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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., (© 2024 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

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;Trpv4 fl/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., (Copyright © 2024 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© 2024. The Author(s).)

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., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

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., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

Intervertebral disc degeneration (IDD) and osteoarthritis (OA) affecting the facet joint of the spine are biomechanically interdependent, typically occur in tandem, and have considerable epidemiological and pathophysiological overlap. Historically, the distinctions between these degenerative diseases have been emphasized. Therefore, research in the two fields often occurs independently without adequate consideration of the co-dependence of the two sites, which reside within the same functional spinal unit. Emerging evidence from animal models of spine degeneration highlight the interdependence of IDD and facet joint OA, warranting a review of the parallels between these two degenerative phenomena for the benefit of both clinicians and research scientists. This Review discusses the pathophysiological aspects of IDD and OA, with an emphasis on tissue, cellular and molecular pathways of degeneration. Although the intervertebral disc and synovial facet joint are biologically distinct structures that are amenable to reductive scientific consideration, substantial overlap exists between the molecular pathways and processes of degeneration (including cartilage destruction, extracellular matrix degeneration and osteophyte formation) that occur at these sites. Thus, researchers, clinicians, advocates and policy-makers should consider viewing the burden and management of spinal degeneration holistically as part of the OA disease continuum., (© 2023. Springer Nature Limited.)

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., (© 2022. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Herrera Quijano, Sharma, Morissette Martin, Séguin and Flynn.)