Characterization of the human intervertebral disc cartilage endplate at the molecular, cell, and tissue levels

Abstract

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

Characterization of bovine and canine animal model cartilage endplates and comparison to human cartilage endplate structure, matrix composition, and cell phenotype

“…To characterize and compare the CEP cell phenotype further, bovine and canine CEP, NP, AF, and AC cells were evaluated for IVD joint matrix and IVD/AC cell marker gene expression (Figure 5). In addition, these comparisons would also ensure there was no cross‐contamination during dissection and isolation, validating our previous method of CEP isolation for human cells 20,21 . There was significantly higher COL1, COL2, and ACAN, and COL1 and COL2 expression in the bovine AF and AC compared to the CEP, respectively.…”

“…We have previously evaluated expression of T, KRT19, MKX, and FBLN1 in human CEP cells and found human CEP cells express T and KRT19, MKX, or FBLN1 at equal or greater levels than human NP, AF, or AC, respectively. Furthermore, we previously demonstrated significantly higher COLX expression in the human CEP compared to NP on the protein level 20,21 . There was lower expression of T, FBLN1, and COLX in bovine CEP cells and lower expression of KRT19, MKX, FBLN1, and COLX in canine CEP cells compared to human CEP cells.…”

“…NP markers T and KRT19, 26 AF marker MKX, 27 AC marker FBLN1, 28 and potential CEP marker COLX 20,21 were examined in bovine and canine CEP cells compared to human. We have previously evaluated expression of T, KRT19, MKX, and FBLN1 in human CEP cells and found human CEP cells express T and KRT19, MKX, or FBLN1 at equal or greater levels than human NP, AF, or AC, respectively.…”

Investigating the role of culture conditions on hypertrophic differentiation in human cartilage endplate cells

“…In Extreme care taken when separating CEP, AF and NP tissue during dissection, and previously observed differences in marker expression at the gene level between cells using the same isolation technique, suggest isolation of distinct cell populations 36,37 . Nevertheless, crosscontamination with additional cell types, such as inflammatory cells, could still be a minimal concern.…”

“…Addition of an articular chondrocyte control to directly compare CEP and articular chondrocyte cell behavior in vitro and increasing sample size in order to further validate the effect of culture conditions and oxygen tension on sex and age‐related differences in human CEP samples would enhance the rigor of the current study. Extreme care taken when separating CEP, AF, and NP tissue during dissection, and previously observed differences in marker expression at the gene level between cells using the same isolation technique, suggest isolation of distinct cell populations 36,43 . Nevertheless, cross contamination with additional cell types, such as inflammatory cells, could still be a minimal concern.…”