Given the importance of the cartilage endplate (CEP) in low back pain (LBP), there is a need to characterize the human CEP at the molecular, cell, and tissue levels to inform treatment strategies that target it. The goal of this study was to characterize the structure, matrix composition, and cell phenotype of the human CEP compared with adjacent tissues within the intervertebral joint: the nucleus pulposus (NP), annulus fibrosus (AF), and articular cartilage (AC). Isolated CEP, NP, AF, and AC tissues and cells were evaluated for cell morphology, matrix composition, collagen structure, glycosaminoglycan content, and gene and protein expression. The CEP contained elongated cells that mainly produce a collagen-rich interterritorial matrix and a proteoglycan-rich territorial matrix. The CEP contained significantly fewer glycosaminoglycans than the NP tissue. Significant differences in matrix and cell marker gene expression were observed between CEP and NP or AF, with the greatest differences between CEP and AC. We were able to distinguish NP from CEP cells using collagen-10 (COLX), highlighting COLX as a potential CEP marker.Our findings suggest that at the cell and tissue levels, the CEP demonstrates both similarities and differences when compared with NP, AF, and hyaline AC. This study highlights a unique structure, matrix composition, and cell phenotype for the human CEP and can help to inform regenerative strategies that target the intervertebral disc joint in chronic LBP. K E Y W O R D S cartilage endplate, cell phenotype, characterization, intervertebral disc, low back pain 1 | INTRODUCTION Low back pain (LBP) is a major source of socioeconomic burden worldwide with an annual cost of greater than $100 billion in the United States. 1 It is the second most frequent reason for visits to primary care physicians 1 and opioid prescription. 2 Despite a lifetime prevalence of LBP at 84%, 3 the underlying mechanisms of LBP remain unknown. Nonspecific LBP is highly associated with lumbar intervertebral disc (IVD) degeneration, 3 but treatment strategies are limited and fail to target the underlying cellular mechanisms implicated in this disease.The healthy IVD is avascular and aneural and provides pain-free movement of the spinal column. It is composed of a gelatinous core of proteoglycans, the nucleus pulposus (NP), concentric rings of type-1 collagen, the annulus fibrosus (AF), and presumably hyaline-like cartilage

Mast cells (MCs) are present in the painful degenerate human intervertebral disc (IVD) and are associated with disease pathogenesis. MCs release granules containing enzymatic and inflammatory factors in response to stimulants or allergens. The serine protease, tryptase, is unique to MCs and its activation of the G-protein coupled receptor, Protease Activated Receptor 2 (PAR2), induces inflammation and degradation in osteoarthritic cartilage. Our previously published work has demonstrated increased levels of MC marker tryptase in IVD samples from discogenic back pain patients compared to healthy control IVD samples including expression of chemotactic agents that may facilitate MC migration into the IVD. To further elucidate MCs’ role in the IVD and mechanisms underlying its effects, we investigated whether (1) human IVD cells can promote MC migration, (2) MC tryptase can mediate up-regulation of inflammatory/catabolic process in human IVD cells and tissue, and (3) the potential of PAR2 antagonist to function as a therapeutic drug in in vitro human and ex vivo bovine pilot models of disease. MC migration was quantitatively assessed using conditioned media from primary human IVD cells and MC migration examined through Matrigel. Exposure to soluble IVD factors significantly enhanced MC migration, suggesting IVD cells can recruit MCs. We also demonstrated significant upregulation of MC chemokine SCF and angiogenic factor VEGFA gene expression in human IVD cells in vitro in response to recombinant human tryptase, suggesting tryptase can enhance recruitment of MCs and promotion of angiogenesis into the usually avascular IVD. Furthermore, tryptase can degrade proteoglycans in IVD tissue as demonstrated by significant increases in glycosaminoglycans released into surrounding media. This can create a catabolic microenvironment compromising structural integrity and facilitating vascular migration usually inhibited by the anti-angiogenic IVD matrix. Finally, as a “proof of concept” study, we examined the therapeutic potential of PAR2 antagonist (PAR2A) on human IVD cells and bovine organ culture IVD model. While preliminary data shows promise and points toward structural restoration of the bovine IVD including down-regulation of VEGFA, effects of PAR2 antagonist on human IVD cells differ between gender and donors suggesting that further validation is required with larger cohorts of human specimens.

There is a need to further explore mechanisms of cartilage endplate (CEP) degeneration, due to its role in the onset and progression of intervertebral disc degeneration and low back pain. Therefore, the goal of this study was to evaluate structure, matrix composition, and cell phenotype between the human and bovine or canine, both clinically relevant animal models currently used to study the intervertebral disc, CEP. This information may be used in addition to other relevant studies, to help determine optimal animal models for use in studying the role of the CEP in intervertebral disc degeneration and back pain. Endplate structure, matrix composition, cell morphology, and gene expression were evaluated using a picrosirius red/alcian blue and hematoxylin and eosin stain, a dimethylmethylene blue assay, and quantitative reverse transcription polymerase chain reaction. The bovine and canine CEPs were thinner with more rounded cells and thicker bony endplates. The canine CEP contained significantly more sulfated glycosaminoglycans. The bovine CEP demonstrated higher expression of ACAN, COL1, and COL2 and lower expression of T, FBLN1, and collagen X (COLX) compared to the human CEP. The canine CEP had higher COL2 and lower COL1, KRT19, MKX, FBLN1, COLX expression compared to human. These similarities and differences between human and bovine or canine CEP are important to consider when evaluating which animal model is most optimal to use in future studies, interpreting research findings using these animal models and assessing translatability to the human condition.

Peripheral nerves allow a bidirectional communication between brain and adipose tissues, and many studies have clearly demonstrated that a loss of the adipose nerve supply results in tissue dysfunction and metabolic dysregulation. Neuroimmune cells closely associate with nerves in many tissues, including subcutaneous white adipose tissue (scWAT). However, in scWAT, their functions beyond degrading norepinephrine in an obese state remain largely unexplored. We previously reported that a myeloid-lineage knockout (KO) of brain-derived neurotrophic factor (BDNF) resulted in decreased innervation of scWAT, accompanied by an inability to brown scWAT after cold stimulation, and increased adiposity after a high-fat diet. These data underscored that adipose tissue neuroimmune cells support the peripheral nerve supply to adipose and impact the tissue’s metabolic functions. We also reported that a subset of myeloid-lineage monocyte/macrophages (Ly6c+CCR2+Cx3cr1+) is recruited to scWAT in response to cold, a process known to increase neurite density in adipose and promote metabolically healthy processes. These cold-induced neuroimmune cells (CINCs) also expressed BDNF. Here we performed RNAseq on CINCs from cold-exposed and room temperature-housed mice, which revealed a striking and coordinated differential expression of numerous genes involved in neuronal function, including neurotrophin signaling and axonal guidance, further supporting that CINCs fulfill a nerve-supporting role in adipose. The increased expression of leukocyte transendothelial migration genes in cold-stimulated CINCs also confirms prior evidence that they are recruited to scWAT and are not tissue resident. We now provide whole-depot imaging of scWAT from LysM-BDNF KO mice, revealing a striking reduction of innervation across the depot fitting with their reduced energy expenditure phenotype. By contrast, Cx3cr1-BDNF KO mice (a macrophage subset of LysM+ cells) exhibited increased thermogenesis and energy expenditure, with compensatory increased food intake and no change in adiposity or body weight. While these KO mice also exhibit a significantly reduced innervation of scWAT, especially around the subiliac lymph node, they displayed an increase in small fiber sympathetic neurite branching, which may underlie their increased thermogenesis. We propose a homeostatic role of scWAT myeloid-lineage neuroimmune cells together in nerve maintenance and neuro-adipose regulation of energy expenditure.