Laagland, Lisanne (Utrecht University)


Low back pain due to intervertebral disc (IVD) degeneration is a major health and socioeconomic problem throughout the world. In the young and healthy IVD, large and vacuolated notochordal cells (NCs) are present1. These cells are, in some species (e.g. humans and dogs), replaced by chondrocyte-like nucleus pulposus cells (NPCs) during maturation and ageing2. In previous studies, porcine NC-derived matrix (NCM), containing matrix and biologic factors secreted by NCs, induced regenerative and anti-inflammatory effects in human, canine, and bovine NPCs in vitro and degenerated canine IVDs in vivo3,4. Since the precise mechanism behind NCM remained elusive, we aimed to determine the mode of action of NCM in the degenerative IVD environment.

Canine and human NPCs were cultured with and without NCM for 6, 24, and 72 hours in monolayers. After 6, 24, and 72 hours, RT-qPCR was performed on inflammatory markers and after 72 hours, targeted proteomics was performed with DigiWest, a proprietary immunoassay technology which transfers Western Blot to a high-throughput bead-based microarray platform5. Lastly, immunohistochemistry was performed on in vivo canine IVDs treated with NCM for 6 months.

RT-qPCR analysis indicated that NCM induced an initial inflammatory response after 6 hours, since IL-6, IL-8 and COX2 mRNA expression was increased in human and canine NPCs. DigiWest analysis showed that NCM mainly induced changes in the Mitogen-activated protein kinase (MAPK) pathway after 72 hours of treatment, i.e. after the initial pro-inflammatory response. The expression of key proteins downstream the MAPK pathway, such as ERK1/2, JNK, and PKC, was mostly inhibited by NCM, whereas expression of proteins that are known to dephosphorylate MAPK key signaling molecules, DUSP1, 5, and 6, was increased in NCM-treated NPCs. Lastly, also expression of KRT19, a healthy NP marker, was induced by 72 hours of NCM treatment. Confirming the DigiWest results, in vivo canine IVDs treated with an intradiscal NCM injection demonstrated increased KRT19 and DUSP5 immunopositivity compared with controls after 6 months of treatment.

Taken together, these results indicate that NCM induces an initial inflammatory response, but thereafter exerts its prolonged anti-inflammatory effects by influencing the MAPK pathway. The latter leads to reduced expression of inflammatory cytokines after prolonged treatment.

1. Bach, F. C. et al. Eur. Cell. Mater. 30, 132–137 (2015).
2. Hunter, C. J., et al. Tissue Eng. 9, 667–677 (2003).
3. Bach, F. C. et al. Oncotarget 9, (2018).
4. de Vries, S., et al. Tissue Eng. A 25, 830–841 (2018).
5. Treindl, F. et al. Nat. Commun. 7, (2016).

The European Union’s Horizon 2020 program (iPSpine; #825925) supported this work."

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