Exploring extracellular vesicles as novel therapeutic agents for intervertebral disc degeneration: delivery, applications, and mechanisms

Abstract

Intervertebral disc degeneration is a multifactorial degenerative disease that poses a significant threat to the health of the elderly population. Current treatments primarily focus on physical therapy, medication, and surgery to alleviate symptoms associated with disc compression but do not address the progression of degeneration. Therefore, this review aimed to explore the potential of extracellular vesicle therapy as a novel preventive strategy to delay degeneration and enhance tissue repair in intervertebral discs. We cover the pathogenic mechanisms underlying intervertebral disc degeneration, including inflammation, apoptosis, pyroptosis, ferroptosis, autophagy dysregulation, and the roles of non-coding RNAs. Subsequently, we discussed the therapeutic potential of extracellular vesicles and their molecular components, such as proteins, RNAs, and lipids, in modulating these pathways to counter intervertebral disc degeneration. We provides a comprehensive review of the significant role of extracellular vesicle cargo in mediating repair mechanisms. It discusses the functional enhancement advantages exhibited by extracellular vesicles under current bioengineering modifications and drug loading. The challenges and future prospects of utilizing extracellular vesicle therapy to treat this degenerative condition are also summarized.

Graphical abstract

Introduction

Low back pain is one of the significant diseases that pose a threat to the global population’s health and well-being [1]. In recent years, the increasing global population, aging society, and changes in lifestyle and work habits have led to a rising incidence of low back pain [2]. This has resulted in significant economic burdens on individuals and healthcare systems. Intervertebral disc degeneration (IVDD) is the most common cause of chronic lower back and leg pain [3, 4]. Its prevalence increases significantly with advancing age and is influenced by various factors such as genetics, mechanical load, nutrition, and aging [5, 6]. Prolonged factors such as sedentary lifestyle, improper heavy lifting, and lumbar muscle strain can lead to increased pressure on the nucleus pulposus (NP), causing fibrosis, altered biomechanics of the spine, facet joint arthritis, osteophyte formation, and, in severe cases, joint dislocation, spinal cord compression, and paralysis [7]. Furthermore, imaging studies have revealed a significant positive correlation between the severity of low back pain and the grading of (IVDD. [8, 9]. Furthermore, the avascular, hypoxic, and acidic nature of the NP presents a significant challenge for the survival of resident and transplanted cells, leading to poor natural healing ability of degenerated intervertebral discs and fostering permanent, difficult-to-reverse damage induced by even minor pathological factors [10]. Similarly, the administration of clinical drugs orally or intravenously has proven challenging in achieving effective drug concentrations within the intervertebral disc [11]. Current drug treatments primarily target symptomatic relief, such as anti-inflammatory and analgesic medications, which do not effectively slow down the degenerative process [12]. Surgical interventions, such as disc replacement or fusion, also fall short of restoring the original structure and mechanical characteristics of the IVD, delaying degeneration, and promoting regeneration.

Currently, there is a focus on pathology prevention and regenerative medicine therapies in the field of IVDD. Stem cell transplantation has emerged as a prominent area of interest. The introduction of autologous stem cells, such as bone marrow-derived mesenchymal stem cells (MSCs), is gaining attention due to their proximity to surrounding tissues and their ability for multilineage differentiation and regeneration [13]. These cells, similar to bone-derived cell populations and bearing a closer resemblance to intervertebral disc tissue, offer advantages in terms of efficient cellular signaling and communication. Through their phenotypic differentiation, damaged tissues can potentially be replaced by induced pluripotent stem cells or other cell types, such as NP and annulus fibrosus (AF), which are difficult to regenerate naturally. Umbilical cord-derived MSCs can be successfully induced to differentiate into chondroprogenitor cells expressing key cartilage cell markers such as SOX9, TGF-β, and Col [14]. Co-culturing MSCs with nucleus pulposus cells (NPCs) reveals intercellular molecular signaling. Nevertheless, the insufficient differentiation potential, limited proliferation, and poor long-term viability in vivo continue to hinder the practical application of stem cell therapy [15]. Cell-based therapy using extracellular vesicles not only exhibits paternal cell-specific markers but also demonstrates effective tolerance to the harsh microenvironment of hypoxic tissues.

Extracellular vesicles, acting as inert carriers of parental tissue cells, have emerged as an effective strategy for intervertebral disc therapy. These lipid bilayer structures facilitate the transfer of small molecules, proteins, nucleic acids, and cytokines to recipient cells, thereby preventing degradation by endogenous proteases and RNAases. Extracellular vesicles are pivotal in facilitating intercellular communication through paracrine, bloodstream, and autocrine mechanisms. Cell-free therapy, a promising yet challenging research focus, holds significant biological potential for soft tissue repair and mechanical restructuring in IVDD.

This review aimed to provide a comprehensive overview of the therapeutic potential of extracellular vesicles in treating IVDD. We first discuss the pathogenic mechanisms underlying disc degeneration, including inflammation, regulated cell death pathways such as apoptosis, pyroptosis and ferroptosis, autophagy disruption, and the roles of coding and non-coding RNAs. Subsequently, we delve into current evidence on how the protein, RNA, and lipid contents of extracellular vesicles can modulate these pathways to exert reparative effects on diseased discs. The specific molecular mechanisms by which extracellular vesicles mediate anti-inflammatory, anti-apoptotic, autophagy-modulatory, and extracellular matrix (ECM) restorative effects are elaborated. Lastly, we examine the obstacles and potential advancements in utilizing extracellular vesicle therapy for addressing IVDD.

Intervertebral disc degeneration

Structure of the intervertebral disc (IVD)

The human spine consists of 26 vertebrae (C1–7 cervical, T1–12 thoracic, L1–5 lumbar, S1–5 sacral, Co1–Co4 coccygeal), extending from the skull to the coccyx. The IVD is a fibrocartilaginous tissue located between the vertebral bodies, consisting of the NP, AF, and cartilaginous endplates (CEP) above and below, serving as a crucial weight-bearing structure. The NP is a white gel-like substance composed primarily of NP cells, type II collagen (Coll-II), proteoglycans (PGs), and water [16]. The high water content (77%) of the NP ensures cell osmotic gradients and cell viability, allowing passive diffusion of small molecules such as glucose, water, and oxygen into the cells. However, with aging, the loss of PGs and glycosaminoglycans results in decreased tissue hydration capacity, leading to an imbalanced osmotic microenvironment [17]. The NP undergoes fibrosis over time, replacing the original combination of collagen and elastin that dispersed vertical pressure and pushing outward toward the annulus fibrosus [18]. The outer annulus fibrosus is defined by a matrix composed of angled collagen fibers and a dispersed arrangement of PGs [19]. The outer layer of the AF is primarily composed of type I collagen fibers, forming a relatively stiff fibrous tissue that provides support and protection. The inner layer is mainly composed of Coll-II fibers, predominantly produced by chondrocytes [20]. The AF not only serves as a physical barrier protecting the NP but also secretes exosomes that can act as molecular inhibitors to suppress vascularization in degenerated NP [21]. The upper and lower cartilaginous endplates, composed of Coll-II, PGs, and water, serve as a physical barrier at the boundary between the NP and the vertebral body. It wraps around the IVD, forming a vascular bed that aids in substance transportation and gas exchange. The high osmotic pressure within the nucleus allows for nutrient absorption from the cartilage and surrounding matrix while also buffering pressure. Elastic fibers, enriched in the AF, act as anchoring proteins and provide reverse elastic force when stretched [22]. Unfortunately, the density of this abundant fibrous network gradually decreases during cell maturation and aging processes. It is precisely this intrinsic dehydration and fibrosis of elastic tissues that can be observed in human body MRI imaging or animal IVD models. With IVDD, the transition zone between the inner and outer AF becomes indistinct, resulting in low signal intensity on T2-weighted images for both the NP and the inner and outer AF. This is in contrast to the NP tissue, which is rich in water content under physiological conditions, as well as the inner AF (Coll-II) which contains a higher water content [23]. However, the impact of purely NP pressurization on ECM or cellular activity levels is much milder compared to inflammatory stimuli such as interleukins [24].

Due to the high density of the ECM and the high mechanical pressure within the IVD, mature IVD tissue contains very few or almost no blood vessels. A small portion relies on endplate marrow arteries for supply, which penetrate through the endplate longitudinally and anastomose into a capillary network at the superior and inferior levels of the endplate [25]. The lactate byproduct of aerobic metabolism diffuses through this vascular network, while severe glucose deprivation in the tissue significantly impairs the synthesis of collagen and PGs. The IVD has limited ways to obtain substances from microvascular sources, relying solely on intercellular diffusion. As a result, the nutrient supply to the inner cells is extremely limited, and when IVDD occurs, the number and proliferative activity of NP cells prominently decline. This combination of low blood perfusion and low cell density limits the reparative capacity of the IVD, leading to permanent and irreparable damage if left untreated. Simultaneously, the calcification of degenerative endplates and increased bone density reduce permeability, further decreasing the supply of oxygen and glucose. Insufficient transportation of metabolites and decreased ECM mobility have a significant impact on mechanical properties [26, 27]. The disruption of the outer barrier caused by degenerated ECM, invasion of nerve fibers, and collection of harmful stimuli transmit sensory information to the dorsal root ganglia, triggering the occurrence of endogenous low back pain [28]. Simultaneously, the progressive degeneration of the intervertebral space, adjacent facet joint contact wear, thickening of the ligamentum flavum, and spinal canal narrowing contribute to chronic low back pain. This combination of mechanical factors and endogenous chemical factors collectively results in the development of chronic lumbar pain. (Fig. 1).

Fig. 1.

The structure and composition of the intervertebral disc

Pathophysiology of IVDD

The degenerative changes in the NP manifest as reduced tissue hydration, leading to decreased load-bearing capacity, diminished buffering effects, and increased pressure on the AF, making it susceptible to stiffening, degeneration, facet instability, and IVDD. Simultaneously, prolonged bending, sitting, and heavy lifting result in a sudden increase in pressure at the IVDs, compressing the anterior edges of the vertebrae and causing the discs to shift backward, leading to the rupture of the tense annular fibers and posterior longitudinal ligament, culminating in lateral and longitudinal compression of the spinal canal nerves and the onset of lower back and leg pain. Disc damage is predominantly a chronic disease process, with acute injuries leading to degeneration being less common. Moreover, pain induces an increase in stress-related cortisol levels, with high concentrations of cortisol shown to induce apoptosis in disc cells, such as dendritic cells, while inhibiting the chondrogenic differentiation of MSCs, thereby reducing ECM formation [29]. Furthermore, in adulthood, the IVD lacks vascularity, making it difficult for cell factors in the blood to reach the NP, resulting in the inability to repair damage effectively after prolonged wear and tear. This leads to dysregulation of ECM degradation and synthesis metabolism, altering the microenvironment of the IVD.

Alternatively, the presence of blood vessels within the intervertebral disc is viewed as a significant pathological characteristic contributing to the advancement of IVDD. [30]. An analysis suggests that the reduction in vacuolated nucleus pulposus (NP) cells is linked to a decrease in load-bearing ability and plays a role in the degenerative progression. [31]. Moreover, the release of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in the IVD induces an inflammatory response [32]. These chemotactic factors recruit macrophages, neutrophils, lymphocytes, and immunocompetent cells, further activating immune-inflammatory responses. ECM degradation is a prominent feature of IVDD, characterized by the loss of Coll-II and PGs. This is attributed to the upregulation of matrix metalloproteinase-3, matrix metalloproteinase-13, and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), which promote the breakdown of Col-II and Aggregating Chondroitin Sulfate Proteoglycan. This degradation of the ECM in the NPCs disrupts homeostasis. Simultaneously, excessive mechanical loading resulting from reduced intervertebral space enhances oxidative stress, increases reactive oxygen species (ROS) levels, and promotes the apoptosis of NPCs [33]. Metabolic imbalance leads to the accumulation of advanced glycation end products (AGEs) in NPCs, causing oxidative stress and further secretion of inflammatory factors, ultimately resulting in the degradation of the ECM. The accumulation of AGEs triggers the reduction of phosphorylation levels of the serine/threonine protein kinase AMPK in NPCs, a key regulator of intracellular energy metabolism in eukaryotes, thereby reducing the activation of downstream protective signaling pathways such as mTOR-ULK1, PGC-1α-SIRT3 [34].

Factors inducing the degeneration of the intervertebral disc

Role of macrophages in IVDD

Macrophages are a vital immunogenic effector cell type derived from the yolk sac during early embryonic development. Depending on their specific locations, macrophages exhibit various roles and are named accordingly. In the brain, they are referred to as microglial cells, in the liver as Kupffer cells, in bone as osteoclasts, and within lymph nodes simply as macrophages. [35]. These macrophages are commonly classified into M1 and M2 subtypes, with M0 representing non-activated macrophages. Macrophages play crucial roles in various musculoskeletal diseases, such as osteoarthritis, spinal cord injury, and IVDD. During the early stage of inflammation, bacteria, viruses, and pro-inflammatory factors such as interferon-γ and bacterial lipopolysaccharide (LPS) induce macrophage differentiation toward the M1 phenotype. M1 macrophages are capable of producing a series of inflammatory mediators such as nitric oxide, IL-6, and TNF-α, thereby promoting the progression of inflammation within IVD [36]. M2 macrophages, also known as alternatively activated macrophages, are triggered by IL-4 and IL-13, inducing them to display anti-inflammatory characteristics and participate in tissue repair processes. [37]. M2 macrophages encompass M2a, M2b, and M2c subtypes. Among them, M2a and M2b play roles in immunomodulation and promotion of type II immune responses, while M2c has functions in immune response suppression and tissue remodeling. The increased vascularization of degenerated intervertebral discs also enhances the likelihood of immune cells, such as macrophages, entering the disc [38]. The macrophage phenotypes M1, M2c, and M2a can be characterized by the expression of macrophage markers CCR7, CD163, and CD206, respectively. In degenerated NP tissue, higher expression levels of CCR7 and CD163 (indicative of M1 and M2c, respectively) were observed [39].

Role of vitamin D in IVDD

Vitamin D comprises a group of fat-soluble hormones, including vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) [40]. These two forms of vitamin D are primarily synthesized in the skin upon exposure to ultraviolet radiation, although they can also be obtained through dietary sources. The primary function of vitamin D is to regulate the absorption of calcium and phosphorus, thereby facilitating the healthy development of bones and teeth [41, 42].

The Vitamin D Receptor (VDR) gene is responsible for encoding the protein that acts as the receptor for vitamin D. This receptor, a nuclear receptor belonging to the steroid/thyroid hormone receptor family, is predominantly found in bone, the intestines, kidneys, the immune system, and various other cell types [43, 44]. Its primary function is to transduce vitamin D signals into the cell, thereby modulating gene expression. Upon binding to vitamin D, the VDR can form complexes with other nuclear receptors and interact with specific DNA sequences known as vitamin D response elements. This interaction facilitates the regulation of gene expression involved in calcium homeostasis [45]. With the evolution of molecular biology techniques, the extensive and significant association between the vitamin D receptor (VDR) and intervertebral disc degeneration (IVDD) has been identified. Situated in the q13.11 region of human chromosome 12, the VDR gene plays a pivotal role in the homeostatic regulation of calcium and phosphate. Furthermore, it exhibits significant associations across diverse ethnic populations. Specifically, VDR TaqI is significantly associated with IVDD in Asian populations, while VDR FokI shows a significant association with IVDD in Caucasians. Additionally, VDR ApaI is significantly associated with IVDD in both Asian and Caucasian populations [46]. Furthermore, a study established a 1,25-dihydroxyvitamin D (1,25(OH)2D) deficiency mouse model by knocking out the Cyp27b1 (1α-hydroxylase) [1α(OH)^ase±] gene. The phenotypes of IVDD in wild-type and 1α(OH)^ase± mice at 8 months of age were observed using X-ray, MRI, and histological staining, confirming the presence of intervertebral disc defects in 1α(OH)^ase± mice. Subsequently, they further created a Sirt1 transgenic mouse model driven by prx1 (Sirt1^Tg) in the 1,25(OH)2D deficiency mouse model (Sirt1^Tg1α(OH)^ase±) and successfully demonstrated the efficacy of Sirt1 in restoring the deficiency of active vitamin D-induced IVDD [47]. Similarly, supplementation with exogenous calcitriol promotes ECM remodeling and reduces the apoptosis of NP cells [48].

Role of non-coding RNAs in IVDD

Non-coding RNAs (ncRNAs) predominantly regulate intracellular and extracellular molecular levels through catalytic and regulatory mechanisms. RNAs serve as a bridge between DNA and proteins, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), and exert significant influence on cellular aging, apoptosis, autophagy, and oxidative stress at the epigenetic modification level [49]. Non-coding RNAs, such as lncRNAs and circRNAs, can competitively bind miRNAs through their response elements, acting as competitive endogenous RNAs (ceRNAs) to regulate the functional levels of miRNAs relative to target gene mRNAs [50]. However, the ceRNA network mechanisms and RNA regulatory roles in IVDD remain largely unexplored, making a comprehensive review of current research progress essential for advancing the understanding of the molecular pathogenesis of IDD and identifying potential targets for RNA-based therapies. Additionally, the signaling pathways between coding and non-coding genes in IVD play a crucial role in the pivotal regulation of IVDD pathogenesis.

MicroRNAs are short non-coding RNAs consisting of approximately 20–24 nucleotides. They primarily regulate gene expression at the transcriptional and post-transcriptional levels in animals and plants by binding to the 3’ UTR region or coding region of target gene mRNAs. This interaction leads to gene transcript cleavage or inhibition of translation, resulting in gene silencing to facilitate the degradation of transcript products [51]. Previous studies have shown significant differences in the expression of miRNAs between healthy individuals and patients with various diseases, indicating their involvement in disease progression. Similar findings have been reported in the context of IVDD. For instance, it has been demonstrated that miR-4450 is highly expressed in degenerate intervertebral disc tissues and positively correlates with Pfirrmann grades. Furthermore, miR-4450 exacerbates NPCs injury by targeting ZNF121 [52]. Additionally, analysis of the GSE63492 database revealed that miR-328-5p, as a highly expressed small molecule miRNA in degenerative human tissues, plays a role in cell apoptosis through enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Construction of a ceRNA network identified WWP2 as a downstream target of miR-328-5p, further confirming the involvement of WWP2 in cellular apoptosis [53].

lncRNAs are large molecules composed of approximately 200 nucleotides that lack protein-coding capacity. However, they play a crucial role in the regulation of miRNA activity, thereby participating in the fundamental process of coordinating gene expression [54]. Cellular autophagy is a programmed cell death process. Previous studies have shown that in patients with intervertebral disc degeneration, high-throughput sequencing of disc tissue revealed a significant upregulation of lncRNA XIST expression. Further investigations revealed that the high expression of lncRNA XIST competitively inhibits the binding site of miR-19 with XIST. This leads to an upregulation of PTEN expression, an important inhibitor of the PI3K/AKT signaling pathway, subsequently resulting in NPC degeneration, induction of excessive cellular autophagy, and acceleration of IVDD progression [55]. Similarly, LINC01121 exhibits significant expression in IVDD tissue and serves as a crucial nucleic acid molecule in IVDD progression. It promotes the expression of MMP-16, MMP-3, Adamts-5, and Ki-67. Additionally, it enhances the levels of IL-6, IL-1β, and TNF-α [56]. Similarly, it has been demonstrated that lncRNA MIR155HG in degenerated nucleus pulposus tissue acts as a sponge for miR-223-3p, mediating NLRP3 inflammasome assembly and inducing nucleus pulposus cell pyroptosis [57]. As an endogenous pathogenic factor, LncRNA OIP5-AS1 has been shown to inhibit apoptosis and reduce extracellular matrix degradation following targeted knockdown. Its aberrant expression can serve as an early molecular biomarker for IVDD diagnosis [58].

CircRNAs are a class of non-coding RNA molecules characterized by a closed loop structure. Due to their lack of a 5’ cap structure and 3’ UTR region, circRNAs exhibit high conservation and stability, rendering them resistant to RNA exonucleases. Additionally, circRNAs can serve as competitive endogenous RNAs, participating in processes such as apoptosis, autophagy, and endoplasmic reticulum stress [59]. CircRNAscontain multiple binding sites for miRNAs, acting as a sponge to regulate miRNA expression. By competitively binding to miRNA binding sites, circRNAs inhibit the negative effects of miRNAs on diseases. Emerging evidence has shown that circRNAs play a significant role in the process of IVDD. Specifically, circularized exons from the syntrophin β2 (SNT β2) gene, circ_0040039 and circ_0004354, competitively sequester miR-345-3p, promoting the expression of TP73 and Fas-associated factor 1 (FAF1), inducing IL-1β release and a mixed type of cell death involving NPC apoptosis and pyroptosis. Circ_0004354 mediates the early inflammatory process of IVDD, while the upregulation of pro-inflammatory factors provides negative feedback on circ_0004354, at which point circ_0040039 comes into play [60]. Circ_0134111 exhibits high expression not only in degenerative human intervertebral disc tissues but also in cell models stimulated by IL-1β and TNF-α. It functions by sequestering downstream miR-578 to regulate the phenotype of NPCs [61].

Clinical treatment strategies for IVDD

Current treatments for IVDD primarily involve reducing factors contributing to damage, both autonomously and through interventions. Non-pharmacological therapies mainly comprise exercises, lumbar muscle training, acupuncture, physiotherapy, etc. [62]. Pharmacological treatments primarily involve the use of non-steroidal anti-inflammatory drugs, opioids, steroids, and hormones to alleviate pain symptoms. However, there are risks of drug contraindications for individuals sensitive to medication. For instance, the use of commonly prescribed non-steroidal anti-inflammatory drugs (NSAIDs) can induce gastrointestinal toxicity in patients, manifesting as symptoms such as nausea, abdominal bloating, and dyspepsia. Concomitantly, this practice elevates cardiovascular risks. Furthermore, habitual NSAID use can lead to renal impairment and, in severe cases, renal failure [63]. In cases of severe and intolerable pain, intravenous administration of corticosteroids can rapidly alleviate inflammation and pain, thereby improving the functional status of patients. However, this may also result in side effects such as weight gain, sodium and water retention, elevated blood pressure, and increased blood glucose levels [64]. The use of anticoagulants in post-operative bedridden patients may lead to bleeding in the skin, mucous membranes, and internal organs [65]. Furthermore, antibiotics employed for the prevention or treatment of post-operative infections can trigger allergic reactions, ranging from rashes and urticaria to more severe hypersensitivity responses [66, 67].

Surgical interventions for treatment include open procedures such as simple NP removal, posterior lumbar decompression with fusion, and total disc replacement. Moreover, minimally invasive endoscopic techniques have emerged as effective approaches to alleviate severe low back and leg pain symptoms associated with IVDD [68]. However, these therapies may come with certain drug side effects and surgical complications. Postoperative infections may occur at the surgical incision site or within internal tissues, with common types including incision infections, discitis, and intervertebral space infections [69]. These infections often result in uncertain surgical outcomes and exacerbation of the patient’s condition. Additionally, intraoperative bleeding and the formation of postoperative hematomas can compress surrounding nerves and tissues, potentially necessitating further surgical intervention for clearance in severe cases. Another significant consideration is the development of deep vein thrombosis (DVT) due to prolonged bed rest after surgery [70]. DVT can lead to swelling and pain in the affected leg, and if dislodged, can travel to the lungs, resulting in severe complications such as chest pain, shortness of breath, and even death. More critically, improper manipulation during the surgical procedure may result in peripheral nerve damage, accompanied by sensory disturbances such as numbness, tingling, and burning sensations. Furthermore, motor dysfunction may manifest as muscle weakness or poor coordination. Sciatica may occur, characterized by pain radiating along the sciatic nerve pathway, which can affect the foot and leg. Cauda equina injury may present with saddle anesthesia and bladder and bowel dysfunction [71, 72]. Therefore, prevention targeting the underlying causes of the disease is crucial. There is an urgent demand for a biological therapy that can slow down the IVDD process, maintain NP cell vitality, and preserve the hydration and elasticity of the AF.

Biological therapies for IVDD

Currently, stem cell therapy, extracellular vesicle therapy, biotherapy, gene editing, and other innovative approaches have emerged as prominent areas of research interest [73, 74]. Stem cell therapy involves the targeted introduction of various sources of MSCs into intervertebral disc tissue, leveraging their potential for multilineage differentiation, including osteogenic, chondrogenic, and adipogenic differentiation, to contribute to ECM repair [75]. For example, NP cell-derived exosomes may induce MSCs to differentiate into nucleus pulposus-like cells in vitro [76]. Similarly, studies have shown that co-culturing adipose-derived stem cells with NP cells leads to a significant increase in NPC marker genes such as SOX-9 and COL-2A1. Furthermore, the mitogen-activated protein kinase (MAPK) signaling pathway and the nuclear factor-kappa B (NF-κB) signaling pathway in activated B cells are significantly inhibited, while the PI3K-AKT signaling pathway is significantly activated. Co-culturing stem cells leads to the downregulation of negative regulatory factors in degenerated NPC cells, thereby mitigating the damaging effects of cytokines [77]. However, the high osmolarity and acidic environment in the IVD pose significant challenges for cell transplantation [78].

Meanwhile, the invasive nature of stem cell harvesting, the immunological rejection associated with allogeneic stem cells, and the ethical concerns surrounding them have presented significant challenges for the clinical application of stem cell therapy, primarily limiting it to basic research. In contrast, therapies based on extracellular vesicles, whether derived from autologous or allogeneic sources, hold great promise. As natural components of the body, extracellular vesicles can serve as drug-delivery vehicles without inducing immune rejection in the host organism [79]. Similarly, in the avascular environment of the IVD, implantation of inert substances such as extracellular vesicles does not compete for nutrients and oxygen with endogenous cells, making them less susceptible to the inflammatory microenvironment of degenerated tissue. This results in more stable therapeutic effects. Additionally, extracellular vesicles can induce the differentiation of endogenous stem cells and supplement the deficiency of endogenous cells. By targeting the relevant pathological targets of NP cells, the utilization of extracellular vesicles represents a breakthrough in the diagnosis and treatment of this chronic disease [80].

Establishment of animal experimental models for IVDD

Therefore, establishing reproducible animal models suitable for experimental medical purposes is essential. The spectrum of animal models encompasses mice, rats, rabbits, dogs, pigs, monkeys, and others [81]. However, Sprague–Dawley rats have emerged as one of the primary choices for IVDD research due to factors such as their size, lifespan, and safety profile. The needle puncture surgery model is a commonly chosen option based on considerations of safety, reliability, and economic feasibility. Typically, following animal anesthesia, the intervertebral space is identified under X-ray guidance, and a fine needle is inserted at the level of the AF. A 22-gauge needle is commonly employed, inserted until reaching the opposite AF. After complete insertion, the needle is rotated twice at 360° and left in place for 30 s before withdrawal [82]. Due to the composition of IVDs consisting of outer cartilaginous layers and inner gelatinous NP tissues, chemical injection methods such as collagenase are employed. Using a microliter syringe, a solution of collagenase is injected into the intervertebral space. This process facilitates the enzymatic degradation of collagen proteins dispersed within the intervertebral space [83]. This enzymatic action results in a blurred boundary between the AF and the NP, thereby compromising the stability and function of the IVD. The annular incision surgery model, involving a dorsal circular incision to access the intervertebral space, entails the removal of a portion of the NP tissue, resulting in a loss of vertebral stability. Due to its complexity and safety considerations, this approach is less frequently utilized and is typically reserved for large animals such as pigs and sheep [84, 85] (Fig. 2).

Fig. 2.

Animal models of intervertebral disc degeneration. Suitable animal model methods for implementing therapeutic strategies for IVDD include the needle puncture surgery model (inducing injury to the intervertebral disc via needle puncture), the collagenase surgery model (injection of collagenase), and the annular incision surgery model (cutting or removing a portion of the IVD tissue)

Extracellular vesicles

Extracellular vesicles consist of three distinct subgroups: exosomes, typically ranging in diameter from 40 to 200 nm microvesicles with sizes spanning from 200 to 2,000 nm and apoptotic bodies, measuring between 500 and 2,000 nm [86]. The formation of exosomes begins with the process of endocytosis, where the plasma membrane invaginates in response to various stimuli. This inward folding is primarily instigated by the engagement of surface receptors with specific ligands, leading to the recruitment of adapter proteins and the assembly of a clathrin or caveolin coat around the developing vesicle. Clathrin-mediated endocytosis is one of the most extensively studied mechanisms. Upon ligand binding, clathrin proteins assemble into a basket-like structure that promotes membrane invagination. Subsequently, dynamin, a GTPase, constricts and pinches off the vesicle from the membrane [87]. Caveolae-Mediated Endocytosis: This involves the formation of flask-shaped invaginations (caveolae) that contain caveolin proteins. Unlike clathrin-mediated endocytosis, caveolae can directly internalize lipids and smaller membrane proteins. Macropinocytosis: This is a form of non-specific endocytosis that results in the uptake of larger volumes of extracellular fluid and solutes [88].

The early endosome, formed upon internalization, undergoes a series of maturation processes to become a late endosome. Early endosomes can fuse with each other and with other vesicular structures, allowing for the mixing of contents and the dilution of internal components, which is critical for subsequent maturation [89]. As the endosome matures, the pH decreases from neutral to acidic, which activates various enzymes and facilitates the sorting of cargo. This pH change is mediated by the activity of the V-ATPase pump, which aids in proton transport across the membrane [90].

During maturation, late endosomes develop a distinct morphology characterized by invaginations of the membrane, forming intraluminal vesicles (ILVs) within the lumen of the MVB [91]. The formation of ILVs is regulated by the ESCRT machinery and lipid and membrane dynamics. The former involves multiple ESCRT complexes (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III), which initially recognize ubiquitinated cargo and subsequently mediate membrane invagination and scission, processes essential for ILV formation [92]. The latter, such as the presence of intracellular lipids, including ceramides, promote membrane curvature, facilitating invagination. Additionally, the recruitment of specific proteins aids in stabilizing newly formed ILVs.

Once MVBs are formed, they can either fuse with lysosomes for degradation or fuse with the plasma membrane, resulting in the release of exosomes into the extracellular environment [93]. The fusion of multivesicular bodies (MVBs) with the cell membrane is mediated by SNARE proteins, such as Syntaxin, SNAP-23, and VAMP proteins. These proteins facilitate the docking and fusion processes, allowing the release of exosomal contents [94]. Additionally, the release of exosomes can be regulated by various external signals, including cytokines and growth factors, which induce changes in cellular activity and promote the fusion of MVBs with the cell membrane [95]. (Fig. 3).

Fig. 3.

The biogenesis, cargo delivery, and receptor-mediated endocytosis properties of exosomes. Exosomes are released from cells through various pathways, including the cell nucleus, early endosomes, late endosomes, and multivesicular bodies. They transport parent cell-specific effector genes into recipient cells, exerting their effects by specifically targeting intracellular organelles or receptor sites within the recipient cells

When apoptosis occurs, apoptotic bodies are gradually formed. Firstly, under the stimulation of apoptotic signals, the chromatin in the cell condenses and marginalizes, and the cell nucleus begins to fragment. At the same time, the cytoskeleton disintegrates, the integrity of the cell membrane is damaged, and the cell loses its original shape, with a blistering phenomenon occurring. These vesicular structures keep expanding, encapsulating cellular contents such as fragmented nuclear fragments and organelles. As the process progresses, the vesicular structures separate from the main body of the cell, and finally, apoptotic bodies are formed [96]. After detaching from the cell, they enter the extracellular environment. Although they were once regarded as remnants of cell death, it has now been discovered that they can participate in intercellular communication and transmit substances and information [97]. In the formation of microvesicles, cells are affected by various stimulating factors. For example, proteins of the Rho family within the small GTPase family play an important role in this process. They can regulate the dynamic changes of the cytoskeleton and the fluidity of the cell membrane, creating conditions for the subsequent budding of the cell membrane and the formation of microvesicles [98]. The activated signaling pathway will trigger the rearrangement of the cytoskeleton. Based on the rearrangement of the cytoskeleton, local areas of the cell membrane deform and start to form bud-like structures. As the bud-like structures continue to grow and develop, their necks gradually become thinner and finally detach from the cell membrane, forming microvesicles [99]. Despite being classified into three major subclasses, there is inevitably an overlap in the functional properties and shared characteristics among these subclasses in the context of cell-secreted communication.

With the advancement of biomedical research, there is increasing evidence indicating the widespread presence of exosomes in various organs, tissues, and cell lines, contributing to the development of a comprehensive exosome-based intercellular communication network. Exosomes are abundantly found in body fluids such as blood, saliva, urine, semen, cerebrospinal fluid, and breast milk. They can also be secreted by a variety of cell types, including myeloid cells, fibroblasts, chondrocytes, MSCs, macrophages, T cells, B cells, and Natural Killer (NK) cells. Virtually all cell types are capable of producing exosomes [100]. Research on exosomes derived from bone marrow, umbilical cord, adipose tissue, and urine represents some of the most widespread and in-depth studies in the field [101]. (Fig. 4).

Fig. 4.

The origin and contents of extracellular vesicles

Surface markers and cargo components of extracellular vesicles

Proteins contained in extracellular vesicles (EVs) can serve as criteria to distinguish subpopulations, with approximately 10⁹–10¹⁰ EVs yielding 1 μg of total protein [102]. Surface proteins and cargo contents of different subpopulations exhibit heterogeneity and can serve as valuable markers for clinical diagnostics. Among them, the exosomal membrane is rich in transmembrane protein families involved in exosome transport (such as CD55, CD59, CD63, CD81, and CD9), adhesion molecules (such as CD29, CD44, and CD73), as well as exosome-positive marker proteins (TSG101, Alix) [103], the heat shock protein family (HSP60, HSP70, HSPA5, CCT2, and HSP90), integrins, antigen presentation proteins, cytokine receptors, and lipoprotein receptors [104]. Other EVs also have characteristic markers that can be used for further distinction. For example, exosomes, derived from the plasma membrane, are significantly associated with proteins such as tetraspanins [105]. In addition, they also contain common proteins such as heat shock proteins, integrin proteins, and cytoskeletal proteins. Apoptotic bodies are cell remnants from apoptotic cell division, with their protein content mainly reflecting the characteristics of the parent cell. (Fig. 4).

EVs are enriched with a wide range of proteins, lipids, and nucleic acids (including genomic DNA, mitochondrial DNA, long non-coding RNA, microRNA, circular RNA, and mRNA), among others. These cargo molecules are encapsulated by the vesicular membrane, primarily composed of phosphatidylserine, phosphatidylcholine, phosphatidylinositol, cholesterol, sphingolipids, and diacylglycerols [106]. This lipid-soluble structure enables EVs to autonomously internalize into the phospholipid bilayer of recipient cells. Moreover, the bilayer membrane structure effectively isolates the cargo from the external environment, acting as a natural carrier. Research has demonstrated the efficacy of vesicle membrane reprogramming as a non-viral delivery system [107]. (Fig. 4).

Function of extracellular vesicles

Originally believed to be cellular remnants or extraneous substances, EVs have emerged as a cell-free active medium [108]. As research delves deeper, it becomes evident that these vesicles exert intercellular regulatory effects through signal transmission and material transport, thereby impeding cell senescence, regulating cell metabolism, and fostering cellular regeneration [109]. This effect is attributed to the delivery of cell factors contained within EVs into recipient cells, primarily achieved through ligand-receptor binding, receptor-mediated endocytosis, and membrane fusion mechanisms [110]. Distinct from the simple transportation of donor cell contents, the bioengineering modification of exosomes represents a strategy to enhance membrane surface stability and optimize targeting capabilities. For instance, utilizing a CD9-based approach, RGD peptides can be integrated into the tetraspanin domain of CD9 to increase the affinity for cancer cells expressing αvβ3. Simultaneously, to minimize the macrophage clearance effect in vivo, the incorporation of CD47p110-130 peptides can help exosomes escape macrophage phagocytosis, thereby partially stabilizing their in vivo concentration [111]. Moreover, exosomes play a significant role in immune responses. Researchers have leveraged this by recruiting and activating endogenous dendritic cells (DCs) to stimulate cross-presentation of tumor antigens, thereby inducing immune responses against neoantigens. As a result, they developed a designed vaccine (DEXP&A&N) that employs exosomes derived from DCs for targeted therapy. These exosomes are conjugated with an immunoadjuvant, high mobility group nucleosome-binding protein 1 (HMGN1), which promotes DC recruitment and activation, as well as peptides and antigenic epitopes targeting hepatocellular carcinoma (HCC). Upon intravenous administration, this vaccine successfully recruited and activated DCs in mouse models of hepatocellular carcinoma, eliciting T-cell immune responses against the tumor and significantly inhibiting tumor growth [112]. Unlike the therapeutic benefits of exosomes, in the realm of oncology research, exosomes are frequently utilized as non-invasive prognostic biomarkers for tumor diagnosis. For instance, in pancreatic cancer, Glypican-1⁺ (GPC1) circulating exosomes (crExos) serve as a promising diagnostic marker. [113]. Similarly, the in vivo clearance of exosomal metabolites also serves as one of the effective means by which exosomes exert their functions. Research by Deng has demonstrated that exosomes released from astrocytes, mediated by ultrasound in vitro, can enhance the clearance of αβ-amyloid peptides [114].

Isolation and enrichment of extracellular vesicles

Isolation and purification of exosomes represent the foundational steps in exosome research, yet to date, a consensus on optimal extraction methods remains elusive. Exosomes are typically harvested from cell culture supernatants, raising concerns about potential contamination from toxins, mycoplasma, and other impurities originating from fetal bovine serum used in production. To mitigate contamination risks, current practices often involve various strategies for depleting serum-derived EVs or serum-free culture protocols [115].

Differential ultracentrifugation is a traditional and widely used method for exosome purification. Based on the differences in particle size between exosomes and other cellular components, such as cells and cellular debris, a series of centrifugation steps are performed. Firstly, a centrifugation at 300 × g for 10 min is carried out to roughly separate the cells. Subsequently, dead cells are removed via centrifugation at 2,000 × g for 10 min, followed by centrifugation at 10,000 × g for 30 min to eliminate cellular debris. The exosome pellet is then obtained through ultracentrifugation, and further purification is achieved by washing with phosphate-buffered saline, followed by another round of ultracentrifugation at 100,000 × g to remove residual proteins, resulting in relatively pure exosomes [116]. However, ultracentrifugation itself can lead to damage to EVs, and the proficiency of the operator can impact both the quantity and quality of EVs during sample preparation. Moreover, due to the inevitable overlap in particle size between EVs and cellular organelles, such as mitochondria, achieving high purity of EVs through simple ultracentrifugation is challenging [117]. Currently, there are alternative methods for EV isolation and purification. One approach involves polymer precipitation, using polyethylene glycol to reduce the solubility of EVs, followed by centrifugation to obtain the precipitated EV pellets. Size-exclusion chromatography is another method based on the principle of size difference, where larger particles are unable to enter the porous gel matrix and are eluted in the flow-through after multiple washes. This technique allows for the isolation of structurally intact and uniformly-sized small molecular EVs, such as commercially available EV purification columns. However, this size-based method may not completely separate EVs from lipoproteins of similar molecular weight. Additionally, immunoprecipitation is a separation technique based on the specific binding of antigen–antibody interactions. It utilizes components on the surface of EVs, such as transmembrane proteins (CD63, CD81), lysosomal-associated membrane protein-2B (LAMP-2B), and adhesion proteins (EpCAM, CD166), to capture EVs using antibody-conjugated beads, followed by elution [118]. Similarly, anion exchange chromatography, which involves binding negatively charged EVs to positively charged elution columns, provides a rapid and efficient purification method [119] (Fig. 5).

Fig. 5.

Isolation and extraction methods for extracellular vesicles. A EV formation and culture. B Low-speed extraction of EVs. C High-speed extraction of EVs

Evaluation of extracellular vesicles

With the advancement of extracellular vesicle research, the analysis and evaluation of sample separation techniques have become essential. Currently, there are two main categories of techniques: physical and chemical analysis. Physical analysis focuses primarily on characteristics such as particle size, vesicle morphology, and concentration. TEM is a visual method for observing EVs, capturing electron transmissions through EVs to generate particle images that show the characteristic bilayer membrane structure of EVs as bright circular rings. NTA, on the other hand, relies on laser-particle interactions to determine diffusion coefficients based on the Brownian motion of particles, thus calculating the size of EV particles within a range of approximately 50–1,000 nm [120]. Dynamic light scattering (DLS) relies on a monochromatic laser beam passing through a solution containing EVs. Utilizing the principles of Brownian motion, DLS is applicable for measuring particles in suspensions. Nano-Flow Cytometry synergizes traditional flow cytometry with nanofluidic technology, leveraging microfluidic channels to direct nanoscale particles, such as exosomes, into the detection zone. This method typically enables the detection of vesicles smaller than 40 nm, accurately quantifying the number of exosomes within a sample. Beyond quantitative analysis, the multi-marker capabilities of Nano-Flow Cytometry allow for the subclassification of exosomes, elucidating their function and origin, while also providing insights into size distribution and morphological characteristics of exosomes [121].

Chemical analysis relies primarily on cellular proteins, surface antigens, etc. Among them, immunoblotting exposes EV membranes by lysing them and exposing the respective marker proteins. After separation by SDS-PAGE, the proteins are transferred onto a polyvinylidene fluoride membrane. Subsequently, a monoclonal or polyclonal primary antibody is used to bind to the protein epitope, followed by detection using a horseradish peroxidase-conjugated secondary antibody. Commonly used EV marker proteins include TSG101, HSP70, CD63, CD81, and others (Fig. 6) [122]. Extracellular vesicle tracking involves binding the specific fluorescent dye PKH67 to the double-layer membrane of EVs. Following PKH67 labeling, co-culturing of recipient cells with labeled EVs for 24–48 h allows observation of internalized vesicles presenting as red fluorescence within the recipient cells under a fluorescence microscope. The aforementioned methods, as early techniques for tracing EVs, have now been supplanted by more advanced technologies. For instance, Single-Particle Tracking (SPT) employs high-resolution microscopy in conjunction with fluorescent labeling to directly monitor the movement of individual EVs within or between cells, thereby capturing detailed information on the dynamic behavior of these vesicles [123]. Single-molecule localization microscopy (SMLM), leveraging high resolution and signal-to-noise ratio, enables the quantitative analysis of the abundance and spatial distribution of different proteins or labeled molecules within EVs. This approach aids in identifying EV-specific markers, which can be utilized for the discovery of biomarkers and the early diagnosis of diseases [124]. Super-resolution microscopy techniques enable the observation of individual EVs and their dynamic interactions with cells, yielding results with exceptionally high spatial resolution [125]. Mass cytometry (CyTOF), which combines mass spectrometry with flow cytometry, allows for high-throughput multiparametric analysis of EVs, enabling the tracking of EV composition and functionality at various time points [126, 127].

Fig. 6.

Extracellular vesicle characterization and identification. A Schematic illustration of hypoxia-pretreated hUCMSC-derived extracellular vesicle isolation. B Spindle-shaped morphology of hUCMSCs observed under light microscopy. C Analysis of surface markers of EVs using flow cytometry. D Determination of EV size via NTA. E Differences between sEVs and HP-EVs were observed via TEM. Scale bar: 100 nm. F Western blot analysis of EV surface marker proteins [128]. Copyright 2023, Wiley. Reproduced with permission

Extracellular vesicles in the treatment of IVDD: applications and mechanisms

Engineering extracellular vesicles for the treatment of IVDD

EVs, as miniature nanovesicles, have gained significant attention in the past decade as a drug-delivery system in the field of molecular science. Besides delivering cell-derived molecular entities, engineered EVs have emerged as a popular approach. The aim is to load specific drugs, cytokines, nucleotide fragments, and other cargo into EV carriers, taking advantage of their molecular compatibility to preserve the integrity of the cargo upon release into recipient cells. For instance, EVs capability of traversing the blood–brain barrier offers significant potential in this regard [129]. The main methods for drug loading include physical (active) and chemical (passive) induction. The physical method encompasses techniques such as electroporation, sonication, freeze–thaw cycles, and extrusion, while the chemical method involves saponins, liposomes, and calcium chloride, among others [130]. However, the short duration of pure exosome bioactivity and their weak targeting ability have led to insignificant therapeutic effects [131, 132]. Currently, surface protein modification of exosomes has emerged as a new approach. For example, lysosome-associated membrane protein 2B (LAMP-2B) is a surface protein of exosomes. In osteoarthritis, to promote chondrogenic differentiation of synovial fluid-derived mesenchymal stem cells (SF-MSCs) within the joints, E7, an MSC-targeting peptide, can bind to LAMP-2B on the exosome surface. By engineering the E7-exosome delivery of Kartogenin (KGN), a small molecule that promotes SF-MSC chondrogenesis, the exosome’s targeting binding capacity can be maximized [133]. Moreover, exosomes deliver intact mRNA fragments, serving as protein synthesis templates for target cells [134]. Currently, the field of biomaterial engineering incorporating EVs has gained popularity. For instance, the design of lactate-polylactic acid microsphere hydrogel carriers for EVs relies on their excellent biocompatibility and mechanical load-bearing support to maximize the benefits of EVs [135]. Similarly, incorporating adipose-derived mesenchymal stem cell microspheres into gelatin microparticles (GMP) coupled with TGF-β3 and matrilin-3 serves a dual purpose. On one hand, TGF-β3 induces microspheres toward chondrogenic differentiation, promoting cartilage tissue repair in the IVD. On the other hand, the presence of matrilin-3 prevents terminal differentiation and hypertrophy of chondrocytes [136]. In the future, the goal is to achieve controlled release of EVs in IVD tissue through material-mediated loading. Additionally, the use of biohydrogels provides support within the intervertebral space, while coupling with surface-specific ligands on EVs enhances their therapeutic potential in the biological system [137]. Moreover, the incorporation of targeted RNA therapy into biomaterial carriers enables the optimal utilization of advantageous molecules within EVs [138] (Fig. 7).

Fig. 7.

A Synthesis and mechanisms of injectable hydrogels containing exosomes in the treatment of IVDD. B Images of NP tissue decellularization and the preparation of hydrogels. C TEM and size distribution analysis of exosomes. Scale bar = 0.1 μm. D X-ray images of the intervertebral disc at 0, 4, 8, and 16 weeks. Reprinted with permission from Springer. [137]Copyright 2021, BMC. Reproduced with permission

The contents of EVs, such as molecular proteins, ncRNAs, etc., play crucial roles in various mechanisms involved in IVDD repair (Table 1). In the following section, the mechanisms of exosomes in the treatment of IVDD are highlighted (Fig. 8).

Table 1.

Extracellular vesicle contents and their specific therapeutic mechanisms

Exosomal substance	Sample source	Mechanism	Mechanisms	References
–	ADSC-EVs	Inflammation	Blocking the nuclear translocation of NF-kB to reduce TNF-induced pro-inflammatory factors	[139]
miR-25-3p	PRP-EVs	Inflammation	Inhibiting SOX4 gene levels and CXCR7 transcription relieved IL-1β-induced cell degeneration	[140]
lncRNA CAHM	BMSC-EVs	Inflammation	Targeting M1 macrophages, inhibiting iNOS, and reducing TNF-α and IL-6	[141]
–	UCMSC-EVs	Apoptosis	Modulating the Bcl-2/Bax ratio, upregulating type II collagen, and proteoglycans	[142]
miR-21	MSC-EVs	Apoptosis	Inhibit PTEN, activate PI3K-AKT, and reduce Bad, Bax, and Caspase-3 activation	[143]
miR-142-3p	BMSC-EVs	Apoptosis	Inhibit the MAPK pathway	[144]
miR-31	BMSC-EVs	Apoptosis	Inhibit NFAT5, activate the Wnt/β-catenin signaling pathway, and reduce TNF-α-induced apoptosis	[145]
miR-199a	BMSC-EVs	Apoptosis	Inhibit GREM1, which activates the TGF-β1 signaling pathway and is detrimental to the intervertebral disc (IVD)	[146]
miR-129-5p	MSC-EVs	Apoptosis	Target LRG1 to inhibit its expression, block the LRG1-dependent p38-MAPK signaling pathway, and delay the polarization of M1 macrophages	[147]
miR-199a	MSC-EVs	Pyroptosis	Reduce levels of NLRP3, GSDMD, and cleaved caspase-1	[148]
miR-141-3p	PRP-EVs	Pyroptosis	Target and downregulate Keap1, upregulate Nrf2, promote Nrf2 nuclear translocation, and effectively block H2O2-induced inflammatory factor production	[149]
–	PRP-EVs	Pyroptosis	Regulate M1 and M2 macrophage polarization through the NF-κB and MAPK signaling pathways, induce ubiquitination, and degrade NLRP3 inflammasomes	[150]
miR-26a-5p	UCMSC-EVs	Pyroptosis	Target METTL14 to reduce IGF2BP2 recruitment and thereby decrease the stability of NLRP3 mRNA	[151]
circ_0072464	BMSC-EVs	Ferroptosis	Upregulate the synthesis of NRF2, then target to downregulate ACSL4, and upregulate GPX4, leading to a decrease in ferritin light chain (FTL) and free iron levels	[152]
circ_0050205	BMSC-EVs	Ferroptosis	Sponge miR-665, leading to increased expression of GPX4 and decreased production of free iron	[153]
–	BMSC-EVs	Autophagy	Activate autophagy initiation through the AKT-mTOR pathway, increasing LCII/I levels	[13]
–	BMSC-EVs	Autophagy	Inhibit autophagy to suppress IL-1β-induced inflammation and apoptosis in AF cells	[154]
–	CESC- EVs	Autophagy	Activate the PI3K-AKT pathway, increasing autophagic proteins LC3B/A and P-AKT while reducing levels of Cleaved-caspase3	[155]
miR-27a	NPC- EVs	Autophagy	Autophagy activation triggers NPCs to release EVs containing miR-27a, selectively inhibiting the activity of the matrix-degrading protein MMP13	[156]
miR-155	BMSC-EVs	Autophagy	Silencing the target protein BACH1 leads to increased expression of downstream HO-1 protein and activation of autophagy	[157]
miR-155-5p	ADSC-EVs	Autophagy	Targeting TGFβR2 activates the AKT/mTOR pathway to enhance NPCs’ autophagy	[158]
–	MSC-EVs	Oxidative Stress	Repair mitochondrial damage and reduce the accumulation of endogenous ROS	[159]
–	PRP-EVs	Oxidative Stress	Upregulate the abundance of PGC1α, decrease ROS levels, stabilize mitochondrial membrane potential, enhance ATP production, and reduce lactate accumulation	[160]
–	BMSC-EVs	Oxidative Stress	Mitigate the high levels of ROS and malondialdehyde induced by high pressure on intervertebral discs, thereby reducing cell apoptosis	[161]
–	BMSC-EVs	Endoplasmic reticulum stress	Inhibiting the expression of ATF6, IRE1α, and ATF4 reduces CHOP transcription, leading to decreased activation of downstream Caspase-3 and Caspase-12	[162]
-	UDSC-EVs	Endoplasmic reticulum stress	Reduced levels of GRP78 and CHOP, as well as apoptosis-related factors Caspase-3 and Caspase-12, under pressure	[163]
–	BMSC-EVs	Extracellular matrix	Induce dendritic cell proliferation, promote the accumulation of proteoglycans and glycosaminoglycans (GAGs), enhance ACAN protein expression, and reduce LDH levels and cell apoptosis	[164]
VASN	MSC-EVs	Extracellular matrix	Directly target Notch1 within the IVD, activate the Notch signaling pathway, and facilitate IVD repair and regeneration	[165]
–	NPC-EVs	Extracellular matrix	NPC-exosomes promote BMSC differentiation toward an NP-like phenotype, while BMSC-exosomes coordinate NPC proliferation, enhancing matrix repair capacity	[166]
miR-17-5p	MSC-EVs	Extracellular matrix	Inhibit the activation of the PI3K-AKT signaling pathway to promote NPC proliferation and ECM repair	[167]
miR-194-5p	MSC-EVs	Extracellular matrix	Targeting TRAF6 to promote NPC proliferation and osteogenic differentiation under TNF-α intervention	[168]
miR-129-5p	BMSC-EVs	Extracellular matrix	Targeting SOX4 inhibits the activation of the Wnt/β-catenin pathway, upregulating Aggrecan and Collagen II	[169]
miR-532-5p	BMSC-EVs	Extracellular matrix	Targeting RASSF5 inhibits collagen degradation and fibrotic accumulation	[170]
miR-124	M2c-EVs	Extracellular matrix	Activating the TGF-β/Smad3 pathway promotes collagen synthesis	[171]
NAMPT	Adipo-sEVs	Cellular senescence	Activation of the nicotinamide adenine dinucleotide (NAD⁺) and Sirt1 pathways restores vigor in aged NPCs	[172]
miR-105-5p	iPSC-EVs	Cellular senescence	Inhibition of the specific cyclic adenosine monophosphate (cAMP) hydrolyzing enzyme PDE4D leads to an increase in cAMP levels, ultimately activating the Sirt6 pathway	[173]
miR-199a	BMSC-EVs	Cellular senescence	Targeted regulation of GREM1 promotes activation of the TGF-β pathway, delaying senescence in bone marrow cells	[146]

ADSC, Adipose-derived mesenchymal stem cell; PRP, Platelet-rich plasma-derived; BMSC, Bone marrow mesenchymal stem cell; UCMSC, umbilical cord mesenchymal stem cell; MSC, marrow mesenchymal stem cell; CESC, Cartilage endplate stem cell; NPC, Nucleus pulposus cell; UDSC, Urinary-derived stem cell; M2c, M2c macrophages; Adipo, adipocytes; Ipsc, Induced pluripotent stem cell; Coll-II: type II collagen

Fig. 8.

Mechanisms of extracellular vesicles repair Intervertebral Disc Degeneration

Inflammation

Degenerative changes in the IVD, associated with aseptic inflammation, correlate with elevated pro-inflammatory factors, including TNF-α, interleukin family members IL-1β, IL-6, IL-10, interferons (IFNs), and prostaglandin E2 (PGE2), among others [174]. These changes further lead to ECM degradation and pathological cell death, involving the activation of immune system cells, including the monocyte-macrophage family, B lymphocytes, and NK cells. In response to inducible inflammatory factors, upregulation of downstream matrix-degrading proteins, such as matrix metalloproteinases (MMPs), platelet-reactive protein motifs, disintegrins, and metalloproteinases (ADAMTS), induces ECM degradation metabolism [175]. A study demonstrated that adipose tissue-derived mesenchymal stem cell exosomes can partially inhibit nuclear translocation of NF-κB, thereby reducing the expression of pro-inflammatory factors induced by TNF in NPCs. Additionally, these exosomes induce the accumulation of nerve growth factor and brain-derived neurotrophic factor in IVDD [139]. Furthermore, EVs derived from platelet-rich plasma (PRP), via miR-25-3p, inhibit the expression levels of the SOX4 gene, consequently suppressing the transcription of CXCR7 and alleviating cell degeneration induced by IL-1β [140]. Bone marrow MSCs can directly transfer lncRNA CAHM targeting M1 macrophages through exosomes, inhibiting the expression of the M1 phenotype marker iNOS and ultimately reducing the infiltration of inflammatory factors TNF-α and IL-6 in the IVD [141].

Apoptosis

Cell apoptosis is a programmed cell death process triggered by intrinsic and extrinsic pathways. The intrinsic pathway involves mitochondrial signaling, where BAK/BAX oligomers induce permeabilization of the mitochondrial membrane, leading to the release of cytochrome c (cyt c) into the cytoplasm. This event recruits various members of the Caspase family, ultimately forming apoptotic bodies. [176]. Umbilical cord mesenchymal stem cell (UCMSC)-derived exosomes modulate the Bcl-2/Bax ratio, thereby upregulating Coll-II and proteoglycan levels, which attenuates high glucose-induced damage to NPCs [142]. Research has indicated that EVs derived from MSCs are rich in miR-21. This microRNA inhibits PTEN, activating the PI3K-AKT pathway in NPCs. Consequently, the reduced activation of downstream apoptotic factors Bad, Bax, and Caspase-3 inhibits apoptosome formation, thereby alleviating TNF-α-induced damage in NPCs [143]. Similarly, miR-142-3p, derived from bone marrow MSC exosomes, inhibits MAPK pathway activation, exerting a similar effect [144]. Moreover, miR-31, an important miRNA derived from bone marrow MSCs, activates the Wnt/β-catenin signaling pathway to alleviate TNF-α-induced apoptosis [145]. This is achieved by targeting and inhibiting NFAT5, a member of the NFAT family associated with NF-κB enhancement [177]. Furthermore, miR-199a found in EVs derived from bone marrow MSCs significantly inhibits cell apoptosis by sponging GREM1, which can negatively impact the TGF-β1 signaling pathway, detrimental to IVD function [146]. Research has shown that the M1 macrophage polarization marker CD86 is upregulated in degenerated IVD tissues. Similarly, LRG1 participates in the p38-MAPK signaling pathway, promoting cell apoptosis and ECM degradation, and is associated with macrophage polarization. Interestingly, miR-129-5p derived from MSCs inhibits LRG1 expression by targeting the 3' UTR region of LRG1, thereby blocking the activation of the LRG1-dependent p38-MAPK signaling pathway, delaying M1 macrophage polarization, and alleviating the progression of IVDD [147].

Pyroptosis

Cell pyroptosis is an inflammatory vesicle-mediated form of cell death, dependent on cysteine proteases and the gasdermin-D (GSDMD) family. The canonical inflammasome multiprotein complex comprises NLRP3, the adapter protein ASC, and pro-caspase-1 [178]. Upon assembly of the inflammasome, pro-caspase-1 is cleaved to caspase-1, which then cleaves the GSDMD protein, releasing its N-terminal domain. This domain binds to membranes, leading to membrane permeabilization, cell rupture, and the release of inflammatory factors such as IL-1β and IL-18, triggering a potent intracellular inflammatory response [179]. The assembly of the NLRP3 inflammasome can be triggered by the NF-κB signaling pathway, infectious stimuli (such as influenza virus, Candida albicans, and Staphylococcus aureus), and sterile stimuli (mitochondrial damage, DNA accumulation) [180]. Viral, bacterial, and LPS stimuli, among others, activate the pattern recognition receptors (PRRs), such as toll-like receptors, which upregulate the NF-κB signaling pathway, promoting the transcription of inflammatory factors such as NLRP3, IL-1β, and IL-18 [181]. Therefore, in degenerative disc diseases, cell pyroptosis occupies an important form.

EVs derived from mesenchymal stem cells (MSC-eVs) exhibit a remarkable capacity to significantly reverse the levels of NLRP3, GSDMD, and cleaved caspase-1 in IVDD. This effect is achieved through the abundant presence of miR-199a within the vesicular cargo [148]. Similarly, research has shown that Evs derived from PRP can target and downregulate Keap1, upregulate Nrf2, and promote the nuclear translocation of Nrf2 by delivering abundant miR-141-3p. Through the creation of Nrf2 knockout models, it has been demonstrated that the specific reduction of NLRP3 expression in the nucleus effectively blocks the H₂O₂-induced inflammatory cytokine storm in NPCs [149]. Likewise, platelet-derived exosomes can induce the ubiquitination and degradation of NLRP3 inflammasomes in the IVD, regulating M1 and M2 macrophage polarization through the NF-kb and MAPK signaling pathways [150]. M6A methylation modification is also involved. The M6A methyltransferase METTL14 is highly expressed in degenerated tissues, enhancing the stability of NLRP3 mRNA by recruiting the methylation partner IGF2BP2. NLRP3, a crucial component of inflammasomes, plays an important role in cellular pyroptosis. Additionally, exosomes derived from human umbilical cord MSCs disrupt endogenous M6A modification targeting METTL14 through miR-26a-5p [151].

Ferroptosis

An iron-dependent oxidative cell death pathway, ferroptosis involves dysregulation of intracellular ROS homeostasis, lipid peroxidation, and disruption of endogenous antioxidant defenses. This process is involved in the pathogenesis of musculoskeletal disorders in orthopedics, such as osteoarthritis, spinal cord injury, rheumatoid arthritis, IVD degeneration, etc. EVs have been demonstrated to effectively regulate intracellular iron-mediated cell death. For instance, EVs from bone marrow MSCs can upregulate the synthesis of NRF2, which in turn downregulates ACSL4 and upregulates glutathione peroxidase 4 (GPX4). This leads to a decrease in levels of ferritin light chain (FTL) and free iron, stabilizing mitochondrial membranes and maintaining mitochondrial morphology. This inhibitory effect is attributed to their abundant circ_0072464 content, as evidenced by a significant correlation between levels of circ_0072464 and iron-mediated cell death through both overexpression and suppression experiments [152]. A recent study shows that circ_0050205 in EVs from bone marrow MSCs participates in oxidative stress-induced iron-mediated cell death. circ_0050205 transfers to NP cells and acts as a sponge for miR-665, leading to increased GPX4 expression and decreased free iron production. This reveals a potential therapeutic target for managing iron-mediated cell death [153]. As a member of the glutathione peroxidase family, GPX4 possesses the ability to eliminate lipid peroxides, with lipid peroxidation serving as a critical factor in iron-mediated cell death [182].

Autophagy

Cellular autophagy is a self-cleaning mechanism within cells, primarily accomplished through the engulfment of damaged organelles or proteins by autophagolysosomes, thereby maintaining cellular homeostasis [183]. Autophagosomes, double-membrane vesicles that enclose cytoplasmic contents, fuse with lysosomes to form autolysosomes [184]. The Unc-51-like kinase 1 (ULK1), along with the ATG family members ATG13, ATG101, and FIP200, are involved in initiating the process of autophagy [185]. In the early stages of IVDD, activation of the intracellular AMPK pathway promotes the formation of ULK complexes and inhibits the activity of mTORC1, thereby enhancing NP cell autophagy. Research has revealed that EVs derived from bone marrow MSCs significantly elevate the LCII/I levels in NPCs, triggering autophagic initiation via the AKT-mTOR pathway. This phenomenon was found to be inhibited by the autophagy inhibitor 3-methyladenine (3-MA) [13]. Similarly, in AF cells, activation of the AKT-mTOR pathway mediated by bone marrow stromal cell (BMSC) EVs is associated with autophagy [154]. Furthermore, EVs derived from cartilage endplate stem cells effectively suppress apoptosis in NPCs by activating the PI3K-AKT signaling pathway, significantly increasing the levels of autophagic proteins LC3B/A and P-AKT while reducing Cleaved-caspase3 levels. These effects can be blocked by the AKT inhibitor LY294002 [155]. On the contrary, rapamycin, a classic autophagy inducer, targets the protein mTOR, a serine/threonine protein kinase in the phosphatidylinositol 3-kinase-related kinases (PIKK) family, binding to the FRB domain and acting as a negative regulator involved in autophagic activation. Moreover, autophagy significantly increases the release of EVs from NPCs, containing abundant miR-21a that selectively inhibits the activation of matrix-degrading protein MMP13, thereby suppressing ECM degradation [156]. In addition, miR-155 delivered by BMSCs-exo activates the autophagic flux in NPCs. Subsequent validation revealed the silencing of its target protein BACH1 expression, leading to increased downstream HO-1 protein expression and activation of autophagy [157]. Transferred from human adipose tissue-derived stem cells, miR-155-5p targets TGFβR2 in LPS-induced NPCs, activating the AKT/mTOR pathway, enhancing autophagy, and suppressing the secretion of pro-inflammatory cytokines [158].

Oxidative stress

Oxidative stress arises from an imbalance between oxidative processes and antioxidant protection in the body. Oxidative stress-induced cell damage is significantly associated with the progression of IVDD and manifests primarily as inhibition of cell proliferation, collagen matrix degradation, and promotion of cell death. Local oxidative stress is accompanied by cellular senescence and AGEs. EVs derived from MSCs can provide essential proteins to mitochondria, repairing mitochondrial damage and reducing endogenous ROS accumulation [159]. Excessive inflammation and oxidative stress play a crucial role in IVD degeneration. PRP is a cytoplasmic fraction derived from autologous marrow megakaryocyte lysis. The released eVs act as lipid bodies, containing numerous cytokines. Studies indicate that eVs released from PRP can upregulate the abundance of the mitochondrial biogenesis regulator PGC1α, simultaneously reduce ROS levels, stabilize mitochondrial membrane potential, enhance ATP production, and decrease lactate accumulation [160]. Similarly, EVs derived from bone marrow MSCs can partially alleviate the high levels of ROS induced by intervertebral high pressure, thereby reducing cellular apoptosis [161].

ER stress

The ER is a membranous network consisting of cisternae, vesicles, and tubules, widely present in eukaryotic cells as a vital organelle. It primarily participates in the processes of protein synthesis, processing, and transport while also regulating intracellular calcium homeostasis [186]. The ER is categorized into rough and smooth ERs based on the presence or absence of ribosomes on its cytoplasmic surface. Impaired ER function in degenerated aging tissues, accompanied by inflammation and metabolic dysfunction, leads to the formation of AGEs [187].

Studies have indicated a positive correlation between the AGEs in degenerated intervertebral disc tissues and the progression of IVDD, which promotes mitochondrial dysfunction and loss of NP cell activity [188].BMSC-exos derived from bone marrow MSCs were found to significantly inhibit the expression of ATF6, IRE1α, and ATF4 in NPCs under stimulation by AGEs.This inhibition led to reduced CHOP transcription, decreased activation of downstream Caspase-3 and Caspase-12, and improved cell apoptosis under ER stress, suggesting a potential therapeutic role of BMSC-exos in alleviating cellular dysfunction induced by AGEs in NPCs [162]. Under prolonged stress, NP cells exhibit enhanced ER stress, increased expression levels of GRP78 and CHOP, as well as corresponding upregulation of Caspase-3 and Caspase-12 proteins. Co-culturing with exosomes derived from urine-derived stem cells reverses the above conditions, with the degree of reversal showing a dose-dependent increase in correlation with exosome content [163].

ECM reconstruction

The ECM primarily consists of collagen and PGs, which are typically secreted by NP cells. Collagen constitutes the majority, providing structural support and elasticity, while PGs maintain the hydration and stability of the extracellular space [189]. Additionally, fibronectin and collagen fibers are crucial components, with the former forming fibrous structures responsible for intracellular signal transduction and the latter providing elastic support to maintain tissue structure [190]. The ECM of the NPCs relies primarily on the balance between synthesis and degradation to maintain NP cell activity. In the degenerated NP of the IVD, the expression of matrix metalloproteinases (MMP-1, MMP-3, MMP-7, MMP-9) and A disintegrin-like and metalloprotease with thrombospondin type-1 motif (ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-15) is significantly upregulated [191]. Concurrently, various adverse intracellular environments lead to insufficient synthesis of Coll-II and PGs in the ECM.

Research has shown that EVs from human bone marrow MSCs can induce dendritic cell proliferation, promote the accumulation of PGs and glycosaminoglycans (GAGs), and increase the expression of ACAN protein while reducing the levels of cell death markers lactate dehydrogenase (LDH) and apoptotic levels [164]. Furthermore, the transmembrane protein Vasorin (VASN) is abundantly expressed on the membrane of mesenchymal stem cell-derived EVs. Upon delivery via thermoresponsive hydrogels, VASN can directly target Notch1 within the IVD, activate the Notch signaling pathway, and facilitate IVD repair and regeneration [165]. Similarly, intercellular communication between NPC and BMSCs is mediated by EVs. NPC-derived exosomes promote BMSC differentiation toward a NP-like phenotype, while BMSC-derived exosomes coordinate NPC proliferation and enhance matrix repair capabilities [166]. By delivering miR-17-5p targeting Toll-like receptor 4 (TLR4) into NPC, hypoxic mesenchymal stem cell-derived EVs inhibit the activation of the PI3K-AKT signaling pathway, thereby promoting NPC proliferation and ECM repair [167]. Similarly, TRAF6 has been shown to participate in the progression of IVDD. Derived from mesenchymal stem cell-derived EVs (MSCs-eVs), miR-194-5p targets the 3' UTR region of TRAF6, ultimately promoting NPC proliferation and osteogenic differentiation under TNF-α intervention [168]. Furthermore, miR-129-5p derived from bone marrow MSCs inhibits the activation of the Wnt/β-catenin pathway by sequestering SOX4, a protein that is upregulated in degenerated IVD tissues. This leads to upregulation of Aggrecan and Collagen II expression, promoting NPC proliferation and ECM synthesis [169]. Similarly, EVs derived from bone marrow MSCs transport miR-532-5p, which effectively targets RASSF5, thereby significantly inhibiting collagen degeneration and fibrotic accumulation in NPCs [170]. Furthermore, EVs derived from M2c macrophages play a significant role in promoting ECM synthesis in NP cells by delivering mir-124, which regulates cartilage intermediate layer protein(CILP) and enhances the TGF-β/Smad3 pathway [171].

Cellular senescence

Cellular senescence involves the gradual decline in function and vitality within the cellular life cycle, culminating in the cessation of metabolic activities and eventual demise. Various factors, including DNA mutations, oxidative stress, telomere dysfunction, and epigenetic modifications, may instigate the process of senescence [192]. Cellular senescence is commonly characterized by the upregulation of senescence secretion markers P53, P21, andp16 and a decrease in the expression levels of the MKI67/Ki67 protein. These alterations trigger the activation of damage signaling pathways such as p38 MAPK and NF-κB, as well as pathways involving p16INK4a-Rb or p53-p21CIP1-Rb, resulting in the expression and secretion of inflammatory cytokines and chemokines. [193]. In NP cells, these changes are moderately correlated with the degree of IVDD (based on Pfirrmann grading) [194].

Studies indicate that Sirt1, in distinction to its counterparts Sirt3 and Sirt5, plays a pivotal role in orchestrating the postponement of NP cell senescence and the establishment of a concurrent calcification blockade within adipocyte-derived extracellular vesicles (Adipo-sEVs). Within these EVs, nicotinamide phosphoribosyltransferase (NAMPT) can activate the NAD⁺ and Sirt1 pathways in NPCs to restore cellular vitality [172]. Similarly, Sirt6 has been identified as a crucial gene for repairing DNA damage and protecting against cell senescence. It stimulates MSCs generated from induced pluripotent stem cells (iPSCs) to release EVs containing miR-105-5p, which are subsequently transported into senescent NPCs. This process downregulates the expression of PDE4D, a specific hydrolytic enzyme of cyclic adenosine monophosphate (cAMP), leading to a subsequent increase in cAMP levels and ultimately activating the Sirt6 pathway [173]. The highly enriched miR-199a in BMSCs-eVs, upon internalization into aging NP cells, downregulates GREM1 through targeted mechanisms, thereby promoting the activation of the TGF-β pathway. This process consequently delays senescence in NPCs and facilitates ECM remodeling [146]. Moreover, the exogenous introduction of osteocalcin combined with SPC25-loaded exosomes (exos) has been shown to effectively delay senescence and cell cycle arrest in NP cells. This intervention is associated with reduced expression of inflammatory and apoptotic factors [195].

Challenges and prospects

Currently, no biomaterials have been applied clinically for the treatment of IVDD. Although EVs have shown significant efficacy in both in vitro and in vivo experiments in previous studies, there remains a long road ahead for their clinical translation. Several challenges need to be addressed, including:

Limitations of animal models and treatment methods

Although EVs have demonstrated good therapeutic effects in animal models, most of these studies have been conducted on coccygeal models of rodents (e.g., rats), whose anatomical structures and physiological characteristics differ significantly from those of humans [196]. Thus, there is uncertainty about whether the results of preclinical animal studies can be fully extrapolated to humans. Furthermore, the pathogenesis of IVDD is highly complex, involving various factors such as cell apoptosis, inflammatory responses, oxidative stress, and more. However, most existing preclinical studies focus on specific mechanisms without comprehensively evaluating the overall impact of EVs on the pathological environment. Future studies need to optimize animal models, particularly by using large animal models that more closely resemble human anatomy and pathology, to improve the clinical relevance of the findings.

Standardization of EVs therapy

The biological function of EVs is closely related to their source cells, extraction methods, and preparation processes. Currently, the methods for isolating and purifying EVs are not standardized. Different studies employ various extraction techniques (e.g., ultracentrifugation, size-exclusion chromatography), which can lead to significant differences in EV yield and purity [197, 198]. This not only affects the reproducibility of therapeutic effects but also poses challenges for clinical application. Future research should aim to establish and optimize standardized protocols for EV preparation to ensure consistency in their bioactivity and safety.

Therapeutic dosage and delivery methods

In current preclinical studies, there is considerable variability in both the dosage and delivery methods of EVs, and a standardized approach has yet to emerge [199]. Most studies use local injection to deliver EVs into the intervertebral discs; however, due to the avascular nature and high-pressure environment of the disc, the diffusion and persistence of EVs are limited, making long-term efficacy difficult to achieve [27]. Additionally, there is no clear basis for selecting the appropriate EV dose, with concentrations used in different experiments ranging widely (from 100 to 1000 µg/mL). Both the biosafety and optimal therapeutic dose of EVs have yet to be thoroughly evaluated. Future studies should explore the optimal dosage and delivery strategies, potentially incorporating biomaterials (such as hydrogels) to prolong the retention of EVs and enhance their therapeutic effects.

Cost and safety concerns

The production of EVs is expensive, especially when considering large-scale production for human therapy. Achieving a balance between cost and yield remains a significant challenge. Additionally, the long-term safety of EVs has not been adequately demonstrated, particularly in scenarios involving repeated injections or high doses, where there may be risks of immune reactions or other adverse effects [200]. Therefore, comprehensive safety assessments must be conducted before advancing to clinical trials, and detailed clinical application protocols need to be developed.

Despite these challenges, the potential of EVs for treating IVDD remains promising. With advancements in bioengineering, improvements in EV surface modification and drug-loading techniques could significantly enhance their targeting and therapeutic efficacy. Additionally, combining EVs with biomaterial-based delivery systems offers the potential to extend their therapeutic duration. Moreover, as our understanding of the pathological mechanisms of IVDD deepens, the multifaceted regulatory properties of EVs may provide unique advantages in suppressing inflammation, promoting cell regeneration, and remodeling the extracellular matrix. As preclinical research progresses and clinical trials are gradually initiated, EVs may become a viable biological therapy to slow down or even reverse the progression of intervertebral disc degeneration.

Conclusions

This review summarizes the mechanisms through which EVs counteract pathogenic factors at the cellular and molecular levels. Various cellular deactivation mechanisms, such as apoptosis, necroptosis, autophagy, oxidative stress, and mitochondrial damage in IVDD, have been extensively studied. Their complex nature persists in challenging therapeutic progress in disc degeneration. However, the discovery of EVs offers a potential avenue for delaying IVDD. By mediating intercellular communication processes, EVs can intervene and regulate many physiological and pathological processes existing in the NP tissue, thus potentially serving as a valuable tool for future clinical interventions.

Currently, there is limited understanding of the adverse effects of extracellular vesicle treatment in this field. The majority of experiments use extracellular vesicle concentrations ranging from 100 to 1,000 µg/mL, making it challenging to ascertain their biological safety profile. Future research endeavors should aim to further clarify reliable dosage strategies for extracellular vesicle therapy. Furthermore, it is noteworthy that not all EVs have a beneficial impact on (IVD health. In degenerated tissues, the infiltration of neovascularization leads to the secretion of endothelial microparticles (EMPs), which in turn stimulate AF cells, upregulating the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) families, thereby degrading the ECM [201].

Extracellular vesicle therapy has demonstrated remarkable efficacy at both in vivo experiments and cellular molecular levels. Additionally, the rat tail injection model established in experimental studies effectively showcases the holistic therapeutic effects of EVs in IVDD treatment. Currently, approximately 90% of IVD experiments utilize Sprague–Dawley or Wistar rats, specifically targeting age (8–12 weeks), sex (male), puncture segment (C7–8), and gauge size of the modeling needle (18–22G) [202]. However, the duration of action for standalone extracellular vesicle injection therapy is short, posing a challenge to achieving effective therapeutic concentration. The medical community has not yet reached a consensus on the administration method, concentration, and frequency of EVs. In the process of biological treatment, it is crucial to avoid administering repeated injections of medication that can cause pain for patients in order to achieve therapeutic efficacy. Consequently, the current rate of clinical translation for extracellular vesicle therapy remains relatively low. Moreover, EVs are prone to loss, and their susceptibility to enzyme deactivation induced by external temperatures hinders their direct clinical application.

However, we believe that extracellular vesicles from stem cells have certain effectiveness in the study of intervertebral disc degeneration process, which is a very promising therapeutic strategy.

Abbreviations

ADAMTS-5

A disintegrin and metalloproteinase with thrombospondin motifs 5

AF

Annulus fibrosus

Aggrecan

Aggregating chondroitin sulfate proteoglycan

AGEs

Advanced glycation end products

CEP

Cartilaginous endplates

Coll- II

Type II collagen

DLS

Dynamic light scattering

ECM

Extracellular matrix

ESCRT

Endosomal sorting complex required for transport

EVs

Extracellular vesicles

IL-1β

Interleukin-1 beta

IVD

Intervertebral disc

IVDD

Intervertebral disc degeneration

KGN

Kartogenin

LAMP-2B

Lysosome-associated membrane protein 2B

MAPK

Mitogen-activated protein kinase

MMP-3

Matrix metalloproteinase-3

MMP-13

Matrix metalloproteinase-13

MVBs

Multivesicular bodies

NFs

Nuclear factor-kappa B

NPs

Nucleus pulposus

NSAIDs

Non-steroidal anti-inflammatory drugs

NTA

Nanoparticle tracking analysis

OA

Osteoarthritis

PGs

Proteoglycans

PVDF

Polyvinylidene fluoride

SEC

Size-exclusion chromatography

SF-MSCs

Synovial fluid-derived mesenchymal stem cells

TEM

Transmission electron microscopy

TNF-α

Tumor necrosis factor-alpha

TGF-β

Transforming growth factor-beta

VDR

Vitamin D receptor gene

PGs

Proteoglycans

Author contributions

YXJ, OQW, and ZHC contributed equally. YXJ and OQW drafted the text. ZHC and LJC produced and checked the tables. KZ, QZC, and HJT produced the figures. XZW, MJ, and KYHK corrected the grammar of this text. YML, PM, and XYW reviewed and revised the text. XH and JZ contributed to certain sections of the final manuscript. AMW supervised and funded this work. All authors reviewed the final version of the manuscript. All authors have reviewed and approved the final manuscript.

Funding

The work was supported by the High – level Innovation Team of Wenzhou’s “Ouyue Talent Plan” (No. 2024R3003), the National Natural Science Foundation of China (No. 82472488), the Natural Science Foundation for Distinguished Young Scholars of Zhejiang Province (No. LR25H060001), and the Wenzhou Major Scientific and Technological Innovation Project (No. ZY2022010). General Project of the 2024 School - level Scientific Research Project of Wenzhou Medical University.

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Competing interests

No conflicts of interest exist among the authors of this article.