P8. Responsiveness of patient derived intervertebral disc cells to mechanobiological treatment

Abstract

BACKGROUND CONTEXT Disc degeneration (DD) is associated with elevated levels of pro-inflammatory cytokines, such as TNFα. We have previously shown that pro-inflammatory stimulation of healthy bovine nucleus pulposus (NP) cells leads to significant changes in cellular biomechanics and cell shape, in a mechanism dependent on actomyosin contractility. However, the extent to which the biomechanical properties of intervertebral disc (IVD) cells are disrupted in human disc degeneration has yet to be investigated. PURPOSE The objective of this study is to measure the elastic modulus of single cells isolated from patients with varying degrees of disc degeneration with or without inflammatory stimulation. We also investigate the protective effects of increasing actomyosin contractility in patient cells using the RhoA activator CN03. PATIENT SAMPLE Adult subjects (N=10) undergoing degenerative lumbar surgery were recruited and consented following IRB approval. Discard disc tissue was collected during surgery. OUTCOME MEASURES Single cell elastic modulus. METHODS Adult subjects (N=10) undergoing degenerative lumbar surgery were recruited and consented following IRB approval. Discard disc tissue was collected during surgery. Severity of degeneration was scored on MRI using Pfirrmann grade. Cells were isolated from human IVD tissue without use of enzymes to prevent disruption of cell integrity. Cells were transferred to 1.2% alginate bead to mimic native 3D cell morphology and were randomized into one of four treatment groups for 24 hours: (1) untreated, (2) 10 ng/ml TNFα, (3) 1μg/ml CNO3, (4) 10 ng/ml TNFα + 1μg/ml CNO3. Cells (n=40-50 per donor per treatment group) were tested using an Asylum MFP-3D Bio Infinity Atomic Force Microscope (AFM). Single force indentation curves were obtained for each cell and were fitted to a Hertz model to determine elastic modulus. Statistics: data were analyzed with ANOVA and Fisher LSD post-hoc test. RESULTS Cell elastic modulus significantly decreased with TNFα stimulation in four of 10 patients tested. Additionally, four of 10 patients were classified as responders to increased actomyosin contractility. Six of 10 patients were defined as nonresponders were CN03+TNFα co-treatment showed no significant difference from TNFα alone. Degree of degeneration was not found to correlate with the effect of TNFα on elastic modulus or with responder classification. CONCLUSIONS To our knowledge, this is the first report that evaluates single cell biophysical properties of human IVD cells from patient samples with a focus on determining the effects of inflammatory stimulation or increased actomyosin contractility. Degree of degeneration as measured by Pfirmann Grade did not contribute to the propensity of patients to be categorized as responders. Understanding the underlying factors that predispose a patient to be responsive or nonresponsive to the protective effects of increasing actomyosin contractility will be an important step in evaluating its therapeutic potential. These findings warrant further investigation into other factors that may be influencing responsiveness to CN03 or TNFα. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs. Disc degeneration (DD) is associated with elevated levels of pro-inflammatory cytokines, such as TNFα. We have previously shown that pro-inflammatory stimulation of healthy bovine nucleus pulposus (NP) cells leads to significant changes in cellular biomechanics and cell shape, in a mechanism dependent on actomyosin contractility. However, the extent to which the biomechanical properties of intervertebral disc (IVD) cells are disrupted in human disc degeneration has yet to be investigated. The objective of this study is to measure the elastic modulus of single cells isolated from patients with varying degrees of disc degeneration with or without inflammatory stimulation. We also investigate the protective effects of increasing actomyosin contractility in patient cells using the RhoA activator CN03. Adult subjects (N=10) undergoing degenerative lumbar surgery were recruited and consented following IRB approval. Discard disc tissue was collected during surgery. Single cell elastic modulus. Adult subjects (N=10) undergoing degenerative lumbar surgery were recruited and consented following IRB approval. Discard disc tissue was collected during surgery. Severity of degeneration was scored on MRI using Pfirrmann grade. Cells were isolated from human IVD tissue without use of enzymes to prevent disruption of cell integrity. Cells were transferred to 1.2% alginate bead to mimic native 3D cell morphology and were randomized into one of four treatment groups for 24 hours: (1) untreated, (2) 10 ng/ml TNFα, (3) 1μg/ml CNO3, (4) 10 ng/ml TNFα + 1μg/ml CNO3. Cells (n=40-50 per donor per treatment group) were tested using an Asylum MFP-3D Bio Infinity Atomic Force Microscope (AFM). Single force indentation curves were obtained for each cell and were fitted to a Hertz model to determine elastic modulus. Statistics: data were analyzed with ANOVA and Fisher LSD post-hoc test. Cell elastic modulus significantly decreased with TNFα stimulation in four of 10 patients tested. Additionally, four of 10 patients were classified as responders to increased actomyosin contractility. Six of 10 patients were defined as nonresponders were CN03+TNFα co-treatment showed no significant difference from TNFα alone. Degree of degeneration was not found to correlate with the effect of TNFα on elastic modulus or with responder classification. To our knowledge, this is the first report that evaluates single cell biophysical properties of human IVD cells from patient samples with a focus on determining the effects of inflammatory stimulation or increased actomyosin contractility. Degree of degeneration as measured by Pfirmann Grade did not contribute to the propensity of patients to be categorized as responders. Understanding the underlying factors that predispose a patient to be responsive or nonresponsive to the protective effects of increasing actomyosin contractility will be an important step in evaluating its therapeutic potential. These findings warrant further investigation into other factors that may be influencing responsiveness to CN03 or TNFα.