Indeed our observation that hydraulic permeability and cell size are correlated in untreated cells supports the notion that cell swelling increases hydraulic permeability, possibly by unfolding the cell membrane. In the presence of inflammatory stimulation, we theorize that inflammatory mediators act as triggers for disruption of the actin cytoskeleton, resulting in loss of cellular pre-stress and increased cell size. Increases in cell size due to inflammation may subsequently induce some unfolding of the cell membrane, resulting in increased hydraulic permeability without significant increases in Aqp-1 required. The cytoskeletal disruption findings and the loss of linear correlation between Lp and radius in inflammatory stimulated cells at both time points further confirm that these changes are irreversible in the current model system. Further studies are needed to determine if cell cytoskeletal changes are causative of changes in osmotic biophysical properties. The potential role of the cytoskeleton in the degenerative cascade associated with disc matrix catabolism, and the relationship between Aqp-1 and F-actin structure remain to be elucidated. A potential limitation of the current study is that the use of 2D culture may alter the NP cell phenotype, and may differentially regulate the inflammatory response. However, NPs are known to respond to cytokines both in vivo and in vitro, and our findings on biophysical responses are assessed on cells in 3D-like rounded morphology. Another limitation of the current study is that the NP cells were examined in an isolated, free-swelling environment. In vivo, these cells are surrounded by pericellular and extracellular matrices that confine the cells and contribute to cell volume regulation. Furthermore, these cells actively regulate their volume in vivo by increasing or decreasing the intracellular concentration of osmolytes. It has also been suggested that this process is dependent on the F-actin cytoskeleton, which we found to be disrupted by the inflammatory stimulation in the current study. The downstream consequences of altered cell mechano-sensitivity, including cell viability, metabolism, and ECM turnover, remain to be elucidated. Future studies will characterize the biophysical responses of NP cells to inflammatory stimulation in a 3D microenvironment that accounts for cell-matrix interactions. In summary, we found that the inflammatory stimuli LPS and TNF-a induced significant changes in the volume-response of NP cells to osmotic loading, including an increase in cell hydraulic permeability and cell size. These changes are associated with alterations in cytoskeletal structure in inflammatory-treated cells. Treated NP cells were unable to recover their biophysical properties to that of untreated cells after removal of inflammatory stimulant and return to basal media for 1 week.