Risk factors for osteoarthritis include raised BMI and female gender. Whether these two factors influenced synovial gene expression was investigated using a triangulation and modelling strategy which generated 12 datasets of gene expression in synovial tissue from three knee pathologies with matching BMI groups, obese and overweight, and gender distributions. Intra-operative synovial biopsies were immersed in RNAlater at 4oC before storage at -80oC. Total RNA was extracted using RNAeasy with gDNA removal. Following RT- PCR and quality assessment, cDNA was applied to Affymetrix Clariom D microarray gene chips. Bioinformatics analyses were performed. Linear models were prepared in limma with gender and BMI factors incorporated sequentially for each pathology comparison, generating 12 models of probes differentially expressed at FDR p<0.05 and Bayes number, B>0. Data analysis of differently expressed genes utilized Ingenuity Pathway Analysis and Cytoscape with Cluego and Cytohubba plug-ins.Abstract
Introduction
Methodology
It is increasingly evident that synovium may play a larger role in the aetiology of osteoarthritis. We compared gene expression in whole tissue synovial biopsies from end-stage knee osteoarthritis and knee trauma patients with that of their paired explant cultures to determine how accurately cultured cells represent holistic synovial function. Synovial tissue biopsies were taken from 16 arthroplasty patients and 8 tibial plateau fracture patients with no osteoarthritis. Pairs of whole tissue fragments were either immediately immersed in RNAlater Stabilisation Solution at 4o C before transfer to -80o C storage until RNA extraction; or weighed, minced and cultured at 500mg tissues/5ml media in a humidified incubator at 37oC, 5% CO2. After sub-culturing total RNA was extracted using RNAeasy Plus Mini Kit with gDNA removal. Following RT-PCR and quality assessment, cDNA was applied to Affymetrix Clariom D microarray gene chips. Bioinformatics analyses were performed.Abstract
Introduction
Methodology
Articular cartilage degradation is a defining feature of osteoarthritis. Synovium is a reactive tissue with synovial villae, neoangiogenesis and intimal hyperplasia common to many joint pathologies. The consequences of cartilage debris in osteoarthritis impacting the synovial intima is not well understood. We analysed the immunohistology of synovium from 16 patients with osteoarthritis and 17 patients undergoing knee surgery for non-arthritic pathologies. This data was integrated with imaging and functional scores to correlate synovitis in osteoarthritis. Formalin-fixed paraffin embedded synovial biopsy sections were cut in serial sequence and processed for routine staining (H&E or CD3, CD68, CD20, Vimentin, vWF and PCNA IHC) using standardised Dako monoclonal mouse anti-human antibodies. Digital images scanned at x20 were evaluated for fragments of cartilage and aggregates of inflammatory cells. Clinical data (gender, BMI, KL grade, WOMAC & SF-12 scores) was aligned with histopathological data.Abstract
Introduction
Methodology
Synovitis impacts osteoarthritis symptomatology and progression. The transcription factors controlling synovial gene expression have not been described. This study analyses gene expression in synovium samples from 16 patients with osteoarthritis with 9 undergoing arthroscopic and 8 knee trauma surgery for non-arthritic pathologies. Intra-operative synovial biopsies were immersed in RNAlater at 4oC before storage at -80oC. Total RNA was extracted using RNAeasy. After purification, RT-PCR and quality assessment, cDNA was applied to Affymetrix Clariom D microarray gene chips. Bioinformatics analyses were performed. Linear models were prepared in limma with gender and BMI factors incorporated sequentially for each pathology comparison, generating 12 models of probes differentially expressed at FDR p<0.05 and Bayes number, B>0. Data analysis of differently expressed genes utilized Ingenuity Pathway Analysis and Cytoscape with Cluego and Cytohubba plug-ins.Abstract
Introduction
Methodology
Wear debris from articulating joint implants is inevitable. Small debris particles are phagocytosed by macrophages. Larger particles initiate the fusion of many macrophages into multi-nucleated giant cells for particle encasement. Macrophages are recruited into inflamed tissues from the circulating monocyte population. Approximately 10% of white blood cells are monocytes which after release from the bone marrow circulate for 2–3 days, before being recruited into tissues as inflammatory macrophages or undergoing apoptosis. Circulating MRP8/14 (S100A8/A9) is a measure of monocyte recruitment, part of the monocyte-endothelial docking complex, and shed during monocyte transmigration across the endothelium. The higher the S100A8/A9 the more monocytes being recruited giving an indirect measure of debris production. 2114 blood samples were collected from arthroplasty patients with hip or knee osteoarthritis (primary, post-traumatic and secondary), 589 before their primary arthroplasty, 1187 patients > 1 year post-arthroplasty, 101 patients before revision for aseptic loosening and 237 patients >1 year post-revision. Plasma S100A8/A9 was measured using BMA Biomedicals Elisa kit, normal levels in health adults are 0.5–3 mg/ml. Joint specific scores, WOMAC knee or Oxford Hip adjusted to percent of maximum, together with SF-12 were completed.Introduction:
Methods:
The risk factors for degenerative joint disease are well established: increasing age, obesity, joint abnormalities, trauma and overuse, together with female gender, ethnic and genetic factors. That obesity is a significant risk factor for developing osteoarthritis in non-weight-bearing as well as weight-bearing and joints was one of the first indications that the risk was nor purely that of aberrant biomechanical loading. Low grade chronic systemic inflammation is a component of each of ageing and obesity, atherosclerosis and diabetes, culminating in Metabolic Syndrome. In our study of 1684 patients with joint degeneration 85% were overweight or obese and 65% older than 65 years with 62% being both, 73% of patients were taking medications for serious, ‘non-orthopaedic’ health problems such as cardiovascular or respiratory disease, obesity or NIDDM. Monocytes are a major component of chronic inflammation, approximately 10% of white blood cells are monocytes which circulate for 2–3 days, before being recruited into tissues as inflammatory macrophages or undergoing apoptosis. Circulating S100A8/A9 (MRP8/14) is a measure of monocyte recruitment being shed during monocyte transmigration across the endothelium. The higher the S100A8/A9 the more monocytes being recruited giving an indirect measure of chronic inflammatory status. 2154 blood samples were collected from arthroplasty patients (first or second joint replacement), 1135 Female and 1019 Male, age 29–93 years, body mass index (BMI) 18–56, with hip or knee osteoarthritis (primary, post-traumatic and secondary), 589 before a primary arthroplasty, 1187 patients >1 year post-arthroplasty, 101 patients before revision for aseptic loosening and 237 patients >1 year post-revision. All study patients received metal on UHMWPE implants. Plasma S100A8/A9 was measured using BMA Biomedicals Elisa kit, normal levels in healthy adults are 0.5–3 mg/ml. The data were analysed using SPSS, p values were calculated using Spearman's test.Introduction:
Methods:
Shoulder motion results from a complex interaction between the interconnected segments of the shoulder girdle. Coordination is necessary for normal shoulder function and is achieved by synchronous and coordinated muscle activity. During rotational movements, the humeral head translates on the glenoid fossa in the anterior-posterior plane. Tension developed by the rotator cuff muscles compresses the humeral head into the glenoid fossa. This acts to limit the degree of humeral head translation and establishes a stable GH fulcrum about which the arm can be moved. Previous studies have been limited by the use of contrived movement protocols and muscular coordination has not been previously considered with regard to shoulder rotation movements. This study reports the activation profile and coordination of 13 muscles and 4 muscle groups during a dynamic rotational movement task based on activities of daily living. Eleven healthy male volunteers were included in the study. Electromyography (EMG) was recorded from 13 muscles (10 surface and 3 fine-wire intramuscular electrodes) using a wireless EMG system. EMG was recorded during a movement task in which the shoulder was consecutively rotated internally (phase 1) and externally (phase 2) with a weight in the hand. Muscle group data was calculated by ensemble averaging the activity of the individual component muscles. Mean signal amplitude and Pearson correlation coefficient (PCC) analysed muscle activation and coordination, respectively.Introduction
Methods
The role of matrix metalloproteinases (MMPs) in the aseptic loosening of hip prostheses is well established. Gelatinase MMPs have been identified in the interface membranes and the pseudosynovial tissues in the hips. Little data are available on gelatinase MMPs and their major regulators, including specific tissue inhibitors of matrix metalloproteinases (TIMPs) in the loosening of shoulder prostheses. The objectives of this study were to determine whether A) gelatinase MMPs and their regulators (MMP14, TIMP-1,-2) are produced by periprosthetic tissues in cases of aseptic loosening of shoulder prostheses, and, B) to identify which cell types, in both interface and synovial tissues, localize the enzymes. Interface tissues and synovial tissues were obtained during revision surgery for loose shoulder implants. In 9 patients (6-Total Shoulder Replacement, 3-Hemiarthro-plasty (Bipolar), 9 samples of interface tissues and 8 samples of synovial tissues were obtained. Of the interface tissues 2 were from the interface of the bipolar and the unresurfaced glenoid. Formalin-fixed paraffin embedded sections were stained using primary antibodies for MMP2 (Neomarkers), MMP9 (Oncogene Ltd), TIMP1, TIMP2 &
MMP14 (Chemicon Ltd). Antigen retrieval required pressure cooker treatment for MMP2 and MMP9 and trypsin for TIMP1. Visualisation used a standard DAB chromagen technique (Envision, Dako Ltd.). Appropriate control sections ensured reproducibility of the staining. The antibodies selected bind to both active and inactive forms of the MMPs. Both HDPE and metal debris were seen in both the synovial and interface tissues. Transformation of macrophages to giant cells was associated with PE debris, and was not observed with metal debris alone. The presence of gelatinase MMPs in both interface and synovial tissues in aseptic loosening of shoulder prostheses was demonstrated. Differences between the MMP content of macrophages and giant cells between the tissues was detected, positivity was associated with the presence of metallic and/or HDPE debris. Activation of endothelial MMP2 by both MMP14 and low levels of TIMP2 would support the development of a vascular network.
The overall incidence of cuff tears increases with age, individuals over 80years having a 51% incidence of a tear. Currently, the aetiology of rotator cuff tears remains unclear and successful repair is achieved in only 30% patients. Matrix metalloproteinases (MMPs) have roles in a wide range of physiological processes including placentation and embryogenesis, tissue remodelling and wound healing. However, the ability of MMPs to dissolve extracellular matrix has been linked to a variety of pathological processes including rheumatoid arthritis, osteoarthritis, periodontitis and multiple sclerosis, which involve excessive matrix destruction. Production of gelatinase MMPs by torn rotator cuff has been demonstrated. The objectives of this study were to examine the expression of MMPs and their association with histological changes in full thickness tears of the rotator cuff. Rotator cuff tissue was obtained from ten patients (age 40–80years) undergoing surgical repair. The size of tear was 1–4.5cm; time from presentation to surgery was 1 month (acute) to between 0.5–4years (chronic). Immunohistochemical staining with commercial monoclonal antibodies to a range of MMPs, endothelial, macrophage and fibroblast markers was performed. Production of gelatinase MMPs was measured by gelatin zymography on tissue culture supernatant. Visualisation used a standard DAB chromagen technique. In the acute specimens there was an infiltrate of macrophages with little collagen degeneration; the fibro-blasts were MMP1 positive and endothelial cells MMP2 positive. At 12 months post-tear mature collagen, plump fibroblasts and proliferating endothelial cells were identified adjacent to the resection edge. Towards the torn edge areas of lower cellularity, sparse vascularity and collagen degeneration were observed. Vimentin positive, CD68 negative cells within this matrix were rounded with foamy cytoplasm, and intensely positive for MMP1 and MMP2, and positive for MMP-3, -10, -11, -13 and -14. Tissue culture supernatant demonstrated active and latent MMP2 production in all cases. The prolonged interval between trauma and surgical repair, with potential pharmacological intervention, remedial physiotherapy and disuse immobility, make assessment of the factors contributing to tendon degeneration difficult to determine. Fatty infiltration, dystrophic calcification and patchy collagen degeneration were common. However, clear evidence of cellular activities typical of wound repair were also identified, including fibroblast and endothelial cell proliferation. The most striking finding was the association between areas of poor collagen structure with fibroblasts staining intensely for both MMP1 and MMP2 and positive for other matrix metalloproteinases. The production of MMP1 and MMP2 may contribute to active remodelling of the tendon matrix. Success of repair could be influenced by both the quality of the matrix and the cell types and activities in the tissue at the resection edge.
