Mechanical Injury to Human Intervertebral Discs and Isolated Cells Initiates Events Implicated in Degeneration and Pain
Authors: Haglund Lisbet , Rosenzweig Derek , Krock Emerson , Gawri Rahul , Steffen Thomas , Ouellet Jean , Weber Michael 
Published: May 2015

Introduction

Painful degenerating intact human intervertebral discs (IVDs) produce elevated levels of Toll-like receptors (TLRs), cytokines, and neuropeptides but events regulating their expression are poorly understood. Mechanical overload has been proposed as one potential initiating mechanism. The aim of this study was to investigate the cellular responses in IVDs and isolated disc cells exposed to mechanical overload or strain.

Methods

Lumbar spine segments were harvested from consenting donors via the Transplant Quebec organ donation program in Montreal, Quebec, Canada. A total of nine discs from six donors were used in this study. Intact IVDs were prepared and cells isolated using a standard technique. Intact discs with cartilaginous endplates were loaded with a single ramp compression peaking at either approximately 0.2 MPa (noninjurious) or about 1.5 MPa (injurious) stress applied at a rate of 30% strain per second. Injurious compression protocol consistently yields endplate fracture. Discs were immediately put in low serum containing media after loading. For isolated cell experiments, 2 × 105 nucleus pulposus (NP) and annulus fibrosus (AF) cells were seeded on flexible or static silicone dishes. The flexible dishes were exposed to a 20% magnitude cyclic mechanical strain at 0.0001 Hz was applied to the cultures for 8 h/d over 2 days. Conditioned media was collected and RNA was isolated. Quantitative RT-PCR was performed to assess TLR, NGF, and TNF gene expression. Conditioned media from intact discs and isolated cells was applied to PC12 cells and neurite outgrowth was assessed. Commercially available cytokine arrays were used to investigate protein levels of cytokines released from intact discs and isolated cells.

Results

Elevated levels of proteoglycan, inflammatory cytokines, and pain mediators were released from intact discs exposed to injurious loading. The highest levels were found 3 days postinjury and the conditioned media caused significantly induced neurite outgrowth in PC12 cells compared with that of the control group. About 50% of the cells in the IVDs died as a result of the inflicted trauma. Isolated NP and AF cells were exposed to a high-magnitude strain at low frequency to preserve cell attachments and maintain cell viability. The protocol was applied to evaluate if the adverse mechanical stimulation of the cells or the induced cell death caused by the injurious loading of the intact IVDs was responsible for the increased expression of inflammatory cytokines and pain mediators. Our data showed that high-magnitude low-frequency strain is sufficient to induce expression inflammatory cytokines and pain mediators in the absence of cell death. We also found a small but significant upregulation of TLR 2 and 4 in both NP and AF cells.

Conclusion

Cell death and cytokine production combined with loss of glycosaminoglycan indicate that mechanical injury to intact IVDs and isolated cells initiates cellular processes leading to disc degeneration. Once the proteoglycan content in the matrix has been degraded, elevated levels of NGF could lead to neuronal ingrowth directly causing pain. Increased TLR expression induced by high strain may prime cells toward a degenerate phenotype and could be a novel therapeutic target for degenerative disc disease.