Project description:Aims of study: (1) To identify systemic differences in osteoarthritic (OA) bone that contribute to OA pathogenesis. (2) Identify novel osteoporotic (OP) bone-related disease genes. Study involved comparison of trabecular bone extracted from the intertrochanteric (IT) region of the proximal femur (PF) from OA, OP and normal/control (CTL) individuals. Bone was obtained from OA and OP individuals at surgery for total hip replacement and from CTL individuals at autopsy. Keywords: Bone tissue comparison, diseased versus non-diseased, factorial design, linear modelling
Project description:Aims of study: (1) To identify systemic differences in osteoarthritic (OA) bone that contribute to OA pathogenesis. (2) Identify novel osteoporotic (OP) bone-related disease genes. Study involved comparison of trabecular bone extracted from the intertrochanteric (IT) region of the proximal femur (PF) from OA, OP and normal/control (CTL) individuals. Bone was obtained from OA and OP individuals at surgery for total hip replacement and from CTL individuals at autopsy. Keywords: Bone tissue comparison, diseased versus non-diseased, factorial design, linear modelling Four sets of sample comparisons (39 comparisons in total) were made in this study. These comprised 10 OA-CTL female, 10 OA-CTL male, 10 OA-OP female and 9 OP-CTL female comparisons. A Compugen Human 19K-oligo library spotted onto glass slides by the Adelaide Microarray facility (AMF) was used in this study. The slides were interrogated by competitive hybridisation of Cy3 and Cy5 labelled pairs of OA-CTL, OA-OP or OP-CTL amplified RNA samples. Sample pairs were age-matched as closely as possible. A biological dye-swap strategy was employed. After hybridisation and washing of the slides they were scanned using a GenePix 4000B Scanner driven by GenePix Pro 4.0. All analyses were performed using the statistical programming and graphics environment R. The “SPOT” software package was used to identify spots by adaptive segmentation method and subtract backgrounds utilising morphological opening approach. Data analysis was performed in R using Bioconductor. Loess print tip method was used to correct for dye-bias and intensity within each group of adjacent spots printed by one pin. Linear modelling was performed with the Limma package of Bioconductor. Linear models were used to incorporate all available data into a single analysis. This allowed the use of both direct and indirect comparisons in calculation of expression ratios as well as improved accuracy in estimation of variance for each gene. Factorial design utilised the following factors: medical condition (OA or OP or CTL); sex (male or female) for OA patients only.
Project description:To date, all of the prior osteoarthritic microarray studies in human tissue have focused on the overlying articular cartilage, meniscus, or synovium but not the underlying subchondral bone. In our previous study, our group developed a methodology for high quality RNA isolation from site-matched cartilage and bone from human knee joints, which allowed us to perform candidate gene expression analysis on the subchohndral bone (published on Osteoarthritis and Cartilage on Dec/5/2012 (doi: 10.1016/j.joca.2012.11.016). To the best of our knowledge, the current study is the first to successfully perform whole-genome microarray profiling analyses of human osteoarthritic subchondral bone. We believe our comprehensive microarray results can improve the understanding of the pathogenesis of osteoarthritis and could further contribute to the development of new biomarker and therapeutic strategies in osteoarthritis. Following histological assessment of the integrity of overlying cartilage and the severity of bone abnormality by microcomputed tomography, we isolated total RNA from regions of interest from human OA (n=20) and non-OA (n=5) knee lateral and medial tibial plateaus (LT and MT). A whole-genome profiling study was performed on an Agilent microarray platform and analyzed using Agilent GeneSpring GX11.5. Confirmatory quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis was performed on samples from nine OA individuals to confirm differential expression of 85 genes identified by microarray. Ingenuity Pathway Analysis (IPA) was used to investigate canonical pathways and immunohistochemical staining was performed to validate protein expression levels in samples.
Project description:Bone marrow lesions (BML) are well described in osteoarthritis (OA) using magnetic resonance imaging (MRI) and associate with pain however, little is known about their role in disease process and pattern of gene expression within the lesions. This study evaluated the gene expression profile of OA BML (n=14) in comparison to normal bone (n=10) by microarray profiling. MR imaging and the MOAKS scoring was used to locate lesions and the scaled axial images were used to sample BMLs. The bone tissue controls were harvested from participants undergoing surgery following trauma.
Project description: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 novel study identified metabolites, proteins and extracellular matrix derived neopeptides which provides insightful information on OA pathogenesis, enabling potential translation for clinical markers and possible novel therapeutic targets.
Project description:Osteoarthritis (OA) treatment is limited by the lack of effective non-surgical interventions to slow disease progression. Here, we examined the contributions of the subchondral bone properties to OA development. We used parathyroid hormone (PTH) to modulate bone mass prior to OA initiation and alendronate (ALN) to inhibit bone remodeling during OA progression. We examined the spatiotemporal progression of joint damage by combining histopathological and transcriptomic analyses across joint tissues. The additive effect of PTH pretreatment prior to OA initiation and ALN treatment during OA progression most effectively attenuated load-induced OA pathology. Individually, PTH directly improved cartilage health and slowed the development of cartilage damage, whereas ALN primarily attenuated subchondral bone changes associated with OA progression. Joint damage reflected early transcriptomic changes. With both treatments the structural changes were associated with early modulation of immunoregulation and -response pathways that may contribute to disease mechanisms. Overall, our results demonstrate the potential of subchondral bone-modifying therapies to slow the progression of OA.
Project description:Gene expression profiling of BMMC from patients with rheumatoid arthritis (RA) vs. osteoarthritis (OA). Bone marrow was obtained from patients with RA (n=9) or OA (n=10). The bone marrow samples from the 10 OA patients are used as controls.
Project description:Osteoarthritis (OA) is an age-related degenerative musculoskeletal disease characterised by loss of articular cartilage, synovitis, abnormal bone proliferation and subchondral bone sclerosis. The underlying pathogenesis of OA is yet to be fully elucidated with no OA specific biomarkers in clinical use. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) allow identification of the global metabolome and proteome respectively. During this study, 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 1H NMR metabolic analysis with media samples also undergoing MS 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 metabolites were identified as being differentially abundant between control and treatment groups for 1-2 day, 3-5 day and 6-8 day time points respectively. Culture media proteomics identified 154, 138 and 72 proteins differentially abundant, with > 2 fold change, between control and treatment groups for 1-2 day, 3-5 day and 6-8 day time points respectively. Nine potential novel OA neopeptides were elevated in treated media. This is the first study to use a multi ‘omics’ approach to simultaneously investigate the metabolomic profile of ex-vivo cartilage and metabolomic/proteomic profiles of culture media using the TNF-α/IL-1β ex-vivo OA cartilage model. This study has identified a panel of metabolites, proteins and extracellular matrix derived neopeptides which are differentially abundant during an early phase of the OA model which may provide further information on underlying disease pathogenesis, allow potential translation for clinical markers and possible novel therapeutic targets.