Introduction: Mechanical overloading, catabolic cell response, and degeneration of the water-binding extracellular matrix could result in the disc degeneration. While the intervertebral disc degeneration occurred, the number of nucleus pulposus cells should be generally reduced. However, there were often many proliferating cell clusters in the nucleus and inner annulus regions of degenerative discs. At present, the roles of cell clusters during the disc degeneration was still unknown. Haemoglobin is a known carrier of oxygen in erythrocytes. Therefore, the main purpose is to discover the roles of haemoglobin body in hypoxia adaption of intervertebral discs.
Methods: Firstly, human lumbar disc specimens were collected from patients with lumbar vertebral burst fractures or LBP who underwent anterior vertebral body excision and fusion surgery. All the specimens were extracted, fixed, sectioned, and stained for histologic evaluation. Then, cell clusters were determined using Hematoxylin-eosin staining, safranin O fast green and alcian blue staining. Cytokeratin-8 and vimentin were stained to identify the notochord-like cells or NP cells. CD31, Vwf, neurofilament and PGP9.5 were to identify the neovessels or nerves. Type 2 collagen(ColⅡ) and MMP13 of cell clusters were stained to evaluate the disc degeneration. Haemoglobin-α(HBA) and haemoglobin-β(HBB) were stained to evaluate extra-erythrocyte role of haemoglobin body in intervertebral discs. Finally, we also isolated the notochord-like cells and NP cells from young or old rabbits’ lumbar discs to determine the roles of HBA/HBB and notochord-like cells in inflammatory NP degeneration under hypoxia.
Results: We showed the general structure of cell clusters (especially abundant in Pfirmann grade Ⅲ and grade IV disc) according to the abundant H&E slices of the same specimens. Cell clusters wrapped in the degenerative disc showed various shapes composed of many notochord-like cells (cytokeratin-8 and vimentin) and a few NP cells (vimentin, only) compared to the “normal” group. These cells rarely expressed the neovessel markers (CD31, Vwf) and nerve markers (neurofilament, PGP9.5). Interestingly, they could highly express the catabolic molecule MMP13 and lowly express the anabolic molecule ColⅡ, indicating that degenerative disc couldn’t be completely repaired or rescued. In addition, HBA/HBB-positive cell clusters were clarified, indicating oxygen-carrying capacity in cell clusters promoted cell proliferation. Finally, the conditioned medium of notochord-like cells could partially slow the IL-1β-induced NP degeneration by detecting the gene and protein level of MMP3, MMP13, ColⅡand Sox9.
Discussion: We identified cell types of cell clusters and further confirmed that notochord-like cells were the most major part, not including the neovessel and nerve. We also found the beneficial role of cell clusters including the HBA/HBB-positive cells in the intervertebral disc degeneration. Cell clusters occurred some of degenerative discs (appropriately 30%) and were regionally or unevenly distributed. Regrettably, we couldn’t isolate the cell clusters to further culture in vitro. Notochord-like cells and NP cells from young or old rabbits’ lumbar discs only could be used to verify the inflammation-associated disc degeneration, but not mechanical or extracellular matrix related disc degeneration. The model was urgent for further studying the role of cell clusters in intervertebral disc degeneration.