gms | German Medical Science

Objectives: Proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), play a major role in the progression of degenerative disc disease (DDD). Therefore, therapies that inhibit the expression of proinflammatory cytokines may be a promising therapeutic approach to consider, aiming to reduce inflammation and maintain the IVDs extracellular matrix. To date, no clinically relevant in vitro systems to test new anti-inflammatory therapeutics are available. The purpose of the present study was to design and evaluate a proinflammatory and degenerative ex vivo system in order to simulate the early onset of DDD.

Methods: Bovine IVDs with endplates were harvested and cultured with or without an intradiscal injection of TNF-α (100ng TNF-α/IVD) for 4 days. IVDs were cultured within a bioreactor system under two different loading and medium conditions: (1) physiological loading (0.02-0.2 MPa; 0.2 Hz; 2h/day, 3 days) and high glucose (4.5 g/L) medium (Phy), or (2) degenerative loading (0.32-0.5 MPa; 5 Hz; 2h/day, 3 days) and low glucose (2 g/L) medium (Deg). IVDs were cultured free swelling between dynamic loading cycles. After the first dynamic loading cycle on day 1, TNF-α was injected into the disc nucleus pulposus (NP) tissue. Disc height was recorded daily after load and free swelling (FS) recovery. Conditioned medium was collected for nitric oxide (NO) and Glycosaminoglycan (GAG) analysis. After 4 days, NP and annulus fibrosus (AF) tissue were harvested and gene expression was analyzed using real-time PCR. GAG/DNA and Hydroxy-proline (OHP)/DNA content of the disc tissue were assessed. One-way ANOVA or Kruskal-Wallis tests were used to determine statistical significance.

Results and Conclusion: Degenerative dynamic loading caused significantly higher disc height loss (~20%) compared to physiological dynamic loading (~10%). Intradiscal injection of TNF-α did not further induce disc height loss after loading. TNF-α combined with Deg condition up-regulated NO release from IVD (p<0.01 Phy vs Deg+TNF-α). TNF-α injection also enhanced GAG release in Phy group (p<0.05). Gene expression analysis demonstrated a decrease of type I collagen in AF tissue and type II collagen in NP tissue, caused by Deg condition or TNF-α injection (p<0.05). TNF-α injection combined with Deg condition induced an up-regulation of interleukin-6 and matrix metalloproteinase 1 in NP tissue, as well as increased interleukin-1 β gene expression in AF tissue (p<0.05).

This degenerative and inflammatory model is able to induce catabolism on organ cultured IVDs revealed by a significant upregulation of catabolic and inflammatory gene expression markers in disc tissue, together with enhanced NO release. By utilization of this model, the early onset of DDD can be simulated and novel anti-inflammatory treatment approaches can be investigated under relevant conditions. Future research seeks to analyze the biologic and biomechanical response of selective cytokine inhibitors on organ cultured IVDs as novel therapies for DDD.