Intervertebral disc degeneration is accompanied by elevated levels of inflammatory cytokines that have been implicated in disease etiology and matrix degradation. While the effects of inflammatory stimulation on disc cell metabolism have been well-studied, their effects on cell biophysical properties have not been investigated. The hypothesis of this study is that inflammatory stimulation alters the biomechanical properties of isolated disc cells and volume responses to step osmotic loading. Cells from the nucleus pulposus (NP) of bovine discs were isolated and treated with either lipopolysaccharide (LPS), an inflammatory ligand, or with the recombinant cytokine TNF-α for 24 hours. We measured cellular volume regulation responses to osmotic loading either immediately after stimulation or after a 1 week recovery period from the inflammatory stimuli. Cells from each group were tested under step osmotic loading and the transient volume-response was captured via time-lapse microscopy. Volume-responses were analyzed using mixture theory framework to investigate two biomechanical properties of the cell, the intracellular water content and the hydraulic permeability. Intracellular water content did not vary between treatment groups, but hydraulic permeability increased significantly with inflammatory treatment. In the 1 week recovery group, hydraulic permeability remained elevated relative to the untreated recovery control. Cell radius was also significantly increased both after 24 hours of treatment and after 1 week recovery. A significant linear correlation was observed between hydraulic permeability and cell radius in untreated cells at 24 hours and at 1-week recovery, though not in the inflammatory stimulated groups at either time point. This loss of correlation between cell size and hydraulic permeability suggests that regulation of volume change is disrupted irreversibly due to inflammatory stimulation. Inflammatory treated cells exhibited altered F-actin cytoskeleton expression relative to untreated cells. We also found a significant decrease in the expression of aquaporin-1, the predominant water channel in disc NP cells, with inflammatory stimulation. To our knowledge, this is the first study providing evidence that inflammatory stimulation directly alters the mechanobiology of NP cells. The cellular biophysical changes observed in this study are coincident with documented changes in the extracellular matrix induced by inflammation, and may be important in disease etiology.

Intervertebral disc (IVD) degeneration (DD) is associated with low back pain, the leading cause of disability worldwide. Damage‐associated molecular patterns (DAMPs) that contribute to inflammation and trigger DD have not been well characterized. Extracellular high mobility group box‐1 (HMGB1) protein has been implicated as a potent DAMP and pro‐inflammatory stimulus in the immune system. In this study, we show that HMGB1 and IL‐6 levels increase in patients with advanced DD in comparison to early DD. This study further tested the hypothesis that HMGB1 promotes inflammatory signaling driving DD in human nucleus pulposus (NP) cells and tissue. Immunofluorescence and western blot analysis confirmed the expression of HMGB1 and its extracellular release by NP cells under cell stress. Gene expression and protein quantification indicate that HMGB1 stimulates the expression IL‐6 and MMP‐1 in a dose‐dependent manner. The contributions of toll‐like receptor (TLR) −2, −4 and receptor for advanced glycation end products (RAGE) as receptors mediating HMGB1 signaling was examined using small molecule inhibitors. Inhibition of TLR‐4 signaling, with TAK‐242, completely abrogated HMGB1 induced IL‐6 and MMP‐1 expression, whereas inhibition of TLR‐2, with O‐vanillin, or RAGE, with FPS‐ZM1, had mild inhibitory effects. HMGB1 stimulation activated NF‐ĸB signaling while TAK‐242 co‐treatment abrogated it. Lastly, effects of HMGB1 on matrix deposition was evaluated in a 3D culture system of human NP cells. These results implicate HMGB1 as a potent DAMP that promotes inflammation in NP cells and degradation of NP tissues. TLR4‐HMGB1 axis is a potential major pathway to alleviate disc inflammation and mitigate DD. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Low back pain is a leading cause of disability worldwide and the second most common cause of physician visits. There are many causes of back pain, and among them, disc herniation and intervertebral disc degeneration are the most common diagnoses and targets for intervention. Currently, clinical treatment outcomes are not strongly correlated with diagnoses, emphasizing the importance for characterizing more completely the mechanisms of degeneration and their relationships with symptoms. This review covers recent studies elucidating cellular and molecular changes associated with disc mechanobiology, as it relates to degeneration and regeneration. Specifically, we review findings on the biochemical changes in disc diseases, including cytokines, chemokines, and proteases; advancements in disc disease diagnostics using imaging modalities; updates on studies examining the response of the intervertebral disc to injury; and recent developments in repair strategies, including cell-based repair, biomaterials, and tissue engineering. Findings on the effects of the omega-6 fatty acid, linoleic acid, on nucleus pulposus tissue engineering are presented. Studies described in this review provide greater insights into the pathogenesis of disc degeneration and may define new paradigms for early or differential diagnostics of degeneration using new techniques such as systemic biomarkers. In addition, research on the mechanobiology of disease enriches the development of therapeutics for disc repair, with potential to diminish pain and disability associated with disc degeneration.

Inflammation triggers degradation of intervertebral disc extracellular matrix (ECM), a hallmark of disc degeneration that contributes to back pain. Mechanosensitive nucleus pulposus cells are responsible for ECM production, yet the impact of a proinflammatory microenvironment on cell mechanobiology is unknown. Using gain- and loss-of-function approaches, we show that tumor necrosis factor–α (TNFα)–induced inflammation alters cell morphology and biophysical properties (circularity, contractility, cell stiffness, and hydraulic permeability) in a mechanism dependent on actomyosin contractility in a three-dimensional (3D) culture. We found that RhoA activation rescued cells from TNFα-induced mechanobiological disruption. Using a novel explant-in-hydrogel culture system, we demonstrate that nuclear factor kappa-B nuclear translocation and transcription are mechanosensitive, and its downstream effects on ECM degradation are regulated by actomyosin contractility. Results define a scaling relationship between circularity, contractility, and hydraulic permeability that is conserved from healthy to inflammatory microenvironments and is indicative of cell mechanobiological control across scales in 3D.

We studied the osteoconductive tissue response of hydroxyapatite (HA) nanoparticles functionalized with osteopontin (OPN) in a matrix of poly-d,l-lactic-acid (PDLLA). In a canine endosseus 0.75-mm gap implant model, we tested the osteointegrative impact of the OPN functionalized composite as an implant coating, and a non-functionalized composite was used as reference control. During the four weeks of observation, the OPN functionalized composite coating significantly increased the formation of new bone in the porosities of the implant, but no differences were observed in the gap. The study provides evidence of its potential use either alone or in combination with other osteoconductive compounds.

The intervertebral disc (IVD) exhibits complex structure and biomechanical function, which supports the weight of the body and permits motion. Surgical treatments for IVD degeneration (e.g., lumbar fusion, disc replacement) often disrupt the mechanical environment of the spine which lead to adjacent segment disease. Alternatively, disc tissue engineering strategies, where cell-seeded hydrogels or fibrous biomaterials are cultured in vitro to promote matrix deposition, do not recapitulate the complex IVD mechanical properties. In this study, we use 3D printing of flexible polylactic acid (FPLA) to fabricate a viscoelastic scaffold with tunable biomimetic mechanics for whole spine motion segment applications. We optimized the mechanical properties of the scaffolds for equilibrium and dynamic moduli in compression and tension by varying fiber spacing or porosity, generating scaffolds with de novo mechanical properties within the physiological range of spine motion segments. The biodegradation analysis of the 3D printed scaffolds showed that FPLA exhibits lower degradation rate and thus has longer mechanical stability than standard PLA. FPLA scaffolds were biocompatible, supporting viability of nucleus pulposus (NP) cells in 2D and in FPLA+hydrogel composites. Composite scaffolds cultured with NP cells maintained baseline physiological mechanical properties and promoted matrix deposition up to 8 weeks in culture. Mesenchymal stromal cells (MSCs) cultured on FPLA adhered to the scaffold and exhibited fibrocartilaginous differentiation. These results demonstrate for the first time that 3D printed FPLA scaffolds have de novo viscoelastic mechanical properties that match the native IVD motion segment in both tension and compression and have the potential to be used as a mechanically stable and biocompatible biomaterial for engineered disc replacement.

Cells within cartilaginous tissues are mechanosensitive and thus require mechanical loading for regulation of tissue homeostasis and metabolism. Mechanical loading plays critical roles in cell differentiation, proliferation, biosynthesis, and homeostasis. Inflammation is an important event occurring during multiple processes, such as aging, injury, and disease. Inflammation has significant effects on biological processes as well as mechanical function of cells and tissues. These effects are highly dependent on cell/tissue type, timing, and magnitude. In this review, we summarize key findings pertaining to effects of inflammation on multiscale mechanical properties at subcellular, cellular, and tissue level in cartilaginous tissues, including alterations in mechanotransduction and mechanosensitivity. The emphasis is on articular cartilage and the intervertebral disc, which are impacted by inflammatory insults during degenerative conditions such as osteoarthritis, joint pain, and back pain. To recapitulate the pro-inflammatory cascades that occur in vivo, different inflammatory stimuli have been used for in vitro and in situ studies, including tumor necrosis factor (TNF), various interleukins (IL), and lipopolysaccharide (LPS). Therefore, this review will focus on the effects of these stimuli because they are the best studied pro-inflammatory cytokines in cartilaginous tissues. Understanding the current state of the field of inflammation and cell/tissue biomechanics may potentially identify future directions for novel and translational therapeutics with multiscale biomechanical considerations.

Cell-based therapies offer a promising approach to treat intervertebral disc (IVD) degeneration. The impact of the injury microenvironment on treatment efficacy has not been established. This study used a rat disc stab injury model with administration of mesenchymal stromal cells (MSCs) at 3, 14, or 30 days post injury (DPI) to evaluate the impact of interventional timing on IVD biochemistry and biomechanics. We also evaluated cellular localization within the disc with near infrared imaging to track the time and spatial profile of cellular migration after in vivo delivery. Results showed that upon injection into a healthy disc, MSCs began to gradually migrate outwards over the course of 14 days, as indicated by decreased signal intensity from the disc space and increased signal within the adjacent vertebrae. Cells administered 14 or 30 DPI also tended to migrate out 14 days after injection but cells injected 3 DPI were retained at a significantly higher amount versus the other groups (p < 0.05). Correspondingly the 3 DPI group, but not 14 or 30 DPI groups, had a higher GAG content in the MSC injected discs (p ¼ 0.06). Enrichment of MSCs and increased GAG content in 3 DPI group did not lead to increased compressive biomechanical properties. Findings suggest that cell therapies administered at an early stage of injury/disease progression may have greater chances of mitigating matrix loss, possibly through promotion of MSC activity by the inflammatory microenvironment associated with injury. Future studies will evaluate the mode and driving factors that regulate cellular migration out of the disc. Keywords: intervertebral disc; mesenchymal stem cells; inflammation; animal model; spine Disc degeneration (DD) is associated with low back pain that affects 40% of the U.S. adult population and accounts for over $100 billion annually in medical cost and disability. 1 DD is characterized by gradual loss and/or senescence of disc cells that contribute to catabolic breakdown of the disc extracellular matrix (ECM). 2 Cell based therapies for treating DD has gathered considerable attention over the past decade, where cells are delivered into the disc in order to recover lost cells and increase proteoglycan (PG) and collagen content. Mesenchymal stem/stromal cells (MSCs) may be a promising therapy in the treatment of DD. MSCs are a heterogeneous population of multipotent cells capable of differentiating along the chondrogenic, osteogenic, and adipogenic lineages but not the hematopoietic lineage. 3 There are many in vitro and animal studies describing successful intervertebral disc (IVD) repair/regeneration with the use of MSC therapy. 4 Pre-clinical evaluations of the efficacy of MSC treatment of injured IVDs indicate that MSCs can survive and differentiate into NP like cells, 5-7 and promote recovery of disc height and radiological indicators of disc integrity. [6][7][8]9 Efficacy, however, depends on the choice of animal species, degeneration/injury model, timing of treatment, and stem cell source. 7,8,10 A recent...