title: A Degenerative/Proinflammatory Intervertebral Disc Organ Culture: An Ex Vivo Model for Anti-inflammatory Drug and Cell Therapy.
abstract: Resolution of intervertebral disc (IVD) degeneration-associated inflammation is a prerequisite for tissue regeneration and could possibly be achieved by strategies ranging from pharmacological to cell-based therapies. In this study, a proinflammatory disc organ culture model was established. Bovine caudal disc punches were needle punctured and additionally stimulated with lipopolysaccharide (10 μg/mL) or interleukin-1β (IL-1β, 10-100 ng/mL) for 48 h. Two intradiscal therapeutic approaches were tested: (i) a nonsteroidal anti-inflammatory drug, diclofenac (Df) and (ii) human mesenchymal stem/stromal cells (MSCs) embedded in an albumin/hyaluronan hydrogel. IL-1β-treated disc organ cultures showed a statistically significant upregulation of proinflammatory markers (IL-6, IL-8, prostaglandin E2 [PGE2]) and metalloproteases (MMP1, MMP3) expression, while extracellular matrix (ECM) proteins (collagen II, aggrecan) were significantly downregulated. The injection of the anti-inflammatory drug, Df, was able to reduce the levels of proinflammatory cytokines and MMPs and surprisingly increase ECM protein levels. These results point the intradiscal application of anti-inflammatory drugs as promising therapeutics for disc degeneration. In parallel, the immunomodulatory role of MSCs on this model was also evaluated. Although a slight downregulation of IL-6 and IL-8 expression could be found, the variability among the five donors tested was high, suggesting that the beneficial effect of these cells on disc degeneration needs to be further evaluated. The proinflammatory/degenerative IVD organ culture model established can be considered a suitable approach for testing novel therapeutic drugs, thus reducing the number of animals in in vivo experimentation. Moreover, this model can be used to address the cellular and molecular mechanisms that regulate inflammation in the IVD and their implications in tissue degeneration.
authors.affiliation: 1 Institute of Orthopaedic Research and Biomechanics, Center for Musculoskeletal Research, University of Ulm , Ulm, Germany .|2 Instituto de Investigação e Inovação em Saúde, Universidade do Porto , Porto, Portugal .|3 Instituto de Engenharia Biomédica (INEB), Universidade do Porto , Porto, Portugal .|4 Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto , Porto, Portugal .
authors.forename: Graciosa Q
authors.initials: GQ
authors.lastname: Teixeira
authors.affiliation: 1 Institute of Orthopaedic Research and Biomechanics, Center for Musculoskeletal Research, University of Ulm , Ulm, Germany .
authors.forename: Antje
authors.initials: A
authors.lastname: Boldt
authors.affiliation: 1 Institute of Orthopaedic Research and Biomechanics, Center for Musculoskeletal Research, University of Ulm , Ulm, Germany .
authors.forename: Ines
authors.initials: I
authors.lastname: Nagl
authors.affiliation: 2 Instituto de Investigação e Inovação em Saúde, Universidade do Porto , Porto, Portugal .|3 Instituto de Engenharia Biomédica (INEB), Universidade do Porto , Porto, Portugal .|4 Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto , Porto, Portugal .
authors.forename: Catarina Leite
authors.initials: CL
authors.lastname: Pereira
authors.affiliation: 5 Natural and Medical Sciences Institute (NMI), University of Tuebingen , Reutlingen, Germany .
authors.forename: Karin
authors.initials: K
authors.lastname: Benz
authors.affiliation: 1 Institute of Orthopaedic Research and Biomechanics, Center for Musculoskeletal Research, University of Ulm , Ulm, Germany .
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Wilke
authors.affiliation: 1 Institute of Orthopaedic Research and Biomechanics, Center for Musculoskeletal Research, University of Ulm , Ulm, Germany .
authors.forename: Anita
authors.initials: A
authors.lastname: Ignatius
authors.affiliation: 2 Instituto de Investigação e Inovação em Saúde, Universidade do Porto , Porto, Portugal .|3 Instituto de Engenharia Biomédica (INEB), Universidade do Porto , Porto, Portugal .|4 Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto , Porto, Portugal .
authors.forename: Mário A
authors.initials: MA
authors.lastname: Barbosa
authors.affiliation: 2 Instituto de Investigação e Inovação em Saúde, Universidade do Porto , Porto, Portugal .|3 Instituto de Engenharia Biomédica (INEB), Universidade do Porto , Porto, Portugal .
authors.forename: Raquel M
authors.initials: RM
authors.lastname: Gonçalves
authors.affiliation: 1 Institute of Orthopaedic Research and Biomechanics, Center for Musculoskeletal Research, University of Ulm , Ulm, Germany .
authors.forename: Cornelia
authors.initials: C
authors.lastname: Neidlinger-Wilke
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000008:administration & dosage; D002478:Cells, Cultured; D004008:Diclofenac / Q000008:administration & dosage*; D004195:Disease Models, Animal; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D066298:In Vitro Techniques; D007403:Intervertebral Disc / Q000187:drug effects / Q000254:growth & development* / Q000473:pathology; D055959:Intervertebral Disc Degeneration / Q000139:chemically induced / Q000276:immunology / Q000628:therapy*; D008070:Lipopolysaccharides; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D009924:Organ Culture Techniques / Q000295:instrumentation* / Q000379:methods; D013552:Swine; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods; D016896:Treatment Outcome
chemical_list: D000893:Anti-Inflammatory Agents; D008070:Lipopolysaccharides; D004008:Diclofenac