Complex mechanical loading and inflammation in intervertebral disc degeneration

Crump, Katherine B.; de Graaf, Kim; Bermudez-Lekerika, Paola; Le Maitre, Christine; Noailly, Jérôme; Gantenbein, Benjamin (2024). Complex mechanical loading and inflammation in intervertebral disc degeneration (Unpublished). In: 29th Congress of the European Society of Biomechanics. 3 July 2024.

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Introduction

Intervertebral disc (IVD) degeneration is the cause of around half of all low back pain cases in young adults, however the initiating and risk factors are poorly understood, limiting development of personalized therapies. [1] Although the effects of pro-inflammatory cytokines and mechanical loading has been investigated within the IVD, [2] it is unknown how IVD response to complex mechanical loading is affected by the presence of cytokines. Thus, we aimed to investigate the combined effects of dynamic compression and torsion with catabolic cytokine interleukin 1 beta (IL-1β) and inhibitory cytokine interleukin 1 receptor antagonist (IL-1Ra), on bovine IVDs using ex vivo culture, magnetic resonance imaging (MRI), and finite element (FE) modeling.

Methods

Whole bovine IVDs obtained within 3-4 hours portmortem from a local abattoir were isolated and the IVDs cultured in a customized two degrees-of-freedom bioreactor applying diurnal dynamic compression (0.1-

0.5 MPa) and torsion (6°) under normal (HG DMEM), catabolic (10 ng/ml IL-1β) and inhibitory (10 ng/ml IL1Ra) media conditions for one week. Static compression (0.1 MPa) under the same media conditions were used as a control. Before and after culture, the IVDs were imaged using 3T MRI, which was used to create subjectspecific FE models. Downstream analyses included height measurement, qPCR, glycosaminoglycan (GAG) quantification, and cell metabolic activity. For statistical analysis (when n>3), nonparametric distribution was assumed and a Kruskal–Wallis test then Dunn’s multiple comparisons test were performed, and a P <

0.05 considered statistically significant.

Results

Following one week of culture, IVD height decreased in all conditions (Figure 1a & b), however, this disc height decreases was less pronounced within IVDs stimulated with IL-1Ra. Cellular metabolic activity in the nucleus pulposus (NP) decreased in all conditions, but the difference was only significant in IVDs stimulated with IL-1β and complex dynamic loading (Figure 1c). Similarly, the GAG content decreased in the NP of all conditions, but was not significant. Gene expression of anabolic genes, i.e. collagen type II (COL II) decreased (Figure 1e), while expression of catabolic genes, i.e. matrix metalloproteinase 3 (MMP3) increased in the NP tissue for all conditions except in control static IVDs and in IL-1Ra stimulated IVDs, respectively (Figure 1d). Introduction

Intervertebral disc (IVD) degeneration is the cause of around half of all low back pain cases in young adults, however the initiating and risk factors are poorly understood, limiting development of personalized therapies. [1] Although the effects of pro-inflammatory cytokines and mechanical loading has been investigated within the IVD, [2] it is unknown how IVD response to complex mechanical loading is affected by the presence of cytokines. Thus, we aimed to investigate the combined effects of dynamic compression and torsion with catabolic cytokine interleukin 1 beta (IL-1β) and inhibitory cytokine interleukin 1 receptor antagonist (IL-1Ra), on bovine IVDs using ex vivo culture, magnetic resonance imaging (MRI), and finite element (FE) modeling.

Methods

Whole bovine IVDs obtained within 3-4 hours portmortem from a local abattoir were isolated and the IVDs cultured in a customized two degrees-of-freedom bioreactor applying diurnal dynamic compression (0.1-

0.5 MPa) and torsion (6°) under normal (HG DMEM), catabolic (10 ng/ml IL-1β) and inhibitory (10 ng/ml IL1Ra) media conditions for one week. Static compression (0.1 MPa) under the same media conditions were used as a control. Before and after culture, the IVDs were imaged using 3T MRI, which was used to create subjectspecific FE models. Downstream analyses included height measurement, qPCR, glycosaminoglycan (GAG) quantification, and cell metabolic activity. For statistical analysis (when n>3), nonparametric distribution was assumed and a Kruskal–Wallis test then Dunn’s multiple comparisons test were performed, and a P <

0.05 considered statistically significant.

Results

Following one week of culture, IVD height decreased in all conditions (Figure 1a & b), however, this disc height decreases was less pronounced within IVDs stimulated with IL-1Ra. Cellular metabolic activity in the nucleus pulposus (NP) decreased in all conditions, but the difference was only significant in IVDs stimulated with IL-1β and complex dynamic loading (Figure 1c). Similarly, the GAG content decreased in the NP of all conditions, but was not significant. Gene expression of anabolic genes, i.e. collagen type II (COL II) decreased (Figure 1e), while expression of catabolic genes, i.e. matrix metalloproteinase 3 (MMP3) increased in the NP tissue for all conditions except in control static IVDs and in IL-1Ra stimulated IVDs, respectively (Figure 1d). Introduction

Intervertebral disc (IVD) degeneration is the cause of around half of all low back pain cases in young adults, however the initiating and risk factors are poorly understood, limiting development of personalized therapies. [1] Although the effects of pro-inflammatory cytokines and mechanical loading has been investigated within the IVD, [2] it is unknown how IVD response to complex mechanical loading is affected by the presence of cytokines. Thus, we aimed to investigate the combined effects of dynamic compression and torsion with catabolic cytokine interleukin 1 beta (IL-1β) and inhibitory cytokine interleukin 1 receptor antagonist (IL-1Ra), on bovine IVDs using ex vivo culture, magnetic resonance imaging (MRI), and finite element (FE) modeling.

Methods

Whole bovine IVDs obtained within 3-4 hours portmortem from a local abattoir were isolated and the IVDs cultured in a customized two degrees-of-freedom bioreactor applying diurnal dynamic compression (0.1-

0.5 MPa) and torsion (6°) under normal (HG DMEM), catabolic (10 ng/ml IL-1β) and inhibitory (10 ng/ml IL1Ra) media conditions for one week. Static compression (0.1 MPa) under the same media conditions were used as a control. Before and after culture, the IVDs were imaged using 3T MRI, which was used to create subjectspecific FE models. Downstream analyses included height measurement, qPCR, glycosaminoglycan (GAG) quantification, and cell metabolic activity. For statistical analysis (when n>3), nonparametric distribution was assumed and a Kruskal–Wallis test then Dunn’s multiple comparisons test were performed, and a P <0.05 considered statistically significant.

Results

Following one week of culture, IVD height decreased in all conditions (Figure 1a & b), however, this disc height decreases was less pronounced within IVDs stimulated with IL-1Ra. Cellular metabolic activity in the nucleus pulposus (NP) decreased in all conditions, but the difference was only significant in IVDs stimulated with IL-1β and complex dynamic loading (Figure 1c). Similarly, the GAG content decreased in the NP of all conditions, but was not significant. Gene expression of anabolic genes, i.e. collagen type II (COL II) decreased (Figure 1e), while expression of catabolic genes, i.e. matrix metalloproteinase 3 (MMP3) increased in the NP tissue for all conditions except in control static IVDs and in IL-1Ra stimulated IVDs, respectively (Figure 1d).Discussion

We hypothesized that catabolic cytokines, i.e. IL-1β, in the microenvironment of the IVD are sufficient to negatively alter the cellular response to complex loading, leading to further downstream degeneration. However, markers of degeneration were found in all conditions, which could indicate that loading was supraphysiological and catabolic alone. This will be investigated further using subject-specific FE models developed from the MRI images. Additionally, short half-lives of cytokines could have prevented them from diffusing through the IVD effectively, thus limiting the cellular response. This will be further investigated using immunohistochemistry and ELISA. Nevertheless, the results convey that static or complex dynamic loading is sufficient to induce catabolism with or without cytokine stimulation.

References

1. Baumgartner L et al., J. Int J Mol Sci. 2021; 22(2):703.
2. Walter BA et al., PLOS ONE. 2015; 10(3): e0118358.5.

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