Preclinical ex-vivo Testing of Anti-inflammatory Drugs in a Bovine Intervertebral Degenerative Disc Model

Abstract

Discogenic low back pain (LBP) is a main cause of disability and inflammation is presumed to be a major driver of symptomatic intervertebral disc degeneration (IDD). Anti-inflammatory agents are currently under investigation as they demonstrated to alleviate symptoms in patients having IDD. However, their underlying anti-inflammatory and regenerative activity is poorly explored. The present study sought to investigate the potential of Etanercept and Tofacitinib for maintaining disc homeostasis in a preclinical intervertebral disc (IVD) organ culture model within IVD bioreactors allowing for dynamic loading and nutrient exchange. Bovine caudal IVDs were cultured in a bioreactor system for 4 days to simulate physiological or degenerative conditions: (1) Phy-physiological loading (0.02-0.2 MPa; 0.2 Hz; 2 h/day) and high glucose DMEM medium (4.5 g/L); (2) Deg+Tumor necrosis factor α (TNF-α)-degenerative loading (0.32-0.5 MPa; 5 Hz; 2 h/day) and low glucose DMEM medium (2 g/L), with TNF-α injection. Etanercept was injected intradiscally while Tofacitinib was supplemented into the culture medium. Gene expression in the IVD tissue was measured by RT-qPCR. Release of nitric oxide (NO), interleukin 8 (IL-8) and glycosaminoglycan (GAG) into the IVD conditioned medium were analyzed. Cell viability in the IVD was assessed using lactate dehydrogenase and ethidium homodimer-1 staining. Immunohistochemistry was performed to assess protein expression of IL-1β, IL-6, IL-8, and collagen type II in the IVD tissue. Etanercept and Tofacitinib downregulated the expression of IL-1β, IL-6, IL-8, Matrix metalloproteinase 1 (MMP1), and MMP3 in the nucleus pulposus (NP) tissue and IL-1β, MMP3, Cyclooxygenase-2 (COX2), and Nerve growth factor (NGF) in the annulus fibrosus (AF) tissue. Furthermore, Etanercept significantly reduced the IL-1β positively stained cells in the outer AF and NP regions. Tofacitinib significantly reduced IL-1β and IL-8 positively stained cells in the inner AF region. Both, Etanercept and Tofacitinib reduced the GAG loss to the level under physiological culture condition. Etanercept and Tofacitinib are able to neutralize the proinflammatory and catabolic environment in the IDD organ culture model. However, combined anti-inflammatory and anabolic treatment may be required to constrain accelerated IDD and relieving inflammation-induced back pain.

Angiotensin II Type 1 Receptor Antagonist Losartan Inhibits TNF-α Induced Inflammation and Degeneration Processes in Human Nucleus Pulposus Cells

“…In contrast to our experiments, these studies used lipopolysaccharides to stimulate inflammation in the respective cell cultures and might not be fully comparable. However, our workgroup recently validated the superior potential of TNF-α-induced inflammatory cell culture models to examine degenerative disc diseases Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 November 2020 doi:10.20944/preprints202011.0456.v1 [14]. Further, we showed that PPARγ pathway stimulation through losartan has protective effects on human NP cells for the first time.…”

“…These strategies try to reduce the inflammatory microenvironment of the IVD in order to slow down the progressive degenerative cascade [12][13][14][15].…”

Investigating the physiological relevance of ex vivo disc organ culture nutrient microenvironments using in silico modeling and experimental validation

“…However, ensuring a nutrient‐deprived ECM in whole disc culture may require more consideration and sophistication. A number of recent ex vivo disc organ studies have combined mechanical loading with low glucose culture medium to induce a catabolic niche and pro‐inflammatory cell response 11,15,23,35 . Another used short‐term glucose deprivation before refreshing the medium with a higher concentration of glucose 37 .…”

Annular Defects Impair the Mechanical Stability of the Intervertebral Disc

“…First, bovine caudal IVDs were used in this experiment, although several studies have noted that the mechanical performance and elastic modulus of bovine caudal IVDs and human lumbar IVDs are quite similar. [17][18][19][20][21] Second, the real-time range of motion of the IVD and the mechanical changes in different regions of the IVD were not measured during testing. Last, but not least, it would be ideal to perform a histological examination of the IVDs to observe the histological changes in the AF, endplate and NP after the biomechanical testing.…”