Effects of Dynamic Compression on Cartilage Endplate Cells in Agarose

Crump, K.B.; Alminawi, A.; Bermudez-Lekerika, Paola; Croft, A. S.; Geris, L.; Noailly, J.; Gantenbein, B. (2022). Effects of Dynamic Compression on Cartilage Endplate Cells in Agarose. In: eCM20: Cartilage and Disc Repair and Regeneration. Davos, Switzerland. 15-18 June.

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INTRODUCTION: Intervertebral disc (IVD) degeneration is one of the main causes of low back pain. However, the initiating and risk factors of IVD degeneration are poorly understood1 . The cartilage endplate (CEP) covers the top and bottom of the IVD and acts as a semipermeable barrier between the discs and the vertebrae2 . However, with aging and degeneration the CEP experiences structural changes with altered permeability3 . Although most research focuses on the nucleus pulposus and annulus fibrosis, early CEP degeneration is believed to be a cause of nucleus dehydration and play a key role in IVD degeneration4 .

METHODS: Bovine tail CEP cells were expanded until passage three. Afterwards, a 1:1 mixture of CEP cells (2 x 10 7 cells/ml) and 4% agarose was pipetted into silicon molds to create constructs of 6 mm in diameter and 3 mm in thickness and then cultured for 2 days for phenotype recovery. Cell-agarose constructs were placed in custom-made chambers, stimulated with 10 ng/ml TGF-β1 throughout the entirety of the experiment and dynamically compressed at 7% strain for 1 hour every day for up to 14 days. Those that were not dynamically loaded were considered to be statically loaded as they had the constant weight of a lid (~18.8 g) imposed. Constructs were collected on Days 0, 7, and 14 for downstream analysis of cell viability and gene expression.

RESULTS: After seven and 14 days of culture, the cell-agarose constructs in all conditions demonstrated increased expression of the anabolic genes aggrecan (ACAN) and collagen II (COL II), alongside decreased expression of catabolic gene matrix metalloproteinase 3 (MMP3) (Fig. 1a-c). Cell viability was unchanged after seven days but decreased after 14 days for all conditions, except for the dynamically loaded chondrogenic constructs which kept increasing after 14 days (Fig. 1d). Cell viability was generally higher at the edge of the construct in comparison to the centre.

Fig. 1: Relative gene expression of (a) ACAN, (b) COL II, (c) MMP13 in CEP cells, and (d) cell viability at the edge of the cell-agarose constructs. Shown are the means, N=2.

DISCUSSION & CONCLUSIONS: Although the data presented is preliminary and additional biological replicates are needed to make any significant conclusions, increased expression of anabolic genes ACAN and COL II convey that the agarose constructs provided an anabolic environment for CEP cells under static and dynamic compression. However, it appeared that the chondrogenic media and type of compression did not affect gene expression. Future work will quantify glycosaminoglycan content and then assess its distribution using alcian blue and histology. Likewise, possible boundary effects shall be quantified.

ACKNOWLEDGEMENTS: This project received funds from the European Commission (H2020-MSCA-ITN-ETN-2020-955735).

REFERENCES: 1 L. Baumgartner et al (2021) Int J Mol Sci 22(2):703. 2 Z. Sun et al (2020) Int J Med Sci 17(5):685-692. 3 S. Roberts et al (1989) Spine 14(2):166-174. 4 C. Ruiz Wills et al (2018) Front Phys 9:1210.

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