Anat Cell Biol.  2023 Sep;56(3):382-393. 10.5115/acb.23.067.

Cell clusters in intervertebral disc degeneration: an attempted repair mechanism aborted via apoptosis

• Lama P
• Tiwari J
• Mutreja P
• Chauhan S
• Harding IJ
• Dolan T
• Adams MA
• Maitre CL

• KMID: 2546467
• DOI: http://doi.org/10.5115/acb.23.067

Abstract

Cell clusters are a histological hallmark feature of intervertebral disc degeneration. Clusters arise from cell proliferation, are associated with replicative senescence, and remain metabolically, but their precise role in various stages of disc degeneration remain obscure. The aim of this study was therefore to investigate small, medium, and large size cell-clusters. For this purpose, human disc samples were collected from 55 subjects, aged 37–72 years, 21 patients had disc herniation, 10 had degenerated non-herniated discs, and 9 had degenerative scoliosis with spinal curvature <45°. 15 non-degenerated control discs were from cadavers. Clusters and matrix changes were investigated with histology, immunohistochemistry, and Sodium dodecyl sulphate polyacrylamide gel electrophoresis (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 and slightly degenerated human discs, while large cell clusters were typically more abundant in severely degenerated and herniated discs. Large clusters associated with matrix fissures, proteoglycan loss, matrix metalloproteinase-1 (MMP-1), and Caspase-3. Spatial association findings were reconfirmed with SDS-PAGE that showed presence to these target markers based on its molecular weight. Controls, slightly degenerated discs showed smaller clusters, less proteoglycan loss, MMP-1, and Caspase-3. In conclusion, cell clusters in the early stages of degeneration could be indicative of repair, however sustained loading increases large cell clusters especially around microscopic fissures that accelerates inflammatory catabolism and alters cellular metabolism, thus attempted repair process initiated by cell clusters fails and is aborted at least in part via apoptosis.

Keyword

Intervertebral disc; Intervertebral disc degeneration; Disc herniaton; Cell clusters; Apoptosis

Figure

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  Fig. 1 Comparisons between (A) control non-degenerated cadaveric human disc with extremely flattened annulus fibrosus (AF) cells (arrows) and (B) herniated disc, arrows shows inflammatory cells, and (C) degenerated disc with displaced lamellae and large cell clusters (arrows). Scale bar: 100 μm.

• 

  Fig. 2 Cell clusters comparisons between (A) control non-degenerated human cadaveric disc with small size cell clusters (2, 3, 6 cells) and (B) herniated disc with large sized cell clusters (50–60 cells, arrow). Scale bar: 100 μm (A) and 50 μm (B). (C) Toluidine blue stained control NP/IAF region without clusters and (D) herniated disc tissue with large cluster (arrow) stained with toluidine blue. Scale bar: 50 μm. (E) Cell cluster scores (0–5) and increasing pf grade of disc degeneration. (F) Proteoglycan staining score (0–3) and increasing pf grade of disc degeneration. NP, nucleus pulposus; IAF, inner annulus fibrosus; pf, Pfirrmann grade of degeneration; PG, proteoglycans.

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  Fig. 3 Small, medium, and large sized cell clusters of the ‘control’ and its comparisons to ‘degenerated’ and ‘herniated’ discs for (A) MMP-1, (B) Caspase-3, and (C) Ki-67. Scale bar: 50 μm. MMP-1, matrix metalloproteinase-1.

• 

  Fig. 4 Medium and large sized cell clusters stained with (A) Caspase-3 (red stain, blue indicates DAPI stained cell nuclei), (B) MMP-1 (red stain, blue indicates DAPI stain cell nuclei) with phase contrast background. Scale bar: 100 μm. (C) Caspase-3 positive cell clusters count and increasing pf grade of disc degeneration. MMP-1, matrix metalloproteinase-1; DAPI, 4’,6-diamidino-2-phenylindole; pf, Pfirrmann.

• 

  Fig. 5 Western blot analysis on a 10% gel. Lane were loaded as C, ‘non-degenerated cadaveric disc’ followed by disc specimens categorised according to pf grades degeneration in the subsequent lanes as pf grade 1, 2, 3, 4, and 5 discs. (A) MMP-1 core protein at 54 kDa. (B) Expression level of control ‘c’, and grade ‘1–5 discs’ shown with grade 3 discs at the highest expression level. (C) Ki-67 core protein with a molecular weight of 250 kDa. (D) Weak expression level for degenerated grade 3–5 discs. (E) PCNA core protein with a molecular weight of 36 kDa. (F) Bands expressed higher values for grade 2 and 3 discs in comparisons to other grades. (G) Caspase-3 core protein at 22 kDa. (H) Strongly expressed for the severely degenerated grade 4 and 5 discs. GAPDH as loading control is at 36 kDa. C, control; pf, Pfirrmann; MMP-1, matrix metalloproteinase-1; PCNA, proliferating cell nuclear antigen; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Reference

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