Therapeutic strategies for intervertebral disc degeneration: Extracellular vesicles and microRNAs derived from mesenchymal stem cells

Abstract

Intervertebral disc degeneration (IDD) results from an imbalance within the intervertebral disc, leading to alterations in extracellular matrix composition, loss of nucleus pulposus cells, increased oxidative stress, and inflammatory cascade. While IDD naturally progresses with age, some factors such as mechanical trauma, lifestyle choices, and genetic abnormalities can elevate the risk of symptomatic disease progression. Current treatments, including pharmacological and surgical interventions, fail to halt disease progression or restore IDD function. Although biological therapies have been evaluated, their effectiveness in reversing long-term disc degeneration remains inconsistent. Mesenchymal stem cell-based therapies have demonstrated potential for IDD regeneration but are hindered by biological limitations, ethical issues, etc. To date, mesenchymal stem cell-derived extracellular vesicles (EVs) have emerged as promising therapeutic agents for regeneration and anti-inflammation. Their therapeutic effects are attributed to several mechanisms, such as the induction of regenerative phenotype, apoptosis mitigation, and immunomodulation. In addition, the abundance of microRNAs within EVs play a crucial role in modulating the disc degeneration. Due to the problems in clinical use, however, the efficiency of the EVs should be overcome further by optimizing cell culture conditions, engineering them to deliver drugs and targeting molecules, etc.

Key Words: Intervertebral disc; Degeneration; Regeneration; Therapy; Stem cell; Extracellular vesicles; MicroRNA; Secretome

Core Tip: Intervertebral disc degeneration (IDD) has been focused on the condition that is linked to several pathological condition, but no effective treatment has been reported so far. Due to the limitations of mesenchymal stem cell-based trials for IDD regeneration, extracellular vesicle or microRNAs derived from mesenchymal stem cell have been evaluated as the next generation of therapeutic option for IDD. Thus, this review aims to provide a comprehensive overview regarding the IDD including anatomical considerations and therapeutic trials with a particular focus on extracellular vesicles and microRNAs. The authors also discuss the challenges and future directions in translating these innovative therapies into clinical practice.

INTRODUCTION

Intervertebral disc degeneration (IDD) is a leading cause of chronic low back pain, a condition that significantly affects global health and economic productivity[1,2]. Characterized by the progressive deterioration of the extracellular matrix (ECM) and the loss of disc cell function, IDD is influenced by a combination of mechanical, genetic, and biochemical factors. Conventional treatments, such as pain management and physical therapy, primarily focus on symptom relief rather than addressing the underlying pathophysiology[3]. Consequently, there is a growing interest in regenerative therapies aimed at restoring disc structure and function, with emerging strategies involving stem cells, extracellular vesicles (EVs), microRNAs (miRNAs), and other approaches.

Stem cell-based therapies have shown promise for IDD regeneration. In particular, mesenchymal stem cells (MSCs) have the ability to differentiate into nucleus pulposus (NP)-like cells and secrete bioactive factors that promote tissue repair and modulate inflammation[4]. Additionally, EVs, including exosomes derived from stem cells, have garnered attention as cell-free therapeutic agents[5]. These nanosized vesicles play a crucial role in intercellular communication, carrying proteins, lipids, and nucleic acids, such as miRNAs, that influence key cellular processes[6,7]. Notably, miRNAs, small noncoding RNAs that regulate gene expression post-transcriptionally, serve as critical modulators in IDD. They are implicated in pathways governing cell proliferation, apoptosis, ECM metabolism, and inflammatory responses. Harnessing miRNAs delivered through stem cells or their derived EVs and exosomes presents a promising avenue for enhancing the effectiveness of regenerative therapies.

This review aims to examine the anatomical aspects of the intervertebral disc and its surrounding structure in the context of degeneration and regeneration. Additionally, it provides a comprehensive overview of current advancements in regenerative strategies for IDD, with a particular focus on stem cell therapies, EVs, and miRNA-based approaches. We will discuss the underlying mechanisms, preclinical and clinical evidence, as well as the challenges and future directions in translating these innovative therapies into clinical practice.

ANATOMIC CONSIDERATION OF THE DISC AND SURROUNDING STRUCTURES

The intervertebral disc is a crucial component of the spinal column, serving as a mechanical buffer that absorbs compressive loads while enabling spinal flexibility. It consists of three primary structures: The NP, the annulus fibrosus (AF), and the cartilaginous endplates (CEPs). The NP, a gelatinous core rich in proteoglycans, collagen type II, and water, provides the disc with viscoelastic properties essential for withstanding compressive forces. Surrounding the NP, the AF comprises concentric lamellae of type I and type II collagen fibers arranged at oblique angles to resist tensile and shear stresses. The CEPs, thin layers of hyaline cartilage, interface the disc with adjacent vertebral bodies and facilitate nutrient exchange through diffusion, compensating for the disc’s avascular nature. With aging and mechanical stress, the intervertebral disc undergoes degenerative changes characterized by a decline in proteoglycan content, reduced NP hydration, and structural disruption of the AF. These changes contribute to a loss of disc height and biomechanical dysfunction, making the disc susceptible to IDD, which is often associated with low back pain and other specific disorders[8].

Surrounding structures play a crucial role in maintaining intervertebral disc health and influencing its susceptibility to degeneration. The vertebral bodies and endplates are essential for disc integrity, with bony endplates acting as conduits for nutrient and metabolite exchange. Degeneration calcification of the endplates impairs this diffusion, exacerbating cell death and ECM degradation within the disc. Ligaments such as the anterior and posterior longitudinal ligaments provide spinal stability, protecting the disc from excessive motion. However, stiffness due to ossification or degeneration changes in these ligaments disrupt spinal biomechanics and increases stress on the discs. Additionally, paraspinal muscles contribute to spinal stabilization and reduce axial loads on the intervertebral disc. Muscle atrophy or fatty degeneration, commonly observed in individuals with chronic back pain, disrupts this equilibrium, further amplifying degeneration. The limited innervation of the outer AF and the restricted vascularization of the outermost layers of the AF and CEPs make the disc highly dependent on diffusion for nutrient delivery. This unique anatomical feature renders the disc vulnerable to nutrient deprivation and mechanical overload, key factors in degeneration[9].

IDD is a multifactorial process involving mechanical, biochemical, and cellular alterations that collectively impair disc function. The pathogenesis begins with alterations in the ECM of the NP, primarily the depletion of proteoglycans and collagen degradation. This loss diminishes the disc’s water-binding capacity, leading to decreased hydrostatic pressure and disc height. Concurrently, microtears in the AF weaken its structural integrity, facilitating the extrusion of NP material. These structural changes initiate an inflammatory response characterized by the release of pro-inflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor-alpha, and matrix metalloproteinases, which further degrade ECM components. The resulting acidic and hypoxic microenvironment accelerates cellular apoptosis and senescence, further impairing matrix synthesis. Additionally, calcification of the CEPs restricts nutrient and oxygen diffusion, creating a vicious cycle that worsens degeneration[10,11].

The cellular mechanisms underlying IDD are complex, with significant contributions from oxidative stress and mitochondrial dysfunction. Reactive oxygen species accumulate due to mechanical overload and impaired antioxidant defenses, leading to DNA damage and mitochondrial dysfunction in disc cells. These alterations activate catabolic pathways, including the nuclear factor kappa B signaling pathway, which promotes inflammation and matrix degradation. Dysregulation of signaling pathways, such as transforming growth factor-beta (TGF-β) and Wnt/β-catenin further impairs matrix repair. Recent studies suggest that certain signaling pathways, including the protein kinase RNA-like endoplasmic reticulum kinase/activating transcription factor 4/IL-10 pathway and Kelch-like ECH-associated protein 1/NF-E2-related factor 2/heme oxygenase-1 pathway, play pivotal role on initiating and exacerbating disc degeneration[12,13]. The chronic inflammatory state also sensitizes nerve endings in the outer AF, contributing to pain. Understanding these pathways provides a foundation for developing targeted interventions, such as antioxidants, cytokine inhibitors, and gene therapies, to mitigate disc degeneration and promote regeneration[14].

A comprehensive understanding of the intervertebral disc and its surrounding anatomy is essential for developing effective regenerative strategies aimed at restoring disc structure and function. Cell-based approaches, particularly MSCs, seek to repopulate the degenerated disc with cells capable of producing a functional ECM. Growth factors such as TGF-β and bone morphogenetic proteins (BMPs), along with cytokines and genetic factors like miRNAs, are often incorporated to reduce the inflammation and enhance matrix synthesis and cellular proliferation[15]. In addition, biomaterial-based therapies as being explored to provide structural stability and support for regeneration. Hydrogels and 3D-printed bio-mimic structures are designed to replicate the biomechanical properties of the NP, promoting cell growth and ECM synthesis. However, significant challenges remain, particularly in overcoming the harsh microenvironment of the degenerated disc, characterized by low oxygen tension, acidic pH, and inflammation.

EFFECTS AND LIMITATIONS OF MSCS IN DISC DEGENERATION

In disc degeneration, inflammatory cytokines are upregulated, triggering regulated cell death[16-18]. This leads to apoptosis and cellular transformation into chondrocytes, contributing to ECM fibrosis[19-22]. Cellular aging plays a pivotal role in disc degeneration[23-25] by inducing cell proliferation arrest, chronic inflammation, and ECM degradation. Inflammation also exerts nociceptive effects, with increased levels of nerve growth factor and brain-derived neurotrophic factor observed in degenerated discs[26]. MSC therapy for disc degeneration is influenced by multiple factors, including its anti-inflammatory, anti-apoptotic, anti-pyretic effects, and increased cell numbers and promote ECM production through MSC differentiation[27]. Studies have demonstrated that bone marrow-derived MSCs (BMSCs) promote the proliferation of notochordal cells in the NP[28]. Additionally, umbilical cord-derived MSCs restore stem cell capacity by upregulating octamer-binding transcription factor 4, Nanog, and TIE2 while increasing CD29 and CD105 expression in NP-derived MSCs[29]. These effects have been evaluated using conditioned media from umbilical cord-derived MSCs, demonstrating the role of EVs in enhancing cell proliferation and chondrocyte differentiation. Furthermore, extracellular secretion and endocytosis facilitate the transfer of intracellular components from MSCs to NP cells (NPCs), leading to functional changes in NPCs[30] (Figure 1).

Figure 1 Schematic diagram of the effects of mesenchymal stem cells and extracellular vesicles on disc degeneration. Mesenchymal stem cells are initially isolated and cultured from tissue samples, and during culture, extracellular vesicles are generated in the medium, collected, isolated and purified. Resuspended extracellular vesicles are delivered to degenerating discs via intradiscal injection. MSC: Mesenchymal stem cell; NPC: Nucleus pulposus cell; ECM: Extracellular matrix.

Several factors contribute to the therapeutic efficacy of MSCs in disc degeneration. The TGF-β family is crucial in MSC differentiation, with TGF-β3 included in chondrogenic differentiation media[31,32]. Research has shown that BMSCs cultured with TGF-β3, dexamethasone, and ascorbic acid form spheroids that positively express type II collagen and genes such as ACAN, DCD, FMOD, and COMP, expression levels comparable to those in NP tissues[33]. The effects of TGF-β3 are further enhanced by growth factors like BMP2 and insulin-like growth factor (IGF)-1. When cultured with TGF-β3, BMP2, and IGF-1 BMSCs undergoes chondrocyte differentiation and activates the mitogen-activated protein kinases/extracellular signal-regulated kinase signaling pathway, leading to differentiation into NPCs[34,35]. Additionally, TGF-β1 has shown to promote MSC differentiation into NPCs[36,37] under hypoxic conditions[38] by upregulating sry-box transcription factor 9, ACAN, and collagen type 2 gene expression through mitogen-activated protein kinase activity[38]. The combination of TGF-β1 and growth differentiation factor 5 in adipose-derived MSCs promote NPC differentiation via the Smad2/3 signaling pathway and ECM production. Platelet-rich plasma has been explored as a potential inducer of chondrocyte differentiation and MSC differentiation into NPs[39-41]. However, studies indicate that when MSCs are cultured with platelet-rich plasma, differentiation at the gene and protein levels may not be optimal[39].

Members of the TGF-β superfamily play essential roles in the MSC into NP or chondrocytes[42]. BMP2, when incorporated into chondrocyte differentiation media, has demonstrated a positive effect on NPMSC differentiation[32], significantly enhancing the chondrogenic differentiation of BMSCs when combined with TGF-β3[34]. BMP3 has been shown to influence MSC proliferation and chondrogenic differentiation[43]. Additionally, BMP2 and BMP7 induce osteogenic and chondrogenic differentiation, respectively, via the Smad pathway[44,45]. BMP family members increase the expression of Runx2, SPP1, and ACAN[44,46,47], suggesting that BMP2 promotes osteogenic differentiation. Beyond BMPs, other growth factors also contribute to MSC differentiation. IGF1 has been used to induce NPC differentiation[48,49], while fibroblast growth factor 2 has been shown to support NPC differentiation through TGF-β1[50]. Table 1 summarizes the above[31,32,34,35,38,44,48-57].

Table 1 Effectiveness of mesenchymal stem cell biological therapy in various studies.

Biological therapies	Effects	Ref.
TGF-β1	MSC differentiation toward NP/chondrogenic phenotype and upregulation of ERK1/2 activity	[38,51]
TGF-β3	MSC differentiation to NPC and AFC in combination with BMP-2 and IGF-1	[31,32,34,35,52,53]
BMP2	Differentiation into NPCs with TGF-β3 and PRP	[32,34,44,54]
BMP7	MSC differentiation into NP-like cells via the Smad pathway	[55]
IGF1	MSC differentiation into NPC-like cells	[48,49,56]
FGF2	MSC differentiation into NPC-like cells	[48,50,57]

TGF: Transforming growth factor; MSC: Mesenchymal stem cell; NP: Nucleus pulposus; ERK: Extracellular signal-regulated kinase; NPC: Nucleus pulposus cell; AFC: Annulus fibrosus cell; BMP: Bone morphogenetic protein; PRP: Platelet-rich plasma; IGF: Insulin-like growth factor; FGF: Fibroblast growth factor.

Although stem cells hold great potential for regenerating damaged tissue and modulating immunological responses, significant limitations and challenges remain in research and therapeutic development. One major obstacle is the difficulty in MSC survival after transplantation. The condition, cell niche, and communication between cells determine the survival of stem cells. Second, MSCs exhibit heterogeneity due to donor variability, cell type differences, differentiation potential, and intercellular factors. Their sensitivity to environmental conditions can negatively impact disease control, particularly in cases of severe inflammation or active osteoarthritis. They are also unstable, which limits their storage duration even at ultra-low temperatures. Furthermore, the entire manufacturing process of MSCs, including ex vivo expansion, extraction technology, and culture method, has not yet been standardized. To address these limitations, EVs have recently gained attention as an alternative therapeutic strategy in regenerative medicine, with active research being conducted across various fields.

EFFECTS AND CONSIDERATIONS ON DISC DEGENERATION IN EVS

EVs are lipid bilayer-enclosed nanoparticles originating from multivesicular endosomes[58], typically ranging in diameter from 50 to 150 nm, with an average diameter of 100 nm[59,60]. Although discovered in the late 1980s, EVs have only recently been recognized as crucial mediators of cell-to-cell communication[61]. Their lipid bilayers withstand freeze-drying and extreme conditions while maintaining biological activity, immunotolerance, and therapeutic efficacy[62]. Secreted by various cells, EVs can be isolated from small amounts of biological fluids, including blood, semen, urine, breast milk, and bile, and they facilitate intercellular communication by delivering biologically active molecules, miRNA, and proteins[63,64].

EVs exhibit significant therapeutic potential in tissue repair by inhibiting apoptosis, enhancing regenerative phenotypic characteristics, and stimulating angiogenesis through immune modulation[65,66]. They contribute to tissue by influencing gene expression and modifying the phenotype of damaged cells through molecular delivery[67,68]. The effects of stem cell-derived EVs have been evaluated in disc degeneration research[69-71]. For instance, BMSC-EVs have been shown to reduce IL-1 induced proinflammatory cytokine secretion and mitogen-activated protein kinases signaling by targeting mixed lineage kinase 3 via miR142-3p[72]. Another study found that miR-532-5p in BMSC-EVs targets Ras association domain family member 5[73]. In vitro, human bone marrow stem cell-derived EVs demonstrated a 50% increase in cell proliferation and a significant reduction in apoptosis in a condition of intervertebral disc cell cultures[7]. Additionally, NP-derived EVs were found to promote MSC differentiation into NP-like cells while downregulating of the Notch1 pathway[74,75]. Age-related declines in NPC numbers are closely associated with the onset of disc degeneration, and treatment with NPC-derived EVs has been shown to increase the DNA and glycosaminoglycan content of human NPC microaggregates[76,77] (Table 2).

Table 2 Effectiveness of mesenchymal stem cells-derived extracellular vesicles in biological therapy across various studies.

Biological therapies	Effects	Ref.
NLRP3	Exosomes play an anti-inflammatory role in pathological NPCs by suppressing inflammatory mediators and NLRP3 inflammasome activation	[78]
MLK3	Bone marrow MSCs-derived exosomes-packaged miR-142-3p alleviates NPCs injury through suppressing MAPK signaling by targeting MLK3	[72]
RASSF5	MSCs may suppress TNF-α-induced apoptosis, ECM degradation, and fibrosis deposition in NPCs through the delivery of miR-532-5p via targeting RASSF5	[73]
Notch1	Inhibition of the Notch1 pathway facilitates NPC exosome-induced differentiation of MSCs into NP-like cells in vitro	[74]
PI3K and AKT	Exosomal miR-21 restrains PTEN and thus activates PI3K/AKT pathway in apoptotic NPCs	[80]
AKT and ERK	MSC-exosomes could attenuate ER stress-induced apoptosis by activating AKT and ERK signaling	[81]
ZNF121	Exo-antagomiR-4450 retarded damage of NPCs in vitro, alleviated IDD damage, and ameliorated gait abnormality in vivo	[82]
ATF6	MSC-exosomes reduced apoptosis and calcification of EPCs via regulation of the miR-31-5p/ATF6/ER stress pathway	[83]

NLRP3: Nod-like receptor protein 3; MLK3: Mixed-lineage kinase 3; MSC: Mesenchymal stem cell; NPC: Nucleus pulposus cell; MAPK: Mitogen-activated protein kinases; RASSF5: Ras association domain family member 5; TNF: Tumor necrosis factor; ECM: Extracellular matrix; PI3K: Phosphatidylinositol-3-kinase; AKT: Protein kinase B; PTEN: Phosphatase and tensin homolog; ERK: Extracellular signal-regulated kinase; Exo: Exosomes; IDD: Intervertebral disc degeneration; ZNF121: Zinc finger protein-121; EPC: Epithelioma papulosum cyprini; ATF6: Activating transcription factor 6; ER: Endoplasmic reticulum.

In a recent in vivo study on stem cell-derived EVs, it has shown that EVs prevented the progression of degenerative diseases in a rabbit model, suggesting that injected EVs may be a viable therapeutic strategy targeting the Nod-like receptor protein 3 inflammasome[78,79]. EVs could supply mitochondrial proteins to NPCs and recover damaged mitochondria in degenerative discs[79] (Figure 1). In a rat model, intradiscal injection of BMSC-EVs alleviated NPC apoptosis and slowed disc degeneration, with miR-21 playing a key role by activating the phosphatidylinositol-3-kinase/protein kinase B pathway through the inhibition of phosphatase and tensin homolog[80]. MSC-EVs may further reduce apoptosis by modulating the protein kinase B and extracellular signal-regulated kinase signaling pathways[81]. Human placental MSC-derived EVs have also demonstrated therapeutic effects in in vivo and in vitro models of NPC degeneration. The inhibition of miR-4450 by these EVs upregulates zinc finger protein-121, alleviating inflammation, apoptosis, and damage in NPCs[82]. Studies in a mouse model suggest that MSC-EVs injections improve disc degeneration by regulating miR-31-5p and activating transcription factor 6-related endoplasmic reticulum stress[83] (Figure 2 and Table 2).

Figure 2 Mechanism of extracellular vesicle to treat intervertebral disc degeneration. Extracellular vesicle inhibits apoptosis of nucleus pulposus and induces differentiation into nucleus pulposus cells for the treatment of intervertebral disc degeneration through microRNA and specific mechanisms. NLRP3: Nod-like receptor protein 3; AKT: Protein kinase B; ERK: Extracellular signal-regulated kinase; MLK3: Mixed-lineage kinase 3; RASSF5: Ras association domain family member 5; PTEN: Phosphatase and tensin homolog; NP: Nucleus pulposus; IDD: Intervertebral disc degeneration.

CURRENT LIMITATIONS AND FUTURE PERSEPCTIVES

Unsurprisingly, MSCs have emerged as a promising therapeutic approach at the forefront of neurodegenerative disease research[84]. In fact, MSCs have the ability to migrate to damaged tissues, differentiate into new cell types even in vitro over long periods of time, and promote anti-inflammation and/or regeneration[85-88]. Although there are many therapeutic indications and evidence for using MSCs[89-96], there is a consensus that MSCs are safe to use, despite the relatively limited number of human studies conducted so far. However, a recent meta-analysis on this topic found that MSC administration would lead to some side effects such as short-term fever, inflammatory effects at the injection site, constipation, fatigue, and insomnia in groups suffering from various diseases[97]. Aging of autologous and transplanted MSCs may contribute to unsatisfactory therapeutic outcomes[98], as one example is that transplantation of adipose-derived MSCs from aged mice causes physical dysfunction in recipients[99], and adverse effects of aged MSCs have also been demonstrated in vitro[100]. The proliferation of BMSCs decreases their osteogenic potential during aging[101,102], which may contribute to age-related diseases. Specifically, aged MSCs exhibit decreased cell proliferation and osteogenic activity measured by alkaline phosphatase activity, ECM mineralization, and osteogenesis-related genes, and increased adipocyte differentiation capacity in accordance with adipocyte protein 2, resistin, and lipid accumulation[103]. These mechanisms by senescent MSCs lead to decreased osteogenesis, resulting in osteoporosis and poor osseointegration capacity[104-108].

Cellular senescence affects the composition of the EVs. EVs released from aged cells contain unpredictable growth factors and cytokines. These factors will cause a “domino effect” that will cause negative effects from cellular to tissue levels[109]. Especially in the context of IDD, the senescent EVs are secreted extracellularly, including inflammatory cytokines, degradative enzymes, and senescence-related miRNAs, which can accelerate the degeneration of disc tissue by inducing inflammatory responses and cellular senescence in surrounding cells. Therefore, when designing or using therapeutic EVs, it is very important whether the mother cell is senescent or not, and it could expect a potential side effect from the use of aged EVs that may neither affect the tissue at all nor rather worsen tissue degeneration. Hao et al[110] in 2022 reported that MSC-derived EVs can regulate the senescence of intervertebral disc cells through miR-217, suggesting that EVs may have opposing biological effects depending on their molecular composition. In this regard, EV-based therapeutic approaches need to be combined with strategies that closely analyze the biological properties of EVs and preemptively remove or inhibit senescence-related signals[110].

CONCLUSION

For several decades, extensive efforts have been made to effectively restore degenerated intervertebral discs to their normal state through regeneration processes, various sources and materials, ranging from stem cells to engineered acellular vesicles and miRNAs. Despite numerous attempts to regenerate the intervertebral disc, the complexity of its anatomical structure and the mechanism involved in maintaining homeostasis continue to pose significant challenges in fully understanding the disc and identifying the ideal regeneration method. With developments in 3D-printing-based scaffolds and the discovery of novel sources, the prospect of achieving normal disc regeneration is now closer than ever. Furthermore, in the near future, these developments may lead to the creation of artificial disc structures that mimic natural discs. In addition to technological advancements, it is crucial to unravel the underlying mechanisms governing both degeneration and regeneration processes in the intervertebral disc.