Project description:The aim of this study was threefold: Firstly, to carry out microarray hybridisations using human AC and NP cells to identify NP and AC specific markers. Secondly, to further investigate their ability to characterise adult derived stem cells differentiating towards an NP phenotype using an in vitro differentiation system and thirdly, in their application to compare the suitability of BMSC and ADRCs as a stem cell source for tissue engineering of the IVD. Using microarray technology we have identified several novel human AC and NP markers and have demonstrated that these markers are capable of identifying the differentiation of adult derived stem cells towards an NP rather than an AC phenotype. In addition these markers suggest that ADRCs provide a more suitable cell source for tissue engineering of the intervertebral disc.
Project description:Low back pain is a major cause of disability especially for people between 20 and 50 years of age. As a costly healthcare problem, it imposes a serious socio-economic burden. Current surgical therapies have considerable drawbacks and fail to replace the normal disc in facilitating spinal movements and absorbing load. Therefore, the focus of regenerative medicine is on identifying biomarkers and signalling pathways to improve our understanding about the cascades of disc degeneration and allow for the design of specific therapies. We hypothesized that comparing microarray profiles from degenerative and non-degenerative discs will lead to the identification of dysregulated signalling and pathophysiological targets. Microarray data sets were generated from human annulus fibrosus cells and analysed using IPA ingenuity pathway analysis system. Gene expression values were validated by qRT-PCR, and respective proteins were identified by immunohistochemistry. Microarray analysis revealed 17 dysregulated molecular markers and various dysregulated cellular functions, including cell proliferation and inflammatory response, in the human degenerative annulus fibrosus. The most significant canonical pathway induced in degenerative annulus fibrosus was found to be the interferon signalling pathway. In conclusion, this study indicates interferon-alpha signalling pathway activation with IFIT3 and IGFBP3 up-regulation which may affect cellular function in human degenerative disc. 48 samples of intervertebral disc tissue - annulus fibrosus and nucleus pulposus - displaying varying degrees (grades) of degeneration
Project description:Responses of human adult articular chondrocytes under conditions of lack or excess of bone morphogenetic protein-7 also called osteogenic protein 1. Original processed data file is in E-MTAB-571.additional.zip archive.
Project description:Articular cartilage is deprived of blood vessels and nerves, and the only cells residing in this tissue are chondrocytes. The molecular properties of the articular cartilage and the architecture of the extracellular matrix demonstrate a complex structure that differentiates on the depth of tissue. Osteoarthritis (OA) is a degenerative joint disease, the most common form of arthritis, affecting the whole joint. It is associated with ageing and affects the joints that have been continually stressed throughout life including the knees, hips, fingers, and lower spine region. OA is a multifactorial condition of joint characterised by articular cartilage loss, subchondral bone sclerosis, and inflammation leading to progressive joint degradation, structural alterations, loss of mobility and pain. Articular cartilage biology is well studied with a focus on musculoskeletal diseases and cartilage development. However, there are relatively few studies focusing on zonal changes in the cartilage during osteoarthritis.
Project description:Glucosamine proved to be a potent, broad-spectrum inhibitor of IL-1beta. Of the 2,813 genes whose transcription was altered by IL-1beta stimulation (p<0.0001), glucosamine significantly blocked the response in 2,055 (~73%). Glucosamine fully protected the chondrocytes from IL-1-induced expression of inflammatory cytokines, chemokines and growth factors as well as proteins involved in PGE2 and NO synthesis. It also blocked the IL-1-induced expression of matrix specific proteases such as MMPs -3,-9,-10,-12 and ADAMTS-1. Experiment Overall Design: Articular cartilage was isolated from the femoral heads of male Wistar rats under aseptic conditions. Chondrocytes were obtained by sequential digestion of the cartilage with pronase and type II collagenase. After filtration to remove tissue debris, the cells were cultured in 75-cm2 flasks in complete Dulbeccoâs Modified Eagle Medium (DMEM; supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Experiments were subsequently performed with second-passage cultures whereby the cells from the large cultures were trypsinized, pooled and seeded into twenty 25 cm2 flasks. These were then divided into 4 treatment groups to evaluate the effects of glucosamine and IL-1 on global transcription patterns.To the culture medium in half of the flasks, glucosamine, HCl was added to a final concentration of 20 mM. Six hours later, IL-1beta was added at 10 ng/ml to 5 of the flasks receiving glucosamine and to 5 of the untreated flasks. Fourteen hours post IL-1beta stimulation, total RNA was isolated individually from the respective cultures and microarray experiments processed.
Project description:To study chondrogenesis, we used a chicken limb bud model: We used RNA sequencing, and examined the differences between gene expression patterns during cartilage formation in micromass cultures of embryonic limb bud-derived progenitors. We sequenced in triplicate at Day 0,1,2,3,4,6,10,15 and also from mature birds.
Project description:Endochondral bone formation is orchestrated by growth factors produced by chondrocytes and deposited in cartilage matrix. Some of these factors were identified, but their complete list and relationship remains unknown. In this work the growth factors were isolated from calcified cartilage of costochondral junctions. The isolation involved hyaluronidase digestion, guanidinium hydrochloride (GuHCl) extraction, HCl decalcification and GuHCl extraction of decalcified matrix. Growth factors were purified by heparin chromatography and estimated by enzyme-linked immunosorbent assay (ELISA) or used for protein sequence analysis. Bone morphogenetic protein-7 (BMP-7), Growth/differentiation factor 5 (GDF-5), and NEL-like protein 1 (NELL-1) quantitatively dominated in material obtained in all steps of isolation. During ossification perivascular cells and septoclasts enter the cartilage and release growth factors stimulating osteoclastogenesis. Osteoclasts dissolve calcified cartilage and transport released factors needed for stimulation of osteoprogenitor cells. Presence of BMP-7, GDF-5 and NELL-1 at the site of initial bone formation may suggest that their synergistic action favors osteogenesis.
Project description:The main aim was to determine how polyadenylation site usage is affected by osteoarthritis. Age matched tissue samples were obtained from both healthy individuals and those with late stage osteoarthritis, total RNA was isolated and then QuantSeq Reverse library produced using commercial kit (Lexogen) before sequencing
Project description:The aim of this transcription profiling study was to identify novel genes that could be used to distinguish bovine Nucleus pulposus (NP) cells from articular cartilage (AC) and annulus fibrosus (AF) cells and to further determine their expression in normal and degenerate human intervertebral disc (IVD). This study has identified a number of novel genes that characterise the bovine and human NP and IVD cell phenotypes and allows for discrimination between AC, AF and NP cells.<br><br>
Project description:We have previously demonstrated that a mixture of curcuminoids extract, hydrolyzed collagen and green tea extract (COT) inhibited inflammatory and catabolic mediatorâ??s synthesis by osteoarthritic (OA) human chondrocytes. The objectives of this study were to identify new targets of COT using genomic approaches. We compared gene expression profiles of chondrocytes treated with COT and/or with interleukin(IL)-1β. The proteins coded by the most important COT sensitive genes were then quantified by specific immunoassays. Cartilage specimens were obtained from 12 patients (10 women and 2 men; mean age 67 years old, range 54-76 years old) with knee OA. Primary human chondrocytes were cultured in monolayer until confluence and then incubated for 24 hours in the absence or in the presence of human IL-1β (10e-11M) and with or without COT, each compound at the concentration of 4 µg/ml. Microarray gene expression profiling between control, COT, IL-1β and COT IL-1β conditions was performed.