Loading induced stress response in the intervertebral disc

Abstract

Mechanical loading is one of the risk factors that lead to disc degeneration disease (DDD). A lot of pathological changes including stress protein expression have been identified in DDD but causal relationship between stress response and mechanical loading has not been established. There is no investigation in whether the IVD cells perceive mechanical loading as stress and respond by expression of stress response proteins such as heat shock proteins (HSP) and endoplasmic reticulum (ER) stress markers. This study aims to study the stress response in the IVD towards compressive loading. Study was divided into two parts: organ culture and in vitro culture. Bovine caudal tail organ culture was used to study the effect of static loading and dynamic loading in a physiological environment. Cell activity, gene expression and immunofluorescence staining were used to analyze the cell response in terms of heat shock response and matrix remodeling. In vitro 3D culture was conducted to further study the loading induced stress response by minimizing individual and anatomical variation in organ culture. Effect of different loading factors (loading strain, loading type, loading duration, incubation duration post- loading) was studied to determine whether these factors will induce stress response in nucleus pulposus cells (NPCs). Cell viability, gene expression and immunofluorescence staining were performed to evaluate two cellular stress responses: heat shock response (HSR: HSP70, HSF1, HSP27 & HSP90) and unfolded protein response (UPR: GRP78, GRP94, ATF4 & CHOP). Upregulation of HSP70 was observed in organ culture during physiological loading, suggesting that stress response was induced by loading without significantly changing cell activity and upregulating matrix remodeling. In the in vitro culture model, stress response of NPCs induced by loading was shown to depend on multiple factors. Different expression patterns were observed due to loading type throughout the experiments where static loading in general induced higher stress response than dynamic loading. Increasing loading strain in short duration did not increase stress response genes significantly while increasing loading duration upregulated expressions of HSP70 and HSP27 to about two-fold. When the loading duration is prolonged and when a “resting period” or post-loading incubation is allowed, a loading strain dose-dependent stress response was demonstrated. High association between expressions and incubation duration was observed after load removal. The upregulation in HSR genes and downregulation in UPR genes with incubation duration after high static strain indicates that the stress response genes play a role in cell survival and protein synthesis. This study on loading induced stress response in IVD cells establishes direct causal relationship between mechanical loading and various types of stress responses and provides useful information for understanding how IVD cells cope with mechanical stress.