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.

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
<ol>
	<li>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 desired size of the IVDs. Considering the desired diameter between 15-20 mm, we used 12 bovine tails with 5 IVDs per tail for the experiments.</li>
	<li>Immerse the whole tail in a box containing 1% betadine solution for 10 min. Briefly dry the tail with sterile gauze and place it on a sterile drape. 
	NOTE: During dissection of the disc, humidify the tails with Ringer&#39;s solution wetted gauze to prevent dehydration. Store the tails (or left-over segments) wrapped in wet gauze until the whole dissection procedure is completed.</li>
	<li>Use a scalpel (No. 20) to remove the soft tissue as completely as possible from the caudal spine to facilitate the identification of the IVDs. Remove the spinous and transverse processes of the vertebrae with bone removal pliers. 
	NOTE: Select IVDs with the desired diameter. IVDs with a diameter range of 15-20 mm were used in the current study.</li>
	<li>Cut transversely with bone pliers through the middle of each vertebral body to obtain individual motion segments. Put motion segments in a Petri dish with gauze wetted with Ringer&#39;s solution.</li>
	<li>Locate the IVD and vertebra by palpation and by moving the motion segments gently. Make two parallel cuts with the band saw in the growth plate of the IVDs, one on each side of the IVD. Identify the location of the growth plate by touching and finding the convex site of the bony endplate part (hard) adjacent to the disc (soft) with a safety distance of approximately 0.5-1 mm from the IVD towards the vertebra. Ensure that the blade of the band saw is cooled with Ringer&#39;s solution while cutting the vertebrae.</li>
	<li>Transfer IVDs in a clean Petri dish with clean gauze wetted with Ringer&#39;s solution. 
	NOTE: The gauze should be moistened and not too wet to prevent swelling of the IVDs,</li>
	<li>Use the scalpel blade to scrape off the vertebral body (red/pink bone), growth plate (white cartilage), leave the endplate intact (yellow-pink). Make the two surfaces flat and parallel for the loading procedure. Transfer scraped IVDs to a fresh Petri dish with gauze wetted with Ringer&#39;s solution. 
	NOTE: Wear a chainmail glove to protect the hand while holding the IVD and scraping.</li>
	<li>Measure the disc height and diameter with a caliper. Clean the blood clots in the vertebrae bone with Ringer&#39;s solution using a jet lavage system.</li>
	<li>Transfer the IVDs to 50 mL plastic tubes, one IVD per tube. Add 25 mL of Phosphate-Buffered Saline (PBS) + 10% Penicillin/Streptomycin (P/S) per IVD and leave it shaking for 15 min on an orbital shaker at room temperature.</li>
	<li>Aspirate the supernatant and add 10 mL of PBS + 1% P/S per IVD for 2 min to rinse the IVDs.</li>
</ol>
2. IVD culture and loading
<ol>
	<li>Transfer discs to IVD chambers and add IVD culture medium (Dulbecco&#39;s Modified Eagle Medium (DMEM, 4.5 g/L high glucose DMEM for the physiological group and 2 g/L low glucose DMEM for the pathological group) + 1% P/S + 2% fetal calf serum + 1% ITS (contains 5 &#181;g/mL insulin, 6 &#181;g/mL transferrin, and 5 ng/mL selenious acid) + 50 &#181;g/mL ascorbate-2-phosphate + 1% non-essential amino acid + 50 &#181;g/mL antimicrobial agent&#160;for primary cells) and place in an incubator at 37 &#176;C, 85% humidity and 5% CO2.</li>
	<li>Culture the discs for 4 days within a bioreactor system according to experimental groups16. In the pathologic group, maintain degenerative loading conditions at 0.32-0.5 MPa, 5 Hz for 2 h/day. In the physiological control group, use a loading protocol of 0.02-0.2 MPa, 0.2 Hz for 2 h/day. 
	NOTE: Position the IVDs in chambers containing 5 mL of IVD medium during the loading procedures. The volume depends on the size of the bioreactor&#39;s loading chambers. Between the loading procedures, place the IVDs in six-well plates with 7 mL of IVD culture medium for free-swelling recovery.</li>
	<li>For analyzing the changes in disc height during the experimental period, measure the disc height with a caliper after IVD dissection (baseline) and then daily after the free swelling period and after dynamic loading for the experimental duration.</li>
</ol>
3. Intradiscal tumor necrosis factor-alpha (TNF-&#945;) injection
<ol>
	<li>Directly after the first dynamic loading cycle on day 1, place the IVDs in a Petri dish in a vertical position and stabilize the IVDs with a tweezer.</li>
	<li>Inject recombinant TNF-&#945; (100 ng in 70 &#181;L of PBS per IVD) with a 30-gauge insulin needle into the nucleus pulposus tissue of the pathological group17. Inject slowly at a speed of approximately 70 &#181;L in 1 min.</li>
	<li>After injection, pull the syringe halfway back within the IVD and pull the syringe plunger to create a vacuum that prevents the injected solution from leaking back, before removing the needle and syringe completely from the IVD. 
	&#8203;NOTE: Perform a pilot experiment by injecting PBS containing trypan blue dye to evaluate the distribution of the injected solution after loading and overnight culture.</li>
</ol>
4. Gene expression
<ol>
	<li>Harvest the IVDs on day 4. Collect the nucleus pulposus (NP) tissue (gelly part in the middle of IVD) with a biopsy punch. Collect the outer annulus fibrosus (AF) with a scalpel blade (No.20). 
	NOTE: For the baseline reference at day 0, collect&#160;tissues immediately after dissection for RNA extraction.</li>
	<li>Use the amount of NP or AF tissue needed for gene expression analysis, depending on the experimental design. 
	NOTE: For the present experiments, approximately 150 mg tissue was used. The ratio of RNA isolation solution to tissue mass should be at least 2 mL per 100-150 mg tissue for efficient extraction.</li>
	<li>Digest the NP or AF tissue with the digestion solution (0.2% pronase in DMEM, filter sterilized) and incubate for 1 h at 37 &#176;C with magnetic stirring18.</li>
	<li>Flash-freeze the tissue samples using liquid nitrogen and pulverize to a fine powder. Divide the pulverized tissue powder equally into two 2 mL tubes each containing 1 mL of guanidine thiocyanate and phenol in a monophase solution (RNA isolation solution).</li>
	<li>Perform the homogenization in 2 mL tubes containing the RNA isolation solution and the pulverized tissue powder. Homogenize the tissue powder 5x with an 8 mm stainless steel ball and a tissue-lyzer at 30 Hz for 3 min. Centrifuge at 12,000 x g, 4 &#176;C for 10 min and transfer the supernatant to a fresh tube. The supernatants can be stored at -80 &#176;C for at least one month.</li>
	<li>Add 0.1 mL of 1-bromo-3-chloropropane (BCP) per 1 mL of RNA isolation solution and shake vigorously for 15 s. Store the resulting mixture at room temperature on an orbital shaker for 15 min and centrifuge at 12,000 x g for 15 min at 4 &#176;C. 
	NOTE: The RNA remains exclusively in the upper aqueous phase.</li>
	<li>Transfer the aqueous phase into a fresh tube and precipitate RNA with 0.25 mL of isopropanol and 0.25 mL of high salt precipitation solution per 1 mL of RNA isolation solution used for initial homogenization. Store the samples at room temperature for 15 min on an orbital shaker and centrifuge at 12,000 x g for 8 min at 4 &#176;C. 
	&#8203;NOTE: Alternatively, use the column-based RNA extraction method which generally leads to higher RNA purity but lower RNA yield.</li>
	<li>Remove the supernatant and wash the RNA pellet with 1 mL of 75% ethanol per 1 mL of RNA isolation solution used for initial homogenization. Centrifuge at 7,500 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the ethanol wash and briefly air-dry RNA pellet for 3-5 min. Dissolve the RNA in 20 &#181;L of diethylpyrocarbonate (DEPC) treated water by passing the solution a few times through a pipette tip and incubating for 10-15 min at 55-60 &#176;C.</li>
	<li>Measure the absorbance at 230 nm, 260 nm, and 280 nm (A230, A260 and A280 respectively). A260 of 1.0 corresponds to 40 &#181;g/mL RNA. An A260/A280 ratio of 1.6-1.9 is expected, whereas contamination results in a A260/A280 ratio of &#60;1.6.</li>
	<li>Prepare a reverse transcriptase (RT) reaction mix for a 20 &#181;L reaction volume. The mix contains RT enzyme mix, ribonuclease inhibitor, helper protein, primers, dNTPs, MgCl2, RNase free water, and 0.4 &#956;g of RNA sample.</li>
	<li>Briefly centrifuge the RT tubes to mix all the components at the bottom of the tube.</li>
	<li>Place the samples in the thermocycler instrument. Select the appropriate program for the RT. Run the RT for 10 min at 25 &#176;C, followed by the reverse transcription step for 120 min at 42 &#176;C and inactivation of the reverse transcriptase for 5 min at 85 &#176;C, cooling it down to 4 &#176;C at the end.</li>
	<li>Dilute the resulting cDNA with Tris(hydroxymethyl)aminomethane (Tris)-ethylenediaminetetraacetic acid (EDTA) (TE) buffer (10 mM Tris with 1 mM EDTA) to a final concentration of 0.4 &#181;g RNA used for RT per 100 &#181;L cDNA solution. Store the cDNA samples at -20 &#176;C.</li>
	<li>Perform real-time polymerase chain reaction (PCR) using a 10 &#181;L reaction volume. The reaction volume contains the master mix (containing DNA polymerase, uracil-DNA glycosylase, dNTPs with dUTP, passive reference and optimized buffer components), forward primer 45 &#181;M, reverse primer 45 &#181;M, probe 12.5 &#181;M (containing a reporter dye linked to the 5&#39; end of the probe, a minor groove binder at the 3&#39; end of the probe, and a nonfluorescent quencher at the 3&#39; end of the probe), 2 &#181;L cDNA and DEPC-treated water.</li>
	<li>Run an endogenous control (RPLP0) for relative quantification with the 2-&#916;&#916;CT method19.</li>
	<li>Add samples in duplicates and run a no template control by adding TE-buffer instead of cDNA. Run PCR at standard conditions (2 min at 50 &#176;C, 10 min at 95 &#176;C, 40 cycles of 15 s at 95 &#176;C, and 1 min at 60 &#176;C).</li>
	<li>Perform relative quantification of mRNA targets following the comparative CT method. The amount of mRNA normalized to the baseline sample is calculated as 2-&#916;&#916;CT, whereas &#916;&#916;CT is the difference between the &#916;CT (CT target- CT endogenous control) of sample and &#916;CT (CT target and CT endogenous control) of the baseline sample.</li>
</ol>
5. Quantification of protein content in the IVD medium
<ol>
	<li>Collect the medium conditioned by the IVD samples to measure the protein content in the medium. Perform enzyme-linked immunosorbent assay (ELISA) according to the protocol of the target protein.</li>
	<li>Bovine interleukine-8 (IL-8) is quantified by anti-bovine IL8 ELISA kit according to the manufacturer&#39;s instruction.</li>
</ol>

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<li>Walter, B. A., Illien-Jünger, S., Nasser, P. R., Hecht, A. C., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+validation+of+a+bioreactor+system+for+dynamic+loading+and+mechanical+characterization+of+whole+human+intervertebral+discs+in+organ+culture%5BTitle%5D)+AND+%22Journal+of+Biomechanics%22%5BJournal%5D)">Development and validation of a bioreactor system for dynamic loading and mechanical characterization of whole human intervertebral discs in organ culture.</a> Journal of Biomechanics. 47 (9), 2095-2101 (2014).</li>
<li>Rajan, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toll-Like+Receptor+4+%28TLR4%29+Expression+and+Stimulation+in+a+Model+of+Intervertebral+Disc+Inflammation+and+Degeneration%5BTitle%5D)+AND+%22Spine%22%5BJournal%5D)">Toll-Like Receptor 4 (TLR4) Expression and Stimulation in a Model of Intervertebral Disc Inflammation and Degeneration.</a> Spine. 38 (16), 1343-1351 (2013).</li>
<li>vanden Akker, G. G., Rorije, A. J., Davidson, E. N. B., vander Kraan, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenotypic+marker+genes+distinguish+inner+and+outer+annulus+fibrosus+from+nucleus+pulposus+tissue+in+the+bovine+intervertebral+disc%5BTitle%5D)+AND+%22Osteoarthritis+and+Cartilage%22%5BJournal%5D)">Phenotypic marker genes distinguish inner and outer annulus fibrosus from nucleus pulposus tissue in the bovine intervertebral disc.</a> Osteoarthritis and Cartilage. 25, 402(2017).</li>
<li>Du, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+cell+phenotype+induction+with+TGF-%CE%B21+and+collagen-polyurethane+scaffold+for+annulus+fibrosus+rupture+repair%5BTitle%5D)+AND+%22European+Cells+%26+Materials%22%5BJournal%5D)">Functional cell phenotype induction with TGF-β1 and collagen-polyurethane scaffold for annulus fibrosus rupture repair.</a> European Cells & Materials. 39, 1-17 (2020).</li>
<li>Risbud, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+phenotype+of+young+healthy+nucleus+pulposus+cells%3A+recommendations+of+the+Spine+Research+Interest+Group+at+the+2014+annual+ORS+meeting%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%3A+Official+Publication+of+the+Orthopaedic+Research+Society%22%5BJournal%5D)">Defining the phenotype of young healthy nucleus pulposus cells: recommendations of the Spine Research Interest Group at the 2014 annual ORS meeting.</a> Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 33 (3), 283-293 (2015).</li>
</ol>

The authors have nothing to disclose.

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-&#945; could induce inflammation in both bovine and human NP cells21, which is in accordance with other studies in the field confirming that TNF-&#945; can be used for inflammation simulation in IVD cells22,23. In the present study, a dose of 100 ng TNF-&#945; per IVD was used to induce inflammation. This dose showed effective induction of inflammatory markers when applied in combination with degenerative loading and limited nutrition15,20. In a recent study using TNF-&#945; as the sole initiation factor of IVD degeneration, a threshold of 100 ng TNF-&#945; / cm3 disc volume has been determined as an effective dose for the induction of inflammatory and degenerative changes in the IVD21. It is suggested that the TNF-&#945; dose used to induce inflammation should be normalized to the volume of the disc for reproducible effects. Furthermore, it should be ensured that there is a good distribution of the injection material in the IVD and that this injection will be adequately reproducible in subsequent experiments. As injection pressure may vary between individuals, there might be different distributions of the material among the same study groups in different experiments. One approach we chose to examine the equal distribution was using PBS diluted trypan blue injections. It is recommended to standardize the injection technique, for example, with injection pumps and predefined and reproducible injection rates. As shown previously, one single needle puncture with 22-gauge, 25-gauge, or 30-gauge needles into the IVD did not cause an effect that interacted with study outcomes20,21,24,25. It is recommended to consider the disc size for the volume of the substance to be injected and for the needle size used for injection. Further, it is recommended to use a single injection to avoid inducing potential degenerative effects caused by multiple annular injections21,26.
Some limitations of the present model need to be addressed. The loading of the IVDs requires access to bioreactors, which are currently not widely available in many labs working on IDD. However, the need for preclinical IVD degeneration models is rising. Organ culture models bring ethical benefits of reducing the need for animal models, and the one-time investment on bioreactor costs could be affordable considering the reproducibility of the degenerative stimulation, and the reductions in the costs associated with animal experiments. Nevertheless, some in vivo interactions, such as the role of the immune system, cannot be simulated with the provided organ culture model. Moreover, the measurement of nerve signals and the evaluation of pain that would be possible in animal models can currently not be considered with the current model. Some concerns were raised whether the mechanical properties of tail IVDs could be comparable with human IVDs as the upright spine position is unique in humans28. Anatomical and molecular properties of bovine tail IVDs, such as cell density, lack of notochordal cells, biochemical composition29,30, and mechanical properties of bovine caudal IVDs, such as range of motion in flexion, extension, torsion, and bending31 are very similar to human IVDs. Notably, human IVDs gain more and more attention recently for organ culture models28. Human cadaveric IVDs in ex vivo models are considered highly efficient as they can avoid species differences which are more clinically relevant32. In contrast to bovine IVD tails obtained from the abattoirs, this would require transplantation of human IVDs 24 h post mortem and, thus, ethical approval following local organ transplantation laws28. The present method can also be easily adapted to other species.
One major advantage of the technique compared to other methods of ex vivo cultures is the combination of intradiscal injection, pathological medium conditions, and detrimental loading to simulate the early stage of degenerative discs better. Walter et al.23 used TNF-alpha in the medium of dynamically loaded bovine IVDs instead of injection into the IVDs and showed increased transport of TNF-alpha from the medium into the nucleus pulposus compared to the annulus fibrosus, which is in accordance with our results. As we aimed to simulate the early stages of IDD, which occurs at the cellular level without major disruptions of the discs architecture12, we chose the TNF-alpha concentrations based on previous organ culture studies using TNF-alpha in the medium to simulate the early stages of IDD12,22,23. The use of other inflammatory stimulants and higher concentrations of TNF-alpha could be tested to simulate the desired degenerative disc state depending on the study question. Higher concentrations of TNF-alpha may better compensate the lack of systemic immune regulatory feedbacks present during disc degeneration as proposed by Ponnappan et al.12 The use of detrimental medium conditions with low-glucose was based on previous work showing that nutritionally limiting media and exposure to TNF-alpha mimics molecular change characteristics which are available in early disc degeneration state12, 27. Thus, the approach combines the evidence provided by previous studies in one ex vivo organ culture model of early degenerative disc disease.
This protocol can be further improved in several ways. The duration and extent of the loading and free-swelling period can be chosen based on the desired study plan. Long-term effects of inflammatory or degenerative stimulations on disc degeneration are of high clinical interest and can be accomplished with the current protocol. We used only selected genes considered highly relevant for the early state of IDD11. Additional validation of this model could include a widened assessment of genes and proteins, such as omics approaches. Consequently, other inflammatory factors could be investigated in combination with the degenerative loading, depending on the desired degenerative state. For example, lipopolysaccharide injections have been shown to stimulate inflammation in the IVD33. A comparison of different inflammatory stimulants can be accomplished with the present protocol to find the most appropriate inflammatory stimulant for the desired study question. We have evaluated changes of selected inflammatory markers (IL-6, IL-8), anabolic markers (aggrecan, collagen), and catabolic markers (matrix metallopeptidases, a disintegrin and metalloproteinases with thrombospondin motifs) with the present protocol20. As the whole gene expression profile might change, future examinations could include next-generation RNA sequencing techniques to determine novel biomarkers for disc degeneration diagnostics and therapeutic targets for early biological intervention. Furthermore, other methodologies, such as histology, immunohistochemical staining, biochemical measurements of extracellular matrix components (such as glycosaminoglycans, GAGs), and dynamic compressive tests can be performed to further analyze the biological, biochemical, and biomechanical properties of IVDs with the present model. Another possible analytical way would be to separately analyze the impact on outer AF and NP tissue by using bCol1a1, Col1a2, CD146, SM22&#945;, and MKX as gene expression markers for the outer AF34,35. The Spine Research Interest Group at the 2014 Annual ORS Meeting in New Orleans recommended following healthy NP phenotypic markers: HIF-1alpha, GLUT-1, aggrecan/collagen II ratio &#62;20, Shh, Brachyury, KRT18/19, CA12, and CD2436. The NP marker genes bBrachyury/T and bSecreted frizzled-related protein 2 were the most convincing separating NP from inner and outer AF tissue in the bovine IVD34. Furthermore, the sGAG content of the NP was reported to be significantly higher compared to the outer AF34.
In conclusion, this novel proinflammatory and degenerative IVD organ culture model provides relevant conditions to simulate early-stage IDD in a highly relevant 3D microenvironment. Certainly, the present protocol can be further modified according to the investigator&#39;s objectives. Furthermore, our model is able to reduce the number of needed study animals and thus totally reflects the 3R principles of reduce, replace, refine.

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&#39;s extracellular matrix (ECM), induced and triggered by multiple factors that lead to an accelerated pathology, neurological disorders, and eventually disability. Furthermore, IDD is associated with the release of proinflammatory cytokines, altered spine biomechanics, angiogenesis, and nerve ingrowth, which increases pain sensation, altogether causing chronic LBP (active discopathy)6,8. To date, treatment options include discectomy and subsequent fusion of the adjacent vertebrae, implantation of an IVD prosthesis, or non-surgical approaches, such as non-steroidal anti-inflammatory drugs, opioids, and muscle relaxants for patients with IDD9. Both current standard therapeutic options, surgical and non-surgical, are only partly effective and fail to address the underlying biological problem9,10. Early-stage degenerative disc disease is characterized by an initial inflammatory tissue response, especially an increase in tumor necrosis factor-alpha (TNF-alpha) expression11. These early disc changes primarily occur at the cellular level without disrupting the disc architecture and could previously be mimicked by nutritional deficiency under pro-inflammatory conditions12. Therefore, precise simulation of the in vivo situation to investigate these degeneration mechanisms and find suitable therapeutic targets is crucial. Additionally, to these simulations of molecular properties, the mechanical loading environment of the discs plays a key role in pathological and physiological changes of IVD. Consequently, combining these approaches would bring us one step forward to mimic the complex microenvironment of IVDs in vivo. There are currently no studies considering the aspect of dynamic loading along with the pro-inflammatory and nutritional setting to the best of our knowledge.
Although large animal models allow the investigation of potential relevant in vivo interactions, they are costly and work intensive. Moreover, as the use of animal models in research has long been a matter of controversy, the reduction of the number of animals needed to answer important research questions is of great interest. Finally, there is currently no ideal animal model to mimic IDD in IVD research13,14. Therefore, it is necessary to establish a cost-effective and reliable replacement, such as an organ culture model to simulate IDD and associated inflammatory and degenerative processes. Recently, the application of the present protocol on the establishment of a proinflammatory and degenerative organ culture model to simulate early-stage intervertebral disc disease allowed us to investigate the effect of anti-inflammatory drugs in the IDD organ culture15.
Here, we describe how to obtain bovine intervertebral discs and induce the state of early-stage IDD via a catabolic and proinflammatory microenvironment caused by direct intradiscal injection of tumor necrosis factor-alpha (TNF-&#945;) and degenerative loading in a bioreactor under low nutritive medium conditions. Figure 1 illustrates the experimental model and shows the bioreactor used to simulate degenerative and physiological loading conditions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62100/62100fig01.jpg" /> 
Figure 1: Illustration of the experimental setup. A: bovine tail; B: dissected bovine intervertebral discs; C: transfer of the disc to a well-plate with culture medium; D: loading the simulation in a bioreactor; E: intradiscal injection technique; F: IVD after injection of PBS/trypan blue dye to reveal distribution. IDD: intervertebral disc degeneration. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62100/62100fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

a proinflammatory, degenerative organ culture model to simulate early-stage intervertebral disc disease.

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.

Degenerative loading in low glucose medium combined with TNF-&#945; 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 1&#946; (IL-1&#946;) and TNF-&#945; in NP cells (data not shown). Furthermore, degenerative culture conditions did not alter the gene expression of IL-6 and IL-8 in AF cells.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62100/62100fig02.jpg" /> 
Figure 2: Gene expression levels in the nucleus pulposus (NP) tissue and annulus fibrosus (AF) tissues. These were measured after 4 days of culture under physiological or pathological culture condition, normalized to baseline (day 0). Means &#177; 95% confidence interval n=8, **p&#60;0.01. This figure has been modified from20. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62100/62100fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Consistent with the gene expression findings, the IL-8 protein content in the medium showed a marked increase after 2 days and 4 days compared to the physiological condition (Figure 3). However, the measurements at day 3 (after free-swelling or loading) revealed no significant differences between the study groups, although the results indicated higher values for the degenerative group.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62100/62100fig3v2.jpg" /> 
Figure 3: Quantification of the pro-inflammatory protein IL-8 release in the IVD culture medium after free swelling culture (FS) and dynamic loading (Load). The results are shown as the original concentrations in ng/mL in the medium without normalization. Mean &#177; 95% confidence interval, n=8, **p&#60;0.01. This figure has been modified from20. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62100/62100fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The disc height (DH) changes normalized to the DH after dissection are shown in Figure 4. Whereas DH reductions after loading revealed higher values (i.e., more height reduction) for the degenerative group compared to the physiological groups, no differences in disc height gains after the free-swelling period were seen between the study groups. This indicated that the difference in disc height changes between the pathological and physiological group was higher after the loading procedure. Furthermore, the differences were less pronounced after 1 day compared to the measurements on days 2 and 3, indicating a progressive effect of the degenerative and inflammatory conditions.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62100/62100fig4v2.jpg" /> 
Figure 4: Disc height changes normalized to the baseline values (after dissection). Mean &#177; 95% confidence interval. FS: free-swelling. N=10, ***p&#60;0.001. This figure has been modified from20. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62100/62100fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease. at JoVE.com

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

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 ...

a-proinflammatory-degenerative-organ-culture-model-to-simulate-early

Universidad San Sebastian

Research

JoVE Journal

Medicine

3.6K Views.  AO Research Institute Davos, Davos, Switzerland. Het protocol is bedoeld om in vivo omstandigheden van tussenwervelschijfdegeneratie te simuleren om de pathofysiologie ervan te onderzoeken zonder dat diermodellen nodig zijn. In vergelijking met in vitro celkweek handhaaft de bioreactororgaankweektechniek de cellen in hun inheemse biologische en biomechanische micromilieu naast de levering van een controleerbare en reproduceerbare kweekconditie. Begin met het grondig spoelen van de hele runderstaart met leidingwater om vuil en haar op het op...

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

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Bioengineering

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Ovine Lumbar Intervertebral Disc Degeneration Model Utilizing a Lateral Retroperitoneal Drill Bit Injury

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration have been developed. The ideal animal model should closely mimic the human intervertebral disc with regard to morphology, biomechanical properties and the absence of notochordal cells. The sheep lumbar intervertebral disc model fulfils these criteria. We present an ovine model of intervertebral disc degeneration utilizing a drill bit injury through a lateral retroperitoneal approach. The lateral approach significantly reduces the incision and potential morbidity associated with the traditional anterior approach to the ovine spine. Utilization of a drill-bit method of injury affords the ability to produce a consistent and reproducible injury, of precise dimensions, that initiates a consistent degree of intervertebral disc degeneration. The focal nature of the annular and nucleus pulposus defect more closely mimics the clinical condition of focal intervertebral disc herniation. Sheep recover rapidly following this procedure and are typically mobile and eating within the hour. Intervertebral disc degeneration ensues and sheep undergo necropsy and subsequent analysis at periods from eight weeks. We believe that the drill bit injury model of intervertebral disc degeneration offers advantages over more conventional annular injury models.

Intervertebral disc degeneration is a significant contributor to back pain and a leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration exist. We demonstrate an ovine model of intervertebral disc degeneration, utilizing a drill bit, which achieves a consistent disc injury and reproducible level of disc degeneration.

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous ...

Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To decode disc degeneration and to be able to develop regenerative approaches a thorough understanding of the cellular biology of the IVD is essential. One aspect of this biology that still remains unanswered is the question of how cells are spatially arranged in a physiological state and during degeneration. The biological properties of the IVD and its availability make this tissue difficult to analyze. The present study investigates spatial chondrocyte organization in the anulus fibrosus from early embryonic development to end-stage degeneration. An optical sectioning method (Apotome) is applied to perform high resolution staining analyses using bovine embryonic tissue as an animal model and human disc tissue obtained from patients undergoing spine surgery. From a very high chondrocyte density in the early embryonic bovine disc, the number of cells decreases during gestation, growth, and maturation. In human discs, an increase in cellular density accompanied the progression of tissue degeneration. As had already been demonstrated in articular cartilage, cluster formation represents a characteristic feature of advanced disc degeneration.

We present a method to investigate spatial chondrocyte organization in the anulus fibrosus of the intervertebral disc using an optical sectioning method.

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To ...

Biology

Comparing Transperitoneal Retroperitoneal Access and Percutaneous Puncture for Lumbar Disc Degeneration Modeling in Rabbits

This study compares the efficacy of two methods for inducing intervertebral disc degeneration (IVDD) in rabbits: percutaneous and trans-retroperitoneal puncture of the annulus fibrosus. Fifteen healthy male New Zealand White rabbits were randomly assigned to three groups: sham, percutaneous puncture, and trans-retroperitoneal puncture. A comprehensive assessment, including mortality rates, morphological and histological evaluations, radiological imaging, and biomarker analysis, was conducted to ensure an accurate and detailed comparison between the two methods. The results demonstrate that both puncture techniques successfully induced IVDD in the rabbit model. However, the trans-retroperitoneal approach resulted in more pronounced degenerative changes in the intervertebral discs while maintaining a significantly lower mortality rate compared to the percutaneous method. These findings highlight the advantages of the trans-retroperitoneal approach in IVDD modeling. This study provides valuable insights into the establishment of IVDD models and lays a foundation for future investigations into effective treatment strategies for low back pain, ultimately improving patient outcomes.

This protocol compared percutaneous and trans-retroperitoneal punctures in a rabbit intervertebral disc degeneration (IVDD) model. Both methods induced IVDD; however, the trans-retroperitoneal approach resulted in more extensive changes and lower mortality.

This study compares the efficacy of two methods for inducing intervertebral disc degeneration (IVDD) in rabbits: percutaneous and trans-retroperitoneal ...

Generating a Pro-Inflammatory Organ Culture Model to Simulate an Early Intervertebral Disc Disease

Source: Saravi, B., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62100">A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.</a> J. Vis. Exp. (2021).
This video demonstrates a technique for culturing bovine intervertebral discs in bioreactors to develop a three-dimensional organ culture model. Specific nutrient-deprived media and degenerative pressure conditions are applied to the discs, mimicking the pro-inflammatory and degenerative microenvironment observed during early-stage intervertebral disc disease. The disc heights are measured post-treatment to analyze the effects of nutrient deprivation and degenerative loading.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Dissection of the bovine ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Generating an Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease

Source: Saravi, B. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62100">A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.</a>&#160;J. Vis. Exp. (2021).
This video demonstrates the development of a pro-inflammatory and degenerative organ culture model for early-stage intervertebral disc (IVD) disease using TNF-&#945;. It outlines the procedure for injecting TNF-&#945; into the intervertebral disc and explains the mechanisms through which TNF-&#945; induces inflammation and degradation of the IVD.

Source: Saravi, B. et al., A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.&#160;J. Vis. Exp. ...

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

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