Bioactive Therapies for Degenerative Disc Disease: Microenvironmental Foundations of Disease

Abstract

Intervertebral disc (IVD) degeneration is a common source of back pain. The IVD is a complex structure that consists of an outer annular ring, an inner nucleus pulposus, and flanking cartilaginous endplates, which together allow for daily mobility by distributing loads and acting as a flexible segment within the spine. Both age and mechanical overload can drive the development of a pathologic disc microenvironment that includes alterations in mechanics, solute transport, and inflammation. Such changes in the disc have negative consequences on resident cells that promote their senescence, apoptosis, and contribution to furthering disc degeneration through mitochondrial dysfunction and the release of reactive oxygen species, proteases, and cytokines. This crosstalk between IVD cells and their microenvironment creates a feedback loop that eventually manifests into such clinical conditions as disc height loss, herniations, and total IVD collapse. Developing a holistic understanding of how this feedback loop is initiated and may be halted will enable the development of novel therapeutics that not only provide analgesic benefit but also help rebuild the deteriorated disc.

INTRODUCTION

Around 5% of the global population experiences back pain due to intervertebral disc (IVD) degeneration.^1,2 The main role of the disc is to facilitate daily movement by acting as a shock absorber, load transmitter, and flexible unit. Hence, when disc degeneration occurs, it destabilizes the spine, prevents normal motion, and, most notably, causes pain. In the clinic, degenerated discs are characterized by gradual morphological alterations in structure. Generally, this degeneration is due to normal wear and tear of the tissue that occurs with usage. However, it is further aggravated by aberrant changes in the disc microenvironment that have detrimental effects on resident cells. This review will cover how disc degeneration is a product of changes in both tissue microenvironment (i.e., mechanics, transport, and inflammation) and cell behavior (i.e., apoptosis, mitochondrial dysfunction, oxidative stress, and senescence) that together lead to clinical manifestations of disease. This article is the first in a series of 3 addressing bioactive therapies for degenerative disc disease with the second discussing the current state of the art and clinical applications³ and the third discussing challenges and innovations.⁴

INTERVERTEBRAL DISC STRUCTURE AND FUNCTION

The IVD is a donut-shaped organ with an inner region known as the nucleus pulposus (NP) and an outer region known as the annulus fibrosus (AF). Together, the NP and AF are sandwiched between an inferior and a superior cartilaginous endplate (CEP). The NP is a gel-like substance primarily made up of water, proteoglycans (i.e., aggrecan), and type II collagen.^5,6 Its main function is to absorb and release water to maintain the osmotic pressure within the disc that is necessary to properly distribute applied loads.⁷ The AF is a tough, fibrous tissue primarily consisting of water, type I collagen, and proteoglycans.^6,8 Its main function is to help maintain intradiscal pressure by circumferentially restraining the NP. The CEPs are thin layers of hyaline cartilage that anchor the disc to the adjacent bony vertebrae.⁹ Notably, various other collagens are present within the IVD at lower levels, including collagen IX, which acts as a linker not only among collagens but also between collagens and noncollagen proteins, thus contributing to the disc’s innate strength.¹⁰

Each region (NP, AF, CEPs) of the disc is governed by distinct cell types derived from the embryonic mesoderm that allow for homeostatic maintenance. Briefly, NP cells (NPCs) are chondrocyte-like cells that originate from the notochord. Although various subtypes of NPCs have been identified, proteoglycan/collagen type II—expressing NPCs and fibroblast-like NPCs are postulated to aid in normal tissue maintenance.¹¹ Similar to the NP, the AF and CEPs contain heterogenous populations of cells, originating from the sclerotome, which can be subdivided into various groups.¹² The 2 main groups in the AF are chondrocyte- and fibroblast-like AF cells, while the main group in the CEPs is mesenchymal chondrocytes.^11,12 All regions also contain their respective progenitor cells that allow for cell replenishment. Although these cell populations maintain homeostasis of the IVD, their activity is limited by the lack of significant vasculature within the disc.

The IVD is considered the largest avascular organ in the human body, relying on passive diffusion through adjacent tissues for nutrient delivery and waste disposal.¹³ Its size and reliance on diffusion creates a unique microenvironment that is distinct from most other tissues. The CEPs and outer AF are its only opportunities for solute flux.^13,14 The CEPs serve this purpose due to the blood vessels of the bony vertebrae that penetrate their periphery.¹⁵ The concave shape and thickness gradient of the CEPs make their center more permeable than their outer edges, allowing for solute flux primarily for the NP and inner AF.^16,17 In contrast, the outer AF experiences solute flux through peripheral blood vessels that mainly supply adjacent soft tissues.¹³ Solute concentrations for each area of the disc vary and are transport and cell dependent. Specifically, solute transport is affected by material permeability, diffusion distance, and geometry, while cells vary in solute consumption, density, and distribution within the tissue.¹⁸ Normally, the IVD shows lower oxygen and glucose levels than surrounding tissue, with the NP and inner AF particularly having lower nutrient levels than the outer AF.^19,20 This scarcity in nutrients limits the innate healing capacity of the IVD by restricting cellular growth and metabolism. During degeneration, the limited healing capacity of the disc allows the NP, AF, and CEPs to undergo structural alterations that eventually impede their function. This shift toward a diseased state begins microscopically, where a loss in essential extracellular matrix (ECM) proteins gradually transforms the cellular microenvironment, creating three distinct stages of degeneration.

THE DEGENERATIVE MICROENVIRONMENT

The cellular microenvironment describes the external, local stimuli experienced by cells, including mechanical force, soluble factor signaling, and ECM composition, all of which change dynamically in both health and disease.²¹ Microenvironmental cues such as mechanical and biochemical signals modulate cell behavior through various cell surface receptors.^22,23 In a healthy state, this communication between cells and their environment plays a critical role in physiological tissue remodeling and cell turnover, helping to maintain whole organ structure and function.²⁴ As tissues become pathologic, changes in structure and loading environment trigger a cascade of intrinsic cell signaling events that, in attempts to heal, lead to alterations in cell behavior and phenotype.^24,25 In the scenario where cells are unable to return to homeostasis after injury, the feedback loop between cells and their environment contributes to further deterioration of tissues. Damage propagation occurs as cells communicate stress to each other and their local environment through various damage-related secretory molecules such as proteases, proinflammatory cytokines, and reactive oxygen species. In the context of the healthy IVD, resident cells naturally lie in a nutrient-deprived and hypoxic microenvironment that is constantly subjected to high mechanical loads. With degeneration, the microenvironment progressively worsens as a consequence of its interactions with resident cells, causing the disc to enter a chronic state of disease. In this section, we will discuss exactly how features of the disc microenvironment such as mechanics, solute transport, and inflammation change in early-stage degeneration, after herniation, and in late-stage degeneration (Figure 1).

Figure 1.

Stages of intervertebral disc degeneration. AF, annulus fibrosus; IVD, intervertebral disc; MT, mitochondria; NP, nucleus pulposus; ROS, reactive oxygen species.

Early Stage

Early-stage IVD degeneration has been observed in individuals as young as 20 years of age.²⁶ At this stage, metabolically active, chondrocyte-like cells govern the IVD, and the initiating characteristic of disease is a shift in the biochemical composition of the ECM.^5,11,27 Proteoglycans are the most abundant ECM molecules within the disc and are composed of a large core protein with negatively charged glycosaminoglycan side chains.^5,27 Their structure and charge allow them to attract water. During degeneration, the proteoglycan content within the disc drops, resulting in a loss in water content.⁵ Both mechanical loading and enzymatic degradation contribute to this observed loss in proteoglycans. Naturally, the disc undergoes repetitive loading cycles due to its inherent function as a load distributer and cushion within the spine.²⁸ Such repetitive loading, as well as overloading, can instigate microscale damage in the NP.^28,29 Matrix damage and mechanical loading itself promote elution of proteoglycans out of the NP.²⁹ Biochemically, matrix degrading enzymes of the matrix metalloprotease (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families are considered the primary contributors to this observed degradation in proteoglycans.^30,31 Many MMP and ADAMTS are present within healthy discs and work together with tissue inhibitors of metalloproteinases to control normal ECM turnover.³² However, during degeneration the intricate balance between these catabolic enzymes and their inhibitors is lost, favoring tissue breakdown. Studies have found that changes in the microenvironment of cells cause this shift in their metabolic function. Various mechanical loading regimes in particular exhibit a regulatory influence on the expression of MMPs and ADAMTS.³² Specifically, high magnitude, high frequencies, and/ or long duration of loading in compression, tension, and shear induce catabolism, increasing the expression of these proteolytic enzymes.³² Numerous inflammatory and oxidative stimuli have also been shown to increase the expression of MMPs.^33,34 Besides alterations in proteoglycan content, collagens have also been shown to contribute to disease manifestation. Specifically, genetic variants in collagen IX and XI have been associated with intervertebral disc degeneration.¹⁰ These shifts in the biochemical composition of the disc instigate the transformation of the microenvironment in which resident cells become subjected to increased mechanical stress, nutrient deprivation, and inflammation.

Mechanical Stress.

A major contributor to the initiation and progression of disc disease is an increase in mechanical stress. During early-stage degeneration, increases in hydraulic permeability due to proteoglycan depletion prevents the NP from maintaining intradiscal swelling pressure that allows it to bear load.^3,7 In turn, the NP experiences a decrease in compressive stiffness and an increase in shear modulus, both of which are correlated to the amount of water, proteoglycans, and fibrotic tissue.^35,36 As these changes occur and its behavior becomes more “solid-like,” the NP loses its capacity to dissipate energy,³⁵ impeding its ability to evenly transfer load to the AF and CEPs. Consequently, the AF experiences higher axial and radial strains.³⁶ Increases in strain cause microstructural changes in the collagen fiber structure within the AF. Under stress, healthy AF collagen fibers reorient themselves to towards the applied loading direction allowing for load bearing. As the AF degenerates and becomes more disorganized, this intrinsic ability is lost, inducing and propagating tears.^37,38 Continued mechanical loading can specifically cause circumferential tearing or delamination of the AF, likely as a result of high interlaminar shear stress.^39-41 In addition to the stress placed on the NP and AF during loading, the CEPs undergo tension when the spine is compressed due to their role as a mechanical barrier between the NP and bony vertebra.¹² As proteoglycan content in the CEPs decreases with degeneration and tears form, the CEP’s tensile modulus decreases, making it more susceptible to deformation.⁴² Although not fully understood, shearing, high compressive loading, and tugging of the CEPs by the AF may also instigate damage.^43-45 Additionally, genetic polymorphism in collagens can also impair the mechanical properties of the disc.¹⁰ In summary, alterations in ECM composition affect disc material properties, altering the amount of loading experienced by the IVD. Cells respond to these changes in the mechanical microenvironment via mechanosensing machinery on their surface.²³ Accordingly, changes in mechanical loading of the disc influence how its cells behave, which in the case of degeneration increases inflammatory and oxidative signaling.

Solute Transport.

During early degeneration, CEP fixed charge density increases with accumulation of proteoglycan fragments released from the NP.⁴⁵ Increased proteoglycan content decreases tissue pore size and increases fixed charge density, changes which both limit solute transport by decreasing permeability.^45,46 With age, outer AF vascularity also decreases.⁴⁷ These alterations in permeability and vascularity hinder nutrient transport, creating a more hypoxic and hypoglycemic microenvironment. In addition to being nutrient deprived, degenerated discs have low pH levels due to increases in lactate.^19,48 Lactate is a waste product of glycolysis, the major pathway for energy production in cells with limited oxygen supply.⁴⁹ Increased lactate with no effective routes for waste disposal has been shown to contribute to the development of an acidic microenvironment.⁴⁸ Collectively, nutritional shortages and a decrease in pH have detrimental effects on cell metabolism and survival.⁵⁰

Inflammation.

Early changes in material properties and structure promote inflammation. Specifically, high mechanical strain has been shown to upregulate toll-like receptors (TLRs) in disc cells.⁵¹ TLRs are transmembrane proteins that promote cytokine expression in response to damage-associated molecular patterns such as matrix fragments.⁵¹ Alongside TLRs, multiple inflammatory cytokines, including IL-6, IL-8, IL-15, and tumor necrosis factor-α have also been shown to increase with high mechanical strain.⁵¹ While strain increases the expression of these proteins, matrix fragments alone modulate the expression of inflammatory cytokines such as IL-1 β, IL-6, and IL-8 through TLRs.⁵² These inflammatory mediators are expressed and secreted by both resident chondrocytes and invading immune cells. Immune cell infiltration occurs through cracks and tears that form in the AF, structural breakdown that is more prominent in late-stage degeneration and in cases of herniations (see the following sections). As disc and immune cells secrete inflammatory cytokines and proteases into their surroundings, the ECM continues to deteriorate, further promoting this degenerative phenotype in disc cells.

Herniation

Herniations are a result of direct trauma or the mechanical instability created at early stages of disc degeneration.⁵³ Herniations occur most frequently in individuals between the ages of 30 and 50 years.⁵⁴ There are 3 types of herniations: protrusion, extrusion, and sequestration.⁵⁵ A protrusion describes a focal bulge where the disc remains intact (i.e., the NP is still within its normal confines). However, extrusions are characterized by NP material beginning to migrate out of an AF tear, while sequestrations are characterized by NP fragments traveling completely outside the disc space. The most common area for herniations to occur is in the posterolateral region of the spine, where the disc is thinner and experiences greater strains.³⁶ For this reason, herniations cause pain by placing pressure on the longitudinal ligaments, thecal sac, and/or nerve roots. Although most herniations are resolved with conservative treatment, ~20% of individuals with a herniation will need to undergo removal surgery,⁵⁶ and about 5% of these patients will experience a reherniation.⁵⁷ In terms of the microenvironment, herniated discs are marked by increased solute transport and inflammation.

Solute Transport.

Angiogenesis is an important part of the wound healing and tissue regeneration process. Thus, it is unsurprising that increased vascularity is a common finding in herniated discs. Neovascularization within herniated tissue is attributed to inflammation.⁵⁸ Immune and resident cells promote angiogenesis by recruiting vascular endothelial cells and secreting proangiogenic factors such as vascular endothelial growth factor, transforming growth factor—β, and fibroblast growth factor.^58-61 The consequent growth of new blood vessels allows more immune cells to infiltrate the tissue and increases nutrient transport to disc cells. Although meant to promote proliferation and protein synthesis, angiogenesis has negative consequences on the disc microenvironment and cell behavior. In particular, vascular ingrowth is accompanied by ECM breakdown to create channels for the blood vessels to infiltrate the tissue, which is detrimental for an already deteriorated IVD.⁵⁹ In addition, increased oxygen tension results in a heavier reliance of disc cells on oxidative phosphorylation for energy production, likely inducing mitochondrial stress.⁶²

Inflammation.

With herniations, the IVD becomes exposed to its surrounding environment. Environmental exposure is detrimental to the disc, as NP fragments instigate an immune response.⁶³ As previously highlighted, disc cells produce proinflammatory cytokines in response to various external stimuli. In addition to proinflammatory cytokines, characterization of the inflammatory profile of herniated disc tissue shows increased concentrations of various chemokines particularly from the CC chemokine ligand and C-X-C motif chemokine ligand families.^64-66 These cytokines and chemokines help resident disc cells recruit immunocytes to begin the healing response after injury.^65,67 Notably, samples derived from extruded and sequestered herniations contain a combination of immune and endothelial cells, chondrocytes, and fibroblasts.⁶⁴ Sequestered herniations in particular have an increased abundance of inflammatory cells.⁶⁷ This heterogeneous cell population is likely a result of their infiltration through granulation tissue and blood vessels that form within the ruptured AF.^67,68 Together, resident disc and invading immune cells create a positive feedback loop via secretory factors, which continues to intensify inflammation.⁶⁹ In addition, this immune response also promotes nerve ingrowth within the disc, resulting in the sensation of pain without direct mechanical compression of nerve roots.⁶⁸

Late Stage

As the disc continues to lose key structural components and becomes plagued by lesions, tears, and herniations, it enters late-stage degeneration. Late-stage IVD degeneration has mostly been observed in individuals 40 years of age or older.²⁶ At this stage there is an increase in fibrocartilaginous, inflammatory, and hypertrophic IVD cells, a significant loss of disc volume, a decline of distinguishable AF lamellar layers, and an increase in lamellar thickness overall.^11,70 The adjacent vertebrae also begin to form osteophytes and undergo Modic changes (MC). There are 3 types of MC that occur to the vertebrae. Type I MC is characterized by inflammation, endplate fissuring, and bone marrow edema. In type II and III MC, the bone marrow is infiltrated with fatty tissue, and the subchondral bone thickens.⁷¹ These end-stage changes in motion segments are both a product and a cause of the degenerative microenvironment.

Mechanics.

In late-stage disc degeneration, changes to the AF’s lamellae result in increased interlaminar shear stress,³⁹ while significant narrowing of the disc space destabilizes the motion segment, placing more stress on the surrounding tissue. Mechanical stress results in degenerative changes such as fibrosis, thickening, and calcification of the supporting ligaments of the spine, particularly the longitudinal ligament.⁷² Stress and degeneration promote buckling and subsequent vertebral bone displacement or spondylolisthesis.⁷³ Disc degeneration itself may also result in a complete collapse of the disc space. In both scenarios, spondylolisthesis and complete disc collapse, there is a consequential reduction in mobility. Throughout this process, the CEPs experience increased mechanical stress as disc volume is lost, promoting MC and osteophyte formation.⁷⁴

Inflammation.

During late-stage degeneration, inflammation is catalyzed by tearing and deterioration of the AF and CEPs. Through tears, immune cells infiltrate the IVD, exacerbating inflammation that was initiated by resident cells. Specific fractures in the CEPs lead to MC by allowing disc material such an NP fragments to stimulate the autoimmune response in the vertebral bone marrow.⁷⁵ Further drainage of disc material as well as the presence of immune cells and inflammatory factors contribute to the initiation of MC.⁶⁶ Similar to cases of herniated discs, inflammation and ECM damage in late-stage degeneration promote innervation of the disc, causing pain sensation in the absence of nerve compression.^76,77

Solute Transport.

At late stages of disc degeneration, transport increases due to both the formation of tears and a reduction in essential structural proteins like proteoglycans.⁷⁸ Tissue deterioration promotes vascular ingrowth and increases permeability by opening up channels for solutes to pass through.^46,79 If the CEPs calcify and/or ossify, transport again decreases as pores become occluded.⁸⁰ MC may also help diminish solute transport by transforming the bone marrow vasculature, as in type II, and causing sclerosis, as in type III.⁷⁴

MICROENVIRONMENTAL CONSEQUENCES ON CELL BEHAVIOR

Throughout the degenerative process, changes in the disc microenvironment trigger specific cell responses meant to minimize or heal damage and halt the progression of disease. However, with prolonged stress and injury, these changes in cell behavior ultimately lead to a worsened microenvironment that catalyzes degeneration, cell dysfunction, and apoptosis.

Mitochondrial Dysfunction

Although the IVD is naturally in a state of hypoxia and relies on glycolysis for energy production, mitochondria remain key organelles to help meet bioenergetic demands and maintain normal cell function. Nevertheless, mitochondrial counts have been shown to decrease with degeneration.⁶² As previously discussed, at late stages of degeneration, there is increased nutrient transport as the ECM deteriorates. This results in an increase in oxygen tension that promotes oxidative phosphorylation. Decreased mitochondrial count in combination with a heavier reliance on oxidative phosphorylation increases the bioenergetic demand per organelle.⁶² Consequently, mitochondria may begin showing signs of dysfunction such as oxygen wasting and reactive oxygen species (ROS) overproduction.⁶² Mechanical stress and inflammation have also been shown to negatively impact mitochondrial health by decreasing mitochondrial membrane potential, causing an imbalance in mitochondrial dynamics (i.e., fission/fusion rates), and increasing ROS production.^81,82 Unsurprisingly, oxidative stress is another key characteristic of disc degeneration.

Oxidative Stress

ROS are oxygen radicals, such as the superoxide anion, hydroxyl radical, and hydrogen peroxide, that are generated by NADPH oxidase and mitochondria. At low levels, ROS are beneficial for cells, acting as messengers during cell proliferation, survival, and growth.⁸³ Antioxidants, including superoxide dismutase and glutathione peroxidase, help to maintain redox homeostasis by neutralizing ROS.⁸³ However, inflammation, increased oxygen tension, and mechanical stress, all of which are characteristic features of the degenerative disc microenvironment, stimulate ROS production and disrupt oxidative equilibrium.^84-86 In such cases, ROS damage cells by reacting with proteins, lipids, DNA, and mitochondria.⁸⁷

Multiple groups have found evidence of oxidative stress in degenerative and aging discs. Lipid peroxidation, which causes severe damage to the cell membrane, has been shown to be upregulated in degenerative discs.^88-90 With age, discs also exhibit increased N-(carboxymethyl)-lysine, a type of advanced glycosylation end product (AGE) that is triggered by ROS.⁹¹ AGEs are detrimental to the IVD, as they promote collagen cross-linking, leading to increased stiffness of the ECM. Along with lipid and protein damage, degenerative discs have also been shown to exhibit telomere shortening, a sign of DNA damage that instigates apoptosis and senescence.⁹²

Apoptosis

Apoptosis is deliberate cell death that is meant to prevent the accumulation and propagation of diseased or damaged cells. Both extrinsic and intrinsic signals initiate apoptosis by prompting a cascade of signaling events. Specifically in degenerative discs, apoptosis occurs through the death receptor, mitochondrial, and endoplasmic reticulum (ER) signaling pathways.⁹³ Each pathway is initiated differently but ends in the activation of members in the caspase family that execute cell death by cleaving specific proteins.⁹⁴ Briefly, in the death receptor pathway, extracellular signaling molecules such as Fas-ligand bind to death receptors on the cell surface to activate caspases.⁹⁵ In the mitochondrial pathway, intrinsic cell signals, such as ROS, and DNA damage cause the release of pro-apoptotic mitochondrial molecules, such as cytochrome C, into the cytosol, activating caspases.⁹⁵ Similarly, in the ER signaling pathway, prolonged ER stress due to the accumulation of misfolded proteins within the ER lumen triggers caspase activity.⁹⁵ The death receptor and ER signaling pathways are linked to mild grades of disc degeneration, while the mitochondrial pathway is linked to more severe disc degeneration.^93,95 Generally, apoptosis increases with worsening disc degeneration⁹³ and causes ECM degradation by releasing proteolytic enzymes and causing inflammation.

Senescence

Senescence is the complete and irreversible halting of cell division. There are 2 types of senescence: replicative and premature.⁹⁶ Replicative senescence occurs naturally due to telomere shortening. Telomeres are DNA sequences found at the ends of chromosomes that are meant to keep them from being degraded.⁹⁷ During mitosis, DNA polymerase is unable to replicate the ends of chromosomes, thus telomere length slowly decreases with each additional replicative cycle.⁹⁷ The enzyme telomerase, meant to add telomeric repeats to chromosomes, is not sufficiently active in adult cells, so once telomeres reach a critical length, cells will become senescent to prevent DNA damage or mutations.⁹⁷ Conversely, premature senescence is caused by mechanical, inflammatory, oxidative, and/or metabolic stress signals.⁹⁶ Both types of senescent cells, in addition to growth arrest, will have altered morphology (i.e., large and flattened), be resistant to apoptosis, and present a senescence-associated secretory phenotype. This phenotype is characterized by the secretion of proinflammatory cytokines, proangiogenic factors, and matrix-degrading enzymes that act as signaling molecules to propagate senescence and degrade the ECM.⁹⁸

Senescent cells have been identified at multiple stages of degeneration in the NP, AF, and CEPs. The NP in particular has been shown to have more senescent cells (13% ± 12.7%) than the AF (5% ± 9.8%), and herniations have been shown to have more senescent cells (8.5% ± 8.5%) than contained, degenerated (0.9% ± 3%), and healthy discs (1.4% ± 1.4%).⁹⁹ Senescence has also been demonstrated to increase with degenerative grade in the AF, with severely degenerated samples averaging 54% senescence, while healthy or mildly degenerated samples average ~13% senescence.¹⁰⁰ Similar results were obtained from NP tissue, also showing that telomere length decreases and the fraction of senescent cells increases with age.⁹² Lastly, in the CEPs, degenerated samples show 30% senescence, while mildly degenerated samples show 10% senescence.¹⁰¹

CLINICAL MANIFESTATION OF DISEASE

As degeneration worsens, individuals will experience back or neck pain with or without radiculopathy. In such cases, clinicians will use various imaging techniques to determine whether disc degeneration or herniation is the root cause. Typically, sagittal and axial T2-weighted and T1-rho magnetic resonance imaging (MRI) sequences are used to assess disc hydration, size/height, and proteoglycan content.^102-104 While these pulse sequences are useful for diagnosing disc degeneration, they provide little information regarding the state of the microenvironment. Hence, when symptomatic individuals have no obvious structural changes in their discs, it can be hard to pinpoint the root cause of pain.

Magnetic resonance spectroscopy (MRS) has recently gained interest as a tool that can be used to better differentiate between painful and nonpainful discs.¹⁰⁵ MRS uses magnetic resonance imaging (MRI) data to create spectral plots that can be used to evaluate signal properties (i.e., frequency, shift, and intensity) of a tissue to distinguish between and determine the concentrations of various chemicals that are present.¹⁰⁵ Particularly, MRS has been shown to measure disc levels of carbohydrate/collagen, proteoglycan, alanine, lactic acid, and propionate.¹⁰⁵ These chemicals are useful for determining tissue integrity and pH.¹⁰⁵ As described in previous sections, ECM degradation and a decline in pH are hallmark features of disc degeneration. Use of MRS provides information on these pathologic changes in the disc microenvironment (i.e., alterations in tissue integrity and pH), and consequently cell behavior, that may precede significant structural alterations.

Early Stage

Increased ECM catabolism, decreased nutrient availability, and inflammation characterize the microenvironment of early-stage IVD degeneration. MR images are useful in detecting matrix degradation, as a decrease in T2 and an increase in T1 rho relaxation times signify a loss in water and proteoglycan content.¹⁰⁴ Disc height reduction and blending of the NP and AF boundaries are also key signs of material breakdown that are noticeable in MR images.¹⁰² Unfortunately, disc-associated back pain can be difficult to diagnose at early stages of degeneration when MRI findings are minimal. In such cases, NOCISCAN (Aclarion, Broomfield, Colorado, USA), an MRS-based platform, can be used to determine the state of a disc in greater detail. High NOCI scores indicate a painful disc exhibiting increased acidity and decreased tissue integrity,¹⁰⁵ whereas low NOCI scores indicate a relatively healthy disc with minimal degenerative changes. MRS has shown a total accuracy of 85% and sensitivity of 82% compared to discography.¹⁰⁵

Herniation

Herniations are the most common clinical manifestation of disc degeneration. With T2-weighted MRI, clinicians diagnose a painful herniation when disc content has ruptured through its normal confinement and is causing spinal stenosis. In the absence of noticeable mechanical compression of the spinal cord or nerve roots, MRS may be helpful in determining whether the herniated disc is causing pain through other mechanisms such as nerve ingrowth and/or irritation due to microenvironmental factors such as low pH.

Late Stage

The microenvironment in late-stage disc degeneration is characterized by inflammation and severe changes in tissue composition. T2-weighted MRIs will demonstrate a significant reduction in relaxation time and disc height as well as a complete loss of the NP/AF boundary.^95,102 Changes in signal intensity of the bony vertebrae can also indicate characteristic features of MC such as inflammation.^71,74 In some cases, patients may demonstrate spondylolisthesis or complete disc collapse, results of motion segment destabilization.

CONCLUSIONS

The intricate relationship between cells and their microenvironment introduces layers of complexity when tissues become diseased. As the microenvironment is altered, cells detect and respond to these changes through various sensory machinery on their surface. In the IVD, aging and mechanical trauma are 2 of the main initiators of disease, causing abnormal changes in microenvironmental features of the disc such as mechanics, solute transport, and inflammation. These changes drive degeneration and allow for diseased discs to progress from early stage to herniated and late stage. In response to these degenerative changes, disc cells slowly manifest a degenerative phenotype that promotes catabolism and inflammation. The transformation and frequent death of disc cells subjects their local microenvironment to further damage, initiating a degenerative loop between disc cells and their microenvironment. It is important to note that this review mainly focuses on lumber disc degeneration and that the microenvironments of the cervical and thoracic spines likely influence the degenerative process at these disc levels. In the following reviews,^3,4 we will discuss treatments for disc degeneration, their evolution, and current emphasis on cellular and microenvironmental revitalization.

Abbreviations and Acronyms

ADAMTS

A disintegrin and metalloproteinase with thrombospondin motifs

AF

Annulus fibrosus

CEP

Cartilaginous endplate

ECM

Extracellular matrix

ER

Endoplasmic reticulum

IVD

Intervertebral disc

MC

Modic changes

MMP

Matrix metalloprotease

MR

Magnetic resonance

MRI

Magnetic resonance imaging

MRS

Magnetic resonance spectroscopy

NP

Nucleus pulposus

NPC

Nucleus pulposus cell

ROS

Reactive oxygen species

TIMPS

Tissue inhibitors of metalloproteinases

TLR

Toll-like receptor