Compression Loading Induced Cellular Stress Response of Intervertebral Disc Cells in Organ Culture

Abstract

Introduction Mechanical stress is often associated to interverterbal disc (IVD) degeneration and the effect of mechanical loading on IVD has been studied and reviewed.1,2 Previously, expression of heat shock proteins, HSP70 and HSP27 has been found in pathological discs.3 However, there is no direct evidence on whether IVD cells respond to the mechanical loading by expression of HSPs. The objective of this study is to investigate the stress response of IVD cells during compressive loading in an organ culture. Materials and Methods Fresh adult bovine caudal discs were cultured with compressive loading applied at physiological range. Effect of loading type (static and dynamic) and repeated loading (2 hours per day for 2 days) were studied. Nucleus pulposus (NP) and annulus fibrosus (AF) of the IVD were retrieved at different time points: right after loading and right after resting. Positive control discs were heat shocked (43°C). Cell activity was assessed and expression of stress response genes (HSP70 and HSF1) and matrix remodeling genes (ACAN, COL2, COL1, ADAMTS4, MMP3 and MMP13) were studied. Results Cell activity was maintained in all groups. Both NP and AF expressed high level of HSP70 in heat shock groups, confirming their expression in response to stress. In NP, expression of HSP70 was up-regulated after static loading and dynamic loading with higher fold change was observed after static loading. During repeated loading, HSP70 appeared to be upregulated right after loading and decreased after resting. Such trend was not observed in AF and HSF1 levels. Expressions of matrix remodeling genes did not change significantly with loading except ADAMTS4 decreased in AF during static loading. Conclusion This study demonstrated that NP cells upregulate expression of HSP70 in response to loading induced stress without changing cell activity and matrix remodeling significantly. Acknowledgments This project was funded by AO Spine (AOSPN) (grant number: SRN_2011_14) and a fellowship exchange award by AO Spine Scientific Research Network (SRN). References Chan SC, Ferguson SJ, Gantenbein-Ritter B. The effects of dynamic loading on the intervertebral disc. Eur Spine J 2011;20(11):1796–1812 Neidlinger-Wilke C, Galbusera F, Pratsinis H, et al. Mechanical loading of the intervertebral disc: from the macroscopic to the cellular level. Eur Spine J 2014;23(Suppl 3):S333–S343 Sharp CA, Roberts S, Evans H, Brown SJ. Disc cell clusters in pathological human intervertebral discs are associated with increased stress protein immunostaining. Eur Spine J 2009;18(11):1587–1594

Mitochondrial quality control in intervertebral disc degeneration

“…It was found that longer loading durations significantly upregulated the Hsp70 level and had little influence on the apoptosis ratio of the NP cells, which indicated a protective effect following mechanical stress 65 . In addition, this research team also used adult bovine caudal discs to construct an organ culture model and found that Hsp70 expression appeared to be upregulated immediately after loading and was decreased upon resting following a repeated loading cycle in NP cells but not in AF cells 66 . In addition, a hypoxic or hypertonic microenvironment showed dramatic effects on mitochondrial function in NP cells 21,33,53 , in which the levels and function of the mitochondrial chaperone Hsp70 presented a corresponding response 67,68 .…”

Comprehensive bioinformatics analyses reveal immune genes responsible for altered immune microenvironment in intervertebral disc degeneration

“…The KEGG enrichment results showed that magenta module genes were mainly involved in “Phospholipase D signaling pathway” and “Osteoclast differentiation”. In most cases, the intracellular activity of phospholipase D remains low, unless the cells were stimulated by cellular stress (Bruntz et al 2014 ; Shin et al 2001 ), and we presumed that phospholipase D and associated signaling pathway might be activated in response to various cellular stress in IDD, such as mechanical stress (Chooi et al 2017 ) and oxidative stress (Hou et al 2014 ). Osteoclast is a crucial player in remodeling, maintaining, and repairing of bone tissue (Jayakumar and Di Silvio 2010 ), and was found activated in IVD with Modic changes (normally occur near the degenerated IVD), which could also be partially attributed to mechanical stress (Rahme and Moussa 2008 ; Torkki et al 2016 ).…”