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.

Keywords: 3R; bioreactor; disc degeneration; inflammation; intervertebral disc; organ culture; regeneration; spine.

Figure 1

Scheme of experimental setup.

Figure 2

Gene expression levels of NP tissue. Gene expression levels of NP tissue after 4 days of culture under Deg culture condition, with or without (A) Etanercept intradiscal injection. (B) Tofacitinib application into the medium, normalized to Phy group. n = 8, *p < 0.05, **p < 0.01, ***p < 0.001 Deg vs. Phy, ^#p < 0.05 Drug vs. Phy, min to max (bottom and top bar) with interquartile range (middle box). NP, Nucleus pulposus; IL, Interleukin; MMP, Matrix metalloproteinases; NGF, Nerve growth factor; COX-2, Cyclooxygenase-2.

Figure 3

Gene expression levels of AF tissue. Gene expression levels of AF tissue after 4 days of culture under Deg culture condition, with or without (A) Etanercept intradiscal injection. (B) Tofacitinib application into the medium, normalized to Phy group. n = 8, *p < 0.05, **p < 0.01 Deg vs. Phy, ^##p < 0.01 Drug vs. Phy, min to max (bottom and top bar) with interquartile range (middle box). AF, Annulus fibrosus; IL, Interleukin; MMP, Matrix metalloproteinases; NGF, Nerve growth factor; COX-2, Cyclooxygenase-2.

Figure 4

GAG (A,B), NO (C,D), and IL-8 release (E,F) in IVD culture medium. Cumulative release of GAG, NO, and IL-8 into IVD conditioned medium during 20 h of free swelling culture (FS) and 2 h of dynamic loading (load); D, day; Means ± SEM, n = 8; *p < 0.05 Phy vs. Deg; ^#p < 0.05 Phy vs. Drug; GAG, glycosaminoglycan; NO, nitric oxide.

Figure 5

IL-1β immunohistochemistry staining of IVD tissue from the Etanercept experiments. Representative IL-1β IHC image of NP region, inner AF region (iAF) and outer AF (oAF) region from day 0 control samples (Day0.inj.), IVDs cultured under physiological condition on day 4 (Phy.inj.), IVDs cultured under degenerative condition on day 4 (Deg.inj.), and IVDs cultured under degenerative condition and treated with Etanercept on day 4 (Etanercept.inj.). Scale bar: 100 μm. The percentage of positively stained cells were counted, as presented in the bar graph. n = 8, Means + SEM, *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 6

IL-6 immunohistochemistry staining of IVD tissue from the Etanercept experiments. Representative IL-6 IHC image of NP region, inner AF region (iAF) and outer AF (oAF) region from day 0 control samples (Day0.inj.), IVDs cultured under physiological condition on day 4 (Phy.inj.), IVDs cultured under degenerative condition on day 4 (Deg.inj.), and IVDs cultured under degenerative condition and treated with Etanercept on day 4 (Etanercept.inj.). Scale bar: 100 μm. The percentage of positively stained cells were counted, as presented in the bar graph. n = 8, Means + SEM.

Figure 7

IL-8 immunohistochemistry staining of IVD tissue from the Etanercept experiments. Representative IL-8 IHC image of NP region, inner AF region (iAF) and outer AF (oAF) region from day 0 control samples (Day0.inj.), IVDs cultured under physiological condition on day 4 (Phy.inj.), IVDs cultured under degenerative condition on day 4 (Deg.inj.), and IVDs cultured under degenerative condition and treated with Etanercept on day 4 (Etanercept.inj.). Scale bar: 100 μm. The percentage of positively stained cells were counted, as presented in the bar graph. n = 8, Means + SEM, **p < 0.01.

Figure 8

IL-1β immunohistochemistry staining of IVD tissue from the Tofacitinib experiments. Representative IL-1β IHC image of NP region, inner AF region (iAF) and outer AF (oAF) region from day 0 control samples (Day0.med.), IVDs cultured under physiological condition on day 4 (Phy.med.), IVDs cultured under degenerative condition on day 4 (Deg.med.), and IVDs cultured under degenerative condition and treated with Tofacitinib on day 4 (Tofacitinib.med.). Scale bar: 100 μm. The percentage of positively stained cells were counted, as presented in the bar graph. n = 8, Means + SEM, **p < 0.01, ***p < 0.001.

Figure 9

IL-6 immunohistochemistry staining of IVD tissue from the Tofacitinib experiments. Representative IL-6 IHC image of NP region, inner AF region (iAF) and outer AF (oAF) region from day 0 control samples (Day0.med.), IVDs cultured under physiological condition on day 4 (Phy.med.), IVDs cultured under degenerative condition on day 4 (Deg.med.), and IVDs cultured under degenerative condition and treated with Tofacitinib on day 4 (Tofacitinib.med.). Scale bar: 100 μm. The percentage of positively stained cells were counted, as presented in the bar graph. n = 8, Means + SEM, *p < 0.05.

Figure 10

IL-8 immunohistochemistry staining of IVD tissue from the Tofacitinib experiments. Representative IL-8 IHC image of NP region, inner AF region (iAF) and outer AF (oAF) region from day 0 control samples (Day0.med.), IVDs cultured under physiological condition on day 4 (Phy.med.), IVDs cultured under degenerative condition on day 4 (Deg.med.), and IVDs cultured under degenerative condition and treated with Tofacitinib on day 4 (Tofacitinib.med.). Scale bar: 100 μm. The percentage of positively stained cells were counted, as presented in the bar graph. n = 8, Means + SEM, *p < 0.05.

Figure 11

Collagen type II immunohistochemistry staining of IVD tissue from the Etanercept experiments. Representative COL2 IHC image of NP region, inner AF region (iAF) and outer AF (oAF) region from day 0 control samples (Day0.inj.), IVDs cultured under physiological condition on day 4 (Phy.inj.), IVDs cultured under degenerative condition on day 4 (Deg.inj.), and IVDs cultured under degenerative condition and treated with Etanercept on day 4 (Etanercept.inj.). Scale bar: 100 μm. The staining optical density (OD)/area value was analyzed, as presented in the bar graph. n = 8, Means + SEM, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 12

Collagen type II immunohistochemistry staining of IVD tissue from the Tofacitinib experiments. Representative COL2 IHC image of NP region, inner AF region (iAF) and outer AF (oAF) region from day 0 control samples (Day0.med.), IVDs cultured under physiological condition on day 4 (Phy.med.), IVDs cultured under degenerative condition on day 4 (Deg.med.), and IVDs cultured under degenerative condition and treated with Tofacitinib on day 4 (Tofacitinib.med.). Scale bar: 100 μm. The staining optical density (OD)/area value was analyzed, as presented in the bar graph. n = 8, Means + SEM, *p < 0.05, ***p < 0.001.

Figure 13

Cell viability of IVD tissue from the Etanercept experiments. Representative LDH/ EthD-1 staining image of NP region, inner AF region (iAF) and outer AF (oAF) region from day 0 control samples (Day0.inj.), IVDs cultured under physiological condition on day 4 (Phy.inj.), IVDs cultured under degenerative condition on day 4 (Deg.inj.), and IVDs cultured under degenerative condition and treated with Etanercept on day 4 (Etanercept.inj.). Scale bar: 50 μm. The percentage of alive cells were counted, as presented in the bar graph. n = 8, Means + SEM, ***p < 0.001.

Figure 14

Cell viability of IVD tissue from the Tofacitinib experiments. Representative LDH/ EthD-1 staining image of NP region, inner AF region (iAF) and outer AF (oAF) region from day 0 control samples (Day0.med.), IVDs cultured under physiological condition on day 4 (Phy.med.), IVDs cultured under degenerative condition on day 4 (Deg.med.), and IVDs cultured under degenerative condition and treated with Tofacitinib on day 4 (Etanercept.med.). Scale bar: 50 μm. The percentage of alive cells were counted, as presented in the bar graph. n = 8, Means + SEM, **p < 0.01, ***p < 0.001.

Figure 15

Disc height change normalized to the original dimension after dissection. n = 8, means ± SEM, ***p < 0.001, ****p < 0.0001, FS, Free swelling.