In Situ Cell Signalling of the Hippo-YAP/TAZ Pathway in Reaction to Complex Dynamic Loading in an Intervertebral Disc Organ Culture

Recently, a dysregulation of the Hippo-YAP/TAZ pathway has been correlated with intervertebral disc (IVD) degeneration (IDD), as it plays a key role in cell survival, tissue regeneration, and mechanical stress. We aimed to investigate the influence of different mechanical loading regimes, i.e., under compression and torsion, on the induction and progression of IDD and its association with the Hippo-YAP/TAZ pathway. Therefore, bovine IVDs were assigned to one of four different static or complex dynamic loading regimes: (i) static, (ii) “low-stress”, (iii) “intermediate-stress”, and (iv) “high-stress” regime using a bioreactor. After one week of loading, a significant loss of relative IVD height was observed in the intermediate- and high-stress regimes. Furthermore, the high-stress regime showed a significantly lower cell viability and a significant decrease in glycosaminoglycan content in the tissue. Finally, the mechanosensitive gene CILP was significantly downregulated overall, and the Hippo-pathway gene MST1 was significantly upregulated in the high-stress regime. This study demonstrates that excessive torsion combined with compression leads to key features of IDD. However, the results indicated no clear correlation between the degree of IDD and a subsequent inactivation of the Hippo-YAP/TAZ pathway as a means of regenerating the IVD

Should Degenerated Intervertebral Discs of Patients with Modic Type 1 Changes Be Treated with Mesenchymal Stem Cells?

Low back pain (LBP) has been among the leading causes of disability for the past 30 years. This highlights the need for improvement in LBP management. Many clinical trials focus on developing treatments against degenerative disc disease (DDD). The multifactorial etiology of DDD and associated risk factors lead to a heterogeneous patient population. It comes as no surprise that the outcomes of clinical trials on intradiscal mesenchymal stem cell (MSC) injections for patients with DDD are inconsistent. Intradiscal MSC injections have demonstrated substantial pain relief and significant disability-related improvements, yet they have failed to regenerate the intervertebral disc (IVD). Increasing evidence suggests that the positive outcomes in clinical trials might be attributed to the immunomodulatory potential of MSCs rather than to their regenerative properties. Therefore, patient stratification for inflammatory DDD phenotypes may (i) better serve the mechanisms of action of MSCs and (ii) increase the treatment effect. Modic type 1 changes-pathologic inflammatory, fibrotic changes in the vertebral bone marrow-are frequently observed adjacent to degenerated IVDs in chronic LBP patients and represent a clinically distinct subpopulation of patients with DDD. This review discusses whether degenerated IVDs of patients with Modic type 1 changes should be treated with an intradiscal MSC injection

Direction Dependent Mass Transport Through the Cartilage Endplate

INTRODUCTION: Intervertebral Disc (IVD) Degeneration can result from chemical changes in the Cartilage Endplate (CEP) and it might suggest that pain is related to CEP weaknesses and imperfections. The CEP has a crucial role in keeping the IVD healthy by acting as the main gateway of nutrients and waste in and out of that avascular region. Yet, among the spinal tissues, CEPs receive the least amount of attention in scientific literature. The purpose of this study is to follow a combined in vitro and imaging-driven in silico approach to obtain an improved understanding of the CEP functionality regarding mass transport. METHODS: In vitro: 6 CEPs were harvested from a fresh bovine tail. Eight mm diameter biopsy samples were prepared and enabled to free swell prior to testing in Dulbecco's Modified Eagle Medium (DMEM). Each sample was fitted tightly into a silicone tube with constant inlet and outlet pressures. DMEM was passed for 10min twice in the forward and reverse direction corresponding to the flow directions into and out of the IVD in vivo respectively. The amount of passed fluid was collected to determine the flow rate. In silico: The samples were incubated in 40% Hexabrix, a contrast agent, for 48 hours and then imaged by a GE Nanotom® M nanoCT device. From the reconstructed nanoCT images, 1 mm diameter diameter subsamples were selected to create 3D models of the pore structure at different locations in the CEPs. To date, one model was meshed and imported into OpenFOAM® to perform Computational Fluid Dynamics (CFD) Simulations. The other models are still under development. RESULTS: The in vitro experiment showed that the average flow rate through that CEP samples was 6.86 mm3/sec and 4.84 mm3/sec in the forward and reverse directions respectively meaning that the reverse flow was 70.55% of the forward flow. The CFD analysis on one subsample showed that the flow rates were 15.1 mm3/sec and 10.7 mm3/sec meaning that the reverse flow was 70.86% of the forward flow. DISCUSSION & CONCLUSIONS: The results from both the in vitro experiment and the in silico simulation showed that the CEP has a tendency to resist flow differently according the direction of the flow where flow into the IVD was higher than that out of the IVD. This combination of imagebased in silico modelling with in vitro experiments is a first step towards a better quantification of the mass transport across the CEP in and out of IVDs

In-Vitro and In-Silico Investigation of Dynamic Compression on Cartilage Endplate Cells in Agarose

INTRODUCTION: The cartilage endplate (CEP) covers the top and bottom of the intervertebral disc (IVD) and acts to transmit compressive loads and transport water, nutrients, and waste in and out of the disc. Early cartilage endplate (CEP) degeneration is likely to play a key role in IVD degeneration, but little is known about CEP mechanobiology and its changes in degeneration. Investigating these changes is essential to elucidate how the CEP contributes to IVD pathology. METHODS: In-vitro: Bovine-tail CEP cells were expanded until passage three. Afterwards, a 1:1 mixture of CEP cells and agarose was pipetted into silicon molds to create 2% agarose and 1x107 cells/ml carriers, 6 mm diameter and 3 mm thickness, and cultured two days for phenotype recovery. Cell-agarose carriers were placed in custom-made chambers, stimulated with 10 ng/ml TGF-β1 throughout the entirety of the experiment and dynamically compressed up to 7% strain for one hour at 1.5 Hz every day for up to 14 days. Those not dynamically loaded experienced the constant weight of the chamber lid exerting ~5.1 Pa per carrier. Carriers were collected on Days 0, 7, and 14 for downstream analysis of cell viability, gene expression, and glycosaminoglycan (GAG) content. In-silico: A 2D axisymmetric porohyperelastic, compressible, Neo-Hookean finite element model (FEM) of a cell-agarose carrier was developed in Abaqus using literature-derived material properties and loaded with dynamic compression as in the in-vitro experiment. A previously developed mechanotransduction network model was used to predict protein activation levels by initial mechanoreceptor perturbations standing for dynamic compression (α5β1, αvβ3), physioosmotic pressure (TRPV4), tensile strain (αvβ5), plus chondrogenic media (TGF-β). Predicted protein activation was normalized by baseline conditions. RESULTS: After seven and 14 days of culture, cell-agarose carriers in all conditions demonstrated significantly increased expression of anabolic genes aggrecan (ACAN) 2-200x, collagen II (COL II) 31000x, and GAG/DNA content 2.5-5x, alongside decreased expression of catabolic gene matrix metalloproteinase 3 (MMP3). Reaction forces from FEM (0.07N) matched force data collected during loading (0.06N). The FEM showed that hydrostatic pressure varies from center to edge of carrier. General trends of increased/decreased gene expression and protein activation matched between experimental and network model results. DISCUSSION & CONCLUSIONS: A novel framework coupling 3D cell culture with in-silico methods is presented, in which the FEM provides details about loads experienced at each point within the carrier, while the network model uses these mechanical cues and environmental perturbations to predict protein expression and identify key proteins for future analysis. Keywords: Intervertebral disc / spine and their disorders, Biomechanics / biophysical stimuli and mechanotransductio

Sulfated hydrogels as primary intervertebral disc cell culture systems

INTRODUCTION: Intervertebral disc (IVD) degeneration is a key contributor for low back pain, a leading cause of disability worldwide1. During degeneration, IVD aging is accelerated, leading to progressive structural changes, including blood vessel and nerve ingrowth that promote discogenic pain2. In vitro studies require novel biomaterials that mimic the IVD extracellular matrix (ECM) to provide mechanical support and a reservoir of cytokines and growth factors. As proteoglycans with their attached sulfated glycosaminoglycans (GAGs) are one of the major components of the ECM, the ECM’s sulfation state could be a key factor for IVD cell-fate3. Thus, we aim to explore human NP cell fate using a novel sulfated alginate model with varying degrees of sulfation (DS). METHODS: Primary human NP cells were expanded, mixed with solutions of i) 2.5% of standard alginate, ii) 0.1 DS, and iii) 0.2 DS alginate (4 x 106 cells/ml) and casted in 27 l cylindrical-shaped carriers (4 mm diameter, 2 mm height). Carriers were cultured for two weeks for phenotype recovery and were collected with the culture media on day 0, 7 and 14. RESULTS: A significant decrease of cell density (p<0.05) was observed in 0.2 DS alginate after 7 and 14 days of culture. Similarly, cell viability was significantly reduced (p<0.05) in 0.2 DS alginate after 7 days of culture (N=4). In addition, cell metabolic activity tended to be decreased in 0.2 DS alginate compared to standard alginate after 14 days of culture. Surprisingly, ECM remodeling factors such as MMP2 and TIMP1 were slightly upregulated in the 0.1 DS group (N=1), whereas catabolic cytokines were downregulated in the 0.1% DS group. DISCUSSION & CONCLUSIONS: We demonstrate significant cellular differences between 0.2 DS alginate vs standard alginate and 0.1 DS alginate. Particularly, a significant decrease in cell density, metabolic activity and viability were observed in the 0.2 DS alginate after 7 days of culture. According to the secretome, the sulfated alginate group seems to possess increased catabolic ECM remodeling with lower secretion of catabolic factors, suggesting less responsive NP cells to ECM structural changes. Overall, standard alginate seems to be the best option for NP cell 3D culture models. ACKNOWLEDGEMENTS: This project was supported by the Marie Skłodowska Curie International Training Network “disc4all” under the grant agreement #955735. REFERENCES: 1FY. Wang et al (2020) JOR Spine 5:1186. 2P. Bermudez-Lekerika et al (2022) Front Cell Dev Biol 29(10):924692. 3E. Lazarus et al (2021) Cells 10(12):3568. Keywords: Hydrogels and injectable systems, In vitro microenvironment

Cartilaginous endplates: A comprehensive review on a neglected structure in intervertebral disc research

The cartilaginous endplates (CEP) are key components of the intervertebral disc (IVD) necessary for sustaining the nutrition of the disc while distributing mechanical loads and preventing the disc from bulging into the adjacent vertebral body. The size, shape, and composition of the CEP are essential in maintaining its function, and degeneration of the CEP is considered a contributor to early IVD degeneration. In addition, the CEP is implicated in Modic changes, which are often associated with low back pain. This review aims to tackle the current knowledge of the CEP regarding its structure, composition, permeability, and mechanical role in a healthy disc, how they change with degeneration, and how they connect to IVD degeneration and low back pain. Additionally, the authors suggest a standardized naming convention regarding the CEP and bony endplate and suggest avoiding the term vertebral endplate. Currently, there is limited data on the CEP itself as reported data is often a combination of CEP and bony endplate, or the CEP is considered as articular cartilage. However, it is clear the CEP is a unique tissue type that differs from articular cartilage, bony endplate, and other IVD tissues. Thus, future research should investigate the CEP separately to fully understand its role in healthy and degenerated IVDs. Further, most IVD regeneration therapies in development failed to address, or even considered the CEP, despite its key role in nutrition and mechanical stability within the IVD. Thus, the CEP should be considered and potentially targeted for future sustainable treatments

Immuno-modulatory effects of intervertebral disc cells

Low back pain is a highly prevalent, chronic, and costly medical condition predominantly triggered by intervertebral disc degeneration (IDD). IDD is often caused by structural and biochemical changes in intervertebral discs (IVD) that prompt a pathologic shift from an anabolic to catabolic state, affecting extracellular matrix (ECM) production, enzyme generation, cytokine and chemokine production, neurotrophic and angiogenic factor production. The IVD is an immune-privileged organ. However, during degeneration immune cells and inflammatory factors can infiltrate through defects in the cartilage endplate and annulus fibrosus fissures, further accelerating the catabolic environment. Remarkably, though, catabolic ECM disruption also occurs in the absence of immune cell infiltration, largely due to native disc cell production of catabolic enzymes and cytokines. An unbalanced metabolism could be induced by many different factors, including a harsh microenvironment, biomechanical cues, genetics, and infection. The complex, multifactorial nature of IDD brings the challenge of identifying key factors which initiate the degenerative cascade, eventually leading to back pain. These factors are often investigated through methods including animal models, 3D cell culture, bioreactors, and computational models. However, the crosstalk between the IVD, immune system, and shifted metabolism is frequently misconstrued, often with the assumption that the presence of cytokines and chemokines is synonymous to inflammation or an immune response, which is not true for the intact disc. Therefore, this review will tackle immunomodulatory and IVD cell roles in IDD, clarifying the differences between cellular involvements and implications for therapeutic development and assessing models used to explore inflammatory or catabolic IVD environments

Sulfated Hydrogels as Primary Intervertebral Disc Cell Culture Systems.

The negatively charged extracellular matrix plays a vital role in intervertebral disc tissues, providing specific cues for cell maintenance and tissue hydration. Unfortunately, suitable biomimetics for intervertebral disc regeneration are lacking. Here, sulfated alginate was investigated as a 3D culture material due to its similarity to the charged matrix of the intervertebral disc. Precursor solutions of standard alginate, or alginate with 0.1% or 0.2% degrees of sulfation, were mixed with primary human nucleus pulposus cells, cast, and cultured for 14 days. A 0.2% degree of sulfation resulted in significantly decreased cell density and viability after 7 days of culture. Furthermore, a sulfation-dependent decrease in DNA content and metabolic activity was evident after 14 days. Interestingly, no significant differences in cell density and viability were observed between surface and core regions for sulfated alginate, unlike in standard alginate, where the cell number was significantly higher in the core than in the surface region. Due to low cell numbers, phenotypic evaluation was not achieved in sulfated alginate biomaterial. Overall, standard alginate supported human NP cell growth and viability superior to sulfated alginate; however, future research on phenotypic properties is required to decipher the biological properties of sulfated alginate in intervertebral disc cells

Inhibition of the extracellular signal-regulated kinase pathway reduces the inflammatory component in nucleus pulposus cells.

Intervertebral disc (IVD) degeneration is a spinal disorder that triggers an inflammatory response and subsequent development of spinal pseudoarthrosis. The aim of the present study is to elucidate the role of the extracellular signal-regulated kinase (ERK) pathway in inflammation-induced IVD cells. Inflammatory human nucleus pulposus (NP) cells (NPCs) were induced using tumor necrosis factor-α and the ERK pathway was blocked using a selective molecule-based inhibitor U0126. Gene expression of catabolic and anabolic markers, proinflammatory, and NPCs markers was investigated. The enzymatic activity of matrix metalloproteinases (MMP)2/MMP9 was determined by gelatin zymography and nitrite production was assessed by Griess reaction. The NPC metabolic activity and viability were assessed using resazurin sodium-salt and live/dead assays, and subsequently, the specificity of U0126 on ERK1/2 signaling was determined. The catabolic enzyme MMP3 (p = 0.0001) and proinflammatory cytokine interleukin 6 (p = 0.036) were downregulated by U0126 in NPCs under inflammatory conditions. A significant increase of the cytokeratin 19 (p = 0.0031) was observed, suggesting a partial and possible recovery of the NP phenotype. U0126 does not seem to have an effect on prostaglandin production, aggrecanases, or other anabolic genes. We confirmed that U0126 selectively blocks the ERK phosphorylation and only affects the cell metabolic activity without the reduction of viable cells. Inhibition of ERK signaling downregulates important metalloproteinases and proinflammatory cytokines, and upregulates some NP markers, suggesting its potential to treat IVD degeneration

In situ cell signalling of the hippo-YAP/TAZ pathway in reaction to complex dynamic loading in an intervertebral disc

Introduction: Intervertebral disc (IVD) degeneration (IDD) is a main contributor to chronic low back pain. Recently, a dysregulation of the Hippo-YAP/TAZ pathway has been correlated with IDD, as it plays a key role in cell proliferation, differentiation, regeneration, and cell survival. Therefore, our aim was to investigate the influence of different mechanical loading profiles in two Degree-of-Freedom (2DoF) loading, i.e., under compression and torsion, on the induction and progression of IDD and in particularly the role and context of the Hippo-YAP/TAZ pathway. Methods: Around one-year old coccygeal IVDs were isolated from bovine tails obtained from a local abattoir. All IVDs were excised and prepared for organ culture as previously established and then randomly assigned to one of four different static or complex mechanical loading regimes for seven days, i.e., i) “static” (0.1 MPa compression), ii) “low stress” (up to 0.2 MPa compression and 2° torsion), iii) “intermediate stress” (up to 0.4 MPa compression and 8° torsion), and iv) “high stress” (up to 0.6 MPa compression and 15° torsion) profile using a bioreactor that allows 2DoF loading. After one week of loading, the tissue and the culture medium of each condition was harvested and analysed for their glycosaminoglycan (GAG) content, relative disc height changes, relative gene expression of anabolic, catabolic, and inflammatory markers, as well as the major players in the Hippo-YAP/TAZ pathway. Results: After one week of organ culture, a significant loss of IVD height was observed in every mechanical loading regime, with the most significant height loss observed in the higher stress regimes (up to 27 ± 5% height loss). Furthermore, the high stress condition showed a significant decrease of GAG in the tissue (232 ± 48 vs. up to 314 ± 14 µg GAG / mg dry weight) and a significant increase of GAG in the culture medium (97 ± 14 vs. 44 ± 22 µg GAG / cm3 freshly isolated tissue). Moreover, a higher upregulation of catabolic (MMP13 up to 1805 ± 2284-fold upregulation) and inflammation-related genes (MCP1 up to 121 ± 201-fold upregulation) was generally observed the more stressed the IVDs were. Finally, a significant upregulation of TAZ (up to 49 ± 73-fold upregulation) was found for the high stress condition. Conclusion: Altogether, this study demonstrated that excessive torsion combined with compression leads to key features of IDD. Furthermore, we were able to show that the Hippo-YAP/TAZ pathway reacted differently depending on the applied mechanical loading profile and the degenerated state of the IVD. Hence, this study showed the potential of targeting the Hippo-YAP/TAZ pathway to counteract IDD