Spatiotemporal changes in imaging features associated with diffuse idiopathic skeletal hyperostosis (DISH).

OBJECTIVES: The purpose of our study was to characterise spatiotemporal features of disease progression in people with diffuse idiopathic skeletal hyperostosis (DISH), early-phase DISH, and those not meeting either criterion who had repeated CT scans of the thoracic spine. METHODS: A retrospective study was designed in collaboration with the Rochester Epidemiology Project to evaluate completeness of ectopic bridging across the thoracic spine and corresponding disease status over an average of 2.7 years (range from 0.2 to 15.0 years) in a cohort of 83 female and 74 male individuals. RESULTS: Over 15% of individuals displayed changes in imaging features over time that resulted in a revised diagnosis along the continuum of DISH. Early-phase DISH was marked by new involvement of previously unaffected motion segments, estimated to occur over 2.1 years. Advanced presentations of DISH were marked by increased prevalence of complete bridging (average two of three available motion segments), estimated to occur over 2.6-2.9 years. Localised nodules of ectopic mineralisation external to and within the intervertebral disc were regularly observed in early-phase DISH. CONCLUSIONS: This is the first characterisation of spatiotemporal features across all phases of DISH, indicating that progression of DISH is characterised by distinct features at different phases along the disease continuum. Localised nodules of mineralisation in the spinal ligaments and within the intervertebral discs coincident with early phases of the disease may be a key factor in the pathogenesis of DISH.

Transient receptor potential vanilloid 4 regulates extracellular matrix composition and mediates load-induced intervertebral disc degeneration in a mouse model.

OBJECTIVE: Transient receptor potential vanilloid 4 (TRPV4) is a multi-modally activated cation channel that mediates mechanotransduction pathways by which musculoskeletal tissues respond to mechanical load and regulate tissue health. Using conditional Trpv4 knockout mice, we investigated the role of Trpv4 in regulating intervertebral disc (IVD) health and injury-induced IVD degeneration. METHODS: Col2-Cre;Trpv4fl/f (Trpv4 KO) mice were used to knockout Trpv4 in all type 2 collagen-expressing cells. Effects of gene targeting alone was assessed in lumbar spines, using vertebral bone length measurement, histological, immunohistochemistry and gene expression analyses, and mechanical testing. Disc puncture was performed on caudal IVDs of wild-type (WT) and Trpv4 KO mice at 2.5- and 6.5-months-of-age. Six weeks after puncture (4- and 8-months-of-age at sacrifice), caudal spines were assessed using histological analyses. RESULTS: While loss of Trpv4 did not significantly alter vertebral bone length and tissue histomorphology compared to age-matched WT mice, Trpv4 KO mice showed decreased proteoglycan and PRG4 staining in the annulus fibrosus compared to WT. At the gene level, Trpv4 KO mice showed significantly increased expression of Acan, Bgn, and Prg4 compared to WT. Functionally, loss of Trpv4 was associated with significantly increased neutral zone length in lumbar IVDs. Following puncture, both Trpv4 KO and WT mice showed similar signs of degeneration at the site of injury. Interestingly, loss of Trpv4 prevented mechanically-induced degeneration in IVDs adjacent to sites of injury. CONCLUSION: These studies suggest a role for Trpv4 in regulating extracellular matrix synthesis and mediating the response of IVD tissues to mechanical stress.

Pannexin 3 deletion in mice results in knee osteoarthritis and intervertebral disc degeneration after forced treadmill running.

Pannexin 3 (Panx3) is a glycoprotein that forms mechanosensitive channels expressed in chondrocytes and annulus fibrosus cells of the intervertebral disc (IVD). Evidence suggests Panx3 plays contrasting roles in traumatic versus aging osteoarthritis (OA) and intervertebral disc degeneration (IDD). However, whether its deletion influences the response of joint tissue to forced use is unknown. The purpose of this study was to determine if Panx3 deletion in mice causes increased knee joint OA and IDD after forced treadmill running. Male and female wildtype (WT) and Panx3 knockout (KO) mice were randomized to either a no-exercise group (sedentary; SED) or daily forced treadmill running (forced exercise; FEX) from 24 to 30 weeks of age. Knee cartilage and IVD histopathology were evaluated by histology, while tibial secondary ossification centers were analyzed using microcomputed tomography (µCT). Both male and female Panx3 KO mice developed larger superficial defects of the tibial cartilage after forced treadmill running compared with SED WT mice. Additionally, Panx3 KO mice developed reduced bone volume, and female PANX3 KO mice had lengthening of the lateral tubercle at the intercondylar eminence. In the lower lumbar spine, both male and female Panx3 KO mice developed histopathological features of IDD after running compared to SED WT mice. These findings suggest that the combination of deleting Panx3 and forced treadmill running induces OA and causes histopathological changes associated with the degeneration of the IVDs in mice.

Protocol for co-producing a framework and integrated resource platform for engaging patients in laboratory-based research.

BACKGROUND: Patient engagement in research is the meaningful and collaborative interaction between patients and researchers throughout the research process. Patient engagement can help to ensure patient-oriented values and perspectives are incorporated into the development, conduct, and dissemination of research. While patient engagement is increasingly prevalent in clinical research, it remains relatively unrealized in preclinical laboratory research. This may reflect the nature of preclinical research, in which routine interactions or engagement with patients may be less common. Our team of patient partners and researchers has previously identified few published examples of patient engagement in preclinical laboratory research, as well as a paucity of guidance on this topic. Here we propose the development of a process framework to facilitate patient engagement in preclinical laboratory research. METHODS: Our team, inclusive of researchers and patient partners, will develop a comprehensive, empirically-derived, and stakeholder-informed process framework for 'patient engagement in preclinical laboratory research.' First, our team will create a 'deliberative knowledge space' to conduct semi-structured discussions that will inform a draft framework for preclinical patient engagement. Over the course of several sessions, we will identify actions, activities, barriers, and enablers (e.g. considerations and motivations for patient engagement in preclinical laboratory research, define roles of key players). The resulting draft process framework will be further populated with examples and refined through an international consensus-building Delphi survey with patients, researchers, and other collaborator organizations. We will then conduct pilot field tests to evaluate the framework with preclinical laboratory research groups paired with patient partners. These results will be used to create a refined framework enriched with real-world examples and considerations. All resources developed will be made available through an online repository. DISCUSSION: Our proposed process framework will provide guidance, best practices, and standardized procedures to promote patient engagement in preclinical laboratory research. Supporting and facilitating patient engagement in this setting presents an exciting new opportunity to help realize the important impact that patients can make.

Engaging patients as partners or collaborators in clinical research is becoming more common, but it is still new in preclinical research. Preclinical researchers work in laboratories on cell and animal experiments. They traditionally don't have frequent interactions with patients compared to their clinical research colleagues. Integrating patient engagement in preclinical laboratory research may help ensure that patient perspectives and values are considered. To help preclinical laboratory research align with patient-centred priorities we propose the development of a practical framework. This framework will facilitate patient engagement in preclinical laboratory research. To achieve this, we will first hold in-depth discussions with patient partners, researchers, and other collaborators to understand views on patient engagement in preclinical laboratory research. Together, we will identify key considerations to draft a framework, including motivations for patient engagement in preclinical laboratory research, and defining the roles of those who need to be involved. We will refine the framework through an international survey where we will collect feedback from researchers, patient partners, and other collaborators to make further improvements. The framework will then be tested and refined by preclinical laboratory teams inclusive of patient partners. The finalized framework and other resources to facilitate patient engagement in preclinical laboratory research will be hosted in a 'one-stop-shop' of online resources. Ultimately, this framework will enable partnerships between patients and researchers and provide a roadmap for patient engagement in preclinical laboratory research. This presents an exciting new opportunity for patients and researchers to collaborate and potentially improve translation of laboratory-based research.

Prevalence of diffuse idiopathic skeletal hyperostosis (DISH) and early-phase DISH across the lifespan of an American population.

OBJECTIVES: DISH is a common musculoskeletal disorder; however, the imaging features and disease continuum from early to advanced stages is poorly understood. The purpose of this study was to evaluate the prevalence of DISH and early-phase DISH in an American population and to assess the extent and pattern of ectopic mineralization across the thoracic spine. METHODS: Data were retrieved in collaboration with the Rochester Epidemiology Project. We conducted a retrospective image evaluation of a sample of individuals over 19 years of age with CT of the thoracic spine from a Northern US catchment area. Stratified random sampling by age and sex was used to populate the study. We examined the prevalence and extent of ectopic mineralization along the thoracic spine using previously established criteria. RESULTS: A total of 1536 unique images (766 female and 770 male individuals) including 16 710 motion segments were evaluated for imaging features of the continuum of DISH. Collectively, 40.5% of all motion segments evaluated displayed evidence of ectopic mineralization in the thoracic spine. The prevalence of early-phase DISH was 13.2% (10.4% of female and 15.8% of male individuals). The prevalence of established DISH was 14.2% (7.4% of female and 20.9% of male individuals). Remarkable heterogeneity was detected in individuals within each disease classification, based on the extent of the thoracic spine affected and degree of mineralization. CONCLUSIONS: The continuum of imaging features associated with DISH is detected in more than one in four adults and both sexes in an American population.

Comparative histopathological analysis of age-associated intervertebral disc degeneration in CD-1 and C57BL/6 mice: Anatomical and sex-based differences.

Background: Intervertebral disc (IVD) degeneration is a major contributor to back pain and disability. The cause of IVD degeneration is multifactorial, with no disease-modifying treatments. Mouse models are commonly used to study IVD degeneration; however, the effects of anatomical location, strain, and sex on the progression of age-associated degeneration are poorly understood. Methods: A longitudinal study was conducted to characterize age-, anatomical-, and sex-specific differences in IVD degeneration in two commonly used strains of mice, C57BL/6 and CD-1. Histopathological evaluation of the cervical, thoracic, lumbar, and caudal regions of mice at 6, 12, 20, and 24 months of age was conducted by two blinded observers at each IVD for the nucleus pulposus (NP), annulus fibrosus (AF), and the NP/AF boundary compartments, enabling analysis of scores by tissue compartment, summed scores for each IVD, or averaged scores for each anatomical region. Results: C57BL/6 mice displayed mild IVD degeneration until 24 months of age; at this point, the lumbar spine demonstrated the most degeneration compared to other regions. Degeneration was detected earlier in the CD-1 mice (20 months of age) in both the thoracic and lumbar spine. In CD-1 mice, moderate to severe degeneration was noted in the cervical spine at all time points assessed. In both strains, age-associated IVD degeneration in the thoracic and lumbar spine was associated with increased histopathological scores in all IVD compartments. In both strains, minimal degeneration was detected in caudal IVDs out to 24 months of age. Both C57BL/6 and CD-1 mice displayed sex-specific differences in the presentation and progression of age-associated IVD degeneration. Conclusions: These results showed that the progression and severity of age-associated degeneration in mouse models is associated with marked differences based on anatomical region, sex, and strain. This information provides a fundamental baseline characterization for users of mouse models to enable effective and appropriate experimental design, interpretation, and comparison between studies.

Glycerol induced paraspinal muscle degeneration leads to hyper-kyphotic spinal deformity in wild-type mice.

Degenerative spinal disorders, including kyphotic deformity, are associated with a range of degenerative characteristics of the paraspinal musculature. It has therefore been hypothesized that paraspinal muscular dysfunction is a causative factor for degenerative spinal deformity; however, experimental studies demonstrating causative relationships are lacking. Male and female mice received either glycerol or saline injections bilaterally along the length of the paraspinal muscles at four timepoints, each separated by 2 weeks. Immediately after sacrifice, micro-CT was performed to measure spinal deformity; paraspinal muscle biopsies were taken to measure active, passive and structural properties; and lumbar spines were fixed for analysis of intervertebral disc (IVD) degeneration. Glycerol-injected mice demonstrated clear signs of paraspinal muscle degeneration and dysfunction: significantly (p < 0.01) greater collagen content, lower density, lower absolute active force, greater passive stiffness compared to saline-injected mice. Further, glycerol-injected mice exhibited spinal deformity: significantly (p < 0.01) greater kyphotic angle than saline-injected mice. Glycerol-injected mice also demonstrated a significantly (p < 0.01) greater IVD degenerative score (although mild) at the upper-most lumbar level compared to saline-injected mice. These findings provide direct evidence that combined morphological (fibrosis) and functional (actively weaker and passively stiffer) alterations to the paraspinal muscles can lead to negative changes and deformity within the thoracolumbar spine.

Stiffness and axial pain are associated with the progression of calcification in a mouse model of diffuse idiopathic skeletal hyperostosis.

BACKGROUND: Diffuse idiopathic skeletal hyperostosis (DISH) is characterized by progressive calcification of spinal tissues; however, the impact of calcification on pain and function is poorly understood. This study examined the association between progressive ectopic spine calcification in mice lacking equilibrative nucleoside transporter 1 (ENT1-/-), a preclinical model of DISH, and behavioral indicators of pain. METHODS: A longitudinal study design was used to assess radiating pain, axial discomfort, and physical function in wild-type and ENT1-/- mice at 2, 4, and 6 months. At endpoint, spinal cords were isolated for immunohistochemical analysis of astrocytes (GFAP), microglia (IBA1), and nociceptive innervation (CGRP). RESULTS: Increased spine calcification in ENT1-/- mice was associated with reductions in flexmaze exploration, vertical activity in an open field, and self-supporting behavior in tail suspension, suggesting flexion-induced discomfort or stiffness. Grip force during the axial stretch was also reduced in ENT1-/- mice at 6 months of age. Increased CGRP immunoreactivity was detected in the spinal cords of female and male ENT1-/- mice compared to wild-type. GFAP- and IBA1-immunoreactivity were increased in female ENT1-/- mice compared to wild-type, suggesting an increase in nociceptive innervation. CONCLUSION: These data suggest that ENT1-/- mice experience axial discomfort and/or stiffness and importantly that these features are detected during the early stages of spine calcification.

Development of 2-D and 3-D culture platforms derived from decellularized nucleus pulposus.

Bioscaffolds derived from the extracellular matrix (ECM) have shown the capacity to promote regeneration by providing tissue-specific biological instructive cues that can enhance cell survival and direct lineage-specific differentiation. This study focused on the development and characterization of two-dimensional (2-D) and three-dimensional (3-D) cell culture platforms incorporating decellularized nucleus pulposus (DNP). First, a detergent-free protocol was developed for decellularizing bovine nucleus pulposus (NP) tissues that was effective at removing cellular content while preserving key ECM constituents including collagens, glycosaminoglycans, and the cell-adhesive glycoproteins laminin and fibronectin. Next, novel 2-D coatings were generated using the DNP or commercially-sourced bovine collagen type I (COL) as a non-tissue-specific control. In addition, cryo-milled DNP or COL particles were incorporated within methacrylated chondroitin sulphate (MCS) hydrogels as a 3-D cell culture platform for exploring the effects of ECM particle composition. Culture studies showed that the 2-D coatings derived from the DNP could support cell attachment and growth, but did not maintain or rescue the phenotype of primary bovine NP cells, which de-differentiated when serially passaged in monolayer culture. Similarly, while bovine NP cells remained highly viable following encapsulation and 14 days of culture within the hydrogel composites, the incorporation of DNP particles within the MCS hydrogels was insufficient to maintain or rescue changes in NP phenotype associated with extended in vitro culture based on gene expression patterns. Overall, DNP produced with our new decellularization protocol was successfully applied to generate both 2-D and 3-D bioscaffolds; however, further studies are required to assess if these platforms can be combined with additional components of the endogenous NP microenvironment to stimulate regeneration or lineage-specific cell differentiation.

Tensile strain and altered synovial tissue metabolism in human knee osteoarthritis.

Synovium is critical for maintaining joint homeostasis and may contribute to mechanobiological responses during joint movement. We investigated mechanobiological responses of whole synovium from patients with late-stage knee osteoarthritis (OA). Synovium samples were collected during total knee arthroplasty and assigned to histopathology or cyclic 10% tensile strain loading, including (1) static (control); (2) low-frequency (0.3 Hz); and iii) high-frequency (1.0 Hz) for 30-min. After 6-h incubation, tissues were bisected for RNA isolation and immunostaining (3-nitrotyrosine; 3-NT). RNA sequencing was analyzed for differentially expressed genes and pathway enrichment. Cytokines and lactate were measured in conditioned media. Compared to controls, low-frequency strain induced enrichment of pathways related to interferon response, Fc-receptor signaling, and cell metabolism. High-frequency strain induced enrichment of pathways related to NOD-like receptor signaling, high metabolic demand, and redox signaling/stress. Metabolic and redox cell stress was confirmed by increased release of lactate into conditioned media and increased 3-NT formation in the synovial lining. Late-stage OA synovial tissue responses to tensile strain include frequency-dependent increases in inflammatory signaling, metabolism, and redox biology. Based on these findings, we speculate that some synovial mechanobiological responses to strain may be beneficial, but OA likely disturbs synovial homeostasis leading to aberrant responses to mechanical stimuli, which requires further validation.

Development of a standardized histopathology scoring system using machine learning algorithms for intervertebral disc degeneration in the mouse model-An ORS spine section initiative.

Mice have been increasingly used as preclinical model to elucidate mechanisms and test therapeutics for treating intervertebral disc degeneration (IDD). Several intervertebral disc (IVD) histological scoring systems have been proposed, but none exists that reliably quantitate mouse disc pathologies. Here, we report a new robust quantitative mouse IVD histopathological scoring system developed by building consensus from the spine community analyses of previous scoring systems and features noted on different mouse models of IDD. The new scoring system analyzes 14 key histopathological features from nucleus pulposus (NP), annulus fibrosus (AF), endplate (EP), and AF/NP/EP interface regions. Each feature is categorized and scored; hence, the weight for quantifying the disc histopathology is equally distributed and not driven by only a few features. We tested the new histopathological scoring criteria using images of lumbar and coccygeal discs from different IDD models of both sexes, including genetic, needle-punctured, static compressive models, and natural aging mice spanning neonatal to old age stages. Moreover, disc sections from common histological preparation techniques and stains including H&E, SafraninO/Fast green, and FAST were analyzed to enable better cross-study comparisons. Fleiss's multi-rater agreement test shows significant agreement by both experienced and novice multiple raters for all 14 features on several mouse models and sections prepared using various histological techniques. The sensitivity and specificity of the new scoring system was validated using artificial intelligence and supervised and unsupervised machine learning algorithms, including artificial neural networks, k-means clustering, and principal component analysis. Finally, we applied the new scoring system on established disc degeneration models and demonstrated high sensitivity and specificity of histopathological scoring changes. Overall, the new histopathological scoring system offers the ability to quantify histological changes in mouse models of disc degeneration and regeneration with high sensitivity and specificity.

A perspective on the ORS Spine Section initiative to develop a multi-species JOR Spine histopathology series.

This perspective summarizes the genesis, development, and potential future directions of the multispecies JOR Spine histopathology series.

Ectopic mineralisation of the mandibular symphysis in ENT1 knockout mice: A model of dystrophic calcification.

Equilibrative nucleoside transporter 1 (ENT1) transfers nucleosides, such as adenosine, across plasma membranes. We reported previously that mice lacking ENT1 (ENT1 -/- ) exhibit progressive ectopic calcification of spinal tissues-a phenotype resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Our objective was to investigate potential calcification of orofacial tissues in ENT1 -/- mice. Heads of wild-type mice and ENT1 -/- mice from 3 to 17 months were evaluated using microcomputed tomography (µCT). Some heads were decalcified and processed for histological assessment. Other heads were examined using energy dispersive X-ray spectroscopy and micro X-ray diffraction. Using µCT, ENT1 -/- mice showed extensive radiopaque lesions within the mandibular symphysis, the severity of which increased with advancing age. Histologically, at 6 months these ectopic radiopacities were found to correspond to acellular, amorphous, eosinophilic material, with no evidence of inflammatory cells. Because lesions were localised to the symphysis, we identified early pathological changes at 3 months and observed that lesions initiated specifically within the fibrocartilage pad. Energy-dispersive X-ray spectroscopy of ectopic lesions revealed large amounts of calcium and phosphorous in a molar ratio of ~1.59, and X-ray diffraction profiles matched that of calcium-deficient hydroxyapatite. This is the first characterisation of ectopic calcifications within the mandibular symphysis of ENT1 -/- mice, indicating a role for ENT1 and adenosine metabolism in regulating calcification of fibrocartilaginous tissues. Moreover, these murine lesions resemble areas of dystrophic calcification in the spinal tissues of humans with DISH. Importantly, ectopic calcifications develop in a reproducible temporal pattern within a well-defined anatomical region and, thus, provide a model for determining the cellular and molecular pathways underlying ectopic calcification in DISH and related disorders.

Diet-induced obesity leads to behavioral indicators of pain preceding structural joint damage in wild-type mice.

INTRODUCTION: Obesity is one of the largest modifiable risk factors for the development of musculoskeletal diseases, including intervertebral disc (IVD) degeneration and back pain. Despite the clinical association, no studies have directly assessed whether diet-induced obesity accelerates IVD degeneration, back pain, or investigated the biological mediators underlying this association. In this study, we examine the effects of chronic consumption of a high-fat or high-fat/high-sugar (western) diet on the IVD, knee joint, and pain-associated outcomes. METHODS: Male C57BL/6N mice were randomized into one of three diet groups (chow control; high-fat; high-fat, high-sugar western diet) at 10 weeks of age and remained on the diet for 12, 24, or 40 weeks. At endpoint, animals were assessed for behavioral indicators of pain, joint tissues were collected for histological and molecular analysis, serum was collected to assess for markers of systemic inflammation, and IBA-1, GFAP, and CGRP were measured in spinal cords by immunohistochemistry. RESULTS: Animals fed obesogenic (high-fat or western) diets showed behavioral indicators of pain beginning at 12 weeks and persisting up to 40 weeks of diet consumption. Histological indicators of moderate joint degeneration were detected in the IVD and knee following 40 weeks on the experimental diets. Mice fed the obesogenic diets showed synovitis, increased intradiscal expression of inflammatory cytokines and circulating levels of MCP-1 compared to control. Linear regression modeling demonstrated that age and diet were both significant predictors of most pain-related behavioral outcomes, but not histopathological joint degeneration. Synovitis was associated with alterations in spontaneous activity. CONCLUSION: Diet-induced obesity accelerates IVD degeneration and knee OA in mice; however, pain-related behaviors precede and are independent of histopathological structural damage. These findings contribute to understanding the source of obesity-related back pain and the contribution of structural IVD degeneration.

Innervation of the Human Intervertebral Disc: A Scoping Review.

OBJECTIVE: Back pain is an elusive symptom complicated by a variety of possible causes, precipitating and maintaining factors, and consequences. Notably, the underlying pathology remains unknown in a significant number of cases. Changes to the intervertebral disc (IVD) have been associated with back pain, leading many to postulate that the IVD may be a direct source of pain, typically referred to as discogenic back pain. Yet despite decades of research into the neuroanatomy of the IVD, there is a lack of consensus in the literature as to the distribution and function of neural elements within the tissue. The current scoping review provides a comprehensive systematic overview of studies that document the topography, morphology, and immunoreactivity of neural elements within the IVD in humans. METHOD: Articles were retrieved from six separate databases in a three-step systematic search and were independently evaluated by two reviewers. RESULTS: Three categories of neural elements were described within the IVD: perivascular nerves, sensory nerves independent of blood vessels, and mechanoreceptors. Nerves were consistently localized within the outer layers of the annulus fibrosus. Neural ingrowth into the inner annulus fibrosus and nucleus pulposus was found to occur only in degenerative and disease states. CONCLUSION: While the pattern of innervation within the IVD is clear, the specific topographic arrangement and function of neural elements in the context of back pain remains unclear.

The Role of Panx3 in Age-Associated and Injury-Induced Intervertebral Disc Degeneration.

Pannexin 3 (Panx3) is a mechanosensitive, channel-forming glycoprotein implicated in the progression of post-traumatic osteoarthritis. Despite evidence for Panx3 expression in the intervertebral disc (IVD), its function in this cartilaginous joint structure remained unknown. Using Panx3 knockout mice, this study investigated the role of Panx3 in age-associated IVD degeneration and degeneration induced by annulus fibrosus (AF) needle puncture. Loss of Panx3 did not significantly impact the progression of age-associated histopathological IVD degeneration; however, loss of Panx3 was associated with decreased gene expression of Acan, Col1a1, Mmp13 and Runx2 and altered localization of COLX in the IVD at 19 months-of-age. Following IVD injury in the caudal spine, histological analysis of wild-type mice revealed clusters of hypertrophic cells in the AF associated with increased pericellular proteoglycan accumulation, disruptions in lamellar organization and increased lamellar thickness. In Panx3 knockout mice, hypertrophic AF cells were rarely detected and AF structure was largely preserved post-injury. Interestingly, uninjured IVDs adjacent to the site of injury more frequently showed evidence of early nucleus pulposus degeneration in Panx3 knockout mice but remained healthy in wild-type mice. These findings suggest a role for Panx3 in mediating the adaptive cellular responses to altered mechanical stress in the IVD, which may buffer aberrant loads transferred to adjacent motion segments.

Paraspinal Muscle Contractile Function is Impaired in the ENT1-deficient Mouse Model of Progressive Spine Pathology.

STUDY DESIGN: Basic science study of the relationship between spine pathology and the contractile ability of the surrounding muscles. OBJECTIVE: The aim of this study was to investigate single muscle fiber contractile function in a model of progressive spine mineralization (ENT1-/- mice). SUMMARY OF BACKGROUND DATA: Altered muscle structure and function have been associated with various spine pathologies; however, studies to date have provided limited insight into the fundamental ability of spine muscles to actively contract and generate force, and how this may change in response to spine pathology. METHODS: Experiments were performed on two groups (ENT1-/- [KO] and ENT1+/+ [WT]) of mice at 8 months of age (n = 12 mice/group). Single muscle fibers were isolated from lumbar multifidus and erector spinae, as well as tibialis anterior (a non-spine-related control) and tested to determine their active contractile characteristics. RESULTS: The multifidus demonstrated decreases in specific force (type IIax fibers: 36% decrease; type IIb fibers: 29% decrease), active modulus (type IIax: 35% decrease; type IIb: 30% decrease), and unloaded shortening velocity (Vo) (type IIax: 31% decrease) in the ENT1-/- group when compared to WT controls. The erector spinae specific force was reduced in the ENT1-/- mice when compared to WT (type IIax: 29% decrease), but active modulus and Vo were unchanged. There were no differences in any of the active contractile properties of the lower limb TA muscle, validating that impairments observed in the spine muscles were specific to the underlying spine pathology and not the global loss of ENT1. CONCLUSION: These results provide the first direct evidence of cellular level impairments in the active contractile force generating properties of spine muscles in response to chronic spine pathology.Level of Evidence: N/A.

Protocol for parallel proteomic and metabolomic analysis of mouse intervertebral disc tissues.

The comprehensiveness of data collected by "omics" modalities has demonstrated the ability to drastically transform our understanding of the molecular mechanisms of chronic, complex diseases such as musculoskeletal pathologies, how biomarkers are identified, and how therapeutic targets are developed. Standardization of protocols will enable comparisons between findings reported by multiple research groups and move the application of these technologies forward. Herein, we describe a protocol for parallel proteomic and metabolomic analysis of mouse intervertebral disc (IVD) tissues, building from the combined expertise of our collaborative team. This protocol covers dissection of murine IVD tissues, sample isolation, and data analysis for both proteomics and metabolomics applications. The protocol presented below was optimized to maximize the utility of a mouse model for "omics" applications, accounting for the challenges associated with the small starting quantity of sample due to small tissue size as well as the extracellular matrix-rich nature of the tissue.

Dystrophic calcification and heterotopic ossification in fibrocartilaginous tissues of the spine in diffuse idiopathic skeletal hyperostosis (DISH).

Diffuse idiopathic skeletal hyperostosis (DISH) is a prevalent noninflammatory spondyloarthropathy characterized by ectopic mineral formation along the anterolateral aspect of the vertebral column, yet little is known about its underlying pathogenesis. Our objective was to evaluate the histopathological features and composition of ectopic mineral within spinal tissues affected by DISH in humans. Thoracic spine segments from six embalmed cadaveric donors (one female and five males; median age 82 years) meeting the radiographic diagnostic criteria for DISH were evaluated using radiological, histological, and physical analyses. Overall, the histological features of ectopic mineralization at individual motion segments were heterogeneous, including regions of heterotopic ossification and dystrophic calcification. Heterotopic ossifications were characterized by woven and lamellar bone, multifocal areas of metaplastic cartilage, and bony bridges along the anterior aspect of the intervertebral disc space. Dystrophic calcifications were characterized by an amorphous appearance, a high content of calcium and phosphorus, an X-ray diffraction pattern matching that of hydroxyapatite, and radiodensities exceeding that of cortical bone. Dystrophic calcifications were found within the anterior longitudinal ligament and annulus fibrosus in motion segments both meeting and not meeting the radiographic criteria for DISH. In summary, our findings indicate that in DISH, ectopic mineral forms along the anterior aspect of the spine by both heterotopic ossification and dystrophic calcification of fibrocartilaginous tissues. Although both types of ectopic mineralization are captured by current radiographic criteria for DISH, dystrophic calcification may reflect a distinct disease process or an early stage in the pathogenesis of DISH.

Transcriptional profiling of the murine intervertebral disc and age-associated changes in the nucleus pulposus.

Purpose/Aim: The intervertebral disc (IVD) is composed of cell types whose subtle phenotypic differences allow for the formation of distinct tissues. The role of the nucleus pulposus (NP) in the initiation and progression of IVD degeneration is well established; however, the genes and pathways associated with NP degeneration are poorly characterized.Materials and Methods: Using a genetic strategy for IVD lineage-specific fluorescent reporter expression to isolate cells, gene expression and bioinformatic analysis was conducted on the murine NP at 2.5, 6, and 21 months-of-age and the annulus fibrosus (AF) at 2.5 and 6 months-of-age. A subset of differentially regulated genes was validated by qRT-PCR.Results: Transcriptome analysis identified distinct profiles of NP and AF gene expression that were remarkably consistent at 2.5 and 6 months-of-age. Prg4, Cilp, Ibsp and Comp were increased >50-fold in the AF relative to NP. The most highly enriched NP genes included Dsc3 and Cdh6, members of the cadherin superfamily, and microRNAs mir218-1 and mir490. Changes in the NP between 2.5 and 6 months-of-age were associated with up-regulation of molecular functions linked to laminin and Bmp receptor binding (including up-regulation of Bmp5 & 7), with the most up-regulated genes being Mir703, Shh, and Sfrp5. NP degeneration was associated with molecular functions linked to alpha-actinin binding (including up-regulation of Ttn & Myot) and cytoskeletal protein binding, with the overall most up-regulated genes being Rnu3a, Snora2b and Mir669h.Conclusions: This study provided insight into the phenotypes of NP and AF cells, and identified candidate pathways that may regulate degeneration.