Sulfated hydrogels as primary intervertebral disc cell culture systems

INTRODUCTION: Intervertebral disc (IVD) degeneration is a key contributor for low back pain, a leading cause of disability worldwide1. During degeneration, IVD aging is accelerated, leading to progressive structural changes, including blood vessel and nerve ingrowth that promote discogenic pain2. In vitro studies require novel biomaterials that mimic the IVD extracellular matrix (ECM) to provide mechanical support and a reservoir of cytokines and growth factors. As proteoglycans with their attached sulfated glycosaminoglycans (GAGs) are one of the major components of the ECM, the ECM’s sulfation state could be a key factor for IVD cell-fate3. Thus, we aim to explore human NP cell fate using a novel sulfated alginate model with varying degrees of sulfation (DS). METHODS: Primary human NP cells were expanded, mixed with solutions of i) 2.5% of standard alginate, ii) 0.1 DS, and iii) 0.2 DS alginate (4 x 106 cells/ml) and casted in 27 l cylindrical-shaped carriers (4 mm diameter, 2 mm height). Carriers were cultured for two weeks for phenotype recovery and were collected with the culture media on day 0, 7 and 14. RESULTS: A significant decrease of cell density (p<0.05) was observed in 0.2 DS alginate after 7 and 14 days of culture. Similarly, cell viability was significantly reduced (p<0.05) in 0.2 DS alginate after 7 days of culture (N=4). In addition, cell metabolic activity tended to be decreased in 0.2 DS alginate compared to standard alginate after 14 days of culture. Surprisingly, ECM remodeling factors such as MMP2 and TIMP1 were slightly upregulated in the 0.1 DS group (N=1), whereas catabolic cytokines were downregulated in the 0.1% DS group. DISCUSSION & CONCLUSIONS: We demonstrate significant cellular differences between 0.2 DS alginate vs standard alginate and 0.1 DS alginate. Particularly, a significant decrease in cell density, metabolic activity and viability were observed in the 0.2 DS alginate after 7 days of culture. According to the secretome, the sulfated alginate group seems to possess increased catabolic ECM remodeling with lower secretion of catabolic factors, suggesting less responsive NP cells to ECM structural changes. Overall, standard alginate seems to be the best option for NP cell 3D culture models. ACKNOWLEDGEMENTS: This project was supported by the Marie Skłodowska Curie International Training Network “disc4all” under the grant agreement #955735. REFERENCES: 1FY. Wang et al (2020) JOR Spine 5:1186. 2P. Bermudez-Lekerika et al (2022) Front Cell Dev Biol 29(10):924692. 3E. Lazarus et al (2021) Cells 10(12):3568. Keywords: Hydrogels and injectable systems, In vitro microenvironment

Immuno-modulatory effects of intervertebral disc cells

Low back pain is a highly prevalent, chronic, and costly medical condition predominantly triggered by intervertebral disc degeneration (IDD). IDD is often caused by structural and biochemical changes in intervertebral discs (IVD) that prompt a pathologic shift from an anabolic to catabolic state, affecting extracellular matrix (ECM) production, enzyme generation, cytokine and chemokine production, neurotrophic and angiogenic factor production. The IVD is an immune-privileged organ. However, during degeneration immune cells and inflammatory factors can infiltrate through defects in the cartilage endplate and annulus fibrosus fissures, further accelerating the catabolic environment. Remarkably, though, catabolic ECM disruption also occurs in the absence of immune cell infiltration, largely due to native disc cell production of catabolic enzymes and cytokines. An unbalanced metabolism could be induced by many different factors, including a harsh microenvironment, biomechanical cues, genetics, and infection. The complex, multifactorial nature of IDD brings the challenge of identifying key factors which initiate the degenerative cascade, eventually leading to back pain. These factors are often investigated through methods including animal models, 3D cell culture, bioreactors, and computational models. However, the crosstalk between the IVD, immune system, and shifted metabolism is frequently misconstrued, often with the assumption that the presence of cytokines and chemokines is synonymous to inflammation or an immune response, which is not true for the intact disc. Therefore, this review will tackle immunomodulatory and IVD cell roles in IDD, clarifying the differences between cellular involvements and implications for therapeutic development and assessing models used to explore inflammatory or catabolic IVD environments

Gram positive bacteria within the intervertebral disc and their potential influence on nucleus pulposus cells

Introduction Modic changes (MC) that are vertebral bone marrow lesions visualised on magnetic resonance imaging have been associated with disc degeneration. For MC type I one of the identified aetiologies is infection of the intervertebral disc (IVD) [1]. However, there is controversy whether infection of the IVD is linked to disc degeneration. Even though there is increasing evidence for bacterial presence within the herniated disc, there are limited studies, which determine whether bacteria are present in the intact IVD in vivo or whether they represent contamination. This study aimed to investigate bacterial presence in non-herniated human IVDs and the potential influence of bacterial components on disc cells. Material and Methods Immunohistochemical staining for Gram-positive bacterial membrane component, and specific antibodies for Staphylococcus aureus, Cutibacterium acnes Cellular recognition receptors Toll-like receptor (TLR) 2, TLR4 and NLR family pyrin domain containing 3 (NLRP3) and the pyroptosis marker Gasdermin D were performed on 90 human IVD samples. Furthermore, human nucleus pulposus cells in monolayer were treated with Lipopolysaccharide (LPS) (5-50μg/ml) and Peptidoglycan (PGN) (5-50μg/ml) for 48 hours. Cells in alginate were treated with PGN up to 72 hours. Secretome analysis was performed using Luminex for cytokines, chemokines, matrix degrading enzymes and other secreted factors. Statistical analysis was performed using Kruskal-Wallis and Dunn’s multiple comparison test. Results Gram-positive bacteria were internalized by at least one disc cell in 90 % of the samples. The percentage of cells containing bacteria across the NP was ~3%. Analysis for the abundance for the other factors is ongoing. Furthermore, the correlation between the abundancy of bacteria, the TLR2, TLR4, NLRP3, Gasdermin D and the histological grade of disc degeneration will be investigated. Treatment of NP cells with LPS and PGN resulted in an increase of several catabolic cytokines such as IL-1, TNF, IL-6 and IFN-γ alongside increased production of chemokines, neurotrophic and angiogenic factors associated with IVD degeneration. Figure 1| Presence of bacteria in intervertebral discs and their potential effects A Average cells containing internalized bacteria after immunohistochemical (IHC) staining for Gram-positive bacteria was 4% (ranging 110%). Current work is determining correlations with histological grade of degeneration. B IHC staining for C. acnes lysate with enzyme antigen retrieval at a 1.100 dilution. Nuclei are stained in blue with Hematoxylin, the presence of C. acnes is shown in brown. C Cyto- and Chemokine expression of Nucleus Pulposus (NP) cells in Alginate stimulated with Peptidoglycans (PGN) from Gram-positive bacteria. PGN treatment induced catabolism in the NP cells in a dos dependent manner. Conclusion This study demonstrated that Gram-positive bacteria are present in non-herniated and cadaveric human disc samples. Furthermore, bacterial cell membrane components triggered a catabolic response in human disc cells. Ongoing interaction studies between bacteria and NP cells will give insights into the internalisation mechanisms and potential role in disc degeneration. References 1.Dudli et al. European Spine Journal vol. 25. 3723–3734, 2016. Acknowledgements This Project is part of the Disc4All Training network advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MCA-ITN-ETN2020GA:955735

The metabolic role of IL-4 and IL-10 in Intervertebral Disc Degeneration

Intervertebral disc (IVD) degeneration is a pathological process often associated with chronic back pain and considered a leading cause of disability worldwide1 . During degeneration, progressive structural and biochemical changes occur, leading to blood vessel and nerve ingrowth and promoting discogenic pain2 . In the last decades, several cytokines have been applied to IVD cells in vitro to investigate the degenerative cascade. Particularly, IL-10 and IL-4 have been predicted as important anabolic factors in the IVD according to a regulatory network model based in silico approach3 . Thus, we aim to investigate the potential presence and anabolic effect of IL-10 and IL-4 in human NP cells (in vitro) and explants (ex vivo) under hypoxia (5% O2) after a catabolic induction. Primary human NP cells were expanded, encapsulated in 1.2% alginate beads (4 x 106 cells/ml) and cultured for two weeks in 3D for phenotype recovery while human NP explants were cultured for five days. Afterwards, both alginate and explant cultures were i) cultured for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (single treatments) or ii) stimulated with 0.1 ng/ml IL-1β for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (combined treatments). The presence of IL-4 receptor, IL-4 and IL-10 was confirmed in human intact NP tissue (Fig 1). Additionally, IL-4 single and combined treatments induced a significant increase of proinflammatory protein secretion in vitro (Fig. 2A-C) and ex vivo (Fig. 2D and E). In contrast, no significant differences were observed in the secretome between IL-10 single and combined treatments compared to control group. Overall, IL-4 containing treatments promote human NP cell and explant catabolism in contrast to previously reported IL-4 anti-inflammatory performance4 . Thus, a possible pleiotropic effect of IL-4 could occur depending on the IVD culture and environmental condition. References:1 J. Hartvigsen et al (2018) The Lancet 391:2356-67, 2 PPA. Vergroesen et al (2015) Osteoarthritis and Cartilage 23:1165-77, 3 S. Tseranidou et al, EBS 2023 28t

The role of IL-4 and IL-10 in degenerating intervertebral disc

Introduction Intervertebral disc (IVD) degeneration is a pathological process often associated with chronic back pain and considered a leading cause of disability worldwide [1]. During degeneration, progressive structural changes occur leading to blood vessel and nerve ingrowth that promote discogenic pain [2]. In the last decades, several cytokines have been applied to IVD cells in vitro to investigate the degenerative cascade. Particularly, IL-10 and IL-4 have been predicted as important anabolic factors in the IVD according to a regulatory network model based in-silico approach [3]. Thus, we aim to investigate the potential presence and anabolic effect of IL-10 and IL-4 in human NP cells (in-vitro) and explants (ex-vivo) under hypoxia (5% O2 )