Toward defining the role of the synovium in mitigating normal articular cartilage wear and tear.

Cartilage repair has been studied extensively in the context of injury and disease, but the joint's management of regular sub-injurious damage to cartilage, or 'wear and tear,' which occurs due to normal activity, is poorly understood. We hypothesize that this cartilage maintenance is mediated in part by cells derived from the synovium that migrate to the worn articular surface. Here, we demonstrate in vitro that the early steps required for such a process can occur. First, we show that under physiologic mechanical loads, chondrocyte death occurs in the cartilage superficial zone along with changes to the cartilage surface topography. Second, we show that synoviocytes are released from the synovial lining under physiologic loads and attach to worn cartilage. Third, we show that synoviocytes parachuted onto a simulated or native cartilage surface will modify their behavior. Specifically, we show that synoviocyte interactions with chondrocytes lead to changes in synoviocyte mechanosensitivity, and we demonstrate that cartilage-attached synoviocytes can express COL2A1, a hallmark of the chondrogenic phenotype. Our findings suggest that synoviocyte-mediated repair of cartilage 'wear and tear' as a component of joint homeostasis is feasible and is deserving of future study.

Transient neonatal shoulder paralysis causes early osteoarthritis in a mouse model.

Neonatal brachial plexus palsy (NBPP) occurs in approximately 1.5 of every 1,000 live births. The majority of children with NBPP recover function of the shoulder. However, the long-term risk of osteoarthritis (OA) in this population is unknown. The purpose of this study was to investigate the development of OA in a mouse model of transient neonatal shoulder paralysis. Neonatal mice were injected twice per week for 4 weeks with saline in the right supraspinatus muscle (Saline, control) and botulinum toxin A (BtxA, transient paralysis) in the left supraspinatus muscle, and then allowed to recover for 20 or 36 weeks. Control mice received no injections, and all mice were sacrificed at 24 or 40 weeks. BtxA mice exhibited abnormalities in gait compared to controls through 10 weeks of age, but these differences did not persist into adulthood. BtxA shoulders had decreased bone volume (-9%) and abnormal trabecular microstructure compared to controls. Histomorphometry analysis demonstrated that BtxA shoulders had higher murine shoulder arthritis scale scores (+30%), and therefore more shoulder OA compared to controls. Articular cartilage of BtxA shoulders demonstrated stiffening of the tissue. Compared with controls, articular cartilage from BtxA shoulders had 2-fold and 10-fold decreases in Dkk1 and BMP2 expression, respectively, and 3-fold and 14-fold increases in Col10A1 and BGLAP expression, respectively, consistent with established models of OA. In summary, a brief period of paralysis of the neonatal mouse shoulder was sufficient to generate early signs of OA in adult cartilage and bone.

Three-Dimensional-Printed Flexible Scaffolds Have Tunable Biomimetic Mechanical Properties for Intervertebral Disc Tissue Engineering.

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.

Actomyosin contractility confers mechanoprotection against TNFα-induced disruption of the intervertebral disc.

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.

Avidin grafted dextran nanostructure enables a month-long intra-discal retention.

Low back pain is often the direct result of degeneration of the intervertebral disc. A wide range of therapeutics including anti-catabolic, pro-anabolic factors and chemo-attractants that can stimulate resident cells and recruit endogenous progenitors are under consideration. The avascular nature and the dense matrix of this tissue make it challenging for systemically administered drugs to reach their target cells inside the nucleus pulposus (NP), the central gelatinous region of the intervertebral disc (IVD). Therefore, local intra-discal injection of therapeutic drugs directly into the NP is a clinically relevant delivery approach, however, suffers from rapid and wide diffusion outside the injection site resulting in short lived benefits while causing systemic toxicity. NP has a high negative fixed charge density due to the presence of negatively charged aggrecan glycosaminoglycans that provide swelling pressures, compressive stiffness and hydration to the tissue. This negative fixed charge density can also be used for enhancing intra-NP residence time of therapeutic drugs. Here we design positively charged Avidin grafted branched Dextran nanostructures that utilize long-range binding effects of electrostatic interactions to bind with the intra-NP negatively charged groups. The binding is strong enough to enable a month-long retention of cationic nanostructures within the NP following intra-discal administration, yet weak and reversible to allow movement to reach cells dispersed throughout the tissue. The branched carrier has multiple sites for drug conjugation and can reduce the need for multiple injections of high drug doses and minimize associated side-effects, paving the way for effective clinical translation of potential therapeutics for treatment of low back pain and disc degeneration.

Confocal scanning of intervertebral disc cells in 3D: Inside alginate beads and in native microenvironment.

The interaction between cells and their extracellular matrix (ECM) is crucial to maintain both tissue and cellular homeostasis. Indeed, cell phenotype is significantly affected by the 3D microenvironment. Although highly convenient, isolating cells from the intervertebral disc (IVD) and growing them in 2D on plastic or glass substrates, causes them to rapidly lose their phenotype and consequently alter their gene and protein expression. While characterization of cells in their native or simulated 3D environment is preferred, such approaches are complexed by limitations in phenotypic readouts. In the current article, we describe a detailed protocol to study nucleus pulposus cells in 3D-embedded in alginate as a permeable cell-staining reservoir, as well as adaptation for cell staining and imaging in their native ECM. This method allows for detection of phenotypical and cytoskeletal changes in cells within native tissue or 3D alginate beads using confocal microscopy, without the need for histological processing.

High mobility group box-1 induces pro-inflammatory signaling in human nucleus pulposus cells via toll-like receptor 4-dependent pathway.

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.

Timing of mesenchymal stem cell delivery impacts the fate and therapeutic potential in intervertebral disc repair.

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 = 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. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:32-40, 2017.

Developments in intervertebral disc disease research: pathophysiology, mechanobiology, and therapeutics.

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 induces irreversible biophysical changes in isolated nucleus pulposus cells.

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.