Project 059

Degeneration of the intervertebral disc accounts for up to 40% of all cases of low back pain. This project aims towards an in vitro model for degeneration and inflammation of the human disc.

IL-1β and TNF-α, two major proinflammatory cytokines, were shown to regulate the expression of A Disintegrin and Metalloprotease with Thrombospondin Motifs (ADAMTS), major catabolic enzymes for extracellular matrix deterioration, through activation of MAPK and NF-κB signaling, which leads to tissue degeneration. Regarding the association to pain, current research arises evidence TNF-α being neurotoxic, inducing pain behavioral changes, axonal and myelin injury, intravascular coagulation and increased vascular permeability. Targeting proinflammatory cytokines by minimally invasive application of their respective inhibitors could prevent further degeneration and development of chronic pain.

Since the IVD does not possess a sufficient self-repair capacity, current treatment options for DDD range from conservative treatments to invasive therapies for severe and symptomatic courses of DDD, like spinal fusion or total disc replacement, although long-term results do not show significant differences compared to conservative therapies and complications are common. Additionally, none of the current invasive techniques address the underlying biological pathology.

Therefore therapies that inhibit the expression of TNF-α and IL-1β may be a promising therapeutic approach to consider, aiming to reduce inflammation and maintain IVDs extracellular matrix.

The aim of our study is to (1) develop an IVD organ culture degeneration and inflammation model in order to mimic and investigate the inflammatory pathogenesis in early stage DDD. Additionally (2) we aim to assess the biological and mechanical effect of selective cytokine inhibition as a potential treatment alternative for DDD. The experiments will be conducted with an organ culture system under degenerative dynamic loading and proinflammatory conditions.

Study Aims
Our study aimed to (1) develop an IVD organ culture degeneration and inflammation model to mimic and investigate the inflammatory pathogenesis in early-stage DDD. Additionally (2) we sought to assess the biological and mechanical effect of selective cytokine inhibition as a potential treatment alternative for DDD. The experiments were conducted utilizing an organ culture system under degenerative dynamic loading and proinflammatory conditions.

3R-Principle: Reduce, Replace, Refine
Our group has recently implemented an ex vivo whole IVD organ culture bioreactor system. This model is capable of culturing whole bovine IVDs for up to four weeks under mechanical load, being much more relevant for testing anti-inflammatory treatment than cell culture experiments, and at the same time reducing the need for animal studies. All bovine tails were obtained from our local abattoirs. No calf had to be killed for our project. Our translational model is a highly efficient approach to analyze the interplay between inflammation and intervertebral disc degeneration, the molecular pathways, and biomechanical behavior within a 3D environment.

Projekt summary
Part 1 - Establishment of a proinflammatory and degenerative intervertebral disc organ culture model
Our group previously implemented an ex vivo whole IVD organ culture bioreactor system that has been used to study IVD degeneration and regeneration (Junger et al. 2009, 2010, 2012). Between 2016 and 2017, we successfully modified our already implemented IVD degeneration organ culture model towards a combination of inflammation and degeneration model to mimic and investigate the inflammatory pathogenesis in early-stage DDD (Lang et al., 2018).

Bovine IVDs with endplates were harvested and cultured with or without TNF-α intradiscal injection (100 ng TNF-α / IVD) for 4 days. IVDs were cultured within a bioreactor system under 2 different loading and medium conditions: (1) physiological loading (0.02-0.2 MPa; 0.2 Hz; 2h/day, 3 days) and high glucose (4.5 g/L) medium (Phy), or (2) degenerative loading (0.32-0.5 MPa; 5 Hz; 2h/day, 3 days) and low glucose (2 g/L) medium (Deg). Between dynamic loading cycles, IVDs were cultured free swelling in well plates. TNF-α was injected into the disc nucleus pulposus (NP) tissue after the first dynamic loading cycle on day 1. Disc height was recorded daily after load and free swelling (FS). Conditioned medium was collected for nitric oxide (NO) and Glycosaminoglycan (GAG) analysis. After 4 days, NP and annulus fibrosus (AF) tissue were harvested and gene expression was analyzed using real-time PCR. GAG/DNA and Hydroxy-proline (OHP)/DNA level of the disc tissue was assessed.

Results indicated that a short-term application of degenerative loading increased the disc height loss of bovine IVDs, while TNF-α injection did not. TNF-α injection alone caused a catabolic effect on the organ cultured IVDs, indicated by higher GAG release into conditioned medium and down-regulated COL1 expression. Only under combined TNF-α injection and degenerative culture condition, a significant upregulation of catabolic and inflammatory markers gene expression was observed in disc tissue, together with enhanced NO release, which indicated an additive inflammatory effect. In summary, a proinflammatory and degenerative organ culture model was established by TNF-α intradiscal injection, detrimental loading, and limited nutrition to mimic the early course of DDD (Lang et al., 2018). This model was utilized as a platform for further experiments - screening of therapeutic agents in further pre-clinical studies (Aim 2).

Part 2 - Validation of anti-inflammatory and regenerative drug therapy in a bioreactor-guided intervertebral disc organ culture model
After validation of our degenerative and inflammatory model, we tested novel anti-inflammatory agents such as Anti-IL-1, Anti-IL-6 antibodies and other cytokine inhibitors in the same experimental setup as performed in the initial experiment to investigate whether these novel therapies are able to slow down the progression of DDD. Whereas Anti-IL-1 and Anti-IL-6 agents did not reveal a significant anti-inflammatory impact, Etanercept, a TNF-α inhibitor, and Tofacitinib, a JAK inhibitor, showed the potential to slow down degeneration and reduce inflammation in the organ culture model.

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; 2h/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; 2h/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. In summary, Etanercept and Tofacitinib were able to neutralize the proinflammatory and catabolic environment in the DDD organ culture model. However, combined anti-inflammatory and anabolic treatment may be required to constrain accelerated DDD and relieving inflammation-induced back pain (Li et al., 2020).

Publikations:

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.

Awards, Scholarships, Titles:

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 presentations:

“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

Oral presentations:

“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

---