Abstract
Organ cultures are practical tools to investigate regenerative strategies for the intervertebral disc. However, most existing organ culture systems induce severe tissue degradation with only limited representation of the in vivo processes. The objective of this study was to develop a space- and cost-efficient tissue culture model, which represents degenerative processes of the nucleus pulposus (NP). Intact bovine NPs were cultured in a previously developed system using Dyneema jackets. Degenerative changes in the NP tissue were induced either by the direct injection of chondroitinase ABC (1–20 U/mL) or by the diffusion of interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) (both 100 ng/mL) from the culture media. Extracellular matrix composition (collagens, proteoglycans, water, and DNA) and the expression of inflammatory and catabolic genes were analyzed. The anti-inflammatory and anti-catabolic compound epigallocatechin 3-gallate (EGCG, 10 µM) was employed to assess the relevance of the degenerative NP model. Although a single injection of chondroitinase ABC reduced the proteoglycan content in the NPs, it did not activate cellular responses. On the other hand, IL-1β and TNF-α significantly increased the mRNA expression of inflammatory mediators IL-6, IL-8, inducible nitric oxide synthase (iNOS), prostaglandin-endoperoxide synthase 2 (PTGS2) and matrix metalloproteinases (MMP1, MMP3, and MMP13). The cytokine-induced gene expression in the NPs was ameliorated with EGCG. This study provides a proof of concept that inflammatory NP cultures, with appropriate containment, can be useful for the discovery and evaluation of molecular therapeutic strategies against early degenerative disc disease.
Highlights
The intervertebral disc (IVD) consists of three structurally and functionally different tissues: the outer annulus fibrosus (AF), the inner nucleus pulposus (NP), and two cartilaginous endplates that connect the disc with the adjacent vertebrae [1,2]
The inflammatory cytokines interleukin-1 beta (IL-1β) [5,6] and tumor necrosis factor alpha (TNF-α) [7] are key molecules involved in IVD degeneration
It is challenging to develop a cost-effective organ culture model that contains all main hallmarks of degenerative disc disease and is at the same time biologically relevant
Summary
The intervertebral disc (IVD) consists of three structurally and functionally different tissues: the outer annulus fibrosus (AF), the inner nucleus pulposus (NP), and two cartilaginous endplates that connect the disc with the adjacent vertebrae [1,2]. IVD degeneration is caused by a combination of many events throughout a lifetime, including non-physiological loading, endplate calcification, immune reactions, cell senescence, and cell death [1,3,4]. These events reduce the repair capacity of the IVD, induce inflammation and cause a catabolic shift in the NP tissue. These accelerated pathological processes with structural failure and the development of lower back pain, e.g., due to nerve compression and/or chemical irritation, are together termed degenerative disc disease, a distinct entity to the process of physiological ageing [16,18,19,20]
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