuous cartilage degeneration. ²⁹ In the early stage of OA, chondrocytes aggregate into clusters and proliferate, and cartilage metabolic activity is heightened, with lost chondrocytes self‐replenishing, illustrating to some extent the capacity for self‐repair of cartilage. ³⁰ Nevertheless, researchers have noted that despite chondrocytes being in a proliferative phase, the glycosaminoglycan content within the ECM does not significantly increase and the synthesis of cartilage matrix is suboptimal. This indicates the compensation level of chondrocytes is low, keeping articular cartilage in a state of constant degeneration. ³¹ While in advanced OA, cartilage degeneration is characterized by increased chondrocyte death and loss of large amounts of collagen, glycosaminoglycans, and water, and even formed empty lacunae. ³² Consequently, the mitigation of excessive chondrocyte death and maintenance of chondrocyte density holds positive implications for cartilage repair. ³³ In general terms, as participants in the cell cycle, proliferation, differentiation, and activated autophagy mediate the renewal of senescent cells. EVs contribute to the maintenance of chondrocyte density by inhibiting excessive chondrocyte death, promoting cell proliferation and differentiation, and activating autophagy.

Protect Chondrocytes from Excessive Death

There are many forms of chondrocyte death under pathological conditions, including apoptosis, pyroptosis, ferroptosis, which promote the loss of chondrocytes and damage the endochondral environment. EVs can protect chondrocytes from excessive death.

Normal cell death is beneficial for maintaining human development, facilitating the timely removal of senescent or damaged cells. However, when cell death becomes excessive and disrupts the balance in the regular cell death process, it often signals the onset of diseases. ³⁴ According to the morphology, cell death can be categorized into lytic and non‐lytic, both of which belong to the gene‐controlled programmed cell death process. More specifically, lytic cell death, which usually comprises necroptosis and pyroptosis, can lead to leakage of intracellular components, stimulate the release of inflammatory cytokines, and trigger subsequent inflammatory responses. This is often referred to as inflammatory death or inflammatory necrosis. ³⁵ On the contrary, the non‐lytic form of cell death (usually refers to apoptosis) manifests that phagocytes eliminated cells but without an inflammatory response. ³⁶ The nonprogrammed process refers to cell death (necrosis) that is not controlled by genes but triggered by intense physical or chemical stimulation. This process is characterized as irreversible cell damage and death, manifesting as cell membrane destruction, cellular and organelle swelling, and release of cell contents. ³⁷ Furthermore, ferroptosis, a recently identified form of cell death driven by high iron‐dependent lipid peroxidation, exhibits morphological characteristics of dead cells with smaller mitochondria, denser membranes, fewer cristae, and an unaltered nucleus. ³⁸ These different forms of cell death have been confirmed in OA, and inhibition of different types of chondrocyte death has emerged as a potential therapeutic strategy for OA. In recent years, EVs have made great progress in the chondrocyte death game (Figure 2).

FIGURE 2.

Mesenchymal stem cells‐derived extracellular vesicles (MSCs‐EVs) can attenuate the excessive chondrocyte death by regulating Bcl‐2/Fas‐Fasl‐mediated apoptosis, Caspase1/GSDMD‐mediated pyroptosis, and GPX4‐mediated ferroptosis (Created with BioRender.com).

Apoptosis

Chondrocyte apoptosis is the most common death mode of chondrocytes, which can be observed in physiological and pathological conditions. And anti‐apoptosis is one of the classical strategies for cartilage repair. Apoptosis is a programmed death process regulated by intracellular signals and genomes. It is divided into endogenous and exogenous pathways, also referred to as mitochondria‐dependent and death receptor‐mediated pathways. The caspase family (including initiating and effector caspases), bcl‐2 family, and fas‐fasl are all involved in the process of apoptosis. ³⁹ , ⁴⁰ The process of apoptosis is often induced by proinflammatory factors, as well as oxidative stress. ⁴¹ , ⁴² EVs, however, have demonstrated a significant potential in inhibiting chondrocyte apoptosis and maintaining the homeostasis of the cartilage matrix.

Intra‐articular injection of bone marrow mesenchymal stem cells‐derived EVs (BMSCs‐EVs) can attenuate surgical‐induced cartilage destruction and subchondral bone remodeling in OA rat models. In vitro, BMSCs‐EVs have been shown to inhibit IL‐1β‐induced chondrocyte senescence and apoptosis, with further studies suggesting that exosomal long non‐coding RNA MEG‐3 might play a critical role. ⁴³ Platelet‐rich plasma‐derived EVs (PRP‐EVs) play a similar role by activating Wnt/β‐catenin signaling. ⁴⁴ MiR‐140‐5p‐rich dental pulp stem cells‐derived EVs (DPSCs‐EVs) can reduce chondrocyte apoptosis and increased the expression of aggrecan and COL II by up‐regulating Bcl‐2. ⁴⁵ Similarly, synovial mesenchymal stem cell‐derived EVs (SMSCs‐EVs) enriched with miR‐155‐5p have also been found to perform the same function in OA by regulating the Runx2 pathway. ⁴⁶ Another study on SMSC‐EVs found that exosomal miR‐129‐5p significantly reduced the release of inflammatory factors and maintained chondrocyte phenotype by inhibiting HMGB1. ⁴⁷ In addition, oxidative stress products also pose a threat to chondrocyte survival. In the cyclic tension‐induced OA cell model, EVs derived from human umbilical cord mesenchymal stem cells (HUCMSCs‐EVs) miR‐100‐5p inhibited ROS production and chondrocyte apoptosis by directly targeting NADPH oxidase 4 (NOX4). ⁴⁸ Interestingly, EVs also play a negative role in OA treatment. EVs secreted by vascular endothelial cells (VECs‐EVs) can promote chondrocyte apoptosis and attenuate antioxidant ability by activating ROS. It presents another potential treatment strategy for OA, which means that inhibiting articular vascular endothelial cells secretes exosomes. ⁴⁹ MiR‐100‐5p‐rich exosomes secreted by infrapatellar fat pad MSCs reduced chondrocyte apoptosis and ECM consumption through mTOR both in vitro and in vivo, and they also maintained cartilage homeostasis and improved the unstable gait of OA mice. ⁵⁰

It is worth noting that a large number of in vitro experiments have verified the anti‐apoptosis effect of EVs, but in vivo studies are still few, and there is a lack of large animal or clinical trials to verify the efficacy and safety of EVs.

Pyroptosis

Chondrocyte pyrosis is another form of cell death closely associated with inflammation, in which cells continue to expand until their membranes rupture, resulting in the release of cell contents that activate a strong inflammatory response. Pyroptosis is a type of programmed cell death that is inflammatory, caspase‐dependent, and is mediated by the protein gasdermin. This process often results in the release of pro‐inflammatory cytokines. ³⁵ During OA progression, macrophages activate caspase‐1 and release inflammasomes (such as NLRP3), increasing pro‐inflammatory cytokines in chondrocytes, leading to the aggravation of chondrocyte pyroptosis and inflammation. Given the close relationship between pyroptosis and inflammation, it is important to take inflammation into account when designing therapeutic strategies based on pyroptosis for OA. ⁵¹ In response to the release of pro‐inflammatory cytokines, chondrocytes secrete large amounts of catabolic enzymes, such as MMP13 and ADAMTS5, which degrade the cartilage matrix. This leads to an imbalance in ECM metabolism and ultimately to cartilage destruction. ⁵² In addition, pyroptosis can also lead to increased symptomatic pain, as inflammasomes in macrophages accelerate the production of IL‐1β, IL‐18, and TNF‐α that increase the hurtful input, which is proportional to the level of pain. ⁵³ The result of pyroptosis, such as cartilage loss, osteochondral fissures, osteophyte formation, and synovitis, lead to an increased density of perivascular sensory and sympathetic nerves extending to the synovium, ligaments, or menisci. This increases pain transmission and sensitivity, further exacerbating the pain perception in OA patients with chronic inflammation.

It is well known that NF‐κB, one of the most classic inflammatory signaling pathways, has been proven to mediate inflammatory responses in diseases, including OA. BMSCs‐EVs rich in miR‐326 not only inhibit pyroptosis and inflammation but also increase the expression of aggrecan and COL II to improve OA through regulating HDAC3 and STAT1/NF‐κB p65. ⁵⁴ Xing et al. found that adipose mesenchymal stem cell‐derived EVs (AMSCs‐EVs) engineered into functionalized ECM hydrogels could inhibit pyroptosis, alleviate inflammatory response, and downregulate MMPs to maintain ECM metabolic balance in intervertebral disc degeneration (IDD). ⁵⁵ Another study on IDD found that exosomal miR‐410 can inhibit LPS‐induced pyroptosis of nucleus pulposus cells (NPCs) by regulating NLRP3 both in vivo and in vitro. ⁵⁶

As mentioned earlier, intense inflammatory responses often lead to the onset of pyroptosis. The anti‐inflammatory effect of EVs should be fully considered when treating chondrocyte pyroptosis, which is often manifested as inhibiting the excessive release of inflammatory factors. Therefore, EVs equipped with anti‐inflammatory drugs or tissue engineering products (including hydrogels, chitosan, etc.) may reduce the release of inflammatory factors and further reduce the pyroptosis of chondrocytes.

Ferroptosis

Ferroptosis is a new type of programmed cell death, which is different from apoptosis, necrosis and autophagy. Ferroptosis is characterized by smaller mitochondria, increased membrane density, decreased cristae, and no obvious morphological changes in the nucleus. Ferroptosis is a newly discovered iron‐dependent cell death different from the classic cell death and renewal pathway. It is a process that is mediated by high expression of unsaturated fatty acids in cell membranes and driven by ferrous iron or ester oxygenase, which catalyzes lipid peroxidation to induce cell death. ⁵⁷ A variety of stimulations factors can trigger ferroptosis, including the interference of glutathione peroxidase 4 (GPX4)‐mediated defense against lipid oxidation and cell detachment from the ECM. ⁵⁸ This iron‐dependent cell death phenomenon has been observed in ischemic and degenerative diseases, where it mediates the death of damaged cells and perhaps even cancer cells in cancer. ⁵⁹ , ⁶⁰ , ⁶¹ Recent research suggests that EVs, which can transfer intracellular iron, play a crucial role in driving ferroptosis resistance. ⁶² Most studies on the inhibition of ferroptosis by EVs have focused on the tumor, cardiovascular and cerebrovascular, and liver‐related diseases. ⁶³ , ⁶⁴ , ⁶⁵

Unfortunately, the research on the role of EVs in ferroptosis, particularly in the context of osteoarthritis, is relatively limited compared to the body of work exploring their role in apoptosis and pyroptosis. Nevertheless, studies are currently underway to further explore the potential role of EVs in the treatment of ferroptosis, with the hope of unveiling their underlying mechanisms in the future. Interestingly, recent years have seen phytochemicals and iron chelators demonstrate significant potential in inhibiting chondrocyte ferroptosis. There is a growing interest in the development of engineered EVs bound to these substances, as they could represent a promising new avenue for addressing chondrocyte ferroptosis and potentially improving outcomes for osteoarthritis patients.

Activate Autophagy

Cell renewal is essential for the metabolism of living organisms. Autophagy plays a critical role in this process, which is characterized by the phagocytosis of damaged or senescent cytoplasmic proteins or organelles by vesicles, and degradation under the action of autophagic lysosomes to achieve cell self‐metabolism and organelles renewal. ⁶⁶

In physiological states, autophagy mediates normal cell renewal, whereas in pathological states, the activation of autophagy accelerates the renewal of damaged or senescent cells. ⁶⁷ The activation of autophagy in chondrocytes when they are senescent, damaged, or dead under various adverse conditions appears to be a determinant in the progression of OA. If autophagy is activated, the degradation of damaged chondrocytes is accelerated to maintain cell viability and homeostasis (Figure 3). In the process of autophagy activation, LC3 and LC3‐II in autophagosomes are both regarded as molecular markers, and LC3‐II/I is commonly used to estimate the level of autophagy. ⁶⁸ In addition, Beclin‐1 is also considered one of the key regulatory proteins of autophagy, which is involved in the formation of autophagosome membranes. ⁶⁹ EVs have been proven to regulate the expression of the above markers in OA. ⁷⁰ , ⁷¹

FIGURE 3.

Autophagy in chondrocytes can be activated by drug‐loaded or engineered extracellular vesicles (EVs) from different sources, showing upregulated autophagy markers LC3 and ATG family. When autophagy is activated, damaged or aging chondrocytes are eliminated, and new chondrocytes are produced to achieve their renewal (Created with BioRender.com).

Intra‐articular injection of the modified overexpressed activating transcription factor 4 (ATF4)‐OA‐EVs (ATF4, as an essential factor involved in chondrocyte proliferation and bone formation) can partially restore the inhibited autophagy of knee chondrocytes, inhibit chondrocyte apoptosis, and alleviate articular cartilage degeneration and inflammation in OA mice. ⁷² ADMSC‐EVs not only activated autophagy and increased LC3B expression in OA chondrocytes but also partially reduced oxidative stress induced by IL‐1β, which can accelerate cell senescence and apoptosis. ⁷³ In addition, BMSCs‐EVs protected chondrocytes from death and reduced the expression of ECM degrading enzyme MMPs, which is related to the exosomal regulation of dynein‐associated protein 1 (Drp1)‐mediated autophagy, that is, BMSCs‐EVs activated autophagy by inhibiting the level of LC3‐II/I and Beclin‐1 through up‐regulating Drp1. ⁷¹ Moreover, BMSCs‐EVs inhibited inflammation and annulus fibrosus (AF) cell apoptosis by regulating the classical autophagy signaling axis PI3K/AKT/mTOR in IDD. ⁷⁴ There is another voice that EVs‐activated autophagy can promote the release of EVs, indicating that NPCs can secrete more EVs after autophagy activation, which forms a virtuous circle. Also exosomal miR‐27a activated autophagy, stimulated NPCs to secrete EVs, and inhibited the excessive degradation of ECM by targeting MMP‐13 in IDD. ⁷⁵ A similar cycle of mutual reinforcement was also observed after external treatment. Low‐intensity pulsed ultrasound treatment activated chondrocyte autophagy and promoted the release of MSCs‐EVs in return, which is beneficial to injured cartilage. ⁷⁶

Promote Chondrocytes Proliferation

Cell proliferation, the process by which multicellular organisms generate new cells via division, is another essential strategy for maintaining cell density.

Proliferation is critical for replacing lost cells and maintaining the intracellular environment. However, excessive cell proliferation is detrimental to the outcome of the disease, and malignant proliferating cells often cluster together to limit the ability to tissue repair. ⁷⁷ Proper cell proliferation is undoubtedly significant for the loss of chondrocytes in the progression of OA. EVs have been demonstrated to promote cell proliferation, which provides another idea for maintaining cell number, especially in articular cartilage where there are sparse chondrocytes.

BMSCs‐EVs treatment significantly reversed the inhibited proliferation and migration of OA chondrocytes and enhanced ECM remodeling that promoted COL II and aggrecan synthesis, and reduced the secretion of MMP13 and ADAMTS5. ⁷⁸ Another study also found that BMSCs‐EVs treatment not only improved the inhibited chondrocytes proliferation and impaired cartilage matrix metabolism but also, to a certain extent, reduced the expression of inflammatory factors in vitro. In vivo, BMSCs‐EVs treatment increased glutamate metabolism and significantly improved the impaired exercise ability of OA mice. ⁷⁹ Both EVs derived from synovial mesenchymal stem cells (SMMSCs‐EVs) and induced pluripotent stem cells (ipMSCs‐EVs) have been shown an ability to alleviate OA. The results suggest that ipMSCs‐EVs has a better therapeutic effect than SMMSCs‐EVs in improving chondrocyte migration and proliferation. ⁸⁰ Also, HUCMSCs‐EVs can enhance chondrocyte proliferation and migration, inhibit chondrocyte apoptosis and the secretion of proinflammatory factors, balance the ECM metabolism, and reduce the formation of osteophytes in mice with knee OA. METTL3 was found to be involved in these protective effects, and further studies showed that HUCMSCs‐EVs reduced NLRP3 mRNA m6A level after miR‐1208 targeting METTL3, thereby reducing the release of inflammatory cytokines and preventing OA progression. ⁸¹ As mentioned, PRP‐EVs protected damaged chondrocytes in OA progression (including enhancing chondrocyte proliferation and reducing apoptosis) by regulating the Wnt/β‐catenin axis. Similarly, both BMSCs from congenital polydactyly tissue (pBMSCs) and BMSCs‐EVs injection can alleviate OA, but pBMSCs‐EVs showed a better therapeutic effect than BMSCs‐EVs. Mechanically, higher BMP4 in pBMSCs regulated the chondrogenic migration, proliferation, differentiation potential, and function of MSCs. ⁸²

The modified exosomal KLF4‐AS1 overexpression acted as a competitive endogenous RNA to upregulated GIT1 expression through sponging miR‐206 to ameliorate inhibited proliferation and increased chondrocytes apoptosis. ⁸³ All in all, numerous exosomal microRNAs, LncRNAs, circRNAs, and proteins are involved in chondrocyte protection, which we list in Table 1. ⁴⁶ , ⁸⁴ , ⁸⁵ , ⁸⁶ , ⁸⁷ , ⁸⁸ , ⁸⁹ , ⁹⁰ , ⁹¹ , ⁹² , ⁹³ , ⁹⁴ , ⁹⁵ , ⁹⁶ , ⁹⁷ , ⁹⁸

TABLE 1.

Includes the cell type, category of exosomal miRNAs, LncRNAs, circRNAs and proteins, roles, and related pathways in OA.

Cell type	miR/LncR/circR	Roles	Pathways	Ref
Human synovial MSCs	miR‐140‐5p	Promote chondrocyte proliferation and migration, inhibit apoptosis, and maintain ECM metabolic balance.	Wnt/YAP	84
			Runx2
			miR‐181c‐3p/let‐7b‐3p
				46
	miR‐155‐5p
	circRNA3503
				85
Human MSCs	miR‐92a‐3p	Promote chondrocyte proliferation, and ECM remodeling and enhance cell viability.	WNT5A	86
	miR‐135b		TGF‐β1/SP1
	Lnc‐RNA‐KLF3‐AS1
			Runx2
				87
				88
Chondrocytes	circRNA‐RWD1	Promote chondrocyte proliferation, and inhibit chondrocyte apoptosis, inflammation, and ECM degradation.	miR‐1277/TRAF6	89
	circRNA‐CDK14		miR‐1183/KLF5
				90
Synovial fibroblasts	miRNA‐126‐3p	Promote chondrocyte proliferation and migration, and inhibit chondrocyte excessive apoptosis and inflammation.	IL‐1β/TNF‐α	91
Bone marrow MSCs	miR‐206	Promote chondrocyte proliferation and differentiation, inhibit chondrocyte apoptosis and inflammation, and regulate ECM metabolic balance.	Elf3	92
			DDX20/NF‐κB
			miR‐135b/MAPK6
	miR‐361‐5p		miR‐135b/MAPK/PDGF‐BB
	TGF‐β1
				93
				94
				95
		Promote M2 synovial macrophage polarization.
		Inhibit abnormal subchondral bone angiogenesis, relieve pain and bone resorption.
Human Urine MSCs	miR‐140‐5p	Promote chondrocyte proliferation and migration, attenuate apoptosis, and regulate ECM secretion.	VEGFA	96
Fibroblast‐like Synoviocytes	Lnc‐RNA‐PCGEM1	Inhibit chondrocyte proliferation, promote cell apoptosis and ECM degradation.	miR‐142‐5p/Runx2	97
	Lnc‐RNA H19
			miR‐106b‐5p/TIMP2
				98
		Promote chondrocyte proliferation, inhibit cell apoptosis and ECM degradation.

Notes: The vast majority demonstrated the ability to treat OA potentially. Interestingly, the fibroblast‐like synoviocyte‐derived exosomal lncRNAs have shown conflicting roles. Exosomal Lnc‐RNA‐PCGEM1 accelerated the progression of OA, while exosomal Lnc‐RNA H19 delayed the progression.

Regulate Osteoblasts and Osteoclasts

Osteoblasts and osteoclasts are two cell types critical for maintaining bone health. Osteoblasts are primarily responsible for promoting bone formation, while osteoclasts promote bone resorption. Thus, these two types of cells are integral to bone metabolism. ⁹⁹ The osteochondral unit is composed of articular cartilage and subchondral bone beneath it. Cartilage and subchondral bone are highly related in structure and function. Studies have shown that if cartilage is damaged or diseased, the underlying bone structure will also undergo secondary changes, and the changes in bone often affect the long‐term effect of cartilage injury treatment. Articular cartilage injury is often divided into chondral injury and osteochondral injury. In the latter injury type, osteoclasts are quite active in the subchondral bone. ¹⁰⁰ , ¹⁰¹ , ¹⁰² , ¹⁰³ Various transcription factors, signaling pathways, and co‐regulators (including runt‐related transcription factor 2, Wnt, TGF‐β, bone morphogenetic proteins, and some miRNAs) were revealed that participated in osteoblast generation and regulated the differentiation of osteoblasts into bone mass. ¹⁰⁴ , ¹⁰⁵ , ¹⁰⁶ Osteoclasts regulate bone resorption and often mediate bone destruction. The phenotypic characteristics of subchondral osteoclasts of OA patients are changed, and the phenotype of OA chondrocytes is induced to switch to a hypertrophic state (Figure 4). In addition, a large number of MMPs produced by osteoclasts play an essential role in cartilage degradation. ¹⁰⁷ , ¹⁰⁸

FIGURE 4.

During Osteoarthritis progression, osteoclasts in cartilage and subchondral bone are more than osteoblasts, meaning that bone resorption is more significant than bone formation. When treated with drug‐loaded or engineered extracellular vesicles (EVs), osteoblasts are increased, and osteoclasts are inhibited by regulating RANKL, BMP‐2, and Runx2 (Created with BioRender.com).

Macrophage colony‐stimulating factor (MC‐SF) and nuclear factor‐κB ligand‐receptor activator (RANKL) are the major cytokines that produce osteoclasts. It has been reported that a low level of RANKL can prevent osteoclast formation, while the level of RANKL in osteoclast‐derived EVs was high, which promoted osteoclast formation. ¹⁰⁹ , ¹¹⁰ Therefore, EVs from osteoclasts have been identified as paracrine controllers of osteoclast genesis. ¹¹¹ , ¹¹² In addition, EVs derived from osteoblasts increased osteoclast production to accelerate bone degradation by carrying RANKL, tartrate‐resistant acid phosphatase (TRAP), and osteoprotegerin (OPG), ¹¹³ , ¹¹⁴ while osteoblasts‐derived EVs improved bone regeneration by up‐regulating Runx2 and alkaline phosphatase. ¹¹⁵ EVs derived from MC3T3‐E1 cells (premineralized osteoblasts), can increase the differentiation of bone marrow stromal cells into osteoblasts and promote osteogenesis. Mechanistically, the Wnt/β‐catenin axis is involved in this process of promoting differentiation and bone formation. ¹¹⁶

IL‐1β and TNF‐α are thought to promote osteoclast production and bone resorption due to their inflammatory induction. External vesicles derived from AMSCs alleviated inflammation and oxidative stress stimulated by IL‐1β and TNF‐α that may enhance the anti‐aging ability of osteoblasts. ¹¹⁷ Recent studies on exosomal miRNAs have shown that numerous microRNAs, such as miRNA‐30d‐5p, can inhibit osteoblast differentiation by knocking down Runx2 expression. ¹¹⁸ , ¹¹⁹ Furthermore, miRNA‐140‐5p reduced the activity of osteoblasts by inhibiting BMP‐2, while miRNA‐885‐5p accelerated osteoblast differentiation and mineralization by negatively regulating BMP‐2 expression. ¹²⁰ , ¹²¹ Many of these specific miRNAs have been shown to repair tissue destruction, including cartilage destruction and inflammation, and to correct bone metabolism. As such, these miRNAs can be modified or packaged into EVs to treat OA.

Maintain Extracellular Matrix Balance

ECM comprises various macromolecules (including collagen and proteoglycans) and water. Initially thought to merely provide structural support, the ECM is now understood to contain many molecules that actively regulate cell growth and development, constituting an essential microenvironment for cell production and survival. ¹²² In addition to the nutrient supply, glycoproteins and collagen in ECM combine with water to provide elastic and expansive forces to the articular cartilage to reduce friction and cushion mechanical stress on the joint surface during violent movement. ¹²³ ECM and chondrocytes work symbiotically to maintain a balanced level of ECM synthesis and degradation. When cartilage is damaged, catabolism of the ECM outweighs anabolism, resulting in excessive secretion of matrix‐degrading enzymes and a marked reduction in collagen and proteoglycan content. ²⁸ Matrix metalloproteinases (MMPs) and disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) are expressed at significantly higher levels in OA, contributing to collagen and proteoglycan degradation. ¹²⁴ As such, maintaining the metabolic balance of the ECM may be even more important than preventing excessive chondrocyte death.

Intraarticular injection of human embryonic mesenchymal stem cell‐derived EVs (ESC‐MSCs‐EVs) increased COL II synthesis and reduced ADAMTS5, and as a result, cartilage destruction and matrix degradation were reversed. ¹²⁵ BMSCs‐EVs reinduced ECM remodeling by inhibiting the production of catabolic enzymes (MMP‐13, ADAMTS5) and inflammatory markers (iNOS). Furthermore, BMSCs‐EVs has also been shown to have the potential to inhibit macrophage activation by reducing inflammation and protecting chondrocytes from excessive apoptosis. ¹²⁶ Xia et al. disclosed that BMSCs‐EVs enriched with miR‐125a‐5p could accelerate chondrocyte migration, promote cartilaginous matrix regulators (COL II, aggrecan, and SOX9) expression, and inhibit the matrix degradation enzyme MMP‐13 in mice with traumatic OA. ¹²⁷ Furthermore, exosomal miR‐136‐5p did the same work by targeting ELF3. ¹²⁸ ADMSCs‐EVs decreased the release of inflammatory factors TNF‐α, IL‐6, PGE2, and NO, as well as the catabolic enzymes (especially MMP‐13), and significantly enhanced the production of anti‐inflammatory cytokine IL‐10 by regulating NF‐κB and activator protein‐1. ¹²⁹ Modified or engineered EVs have been shown to promote cartilage repair by inhibiting matrix degradation enzymes' activity from balancing the ECM's metabolism (Figure 5). And the functions and mechanisms of other engineered EVs (mainly exosomal microRNAs, LncRNAs, circRNAs and proteins) in maintaining ECM metabolism balance listed in Table 1.

FIGURE 5.

Extracellular matrix (ECM) mainly comprises glycosaminoglycan, collagen, collagen fiber, aggrecan, proteoglycan, and water. When cartilage damage occurs, the main components of ECM are degraded by MMPs and ADAMTS. After EVs injection therapy, the synthesis of ECM increased, which played a role in maintaining the balance of ECM metabolism (Created with BioRender.com).

Alleviate Inflammation

OA is a systemic inflammatory disease characterized by inflammation‐driven cartilage and subchondral bone destruction, osteophyte formation, synovitis, ligament and meniscus damage, and joint capsule hypertrophy. The primary cause of OA is often biomechanical damage or overloaded stress on the joint, which results in an excessive release of inflammatory mediators. These mediators activate various inflammatory signaling pathways, triggering an inflammatory response in the damaged cartilage. ¹³⁰ In addition, meniscal or ligament injuries, and even intra‐articular fractures resulting from violent exercise, are the leading causative factors in traumatic OA. The shear stress and compression induce the production of inflammatory mediators such as nitric oxide synthase (NOS), IL‐6, and IL‐8, which may give a chance for synovitis development. ¹³¹ Activated fibroblast‐like synovial cells (FLS) promote macrophage activation by secreting cytokines, growth factors, MMPs, and tissue inhibitors of metalloproteinases (TIMP), while activated macrophages secrete proinflammatory mediators that stimulate FLS and chondrocytes to release ECM degrading enzymes, leading to a repeated cycle of inflammation and ECM degradation. ¹³² , ¹³³ As an upstream cytokine involved in OA, IL‐1β can enhance the expression and secretion levels of other proinflammatory cytokines, such as IL‐8 and IL‐6, as well as the production of MMPs. ¹³⁴ It is well known that inflammation can amplify pain sensation by increasing nociceptive input. The severity of synovial inflammation was positively correlated with pain, as well as with cartilage degeneration. For every 0.1 mm reduction in cartilage, the WOMAC pain subscale score increased by 0.32 within two years. ¹³⁵ However, inflammation in OA is never an isolated issue; it is frequently accompanied by other pathological processes such as chondrocyte apoptosis and cartilage matrix destruction. Hence, addressing inflammation alone is not sufficient when developing treatment strategies for OA; a more comprehensive approach is necessary. The anti‐inflammatory effect of EVs are not discussed here and should be discussed with other pathological processes.

Angiogenesis

Articular cartilage, like intervertebral discs, is avascular tissue, largely because blood vessels are susceptible to mechanical stress and vascularization could potentially impair the biomechanics of the cartilage. ¹³⁶ , ¹³⁷ Limited nutrient availability leads to limited self‐repair capacity, making it difficult to delay or reverse the progression of OA, which is the greatest challenge in repairing damaged avascular tissue. Angiogenesis is a process of forming a new vascular system (mainly capillaries) based on the original vascular system. And it is involved in physiological processes (such as body growth, development, tissue repair, and regeneration) and pathological processes (such as cancer genesis and metastasis). ¹³⁸ , ¹³⁹ Angiogenesis and following neuroinvasion are considered significant factors in the formation and exacerbation of clinical symptoms of OA. ¹⁴⁰ , ¹⁴¹ However, the role of angiogenesis in OA progression is still a topic of debate. It could offer partial nutritional support for cartilage repair, potentially aiding self‐repair, but the accompanying inflammatory response and increased pain may exacerbate OA (Figure 6).

FIGURE 6.

In the progression of Osteoarthritis (OA), MSCs‐EVs promoted the formation of new blood vessels, especially H‐type vessels, which provided nutritional support for the recovery of damaged cartilage. However, the trade‐off for angiogenesis makes it amplify inflammation and pain. Notably, notochord and annulus fibrosus cell‐derived exosomes inhibited angiogenesis during disc degeneration, showing the opposite effect to MSCs‐EVs, which deserves further discussion (Created with BioRender.com).

On the one hand, angiogenesis promotes the nutrient supply required for cartilage repair, which is crucial. MSCs seed scaffold transplantation has been applied in treating traumatic cartilage injury and achieved good results. The defect cartilage is repaired by filling new tissue, probably because of angiogenesis that promotes tissue integration. ¹⁴² IpMSCs‐EVs enhanced angiogenesis and osteogenesis to promote bone regeneration in ovariectomized rat models. ¹⁴³ BMSCs‐EVs regulated the crosstalk of BMP‐2/Smad1/RUNX2 and HIF‐1α/VEGF axis to induce angiogenesis that provides essential nutritional support for fracture healing and accelerate fracture repair in femoral nonunion models. ¹⁴⁴ Moreover, HUCMSCs‐EVs also promoted the healing of stable fractures induced by surgery, and histological results showed increased angiogenesis, which was associated with HIF‐1α‐induced VEGF expression. ¹⁴⁵ Studies also revealed that cartilage regeneration can be enhanced by limiting the secretion of anti‐angiogenic molecules (Indian Hedgehog and Serpin E1) that regulate cartilage formation. Blocked Indian Hedgehog and Serpin E1 lead chondrogenically differentiated BMSCs to promote endothelial cell proliferation, which is beneficial for articular cartilage repair. ¹⁴⁶

On the other hand, some studies present a completely different perspective on angiogenesis. Notochord cells (NC) promote disc development and homeostasis. Transferring NC‐EVs rich in miR‐140‐5p to endothelial cells inhibited angiogenesis via regulating the Wnt/β‐catenin axis. ¹⁴⁷ In addition, EVs derived from annulus fibrosus of normal discs (AF‐EVs) have shown the potential to reduce angiogenesis in degenerative discs via regulating VEGF. ¹⁴⁸ EVs promote angiogenesis, which is beneficial in many diseases. However, their value in OA needs to be reevaluated. Gingival mesenchymal stem cell‐derived EVs (GMSCs‐EVs) hydrogels repairs wounded skin effectively by reinducing re‐epithelialization, collagen deposition, promoting angiogenesis and inward neuronal growth in diabetic rats. ¹⁴⁹ Abnormal nerve growth or invasion is detrimental to the development of OA, which can amplify and aggravate the pain experienced by OA patients. BMSCs‐EVs alleviated cartilage and subchondral bone injury in the lumbar facet joint OA model to a large extent. In addition, the pain feeling of mice can also is alleviated by inhibiting the abnormal formation of H‐type blood vessels and nerve invasion in the subchondral bone. ¹⁵⁰ The further study disclosed that the BMSCs‐EVs‐TGF‐β1 might play the above role by regulating platelet‐derived growth factor‐BB in anterior cruciate ligament transection mice. ⁹⁵ The pathogenesis of OA lacks angiogenesis regulation. As a result, establishing a positive and negative feedback mechanism of EVs will be a potential strategy to regulate the degenerative progression. However, further studies on the modulation of this effect must be performed cautiously, as angiogenesis may deform the joint and accelerate the progression of OA. In OA, EVs‐mediated regulation of angiogenesis provides diverse insights into mechanisms that remain understudied. Thus, a better understanding of angiogenesis in OA may unlock great therapeutic potential.

Preclinical and Clinical Practice of EVs

It is well known that EVs, an emerging cell‐free therapy in recent years, have shown repair potential in diseases of multiple systems throughout the body. Many preclinical or clinical trials of EVs therapy are in full swing, which is an essential step from the laboratory to the bedside. It is not difficult to find that a large number of preclinical or clinical applications first appeared in the field of medical cosmetology due to its excellent regenerative and repair capabilities. ¹⁵¹ , ¹⁵² Studies have confirmed that HUCMSCs‐EVs and AMSCs‐EVs have shown a better potential in wound healing and injured skin regeneration, which mainly plays anti‐inflammatory, antioxidant, cell proliferation, and matrix regeneration roles in the repair process. ¹⁵³ , ¹⁵⁴ Moreover, EVs combined with tissue‐engineered hydrogels or chitosan showed stronger repair ability. ¹⁵⁵ , ¹⁵⁶ , ¹⁵⁷ Interestingly, drug‐pretreated EVs promoted local angiogenesis in the process of skin healing and regeneration, which improved local blood supply and nutritional support. ¹⁵⁸ , ¹⁵⁹ , ¹⁶⁰ The functions and mechanisms of EVs in skin repair and wound healing are similar to those in cartilage repair. They may be closely related, providing valuable experience to develop EVs products in OA treatment.

A systematic review of MSCs‐EVs in cartilage repair identified 13 preclinical animal studies involving 434 animals, most of which were mice or rats (378, 87.1%) and a small number of rabbits (56, 12.9%). The data suggested that MSCs‐EVs can effectively enhances cartilaginous defects to repair and regeneration. Among those studies, animals with EVs treated showed increased chondrocyte proliferation, enhanced matrix remodeling, and improved histological scores. ¹⁶¹ In addition, EVs drug loading, as a route of drug delivery, shows a non‐negligible potential. Berberine encapsulated in PRP‐EVs (PRP‐EVs‐ber) promoted BMSCs proliferation and induced BMSCs into chondrogenic differentiation in vitro. And PRP‐EVs‐ber also increased the migration activity of BMSCs and the expression of ECM‐related proteins (including COL II, Sox9, and aggrecan). Further study revealed that PRP‐EVs‐ber treatment significantly increased the expression of β‐catenin. And the activated Wnt/β‐catenin axes lead to increased COL II, SOX9, and aggrecan expression, indicating the potential of PRP‐EVs‐ber in promoting BMSCs chondrogenic differentiation and cartilage matrix deposition. ¹⁶²

Based on the published papers, current research is mainly focused on cells and small animals but few on large animals and clinical trials, which limits the clinical application of EVs in the treatment of OA. As mentioned above, many in vivo and in vitro studies have gradually uncovered the mechanism of EVs in the treatment of OA or cartilage injury, such as inhibiting chondrocyte death, anti‐inflammatory, and reducing ECM degradation. However, there are still many statements that need to be further explored and confirmed, such as the source of EVs with good therapeutic effect, the efficient way of EVs acquisition and preservation, the appropriate therapeutic dose, route and duration of administration, and the side effects and risks. ¹⁶³ , ¹⁶⁴ Ultrafiltration was the most used method to obtain EVs from different sources. To standardize the study of EVs, the researchers used transmission electron microscopy (TEM) to observe the morphology and detect the surface biomarkers of EVs to identify and standardize EVs. The main route of administration is intra‐articular injection. The dose of EVs differs in various types of animals, but most doses are 50 or 100μl. The therapeutic effect was mainly evaluated by histology and imaging, and almost all EVs showed a positive role in the treatment of OA (Table 2). The dosage of EVs is directly related to its therapeutic effect and safety and is largely determined by the method of isolation and extraction. For example, the method of separation will affect the protein markers, nucleic acids, and receptors expressed on the particles, which directly affects their targeting. ¹⁶⁵ Furthermore, the researchers found that the difference in the therapeutic dose of EVs depends more on the type of disease model, and the choice of therapeutic dose should fully consider the proven or published EVs pharmacokinetics or biological distribution patterns. Researchers should focus on the efficacy of the treatment cargo entities to ensure consistency across batches of EVs and to improve the repeatability of treatment results, which can be achieved by unifying isolation and extraction criteria. ¹⁶⁶

TABLE 2.

Presents some experimental data from different animal experiments included in this review (including animal model type, EVs characteristics, source, extract methods, therapeutic dose, route of administration, and treatment outcomes).

Animal model	MSC‐EVs characteristic	Source of EVs	Extract methods	Dose	Administration route	Treatment outcomes	Ref
Micropig	Diameter: 147.4 nm	Human embryonic MSCs	Ultrafiltration	1 mL	Joint cavity injection	Promoted cartilage function and subchondral bone repair, and significantly improved morphological, histological, and biomechanical outcomes	14
	Biomarkers: CD81, ALIX, and TSG101
Rat	Diameter: 50–150 nm	BMSCs	Ultracentrifugation	100 μL	Joint cavity injection	Improved histological scores and subchondral bone remodeling	42
Rabbit	Diameter: 145.6 ± 50.4 nm	PRP	Ultracentrifugation	100 μL	Joint cavity injection	Improved histological scores and subchondral bone remodeling	43
	Biomarkers: CD9, CD63, CD81 and HPS101
Rat	Diameter: 134 ± 29 nm	Human DPSCs	Ultracentrifugation	50 μL	Joint cavity injection	Improved histological scores and the joint cavity structure and reduced the formation of osteophytes, articular surface irregularity and osteosclerosis	44
	Biomarkers: CD9 and CD63
Mouse	Diameter: 100–120 nm	Synovial MSCs	Total Exosome Isolation Reagent (from cell culture media)	30 μL	Joint cavity injection	Improved histological scores and increased collagen II expression	45
	Biomarkers: CD63 and CD81
Mouse	Diameter: 90–180 nm	Vascular endothelial cells	Total Exosome Isolation Reagent(from cell culture media)/Ultrafiltration	100 μL	Tail vein injection	Reduced histological and synovitis scores, as well as the resistance of chondrocytes to oxidative stress, and increased cell apoptosis	48
	Biomarkers: CD81, CD9, Alix and Tsg101
Mouse	Diameter: 30–150 nm	Infrapatellar fat pad MSCs	ExoQuick reagent kit/Ultrafiltration	10 μL	Joint cavity injection	Improved histological scoring, reduced apoptosis, and delayed ECM degradation	49
	CD81, CD9 and CD63
Rat	Diameter: Ranged around 123 nm	BMSCs	ExoQuick‐TC™ system	100 μL	Joint cavity injection	Improved histological score and reduced pyroptosis of chondrocytes and the expression of pyroptosis‐related proteins	53
	Biomarkers: CD9, CD63 and CD81

It is worth noting that while the therapeutic potential of EVs has been widely reported, these findings are mostly based on cell or animal experiments. The clinical efficacy and safety of EVs have yet to be confirmed. It is expected that comprehensive clinical trials will be conducted to evaluate the efficacy of EVs in treating OA, just as PRP does for OA. ⁷ There have been thousands of registered clinical trials for the treatment of OA by searching for https://clinicaltrials.gov/, but few clinical trials about EVs or exosomes, we listed them in Table 3. We desire to conduct more large clinical trials in the future and reveal the ability of EVs to treat OA.

Table 3.

Presented the registered clinical trials of EVs or exosomes treating OA by searching https://clinicaltrials.gov/ .

Clinical trial Identifier	Source of Exo/Ev	Disease	Included patients	Intervention	Phase
NCT05060107	Allogeneic mesenchymal stromal cells	Knee OA	10	Exosomes	Phase I
NCT04223622	AMSCs	Knee OA	24	EVs	Phase I
NCT05261360	Synovial fluid MSCs	Degenerative meniscal injury	30	Exosomes	Phase II
NCT04849429	PRP	Disc degeneration	30	Exosomes	Phase I
NCT04719793	Umbilical cord MSCs	Knee OA	12	Exosomes	Phase I
NCT04711304	Umbilical cord MSCs	Knee OA	168	Exosomes/EVs	Phase I

As the field of EV research continues to mature, and as various EV products, such as genetically modified EVs, drug‐loaded EVs, and EV hydrogels, progress into clinical trials, there is growing optimism about the potential of EVs in challenging areas such as cartilage repair.

Challenges and Prospects

Extracellular vesicles (EVs) hold remarkable promise in the field of regenerative medicine and disease treatment, however, there remain several barriers to their widespread clinical implementation. The following limitations need to be addressed: (i) lack of satisfactory parental cells; (ii) lack of cost‐effective EVs extraction process, especially lack of standardized operating specifications to ensure EVs production, purity, and function with good reproducibility; (iii) lack efficient EVs drug loading methods; (iv) lack of mature EVs storage and transport system; and (v) the safety of EVs in vivo is still controversial. Therefore, it is necessary to develop a series of advanced and reasonable EVs isolation, extraction and identification systems to standardize the study of EVs, which will help to form and maintain GMP standards ¹⁶⁷ , ¹⁶⁸ Additionally, naturally derived EVs presented with the characteristics of weak targeting and quick clearance, leading to poor therapeutic effects. ¹⁶⁹ To overcome the challenges that block the clinical applications of EVs, they are often modified into engineered EVs. ¹⁷⁰ At present, the main way of engineering EVs is EVs drug loading. There are two common ways to load a drug into EVs pre‐secretion and post‐secretion drug loading. In the pre‐secretion drug loading method, parental cells are usually co‐incubated with drugs (generally, transfection reagents are required) to make the drugs enter the cytoplasm. The drugs in the cytoplasm are sorted into EVs actively or passively, and then the drug‐loaded EVs can be obtained by appropriate extraction methods. While the post‐secretion drug loading method is the most commonly used EVs drug loading strategy, which usually requires the isolation and purification of EVs before loading drugs into them.

Treatment based on stem cell‐derived EVs can effectively avoid immune rejection and tumorigenicity, but the risk of EVs from allogeneic sources should to be taken seriously and further explored. Recent developments in polymer biomaterials like hydrogels and chitosan have promoted the compatibility between allogeneic materials and receptors, enhancing the repair of cartilage with fewer negative effects. The combination of EVs with them in the future may yield more excellent results. When it comes to modified EVs, the difference between gene‐modified EVs and protein‐modified EVs cannot be ignored. In cartilage repair, EVs‐miRNAs regulate target mRNA and play a role. However, miRNA levels in EVs are insufficient to regulate target mRNA in the recipient cells during EV‐mediated delivery or that EV‐containing RNA molecules do not function in the recipient cells. ¹⁷¹ And miRNA or protein‐induced bioactivity suggested that exosomal proteins might be the main drivers of MSCs‐EVs therapeutic activity. The presence of specific miRNAs is not sufficient to indicate a mechanism without proper consideration of its concentration, miRNA structure, and availability of helper proteins. ¹⁷² Therefore, the therapeutic effects of EVs‐miRNAs deserves further confirmation. In addition, the binding of miRNA and target genes is not completely complementary, showing that one miRNA can regulate multiple targets, and one target can be regulated by multiple miRNAs, resulting in low specificity of miRNA. ¹⁷³

In terms of EVs in cartilage repair, increasing studies on the treatment of OA with EVs prove one thing: EVs have great potential to be applied in OA treatment. Indeed, EVs have been shown to be involved in the entire pathological process of OA that has been revealed so far (such as chondrocyte death, ECM degradation, inflammation, and abnormal angiogenesis, etc.), which is a pleasing result. However, it should be noted that no single study disclosed that EVs are involved in the entire degenerative process. Although BMSCs and AMSCs‐derived EVs demonstrated their comprehensive therapeutic effects, the promising results came from multiple studies, not just one. Also, despite positive results from animal studies, there is a lack of clinical trials based on OA patients. Given the significant differences between humans and animal models, more extensive clinical trials are crucial. In conclusion, while EVs represent a promising new frontier in the field of cartilage repair and OA treatment, there is still a considerable gap between laboratory research and clinical application. However, given the impressive therapeutic potential of EVs, which can not only relieve symptoms but also fundamentally delay the progression of OA and restore the structure of articular cartilage, it is still worth expecting.

Author Contributions

Shanjun Huang, Yujiao Liu, and Chenglong Wang designed the idea and drew the manuscript of this review; Wei Xiang and Nianwu Wang performed literature retrieval and collation, Li Peng and Xuanang Jiang summarized the part of the literature, and presented their work in the form of figures and tables; and Xiaomin Zhang and Zhijiang Fu assisted Shanjun Huang, Yujiao Liu, and Chenglong Wang in revising the first draft and provided language support.

Funding Information

This work was supported by grants from the Construction Project of Traditional Technical Key Talents Inheritance Studio and the Sichuan Luzhou People's Government–Southwest Medical University‐Key Project of Strategic Cooperation in Science and Technology (2019LZXNYDZ13).

Conflict of Interest Statement

The authors declare that they have no competing interests.