Mechanotransduction and cell biomechanics of the intervertebral disc

Mechanical loading of the intervertebral disc (IVD) initiates cell‐mediated remodeling events that contribute to disc degeneration. Cells of the IVD, nucleus pulposus (NP) and anulus fibrosus (AF), will exhibit various responses to different mechanical stimuli which appear to be highly dependent on loading type, magnitude, duration, and anatomic zone of cell origin. Cells of the NP, the innermost region of the disc, exhibit an anabolic response to low‐moderate magnitudes of static compression, osmotic pressure, or hydrostatic pressure, while higher magnitudes promote a catabolic response marked by increased protease expression and activity. Cells of the outer AF are responsive to physical forces in a manner that depends on frequency and magnitude, as are cells of the NP, though they experience different forces, deformations, pressure, and osmotic pressure in vivo. Much remains to be understood of the mechanotransduction pathways that regulate IVD cell responses to loading, including responses to specific stimuli and also differences among cell types. There is evidence that cytoskeletal remodeling and receptor‐mediated signaling are important mechanotransduction events that can regulate downstream effects like gene expression and posttranslational biosynthesis, all of which may influence phenotype and bioactivity. These and other mechanotransduction events will be regulated by known and to‐be‐discovered cell‐matrix and cell‐cell interactions, and depend on composition of extracellular matrix ligands for cell interaction, matrix stiffness, and the phenotype of the cells themselves. Here, we present a review of the current knowledge of the role of mechanical stimuli and the impact upon the cellular response to loading and changes that occur with aging and degeneration of the IVD.

pressurization, a decreased T2 MRI signal, and changes to the vertebral EPs, all of which are believed to contribute to altered IVD function.
Increasingly, evidence suggests that the cell-mediated and biological remodeling responses to mechanical stimuli generated by spinal motions are a large contributor to degenerative and pathological changes 3 . These cell-level responses to loading environment occur decades before disc degeneration manifests 4,5 . For these reasons, studies of mechanobiology and the interactions between mechanical stimuli and biological processes are performed to understand the development of disc degeneration. Many studies have documented the biological responses of IVD cells to mechanical stimuli, but only recently have studies begun to elucidate mechanisms governing these observed mechanobiologic responses in the IVD. In this article, we present a summary of the current knowledge of IVD mechanobiology and its relevance to disc degeneration, and identify several signaling events and mechanisms that govern cellular responses to mechanical stimuli.

| STRUCTURE, MORPHOLOGY, AND BIOLOGY OF IVD CELLS
The disc consists of structurally distinct, anatomical regions, which dictate the range of IVD cell phenotypic characteristics, including unique physiological and biological responses to mechanical stimuli 6,7 .
The innermost NP is a highly hydrated, gelatinous tissue containing large quantities of proteoglycans, collagens, and noncollagenous proteins 8,9 . Due to the large amounts of the proteoglycan aggrecan, which carries a net negative and "fixed" charge through its compositionally major sulfated-glycosaminoglycans, the NP is subject to high interstitial swelling and osmotic pressures during joint loading 10,11 .
The notochordal-derived cells of the NP can be present with large vacuoles [12][13][14][15] and young, immature NP cells display clustered, tightly packed cells with smaller compacted nuclei, and surrounded by dense pockets of ECM ( Figure 1A). The ratio of large vacuolated notochordal to small nonvacuolated cells in the NP region declines with maturity of the human IVD 15 . With age, these phenotypically unique cells are rarely seen and instead an increased presence of chondrocyte-like or fibroblast-like cells is observed 16   Insets (right) show matrix organization (scanning electron microscopy, gray) and cell organization (fluorescence) for anulus fibrosus (AF) and nucleus pulposus (NP) regions. AF matrix is composed of aligned collagen I fiber bundles, while a much random distribution of collagen II bundles and proteoglycan is observed in the NP. Fluorescence insets are rat IVD cells stained for collagen VI (green) and nucleus (red) revealing the different cellular arrangement in each tissue area. Adapted from Cao et al 174 . (B) Mechanical deformation of the IVD. Axial compression can be either catabolic or anabolic depending on mode (static, dynamic), magnitude, frequency, and duration. Loading of the hydrated NP matrix results in hydrostatic and osmotic pressures. When the disc is axially loaded, the NP region becomes compressed and the AF undergoes radial and circumferential tension to limit overall disc expansion in the transverse plane. Adapted from Setton et al 195 may be a more appropriate fit for addressing early events that can lead to degeneration, while older samples may give more accurate information on the degenerate or aged cellular response to further environmental challenges.
Surrounding and constraining the NP is the AF, a lamellar and fibrocartilaginous structure containing highly organized, distinct lamellae 19,20 and largely aligned collagen fiber bundles [21][22][23] . Specifically, AF is constructed of concentric lamellae of alternating alignment, with each lamella composed of parallel collagen fibril bundles 24 (Figure 1A).
Sheaths of elastic fibers, formed by elastin, fibrillin, and other ECM proteins, enclose these collagen fibril bundles 25 , resembling what has been described for tendon and muscle. Elastin appears to maintain the integrity of the collagen lamellae, allowing its recoiling after deformation and to contribute to the anisotropy of the AF stiffness in response to shear 26,27 . Cellular interactions with ECM of the AF and other regions can be quite complex, however, with evidence of cellular projections into lamella of the AF that can contribute to stellatelike cells and some documented evidence of cellular "gliding" and lamellar cross bridges forming during simulated motion 28,29 . Containing bundles of collagen I, the outer regions of the AF help resist tensile loads arising from physiological joint motions and swelling effects that give rise to annular bulging and deformation ( Figure 1B) 30 . Cells of the AF, which are derived from the mesenchyme, display a more ellipsoidal morphology with many characteristics of fibroblasts and chondrocytes 8,9,31 . The inner region of the AF transitions to a more collagen II-rich niche with increased proteoglycan content, allowing it to contribute to the adjacent NP's ability to support compressive loads. Similar to NP, cells of the inner AF (IAF) are more rounded and sparsely distributed as compared to cells of the outer AF (OAF; Figure 2). Containing bundles of collagen I, the OAF help resist tensile loads arising from physiological joint motions and swelling effects that give rise to annular bulging and deformation ( Figure 1B). Cells residing in the OAF exhibit a flatter morphology, with major cell radii closely aligned with collagen fibers of the organized lamellae ( Figure 2). Therefore, IAF cells appear to have greater similarity with NP over OAF, in terms of morphology and gene expression 32 . The elastic fiber network also differs between NP, IAF, and OAF. In OAF, microfibrils colocalize more with elastin fibrils when compared to IAF. In NPs, microfibrils surround the cell while elastin preferentially localizes in the interterritorial ECM. Moreover, the elastin content increases with the degree of degeneration and with age in NP, OAF, and IAF, with a higher increase in the latter 33 .
The avascular nature of the IVD largely dictates the metabolism of disc cells. The primary means by which nutrients and metabolites reach the disc is diffusion through the vertebral EP and AF, which causes relatively low oxygen tension within the disc and cell metabolism largely relies on anaerobic glycolysis 34,35 . This further contributes to high concentrations of lactate, the end product of glycolysis, and low pH conditions. When oxygen tension is low, cells will rely completely on glycolysis for ATP production and energy generation 36 .
Disc cells are highly adapted to such a hypoxic environment and maintain maximum cell survival when oxygen tension is below 5% 37,38 . The hypoxic demands of the disc thus require high amounts of glucose for glycolysis-dependent ATP production and cell viability becomes compromised if glucose levels fall below physiologic levels (0.2 mM) [39][40][41] .
The resulting lactate accumulation can lead to a reduced pH, if lactate is not properly cleared, and can be detrimental to cell survival. Acidic pH levels as low as 6.1 have been predicted, 42 that is exacerbated during degeneration because the disc has diminished diffusion abilities to remove metabolic waste products. Degeneration-induced calcified EPs block not only nutrient supply, but also clearance of metabolites, leading to lactic acid buildup and an increasingly acidic environment 43 .
Few studies have compared metabolism of NP and AF in response to loading, though AF and NP cells exhibit different metabolic pathways.
NP cells have higher adenosine triphosphate content, while AF cells have higher lactate production and glucose consumption 44 . The same study also showed that dynamic loading affects energy metabolism, with the effect being greater in AF.
Pronounced changes occur with aging in the IVD, where the already low cell density decreases further, along with marked changes in the ECM composition 45 . A reduction in aggrecan within the NP results in a more dehydrated tissue with a diminished ability to properly distribute and transmit mechanical loads 46,47 . This in turn leads to altered AF mechanical properties as well, due to these forces being unnaturally transferred to the adjoining AF. An important FIGURE 2 Representative images of isolated porcine cells from outer AF (OAF), inner AF (IAF), and nucleus pulposus (NP) regions. (A) Differential interference contrast (DIC, top), confocal laser scanning microscopy (CLSM, middle) depicting F-actin (phalloidin) staining and scanning electron microscopy (SEM, bottom). Porcine NP cells are larger than OAF and IAF cells, and have a vacuolated morphology. F-actin is localized to the cell cortex in OAF and IAF cells, but was found in abundance throughout NP cells, including the perinuclear region. SEM showed the presence of significant membrane ruffles and folds. Scale bar = 5 μm. Adapted from Guilak et al 196 . (B) Immunofluorescence of actin microfilaments (green) and the cell nucleus (red). OAF cells in situ are elongated and distributed in concentric layers mimicking the collagen arrangement. IAF cells in situ appear rounder than OAFs. Bovine NP cells (in alginate) have a dense network of punctuated cortical actin microfilaments and lack stress fibers, while OAF and IAF cells have a cortical actin distribution. Scale bar = 5 μm. Adapted from Setton et al 195 characteristic of IVD degeneration is the pathological infiltration of other cell types, namely, nerve fibers, immune cells including macrophages, lymphoid-derived T cells, Schwann cells, endothelial cells, and fibroblasts, the latter of which may be associated with increased vasculature 4,[48][49][50][51] . Along with this aberrant vasculature and subsequent blood supply, there is also a reduction in nutrient diffusion 52,53 , all of which are believed to support a particularly adverse cellular environment where matrix-degrading metalloproteinases (MMPs) and proinflammatory cytokines such as interleukin(IL)-1, IL-8, and tumor necrosis factor (TNF)-α, exacerbate and further the progression of degeneration 4,48,54,55 .

| MECHANICAL LOADING EFFECTS ON IVD CELLS
During loading, cells of the IVD experience compressive, tensile and shearing deformation, fluid flows, pressures, and electrokinetic effects, all of which represent important regulators of cell metabolism in the IVD ( Figure 1B). Cells of the separate anatomical regions are exposed to a range of mechanical loads and the biological response to a particular stimulus may depend on tissue location, magnitude, and frequency of loading 3,56,57 .

| Compression
The differential response of IVD cells to loading has been most studied for compressive stimuli. Evidence consistently suggests that there is a threshold effect observed in IVD compression studies in both NP and AF that is dependent upon magnitude, duration, and frequency.
Static compressive loading has been shown to induce changes in cell biosynthesis and gene expression for collagens and proteoglycans, and protease activation, as well as cell death when applied in vivo and to explants or isolated cells [58][59][60][61][62][63][64][65] . Static compressive loading is thought to inhibit nutrient transport and necessary gas exchange that may be essential for promoting cellular biosynthesis and maintaining cell survival, whereas dynamic compression promotes convection that increases large molecular weight nutrient uptake into the disc 42,66 .
Interestingly, transport of small molecules such as oxygen and glucose are predominantly governed by diffusion rather than convection that are modulated under dynamic loading 42,[67][68][69][70] . In terms of gene expression, one study showed 72 hours of immobilization followed by 2 hours of dynamic loading can produce damaging effects, measured by a decrease in anabolic gene expression and an upregulation of catabolic gene expression, that were not rescued by the brief period of dynamic loading 71 . In contrast, shorter periods of static loading can lead to an anabolic response in IVD cells, shown through increases in proteoglycan and collagen synthesis in a bovine explant culture system (5-10 kg,~0.2-0.4 MPa) 65 . A similar effect was observed for AF cells isolated from tissue and embedded in alginate matrix, demonstrating elevated gene expression of collagens I and II, aggrecan, decorin, and biglycan for short periods of loading 64 . As has been shown for other mechanosensitive tissues and associated cell types, long periods of matrix compression give rise to altered physical signals to IVD cells and also disrupt gas and nutrient transport that is essential for maintaining metabolism. Transport properties of human AF are dependent on the magnitude of applied compressive strain, where transport decreased with increasing deformation, likely due to fluid exudation and reduction in pore size with compression 72,73 . AF diffusivity is also anisotropic, with lower diffusivity in the radial direction than in the axial or circumferential directions, indicating that nutrient transport in human AF is anisotropic. This behavior is likely a consequence of the layered structure, unique collagen architecture, elastic fibrils arrangement, and high complexity of the ECM of AF tissue 73,74 .
Dynamic compressive loads change the cell-specific expression of crucial matrix genes (eg, collagens I and II, aggrecan) and catabolic genes (MMP-1, -3, -13, ADAMTS-4), but the degree of response is conditional upon frequency and magnitude 71,75,76 . Overall, an anabolic response is more likely elicited from dynamic loading, whereas static loading tends to produce a catabolic response. In a bovine IVD organ culture study, it was found that dynamic compression (0.2-1 MPa) increased metabolic rates and biosynthesis of ECM molecules, without any structural changes or impaired mechanical properties, indicating there is some degree of healthy loading conditions required for all regions of the IVD 77 . Lower loading rates to cells in IVD matrix and as isolated cells (<0.5 Hz) have been shown to maintain proteoglycan content 75,78 and support normal disc metabolism 79 , whereas increased loading frequencies result in higher amounts of apoptotic cells 80 . Cell death has also been noted in cells in the NP and AF when the dynamic compressive magnitude reaches levels greater than 1.3 MPa 76 , although low frequency of loading and long periods of duration (>24 hours) also modulate this effect 59,60,63,71,76 . One study used a rat tail dynamic compression model to demonstrate that discs loaded for 2 weeks at 8 hours/day expressed higher levels of anabolic and anti-catabolic genes compared to static compression 81 . However, when discs were loaded at the same magnitude for 8 hours/day out to 8 weeks, early signs of degeneration were noted through decreased TIMP-3 and increased ADAMTS-4 expression, which can promote matrix degradation. Thus, dynamic compression leads to an anabolic response when applied daily, but initial signs of degeneration may begin with extended dynamic loading duration.
Compressive loading also regulates the synthesis and consumption of glucose and ATP in the IVD 42,44,65,82,83 . Both static and dynamic loading conditions have been shown to significantly increase lactate accumulation in the AF and NP regions, likely a consequence of lactate production, occurring contemporaneously with increased ATP content and decreased pH 84 . It was also noted that lactate accumulation was higher in the 1 Hz compression group compared to the 0.1 Hz compression group, suggesting frequency-dependent differences. Isolated porcine NP and AF cells in a three-dimensional (3D) agarose culture model that were subjected to various mechanical loads (which had previously shown the greatest changes in biosynthetic activity by mesenchymal stem cells [MSCs]) demonstrated increased glucose consumption and lactate production and were further shown to produce higher levels of ATP compared to cells exposed to decreased loading conditions 82 . Given the fact that proteoglycan synthesis is a highly ATP-demanding process in which it participates as an energy source and as a building block, it seems likely the increased ATP found under certain conditions may interact with proteoglycan synthesis 85,86 . ATP regulates various cellular activities through purinergic signaling pathways, specifically by way of ATP hydrolysis to produce ADP and adenosine, which directly modulate cellular activity through purinergic P2Y and P1 receptors [87][88][89][90] . Isolated porcine NP and AF cells have been shown to express P2X 4 purinergic receptors and when this receptor is inhibited, disc cells lose the ATP-induced membrane potential response observed when stimulated with exogenous ATP 91,92 . Thus, influence of mechanical loading on disc cell energy metabolism and bioactivity may affect mechanotransduction pathways.
Dynamic loading has been shown to modulate cellular responses and mechanotransduction for many cell-laden 3D tissues such as cartilage and IVD. For the IVD, finite element modeling of dynamic compression is associated with increased oxygen concentration throughout the tissue 42 that will be associated with elevated metabolism, confirming many of the experimental observations described.
This elevated transport of oxygen to cells in the IVD is associated with increased glucose consumption and lactate production (ie, energy conversion) that may be associated with elevated proteoglycan and collagen synthesis 93 . While oxygen transport has been reported to be sensitive to loading frequency and amplitude, the effect of dynamic loading on transport is dependent on the molecular weight of the solute. In dense hydrated tissues, small molecules are less sensitive to dynamic loading and associated changes in pore size, whereas larger molecular weight solutes, such as growth factors which dramatically influence anabolism (eg, IGF-1, TGF-β, FGF, PDGF), undergo enhanced nutrient transport under dynamic loading that is mediated by enhanced convection [94][95][96][97][98] . Thus, many of the reported effects for dynamic compressive loading on metabolism may in fact derive from dynamic compression-related regulation of nutrient transport and associated receptor signaling 99 .
The distinct anatomical regions of the IVD are known to respond differently to compression, as will isolate cells from their respective tissue in terms of health and age. Many responses are similar for the IAF and NP tissues; however both short-and long-term studies show that the more fibrocartilaginous OAF is not equally responsive to low to moderate loading magnitudes 61,62,65 . Rodent tail models have shown increased protease activation (MMP-2) and decreased protein gene expression (collagen II and aggrecan) after 4 to 7 days of an in vivo loading protocol that was observed in the IAF and NP ( Figure 3A) 58,79 . The NP exhibited a frequency-dependent response while the AF had a magnitude-dependent response in collagen synthesis, which is believed to be due to the separate regions experiencing differences in hydrostatic and deformation-related stimuli 79,100 .
The observed cell-specific effects likely apply to duration of loading as well, where longer periods of loading (8 hours/day) can be beneficial for NP but damaging for AF 81 . Distinctions between the biological responses to load are related to the different magnitudes of deformation, fluid-flow, and associated electrokinetic effects for cells in different tissue regions 24,42,44,70,101 . Age is also known to play a role in the response of NP and AF cells to dynamic compression, where mature cells lose the ability to maintain homeostasis to the degree that young cells do 83 . Overall, little is known of the mechanisms that govern these responses but could in part be attributed to transport of chemokines like growth factors, activation of mechanoreceptors, related ion channel activation, and cytoskeletal reorganization. These specific signaling mechanisms that contribute to the observed anabolic and catabolic mechanobiological responses of NP and AF cells have been little studied in the context of healthy, and more so in degenerated IVDs. A summary of known and proposed mechanisms regulating IVD mechanobiology is presented in Section 4.

| Tension
Most mechanical loading conditions present tensile strains in the AF region of the IVD, though the magnitude and response of these stimuli likely varies depending on the anatomic zone. in vivo 102,103 and explant studies have previously demonstrated tension will lead to a decrease in proteoglycan and collagen synthesis in the IVD; however, this effect may be magnitude dependent with modest inhibitory effects of physiological magnitudes of tension on isolated AF cell biosynthesis 104 . The OAF region has been shown to experience biaxial stretch during functional loading, with magnitudes between 4% and 6% during flexion and extension with minimal AF biologic response 105,106 . More studies have shown that isolated AF cells at low magnitudes (1%) and physiologic frequencies (1 Hz) of tensile strain maintain proteoglycan production, consistent with a modest response to strain 107 . Loading frequencies outside of this physiologic range, however, can cause a catabolic response and upon exposure to frequencies greater or less than 1 Hz, AF cells lose the ability to maintain important matrix production 108,109 . Increasing strains (5%-20%) as well can lead to a catabolic response as shown through increased MMP-3, COX2, nitric oxide (NO), TNFα, and decreased proteoglycan production ( Figure 3B) 107,109,110 . It has been suggested that this transition from anabolic to catabolic response occurs during periods of continuous strain and may be explained by the cells' loss of energy production necessary to maintain biologic processes.
In contrast, the NP is not believed to experience high tensile strains under physiological loading, with no major changes in the NP observed (with the exception of ADAMTS-4, which was also confirmed in a separate study 111 ). Under hyperphysiologic stretch of 10% (applied at 0.5 Hz) or 20% (applied at 0.05 Hz), NP cells respond with increasing cell proliferation and collagen synthesis 112 . Short-term hyperphysiologic stretch (20%) of NP cells also activates innate immune receptors, such as Toll-like receptors (TLRs), in vitro 110 . It is believed that these differential responses of NP and AF cells, likely reflect cytoskeletal adaptation to their respective in situ mechanical demands, with NP cells experiencing hydrostatic and/or compressive pressure and AF cells subjected to tensile strains.

| Hydrostatic pressure
The NP is highly hydrated, and therefore cells within the NP experience hydrostatic pressure when loaded. Hydrostatic pressure is distinct from other loading exposures due to the presumed conservation of cell and pericellular matrix volumes. Hydrostatic pressure magnitude also varies diurnally in a manner that depends on spinal alignment and physical activity 113,114 . Both experimental and computational methods have been used to estimate the magnitude of hydrostatic pressure that disc cells are exposed to during activity.
Hydrostatic pressure magnitudes range from 0.1to over 3 MPa, with baseline magnitude of around 0.1 MPa applied at all times regardless of posture or activity 115,116 . With aging, hydrostatic pressure magnitude is likely reduced due to lower proteoglycan and water content of the disc 17,117 . Exposure of disc cells to hydrostatic pressure is known to influence disc cell biologic responses in duration, magnitude, and frequency-dependent manner, with some species-specific variations observed. In general, higher magnitudes of hydrostatic pressure (>3 MPa) can lead to degenerative responses in IVD cells mediated by an increase in MMPs and reduction in ECM expression and synthesis when applied to NP cells seeded within a 3D culture system or within tissue explants 113,118 . Hydrostatic pressure overloading of 3 to proteoglycan synthesis, while others show a downregulation in ECM and upregulation of catabolic signaling ( Figure 3C) [124][125][126][127][128]

| Osmotic pressure
The osmotic environment of the IVD, resulting from a combination of mechanical deformations and fluid redistribution, can vary locally depending on biochemical composition and local volumetric change 7 .
Osmotically induced swelling due to hypotonic conditions draws water into the disc tissue to maintain hydration, and causes volumetric

| Cytoskeletal responses in mechanical loading of IVD cells
Studies of isolated IVD cell to specific physical stimuli reveal broad changes in cell morphology and suggest that cytoskeletal  In contrast, NP cells were not responsive to the same mechanical loading conditions. These distinctions are likely attributed to differences in strain magnitudes experienced in situ, but may also be due to the presence of a more diffuse and stiff cytoskeleton in the notochordal NP cells, which may restrict deformations or cell shape changes with loading. 64  In evaluation of regional variations of integrins, the expression of  163 to promote cell attachment, as well as upregulated proteoglycan synthesis and elevated mRNA for a subset of NP ECM molecules. It has further been shown that certain laminin-mimetic peptides induce higher levels of biosynthetic activity compared to others ( Figure 5B) and can even express statistically higher amounts of NP markers despite a stiff substrate presentation ( Figure 5C). The staining of laminin seems to decrease with age in pathways.

| Cell-matrix interactions
An additional role for integrins is associated with the release and activation of latent TGF-β that is stored in the ECM, bound to a peptide 167 . Some integrin subunits have been shown to participate as docking points to allow proteolytic cleavage of immobilized TGF-β from the docking peptide, and to enable generation of cell traction that will mechanically separate TGF-β to enable its activation 167 . TGFβ is critical for the disc development at embryonic stage and for its postnatal maintenance 168,169 and regulates connective tissue growth factor CCN2, implicated in synthesis of aggrecan 170 . Both excess and deficiency of TGF-β activation can be detrimental for IVD 157,169,171,172 . In this manner, TGF-β-receptor-mediated signaling could be regulated by integrin engagement with the ECM; however, investigations on changes in TGF-β activation with disc aging and changes in ECM stiffness may provide further insight into disease related changes.

| Cell-cell interactions and the cadherin-catenin pathway
Cells of the NP and AF interact with neighboring cells in a manner that varies with age, and that is distinctly different between these two regions. Some cells of the AF form elongated cell "arrays" or may spread into flattened cells as in the case of the stellate interlamellar AF cell. These cells may interact with adjacent cells through Ecadherin mediated junctions that are known to be key regulators of cell signaling in multiple cell types. AF expression of E-cadherin appears to be independent of age and matrix stiffness 173 , and little is known about the relationship between E-cadherin expression and cellular responses to physical stimuli.

CELL-MATRIX INTERACTIONS
sGAG/DNA µg/µg (A)   sGAG  type II collagen,  aggrecan, T,  n-cadherin   sGAG  type II  collagen,  aggrecan, T signaling. More work will be needed to isolate factors of cell morphology as distinct from N-or E-cadherin mediated signaling in order to isolate the specific signaling events and their relationship to IVD cell phenotype.

| Substrate stiffness effects
We and others have found an effect of substrate stiffness on mechanobiology in IVD cells, as shown through mRNA expression and ECM biosynthesis 117,151,158,161,162,173,175,176 , but the degree of response seems to depend largely on cell type. This is also true for chondrogen- to be active and present in primary nucleus pulposus (NP) and anulus fibrosus (AF) cells, where they are believed to be regulated by F-actin turnover and Rho/ROCK signaling. On stiff substrates, increased polymerized F-actin form contractile complexes with focal adhesions and actomyosin to sustain Rho/ROCK signaling to allow nuclear translocation of coactivator YAP/TAZ to bind TEAD and drive transcription of proliferation-and differentiation-related genes. Similarly, coactivator MRTF is able to localize to the nucleus due to the decreased presence of monomeric actin (G-actin) where it will bind and activate SRF to transcribe fibrotic response-related genes. Soft substrates allow for liberation of G-actin which sequesters MRTF in the cytosol and decreased Rho/ROCK signaling, due to the loss of the contractile actomyosin/F-actin complex, prevents YAP/TAZ nuclear localization, thus keeping both SRF and TEAD transcriptionally silent. (B) Verteporfin (VP), a known inhibitor of YAP/TAZ, and Latrunculin B (Lat. B), which disrupts polymerized F-actin, were cultured with SRF transcriptional reporter primary human NP cells, which resulted in significantly decreased transactivation of SRF with both Lat. B and VP treatment. (C) When TEAD transcriptional reporter NP cells were treated with Lat. B and VP, a similar decrease in transcriptional activity was observed, suggesting inhibition of a contractile cytoskeleton can prevent expression of fibrotic and aberrant cell cycle genes signaling responses and associated catabolic upregulation in inflammatory environments are thought to mimic the degenerative milieu of the IVD. Evidence suggests that the response of cells from degenerate tissue to applied loading differs from cells isolated from normal IVD tissue. AF cells from degenerative tissues demonstrated a more proinflammatory response to applied stretch (6% elongation, 0.1 Hz) compared to cells from nondegenerate discs, suggesting that degenerate AF cells are more reactive to exogenous stimuli. Moreover, the short-lived beneficial effects of mechanical stretch seen in normal AF cells do not appear when degenerate AF cells are similarly stretched.
Indeed, increasing evidence suggests that interactive effects of tensile stretch and inflammatory signaling in AF cells 109,185,186 . This may be mediated by altered cytoskeletal mechanotransduction. In AF cells, TNFα treatment increased F-actin stress fibers α-tubulin, which potentially sensitized AF cells to mechanical strain 185  All these studies point to a positive feedback of degenerative microenvironments, mediated primarily by pro-inflammatory cytokines, which alter cytoskeletal remodeling and related mechanotransduction.

| Cell-ECM interactions
Although one study found that physiological level of dynamic compression induced integrin α5β1 expression in rat disc explants 187 57 . This suggests that mechanotransduction signaling is altered in degenerated tissue, in that integrins may not be directing response of degenerate cells to loading or that there are changes in the mechanosensitivity of integrins to mechanical loading in degenerate cells 57 . Studies of human AF cells derived from degenerated and nondegenerated tissue and exposed to 10% strain, 1 Hz for 20 minutes also respond differently. Specifically, there was a decrease in expression of ADAMTS4 for nondegenerated cells and a decrease in expression of collagen I for degenerated cells. Blocking integrin binding transiently prevented a response to loading in nondegenerated cells that was mediated by focal adhesion kinase; however, it failed to affect the mechanotransduction in degenerated AF cells. These results suggest that integrins participate in mechanotransduction of AF cells from nondegenerated tissue, and that alternative mechanotransduction pathways mediate responses of degenerate cells 190 .

| ER stress
Cells also protect themselves from stresses through cellular stress responses. In one study, Chooi and Chan 191 found differential gene expression patterns based on compression loading regimens that suggest an increase in ER stress pathway genes as a form of cell survival.
In a 3D collagen model with bovine NP cells, compressive loading was applied for various durations and assessed the heat shock protein response (HSR) and the unfolded protein response (UPR). Although an increase in both UPR and HSR stress response genes were observed in the highest group of static compression, the authors note that upon load removal, HSR genes remain upregulated while UPR genes become downregulated, which may suggest HSR genes' continual expression is a protective mechanism to allow cells to recover and avoid apoptosis. This was further confirmed by showing HSR geneexpressing cells do not become apoptotic and those that did undergo apoptosis did not express these genes postloading. This points to a master regulator of cell survival and biosynthesis following periods of static compressive loading. Similarly, hyperphysiologic cyclic stretch has been shown to increase the production of ER stress markers CHOP, GRP78, and caspase-12 in AF cells 106,192,193 . In one study, the addition of caspase inhibitors was able to partially suppress the stretch-induced apoptosis as well as NO overproduction 192 . It has been suggested that cyclic stretch-induced apoptosis may occur through mitochondrial pathways, as cyclic stretch has been noted to cause reduction of the mitochondrial membrane potential, though other cell death pathways are likely involved.

| CONCLUSION
In conclusion, cells of the NP and AF have been observed to respond to compression, tension, osmotic stimuli, and hydrostatic pressure with changes in ECM, and regulators of ECM synthesis, in a manner that differs between these two cell types. Cellular studies reveal a role for differential regulation of cytoskeletal remodeling between AF and NP cells in contributing to these differential responses, with the remodeling being specific to actin, vimentin or IF reorganization and/or expression specific to the stimuli. Cytoskeletal reorganization can activate downstream effects not only through ROCK signaling and coactivators such as YAP/TAZ, but also through volumetric regulation that may affect mechanosensitive ion channels as observed through calcium signaling. Whether the cell is permitted to interact with adjacent cells or with ECM ligands will affect cell morphology and cytoskeletal organization, but may also activate β-catenin and ERK phosphorylation events that are known regulators of gene expression and downstream biosynthesis. While cell morphology, cytoskeletal reorganization, ion channel activation, and cell-ligand mediated events are linked in many cell types, the compelling differences shown between NP and AF cells within a single IVD structure motivate studies to reveal the precise mechanisms that govern mechanobiology in these systems.
In the process of performing a comprehensive review of the literature, we have identified some gaps in knowledge regarding topics related to mechanobiology of the IVD. Here, we briefly summarize key gaps, with the hope that future studies will inform some of the open questions in the field:

| Development and aging
• Mechanical factors that drive phenotypic changes are not well understood in the developing, adult and aging IVD. There is a need for in vitro and ex vivo studies that identify relationships between magnitudes of spinal motion and loading conditions in vivo with long-term changes in cellular morphology, phenotype, and metabolism.
• Tracing of cell fate through the continuum of notochord to chondrocyte-like phenotype is needed to confirm standing hypotheses about phenotypic transition and/or transformation with aging. Nevertheless, the use of cells from younger specimens are better suited for studying early events that can lead to degeneration, whereas cells from older specimens give insight into how aged or degenerate cells react to further damage and inflammation.

| Response of cells to loading and microenvironmental changes
• Overall, little is known about the mechanisms that govern response of cells to loading and changes in physiochemical microenvironments.
• While cell response to loading could in part be attributed to transport of chemokines like growth factors, activation of mechanoreceptors, related ion channel activation, and cytoskeletal reorganization, detailed mechanistic studies are needed to assess first-order mechanisms. These specific signaling mechanisms that contribute to the observed anabolic and catabolic mechanobiological responses of NP and AF cells have been little studied in the context of degenerated IVDs.
• The response to altered osmolality, pH, and oxygen tension is regulated at the transcriptional level, yet the exact mechanisms by which microenvironmental changes (alone and in combinations) modulate IVD cell fate, metabolism, and interactions with immune cell infiltrates are not known.
• Knowledge of the effects of dynamic and static loading upon nutrient transport to varying compartments and substructures of the IVD. Modern approaches to monitoring tracers and contrast agents could be employed to better track transport and fluid flow to varying substructures and thus to varying cell types.

| Cell-ECM interactions and cell-cell signaling
• Future studies are required to verify the precise role of ligand interactions with integrins on NP and AF cells, and to confirm a role for ligand density and specificity in activating cell signaling through known and to be discovered pathways. Emphasis on changes in cell-ECM interactions with growth factor and chemokine signaling is also warranted.
• Cellular interactions with ECM, both visualization of and knowledge of the biochemical nature of these interactions, is needed.
The role of integrin and nonintegrin matrix protein receptors, aquaporins, ion channels, and mechanosensitive channels in interacting with ECM are only limitedly studied.
• Investigations on changes in growth factor (eg, TGF-β) activation with disc aging and changes in ECM stiffness may provide further insight into disease-related changes.
• The mechanism by which substrate stiffness information is transduced in IVD cells is not completely understood, but it likely relates to cytoskeletal organization and related transcriptional activators.
• Given the close relationship between morphological confirmation and cell signaling through cell-cell interactions, more work will be needed to isolate factors of cell morphology from cell-cell mediated signaling in relationship to IVD cell phenotype.
• There is a need for in vitro culture environments that can reproduce the anaerobic metabolism and cell-ECM interactions of the native tissue to address some of these gaps.

| Mechanotransduction
• Only a limited number of studies have investigated ion channels, including mechanosensitive channels, and their role in IVD mechanotransduction. Investigations into the link between mechanosensitive channels, cell mechanics, and downstream transduction pathways will provide insight into physiological disc cell function.
• Evidence suggests that alternative mechanotransduction pathways mediate responses of degenerate cells to loading. Comprehensive approaches are needed to characterize and delineate alternative mechanisms of mechanotransduction with degeneration.
• Cell stress response including role of mitochondrial function in recovery from or protection against overloading are also needed.

| Organ-level interactions
• Knowledge of the cross talk between IVD substructures is not well-described, particularly cross talk between the vertebral body, EP, and AF or NP. Aging, or a lifetime of spinal motion and loading, is often associated with vertebral body changes that can include EP calcification, EP thinning, or evidence of bony microdamage. Factors that drive osteoblast and osteoclast remodeling in the vertebral body, and the formation of osteophytes where relevant, would be important to determine.
• The mechanism(s) underlying the biochemical changes of the cartilage EP in disc degeneration are currently unknown, though some recent studies are beginning to address these questions 175,194 . Further investigations are needed to assess related mechanism and to unveil effect of cartilage EP changes on cell mechanobiology, mediated through the cytoskeleton and other pathways. Changes in structure and cell mechanics could help in understanding the induction of mineralization, reduction in diffusion and onset of tissue damage that could result in herniation through the EP.
• Other connective tissues in the spine are also mechanosensitive and warrant consideration, including cartilage EP, vertebrae-IVD interactions, and spinal ligaments.
To conclude, it is expected that mechanistic studies of mechanobiology of IVD cells in healthy and disease conditions may lead to novel concepts, and potential targets, for mitigating or reversing the effects of altered biomechanics on biological functions of IVD cells.
Such findings will expand the basic understanding and drive the development of therapeutic strategies for recovering "physiological" mechanotransduction of the disc.