A Degenerative/Proinflammatory Intervertebral Disc Organ Culture: An Ex Vivo Model for Anti-inflammatory Drug and Cell Therapy.

Resolution of intervertebral disc (IVD) degeneration-associated inflammation is a prerequisite for tissue regeneration and could possibly be achieved by strategies ranging from pharmacological to cell-based therapies. In this study, a proinflammatory disc organ culture model was established. Bovine caudal disc punches were needle punctured and additionally stimulated with lipopolysaccharide (10 µg/mL) or interleukin-1ß (IL-1ß, 10-100 ng/mL) for 48 h. Two intradiscal therapeutic approaches were tested: (i) a nonsteroidal anti-inflammatory drug, diclofenac (Df) and (ii) human mesenchymal stem/stromal cells (MSCs) embedded in an albumin/hyaluronan hydrogel. IL-1ß-treated disc organ cultures showed a statistically significant upregulation of proinflammatory markers (IL-6, IL-8, prostaglandin E2 [PGE2]) and metalloproteases (MMP1, MMP3) expression, while extracellular matrix (ECM) proteins (collagen II, aggrecan) were significantly downregulated. The injection of the anti-inflammatory drug, Df, was able to reduce the levels of proinflammatory cytokines and MMPs and surprisingly increase ECM protein levels. These results point the intradiscal application of anti-inflammatory drugs as promising therapeutics for disc degeneration. In parallel, the immunomodulatory role of MSCs on this model was also evaluated. Although a slight downregulation of IL-6 and IL-8 expression could be found, the variability among the five donors tested was high, suggesting that the beneficial effect of these cells on disc degeneration needs to be further evaluated. The proinflammatory/degenerative IVD organ culture model established can be considered a suitable approach for testing novel therapeutic drugs, thus reducing the number of animals in in vivo experimentation. Moreover, this model can be used to address the cellular and molecular mechanisms that regulate inflammation in the IVD and their implications in tissue degeneration.

Molecular interactions between human cartilaginous endplates and nucleus pulposus cells: a preliminary investigation.

STUDY DESIGN: Conditioned media (CM) of cartilaginous endplates (CEPs) of intervertebral discs were analyzed in a bioassay with regard to their influence on matrix turnover and inflammatory factors on nucleus pulposus (NP) cells of the same patient. CEP tissue underwent further histological and ultrastructural analysis. OBJECTIVE: To identify possible interactions between the CEP and the disc via molecular factors that may influence disc matrix degradation and to determine degenerative changes of CEP tissue. SUMMARY OF BACKGROUND DATA: Impaired endplate perme-ability due to degeneration and calcification is considered to be a key contributor to disc degeneration. An upregulation of metalloproteinases and inflammatory cytokines has been observed in degenerated intervertebral discs. Possibly, the CEP contributes to the regulation of disc matrix degradation via molecular interactions with the disc tissue. METHODS: CEP and NP cells from the same patients (n = 6) were investigated in a bioassay with regard to their influence on matrix turnover and inflammatory factors. We determined gene expression of NP cells in alginate beads that were exposed to CM of CEP punches (CEP-CM) from the same patients. The CEP-CMs were analyzed by protein array for inflammatory cytokines. Further CEP samples underwent histological (n = 15) and ultrastructural analysis (n = 8) to determine alterations of cell and matrix structure. RESULTS: NP cells exposed to their donor-corresponding CEP-CM significantly upregulated interleukins (IL-6, IL-8) and matrix metalloproteinase (MMP-3, MMP-13) expression, and significantly decreased aggrecan and collagen type 2 expression. Proinflammatory cytokines were identified in the CEP-CM. The occurrence of apoptotic cells and degraded matrix fragments varied strongly between donors. CONCLUSION: Our results indicate interactions between the CEP and the NP tissue via molecular factors that upregulate matrix degrading enzymes and inflammatory cytokines and thereby influence the pathophysiology of disc degeneration. Ongoing investigations will further identify the regulative role of potential molecular factors that are responsible for these degenerative alterations. LEVEL OF EVIDENCE: N/A.