The biological and mechanical effects of selective pro-inflammatory cytokine inhibition in degenerative disc disease

Low back pain (LBP) is the most common cause of work disability worldwide and has enormous socio-economic implications. More than 40% of LBP cases are attributable to ‘degenerative disc disease’ (DDD), which is characterised by accelerated catabolism of the extracellular matrix (ECM) of the intervertebral disc (IVD). This degradation is induced and triggered by mechanical stress, trauma, genetic predisposition and inflammation, and can lead to immobilising pain, dysfunctional spinal biomechanics and neurological complications.

Current treatment methods include not only conservative therapies but also spinal fusion or the implantation of an artificial disc prosthesis. In addition to unsatisfactory long-term outcomes and frequent complications, none of these procedures addresses the underlying pathology of disc-related back pain. Pro-inflammatory cytokines such as TNF-α and IL-1β play a key role in the progression and chronicity of DDD, as they accelerate disc catabolism by stimulating enzymes that degrade the extracellular matrix and contribute to pain sensitisation. Anti-inflammatory therapies using disease-modifying antirheumatic drugs (DMARDs) could offer a less invasive alternative for DDD. Currently, DMARDs are primarily used in the treatment of chronic inflammatory conditions such as rheumatoid arthritis or Crohn’s disease. However, recent studies suggest that cytokine inhibitors may also have significant therapeutic potential in DDD.

Objective:

The aim of this study was (1) to develop an inflammatory model to simulate the early stages of degenerative disc disease and (2) to evaluate the biological and mechanical effects of specific cytokine inhibitors as potential therapeutic alternatives. The experiments were conducted in an organ culture system under degenerative dynamic loading and pro-inflammatory conditions. 3R principle: Reduce, Replace, Refine: Our research group has recently implemented an ex vivo intervertebral disc organ culture bioreactor. This model is capable of culturing bovine intervertebral discs for up to four weeks under (dynamic or static) mechanical loading and under pro-inflammatory conditions. This method is far more realistic and relevant for investigating the pathogenesis of DDD and new anti-inflammatory treatment strategies than conventional cell culture experiments. At the same time, it reduces the need for in vivo experiments. All calf tails are sourced from local abattoirs. No calves had to be killed specifically for this project. Bovine intervertebral discs are animal by-products that are not used for human consumption during the slaughter process. The model developed in this project is a highly efficient approach for analysing the interplay between disc degeneration and inflammation, as well as the molecular mechanisms and biomechanical properties within a 3D environment. For every experiment carried out as part of these ex vivo studies (for which two calf tails are required), the killing of two nearly fully grown large animals in an in vivo experiment can be avoided.

Project timeline:

Part 1 – Development of an inflammatory model to simulate the early stages of degenerative disc disease Over the past few years, the research group at the AO Research Institute in Davos has implemented an ex vivo intervertebral disc organ culture bioreactor and optimised the culture conditions (Junger et al. 2009, 2010, 2012). As part of the InflamoDisc project, the established bovine intervertebral disc organ culture system was successfully further developed between 2016 and 2017 to realistically simulate the early stages of DDD under degenerative and pro-inflammatory conditions (Lang et al., 2018). To promote accelerated degeneration, the intervertebral discs were cultured in limited nutrient solution and subjected to high-frequency dynamic axial loading for 2 hours per day (0.32–0.5 MPa; 5 Hz) for 4 days. To induce an inflammatory process, the degenerative intervertebral discs were additionally cultured under pro-inflammatory conditions by injecting 100 ng TNF-α per disc for 4 days.
A specialised bioreactor (Fig. 2-B) was used to apply sinusoidal dynamic compression to the intervertebral discs (IVDs). For this purpose, the IVDs are housed in sealed chambers under sterile conditions. The bioreactor units are maintained in an incubator at 37°C, 5% CO₂ and 85% humidity. After 4 days, the IVD tissue was resected for biochemical analysis. Cell viability and the DNA, glycosaminoglycan and collagen content were measured. The gene expression of relevant anabolic and catabolic IVD EZM factors was investigated. Finally, Safranin O/Fast Green staining was performed for histological analysis. The results of our work showed that, through the combined use of TNF-α and degenerative culture conditions, we were able to create and reproduce a catabolic and pro-inflammatory environment corresponding to the early phase of DDD. Typical inflammatory mediators involved in DDD were also expressed at higher levels in our model. The successful further development of the organ culture model was published in 2018 and served as a platform for all subsequent experiments in the project.

Part 2 – Evaluation of the biological and mechanical effects of specific cytokine inhibitors as potential therapeutic alternatives for DDD First, the protective effects of the IL-1 antagonist anakinra and the IL-6 antagonist tocilizumab were investigated using our degenerative and pro-inflammatory intervertebral disc organ culture system. Neither IL-1 nor IL-6 inhibition showed any significant effect against the catabolic environment, which is intended to realistically represent the early stage of DDD. However, we were able to verify on multiple occasions that our modified model could reliably and reproducibly simulate the catabolic and inflammatory conditions. Following extensive research, we investigated the anti-inflammatory and anti-degenerative effects of the TNF-α inhibitor etanercept and the Janus kinase (JAK) inhibitor tofacitinib. In the treatment group, the TNF-α/JAK3 inhibitors were applied to the intervertebral disc or added to the culture medium. Intact IVDs served as the positive control. IVDs without treatment served as the negative control. IVD measurements were recorded at various time points before and after loading. After 4 days, the IVD tissue was resected for biochemical analysis. Cell viability and the DNA, glycosaminoglycan and collagen content were measured. DThe gene expression of relevant anabolic and catabolic IVD ECM factors was investigated. Finally, Safranin O/Fast Green staining was performed for histological analysis. Our work demonstrated that the inhibition of TNF-α and Janus kinase (JAK) shows promise. The use of etanercept and tofacitinib reduced the expression of pro-inflammatory and catabolic mediators and neutralised the degenerative environment within the organ culture model. However, a combination of anabolic and anti-inflammatory agents is likely to be necessary for clinical use. We were able to publish the successful findings in 2020 (Li et al., 2020).

Publikationen:
Lang G, Liu Y, Geries J, Zhou Z, Kubosch D, Südkamp NP, Richards RG, Alini M, Grad S, Li Z. An intervertebral disc whole organ culture system to investigate proinflammatory and degenerative disc disease condition. J Tissue Eng Regen Med 12 (4), e2051-e2061.
Pfannkuche JJ, Guo W, Cui S, Ma J, Lang G, Peroglio M, Richards RG, Alini M, Grad S, Li Z (2019) Intervertebral disc organ culture for the investigation of disc pathology and regeneration - benefits, limitations, and future directions of bioreactors. Connect Tissue Res:1-18. doi: 10.1080/03008207.2019.1665652.
Du J, Pfannkuche JJ, Lang, G, Haeckel, S, Creemers L, Alini M, Grad S, Li Z (2020). Proinflammatory intervertebral disc cell and organ culture models induced by tumor necrosis factor alpha (JOR Spine, 2020)
Li Z, Gehlen Y, Heizmann F, Grad S, Alini A, Richards RG, Kubosch D, Südkamp NP, Izadpanah K, Kubosch EJ, Lang G (2020). Preclinical ex-vivo testing of anti-inflammatory drugs in a bovine intervertebral degenerative disc model. Frontiers in Bioengineering and Biotechnology, section Tissue Engineering and Regenerative Medicine.
Preise/Stipendien/Auszeichnungen/Titel:
2017 DWG Nachwuchspreis for M.D. candidate Yishan Liu - best abstract and oral presentation award from German Spine Congress 2017 for “An intervertebral disc whole organ culture system to investigate proinflammatory and degenerative disc disease condition"
2017 DGOOC Student scholarship for M.D. candidate Yishan Liu for her doctoral thesis “An intervertebral disc whole organ culture system to investigate proinflammatory and degenerative disc disease condition"
2017 Finalist of best poster award on ORS 2017: A proinflammatory and degenerative intervertebral disc organ culture model by combining TNF-α intradiscal injection and detrimental dynamic loading
2018 DGOOC Student scholarship for M.D. candidate Yannik Gehlen for his doctoral thesis "The effect of the selective JAK3- Inhibitor Tofacitinib in degenerative disc disease"
2018 Finalists of best paper award DKOU ORS Travel Award 2018: “Anti-inflammatory and regenerative drug therapy as biological treatment for degenerative disc disease”
2018 Kurt-Steim-Award from Albert Ludwigs University Freiburg for “An intervertebral disc whole organ culture system to investigate proinflammatory and degenerative disc disease condition”
2018 Finalist of ON/Eurospine Award for „Validation of anti-inflammatory and anti-degenerative drug therapy using a bioreactor- guided intervertebral Disc organ culture model “, EuroSpine 2018, Barcelona, Spain
2019 Berta-Ottenstein-Fellowship 2019 for Advanced Clinician Scientists – Gernot Lang
2020 Habilitation Gernot Lang - „Staged Therapeutic Concepts in Degenerative Disc Disease Including Biological and Minimally Invasive Treatment Approaches“

Poster-Präsentationen:
“A proinflammatory and degenerative intervertebral disc organ culture model by combining TNF-α intradiscal injection and detrimental dynamic loading”, ORS 2017 Annual Meeting, San Diego, CA, USA, 19.-22.03.2017
“Anti-inflammatory and regenerative drug therapy for the treatment of degenerative disc disease: Validation in organ culture model”. 2017 ORS PSRS 4th International Spine Research Symposium (poster), Philadelphia, PA, USA
„Validation of anti-inflammatory and anti-degenerative drug therapy using a bioreactor-guided intervertebral Disc organ culture model“, EuroSpine 2018, Barcelona, Spain
Vorträge:
“Proinflammatory cytokines in DDD – our next target?”, Spine Research Group meeting, Icahn School of Medicine at Mount Sinai, New York, September 26, 2016
“Inflammation in DDD”, Spine Research meeting, Shriners Hospitals for Children, The Orthopaedic Research Laboratory, McGill University, Montreal, Canada, September 28, 2016
“Biological approaches for disc repair and regeneration”, 10th New York City Minimally Invasive Spine and Navigation Course: Case-Based and Hands-On, December 12/16/2016, New York
“A proinflammatory and degenerative intervertebral disc organ culture model to investigate novel anti-inflammatory treatment approaches for degenerative disc disease“, Global Spine Congress 2017, Milano, Italy
“An intervertebral disc organ culture model mimicking proinflammatory and degenerative disease condition”, TERMIS EU 2017, Davos, Switzerland
“Establishment of a proinflammatory and degenerative intervertebral disc ex vivo system to investigate anti-inflammatory therapies for degenerative disc disease”, DKOU 2017, Berlin, Germany
"Entwicklung eines inflammatorischen und degenerativen Bandscheiben-Organkulturmodells zur Simulation der Frühphase der degenerativen Bandscheibenerkrankung", 12. German Spine Congress (DWG) 2017, Stuttgart, Germany - DWG Nachwuchspreis for Yishan Liu
“Short- and mid-term effect of TNF-α intradiscal injection and detrimental dynamic loading in intervertebral disc organ culture”, 2017 AOSpine Masters Symposium, Bern, Switzerland
“An intervertebral disc organ culture model mimicking proinflammatory and degenerative disease condition, 2017 TERMIS-EU, Davos, Switzerland
“Establishment of a proinflammatory and degenerative intervertebral disc organ culture model”, 2017 Biospine, Berlin, Germany
“Anti-inflammatory and regenerative drug therapy as biological treatment for degenerative disc disease”, DKOU 2018, Berlin, Germany
„Validation of anti-inflammatory and regenerative drug therapy in a bioreactor-guided intervertebral disc organ culture model“, ISSLS Annual Meeting 2018 in Banff, Canada
"Validation of anti-inflammatory and regenerative drug therapy in an intervertebral disc organ culture model", ECM, 2018, Davos, Switzerland
"Pre-clinical testing of anti-inflammatory compounds in an intervertebral disc organ culture model", BioSpine, 2019 Rome, Italy
"Preclinical Testing of Anti-Inflammatory Compounds Using a Whole Intervertebral Disc Organ Culture Model", Global Spine Congress, 2019, Toronto, Canada
"In vitro Evaluation der protektiven Wirkung von Losartan in der Behandlung der degenerativen Bandscheibenerkrankung" , 14. Deutscher Wirbelsäulenkongress, 28. – 30. November 2019, München