Effects of compression and TNF on human effects of compression on human cartilaginous endplate cells in 3D agarose culture

Abstract

Intervertebral disc (IVD) degeneration is the main cause of low back cases in young adults [1]. However, the initiating risk factors are poorly understood as it is a highly multifactorial disease. The cartilage endplate (CEP) covers the cranial and caudal surfaces of the IVD and acts to transmit compressive loads and transport water, nutrients, and waste in and out of the IVD. [2] Early CEP degeneration is likely to play a key role in IVD degeneration, but little is known about CEP mechanobiology and its changes in degeneration. [3,4]. Investigating these changes is essential to elucidate how the CEP contributes to IVD pathology. It was hypothesized that CEP cells would behave similarly to articular chondrocytes. Thus, it was predicted that dynamic compression would be su!cient to induce anabolism, while stimulation with pro-inflammatory cytokines would induce catabolism.

METHODS

Human CEP cells were expanded until passage 3 or 4, then seeded at a density of 7.5x10 6 cells/ml into 2% agarose carriers (dimensions: 6 mm ⌀ and 3 mm height) and cultured for 5 days for

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phenotype recovery. Cell-agarose carriers were placed in custom-made chambers, stimulated with 10 ng/ml TNF throughout the entirety of the experiment and dynamically compressed to ~7% strain for one hour at 1.5 Hz daily for up to 14 days. Carriers were collected on Days 0, 7, and 14 for downstream analysis of cell viability, metabolism, gene expression, and glycosaminoglycan (GAG) content. For statistical analysis, nonparametric distribution was assumed and a Kruskal–Wallis test then Dunn’s multiple comparisons test was done, and a p < 0.05 was considered statistically significant.

RESULTS

After 14 days of culture, TNF-stimulated cell-agarose carriers showed a trend (p=0.1) towards decreased expression of anabolic gene aggrecan (ACAN). Specifically, the dynamically loaded TNFstimulated condition showed significantly less ACAN expression (p=0.0436) than the dynamically loaded control condition after 14 days, but not after 7 days (Fig 1a). While there was no change in expression of collagen II (COLII), expression of collagen I (COLI) trended towards lower expression in TNF-stimulated carriers after 7 and 14 days (p=0.0663 and p=0.0549, respectively) (Fig 1b). Catabolic genes matrix metalloproteinase 3 (MMP3) and interleukin 6 (IL-6) showed a trend towards increased expression in TNF-stimulated dynamic carriers when compared to the controls (p=0.2169 and p=0.1240, respectively) (Fig 1c and 1d). However, the GAG/DNA content in the carriers and GAG released in the media stayed consistent throughout the entirety of the experiment for all conditions. The TNF-stimulated carriers also showed a trend towards higher cell metabolic activity.

DISCUSSION

This study demonstrated that TNF was sufficient to induce a catabolic response in human CEP cells through the downregulation of ACAN and the upregulation of MMP3 and IL6. Interestingly, these results suggest TNF has a greater e#ect on ACAN and COL1 than COL2 within the CEP. Further, the response to TNF appeared to be enhanced by dynamic compression. Additionally, significant changes did not happen until after 14 days of culture, thus demonstrating a time dependent response to TNF-stimulation and dynamic compression.