Vertebral endplate trauma induces disc cell apoptosis and promotes organ degeneration in vitro

Abstract

There is a major controversy whether spinal trauma with vertebral endplate fractures can result in posttraumatic disc degeneration. Intervertebral discs, which are adjacent to burst endplates, are frequently removed and an intercorporal spondylodesis is performed. In any case, the biological effects within the discs following endplate factures are poorly elucidated to date. The aim of our investigations was therefore to establish a novel disc/endplate trauma culture model to reproducibly induce endplate fractures and investigate concurrent disc changes in vitro. This model is based on a full-organ disc/endplate culture system, which has been validated by the authors before. Intervertebral disc/endplate specimens were isolated from Burgundy rabbits and cultured in standard media (DMEM/ F12, 10%FCS). Burst endplate fractures were induced in half of the specimens with a custom-made fracture device and subsequently cultured for 9 days. The biological effects such as necrotic or apoptotic cell death and the expression of pro-apoptotic genes and other genes involved in organ degeneration, e.g. matrix metalloproteinases (MMPs) were analyzed. Cell damage was assessed by quantification of the lactate dehydrogenase (LDH) activity in the supernatant. The expression of genes involved in the cellular apoptotic pathway (caspase 3) and the pro-apoptotic proteins FasL and TNF-a were monitored. The results demonstrate that LDH levels increased significantly post trauma compared to the control and remained elevated for 3 days. Furthermore, a constant up-regulation of the caspase 3 gene in both disc compartments was present. The pro-apoptotic proteins FasL and TNF-a were up regulated predominantly in the nucleus whereas the MMP-1 and -13 transcripts (collagenases) were increased in both disc structures. From this study we can conclude that endplate burst fractures result in both necrotic and apoptotic cell death in nucleus and annulus tissue. Moreover, FasL and TNF-a expression by nucleus cells may lead to continued apoptosis induced by Fas-and TNF-a receptor bearing cells. In addition TNF-a over-expression has potentially deleterious effects on disc metabolism such as overexpression of matrix proteinases. Taken together, the short term biological response of the disc following endplate fracture exhibits characteristics, which may initiate the degeneration of the organ.

TNF-α and IL-1β Promote a Disintegrin-like and Metalloprotease with Thrombospondin Type I Motif-5-mediated Aggrecan Degradation through Syndecan-4 in Intervertebral Disc

“…In human as well as animal models of degenerative disc disease there are increased levels of inflammatory cytokine such as TNF-␣ and IL-1␤; our studies confirmed and extended these earlier findings (7,8,(23)(24)(25)(26)(27)(28). Likewise, in both mouse and human articular cartilage, cytokines and SDC4 expression is elevated during osteoarthritis (18).…”

The NLRP3/Caspase-1/Interleukin-1β Axis Is Active in Human Lumbar Cartilaginous Endplate Degeneration

“…Some studies have shown that intervertebral disc degeneration is inseparably linked with cartilaginous endplate degeneration [14,31] and Modic changes [47]. The cartilaginous endplate is indispensable to maintain the structural and functional integrity of local structures in the lower lumbar vertebral bodies [34].…”

Effects of mechanical strain on ANK, ENPP1 and TGF-β1 expression in rat endplate chondrocytes in�vitro

“…The endplate cartilage was cut with ophthalmic scissors into pieces of approximately 1 mm 3 in size. Endplate cartilage pieces were collected aseptically, and chondrocytes were obtained by sequential digestion with Trypsin (Gibco) and Collagenase II (Gibco) as described previously (1 Application of cyclic strain. Chondrocytes (the second passage) were seeded on an elastic silicone membrane coated with collagen I (BioFlex, Flexcell International) at a density of 1x10 5 cells/well (diameter 35 mm) for culture.…”

“…However, the exact cause of this is unclear. Studies have found that disc degeneration begins in the cartilage endplate (1). Previous studies have shown that the process of cartilage disc degeneration in the vertebral endplate occurs with different degrees of calcification and that the degree of disc degeneration is positively correlated with calcification.…”