TNFα Transport Induced by Dynamic Loading Alters Biomechanics of Intact Intervertebral Discs

March TNF Transport Induced by Dynamic Loading Alters Biomechanics of Intact Intervertebral Discs Benjamin A. Walter 0 1 Morakot Likhitpanichkul 0 1 Svenja Illien-Junger 0 1 Peter J. Roughley 0 1 Andrew C. Hecht 0 1 James C. Iatridis 0 1 0 1 Leni & Peter W. May Department of Orthopaedics at the Icahn School of Medicine at Mount Sinai , New York, NY , United States of America, 2 Department of Biomedical Engineering, The City College of New York , New York, NY , United States of America, 3 Shriners Hospital for Children , Montreal QC , Canada 1 Academic Editor: Christoph Englert, Leibniz Institute for Age Research-Fritz Lipmann Institute (FLI) , GERMANY Intervertebral disc (IVD) degeneration is an important contributor to the development of back pain, and a key factor relating pain and degeneration are the presence of pro-inflammatory cytokines and IVD motion. There is surprisingly limited understanding of how mechanics and inflammation interact in the IVD. This study investigated interactions between mechanical loading and pro-inflammatory cytokines in a large animal organ culture model to address fundamental questions regarding (i.) how inflammatory mediators arise within the IVD, (ii.) how long inflammatory mediators persist, and (iii.) how inflammatory mediators influence IVD biomechanics. Bovine caudal IVDs were cultured for 6 or 20-days under static & dynamic loading with or without exogenous TNF in the culture medium, simulating a consequence of inflammation of the surrounding spinal tissues. TNF transport within the IVD was assessed via immunohistochemistry. Changes in IVD structural integrity (dimensions, histology & aggrecan degradation), biomechanical behavior (Creep, Recovery & Dynamic stiffness) and pro-inflammatory cytokines in the culture medium (ELISA) were assessed. - Funding: Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR057397 and R01AR064157. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. TNF was able to penetrate intact IVDs when subjected to dynamic loading but not static loading. Once transported within the IVD, pro-inflammatory mediators persisted for 48 days after TNF removal. TNF exposure induced changes in IVD biomechanics (reduced diurnal displacements & increased dynamic stiffness). This study demonstrated that exposure to TNF, as might occur from injured surrounding tissues, can penetrate healthy intact IVDs, induce expression of additional pro-inflammatory cytokines and alter IVD mechanical behavior. We conclude that exposure to pro-inflammatory cytokine may be an initiating event in the progression of IVD degeneration in addition to being a consequence of disease. Inflammation is emerging as an important contributor to the pathogenesis of painful intervertebral disc (IVD) degeneration [1, 2], however, the specific role it plays in disease progression remains unclear. Pro-inflammatory cytokines can induce cellular changes that are characteristic of degeneration [38] and the expression of pro-inflammatory cytokines is correlated with aging and the severity of IVD degeneration [911]. It remains unclear how pro-inflammatory cytokines arise during disease and whether their presence is a contributor to, or consequence of, the disease process. The overall goal of this study was to investigate the fundamental questions regarding how inflammatory mediators arise within the IVD, how long inflammatory mediators persist, and how inflammatory mediators influence IVD biomechanics. Injury and/or inflammation of spinal structures surrounding the IVD (i.e. spinal ligaments, vertebrae, and facet joints) are associated with spinal pathology [1216] yet it remains unknown if inflammatory mediators, possibly resulting from inflamed spinal tissues, can penetrate intact IVDs. The acute response to tissue injury involves the expression of multiple pro-inflammatory cytokines including TNF, IL-1 and IL-6 [17, 18]. This local increase in the concentration of inflammatory mediators immediately surrounding the IVD may provide another source of elevated inflammatory mediators within the IVD, as the concentration gradient would favor transport into the IVD. However, it is not known whether pro-inflammatory cytokines outside the IVD can penetrate a healthy IVD, which is considered immune-privileged due to its lack of vasculature and slow transport kinetics. Mechanical factors are also known to contribute to the progression of IVD degeneration [19] and may interact with the inflammatory component of the disease through enhancing transport of pro-inflammatory cytokines. The dominant mode of transportation for pro-inflammatory cytokines within the IVD remains unclear, however dynamic mechanical loading plays an important role in enhancing molecular transport of large solutes within cartilaginous tissues, through the addition of convective fluid flow [20, 21]. Solute size is an important factor in determining which mode of transport (convection or diffusion) dominates within the IVD and modelling studies have suggested that pro-inflammatory cytokines are of sufficient size (TNF *17.5kDa, IL-1 *17.3kDa) that they may be enhanced by convective fluid flow [20], yet experimentally it is less clear which mode dominates the intradiscal transport of pro-inflammatory cytokines. A recent study found that exogenously added pro-inflammatory cytokines were able to penetrate intact rat IVDs when cultured under free swelling (diffusion) conditions [22, 23]. However, another study found that diffusion alone was insufficient to transport exogenously-added dextran (MW: 3kDa), which was an order of magnitude smaller than TNF, into the nucleus pulposus of ovine caudal IVDs [24]. Together, this suggests that both solute and IVD size are important factors in accurately modeling transport phenomenon relevant to the human condition. The persistence of inflammatory mediators within the IVD is dictated by the balance between what is being produced and metabolized within the IVD and what is being transported in/out of the tissue. Therefore, in order to accurately investigate how long an elevated presence of inflammatory cytokines persists within the IVD, a model must incorporate both the native cell population and dynamic physiological loading. We previously demonstrated in a bovine caudal organ culture model that the IVD could not recover from a transient exposure to TNF under static loading conditions [3], suggesting that inflammatory mediators may have persisted throughout the 21-day experiment. However, the experimental conditions in that model may not have been conducive to recovery since vertebral endplates were removed to promote cell viability and static loading was applied. Given the complex nature of transport and cytokine expression, as well as th (...truncated)