Dataset Information

A Three-Dimensional Chondrocyte-Macrophage Coculture System to Probe Inflammation in Experimental Osteoarthritis.

ABSTRACT: The goal of the present study was to develop a fully three-dimensional (3D) coculture system that would allow for systematic evaluation of the interplay between activated macrophages (AMs) and chondrocytes in osteoarthritic disease progression and treatment. Toward this end, our coculture system was first validated against existing in vitro osteoarthritis models, which have generally cultured healthy normal chondrocytes (NCs)-in two-dimensional (2D) or 3D-with proinflammatory AMs in 2D. In this work, NCs and AMs were both encapsulated within poly(ethylene glycol) diacrylate hydrogels to mimic the native 3D environments of both cell types within the osteoarthritic joint. As with previous studies, increases in matrix metalloproteinases (MMPs) and proinflammatory cytokines associated with the early stages of osteoarthritis were observed during NC-AM coculture, as were decreases in protein-level Aggrecan and collagen II. Thereafter, the coculture system was extended to osteoarthritic chondrocytes (OACs) and AMs to evaluate the potential effects of AMs on pre-existing osteoarthritic phenotypes. OACs in coculture with AMs expressed significantly higher levels of MMP-1, MMP-3, MMP-9, MMP-13, IL-1?, TNF-?, IL-6, IL-8, and IFN-? compared to OACs in mono-culture, indicating that proinflammatory macrophages may intensify the abnormal matrix degradation and cytokine secretion already associated with OACs. Likewise, AMs cocultured with OACs expressed significantly more IL-1? and VEGF-A compared to AM mono-culture controls, suggesting that OACs may intensify abnormal macrophage activation. Finally, OACs cultured in the presence of nonactivated macrophages produced lower levels of MMP-9 and proinflammatory cytokines IL-1?, TNF-?, and IFN-? compared to OACs in the OAC-AM system, results that are consistent with anti-inflammatory agents temporarily reducing certain OA symptoms. In summary, the 3D coculture system developed herein captures several key features of inflammatory OA and may prove useful in future screening of therapeutic agents and/or assessment of disease progression mechanisms.

SUBMITTER: Samavedi S

PROVIDER: S-EPMC5312455 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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A Three-Dimensional Chondrocyte-Macrophage Coculture System to Probe Inflammation in Experimental Osteoarthritis.

Samavedi Satyavrata S Diaz-Rodriguez Patricia P Erndt-Marino Joshua D JD Hahn Mariah S MS

Tissue engineering. Part A 20161118 3-4

The goal of the present study was to develop a fully three-dimensional (3D) coculture system that would allow for systematic evaluation of the interplay between activated macrophages (AMs) and chondrocytes in osteoarthritic disease progression and treatment. Toward this end, our coculture system was first validated against existing in vitro osteoarthritis models, which have generally cultured healthy normal chondrocytes (NCs)-in two-dimensional (2D) or 3D-with proinflammatory AMs in 2D. In this ...[more]

PMID: 27736317

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Project description:Mechanical stress damage and insufficient self-repair can contribute to osteoarthritis (OA) in the affected joint. As the effects of stress on chondrocyte metabolism can regulate cartilage homeostasis, the specific stress-response condition is therefore a key to the generation of an OA disease model. We aimed to produce a specific stress- and cell-based OA model after evaluating the metabolic responses of chondrocytes in response to a series of static and cyclic compression stressors. A static load exceeding 40 psi initiated extracellular matrix (ECM) degradation through a decrease in the sulphated-glycosaminoglycan (GAG) content, upregulation of catabolic matrix metalloproteinase (MMP)-13 encoding gene expression, and downregulation of the ECM-related aggrecan and type II collagen encoding genes within 24?h. Indicators of pro-inflammatory events and oxidative stress were found to correlate with increased IL-6 expression and reactive oxygen species (ROS) production, respectively. However, chondrocytes stimulated by moderate cyclic loading (30-40 psi) exhibited increased ECM-related gene expression without significant changes in catabolic and pro-inflammatory gene expression. BMP-7 expression increased at cyclic loading levels above 30-60 psi. These results demonstrated that static compression exceeding 60 psi is sufficient to produce OA-like chondrocytes that exhibit signs of ECM degradation and inflammation. These OA-like chondrocytes could therefore be used as a novel cell-based drug screening system. Impact statement The lack of an effective treatment for osteoarthritis (OA) reflects the great need for alternative therapies and drug discovery. Disease models can be used for early-stage compound screening and disease studies. Chondrocytes are solely responsible for the maintenance of the articular cartilage extracellular matrix. Our strategy involved the generation of a cell-based model of OA, a more readily studied disease. Instead of using animal cartilage explants, we incorporated isolated porcine chondrocytes with hydrogel to form three-dimensional assemblies. We could identify the specific magnitude-dependent metabolic responses of chondrocytes by applying a series of static and cyclic compression, and therefore successfully generated a novel OA-like cell-based model for drug screening.

Project description:Osteoarthritis (OA) is the most prevalent joint disease and uncontrolled inflammation is now recognized to play vital roles in OA development. Targeting the endogenous counterpart of inflammation may develop new therapeutic approaches in resolving inflammation persistence and treating inflammatory disease including OA. The orphan nuclear receptor 4A1 (NR4A1) is a key negative regulator of inflammatory responses but its role in osteoarthritis remains unclear. In the present study, we found that the NR4A1 expression was elevated in human osteoarthritis cartilage and in vitro OA model, which could be blocked by NF-?B signal inhibitor JSH23. The overexpression of NR4A1 inhibited, whereas knockdown of NR4A1 enhanced IL-1? induced COX-2, iNOS, MMP3, MMP9 and MMP13 expression, and luciferase reporter activity of NF-?B response element. Though NR4A1 was upregulated in inflammatory stimulation and creates a negative feedback loop, persistent inflammatory stimulation inhibited NR4A1 expression and activation. The expression of NR4A1 declined rapidly after an initial peak in conditions of chronic IL-1? stimulation, which could be partially restored by HDACs inhibitor SAHA. The phosphorylation of NR4A1 was increased in human osteoarthritis cartilage, and p38 inhibitor SB203580, JNK inhibitor SP600125 and ERK inhibitor FR180204 could significantly inhibited IL-1? induced NR4A1 phosphorylation. Reactivation of NR4A1 by its agonist cytosporone B could inhibit IL-1? induced chondrocyte inflammation and expression of COX-2, iNOS, MMP3, MMP9, and MMP13. In rat OA model, intra-articular injection of cytosporone B protected cartilage damage and ameliorated osteoarthritis. Thus, our study demonstrated that the NR4A1 is a key endogenous inhibitor of chondrocyte inflammation, which was relatively inactivated under chronic inflammatory stimulation through HDACs mediated transcriptional suppression and MAKP dependent phosphorylation in osteoarthritis. NR4A1 agonist cytosporone B could reactivate and restore the inhibitory regulatory ability of NR4A1, prevent excessive inflammation, and ameliorates osteoarthritis.

Dataset Information

A Three-Dimensional Chondrocyte-Macrophage Coculture System to Probe Inflammation in Experimental Osteoarthritis.

Publications

A Three-Dimensional Chondrocyte-Macrophage Coculture System to Probe Inflammation in Experimental Osteoarthritis.

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