Ex Vivo Equine Cartilage Explant Osteoarthritis Model: A Metabolomics and Proteomics Study

Abstract

Osteoarthritis is an age-related degenerative musculoskeletal disease characterized by loss of articular cartilage, synovitis, and subchondral bone sclerosis. Osteoarthritis pathogenesis is yet to be fully elucidated with no osteoarthritis-specific biomarkers in clinical use. Ex vivo equine cartilage explants ( n = 5) were incubated in tumor necrosis factor-α (TNF-α)/interleukin-1β (IL-1β)-supplemented culture media for 8 days, with the media removed and replaced at 2, 5, and 8 days. Acetonitrile metabolite extractions of 8 day cartilage explants and media samples at all time points underwent one-dimensional (1D) 1 H nuclear magnetic resonance metabolomic analysis, with media samples also undergoing mass spectrometry proteomic analysis. Within the cartilage, glucose and lysine were elevated following TNF-α/IL-1β treatment, while adenosine, alanine, betaine, creatine, myo-inositol, and uridine decreased. Within the culture media, 4, 4, and 6 differentially abundant metabolites and 154, 138, and 72 differentially abundant proteins were identified at 1–2, 3–5, and 6–8 days, respectively, including reduced alanine and increased isoleucine, enolase 1, vimentin, and lamin A/C following treatment. Nine potential novel osteoarthritis neopeptides were elevated in the treated media. Implicated pathways were dominated by those involved in cellular movement. Our innovative study has provided insightful information on early osteoarthritis pathogenesis, enabling potential translation for clinical markers and possible new therapeutic targets.

Synovial Fluid Proteomics From Serial Aspirations of ACL-Injured Knees Identifies Candidate Biomarkers

“…Vimentin (VIM) was another protein elevated in aspiration 2, whereas a previous study reported lower levels of VIM in synovial fluid from knees with subacute ACL tears compared with those with acute ACL tears. 12 In an equine explant model of OA, ex vivo treatment of cartilage explants with tumor necrosis factor–α/interleukin 1β (IL-1β) resulted in elevated VIM levels, 4 consistent with our current findings. A multifunctional intermediate filament protein, 22 VIM is thought to be involved in mechanotransduction in cartilage.…”

Aggrecan and Hyaluronan: The Infamous Cartilage Polyelectrolytes – Then and Now

“…Other mechanical perturbances, such as cyclic mechanical stretch or strain, shear stress, surface motion or mechanical injury [63, 119,138,142,210,254,298] imposed on connective tissue cells, including fibrochondrocytes and articular chondrocytes, have also been shown to modulate HA production. The later studies have not provided any information on potential transduction pathways for stimulated HA production, but likely mechanisms could come from the newly emerging databases on cartilage "metabolomics' [5,50,232,236,243]. Key regulatory points would include glucose transport [168,169,241], subsequent Glc6P shunting to aerobic [113] or anaerobic glycolysis [292,302] for energy production, and/or synthesis of UDP-GlcNAc and UDP-GlcUA to regulate HAS activities.…”

A Progress Report and Roadmap for Microphysiological Systems and Organ-On-A-Chip Technologies to Be More Predictive Models in Human (Knee) Osteoarthritis

“…Explant models provide native tissue architecture and in vivo- like cell composition and thus feature the most authentic and “natural” microenvironment. Explant mono- and cocultures of equine ( Haltmayer et al, 2019 ; Anderson et al, 2020 ) and porcine ( Ding et al, 2014 ; Vernon et al, 2014 ) as well as human tissues ( Topoluk et al, 2018 ; Dolzani et al, 2019 ) have been used to elaborate on the structure-function relationship between tissue architecture, the extracellular matrix composition and cell phenotypes in OA pathophysiology, but they are difficult to standardize due to the large interindividual variation. To improve standardization and thus comparability of data, pellet and micromass cultures, with and without natural (e.g., alginate, hyaluronan, collagen) or synthetic [e.g., polylactic acid, poly(ethyelene glycol)‐terephthalate] hydrogel-based scaffolds, have been developed ( Smeriglio et al, 2015 ; Mouser et al, 2020 ).…”