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

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.

Cartilage endplate degeneration/calcification has been linked to the onset and progression of intervertebral disc degeneration and there is a critical need to understand mechanisms, such as hypertrophic differentiation, of cartilage endplate degeneration/calcification to inform treatment strategies for discogenic back pain.In vitro cell culture conditions capable of inducing hypertrophic differentiation are used to study pathophysiological mechanisms in articular chondrocytes, but culture conditions capable of inducing a hypertrophic cartilage endplate cell phenotype have yet to be explored. The goal of this study was to investigate the role of culture conditions capable of inducing hypertrophic differentiation in articular chondrocytes on hypertrophic differentiation in human cartilage endplate cells.Isolated human cartilage endplate cells were cultured as pellets for 21 days at either 5% O 2 (physiologic for cartilage) or 20.7% O 2 (hyperoxic) and treated with 10% fetal bovine serum or Wnt agonist, two stimuli used to induce hypertrophic differentiation in articular chondrocytes. Cartilage endplate cells did not exhibit a hypertrophic cell morphology in response to fetal bovine serum or Wnt agonist but did display other hallmarks of chondrocyte hypertrophy and degeneration such as hypertrophic gene and protein expression, and a decrease in healthy proteoglycans and an increase in fibrous collagen accumulation. These findings demonstrate that cartilage endplate cells take on a degenerative phenotype in response to hypertrophic stimuli in vitro, but do not undergo classical changes in morphology associated with hypertrophic differentiation regardless of oxygen levels, highlighting potential differences in the response of cartilage endplate cells versus articular chondrocytes to the same stimuli.

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