Evidence-Based Network Modelling to Simulate Nucleus Pulposus Multicellular Activity in Different Nutritional and Pro-Inflammatory Environments.

Initiation of intervertebral disc degeneration is thought to be biologically driven. This reflects a process, where biochemical and mechanical stimuli affect cell activity (CA) that compromise the tissue strength over time. Experimental research enhanced our understanding about the effect of such stimuli on different CA, such as protein synthesis or mRNA expression. However, it is still unclear how cells respond to their native environment that consists of a "cocktail" of different stimuli that might locally vary. This work presents an interdisciplinary approach of experimental and in silico research to approximate Nucleus Pulposus CA within multifactorial biochemical environments. Thereby, the biochemical key stimuli glucose, pH, and the proinflammatory cytokines TNF-α and IL1ß were considered that were experimentally shown to critically affect CA. To this end, a Nucleus Pulposus multicellular system was modelled. It integrated experimental findings from in vitro studies of human or bovine Nucleus Pulposus cells, to relate the individual effects of targeted stimuli to alterations in CA. Unknown stimulus-CA relationships were obtained through own experimental 3D cultures of bovine Nucleus Pulposus cells in alginate beads. Translation of experimental findings into suitable parameters for network modelling approaches was achieved thanks to a new numerical approach to estimate the individual sensitivity of a CA to each stimulus type. Hence, the effect of each stimulus type on a specific CA was assessed and integrated to approximate a multifactorial stimulus environment. Tackled CA were the mRNA expressions of Aggrecan, Collagen types I & II, MMP3, and ADAMTS4. CA was assessed for four different proinflammatory cell states; non-inflamed and inflamed for IL1ß, TNF-α or both IL1ß&TNF-α. Inflamed cell clusters were eventually predicted in a multicellular 3D agent-based model. Experimental results showed that glucose had no significant impact on proinflammatory cytokine or ADAMTS4 mRNA expression, whereas TNF-α caused a significant catabolic shift in most explored CA. In silico results showed that the presented methodology to estimate the sensitivity of a CA to a stimulus type importantly improved qualitative model predictions. However, more stimuli and/or further experimental knowledge need to be integrated, especially regarding predictions about the possible progression of inflammatory environments under adverse nutritional conditions. Tackling the multicellular level is a new and promising approach to estimate manifold responses of intervertebral disc cells. Such a top-down high-level network modelling approach allows to obtain information about relevant stimulus environments for a specific CA and could be shown to be suitable to tackle complex biological systems, including different proinflammatory cell states. The development of this methodology required a close interaction with experimental research. Thereby, specific experimental needs were derived from systematic in silico approaches and obtained results were directly used to enhance model predictions, which reflects a novelty in this research field. Eventually, the presented methodology provides modelling solutions suitable for multiscale approaches to contribute to a better understanding about dynamics over multiple spatial scales. Future work should focus on an amplification of the stimulus environment by integrating more key relevant stimuli, such as mechanical loading parameters, in order to better approximate native physiological environments.

Differential regulation of TRP channel gene and protein expression by intervertebral disc degeneration and back pain.

Intervertebral disc (IVD) degeneration and consequent low back pain (LBP) are common and costly pathological processes that require improved treatment strategies. Transient Receptor Potential (TRP) channels constitute a family of multimodal ion channels that have recently emerged as contributors to disc pathologies and were thus proposed as potential therapeutic targets, although limited data on their presence and function in the IVD exist. The purpose of this study was to determine the mRNA and protein expression of TRP channels in non-degenerated and degenerated human IVD tissue (with different pain intensity and chronicity) using gene array, conventional qPCR and immunohistochemistry. We could demonstrate that 26 out of 28 currently known TRP channels are expressed in the IVD on the mRNA level, thereby revealing novel therapeutic candidates from the TRPC, TRPM and TRPML subfamilies. TRPC6, TRPM2 and TRPML1 displayed enhanced gene and protein expression in degenerated IVDs as compared to non-degenerated IVDs. Additionally, the gene expression of TRPC6 and TRPML1 was influenced by the IVD degeneration grade. Pain intensity and/or chronicity influenced the gene and/or protein expression of TRPC6, TRPM2 and TRML1. Interestingly, decreased gene expression of TRPM2 was observed in patients treated with steroids. This study supports the importance of TRP channels in IVD homeostasis and pathology and their possible application as pharmacological targets for the treatment of IVD degeneration and LBP. However, the exact function and activation of the highlighted TRP channels will have to be determined in future studies.

Osmosensing, osmosignalling and inflammation: how intervertebral disc cells respond to altered osmolarity.

Intervertebral disc (IVD) cells are naturally exposed to high osmolarity and complex mechanical loading, which drive microenvironmental osmotic changes. Age- and degeneration-induced degradation of the IVD's extracellular matrix causes osmotic imbalance, which, together with an altered function of cellular receptors and signalling pathways, instigates local osmotic stress. Cellular responses to osmotic stress include osmoadaptation and activation of pro-inflammatory pathways. This review summarises the current knowledge on how IVD cells sense local osmotic changes and translate these signals into physiological or pathophysiological responses, with a focus on inflammation. Furthermore, it discusses the expression and function of putative membrane osmosensors (e.g. solute carrier transporters, transient receptor potential channels, aquaporins and acid-sensing ion channels) and osmosignalling mediators [e.g. tonicity response-element-binding protein/nuclear factor of activated T-cells 5 (TonEBP/NFAT5), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)] in healthy and degenerated IVDs. Finally, an overview of the potential therapeutic targets for modifying osmosensing and osmosignalling in degenerated IVDs is provided.

The role of transient receptor potential channels in joint diseases.

Transient receptor potential channels (TRP channels) are cation selective transmembrane receptors with diverse structures, activation mechanisms and physiological functions. TRP channels act as cellular sensors for a plethora of stimuli, including temperature, membrane voltage, oxidative stress, mechanical stimuli, pH and endogenous, as well as, exogenous ligands, thereby illustrating their versatility. As such, TRP channels regulate various functions in both excitable and non-excitable cells, mainly by mediating Ca2+ homeostasis. Dysregulation of TRP channels is implicated in many pathologies, including cardiovascular diseases, muscular dystrophies and hyperalgesia. However, the importance of TRP channel expression, physiological function and regulation in chondrocytes and intervertebral disc (IVD) cells is largely unexplored. Osteoarthritis (OA) and degenerative disc disease (DDD) are chronic age-related disorders that significantly affect the quality of life by causing pain, activity limitation and disability. Furthermore, currently available therapies cannot effectively slow-down or stop progression of these diseases. Both OA and DDD are characterised by reduced tissue cellularity, enhanced inflammatory responses and molecular, structural and mechanical alterations of the extracellular matrix, hence affecting load distribution and reducing joint flexibility. However, knowledge on how chondrocytes and IVD cells sense their microenvironment and respond to its changes is still limited. In this review, we introduced six families of mammalian TRP channels, their mechanisms of activation, as well as, activation-driven cellular consequences. We summarised the current knowledge on TRP channel expression and activity in chondrocytes and IVD cells, as well as, the significance of TRP channels as therapeutic targets for the treatment of OA and DDD.

Epigallocatechin 3-gallate suppresses interleukin-1ß-induced inflammatory responses in intervertebral disc cells in vitro and reduces radiculopathic pain in rats.

Intervertebral disc (IVD) disease, which is characterised by age-related changes in the adult disc, is the most common cause of disc failure and low back pain. The purpose of this study was to analyse the potential of the biologically active polyphenol epigallocatechin 3-gallate (EGCG) for the treatment of painful IVD disease by identifying and explaining its anti-inflammatory and anti-catabolic activity. Human IVD cells were isolated from patients undergoing surgery due to degenerative disc disease (n = 34) and cultured in 2D or 3D. An inflammatory response was activated by IL-1ß, EGCG was added, and the expression/activity of inflammatory mediators and pathways was measured by qRT-PCR, western blotting, ELISA, immunofluorescence and transcription factor assay. The small molecule inhibitor SB203580 was used to investigate the involvement of the p38 pathway in the observed effects. The analgesic properties of EGCG were analysed by the von Frey filament test in Sprague-Dawley rats (n = 60). EGCG significantly inhibited the expression of pro-inflammatory mediators and matrix metalloproteinases in vitro, as well as radiculopathic pain in vivo, most probably by modulation of the activity of IRAK-1 and its downstream effectors p38, JNK and NF-κB.