Physical disruption of intervertebral disc promotes cell clustering and a degenerative phenotype

Abstract

To test the hypothesis that physical disruption of an intervertebral disc disturbs cell-matrix binding, leading to cell clustering and increased expression of matrix degrading enzymes that contribute towards degenerative disc cell phenotype. Lumbar disc tissue was removed at surgery from 21 patients with disc herniation, 11 with disc degeneration, and 8 with adolescent scoliosis. 5 μm sections were examined with histology, and 30-µm sections by confocal microscopy. Antibodies were used against integrin α5beta1, matrix metalloproteinases (MMP) 1, MMP-3, caspase 3, and denatured collagen types I and II. Spatial associations were sought between cell clustering and various degenerative features. An additional, 11 non-herniated human discs were used to examine causality: half of each specimen was cultured in a manner that allowed free ‘unconstrained’ swelling (similar to a herniated disc in vivo), while the other half was cultured within a perspex ring that allowed ‘constrained’ swelling. Changes were monitored over 36 h using live-cell imaging. 1,9-Di-methyl methylene blue (DMMB) assay for glycosaminoglycan loss was carried out from tissue medium. Partially constrained specimens showed little swelling or cell movement in vitro. In contrast, unconstrained swelling significantly increased matrix distortion, glycosaminoglycan loss, exposure of integrin binding sites, expression of MMPs 1 and 3, and collagen denaturation. In the association studies, herniated disc specimens showed changes that resembled unconstrained swelling in vitro. In addition, they exhibited increased cell clustering, apoptosis, MMP expression, and collagen denaturation compared to ‘control’ discs. Results support our hypothesis. Further confirmation will require longitudinal animal experiments.

A bovine nucleus pulposus explant culture model

“…This was also consistent with the observation on H&E stained sections in NCM‐treated explants where cells appeared in clusters (indication of cloning) which we interpret as proliferation. However, cell clustering is also described in physically disrupted discs and is thought to arise from disturbed cell–matrix binding 41 . Thus, although we could not observe a direct biomechanical augmentation due to leakage of injected sGAG, potential metabolic effects were evident.…”

“…Cellular reactions might be due to an osmotic shock or due to degrading products of CS resulting from chABC cleavage, as the latter were found to change morphology and induce catabolic expression in chondrocytes in 3D culture 40 . Furthermore, a distorted physical and biochemical microenvironment might be a trigger for resident cells to go into a “pro‐inflammatory state.” 41 In this study, cells remained viable in all tested conditions. Nevertheless, the long‐term response of cells to chABC injection remains unclear.…”

“…However, cell clustering is also described in physically disrupted discs and is thought to arise from disturbed cell-matrix binding. 41 Thus, although we could not observe a direct biomechanical augmentation due to leakage of injected sGAG, potential metabolic effects were evident.…”

Development of a standardized histopathology scoring system for human intervertebral disc degeneration: an Orthopaedic Research Society Spine Section Initiative

“…Innervation is also greater in painful IVDs with annulus fissures, 17 which may provide a chemically and mechanically favorable environment for nerve ingrowth, 51 with nerve fibers found to migrate into the NP associated with loss of proteoglycans and ECM fissures 17 . Likewise, elevated levels of pro‐inflammatory cytokines measured in these IVD and endplate tissues, 32,80 in particular associated with cellular clusters, 81 suggest these cytokines may play an important role in promoting degeneration and pain 82,83 …”

The human intervertebral disc as a source of DNA for molecular identification

“…A variation between samples (within one stage of decay) may be due to the distribution of cells (e.g. clusters, single cells) [ 29 , 30 ], a varying thickness (i.e. height) of the removed IVDs, or differences in bacterial colonization [ 31 ].…”