Abstract
Osteoarthritis (OA) is an age-related degenerative musculoskeletal disease characterised by loss of articular cartilage, synovitis, abnormal bone proliferation and subchondral bone sclerosis. Underlying OA pathogenesis is yet to be fully elucidated with no OA specific biomarkers in clinical use. Ex-vivo equine cartilage explants (n=5) were incubated in TNF-α/IL-1β supplemented culture media for 8 days, with 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 1D 1H nuclear magnetic resonance metabolomic analysis with media samples also undergoing mass spectrometry proteomic analysis. Within the cartilage, metabolites glucose and lysine were elevated following TNF-α/IL-1β treatment whilst adenosine, alanine, betaine, creatine, myo-inositol and uridine levels decreased. Within the culture media, four, four and six differentially abundant metabolites and 154, 138 and 72 differentially abundant proteins, with > 2 fold change, were identified for 1-2 day, 3-5 day and 6-8 day time points respectively. Nine potential novel OA neopeptides were elevated in treated media. Our innovative study has identified differentially abundant metabolites, proteins and extracellular matrix derived neopeptides, providing insightful information on OA pathogenesis, enabling potential translation for clinical markers and possible new therapeutic targets.
Footnotes
Abbreviations
- ADAMTS
- A disintegrin and metalloproteinase with thrombospondin motifs
- CPMG
- Carr-Purcell-Meiboom-Gill
- COMP
- Cartilage oligomeric matrix protein
- DMEM
- Dulbecco’s modified Eagle’s medium
- EDTA
- Ethylenediaminetetraacetic acid
- ECM
- Extracellular matrix
- FDR
- False discovery rate
- FCS
- Foetal calf serum
- GLUT
- Glucose transporter
- HRMAS
- High resolution magical angle spinning
- IL-1β
- Interleukin-1β
- MS
- Mass spectrometry
- MMP
- Matrix metalloproteinase
- MSI
- Metabolomics Standards Initiative
- NMR
- Nuclear magnetic resonance
- 1D SDS PAGE
- One dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis
- OA
- Osteoarthritis
- PBS
- Phosphate buffered saline
- PCA
- Principal component analysis
- PC1
- Principal component 1
- PQN
- Probabilistic quotient normalisation
- RA
- Rheumatoid arthritis
- RMS
- Root mean square
- SF
- Synovial fluid
- TIC
- Total ion current
- TFA
- Trifluoroacetic acid
- TSP
- Trimethylsilyl propionate
- TNF-α
- Tumour necrosis factor-α