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Inflammatory stimulation and mechanical loading lead to a weakening of the trans-lamellar bridging network of the anulus fibrosis
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Veröffentlicht: | 6. November 2018 |
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Objectives: Investigate the effects of inflammatory stimulation and mechanical overloading of the trans-lamellar bridging network (TLBN). Anulus fibrosus (AF) disruption can lead to prolapse or herniation of intervertebral discs (IVDs) (Adams et al. Spine 2006). The TLBN is a proteoglycan and elastic fiber-rich structure which separates the lamellae, as well as forming transverse bridges which connect the layers of the AF (Schollum et al. J Anat 2009). Little is known about how mechanobiological factors influence the TLBN and anular wall integrity (Schollum et al. J Anat 2009; Han et al. J Orthop Res 2015; Yu et al. Spine 2015). The current study established a bovine AF organ culture model (AF-OC) which allows defined cyclic strains to be applied to the tissue in vitro. Additionally, IL-1 β media supplementation was used as a concurrent pro-inflammatory stimulus.
Methods: Methods Standardized AF rings were cut and punched from bovine caudal discs. A custom-made electro-mechanical device (Figure 1A [Fig. 1]) was used to exposed the AF-organ cultures (AF-OCs) to high physiological cyclic tensile strain (CTS) for 5 days (9% 1Hz, 3 hours/day). A sub-group was cultured in the presence of IL-1 β (10 ng/ μ L) to simulate a pro-inflammatory environment. PGE2 release and the expression pattern of COX-2, IL-6, and MMP3 in the tissue were assessed by ELISA and immunohistochemistry (IHC) respectively. The mechanical strength of TLBN was evaluated using a peel-test. AF segments were dissected into a 'Y' configuration, and a uniaxial material testing machine was used to measure the force required to separate the AF segment along a lamellar boundary (Gregory et al 2012).
Results: The combination of cyclic strain and IL-1 β (CTS+IL-1 β) resulted in a significant 25-fold increase in PGE2 release compared to controls (p=0,001) (Figure 1B [Fig. 1]). IHC analyses showed that the CTS+IL-1 β group was associated with a higher expression of IL-6 and MMP3 within the TLBN regions compared to adjacent lamellar matrix. Mechanical testing found a significant decrease in the adhesive strength for the CTS+IL1 β group compared to control (p=0,02) (Figure 1C [Fig. 1]).
Conclusion: Our unique organ-culture and mechanical loading approach enables investigation of the AF and TLBN structure-function relationship. Our findings suggest that the combination of cyclic-strain and IL-1 β act synergistically to increase pro-inflammatory and catabolic molecules within the AF, particularly the TLBN matrix, which consequently leads to a weakening of the tissue structure. This new AF-OC model will contribute to a better understanding of the pathomechanism of disc herniation.
Acknowledgements: Supported by the German Spine Foundation.