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Abstract

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Materials

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Medicine

A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.

Published: February 14th, 2021

DOI:

10.3791/62100

1AO Research Institute Davos, Davos, Switzerland, 2Department of Orthopedics and Trauma Surgery, Medical Centre-Albert-Ludwigs-University of Freiburg, Faculty of Medicine, Albert-Ludwigs-University of Freiburg, Freiburg, Germany
* These authors contributed equally

This protocol presents a novel experimental model of proinflammatory, degenerative bovine organ culture to simulate early-stage intervertebral disc degeneration.

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due to the lack of causative therapies, there is an urgent need for innovative experimental organ culture models to study the mechanisms involved in the progression of the disease, find therapeutic targets, and reduce the need for animal models. We here present a novel, three-dimensional organ culture model protocol mimicking the proinflammatory and catabolic microenvironment, which is present during IDD.

Initially, bovine caudal IVDs were dissected, cleaned, and cultured in the tissue culture medium. Dynamic physiologic or pathologic loading was applied in a custom-made bioreactor for 2 hours per day. IVDs were assigned to a control group (high glucose medium, physiological loading, phosphate-buffered saline injection) and a pathological group (low glucose medium, pathological loading, tumor necrosis factor-alpha injection) for four days. Gene expression analysis from collected nucleus pulposus cells of the IVDs and enzyme-linked immunosorbent assay of the conditioned organ culture media was performed.

Our data revealed a higher expression of inflammatory markers and reduced disc heights after loading in the pathological group compared to the control group. This protocol is reliable to simulate IVD inflammation and degeneration and can be further expanded to broaden its application scope.

Low back pain (LBP) can affect individuals of all ages and is a leading cause for disability worldwide1,2,3. The total cost associated with LBP exceeds $100 billion per year4,5. Symptomatic intervertebral disc (IVD) degeneration (IDD), a condition characterized by inflammation and tissue degradation, is a major cause of LBP6,7. Specifically, IDD is characterized by a gradually evolving breakdown of the IVD's extracellular matrix (ECM), induced and triggere....

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Experiments were performed using bovine tails obtained from local abattoirs. The biological materials used in the current study are taken from the food chain and require no ethical approval in Swiss and European law.

1. Dissection of the bovine intervertebral disc

  1. Rinse the whole tail thoroughly with tap water to remove dirt and hair on the surface.
    NOTE: With intact, distal ends, a maximum of 9 IVDs (coccygeal 1-9) per tail can be used for the experiments depending on the d.......

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Degenerative loading in low glucose medium combined with TNF-α injection caused a significant increase of the gene expression of proinflammatory markers interleukin 6 (IL-6) and interleukin 8 (IL-8) compared to the physiological control group in NP cells after 4 days of culture (Figure 2). In contrast, we did not observe significant changes for the proinflammatory genes interleukin 1β (IL-1β) and TNF-α in NP cells (data not shown). Furthermore, degenerative culture condit.......

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We here provided a detailed protocol to simulate degenerative and inflammatory IVDD. This protocol can be applied for detailed examinations of inflammatory pathways leading to the destructive effects on the disc. Moreover, the protocol can help to determine promising therapeutic targets involved in the progression of the disease.

We recently showed that human recombinant TNF-α could induce inflammation in both bovine and human NP cells21, which is in accordance wit.......

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This work was supported by AO Foundation and AOSpine International. Babak Saravi received fellowship support from the German Spine Foundation and the German Osteoarthritis Foundation. Gernot Lang was supported by the Berta-Ottenstein-Programme for Advanced Clinician Scientists, Faculty of Medicine, University of Freiburg, Germany.

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Name Company Catalog Number Comments
1-Bromo-3-chloropropane(BCP) Sigma-Aldrich, St. Louis, USA B9673
Ascorbate-2-phosphate Sigma-Aldrich, St. Louis, USA A8960
Band saw Exakt Apparatebau, Norderstedt, Germany model 30/833
Betadine Munndipharma, Frankfurt, Germany
Bovine IL-8 Do.it-Yourself ELISA Kingfisher Biotech, St. Paul, USA DIY1028B-003
Corning ITS Premix Corning Inc., New York, USA 354350
DMEM high glucose Gibco by life technologies, Carlsbad, USA 10741574
DMEM low glucose Gibco by life technologies, Carlsbad, USA 11564446
Ethanol for molecular biology Sigma-Aldrich, St. Louis, USA 09-0851
Fetal Bovine Serum (FBS) Gibco by life technologies, Carlsbad, USA A4766801
Non-essential amino acid solution Gibco by life technologies, Carlsbad, USA 11140050
Penicillin/Streptomycin(P/S) gibco by life technologies, Carlsbad, USA 11548876
Phosphate Buffer Solution, tablet Sigma-Aldrich, St. Louis, USA P4417
Pronase Sigma-Aldrich, St. Louis, USA 10165921001
Primocin InvivoGen, Sandiego, USA ant-pm-05
Pulsavac Jet Lavage System Zimmer, IN,USA
TissueLyser II Quiagen, Venlo, Netherlands 85300
Streptavidinn-HRP Kingfisher Biotech, St. Paul, USA AR0068-001
Superscript VILO Invitrogen by life Technologies, Carlsbad, USA 10704274
cDNA Synthesis Kit Applied Biosystems by life technologies 10400745
TaqMan Universal Master Mix Applied Biosystems by life technologies
TNF-alpha, recombinant human protein R&D systems, Minnesota, USA 210-TA-005
TRI Reagent Molecular Research Center, Cincinnati, USA TR 118
Tris-EDTA buffer solution sigma-Aldrich, St. Louis, USA 93283
Gene bIL-6 Applied Biosystems by life technologies Custom made probes Primer fw (5′–3′) TTC CAA AAA TGG AGG AAA AGG A
Primer rev (5′–3′) TCC AGA AGA CCA GCA GTG GTT
Probe (5′FAM/3′TAMRA) CTT CCA ATC TGG GTT CAA TCA GGC GATT
Gene bIL8 Applied Biosystems by life technologies Bt03211906_m1
Gene bTNF-alpha Applied Biosystems by life technologies Custom made probes Primer fw (5′–3′) CCT CTT CTC AAG CCT CAA GTA ACA A
Primer rev (5′–3′) GAG CTG CCC CGG AGA GTT
Probe (5′FAM/3′TAMRA) ATG TCG GCT ACA ACG TGG GCT ACC G
GENE bIL1beta Applied Biosystems by life technologies Custom made probes Primer fw (5′–3′) TTA CTA CAG TGA CGA GAA TGA GCT GTT
Primer rev (5′–3′) GGT CCA GGT GTT GGA TGC A
Probe (5′FAM/3′TAMRA) CTC TTC ATC TGT TTA GGG TCA TCA GCC TCA A
RPLP0 Applied Biosystems by life technologies Bt03218086_m1

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