Cell clustering in intervertebral disc degeneration: An attempted repair mechanism aborted via apoptosis?

Cell clusters are a histological hallmark feature of adversely loaded aging intervertebral discs. Clusters arising from cell proliferation are associated with replicative senescence, remain metabolically active and accumulate over time but their precise role in increasing grades of intervertebral disc degeneration remains obscure. The aim of this study was therefore to investigate small, medium, and large size cell-clusters during degeneration, or innate tissue repair response. For this purpose, human disc samples were collected from 55 subjects, aged 37-72 years, twenty-one patients had disc herniation, ten had degenerated non-herniated discs, and nine had degenerative scoliosis, spinal curvature < 45°. Fifteen non-degenerated control discs were collected from cadavers. To corelate, the influence of abnormal loading on clustering process, 18-month-old, 10 male and 10 female sprague dawley rat spines were compressed with a custom-built loading device to induce tissue damage and stimulate attempted repair response during sustained loading. Clusters and matrix changes were investigated with histology, immunohistochemistry, and SDS-PAGE. Data obtained were analyzed with spearman rank correlation and ANOVA. Results revealed, small and medium-sized clusters were positive for cell proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA) in control, slightly degenerated human, and rat discs loaded for <= 7 days. Large cell clusters were typically more abundant in severely degenerated and herniated human discs, and in rat discs loaded for <=30 days. Large clusters spatially associated with matrix fissures, proteoglycan loss, MMP-1, and caspase-3. Spatial associations findings were reconfirmed with SDS-PAGE that showed the presence of target molecular markers thorough its weight within the dense matrix. Caspase-3 activity amplified with sustained loading and were localised in larger clusters. Controls and slightly degenerated discs showed fewer cell clusters, less proteoglycan loss, MMP-1 and caspase 3 activity. In conclusion, this study provides the first direct evidence on cell clusters in the early stages of disc degeneration as indicative of a reparative phenotypes, however sustained physical loading, inflammatory catabolism alters cell metabolism facilitating formation of large clusters, thus attempted repair process fails and is aborted at least in part via apoptosis, which contributes towards further matrix dysregulation, and can perhaps be halted by senolytic drugs.