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Nucleus pulposus cells synthesize a functional extracellular matrix and respond to inflammatory cytokine challenge following long-term agarose culture.

ABSTRACT: Intervertebral disc degeneration is characterized by a cascade of cellular, biochemical and structural changes that may lead to functional impairment and low back pain. Interleukin-1 beta (IL-1?) is strongly implicated in the etiology of disc degeneration, however there is currently no direct evidence linking IL-1? upregulation to downstream biomechanical changes. The objective of this study was to evaluate long-term agarose culture of nucleus pulposus (NP) cells as a potential in vitro model system to investigate this. Bovine NP cells were cultured in agarose for 49 days in a defined medium containing transforming growth factor-beta 3, after which both mechanical properties and composition were evaluated and compared to native NP. The mRNA levels of NP cell markers were compared to those of freshly isolated NP cells. Glycosaminoglycan (GAG) content, aggregate modulus and hydraulic permeability of mature constructs were similar to native NP, and aggrecan and SOX9 mRNA levels were not significantly different from freshly isolated cells. To investigate direct links between IL-1? and biomechanical changes, mature agarose constructs were treated with IL-1?, and effects on biomechanical properties, extracellular matrix composition and mRNA levels were quantified. IL-1? treatment resulted in upregulation of a disintegrin and metalloproteinase with thrombospondin motifs 4, matrix metalloproteinase-13 and inducible nitric oxide sythase, decreased GAG and modulus, and increased permeability. To evaluate the model as a test platform for therapeutic intervention, co-treatment with IL-1? and IL-1 receptor antagonist (IL-1ra) was evaluated. IL-1ra significantly attenuated degradative changes induced by IL-1?. These results suggest that this in vitro model represents a reliable and cost-effective platform for evaluating new therapies for disc degeneration.

SUBMITTER: Smith LJ

PROVIDER: S-EPMC3424069 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Nucleus pulposus cells synthesize a functional extracellular matrix and respond to inflammatory cytokine challenge following long-term agarose culture.

Smith Lachlan J LJ Chiaro Joseph A JA Nerurkar Nandan L NL Cortes Daniel H DH Horava Sarena D SD Hebela Nader M NM Mauck Robert L RL Dodge George R GR Elliott Dawn M DM

European cells & materials 20111120

Intervertebral disc degeneration is characterized by a cascade of cellular, biochemical and structural changes that may lead to functional impairment and low back pain. Interleukin-1 beta (IL-1β) is strongly implicated in the etiology of disc degeneration, however there is currently no direct evidence linking IL-1β upregulation to downstream biomechanical changes. The objective of this study was to evaluate long-term agarose culture of nucleus pulposus (NP) cells as a potential in vitro model sy ...[more]

PMID: 22102324

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Project description:Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect.BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3×10(6) cells/mL) and NPCs+BMSCs (6×10(6) cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10?ng/mL transforming growth factor-?1 (TGF-?1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9, A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR.NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone.NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. In the current study, no synergistic effect of adding BMSCs was observed.

Dataset Information

Nucleus pulposus cells synthesize a functional extracellular matrix and respond to inflammatory cytokine challenge following long-term agarose culture.

Publications

Nucleus pulposus cells synthesize a functional extracellular matrix and respond to inflammatory cytokine challenge following long-term agarose culture.

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