An Inflammatory Nucleus Pulposus Tissue Culture Model to Test Molecular Regenerative Therapies: Validation with Epigallocatechin 3-Gallate

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.

An intervertebral disc whole organ culture system to investigate proinflammatory and degenerative disc disease condition

“…These results indicate that the current model may mimic a “degenerative”, while not “degenerated” IVD condition. To simulate a degenerated IVD with loss of ECM content, longer term culture or enzyme injection (Chan, Burki, Bonel, Benneker, & Gantenbein‐Ritter, ; Jim, Steffen, Moir, Roughley, & Haglund, ; Krupkova et al, ) would be needed to deplete the ECM components.…”

“…Several proinflammatory degenerative culture systems have been developed using explant/organ culture (Walter, Likhitpanichkul, et al, ; Krupkova et al, ; Ponnappan et al, ; Purmessur et al, ; Teixeira et al, ). Though, many studies lack the 3D micro‐environment of the IVD and/or cyclic dynamic loading condition, and therefore might fail to represent relevant biological and biomechanical alterations.…”

Polydatin suppresses nucleus pulposus cell senescence, promotes matrix homeostasis and attenuates intervertebral disc degeneration in rats

“…The administration of TNF‐α aggravates the NPCs senescence by promoting levels of senescence markers p53 and p16 . TNF‐α also disturbs the balance between synthesis and degradation of ECM by up‐regulating MMP‐3, MMP‐13 and ADAMTS‐4, as well as down‐regulating aggrecan and collagen II . Moreover, it is widely recognized that TNF‐α induces mitochondrial dysfunction by increasing ROS production, which may further aggravate senescence and ECM catabolism in NPCs .…”

“…6 TNF-α also disturbs the balance between synthesis and degradation of ECM by up-regulating MMP-3, MMP-13 and ADAMTS-4, as well as down-regulating aggrecan and collagen II. 7 Moreover, it is widely recognized that TNF-α induces mitochondrial dysfunction by increasing ROS production, [8][9][10] which may further aggravate senescence and ECM catabolism in NPCs. 11,12 From these studies, it is obvious that maintaining mitochondrial homeostasis would help to reverse senescence and ECM degradation in NPCs against IVDD.…”

The potential of CRISPR/Cas9 genome editing for the study and treatment of intervertebral disc pathologies

“…It has been demonstrated that in vitro organ cultures can be more suitable than cells to study IVD biology and therapeutic testing as they retain biological and biomechanical properties similar to the IVD in vivo . However, hallmarks of IVD degeneration are usually not present in organ cultures and have to be induced by nonphysiological insults like injections of proteases or high concentrations of proinflammatory cytokines . Generating organ cultures with multiple edited genes (aggrecan, collagen, MMPs, ADAMTS, ILs) might simulate IVD pathologies more naturally.…”