Celecoxib alleviates nociceptor sensitization mediated by interleukin-1beta-primed annulus fibrosus cells.

PURPOSE: This study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization. METHODS: hAFCs from spinal trauma patients were stimulated with TNF-α or IL-1ß. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 µM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model. RESULTS: IL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1ß treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation. CONCLUSION: Cxb can inhibit PGE-2 production in hAFCs in an IL-1ß-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM.

Small molecule-based treatment approaches for intervertebral disc degeneration: Current options and future directions.

Low back pain (LBP) is a major reason for disability, and symptomatic intervertebral disc (IVD) degeneration (IDD) contributes to roughly 40% of all LBP cases. Current treatment modalities for IDD include conservative and surgical strategies. Unfortunately, there is a significant number of patients in which conventional therapies fail with the result that these patients remain suffering from chronic pain and disability. Furthermore, none of the current therapies successfully address the underlying biological problem - the symptomatic degenerated disc. Both spinal fusion as well as total disc replacement devices reduce spinal motion and are associated with adjacent segment disease. Thus, there is an unmet need for novel and stage-adjusted therapies to combat IDD. Several new treatment options aiming to regenerate the IVD are currently under investigation. The most common approaches include tissue engineering, growth factor therapy, gene therapy, and cell-based treatments according to the stage of degeneration. Recently, the regenerative activity of small molecules (low molecular weight organic compounds with less than 900 daltons) on IDD was demonstrated. However, small molecule-based therapy in IDD is still in its infancy due to limited knowledge about the mechanisms that control different cell signaling pathways of IVD homeostasis. Small molecules can act as anti-inflammatory, anti-apoptotic, anti-oxidative, and anabolic agents, which can prevent further degeneration of disc cells and enhance their regeneration. This review pursues to give a comprehensive overview of small molecules, focusing on low molecular weight organic compounds, and their potential utilization in patients with IDD based on recent in vitro, in vivo, and pre-clinical studies.

Proinflammatory intervertebral disc cell and organ culture models induced by tumor necrosis factor alpha.

Inflammation plays an important role in the pathogenesis of intervertebral disc (IVD) degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has shown markedly higher expression in degenerated human disc tissue compared with healthy controls. Anti-inflammatory treatment targeting TNF-α has shown to alleviate discogenic pain in patients with low back pain. Therefore, in vitro and ex vivo inflammatory models utilizing TNF-α provide relevant experimental conditions for drug development in disc degeneration research. The current method article addressed several specific questions related to the model establishment. (a) The effects of bovine and human recombinant TNF-α on bovine nucleus pulposus (NP) cells were compared. (b) The required dose for an inflammatory IVD organ culture model with intradiscal TNF-α injection was studied. (c) The effect of TNF-α blocking at different stages of inflammation was evaluated. Outcomes revealed that bovine and human recombinant TNF-α induced equivalent inflammatory effects in bovine NP cells. A bovine whole IVD inflammatory model was established by intradiscal injection of 100 ng TNF-α/ cm3 disc volume, as indicated by increased nitric oxide, glycosaminoglycan, interleukin 6 (IL-6), and interleukin 8 (IL-8) release in culture media, and upregulation of MMP3, ADAMTS4, IL-8, IL-6, and cyclooxygenase (COX)-2 expression in NP tissue. However, results in human NP cells showed that the time point of anti-inflammatory treatment was crucial to achieve significant effects. Furthermore, anticatabolic therapy in conjunction with TNF-α inhibition would be required to slow down the pathologic cascade of disc degeneration.

Intervertebral disc organ culture for the investigation of disc pathology and regeneration - benefits, limitations, and future directions of bioreactors.

Low back pain is the leading cause of disability worldwide and in many patients the source of pain can be attributed to pathological changes within the intervertebral disc (IVD). As present treatment options fail to address the underlying biological problem, novel therapies are currently subject to intense research. The physiologic IVD microenvironment features a highly complex interaction of biochemical and mechanical factors influencing cell metabolism and extracellular matrix turnover and is therefore difficult to simulate for research purposes on IVD pathology. The first whole organ culture models were not able to sufficiently replicate human in vivo conditions as mechanical loading, the predominant way of IVD nutrient supply and waste exchange, remained disregarded. To mimic the unique IVD niche more realistically, whole organ culture bioreactors have been developed, allowing for dynamic loading of IVDs and nutrient exchange. Recent advancements on bioreactor systems have facilitated whole organ culture of various IVDs for extended periods. IVD organ culture bioreactors have the potential to bridge the gap between in vitro and in vivo systems and thus may give valuable insights on IVD pathology and/or potential novel treatment approaches if the respective model is adjusted according to a well-defined research question. In this review, we outline the potential of currently utilized IVD bioreactor systems and present suggestions for further developments to more reliably investigate IVD biology and novel treatment approaches.