Aims. Knee osteoarthritis (OA) involves a variety of tissues in the joint.
Objectives. The molecular mechanism of rheumatoid arthritis (RA) remains elusive. We conducted a protein-protein interaction network-based integrative analysis of genome-wide association studies (GWAS) and
Effects of insulin-like growth factor 1 (IGF1), fibroblast growth
factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression
of genes involved in the proliferation and differentiation of osteoblasts
in culture were analysed. The best sequence of growth factor addition
that induces expansion of cells before their differentiation was
sought. Primary human osteoblasts in Objectives
Methods
Aims. This study aimed to explore the role of small colony variants (SCVs) of Staphylococcus aureus in intraosseous invasion and colonization in patients with periprosthetic joint infection (PJI). Methods. A PJI diagnosis was made according to the MusculoSkeletal Infection Society (MSIS) for PJI. Bone and tissue samples were collected intraoperatively and the intracellular invasion and intraosseous colonization were detected. Transcriptomics of PJI samples were analyzed and verified by polymerase chain reaction (PCR). Results. SCVs can be isolated from samples collected from chronic PJIs intraoperatively. Transmission electron microscopy (TEM) and immunofluorescence (IF) showed that there was more S. aureus in bone samples collected from chronic PJIs, but much less in bone samples from acute PJIs, providing a potential mechanism of PJI. Immunofluorescence results showed that SCVs of S. aureus were more likely to invade osteoblasts in vitro. Furthermore, TEM and IF also demonstrated that SCVs of S. aureus were more likely to invade and colonize in vivo. Cluster analysis and principal component analysis (PCA) showed that there were substantial differences in
Aims. This study aimed to explore the biological and clinical importance of dysregulated key genes in osteoarthritis (OA) patients at the cartilage level to find potential biomarkers and targets for diagnosing and treating OA. Methods. Six sets of
Aims. Osteoarthritis (OA) is a common degenerative joint disease. The osteocyte transcriptome is highly relevant to osteocyte biology. This study aimed to explore the osteocyte transcriptome in subchondral bone affected by OA. Methods.
Aims. This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD). Methods. The
Aims. This study aimed, through bioinformatics analysis, to identify the potential diagnostic markers of osteoarthritis, and analyze the role of immune infiltration in synovial tissue. Methods. The
RNA-Seq or whole transcriptome shotgun sequencing has been adopted in the last years as a reference technique to determine the presence and the quantity of different species of RNA in determined biological samples, thanks to it allows the identification every single RNA species transcribed from a reference genome. Meta-profiling takes advantage of the public availability of an increasing set of RNA-Seq data produced by different laboratories to summarize the expression levels of the different RNA species of many samples according to their biological context, giving the opportunity to perform comparisons on the
Stem cells are known to have low levels of intracellular reactive oxygen species (ROS) and high levels of glutathione. ROS are thought to interact with several pathways that affect the transcription machinery required for stem cell differentiation, and are critical for maintaining stem cell function. In this study, we are developing a new fluorescent probe that rapidly and reversibly reacts with glutathione (GSH), the most abundant non-protein thiol in living cells that acts as an antioxidant and redox regulator. Multipotent perivascular progenitor cells derived from human ESCs (hESC-PVPCs): Differentiated ESCs as embryoid bodies in the presence of BMP4 to induce mesoderm differentiation followed by a simple cell selection strategy using attachment of single cells onto collagen-coated dishes. Differential
Complement C5a receptor 1 (C5aR1) has crucial functions in host defense against danger molecules, as does toll-like receptor 2 (TLR2). Both innate immunity receptors interact in immune cells in the context of infectious inflammatory diseases often associated with bone loss, such as periodontitis. C5aR1 plays an important role in bone, as it is expressed on bone cells and strongly upregulated due to bone injury. Importantly, C5aR1-ko mice are protected against arthritis and C5aR1 contributes to bone loss in periodontitis. In contrast, less data exist on the role of TLR2 on osteoblasts, however, it is known that TLR2 is expressed on osteoblasts and contributes to bacterial-induced bone resorption. The aim of this study was to evaluate the interaction of C5aR1 and TLR2 in osteoblasts, including intracellular signaling pathways and gene expression patterns. Primary osteoblasts were isolated from 8–12 week-old WT mice and differentiated for 14 days. Osteoblasts were assessed for expression of C5aR1 and TLR2. Phosphorylation of mitogen-activated protein kinases (MAPK) in response to C5a and Pam3CSK4 (TLR2 agonist) was analyzed by immunoblotting.
Objectives. In order to screen the altered
The poor prognosis of patients with soft-tissue sarcoma as not changed in the past several decades, highlighting the necessity for new therapeutic approaches. T-cell based immunotherapies are a promising alternative to traditional cancer treatments due to their ability to target only malignant cells, leaving benign cells unharmed. The development of successful immunotherapy requires the identification and characterization of targetable immunogenic tumor antigens. Cancer-testis antigens (CTA) are a group of highly immunogenic tumor-associated proteins that have emerged as potential targets for CD8+ T-cell recognition. In addition to identifying a targetable antigen, it is crucial to understand the tumor immune microenvironment. The level of immune infiltration and mechanisms of immune suppression within the tumor play important roles in the outcome of immunotherapy. The goal of this study is to identify targetable immunogenic antigens for T-cell based immunotherapy and to characterize the tumor immune microenvironment in human dedifferentiated liposarcoma (DDLS) by Nanostring and IHC. To assess the complexity of the human DDLS tumor immune microenvironment and to identify target antigens we used the nCounter NanoString platform to generate a
Breast cancer is the most frequent malignancy in women with an estimation of 2.1 million new diagnoses in 2018. Even though primary tumours are usually efficiently removed by surgery, 20–40% of patients will develop metastases in distant organs. Bone is one of the most frequent site of metastases from advanced breast cancer, accounting from 55 to 58% of all metastases. Currently, none of the therapeutic strategies used to manage breast cancer bone metastasis are really curative. Tailoring a suitable model to study and evaluate the disease pathophysiology and novel advanced therapies is one of the major challenges that will predict more effectively and efficiently the clinical response. Preclinical traditional models have been largely used as they can provide standardization and simplicity, moreover, further advancements have been made with 3D cultures, by spheroids and artificial matrices, patient derived xenografts and microfluidics. Despite these models recapitulate numerous aspects of tumour complexity, they do not completely mimic the clinical native microenvironment. Thus, to fulfil this need, in our study we developed a new, advanced and alternative model of human breast cancer bone metastasis as potential biologic assay for cancer research. The study involved breast cancer bone metastasis samples obtained from three female patients undergoing wide spinal decompression and stabilization through a posterior approach. Samples were cultured in a TubeSpin Bioreactor on a rolling apparatus under hypoxic conditions at time 0 and for up to 40 days and evaluated for viability by the Alamar Blue test,
Aim. In vivo biofilm models play major role to study biofilm development, morphology, and regulatory molecules involve in biofilm. Due to ethical restrictions, the use mammalian models are replaced with other alternative models in basic research. Recently, we have developed insect infection model G. mellonella larvae to study implant associated biofilm infections. This model organism is easy to handle, cheap and ethical restriction free and could be used for the high through put screening of antimicrobial compounds to treat biofilm. To promote the use of this model in basic research we aimed to validate this based on the typical biofilm features such as less susceptible to the antibiotics, complexity of the biofilm structure and
Introduction. Improvements in material properties of total joint prostheses and methods of fixation mean that arthroplasty is the most effective means of restoring mobility in osteoarthritic patients. Aseptic loosening is the major cause of long-term failure of prostheses. Cobalt particles may act directly on osteoblasts, decreasing bone formation and potentially playing a role in osteolysis and aseptic loosening. Objectives. To assess
Chondrosarcomas are hyaline cartilage-forming tumours which can be classified according to malignancy through histological grading. Grade I chondrosarcomas rarely metastasize whereas in grade III chondrosarcoma metastasis is observed in 71% of cases. There is, so far, no clear molecular marker allowing an objective classification of chondrosarcoma. The aim of this project was to identify such marker genes through the comparison of gene expression of chondrosarcoma and normal hyaline cartilage and through the correlation of expression profiles to histological grading. The mRNA of 19 chondrosarcomas with different histological grades and of eight normal cartilage samples was analysed.
Abstract. Objective. Mesenchymal stem cells (MSCs) and chondrocytes have both been crucial in trials for cartilage repair, and there has been growing interest into their respective secretomes owing to their role in chondrogenic crosstalk. This has been studied by in vitro co-culture studies, yet the optimal ratio of seeding MSCs in co-culture has been understudied. Methods. Our study utilised an in vitro autologous co-culture of p0 adipose-derived MSCs (AMSCs) and articular chondrocytes derived from Kellgren-Lawrence Grade III/IV osteoarthritic knee joints (n=5). To investigate whether a large proportion of MSCs could be stimulated by a small number of chondrocytes, we seeded these MSCs at increasing logarithmic ratios to the number of chondrocytes at 1:1, 10:1, and 100:1. The AMSCs were phenotyped by a panel of MSC surface markers in flow cytometry, and allowed to undergo trilineage differentiation. Gene expression following in vitro co-culture was quantified by RT-qPCR with a panel comprising COL1A1, COL2A1, COL10A1, L-SOX5, SOX6, SOX9, ACAN, HSPG2, and COMP for chondrogenesis. Experiments were performed in triplicate. Results. The AMSCs had CD105, CD73, CD90, and heterogenous CD34 expression but not CD45, CD14, CD19, and HLA-DR expression in flow cytometric phenotyping, and demonstrated differentiation into chondrogenic, osteogenic, and adipogenic lineages. The chondrogenic
Summary Statement. Paraspinal muscle contain higher proportion of slow-twich fibers. The fixation of the rat tail induced transition of muscle fiber types in the paravertebral muscles characterised by the decrease in the proportion of the slow type myosin heavy chain. Introduction. Lumbar degenerative kyphosis often accompanies back pain, easy fatigability, fatty degeneration and atrophy of back muscles. There are two types of skeletal muscle fibers according to oxidative activities: slow-twich (Type 1) and fast-twitch (Type 2) fibers. Type 2 fibers were subdivided into three types: Type 2A, 2B and 2D/X. Each fiber type primarily expresses a specific isoform of myosin heavy chain (MHC). It has been known that back muscles contain higher proportion of MHC type 1. However, the impact of kyphosis on the proportion of fiber types in the paravertebral muscles has not been fully understood. The aim of this study is to analyze the transition of muscle fiber types after kyophotic or straight fixation using a rat tail model. Methods. A rat tail was fixed in straight or kyphotic position (straight or kyphosis group) by a custom-made external fixator and wires. A group of animals which underwent only pierced wounds in their tails served as control. The
Summary Statement. A biomimetic tissue engineering strategy involving culture on bone scaffolds in perfusion bioreactors allows the construction of stable, viable, patient-specific bone-like substitutes from human induced pluripotent stem cells. Introduction. Tissue engineering of viable bone substitutes represents a promising therapeutic strategy to mitigate the burden of bone deficiencies. Human induced pluripotent stem cells (hiPSCs) have an excellent proliferation and differentiation capacity, and represent an unprecedented resource for engineering of autologous tissue grafts, as well as advanced tissue models for biological studies and drug discovery. A major challenge is to reproducibly expand, differentiate and organize hiPSCs into mature, stable tissue structures. Based on previous studies (1,2,3), we hypothesised that the culture conditions supporting bone tissue formation from adult human mesenchymal stem cells (hMSCs) and human embryonic stem cell (hESC)-derived mesenchymal progenitors could be translated to hiPSC-derived mesenchymal progenitors. Our objectives were to: 1. Derive and characterise mesenchymal progenitors from hiPSC lines. 2. Engineer bone substitutes from progenitor lines exhibiting osteogenic potential in an osteoconductive scaffold – perfusion bioreactor culture model. 3. Assess the molecular changes associated with the culture of hiPSC-progenitors in perfusion bioreactors, and evaluate the stability of engineered bone tissue substitutes in vivo. Methods. hESC and hiPSC lines (derived using retroviral vectors, Sendai virus and episomal vectors) were karyotyped, characterised for pluripotency and induced into the mesenchymal lineage. Mesenchymal progenitors were evaluated for growth potential, expression of surface markers and differentiation potential. Progenitors exhibiting osteogenic potential were cultured on decellularised bovine bone scaffolds in perfusion bioreactors for 5 weeks as previously (3). Global
