Abstract
Introduction
Intervertebral disc (IVD) degeneration (DD) is characterized by increased catabolic activity, ECM breakdown, and elevated levels of pro-inflammatory cytokines, particularly TNF-α and IL-1β. DD also alters hydrostatic pressure and biomechanical loading in the nucleus pulposus (NP), potentially leading to an altered biomechanical microenvironment around the NP cells. In a previous study we have shown that stimulation of isolated NP cells with the inflammatory cytokine TNFα, or activation of innate immune signaling via toll-like-receptor-4 (TLR4) with the ligand LPS, causes significant increases in both cell volume and hydraulic permeability of the cell. The goal of the current study is to examine the effects of anti-inflammatory drugs on mitigation of inflammatory induced biophysical changes of NP cells.
Material and Methods
NP tissue was isolated from bovine lumbar discs. Inflammatory stimulation was performed with either TNFα (10ng/ml) or LPS (0.1 µg/ml) for 24 hours. To inhibit the effects of TNFα, cells were pre-treated with dexamethasone (DEX, 1 µg/mL) for 90 minutes prior to inflammatory stimulation. For TLR4 inhibition groups, cells were pre-treated with TAK-242 (1µM), an intracellular inhibitor of TLR4, for 1 hour prior to addition of LPS. Supernatants were collected and nitrite (NO) concentration was measured. Cell osmotic properties were also measured using a custom Y-shaped microfluidic channel. Cells were equilibrated in a 333 mOsm/L NaCl solution after which a single hyper-osmotic loading followed by a hypo-osmotic loading step was applied with NaCl solutions at 466mOsm/L followed by 333 mOsm/L. During osmotic loading, cells were imaged using DIC and volume response was computed over time for each cell. Volume response was curve fitted to a mixture theory framework to determine biophysical properties for each cell including membrane hydraulic permeability (Lp), and reference intracellular water content (Φir). Data was analyzed with ANOVA and Fisher LSD post-hoc test (p < 0.05 considered significant).
Results
DEX and TAK-242 significantly inhibited TNFα and LPS induced nitrite release, respectively. Both TNFα and LPS stimulation significantly increased cell size at 24 hours post treatment. Hydraulic permeability (Lp) of cells significantly increased for both osmotic steps. Treatment of cells with anti-inflammatory drugs significantly reduced Lp back to baseline levels in both TNFα and LPS groups. No significant changes in Φir were observed due to inflammatory simulation.
Conclusion
The goal of this study was to investigate the effects of anti-inflammatory drugs on the biophysical properties of isolated NP cells. DEX, an anti-inflammatory glucocorticoid, and TAK-242, a small molecule TLR4 inhibitor, were both found to decrease TNFα and LPS induced NO release respectively. TNFα and LPS each increased Lp in NP cells, consistent with our previous findings. Interestingly, treatment of cells with anti-inflammatory drugs inhibited TNFα and LPS induced changes in Lp, where Lp was found to be comparable to baseline control levels. These findings indicate that anti-inflammatory drugs may prevent alterations in cell biomechanical properties and protect the mechanobiological function of NP cells from inflammatory changes. Our findings identify potential targets and therapeutic biologics agents for further consideration in the treatment of disc degeneration.