Compression Induced Stress Response of Nucleus Pulposus Cells in 3D Collagen Gel (2016)

Introduction The cause of degenerative disc disease is multi-factorial and is often associated with mechanical loading. However, mechanism of the cells' response to compressive loading is not fully known. It was demonstrated that in mechanical force can induce chondrocytes to upregulate expression of heat shock proteins (HSPs) as a form of cellular stress response. 1 Similarly, stretching annulus fibrosus cells was found to upregulate endoplasmic reticulum stress markers. 2 However, it is unclear that whether cells in the intervertebral disc (IVD) would experience compressive force as cellular stress in 3D environment. The objective of this study is to investigate the stress response of nucleus pulposus cells (NPCs) during compression in 3D culture environment. Materials and Methods To simulate the condition in the IVD, NPCs were isolated from bovine caudal disc and encapsulated in 3D collagen gel as described previously. 3 The culture system was shown to be able to maintain NPCs' phenotype. 4 The cell-encapsulated gels were cultured for 4 days and then compressed with different types of loading (static and dynamic loading), different strains (10% - 70%) and different durations (2h - 8h). Samples were retrieved to study the expressions of stress response genes in two groups: Heat Shock Response (HSP70, HSP27, HSP90 and HSF1) and Unfolded Protein Response (GRP78, GRP94, ATF4 and CHOP). Results Encapsulation in 3D collagen gel was found to upregulate HSP70 in NPCs. Compression further induced stress response of NPCs which depended on multiple factors. The upregulation of stress response genes was generally insignificant with increasing loading strain in short duration. However the upregulations increased with longer duration where increasing loading duration has a larger effect in the upregulation of HSP70 and HSP27. When high static strain was applied, the expressions of Heat Shock Response genes were continued to be upregulated with time after the load was removed while Unfolded Protein Response genes were downregulated with incubation duration. The changes in expression with incubation duration show that cellular stress response may play a role in NPC survival and protein homeostasis post-loading. Conclusion The NPCs demonstrated cellular stress response during compression in collagen gel. The expression of stress response genes were mainly affected by loading duration and incubation duration after loading. This study helps us to understand how the NPCs cope with mechanical stress. Acknowledgments This project was funded by TBRS (T12–708/12-N). References Kaarniranta K, Elo M, Sironen R, et al. Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation. Proc Natl Acad Sci U S A 1998;95(5):2319–2324 Zhang YH, Zhao CQ, Jiang LS, Dai LY. Cyclic stretch-induced apoptosis in rat annulus fibrosus cells is mediated in part by endoplasmic reticulum stress through nitric oxide production. Eur Spine J 2011;20(8):1233–1243 Chan BP, Hui TY, Yeung CW, Li J, Mo I, Chan GC. Self-assembled collagen-human mesenchymal stem cell microspheres for regenerative medicine. Biomaterials 2007;28(31):4652–4666 Yuan M, Leong KW, Chan BP. Three-dimensional culture of rabbit nucleus pulposus cells in collagen microspheres. Spine J 2011;11(10):947–960