Objectives. In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis. Methods. We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. Results. A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Conclusion. Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as MAPK and calcium, may involve in osteoporosis through osteoblastic differentiation and bone formation. Cite this article: J. J. Li, B. Q. Wang, Q. Fei, Y. Yang, D. Li. Identification of candidate genes in osteoporosis by integrated microarray analysis. Bone Joint Res 2016;5:594–601. DOI: 10.1302/2046-3758.512.BJR-2016-0073.R1

Introduction: In attempting to unravel the complex cellular responses leading to prosthetic loosening investigators have been limited to studying gene expression of extracellular molecules about which most is known whereas new microarray technology allows simultaneous expression profiling of thousands of genes from a complex sample such as the membrane formed around loosened hip prostheses. Methods: Two groups of 8 patients were recruited who have undergone primary total hip arthroplasty for osteoarthritis and subsequently developed either septic or aseptic loosening +/− osteolysis. The control group consisted of one group of 5 patients with the same initial diagnosis who had undergone identical procedures, developed no clinical or radiological signs of aseptic or septic loosening, but had come to revision surgery for other complications as defined by the Swedish Hip register: fracture without previous osteolysis, dislocation, technical error, implant fracture, polyethylene wear or pain. Periprosthetic membrane was harvested at the time of revision surgery and subjected to RNA extraction. cDNA was then synthesized and hybridised to a Human Genome u95 Genechip ® array which contains a complete set of known human genes. Data normalisation, data filtering and pattern identification was performed using Genechip®3.1 software (Affymetrix, Santa Clara, CA). Results: This has revealed the involvement of a large number of genes coding for transcriptional regulators upstream from the extracellular and cell-cell signalling molecules already known to be involved in osteolysis and deep infection and which may ultimately control the responses to wear particles and bacterial challenge. Differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation and immune response was found. Most interestingly pathways for control of local bone resorption and inflammatory response have been shown to be highly activated. Conclusions: The identification of these new pathogenetic mechanisms of total hip replacement failure make new indicators of disease susceptibility and prognosis plus new drug targets direct possibilities

Aims. To assess the effect of physical exercise (PE) on the histological and transcriptional characteristics of proteoglycan-induced arthritis (PGIA) in BALB/c mice. Methods. Following PGIA, mice were subjected to treadmill PE for ten weeks. The tarsal joints were used for histological and genetic analysis through microarray technology. The genes differentially expressed by PE in the arthritic mice were obtained from the microarray experiments. Bioinformatic analysis in the DAVID, STRING, and Cytoscape bioinformatic resources allowed the association of these genes in biological processes and signalling pathways. Results. Arthritic mice improved their physical fitness by 42.5% after PE intervention; it induced the differential expression of 2,554 genes. The bioinformatic analysis showed that the downregulated genes (n = 1,371) were significantly associated with cellular processes that mediate the inflammation, including Janus kinase-signal transducer and activator of transcription proteins (JAK-STAT), Notch, and cytokine receptor interaction signalling pathways. Moreover, the protein interaction network showed that the downregulated inflammatory mediators interleukin (IL) 4, IL5, IL2 receptor alpha (IL2rα), IL2 receptor beta (IL2rβ), chemokine ligand (CXCL) 9, and CXCL12 were interacting in several pathways associated with the pathogenesis of arthritis. The upregulated genes (n = 1,183) were associated with processes involved in the remodelling of the extracellular matrix and bone mineralization, as well as with the processes of aerobic metabolism. At the histological level, PE attenuated joint inflammatory infiltrate and cartilage erosion. Conclusion. Physical exercise influences parameters intimately linked to inflammatory arthropathies. Research on the effect of PE on the pathogenesis process of arthritis is still necessary for animal and human models. Cite this article:Bone Joint Res. 2020;9(1):36–48

Objectives. Osteoporosis is a metabolic disease resulting in progressive loss of bone mass as measured by bone mineral density (BMD). Physical exercise has a positive effect on increasing or maintaining BMD in postmenopausal women. The contribution of exercise to the regulation of osteogenesis in osteoblasts remains unclear. We therefore investigated the effect of exercise on osteoblasts in ovariectomized mice. Methods. We compared the activity of differentially expressed genes of osteoblasts in ovariectomized mice that undertook exercise (OVX+T) with those that did not (OVX), using microarray and bioinformatics. Results. Many inflammatory pathways were significantly downregulated in the osteoblasts after exercise. Meanwhile, IBSP and SLc13A5 gene expressions were upregulated in the OVX+T group. Furthermore, in in vitro assay, IBSP and SLc13A5 mRNAs were also upregulated during the osteogenic differentiation of MC3T3-E1 and 7F2 cells. Conclusion. These findings suggest that exercise may not only reduce the inflammatory environment in ovariectomized mice, indirectly suppressing the overactivated osteoclasts, but may also directly activate osteogenesis-related genes in osteoblasts. Exercise may thus prevent the bone loss caused by oestrogen deficiency through mediating the imbalance between the bone resorptive activity of osteoclasts and the bone formation activity of osteoblasts. Cite this article: W-B. Hsu, W-H. Hsu, J-S. Hung, W-J. Shen, R. W-W. Hsu. Transcriptome analysis of osteoblasts in an ovariectomized mouse model in response to physical exercise. Bone Joint Res 2018;7:601–608. DOI: 10.1302/2046-3758.711.BJR-2018-0075.R2

The concept of translational research is always hampered by the problem that most of the disease phenotypes do not have a mono causal origin. Therefore most treatment schemes based on one to three drugs are not really productive for most of the patients even if the patients are carefully selected from the responder group. Here the array techniques has inspired many research groups to develop algorithms deriving interaction networks or regulatory networks from this type of data to better get rid of the complexity of the biochemical interactions. The challenge is to find networks and to select the group of master nodes which might be good targets for a balanced multi-drug treatment. This means not only to measure one data type with array techniques but to join array data from multiple platforms and different data levels. Our goal is to integrate these data types to form networks with a predictive character for osteosarcomas.

The existing web platform CAPweb/VAMP from the Institute Curie is based on a Java web-client and R. This platform is focused on array data analysis and visualisation, can be extended by additional R modules and is therefore an excellent choice to implement further algorithms for data integration and network prediction. We are now establishing algorithms beyond a pure association of effects like permutation procedures for optimal rank orders of effects in a given subset of 16 factors which can be assembled to bigger units and selection procedures of gene expression signals by gene dosage concepts.

The presented approach is sustainable because the platform can be constantly extended and improved. On the other hand this platform is end-user suitable. This is the best way to bring theoretical concepts to the bench scientist. As a consequence translational research will become more real and complex systems more feasible.

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 gene expression profiles of RA.

Aims. This study explored the shared genetic traits and molecular interactions between postmenopausal osteoporosis (POMP) and sarcopenia, both of which substantially degrade elderly health and quality of life. We hypothesized that these motor system diseases overlap in pathophysiology and regulatory mechanisms. Methods. We analyzed microarray data from the Gene Expression Omnibus (GEO) database using weighted gene co-expression network analysis (WGCNA), machine learning, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify common genetic factors between POMP and sarcopenia. Further validation was done via differential gene expression in a new cohort. Single-cell analysis identified high expression cell subsets, with mononuclear macrophages in osteoporosis and muscle stem cells in sarcopenia, among others. A competitive endogenous RNA network suggested regulatory elements for these genes. Results. Signal transducer and activator of transcription 3 (STAT3) was notably expressed in both conditions. Single-cell analysis pinpointed specific cells with high STAT3 expression, and microRNA (miRNA)-125a-5p emerged as a potential regulator. Experiments confirmed the crucial role of STAT3 in osteoclast differentiation and muscle proliferation. Conclusion. STAT3 has emerged as a key gene in both POMP and sarcopenia. This insight positions STAT3 as a potential common therapeutic target, possibly improving management strategies for these age-related diseases. Cite this article: Bone Joint Res 2024;13(8):411–426

Objectives. Diabetes mellitus (DM) is known to impair fracture healing. Increasing evidence suggests that some microRNA (miRNA) is involved in the pathophysiology of diabetes and its complications. We hypothesized that the functions of miRNA and changes to their patterns of expression may be implicated in the pathogenesis of impaired fracture healing in DM. Methods. Closed transverse fractures were created in the femurs of 116 rats, with half assigned to the DM group and half assigned to the control group. Rats with DM were induced by a single intraperitoneal injection of streptozotocin. At post-fracture days five, seven, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture days five and 11. For further analysis, real-time polymerase chain reaction (PCR) analysis was performed at each timepoint. Results. Microarray analysis showed that there were 14 miRNAs at day five and 17 miRNAs at day 11, with a greater than twofold change in the DM group compared with the control group. Among these types of miRNA, five were selected based on a comparative and extended literature review. Real-time PCR analysis revealed that five types of miRNA (miR-140-3p, miR-140-5p, miR-181a-1-3p, miR-210-3p, and miR-222-3p) were differentially expressed with changing patterns of expression during fracture healing in diabetic rats compared with controls. Conclusions. Our findings provide information to further understand the pathology of impaired fracture healing in a diabetic rat model. These results may allow the potential development of molecular therapy using miRNA for the treatment of impaired fracture healing in patients with DM. Cite this article: S. Takahara, S. Y. Lee, T. Iwakura, K. Oe, T. Fukui, E. Okumachi, T. Waki, M. Arakura, Y. Sakai, K. Nishida, R. Kuroda, T. Niikura. Altered expression of microRNA during fracture healing in diabetic rats. Bone Joint Res 2018;7:139–147. DOI: 10.1302/2046-3758.72.BJR-2017-0082.R1

The pathophysiological basis of alterations in trabecular bone of patients with osteonecrosis of the femoral head (ONFH) remains unclear. ONFH has classically been considered a vascular disease with secondary changes in the subchondral bone. However, there is increasing evidence suggesting that ONFH could be a bone disease, since alterations in the functionality of bone tissue distant from the necrotic lesion have been observed. We comparatively studied the transcriptomic profile of trabecular bone obtained from the intertrochanteric region of patients with ONFH without an obvious aetiological factor, and patients with osteoarthritis (OA) undergoing total hip replacement in our Institution. To explore the biological processes that could be affected by ONFH, we compared the transcriptomic profile of trabecular bone from the intertrochanteric region and the femoral head of patients affected by this condition. Differential gene expression was studied using an Affymetrix microarray platform. Transcriptome analysis showed a differential signature in trabecular bone from the intertrochanteric region between patients with ONFH and those with OA. The gene ontology analyses of the genes overexpressed in bone tissue of patients with ONFH revealed a range of enriched biological processes related to cell adhesion and migration and angiogenesis. In contrast, most downregulated transcripts were involved in cell division. Trabecular bone in the intertrochanteric region and in the femoral head also exhibited a differential expression profile. Among the genes differentially expressed, we highlighted those related with cytokine production and immune response. This study identified a set of differently expressed genes in trabecular bone of patients with idiopathic ONFH, which might underlie the pathophysiology of this condition. Acknowledgements: This work was supported by grants PI18/00643 and PI22/00939 from ISCIII-FEDER, Ministerio de Ciencia, Innovación y Universidades (MICINN)-AES

In osteoarthritis, chondrocytes acquire a hypertrophic phenotype that contributes to matrix degradation. Inflammation is proposed as trigger for the shift to a hypertrophic phenotype. Using in vitro culture of human chondrocytes and cartilage explants we could not find evidence for a role of inflammatory signalling activation. We found, however, that tissue repair macrophages may contribute to the onset of hypertrophy (doi: 10.1177/19476035211021907) Intra-articularly injected triamcinolone acetonide to inhibit inflammation in a murine model of collagenase-induced osteoarthritis, increased synovial macrophage numbers and osteophytosis, confirming the role of macrophages in chondrocyte hypertrophy occurring in osteophyte formation (doi: 10.1111/bph.15780). In search of targets to inhibit chondrocyte hypertrophy, we combined existing microarray data of different cartilage layers of murine growth plate and murine articular cartilage after induction of collagenase-induced osteoarthritis. We identified common differentially expressed genes and selected those known to be associated to inflammation. This revealed EPHA2, a tyrosine kinase receptor, as a new target. Using in silico, in vitro and in vivo models we demonstrated that inhibition of EPHA2 might be a promising treatment for osteoarthritis. Recently, single cell RNA-seq. has revealed detailed information about different populations of chondrocytes in articular cartilage during osteoarthritis. We re-analysed a published scRNA-seq data set of healthy and osteoarthritic cartilage to obtain the differentially expressed genes in the population of hypertrophic chondrocytes compared to the other chondrocytes, applied pathway analyses and then used drug databases to search for upstream inhibitors of these pathways. This drug repurposing approach led to the selection of 6 drugs that were screened and tested using several in vitro models with human chondrocytes and cartilage explants. In this lecture I will present this sequence of studies to highlight different approaches and models that can be used in the quest for a disease modifying drug for osteoarthritis

Abstract. Introduction. 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. Methodology. 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. Results. Expression of 453 synovial genes was influenced by BMI and gender, 360 encode proteins such as HIF-1a, HSF1, HSPA4, HSPA5. Top canonical pathways include Unfolded protein response, Protein Ubiquiitation and Clathrin mediated endocytosis signalling linked by modulation of heat shock proteins, comparable to pathology dependent regulation. In addition BMI and gender modulate gene expression in the NRF2-mediated oxidative stress response pathway with down regulation of Glutathione-S-transferases potentially down regulating antioxidant defences. Conclusion. The enhanced risk of osteoarthritis induced by an elevated BMI and female gender maybe include differential expression of heat shock proteins and genes in the NRF2 pathway

Abstract. Introduction. 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. Methodology. 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. Results. PCA analysis illustrates the clear separation of expression array data from cultured cells compared with their parental whole tissues and no segregation between cells derived from osteoarthritic or trauma tissues. A differentially expressed gene heat map demonstrated the hierarchical independence of cultured cells from their paired sample parental tissues. The biological pathways enriched by these gene expression differences emphasise the activities of macrophages and lymphocytes lost from culture. Conclusion. Adherent synovial cells grown from different knee pathologies lose the expression patterns characteristic of their originating pathology. Interpretation of data needs caution as the cells are not representative of whole synovium

Abstract. Introduction. 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. Methodology. 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. Results. Amongst the 2084 genes with significantly differential expression (DEG), 135 had transcription regulator capabilities and 121 a nuclear location. IPA analysis of OATKR and arthroscopic tissue comparison DEG identified 12 nuclear transcription factors linked to 31 DEG whose encoded proteins located within cytoplasmic and cell membrane compartments. All 12 were significantly up-regulated and acting in pathways up-regulating transcription of DNA and RNA, cell survival and angiogenesis while down-regulating senescence and apoptosis. NFE2L2, integral to the TGF-beta signalling pathway, was identified as a bottleneck gene. Conclusion. This analysis indicates the complexity of synovial gene expression regulation and offers target genes and pathways for evaluation during osteoarthritis pathogenesis

Osteoarthritis (OA) is the most common form of arthritis and one of the ten most disabling diseases in developed countries. Total joint replacement (TJR) is considered by far as the most effective treatment for end-stage OA patients. The majority of patients achieve symptomatic improvement following TJR. However, about 22% of the TJR patients either do not improve or deteriorate after surgery. Several potential non-genetic predictors for the TJR outcome have been investigated. However, the results were either inconclusive or had very limited predictive power. The aim of this study was to identify genetic variants for the poor outcome of TJR in primary OA patients by a genome-wide association study (GWAS). Study participants were total knee or hip replacement patients due to primary OA who were recruited to the Newfoundland Osteoarthritis Study (NFOAS) before 2017. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) was used to assess pain and functional impairment pre- and 3.99±1.38 years post-surgery. Two non-responder classification criteria were used in our study. One was defined by an absolute WOMAC change score. Participants with a change score less than 7/20 points for pain were considered as pain non-responders; and those with less than 22/68 points for function were classified as function non-responders. The second one was the Outcome Measures in Arthritis Clinical Trials and the Osteoarthritis Research Society International (OMERACT-OARSI) criteria. Blood DNA samples were genotyped using the Illumina GWAS microarrays genotyping platform. The quality control (QC) filtering was performed on GWAS data before the association of the genetic variants with non-responders to TJR was tested using the GenABEL package in R with adjustment for the relatedness of the study population and using the commonly accepted GWAS significance threshold p < 5*10. −8. to control multiple testing. In total, 316 knee and 122 hip OA patients (mean age 65.45±7.62 years, and 58% females) passed the QC check. These study participants included 368 responders and 56 non-responders to pain, and 364 responders and 68 non-responders to function based on the absolute WOMAC point score change classification. While 377 responders and 56 non-responders to pain, and 366 responders and 71 non-responders to function were identified by the OMERACT-OARSI classification criteria. Interestingly, the same results were obtained by both classification methods, and we found that the G allele of rs4797006 was significantly associated with pain non-responders with odds ratio (OR) of 5.12 (p<7.27×10. -10. ). This SNP is in intron one of the melanocortin receptor 5 (MC5R) gene on chr18. This gene plays central roles in immune response, pain sensitivity, and negative regulation of inflammatory response to antigenic stimulus. The A allele of rs200752023 was associated with function non-responders with OR of 4.41 (p<3.29×10. -8. ). The SNP is located in intron three of the RNA Binding Fox-1 Homolog 3 (RBFOX3) gene on chr17 which has been associated with numerous neurological disorders. Our data suggested that two chromosomal regions are associated with TJR poor outcomes and could be the novel targets for developing strategies to improve the outcome of the TJR

Objectives. MicroRNAs (miRNAs) have been reported as key regulators of bone formation, signalling, and repair. Fracture healing is a proliferative physiological process where the body facilitates the repair of a bone fracture. The aim of our study was to explore the effects of microRNA-186 (miR-186) on fracture healing through the bone morphogenetic protein (BMP) signalling pathway by binding to Smad family member 6 (SMAD6) in a mouse model of femoral fracture. Methods. Microarray analysis was adopted to identify the regulatory miR of SMAD6. 3D micro-CT was performed to assess the bone volume (BV), bone volume fraction (BVF, BV/TV), and bone mineral density (BMD), followed by a biomechanical test for maximum load, maximum radial degrees, elastic radial degrees, and rigidity of the femur. The positive expression of SMAD6 in fracture tissues was measured. Moreover, the miR-186 level, messenger RNA (mRNA) level, and protein levels of SMAD6, BMP-2, and BMP-7 were examined. Results. MicroRNA-186 was predicted to regulate SMAD6. Furthermore, SMAD6 was verified as a target gene of miR-186. Overexpressed miR-186 and SMAD6 silencing resulted in increased callus formation, BMD and BV/TV, as well as maximum load, maximum radial degrees, elastic radial degrees, and rigidity of the femur. In addition, the mRNA and protein levels of SMAD6 were decreased, while BMP-2 and BMP-7 levels were elevated in response to upregulated miR-186 and SMAD6 silencing. Conclusion. In conclusion, the study indicated that miR-186 could activate the BMP signalling pathway to promote fracture healing by inhibiting SMAD6 in a mouse model of femoral fracture. Cite this article: Bone Joint Res 2019;8:550–562

Periprosthetic joint infection (PJI) is a potentially devastating complication of joint replacement surgery. Osteocytes comprise 90–95% of all cells in hard bone tissue, are long-lived and are becoming increasingly recognised as a critical cell type in the regulation of bone and systemic physiology. The purpose of this study was to examine role of these cells in PJI pathophysiology and aetiology, with the rationale that their involvement could contribute to the difficulty in detecting and clearing PJI. This study examined the ability of human osteocytes to become infected by Staphylococcus aureus and the responses of both the host cell and pathogen in this scenario. Several S. aureus (MRSA) strains were tested for their ability to infect human primary osteocyte-like cells in vitro and human bone samples ex vivo. Bone biopsies were retrieved from patients undergoing revision total hip arthroplasty for either aseptic loosening associated with osteolysis, or for PJI. Retrieved bacterial colony number from cell lysates and colony morphology were determined. Gene expression was measured by microarray/bioinformatics analysis and/or real-time RT-PCR. Exposure to planktonic S. aureus (approx. 100 CFU/cell) resulted in intracellular infection of human osteocyte-like cells. We found no evidence of increased rates of osteocyte cell death in bacteria exposed cultures. Microarray analysis of osteocyte gene expression 24h following exposure revealed more than 1,500 differentially expressed genes (fold-change more than 2, false discovery rate p < 0.01). The gene expression patterns were consistent with a strong innate immune response and altered functionality of the osteocytes. Consistent patterns of host gene expression were observed between experimentally infected osteocyte-like cultures and human bone, and in PJI patient bone samples. Internalised bacteria switched to the quasi-dormant small colony variant (SCV) form over a period of 5d, and the ensuing infection appeared to reach a stable state. S. aureus infection of viable osteocytes was also identified in bone taken from PJI patients. We have demonstrated [1] that human osteocytes can become infected by S. aureus and respond robustly by producing immune mediators. The bony location of the infected osteocyte may render them refractory to clearance by immune cells, and osteocytes may therefore be an immune-privileged cell type. The phenotypic switch of S. aureus to SCV, a form less sensitive to most antibiotics and one associated with intracellular survival, suggests that infection of osteocytes may contribute to a chronic disease state. The osteocyte may therefore serve as a reservoir of bacteria for reinfection, perhaps explaining the high prevalence of infections that only become apparent after long periods of time or recur following surgical/medical treatment. Our findings also provide a biological rationale for the recognised need for aggressive bone debridement in the surgical management of PJI

Osteoarthritis (OA) is the fastest growing global health problem, with a total joint replacement being the only effective treatment for patients with end stage OA. Many groups are examining the use of bone marrow or adipose derived mesenchymal stem cells (MSCs) to repair cartilage, or modulate inflammation to promote healing, however, little efficacy in promoting cartilage repair, or reducing patient symptoms over temporary treatments such as micro-fracture has been observed. There is a growing body of literature demonstrating that MSCs derived from the synovial lining of the joint are superior in terms of chondrogenic differentiation and while improvements in clinical outcome measures have been observed with synovial MSCs, results from clinical studies are still highly variable. Based on our results, we believe this variability in clinical studies with MSCs results in part from the isolation, expansion and re-injection of distinct MSCs subtypes in normal vs. OA tissues, each with differing regenerating potential. However, it remains unknown if this heterogeneity is natural (e.g. multiple MSC subtypes present) or if MSCs are influenced by factors in vivo (disease state/stage). Therefore, in this study, we undertook an ‘omics’ screening approach on MSCs from normal and OA knee synovial tissue. Specifically, we characterized their global proteome and genomic expression patterns to determine if multiple MSC from normal and OA joints are distinct at the protein/gene expression level and/if so, what proteins/genes are differentially expressed between MSCs derived from normal and OA synovial tissue. Synovium tissue was collected from OA patients undergoing joint replacement and normal cadaveric knees. The in vitro adipogenic, chondrogenic and osteogenic differentiation potential of the MSCs was analyzed via qPCR and histology. Fully characterized MSC populations where then analyzed through an unbiased shotgun proteomics, and microarray analysis. Synovial MSCs isolated from both OA and normal knees demonstrated similar multipotent differentiation capacity. Likewise, both OA and normal MSCs display the typical MSCs cell surface marker profile in vitro (CD90+, CD44+, CD73+, CD105+). Using shotgun proteomics, 7720 unique peptides corresponding to 2183 proteins were identified and quantified between normal and OA MSCs. Of these 2183 proteins, 994 were equally expressed in normal and OA, MSCs, 324 were upregulated in OA MSCs (with 50 proteins exclusively expressed in OA MSCs), 630 proteins were upregulated in normal MSCs (with 16 proteins exclusively expressed in normal MSCs). Microarray analysis of normal and OA MSCs demonstrated a similar result in where, 967 genes were differentially expressed between normal and OA MSCs, with 423 genes upregulated in OA, and 544 genes upregulated in normal MSCs. In this project, we have demonstrated that although normal and OA synovial derived MSCs demonstrate similar multipotent differentiation potential and cell surface markers expression, these cells demonstrated significant differences at the molecular level (protein and gene expression). Further research is required to determine if these differences influence functional differences in vitro and/or in vivo and what drives this dramatic change in the regulatory pathways within normal vs. OA synovial MSCs

Commonly used alterations of prosthetic surfaces include grit-blasting (GB), plasma-sprayed titanium (Ti) or hydroxyapatite (HA) coating. Systemic concentrations of cobalt (Co) and chromium (Cr) are elevated in patients with metal-on-metal hip replacement, but can occur for all modular hip replacements. Here, we use whole genome microarrays to assess differential gene expression in primary human osteoblasts grown in vitro and on these prosthesis surfaces following exposure to clinically relevant concentrations of Co and Cr. Mesenchymal cells obtained from bone-fragments of 3 patients undergoing joint replacement surgery were differentiated into osteoblasts. Subsequently, cells were cultured in vitro on tissue-culture plates (TCP), or on GB, Ti and HA surfaces (JRI Orthopaedics Ltd, Sheffield, UK). Following 24hr exposure to a combination of clinically equivalent concentrations of Co2+:Cr3+, RNA was extracted and hybridized to SurePrint-G3 Gene Expression Microarray. Probe signals were normalised using ‘Limma’ package on R-Bioconductor and differential gene expression assessed with empirical Bayes approach (Log2FC>1.00, P<0.001 for differentially expressed genes). For cells grown on TCP, 11 genes were upregulated with 500μg/L Co2+:Cr3+. Of these, 4 were associated to HIF-1 signalling based on KEGG pathway analysis (P=5.4e-5). Exposure to 1000μg/L Co2+:Cr3+ altered expression at 164 loci for HA surfaces, and a separate 50 loci for Ti surfaces compared to GB surfaces. Genes for osteoblast differentiation (BMP2 and RGS2) were downregulated on HA surfaces compared to GB, whilst genes for cell-adhesion (ESAM), vesicular trafficking (RAB37) and protection against oxidative damage (NRF2) were upregulated. Ti surfaces caused an upregulation in ERBB3 and CNTF, which are associated with inhibition of osteoblast differentiation and mineralisation, when compared to GB surfaces. This study confirms the role of HIF-1 signalling in response to prosthesis generated metal ions, and is the first to provide a comprehensive genome-wide insight into transcriptional response of osteoblasts at prosthesis surface to clinically equivalent metal exposure

Objectives. Osteoporosis is a chronic disease. The aim of this study was to identify key genes in osteoporosis. Methods. Microarray data sets GSE56815 and GSE56814, comprising 67 osteoporosis blood samples and 62 control blood samples, were obtained from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified in osteoporosis using Limma package (3.2.1) and Meta-MA packages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to identify biological functions. Furthermore, the transcriptional regulatory network was established between the top 20 DEGs and transcriptional factors using the UCSC ENCODE Genome Browser. Receiver operating characteristic (ROC) analysis was applied to investigate the diagnostic value of several DEGs. Results. A total of 1320 DEGs were obtained, of which 855 were up-regulated and 465 were down-regulated. These differentially expressed genes were enriched in Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways, mainly associated with gene expression and osteoclast differentiation. In the transcriptional regulatory network, there were 6038 interactions pairs involving 88 transcriptional factors. In addition, the quantitative reverse transcriptase-polymerase chain reaction result validated the expression of several genes (VPS35, FCGR2A, TBCA, HIRA, TYROBP, and JUND). Finally, ROC analyses showed that VPS35, HIRA, PHF20 and NFKB2 had a significant diagnostic value for osteoporosis. Conclusion. Genes such as VPS35, FCGR2A, TBCA, HIRA, TYROBP, JUND, PHF20, NFKB2, RPL35A and BICD2 may be considered to be potential pathogenic genes of osteoporosis and may be useful for further study of the mechanisms underlying osteoporosis. Cite this article: B. Xia, Y. Li, J. Zhou, B. Tian, L. Feng. Identification of potential pathogenic genes associated with osteoporosis. Bone Joint Res 2017;6:640–648. DOI: 10.1302/2046-3758.612.BJR-2017-0102.R1

It is supposed that disturbed vascularization is a major cause for the development of an atrophic non-union. However, an actual study revealed normal vessel formation in human non-union tissues [1]. An animal study using an atrophic non-union model should clarify the influence of the inhibition of angiogenesis by the inhibitor Fumagillin on bone healing and the underlying processes including inflammation, chondrogenesis, angiogenesis and osteogenesis. For each group and time point (3, 7, 14, 21 and 42 days) 5–6 adult female Sprague Dawley rats were analyzed. The tibia was osteotomized and stabilized intramedullary with a k-wire coated with the drug carrier PDLLA (control group) or PDLLA +10% Fumagillin (atrophy group). Microarrays: Total-RNA were pooled per group, labeled with the Agilent single-color Quick-Amp Labeling Kit Cy3 and hybridized on Agilent SurePrint G3 Rat Gene Expression microarrays. After feature extraction and quantile normalization, relevant biological processes were identified using GeneOntology. Genes with an expression value below the 25. percentile were excluded. Heatmaps were used for visualization. The analysis of inflammatory genes revealed an upregulation of monocyte/macrophage- relevant factors such as the chemokines Ccl2 and Ccl12 and the surface marker CD14. Other factors involved in the early inflammation process such as Il1a, Tnf and Il6 were not affected. Chondrogenic markers including Collagen Type II, -IX, -X, Mmp9, Mmp13, Hapln1, Ucma, Runx2, Sox5 and -9 were downregulated in this group. Furthermore, osteogenic factors were less regulated within the middle stage of healing (day 14–21). This gene panel included Bmps, Bmp antagonists, Bmp- and Tgfb receptors, integrines and matrix proteins. qPCR analysis of angiogenic genes showed an upregulation of Angpt2, Fgf1 and -2, but not for Vegfa over the later healing time points. We demonstrated in a previous study that inhibiting angiogenesis in an osteotomy model led to a reduction in vessel formation and to the development of an atrophic non-union phenotype [2]. The microarray analysis indicated no prolonged inflammatory reaction in the atrophy group. But the upregulation of chemokines together with a delay in hematoma degradation signs to a mismatch between recruitment and demand of macrophages from the vessel system. Furthermore, chondrogenesis was completely blocked, which was shown by a downregulation of chondrogenic but also osteogenic markers being involved in chondrogenic processes. A reduced recruitment of MSCs might be a possible explanation. Although, microarray data revealed only minor expression changes regarding angiogenic genes, validation by q-PCR showed an upregulation of Angpt2, Fgf1 and -2 over the later healing time points. Due to the heterogeneity of the callus tissue it might be that variations of gene expression of a single tissue type will be masked by the expression levels of other tissue types. This issue is even more pronounced when analyzing different time points and by pooling the samples

Introduction and Objective. Senescent bone cell overburden accelerates osteoporosis. Epigenetic alteration, including microRNA signalling and DND methylation, is one of prominent features of cellular senescence. This study aimed to investigate what role microRNA-29a signalling may play in the development of senile osteoporosis. Materials and Methods. Bone biopsy and serum were harvested from 13 young patients and 15 senior patients who required spine surgery. Bone mass, microstructure, and biomechanics of miR-29a knockout mice (miR-29aKO) and miR-29a transgenic mice (miR-29aTg) were probed using mCT imaging and three-point bending material test. Senescent cells were probed using senescence-associated b-galactosidase (SA-b-gal) staining. Transcriptomic landscapes of osteoblasts were characterized using whole genome microarray and KEGG bioinformatics. miR-29a and senescence markers p16. INK4a. , p21. Waf/cipl. and inflammatory cytokines were quantified using RT-PCR. DNA methylome was probed using methylation-specific PCR and 5-methylcytosine immunoblotting. Results. Senescent osteoblast overburden, DNA hypermethylation and oxidative damage together with significant decreases in serum miR-29a levels were present in bone specimens of aged patients. miR-29aKO mice showed a phenotype of skeletal underdevelopment, low bone mineral density and weak biomechanics. miR-29a knockout worsened age-induced bone mass and microstructure deterioration. Of note, aged miR-29aTg mice showed less bone loss and fatty marrow than aged wild-type mice. Transgenic overexpression of miR-29s compromised age-dysregulated osteogenic differentiation capacity of bone-marrow mesenchymal cells. In vitro, miR-29a promoted transcriptomic landscapes of antioxidant proteins in osteoblasts. The microRNA interrupted DNA methyltransferase (Dnmt3b)-mediated DNA methylation, inhibiting reactive oxygen radicals burst, IL-6 and RANKL production, and a plethora of senescent activity, including increased p16. INK4a. , p21. Waf/cipl. signalling and SA-b-gal activity. Conclusions. miR-29a loss is correlated with human age-mediated osteoporosis. miR-29a signalling is indispensable in bone mase homeostasis and microstructure integrity. Gain of miR-29a function is advantageous to delay age-induced bone loss through promoting antioxidant proteins to inhibit DNA hypermethylation-mediated osteoblast senescence. Collective investigations shine light onto the anabolic effects miR-29a signalling to bone integrity and highlight a new epigenetic protection strategy through controlling microRNA signalling to delay osteoblast senescence and senile osteoporosis development

Aim. Osteoarthritis (OA) is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control the expression of genes and are likely to regulate the OA transcriptome. We performed integrative genomic analyses to define methylation-gene expression relationships in osteoarthritic cartilage. Patients and Methods. Genome-wide DNA methylation profiling of articular cartilage from five patients with OA of the knee and five healthy controls was conducted using the Illumina Infinium HumanMethylation450 BeadChip (Illumina, San Diego, California). Other independent genome-wide mRNA expression profiles of articular cartilage from three patients with OA and three healthy controls were obtained from the Gene Expression Omnibus (GEO) database. Integrative pathway enrichment analysis of DNA methylation and mRNA expression profiles was performed using integrated analysis of cross-platform microarray and pathway software. Gene ontology (GO) analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Results. We identified 1265 differentially methylated genes, of which 145 are associated with significant changes in gene expression, such as DLX5, NCOR2 and AXIN2 (all p-values of both DNA methylation and mRNA expression < 0.05). Pathway enrichment analysis identified 26 OA-associated pathways, such as mitogen-activated protein kinase (MAPK) signalling pathway (p = 6.25 × 10-4), phosphatidylinositol (PI) signalling system (p = 4.38 × 10-3), hypoxia-inducible factor 1 (HIF-1) signalling pathway (p = 8.63 × 10-3 pantothenate and coenzyme A (CoA) biosynthesis (p = 0.017), ErbB signalling pathway (p = 0.024), inositol phosphate (IP) metabolism (p = 0.025), and calcium signalling pathway (p = 0.032). Conclusion. We identified a group of genes and biological pathwayswhich were significantly different in both DNA methylation and mRNA expression profiles between patients with OA and controls. These results may provide new clues for clarifying the mechanisms involved in the development of OA. Cite this article: A. He, Y. Ning, Y. Wen, Y. Cai, K. Xu, Y. Cai, J. Han, L. Liu, Y. Du, X. Liang, P. Li, Q. Fan, J. Hao, X. Wang, X. Guo, T. Ma, F. Zhang. Use of integrative epigenetic and mRNA expression analyses to identify significantly changed genes and functional pathways in osteoarthritic cartilage. Bone Joint Res 2018;7:343–350. DOI: 10.1302/2046-3758.75.BJR-2017-0284.R1

Objectives. We have previously investigated an association between the genome copy number variation (CNV) and acetabular dysplasia (AD). Hip osteoarthritis is associated with a genetic polymorphism in the aspartic acid repeat in the N-terminal region of the asporin (ASPN) gene; therefore, the present study aimed to investigate whether the CNV of ASPN is involved in the pathogenesis of AD. Methods. Acetabular coverage of all subjects was evaluated using radiological findings (Sharp angle, centre-edge (CE) angle, acetabular roof obliquity (ARO) angle, and minimum joint space width). Genomic DNA was extracted from peripheral blood leukocytes. Agilent’s region-targeted high-density oligonucleotide tiling microarray was used to analyse 64 female AD patients and 32 female control subjects. All statistical analyses were performed using EZR software (Fisher’s exact probability test, Pearson’s correlation test, and Student’s t-test). Results. CNV analysis of the ASPN gene revealed a copy number loss in significantly more AD patients (9/64) than control subjects (0/32; p = 0.0212). This loss occurred within a 60 kb region on 9q22.31, which harbours the gene for ASPN. The mean radiological parameters of these AD patients were significantly worse than those of the other subjects (Sharp angle, p = 0.0056; CE angle, p = 0.0076; ARO angle, p = 0.0065), and all nine patients required operative therapy such as total hip arthroplasty or pelvic osteotomy. Moreover, six of these nine patients had a history of operative or conservative therapy for developmental dysplasia of the hip. Conclusions. Copy number loss within the region harbouring the ASPN gene on 9q22.31 is associated with severe AD. A copy number loss in the ASPN gene region may play a role in the aetiology of severe AD. Cite this article: T. Sekimoto, M. Ishii, M. Emi, S. Kurogi, T. Funamoto, Y. Yonezawa, T. Tajima, T. Sakamoto, H. Hamada, E. Chosa. Copy number loss in the region of the ASPN gene in patients with acetabular dysplasia: ASPN CNV in acetabular dysplasia. Bone Joint Res 2017;6:439–445. DOI: 10.1302/2046-3758.67.BJR-2016-0094.R1

MicroRNAs (miRNAs ) are small non-coding RNAs that regulate gene expression. We hypothesised that the functions of certain miRNAs and changes to their patterns of expression may be crucial in the pathogenesis of nonunion. Healing fractures and atrophic nonunions produced by periosteal cauterisation were created in the femora of 94 rats, with 1:1 group allocation. At post-fracture days three, seven, ten, 14, 21 and 28, miRNAs were extracted from the newly generated tissue at the fracture site. Microarray and real-time polymerase chain reaction (PCR) analyses of day 14 samples revealed that five miRNAs, miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p, were highly upregulated in nonunion. Real-time PCR analysis further revealed that, in nonunion, the expression levels of all five of these miRNAs peaked on day 14 and declined thereafter. . Our results suggest that miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p may play an important role in the development of nonunion. These findings add to the understanding of the molecular mechanism for nonunion formation and may lead to the development of novel therapeutic strategies for its treatment. Cite this article: Bone Joint J 2015; 97-B:1144–51

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. Gene expression profiling after 30 min and 4 h stimulation of C5a was performed by microarray and candidate genes were validated by quantitative Real-Time PCR (qRT-PCR). Immunoprecipitation was performed using a C5aR1-antibody and C5aR1 and TLR2 were subsequently detected by immunoblotting. Statistics: One way ANOVA p<0.05, n=4–6. We showed that C5aR1 and TLR2 are expressed on osteoblasts and strongly upregulated during differentiation. Via immunoprecipitation, we could show that C5aR1 and TLR2 do physically interact in osteoblasts. We then examined if C5aR1 and TLR2, besides their physical interaction, also act via the same intracellular signaling pathways. Gene expression profiling upon C5a stimulation revealed that the top regulated pathways are related to MAPK and transforming growth factor beta (TGF-β). Respective genes, such as TGF-β (Tgfb1) and its receptor (Tgfbr) were found to be upregulated, and negative MAPK regulators were found to be downregulated, both by microarray analysis and qRT-PCR. Accordingly, we saw a C5aR1- and TLR2-dependent phosphorylation of p38 MAPK. Interestingly, this effect was enhanced and prolonged by costimulation of both receptors. An additive effect of C5aR1 and TLR2 was also seen regarding Cxcl10 levels, which were enhanced compared to C5aR1 or TLR2 stimulation alone. This study shows that C5aR1 and TLR2 interact in osteoblasts, not only physically but also functionally, regarding downstream signaling and target genes. Those data strongly imply a synergistic interplay between the receptors, through which osteoblasts possibly contribute to inflammatory reactions affecting bone

Aims. Animal models have been developed that allow simulation of post-traumatic joint contracture. One such model involves contracture-forming surgery followed by surgical capsular release. This model allows testing of antifibrotic agents, such as rosiglitazone. Methods. A total of 20 rabbits underwent contracture-forming surgery. Eight weeks later, the animals underwent a surgical capsular release. Ten animals received rosiglitazone (intramuscular initially, then orally). The animals were sacrificed following 16 weeks of free cage mobilisation. The joints were tested biomechanically, and the posterior capsule was assessed histologically and via genetic microarray analysis. Results. There was no significant difference in post-traumatic contracture between the rosiglitazone and control groups (33° (standard deviation (. sd. ) 11) vs 37° (. sd. 14), respectively; p = 0.4). There was no difference in number or percentage of myofibroblasts. Importantly, there were ten genes and 17 pathways that were significantly modulated by rosiglitazone in the posterior capsule. Discussion. Rosiglitazone significantly altered the genetic expression of the posterior capsular tissue in a rabbit model, with ten genes and 17 pathways demonstrating significant modulation. However, there was no significant effect on biomechanical or histological properties. Cite this article: M. P. Abdel. Effectiveness of rosiglitazone in reducing flexion contracture in a rabbit model of arthrofibrosis with surgical capsular release: A biomechanical, histological, and genetic analysis. Bone Joint Res 2016;5:11–17. doi: 10.1302/2046-3758.51.2000593

During OA the homeostasis of healthy articular chondrocytes is dysregulated, which leads to a phenotypical transition of the cells, further influenced by external stimuli. Chondrocytes sense those stimuli, integrate them at the intracellular level and respond by modifying their secretory and molecular state. This process is controlled by a complex interplay of intracellular factors. Each factor is influenced by a myriad of feedback mechanisms, making the prediction of what will happen in case of external perturbation challenging. Hampering the hypertrophic phenotype has emerged as a potential therapeutic strategy to help OA patients (Ripmeester et al. 2018). Therefore, we developed a computational model of the chondrocyte's underlying regulatory network (RN) to identify key regulators as potential drug targets. A mechanistic mathematical model of articular chondrocyte differentiation was implemented with a semi-quantitative formalism. It is composed of a protein RN and a gene RN(GRN) and developed by combining two strategies. First, we established a mechanistic network based on accumulation of decades of biological knowledge. Second, we combined that mechanistic network with data-driven modelling by inferring an OA-GRN using an ensemble of machine learning methods. This required a large gene expression dataset, provided by distinct public microarrays merged through an in-house pipeline for cross-platform integration. We successfully merged various micro-array experiments into one single dataset where the biological variance was predominant over the batch effect from the different technical platforms. The gain of information provided by this merge enabled us to reconstruct an OA-GRN which subsequently served to complete our mechanistic model. With this model, we studied the system's multi-stability, equating the model's stable states to chondrocyte phenotypes. The network structure explained the occurrence of two biologically relevant phenotypes: a hypertrophic-like and a healthy-like phenotype, recognized based on known cell state markers. Second, we tested several hypotheses that could trigger the onset of OA to validate the model with relevant biological phenomena. For instance, forced inflammation pushed the chondrocyte towards hypertrophy but this was partly rescued by higher levels of TGF-β. However, we could annihilate this rescue by concomitantly mimicking an increase in the ALK1/ALK5 balance. Finally, we performed a screening of in-silico (combinatorial) perturbations (inhibitions and/or over-activations) to identify key molecular factors involved in the stability of the chondrocyte state. More precisely, we looked for the most potent conditions for decreasing hypertrophy. Preliminary validation experiments have confirmed that PKA activation could decrease the hypertrophic phenotype in primary chondrocytes. Importantly the in-silico results highlighted that targeting two factors at the same time would greatly help reducing hypertrophic changes. A priori testing of conditions with in-silico models may cut time and cost of experiments via target prioritization and opens new routes for OA combinatorial therapies

Total joint replacement (TJR) is by far the most effective therapy for end-stage OA patients. Most of patients achieve joint pain reduction and function improvement following to TJR, however up to 22% of them either do not improve or deteriorate after surgery. The aim of this study was to identify genetic variants to be associated with poor outcome of TJR in primary OA patients by a genome-wide association approach (GWAS). Study participants were primary OA patients from the Newfoundland Osteoarthritis Study (NFOAS) that comprised total knee or hip replacement and recruited before 2016 in St. John's, NL. DNA samples were extracted from patients' blood. Study participants completed their pre-operation and 3.99±1.38 years post-surgery outcome assessment using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). DNA samples were genotyped using the genome-wide Illumina HumanOmni2.58 genotyping microarray containing 2.4 million SNPs. Pre-association quality control filtering was conducted for the raw genotyping data using PLINK 1.7 program, and genotype imputation was performed using the IMPUTE2 algorithm with multiple population reference data from 1000 Genome Project. The imputed data with ∼3.1 million variants was used to test the association with non-responders to TJR using the additive genetic model. Eighty three primary OA patients (44 responders and 39 non-responders) were included in the analysis. Association analysis detected three chromosomal regions on chr5, 7, and 8 to be significantly associated with non-responding to pain. The top SNPs at these loci are intergenic variants that include SNP (rs17118094, p=4.4×10-5) on chr5. This SNP is adjacent to SGCD gene that plays an important role in muscular strength and maintenance. Another associated SNP (rs71572810, p=4.7×10-5) is nearby IMMP2L gene on chr7. This gene is reported to be associated with behavioral abnormalities. Finally, SNP (rs6992938, p=5.8×10-5) on chr8 is located downstream of TRPA1 gene that is known to have a central role in the pain response to endogenous inflammatory mediators. Three loci were also found to be significantly associated with non-responding to function. The lead variant in the locus on chr1 is an intergenic SNP (rs9729377, p=1.7×10-5) falling between CTBS and MCOLN2 genes. CTBS gene is associated with TNF-α, a cytokine that stimulate the inflammation acute phase reaction, and MCOLN2 gene plays a role in the chemokine secretion and macrophage migration in the innate immune response. Other top SNPs in loci on chr2 and 10 harbor CCDC93, INSIG2, and KLF6 genes that are associated with heel bone mineral density, hypercholesterolemia, obesity and BMI. To our knowledge, this project is the first study that investigated the association between genetic factors and TJR non-responders. Our results demonstrated that genes related to muscle strength, behavioral trait, pain response, and inflammation play a significant role in poor outcome of TJR, warranting further investigation

Major drawbacks associated with autologous bone grafting are the risk of donor site morbidity and its limited availability. Sterilized bone allograft, however, lacking osteoinductive properties, carries the risk of graft failure resulting from insufficient osseointegration of the graft. The aim of this study was to vitalize bone allograft with human osteoprogenitor cells under GMP-conform conditions. For this purpose we investigated proliferation, osteogenic differentiation and large-scale gene expression of human MSCs cultured three-dimensionally on peracetic acid (PAA)-treated spongious bone chips. MSCs were isolated from healthy donors (N=5) and seeded onto PAA-treated spongious bone samples (~5×5×5 mm, DIZG, Germany) under GMP-conform conditions. Proliferation (total protein assay), osteogenic differentiation (cell-specific ALP activity assay, quantitative gene expression analysis of selected osteogenic marker genes), and morphology were assessed. RNA was isolated and microarray analysis was performed using the PIQORTM Stem Cell Microarray system (Miltenyi Biotec) including 942 target sequences. Increasing cellularity was observed during the 42 d observation period while cell-specific ALP activity peaked at day 21. Effective proliferation and adhesion of human MSCs on PAA-treated spongious bone was confirmed by histology, scanning electron and confocal laser scanning microscopy. Gene expression of early (Runx-2), intermediate (ALP), and late (osteocalcin) osteogenic marker genes was present during 42 days of cultivation. Microarray analysis of MSCs cultivated on bone allograft versus 2-D tissue culture demonstrated temporal upregulation of genes involved in extracellular matrix synthesis (e.g., matrix metalloproteases, collagens), osteogenesis (e.g., BMPR1b, Runx-2) and angiogenesis (angiopoietin, VEGF). PAA-treated spongious bone allograft is a biocompatible carrier matrix for long-term ex vivo cultivation of MSCs as observed by favorable proliferation, cell distribution, gene expression profile, and persisting osteogenic differentiation. GMP-grade vitalisation of bone allograft by cultivation with autologous MSCs represents a promising clinical application for the treatment of osseous defects

Metallic implants are widely used in orthopedic, oral and maxillofacial surgery. Durable osseous fixation of an implant requires that osteoprogenitor cells attach and adhere to the implant, proliferate, differentiate into osteoblasts, and produce mineralized matrix. We previously observed that human mesenchymal stem cells (MSCs) adherent to smooth tantalum (Ta) surfaces demonstrated superior biocompatibility compared with titanium (Ti) coatings. The aim of the present study was to investigate the interactions between MSCs and smooth surfaces of Ta and by means of whole-genome microarray technology. Immortalized human mesenchymal stem cells were cultivated on smooth surfaces of Ti and Ta. Total RNA was extracted after culturing for 1, 2, 4, and 8 days and hybridized to Affymetrix whole-genome microarrays (N=16). Replicate arrays were averaged and the ratios of gene expression by MSCs cultivated on Ta versus Ti coating were calculated. Absolute fold differences were also calculated and lists of upregulated genes were generated. Moreover, gene Ontology (GO) annotation analysis of differentially regulated genes was performed. For both Ta and Ti coatings, the vast majority of genes were upregulated after 4 d of cultivation. Genes upregulated by MSCs cultivated on Ta coating for 4 d were annotated to relevant GO terms. Ti-regulated GO annotation clusters were predominantly transcription-related. By using the K-means clustering algorithm, 10 clusters containing more than 5 genes were identified. Moreover, various genes related to osteogenesis and cell adhesion were upregulated by MSCs exposed to Ta surface. Microarray analysis of MSCs exposed to smooth metallic surfaces of both Ta and Ti generally showed a huge increase in transcriptional activity after 4 d of cultivation. According to GO annotation analysis Ta coating may induce increased adhesion and earlier differentiation of MSCs compared to Ti surface making Ta a promising biocompatible material for bone implants

Objectives. The goal of this study was to determine whether intra-articular administration of the potentially anti-fibrotic agent decorin influences the expression of genes involved in the fibrotic cascade, and ultimately leads to less contracture, in an animal model. Methods. A total of 18 rabbits underwent an operation on their right knees to form contractures. Six limbs in group 1 received four intra-articular injections of decorin; six limbs in group 2 received four intra-articular injections of bovine serum albumin (BSA) over eight days; six limbs in group 3 received no injections. The contracted limbs of rabbits in group 1 were biomechanically and genetically compared with the contracted limbs of rabbits in groups 2 and 3, with the use of a calibrated joint measuring device and custom microarray, respectively. Results. There was no statistical difference in the flexion contracture angles between those limbs that received intra-articular decorin versus those that received intra-articular BSA (66° vs 69°; p = 0.41). Likewise, there was no statistical difference between those limbs that received intra-articular decorin versus those who had no injection (66° vs 72°; p = 0.27). When compared with BSA, decorin led to a statistically significant increase in the mRNA expression of 12 genes (p < 0.01). In addition, there was a statistical change in the mRNA expression of three genes, when compared with those without injection. . Conclusions. In this model, when administered intra-articularly at eight weeks, 2 mg of decorin had no significant effect on joint contractures. However, our genetic analysis revealed a significant alteration in several fibrotic genes. Cite this article: Bone Joint Res 2014;3:82–8

Introduction. The biological pathways responsible for adverse reactions to metal debris (ARMD) are unknown. Necrotic and inflammatory changes in response to Co-Cr nanoparticles in periprosthetic tissues may involve both a cytotoxic response and a type IV delayed hypersensitivity response. Our aim was to establish whether differences in biological cascade activation exists in tissues of patients with end-stage OA compared to those with aseptic loosening of a metal on polyethylene (MoP) THR and those with ARMD from metal-on-metal (MoM) THR. Patients & Methods. A microarray experiment (Illumina HT12-v4) was performed to identify the range of differential gene expression between 24 patients across 3 phenotypes: Primary OA (n=8), revision for aseptic loosening of MoP THR (n=8) and ARMD associated with MoM THR (n=8). Results were validated using Taqman Low Density Array (TLDA) selecting the top 36 genes in terms of fold-change (FC)>2 and a significant difference (p<0.05) on ANOVA. Pathways of cellular interaction were explored using Ingenuity IPA software. Results. There is a similar pattern of gene expression between MoP and MoM phenotypes versus primary OA across 33,777 genes. One hundred and thirty significantly differentially expressed genes across 3 phenotypes were identified. Fifteen pathways were associated with differentially expressed genes between MoP and MoM phenotypes. TLDA demonstrated qualitative mirroring of the expression pattern observed in the microarray and consistency in the direction of change for individual genes. Discussion. There were no signature pathways in which multiple genes are differentially expressed such that inferences between the contributions of innate macrophage and adaptive T-cell responses can be made. TIMP3 & MMP12 were consistently identified in 15 pathways that were associated with differential gene expression between MoP and MoM phenotypes. Conclusion. Analyses of the expression of individual genes such as PRG4 (lubricin) have demonstrated patterns that may provide avenues for further research into biomarkers for periprosthetic osteolysis and ARMD

Introduction. Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity. It occurs mainly in girls and progresses during pre-pubertal and pubertal growth, which is a crucial period for bone mass acquisition. The cause and molecular mechanisms of AIS are not clear; at present the consensus is that AIS has a multifactor cause, with many genetic factors. During the past 5 years, considerable effort has been devoted to identify a gene or genes that cause a predisposition to AIS. Many loci for this disorder have been mapped to different chromosome regions, but no genes have been clearly identified as being responsible for AIS, and, most importantly, the resulting protein defects remain to be shown. We aimed to identify the gene(s) that could be involved in AIS and to validate their involvement by both genetic and functional analyses. Methods. A large multiplex AIS French family was chosen for this study on the basis of clinical and radiological data. Whole genome genotyping of the 20 members of this family led to the mapping of a dominant disease-causing gene to two critical genomic intervals (Edery and colleagues, Eur J Hum Genet, accepted [2011]), but the causative mutation remains to be identified. In parallel, gene expression profiling was investigated by microarray analysis in RNA samples isolated from osteoblasts derived from healthy individuals and those with AIS. RNA samples were extracted from osteoblasts, purified, fluorescently labelled, and then hybridised to gene expression microarrays with the Illumina expression BeadChips technology containing more than 46 000 probes for the human genome (HumanHT-12). Data analysis in R version 2.10.1 (Bioconductor packages oligo and limma) was done, and genes that had at least 1·5-fold change in expression were considered differentially regulated relative to controls. AIS candidate genes within the critical intervals were selected on the basis of their mRNA expression in AIS individuals and by their known functions. The coding regions of these candidate genes were then sequenced to identify potential mutations. The biological activity of mutant proteins is under evaluation by in-vivo functional studies in zebrafish. Results. In the AIS family, a maximum LOD score of 3·01 was reached on two specific chromosomal regions. The interval lengths of these regions were 7cM and 12cM. These two regions contain several genes that might be responsible for AIS. Microarray analysis showed many genes that are differentially regulated in AIS osteoblasts compared with control osteoblasts. We recorded that 2·6% of the 24000 genes examined were upregulated in AIS osteoblasts, whereas 2·16% of them were downregulated. We observed a roughly 3-fold increase or decrease in the transcripts of many genes in AIS osteoblasts. Some of the differentially regulated genes are located within the two chromosomal candidate regions. The sequencing of the candidate genes' coding sequences was done on the family members. Sequence analysis showed two rare SNPs located on the coding regions of a gene that we called CH5G1. These two SNPs are located on the C-terminal region of the CH5G1 protein and affect its structure and probably its cellular activity and biological process leading to the disease. The C-terminal region of this protein interacts with the mRNA of a gene whose defects cause scoliosis as a secondary phenotype. The pathogenic nature of these SNPs is being investigated in the zebrafish model. The results suggest that CH5G1 gene's defects could be associated with AIS in this family. Conclusions. Identification of susceptibility genes for AIS will facilitate the understanding of underlying biochemical pathways (functional studies) and ultimately the development of specific therapies (pharmacological studies). This is likely to have important implications, since the cause of AIS is unknown. Acknowledgments. This study is supported by the Fondation Yves Cotrel, Institut de France

Purpose and Background. The intervertebral disc is constantly subjected to forces generated by movement. But degeneration can disrupt normal biomechanics, generating uneven and complex loading patterns. Evidence suggests that these forces are converted into voltages through different mechanisms, such as streaming potentials. This implicates voltage-gated ion channels in the biological remodelling response of the disc to loading. These signalling pathways have not been studied, and this incomplete understanding of disc mechanotransduction may hinder regenerative therapies. The purpose of this study is to identify and determine the role of voltage-gated ion channels in the intervertebral disc and to investigate any changes in degeneration. Methods and Results. Primary bovine and human disc cells were cultured in monolayer or alginate beads for experiments. Cells were treated with altered osmolarity alone or in combination with IL-1β. Ion flux was measured through calcium influx and will be further investigated using the xCelligence RTCA CardioECR. Immunohistochemistry was performed on human and bovine discs to evaluate expression levels of ion channels. RNA was extracted from bovine NP cells and will be analysed through PCR/Microarray for gene expression. Conclusions. Preliminary results show that the Ca. v. 2.2 channel is expressed across the human disc, and is altered by degree of degeneration. Treatment with IL-1β may partly hinder the increase in calcium signalling of disc cells in response to lower osmolarity conditions. The presence of voltage-gated ion channels in the disc has been demonstrated for the first time. The role of these channels will be investigated through measuring ion flux with channel inhibitors across different culture treatments. No conflicts of interest exist. This research was supported by funding from the Society for Back Pain Research through the Travel Award 2019 and from the Irish Research Council under the Government of Ireland Postgraduate Scholarship Programme (GOIPG/2018/2416)

Equilibrative nucleoside transporter 1 (ENT1) transfers nucleosides, such as adenosine, across plasma membranes. We reported previously that mice lacking ENT1 (ENT1-KO) exhibit progressive ectopic calcification of spinal tissues, including the annulus fibrosus (AF) of intervertebral discs (J Bone Miner Res 28:1135–49, 2013, Bone 90:37–49, 2016). Our purpose was twofold: (1) to compare ectopic calcifications in ENT1-KO mice with those in human DISH, and (2) to investigate the molecular pathways underlying pathological calcification in ENT1-KO mice. Studies were performed with age-matched wild-type (WT) and ENT1-KO mice, as well as human cadaveric vertebral columns meeting radiographic criteria for DISH. Mouse and human specimens were scanned using high-resolution, micro-computed tomography (micro-CT). As well, some samples were decalcified and processed for histological assessment. Calcified lesions in selected specimens were examined using energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). To investigate molecular changes associated with ectopic calcification, we isolated AF tissue from thoracic intervertebral discs of WT and ENT1-KO mice. Tissues were then subjected to transcriptomic and proteomic analyses. Micro-CT of ENT1-KO mice revealed ectopic calcification of spinal tissues, first appearing in the cervical-thoracic region and extending caudally with advancing age. Histological examination of calcified lesions in mice revealed accumulations of amorphous, eosinophilic, acellular material in paraspinal ligaments and entheses, intervertebral discs, mandibular symphysis, and sternocostal articulations. There was no evidence of inflammation associated with these lesions. EDX of calcified lesions revealed a high content of calcium and phosphorus in a molar ratio of ∼1.6, with hydroxyapatite detected by micro-XRD. Ten human cadaveric spines (three females and seven males, mean age 81 years) that met radiographic criteria for DISH were analysed in detail by micro-CT. Remarkable heterogeneity in the density and morphology of ectopic calcifications was observed. Analyses of calcifications by EDX and XRD again yielded a calcium/phosphorus ratio of ∼1.6 and a crystalline diffraction pattern matching hydroxyapatite. Histological examination of human lesions revealed regions of mature ossification and other areas of irregular amorphous calcification that resembled lesions in ENT1-KO mice. Microarray analysis of AF tissue from WT and ENT1-KO mice showed extensive dysregulation of transcription in affected tissues. Cell cycle-associated transcripts were the most affected, including the E2f family of transcription factors and proliferating cell nuclear antigen. In addition, expression of genes involved in the regulation of mineralization and bone development were dysregulated. Proteomic analyses confirmed transcriptomic changes and revealed alterations in known modulators of biomineralization such as matrix Gla-protein. Many of the characteristics of ectopic calcification in ENT1-KO mice resemble those of DISH in humans. Human lesions were found to be heterogeneous with regions of pathological ossification and amorphous calcification, the latter resembling lesions in the mouse model. Our studies of the molecular events associated with ectopic calcification in ENT1-KO mice may provide insights into the pathogenesis of DISH in humans. ENT1-KO mice may also be useful for evaluating therapeutics for the prevention of ectopic calcification in DISH and related disorders

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.

Ewing Tumors (ET) are highly malignant, localized in bone or soft tissue and are molecularly defined by ews/ets translocations. DNA microarray analysis revealed a relationship of ET to both endothelium and fetal neural crest. We identified expression of histone methyl-transferase Enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be increased in ET. EZH2’s suppressive activity maintains stemness in normal and malignant cells. Here, we found EWS/FLI1 bound to the EZH2 promoter in vivo and induced EZH2 expression in ET and mesenchymal stem cells. Down-regulation of EZH2 by RNA interference in ET suppressed oncogenic transformation by inhibiting clonogenicity in vitro. Similarly, tumor development and metastasis was suppressed in immunodeficient Rag2−/−γC−/− mice. EZH2-mediated gene silencing was shown to be dependent on histone deacetylase (HDAC) activity. Subsequent microarray analysis of EZH2 knock down, HDAC-inhibitor treatment and confirmation in independent assays revealed an undifferentiated phenotype maintained by EZH2 in ET. EZH2 regulated stemness genes such as nerve growth factor receptor (NGFR) as well as genes involved in neuroectodermal and endothelial differentiation (EMP1, EPHB2, GFAP, GAP43). These data suggest that EZH2 might play a central role in Ewing Tumor pathology by shaping the oncogenicity and stem cell phenotype of this tumor

Introduction. Microbiological diagnosis of bone and joint infections (BJIs) currently relies on standard cultures which are time consuming and lack sensitivity. Various molecular approaches have been described and allowed improvement of BJI diagnosis. This study evaluated for the first time the performance of a DNA microarray-based assay (Prove-it™ Sepsis assay, PISA) for the rapid (<6 hours) detection and identification of 50 different species involved in BJI directly from clinical samples. Material and methods. We retrospectively selected 130 bone and joint samples (67 synovial fluids and 63 bone biopsies) including 114 positive and 16 negative samples. The microbiological diagnosis had been previously established either by culture(C+, n=53) or by PCR16S and sequencing when culture was negative (C-/PCR+). The positive samples were selected to match the species targeted on the DNA microarray. DNA extraction was performed before proceeding to PISA amplification and hybridization on every selected sample. Results. Among the 16 negative samples, one was detected positive with S. epidermidis by PISA, result that was secondarily confirmed using specific PCR. Among the 114 positive samples, 62.3% were positive using PISA with highly concordant identification compared to culture and PCR16S/sequencing results. Forty-three samples (37.7%) remained negative, illustrating a defect of sensitivity. However, PISA accurately detected methicillin resistance not only among the 16 C+/PISA+ Staphylococcus species (n=5) but also among the 28 C-/PCR16S+ Staphylococcus species (n=12) offering crucial rapid information to adapt the treatment of staphylococcal BJIs. Seven polymicrobial samples were also identified without extensive experiments. Discussion – Conclusion. Even if the sensitivity deserves to be improved by optimizing DNA extraction and investigating on human DNA interference, these preliminary promising results highlight that this new and simple microarray method could be in the future an alternative to conventional PCR16S for the diagnosis of BJI

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 gene expression profiles of primary human osteoblasts exposed to cobalt ions in a temporal manner, and to identify gene clusters underpinning the osteoblast response to cobalt. Methods. Primary human osteoblasts were exposed to cobalt ions at a concentration of 10ppm. To determine gene expression profiles, comparisons were made between control and 12, 24, 48 and 72 hour time exposures. RNA isolation and cDNA synthesis were performed. Gene profiling was carried out using the Affymetrix Human Genome UI33 Plus 2.0 array. Data was normalised using RMA express and an average expression measure for each time point used to identify alterations in gene expression. Validation was achieved by performing quantitative real time RT-PCR on selected genes. Results. Oligonucleotide microarray profiling identified significant alterations in osteoblast gene expression in response to cobalt exposure. Distinct phase patterns were observed, with significant altered expression following 12, 24 and 48 hours cobalt ion exposure. Of the 22,233 gene sequences represented on the Affymetrix microarray, 4.8% (1077 genes), 4.1% (930 genes), and 2.13% (486 genes) were significantly altered. We identified dysregulation of key functional families in response to cobalt ions, including alterations in cellular proliferation, development and and inflammation. Conclusion. These data will provide novel avenues for exploration to further characterise the molecular mechanisms underpinning the initiation and progression of osteolysis

Summary. PCA-III, a phosphocitrate analog, acts not only as a potent calcification inhibitor but also as a protective agent for extracellular matrices. PCA-III has potential as a disease-modifying drug in the treatment of primary osteoarthritis and posttraumatic osteoarthritis in humans. Introduction. Phosphocitrate (PC) inhibits the development of primary osteoarthritis (OA) in Hartley guineas pigs but not menisectomy-induced OA in rabbits (1). We sought to examine the molecular mechanisms underlying the disease-modifying activity of PC, and evaluate the effect of PCA-III, a PC analog (PCA), on the development of primary and secondary OA. Patients & Methods. Meniscal explant and microarray. OA menisci were obtained from OA patients undergoing joint replacement surgery. OA meniscal explants were cultured in medium containing PC (three wells) and medium without PC (three wells). Total RNA was extracted from the explant, and subjected to microarray analysis. RT-PCR. OA fibroblast-like synoviocytes were treated with basic calcium phosphate (BCP) crystals in the absence or presence of PCA-III. RNAs were extracted, and subjected to semi-quantitative RT-PCR to examine the expression of MMP1 and IL-1b. Micromass culture. A droplet of OA chondrocyte suspension was placed in each well of a 24-well plate. After placing all droplets, the wells were fed with chondrogenesis medium with PCA-III (five wells) and without PCA-III (five wells). The production of proteoglycans was examined by alcian blue staining. Animal treatment. The first group of Hartley guinea pigs (n=5) received injections of PCA-III and the second group received injections of saline as control. Two months later, partial-menisectomy surgery was performed on the right knee of all guinea pigs. After the surgery, injections of PCA-III and saline were resumed. All animal were euthanatised four months later, and both knees were examined. Results. PC inhibited the expression of many genes classified into the molecular function group of MMP activity. Of the 23 genes classified into MMP activity, the expression of 16 genes, including CPM, ADAM28, MMP7, MMP10, MMP1, MMP3, ADAMTS5, ADAMTS1, and ADAMTS9, was inhibited. In contrast, the expression of many genes classified into the molecular function group of extracellular matrix structural constituents, was induced by PC, including COL2A1, COL11A1, COL1A1 and ACAN. PC also inhibited the expression of numerous genes classified into the biological process of inflammatory response (data not shown). PCA-III, similar to PC, inhibited BCP crystals-induced expression of MMP1 and IL-1b). In addition, PC-III strongly stimulated the production of proteoglycans by OA chondrocytes while inhibiting calcium deposition (not shown). Microscopic examination of the Indian ink stained medial tibia plateau of the left knees (non-surgery knee) of the guinea pigs indicated that PCA-III inhibited the development of primary OA in the Hartley guinea pigs. Microscopic examination also indicated that PCA-III inhibited the development of partial-menisectomy-induced OA or posttraumatic OA in the post-operative knees. Discussion/Conclusion. PC is thought to act as a potential structural disease-modifying drug for crystal-associated OA by inhibiting crystal deposition within the OA joints. However, PC and its analogs are not only potent calcification inhibitors, but also protective agents for extracellular matrices. Our findings indicate that PCA-III has potential as a disease-modifying drug for both human crystal-associated OA and posttraumatic OA

Dynamic loading is necessary for the preservation of native cartilage, but mechanical disuse is one major risk factor for osteoarthritis (OA). As post-transcriptional regulators, microRNAs (miRs) represent promising molecules to quickly adjust the cellular transcriptome in a stimulus-dependent manner. Several miR clusters were related to skeletal development, joint homeostasis and OA pathophysiology but whether miRs are associated with mechanosensitivity and regulated by mechanotransduction is so far unknown. We aimed to investigate the influence of mechanical loading on miR expression and to identify mechanosensitive miR clusters characteristic for non-beneficial loading regimes which may serve as future tools for improved diagnosis or intervention during OA development. Loading regimes leading to an anabolic or catabolic chondrocyte response were established based on an increase or decrease of proteoglycan synthesis after loading of human engineered cartilage. miR microarray profiling at termination of loading revealed only small changes of miR expression (7 significantly upregulated miRs) by an anabolic loading protocol while catabolic stimulation produced a significant regulation of 80 miRs with a clear separation of control and compressed samples by hierarchical clustering. Overall regulation of 8/14 miR was confirmed by qRT-PCR with mean amplitudes of up to 2.5-fold for catabolic loading. Cross-testing revealed that 2 miRs were upregulated by both loading conditions and 6 were specifically elevated by the catabolic loading regime. Conclusively, this study defines the first mechanosensitive miR cluster associated with non-beneficial compressive cyclic loading of human engineered cartilage which can now be tested for its diagnostic potential in healthy versus OA-affected human cartilage

Aims

Knee osteoarthritis (OA) involves a variety of tissues in the joint. Gene expression profiles in different tissues are of great importance in order to understand OA.

Aims

Long non-coding RNAs (lncRNAs) act as crucial regulators in osteoporosis (OP). Nonetheless, the effects and potential molecular mechanism of lncRNA PCBP1 Antisense RNA 1 (PCBP1-AS1) on OP remain largely unclear. The aim of this study was to explore the role of lncRNA PCBP1-AS1 in the pathogenesis of OP.

Osteoporosis is a common skeletal disorder characterised by a reduced bone mass and a progressive microarchitectural deterioration in bone tissue leading to bone fragility and susceptibility to fracture. The Wnt/β-catenin pathway is a major signaling cascade in bone biology, playing a key role in regulating bone development and remodeling, with aberrations in signalling resulting in disturbances in bone mass. Our objectives were to assess the gene expression profile of primary human osteoblasts (HOBs) exposed to dexamethasone with a view to identifying key genes driving bone mass regulation and to assess the effects of the Wnt antagonist Dickkopf-1 (Dkk1) on the bone profile of primary human osteoblasts exposed in vitro to dexamethasone. HOBs were cultured in vitro and exposed to 10–8M dexamethasone over a time course of 4hr, 12hr and 24hr. RNA isolation, cDNA synthesis, in vitro transcription and microarray analysis were performed. Microarray data was validated by quantitative real time RT-PCR. Dkk1 expression was silenced using small interfering RNA (siRNA). Quantitative RT-PCR was performed to confirm gene knockdown. Control and Dex-treated HOBs were compared with respect to bone turnover. Markers of bone turnover analyzed included alkaline phosphatase activity, calcium deposition, osteocalcin expression, along with cell proliferation and cellular apoptosis. Global changes in HOB gene expression were elicited by dexamethasone. Development associated gene pathways were co-ordinately dysregulated with the expression profile of key genes of the Wnt Pathway significantly altered. Dkk1 expression in HOBs was increased in response to dexamethasone exposure with an associated reduction in alkaline phosphatase activity, calcium deposition and osteocalcin expression. Silencing of Dkk1 expression, as confirmed by quantitative RT-PCR, was associated with an increase in alkaline phosphatase activity and calcium deposition, along with increased cell proliferation and reduced cellular apoptosis. Dkk1 is an antagonist of Wnt/β-catenin signalling and plays a key role in regulating bone development and remodeling. Silencing the expression of Dkk1 in primary human osteoblasts has been shown to rescue the effects of dexamethasone-induced bone loss in vitro. The pharmacological targeting of the Wnt/β-catenin signaling pathway offers an exciting opportunity for the development of novel anabolic bone agents to treat osteoporosis and disorders of bone mass

Aims

This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD).

Excessive apoptosis has been found in torn supraspinatus tendon1 and mechanically loaded tendon cells2. Following oxidative and other forms of stress, one family of proteins that is often unregulated are Heat Shock Proteins (HSPs). The purpose of this study was to determine if HSPs were unregulated in human and rat models of tendinopathy and to determine if this was associated with increased expression of regulators of apoptosis (cFLIP, Caspases 3& 8). A running rat supraspinatus tendinopathy overuse model 3 was used with custom microarrays consisting of 5760 rat oligonucleotides in duplicate. Seventeen torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from ten patients undergoing arthroscopic stabilisation surgery and evaluated using semiquantative RT-PCR and immunohistochemistry. Rat Microarray: Upregulation of HSP 27 (×3.4) & 70 (×2.5) and cFLIP (×2.2) receptor was noted in degenerative rat supraspinatus tendon subjected to daily treadmill running for 14 days compared to tendons of animals subject to cage activity only. Histological analysis: All torn human supraspinatus tendons exhibited changes consistent with marked tendinopathy. Matched subscapularis tendon showed appearances of moderate-advanced degenerative change. Apoptosis mRNA expression: The expression levels of caspase 3 & 8 and HSPs 27 & 70 were significantly higher in the torn edges of supraspinatus when compared to matched subscapularis tendon and control tendon (p< 0.01). cFLIP showed significantly greater (p< 0.001) expression in matched subscapularis compared to supraspinatus and control tendon. Immunohistochemical analysis: cFLIP, Caspase 3 & 8 and HSP 27 and 70 was confirmed in all samples of torn supraspinatus tendon. Significantly increased immunoactivity of Caspase 3& 8 and HSP 27 & 70 were found in torn supraspinatus (p< 0.001) compared to matched and normal subscapularis. The proteins were localized to tendon cells. The finding of significantly increased levels of Heat Shock Proteins in human and rat models of tendinopathy with the co-expression of other regulators of apoptosis suggests that Heat Shock Proteins play a role in the cascade of stress activated-programmed cell death and degeneration in tendinopathy

Aim: The purpose of this study was to evaluate the cytokine molecules present in a rat tendinopathy model and in the torn edge of human rotator cuff tendon in an attempt to understand their role in tendon degeneration. Methods: A rat tendon overuse model was used with custom microarrays consisting of 5760 rat oligonucleotide features in duplicate. Seventeen torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from patients undergoing arthroscopic shoulder surgery.Control samples of subscapularis tendon were collected from ten patients undergoing arthroscopic stabilisation surgery.Specimens were analysed for the presence of interleukins 18, 15, 12, 11, 6, 2, macrophage inhibitory factor (MIF), and tumour necrosis factor ƒÑ by semiquantitative RT-PCR and immunohistochemistry. Tendinopathy was assessed on a basic histological scale. Results: Rat Microarray analysis: Upregulation of IL-6, IL-11 and IL18 receptor was noted in the degenerated rat supraspinatus tendon. Downregulation of IL-2 was noted. No other cytokine signal was expressed. Histological analysis: All torn human supraspinatus tendons changes consistent with marked tendinopathy. Matched subscapularis tendon showed appearances of moderate-advanced degenerative change. Cytokine mRNA expression: TNF-£\ mRNA expression was found to be significantly elevated (p< 0.01) in subscapularis tendon compared to torn supraspinatus samples. The expression levels of IL-18, IL-15, IL-6 and MIF was significantly higher in the torn edges of supraspinatus when compared to matched subscapularis tendon and normal control tendon (p< 0.001). Immunohistochemical analysis: Presence of IL-18, IL-15, Il-6, MIF and TNF-£\ was confirmed in all samples of torn supraspinatus tendon. Significantly increased levels of IL-18, IL-15, IL-6 and MIF were found in torn supraspinatus. (p< 0.01) compared to matched and normal subscapularis. Conclusions: Cytokines have been shown to promote the intensive production of reactive O2 metabolites . 1. and are potent agonists of protein kinases . 2. Our finding of significantly increased cytokine levels may suggest that these molecules when expressed during the degenerate and healing phases of tendon injury result in the subsequent production of reactive O2 species and protein kinases. 3. causing tendon damage or failure of the normal reparative process. Our finding of marked tendinopathy in matched subscapularis tendon may also provide a useful human tendinopathy model

Introduction: Ischaemic preconditioning (IPC) is a well recognised and powerful phenomenon where a tissue becomes more tolerant to prolonged ischaemia when it is first subjected to short bursts of ischaemia/reperfusion. IPC has been most comprehensively studied in cardiothoracic surgery, to date there has been little use of this powerful phenomenon in orthopaedic surgery. In this study, we report on the first clinical trial of IPC on human skeletal muscle, and show the potential of IPC in orthopaedics using global gene expression analysis. Methods: After local ethics committee approval and informed consent, patients undergoing primary knee arthroplasty were randomly assigned into an IPC group and a control group. Diabetic patients or patients with an ankle/brachial index of less than 1 were excluded. The IPC consisted of three five-minute periods of tourniquet insufflation on the operative limb, interrupted by five minute periods of reperfusion. The tourniquet was again insufflated and the operation started. The control group simply had tourniquet insufflation as normal prior to the start of surgery. Muscle samples were taken from the operative knee of all patients at the immediate onset of surgery (t=0), and again, at one hour into the surgery (t=1). Total RNA was extracted from the muscle samples, and the gene expression profiles were determined using microarray technology. Results: Comparison of IPC and control samples identified 702 transcripts with differences of ≥1.5-fold in their expression. Of these, 137 were altered at t=0 while 565 were altered at t=1. Amongst these changes was an up-regulation in the expression of a number of heat shock proteins (HSPs) in the IPC group as compared to the control group. Notably, there was up-regulation of the well known cytoprotective/anti-apoptotic gene, HSP72, at one hour post IPC (1.5-fold, p=0.039). There was also up-regulation of important oxidative stress defense genes, such as glutathione-S-transferase (1.6-fold, p = 0.021) and superoxide dismutase 2 (3.6-fold, p= 0.048). Microarray analysis also revealed a down-regulation in the expression of genes involved in metabolism, down-regulation of pro-apoptotic genes and up-regulation of genes necessary for transformation to a hypoxia-tolerant state. Discussion: We present convincing evidence that IPC is beneficial to human skeletal muscle and for the first time show that IPC of human skeletal muscle works in the clinical setting. In this study, the protective effect of IPC involved a down-regulation in the expression of genes associated with metabolism, and an up-regulation in the expression of genes that provide protection from cell stress, oxidative stress and apoptosis. HSPs, and especially HSP72, have well documented roles in cell stress protection. Their presence has been cited by other studies as an indicator of cell adaptation to stress

Manufacturing of autologous chondrocytes presents unique challenges, and robust and reliable release assays are required to ensure product quality. We have discovered markers that correctly identify chondrocytes and predict potency. Novel qPCR assays developed with these markers for our Matrix-induced Autologous Chondrocyte Implant product (MACI. ®. implant) are described. An identity assay must distinguish chondrocytes from potentially contaminating cell types, such as synovial fibroblasts. Microarray analysis of more than 47,000 transcripts led to the discovery of two markers, currently aliased “Cart1” and “Synov1”, that have been characterized as the two most differentially expressed mRNAs between chondrocyte and synovial fibroblast cultures. A potency assay must identify cells that have the potential to form hyaline-like cartilage. We examined expression of critical components of hyaline cartilage during the chondrocyte manufacturing process and in re-differentiation assays. From these studies a gene, which we call “Hyaline1”, was identified as a candidate potency marker. Using an assay measuring the ratio of Cart1:Synov1, a large population study of chondrocyte and synovial fibroblast cultures examined the assay’s suitability for identity classification with our proposed Cart1:Synov1 acceptance boundary. In this study, assay specificity and sensitivity were both observed to be 100%. The utility of the assay was further demonstrated in mixing experiments, where a majority of chondrocytes (in mixtures with synovial fibroblasts) was required to pass the assay acceptance. These results indicate that the assay is useful for determination of both culture identity and culture homogeneity, and thus represents a significant improvement over previous identity assays. The potency assay is also a real-time quantitative RT-PCR assay that measures levels of Hyaline1. Characterization of MACI. ®. implants indicated that Hyaline1 is stable in long-term culture of chondrocytes but not fibroblasts on ACI-Maix membrane, and is overexpressed in chondrocytes compared fibroblasts that had been recovered from MACI. ®. implants and tested in various redifferentiation assays. These data suggest that Hyaline1 is predictive of the chondrogenic potential of cells used to manufacture MACI. ®. implants. After comparing may cell strains, a threshold level which indicates product potency was established. The identification of genetic markers that unambiguously identify cultured chondrocytes has been a long-standing challenge. Another challenge has been the ability to predict re-differentiation capacity of cultured chondrocytes. Modern techniques like whole genome microarray analysis have enabled us to develop novel identity and potency assays for quality control of MACI. ®. implants

Aims

Osteoarthritis (OA) is a common degenerative joint disease worldwide, which is characterized by articular cartilage lesions. With more understanding of the disease, OA is considered to be a disorder of the whole joint. However, molecular communication within and between tissues during the disease process is still unclear. In this study, we used transcriptome data to reveal crosstalk between different tissues in OA.

Aims

Rotator cuff tear (RCT) is the leading cause of shoulder pain, primarily associated with age-related tendon degeneration. This study aimed to elucidate the potential differential gene expressions in tendons across different age groups, and to investigate their roles in tendon degeneration.

Introduction: Our group has previously reported on microarray gene expression profiling of failed aseptic and septic THRs. The data obtained from the Affymetrix DNA chips suggested a range of 21 differentially expressed genes between the tissue samples obtained from the control and study patients with failed aseptic THRs. The variation in expression that was demonstrated did not suggest that the basis of the local tissue reaction that occurs in aseptic loosening of THR is primarily inflammatory in nature. In order to validate these results we have performed quantitative real-time polymerase chain reaction (RT-PCR) to analyse the transcriptional levels of genes expression in the samples used in our original study and to formulate a hypothesis of how these candidate genes can be related to aseptic join loosening. Methods: 3 control and 6 aseptic samples of peri-prosthetic membrane were subjected to RNA extraction. RNA quality analysis and quantification were performed. SYBRâ Green I real time quantitative PCR (RT qPCR) assays were designed using Primer Express [Applied Biosystems] and BLAST searching the resulting sequences. The comparative method for quantitation of gene expression levels, which utilizes arithmetic formulas to give the similar results to those achieved with standard curves, was utilised to validate the cDNA microarray data. Results: We were able to devise successful quantitative real-time PCR for 15 of the 21 candidate genes plus the reference gene GAPDH. The genes coding for complement component C4B, Osteonectin , ATP2A2 (an ATPase linked to the regulation of adhesion, differentiation and proliferation in tissue that expresses this gene such as bone) and Phospholipase2A, were all found to be under-expressed whereas SLC2A5 (a solute carrier that can facilitate glucose/fructose transport)and NPC1 (intimately involved in cholesterol and glycolipid trafficking and inversely related to PLA2-mediated release of eicosanoids such as PGE2) were found to be over-expressed. Conclusions: The data from our gene expression and RT-PCR studies have suggested novel pathways that may be intimately involved in the development of peri-prosthetic osteolysis and aseptic loosening that are distinctly different from the currently accepted theory of a proinflammatory cytokine cascade initiated by tissue reaction to particulate wear debris. These include possible alteration in both extra- and intracellular Ca2+ metabolism together with a possible effect upon extra-cellular matrix function. Altered lipid metabolism may also be evident and in particular decreased eicosanoid production. Intriguingly, the pattern of gene expression that is seen our studies would appear to be quite different than that seen in synovial inflammatory arthritidies such as rheumatoid and osteo-arthritis and suggests that previous studies that has used these pathological mechanisms as comparisons or controls may be flawed

Background. While the human embryonic, foetal and juvenile intervertebral disc (IVD) is composed of large vacuolated notochordal cells, these morphologically distinct cells are lost with skeletal maturity being replaced by smaller nucleus pulpous cells. Notochordal cells are thought to be fundamental in maintaining IVD homeostasis and, hence, their loss in humans may be a key initiator of degeneration, leading ultimately to back pain. Therefore, it is essential to understand the human notochordal cell phenotype to enable the development of novel biological/regenerative therapies. Methods. CD24+ notochordal cells and CD24- sclerotomal cells were sorted from enzymatically-digested human foetal spines (7.5–14 WPC, n=5) using FACS. Sorting accuracy was validated using qPCR for known notochordal markers and Affymetrix cDNA microarrays performed. Differential gene expression was confirmed (qPCR) and Interactive Pathway Analysis (IPA) performed. Results. CD24+ve notochordal cells (mean 10.4%) and CD24-ve sclerotomal cells (mean 60.9% CD24-) were successfully sorted. Higher expression of notochordal markers CD24 and brachyury was identified in CD24+ve cells. Hierarchical clustering and PCA mapping revealed distinct differences in the gene expression profile of CD24+ and CD24- cells. Top notochordal markers were CD24, STMN2. RTN1, PRPH and CXCL12. IPA identified IL-1 receptor antagonist (IL-1RN) and noggin as master regulators of notochordal cell phenotype. Conclusions. This study has, for the first time, defined human foetal notochordal cell phenotype and identified important pathways and upstream regulators. In particular, IL-1RN and noggin are of interest as master regulators of notochordal cell function, suggesting vital roles for these molecules in IVD development and homeostasis. Conflicts of interest. No conflicts of interest. Sources of funding. We would like to acknowledge UKRMP Acellular Hub, MRC, NIHR Musculoskeletal BRU and The Rosetrees Trust for funding this research

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This study aimed, through bioinformatics analysis, to identify the potential diagnostic markers of osteoarthritis, and analyze the role of immune infiltration in synovial tissue.

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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.

With around 20–40% of our bodyweight, skeletal muscles are the biggest organ complex of the human body. Being a metabolic active tissue, muscle mass, function and fibertype composition is highly regulated in a tight spatial-temporal manner. In geriatric patients, it is essentially important to understand the underlying mechanisms of the age related losses of fiber size and total number of fibers, as well as fibertype shifting. To date, there have been few studies dealing with gene expression profiling of skeletal muscles, mostly focusing on age related differences in whole-muscle specimen. Being carried out on mouse or rat limb muscles, most other studies do not represent the conditions of human muscle, due to the differences in fibertype composition. Our study provides a fibertype-specific approach for whole-genome expression analysis in human skeletal muscle. 22 fresh frozen biceps brachii and quadriceps femoris muscle samples were acquired from the muscle bank of the Friedrich-Baur-Institut, Department of Neurology, Ludwig-Maximilians-University, Munich, Germany. Consecutive cross-sections were used for immunohistochemical myosine-heavy-chain-staining and individual fibers were acquired by laser-capture-microdissection. Around 100 cells of each fibertype of each biopsy were dissected, reversely transcribed, pre-amplified and labeled for microarray analysis. Fiber type-specific gene expression was analyzed with ANOVA. Correction for multiple testing was performed using the Benjamini-Hochberg procedure with a conservative threshold and the pathway analysis was carried out using the Ingenuity Pathway Analysis program (QIAGEN). By comparing type I vs. type IIa, type I vs. type IIx and type IIa vs. type IIx, we could identify 2855, 2865 and 510 differentially expressed genes. As expected, many differentially regulated genes belong to functional groups like cytoskeleton, muscle contraction and energy metabolism, proving the feasibility of our study. However, many genes that are involved in the response to oxidative stress were also differently regulated, showing distinct mechanisms of the different fiber types, of coping with oxidative stress. In consensus with available literature, the relative proportion of type I fibers seemed to increase with age. Despite higher levels of oxidative stress, type I fibers seem to have more efficient antioxidative mechanisms in comparison to type IIa and IIx fibers, which might explain the higher vulnerability of members of the type II family to oxidative stress. Furthermore, genes that are involved in fibertype specification were also regulated differently. However, we could not verify an age-specific activation of pathways involved in fibertype shifting. Whether fibertype shifting is solely due to disproportionate loss of type II fibers, or also in vivo - transdifferentiation of fibers, has to be investigated further

Dupuytren Disease (DD), the most common connective tissue disease in man, presents as a benign fibromatosis of the hands and fingers resulting in the formation of nodules and cords and often leading to flexion contractures in association with keloids or Peyronie disease. Surgical resection of the fibrotic nodules, and more recently intra-lesional collagenase injection are the main therapeutic options for these patients. While the exact cause of DD is still unknown, linkage and Genome Wide Association Studies (GWAS) showed molecular heterogeneity with at least 10 different susceptibility loci 6 of which are close to genes encoding proteins in the Wnt-signaling pathway. We aim to identify the molecular basis of Dupuytren Disease (DD). Twenty patients with Dupuytren disease (including 3 patients with autosomal dominant inheritance, 1 with keloids and congenital torticollis, 2 with Peronie disease), were included in this study. Chromosome Microarray Analysis (CMA), Whole Exome Sequencing (WES) of gDNA and proteomic analysis by LC-Tandem Mass Spectrometry (LC-MSMS) studies were performed. Expression and Network analysis of LCMSMS results was performed using Principal Component Analysis (PCA), ANOVA and Ingenuity Pathway Analysis (IPA). No pathogenic copy number variants (CNVs) were found in CMA (n = 3). WES showed potentially pathogenic variants in POSTN, WNT11, MMP1 and COL3A1. PCA showed three differentially expressed clusters and network-IPA identified ACTB, BAX, COL3A1, FBN1, FN1, MMP1 as potential biomarkers. Comprehensive multi-OMIC analysis of gDNA and tissue proteins in patients with DD identified several connective tissue biomarkers potentially important in the pathogenesis of DD

Periprosthetic joint infection (PJI) remains an extremely challenging complication. We have focused on this issue more over the last decade than previously, but there are still many unanswered questions. We now have a workable definition that everyone should align to, but we need to continue to focus on identifying the organisms involved. Surgical strategies are evolving and care is becoming more patient-centred. There are some good studies under way. There are, however, still numerous problems to resolve, and the challenge of PJI remains a major one for the orthopaedic community. This annotation provides some up-to-date thoughts about where we are, and the way forward. There is still scope for plenty of research in this area.

Cite this article: Bone Joint J 2022;104-B(11):1193–1195.

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Myokine developmental endothelial locus-1 (DEL-1) has been documented to alleviate inflammation and endoplasmic reticulum (ER) stress in various cell types. However, the effects of DEL-1 on inflammation, ER stress, and apoptosis in tenocytes remain unclear.

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Lumbar spinal stenosis (LSS) is a common skeletal system disease that has been partly attributed to genetic variation. However, the correlation between genetic variation and pathological changes in LSS is insufficient, and it is difficult to provide a reference for the early diagnosis and treatment of the disease.

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Osteoporosis (OP) is a metabolic bone disease, characterized by a decrease in bone mineral density (BMD). However, the research of regulatory variants has been limited for BMD. In this study, we aimed to explore novel regulatory genetic variants associated with BMD.

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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).

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Acquired heterotopic ossification (HO) is a debilitating disease characterized by abnormal extraskeletal bone formation within soft-tissues after injury. The exact pathogenesis of HO remains unknown. It was reported that BRD4 may contribute to osteoblastic differentiation. The current study aims to determine the role of BRD4 in the pathogenesis of HO and whether it could be a potential target for HO therapy.

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Exosomes (exo) are involved in the progression of osteoarthritis (OA). This study aimed to investigate the function of dysfunctional chondrocyte-derived exo (DC-exo) on OA in rats and rat macrophages.

Staphylococcus aureus is a leading cause of implant-associated infections (IAI). The aim of this study was to identify bacterial and/or clinical features involved in the pathogenesis of S. aureus IAI. 57 IAI S. aureus and 31 nasal carriage (NC) S. aureus isolates were studied. S. aureus genetic background was obtained by microarray analysis. Multi-Locus Sequence Typing was performed to determine clonal complexes (CC). The ability of S. aureus isolates to produce biofilm was investigated by resazurin and crystal violet methods. Clinical data were retrospectively collected from the patient's medical records. Fifty-five IAI patients were included. Two of them had two different S. aureus IAI episodes. The median age was 73 years (range: 21–96 years) with 29 women (52.7%). The main diagnosis for arthroplasty was arthrosis (38%). Implants were hip prosthesis (n=35), knee prosthesis (n=18) and osteosynthesis (n=4). Infectious and nasal carriage isolates belonged respectively to 18 and 13 different sequence types (STs) without significant difference. Among IAI isolates, five strains were methicillin resistant. IAI isolates were classified as strong (14%), moderate (42.1%) and weak (43.9%) biofilm producers. For NC isolates, distribution was 12.9%, 25.8% and 61.3% for strong, moderate and weak, respectively. Staphylokinase gene was associated with the occurrence of S. aureus IAI (p<0.001). Patients’ ABO blood group phenotype was associated with IAI S. aureus genetic background (sasG, slpB, lukD and set12/ssl8) (p≤0.01). In vitro, CC8 S. aureus strains produce more biofilm than others (p≤0.0001). Two alleles of bbp gene were significantly associated with CC8 S. aureus strains (p≤0.0001). No specific CC involved in IAI compared to NC S. aureus isolates was revealed. Our results suggested that occurrence of IAI may depend on patients’ ABO blood group and staphylokinase gene detection. We also observed a strong biofilm producer phenotype in CC8 S. aureus. Further studies are needed to prove whether one bbp gene variant is correlated to this phenotype. This study was supported by a grant number WS1106649 from Pfizer, France and by the French “Ministère de l'Enseignement Supérieur et de la Recherche”

Summary Statement. In this study we suggested a possible role of prion proteins genes in osteosarcoma. Therefore, the inhibition of prion proteins expression must be tested because it could represent a new approach to the molecular treatment of osteosarcoma. Introduction. Although osteosarcoma is the most common bone malignancy, the molecular and cellular mechanisms influencing its pathogenesis have remained elusive. Prion proteins (PRNP and PRND), known mostly for its involvement in neurodegenerative spongiform encephalopathies, have been recently demonstrated to be involved in resistance to apoptosis, tumorigenesis, proliferation and metastasis. Patients & Methods. The main aim of research was to study whether prion proteins were over-expressed in human osteosarcoma, and if prion proteins could have a role also in osteosarcomas. We evaluated differential gene expression between 22 cases of osteosarcoma and 40 cases of normal bone specimens through cDNA microarray analysis spanning a substantial fraction of the human genome. Results. PRNP and PRND are significantly over-expressed in osteosarcoma. PRNP and PRND appear involved with some important genes related to tumorigenesis and apoptosis. PRNP is linked to PTK2, RBBP9 and TGFB1 while PRND is linked to TNFSF10, BCL2A1, NFKB2 and TP53RK. Discussion/Conclusion. Increased expression on Affymetrix arrays of prion proteins seems to be associated with the development of osteosarcoma. Prions seem to induce a negative regulation of apoptosis, thus promoting osteosarcoma development and progression. Osteosarcoma is a very aggressive tumor and even after modern chemotherapy and excision of tumors efforts are needed to improve clinical outcome. Since Prion proteins seem to be related to osteosarcoma development, their inhibition could represent a new approach to the molecular treatment of osteosarcoma

Introduction. Our previous study using microarray analysis showed that Rad (Ras associated with diabetes) was highly expressed in nonunion. The purpose of this study is to investigate the gene expression and immunolocalization of Rad, and other Ras-related G proteins: Rem1 and Rem2 in fracture/nonunion site using rat experimental models. Hypothesis. We hypothesized that Rad had a significant role in nonunion formation. Materials & Methods. For standard healing model, K-wire was inserted into the femur and a closed fracture was created. Nonunion model was produced by periosteal cauterization at the fracture site. At post-fracture days 3, 7, 10, 14, 21, and 28, RNA was extracted from callus or fibrous tissue for real-time PCR. At day 14, specimens were harvested for immunohistochemistry. Results. Significant difference of Rad gene expression was not observed between standard healing fracture and nonunion at the earlier time points. In contrast, significantly higher expression in nonunion was observed at the later time points. There were no significant differences between standard healing fracture and nonunion in gene expression of Rem1 and Rem2. In immunohistochemical analysis, Rad and Rem1 were detected in the fracture site, and Rem2 was not detected. On the other hand, Rad was only detected in fibrous tissue in nonunion. Discussion & Conclusion. Our results suggest a significant role of Rad in fracture healing and nonunion formation. Rad may become a target agent for treatment of nonunion

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The aim of this study was to explore the genetic correlation and causal relationship between blood plasma proteins and rheumatoid arthritis (RA).

Aim and purpose. The clinical management of osteosarcoma differs significantly from that of chondrosarcoma;. Therefore it is extremely important to diagnose these two types of bone tumour accurately. In the absence of a specific marker, differential diagnosis by histochemistry is sometimes impossible, especially between chondroblastic osteosarcoma and conventional chondrosarcoma. The aim of the study was to find an useful diagnostic marker, simple to use for distinguishes chondroblastic osteosarcoma from conventional chondrosarcoma. Method. We analysed 165 bone sarcomas by immunohistochemical staining of tissue microarrays for expression of the galectin-1 (GAL1) lectin and by Western Blot experiments. Results. We found that GAL1 was abundant in normal human osteoblasts from benign proliferations and in osteosarcomas, including chondroblastic osteosarcomas, but not in chondrosarcomas. There was a highly significant statistical difference in the percentage of stained cells (p<10-4) and in the staining intensity (p<10-3) of chondroblastic osteosarcomas compared to conventional chondrosarcomas. This discriminatory potential of GAL1 staining for osteosarcoma-derived tumours was confirmed by western blotting. We propose a diagnostic test for bone tumours that takes into account the optimal discriminative values for the percentage of cells stained and the intensity of staining. The positive and negative predictive values were 85.7% (trust interval of 63.7-97%) and 90% (trust interval of 80-95.9%), respectively, demonstrating the pertinence of the test. Conclusion. Altogether, our data indicate that GAL1 is a powerful diagnostic marker that distinguishes chondroblastic osteosarcomas from conventional chondrosarcomas

Summary. Based upon genetic analysis, decorin is an exciting pharmacologic agent of potential anti-fibrogenic effect on arthrofibrosis in our animal model. Introduction. While the pathophysiology of arthrofibrosis is not fully understood, some anti-fibrotic molecules such as decorin could potentially be used for the prevention or treatment of joint stiffness. The goal of this study was to determine whether intra-articular administration of decorin influences the expression of genes involved in the fibrotic cascade ultimately leading to less contracture in an animal model. Material and Methods. Eighteen rabbits had their right knees operated on to form contractures. The left knees served as controls. The 6 right limbs in the experimental group (Group 1) received four 500 ug/ml intra-articular injections of decorin over 8 days starting at 8 week, for a total of 2 mg. The 6 right limbs in the first control group (Group 2) received four intra-articular injections of bovine serum albumin (BSA) over 8 days starting at 8 weeks as well. The 6 six right limbs in the second control group (Group 3) received no injections. The contracted limbs of rabbits in Group 1 were biomechanically and genetically compared to the contracted limbs of rabbits in Groups 2 and 3 with the use of a calibrated joint measuring device and custom microarray, respectively. Results. There was no statistical difference in the flexion contracture angles between those right limbs that received intra-articular decorin versus those that received intra-articular BSA (66° vs. 69°; p = 0.41). Likewise, there was no statistical difference between those right limbs that received intra-articular decorin as opposed to those who had no injection (66° vs. 72°; p = 0.27). The lack of significance remained when the control left limbs were taken into account (p > 0.40). When compared to bovine serum albumin (BSA), decorin led to a statistically significant increase in the mRNA expression of 5 genes: substance P, neuropeptide γ, and neurokinin A, cyclin E2, and MMP-9 (p < 0.001). In addition, there was a statistically significant decrease in fibroblast growth factor receptor-2 (FGFR-2), rho-associated coiled-coil containing protein kinase-1 (ROCK-1), and vascular cell adhesion molecule-1 (VCAM-1) genes when intra-articular decorin was compared to no injection (p < 0.001). Conclusions. In this model, when administered intra-articularly at 8 weeks, 2 mg of decorin had no significant effect on joint contractures. However, our genetic analysis revealed a significant alteration in the expression of several fibrotic genes. Further studies investigating the route of administration, dosing, frequency, and timing are required before definitive conclusions may be drawn on the effects of decorin on joint contractures

Summary Statement. The Dkk3-derived cells represent a branch of the periosteal mesenchymal lineage that produces fibrocartilage as well as regenerating the periosteal structures. Introduction. Mesenchymal progenitor cells are capable of generating a wide variety of mature cells that constitute the connective tissue system. Our Laboratory has been developing SMAA GFP reporter mice to prove to be an effective tool for identifying these cells prior to the expression of markers of differentiation characteristic of bone, fat, muscular blood vessels or fibrocartilage. Dkk3 was chosen as a candidate reporter because microarray of SMAA-sorted cells culture indicated high expression of this non-canonical anti-Wnt factor, which was not anticipated in a culture with strong osteogenic potential. Material and Methods. Fracture healing process was evaluated in 12 week old male mice at 3, 5, 7, 14, 21 and 28days post fracture. A 3 color reporter mouse was generated by crossing SMAA-GFPcherry × Col3.6GFPcyan × Dkk3-eGFP and subjected to tibial fracture. A closed transverse fracture was performed by Einhorn device under isoflurane anesthesia after insertion of intramedullary pinning. Longitudinal 5 mm non-calcified cryosections were stabilised with Cryofilm tape. Results. Three days post fracture, the proliferating SMAA-red cells were also beginning to express either Dkk3 or Col3.6. By day 5 the two populations had diverged with the Dkk3 cells being on the outer surface of the developing callus while the Col3.6 cells were forming bone at the base of the callus. By day 7 when the callus is filled with cartilage, Dkk3 is active in cells that are in transition from elongated cells on the external surface of the callus to fibrocartilagenous cells that now express low levels of Col3.6. The zone of cells that express Dkk3 appear to block the passage of the surrounding vasculature into the underlying cartilage and does not deposit fibronectin. By day 14–21 when the cartilage core is resorbed, the only remaining Dkk3 is located in the newly formed periosteum external to the active endocortical bone forming activity associated with the inward remodeling of the outer cortical shell. Discussion. We interpret these findings that Dkk3 marks a non-osteogenic limb of the SMAA progenitor population that within the fracture partitions the osteogenic signals away from the surrounding skeletal muscle and the underlying differentiating fibrocartilage. It is a progenitor to cells that form fibrocartilage in the fracture zone as well as the tenascin C positive cells that populate the fibrous zone of the periosteum, and it resides in the cambial zone of the periosteum. Knowing the biological and molecular function of these cells should lead to a fuller appreciation of the pro- and anti-osteogenic factors that regulate skeletal repair

Besides conventional chondrosarcoma, several rare chondrosarcoma subtypes are described, comprising about 15% of all chondrosarcomas. Clear cell chondrosarcoma (CCS) is a low-grade malignant tumour, often recurring after curettage, and showing overall survival of about 85%. Mesenchymal chondrosarcoma (MCS) is a highly malignant tumour occurring in bone and soft tissue of relatively young patients. The tumour shows differentiated cartilage mixed with undifferentiated small round cells. It often metastasises and shows a 5-year overall survival of 55%. Dedifferentiated chondrosarcoma (DDCS) is a tumour containing a high-grade non-cartilaginous sarcoma (DD), and a usually low-grade malignant cartilage-forming tumour (WD). The prognosis is poor. The lack of efficacious treatment of these rare tumours emphasises the need to learn more about their characteristics and to unravel potential targets for therapy. We constructed tissue microarrays (TMAs) with 2mm cores of 45 DDCS (WD and DD), 24 CCS, and 25 MCS, in triplicate. Using immunohistochemistry, we investigated protein expression of estrogen-signaling molecules, growth plate-signaling molecules, and other molecules which might be potential targets for therapy. In addition, we gathered genomic information using Agilent 44K oligo arrays. 30% of the WD components were positive for Cox-2. Almost all others were negative. For Bcl2, 88% of the small cells and 32% of the cartilage in MCS were positive. In CCS, WD, and DD 48%, 4%, and 12% were positive, respectively. We demonstrated the presence of ESR1 and aromatase protein in the majority of tumours in all subtypes. Using array CGH, we observed similar aberrations in the two components of DDCS, with additional aberrations in the DD. Celecoxib treatment is not recommended, as most of the tumours are negative for Cox-2. However, the presence of ESR1 and aromatase support a possible effect of anti-estrogen treatment in all subtypes, and application of Bcl2 inhibitors might chemosensitise MCS

The rationale of adoptive T cell therapy is based on the attempt to circumvent pre-existing tolerance mechanisms by stimulating potentially tumor-reactive T cells ex vivo. Efforts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of isolating tumor-reactive T cells present in low numbers in peripheral blood of tumor patients. Furthermore, the development of an effective immunotherapy in the autologous context is hampered by the deficit of an effective T cell repertoire against tumor antigens. We have optimized the techniques for isolating and expanding antigen-specific allogeneic T cells. Following repetitive peptide-driven stimulations with HLA-A*0201 positive dendritic cells the responding HLA-A*0201 negative CD8+ T cells were stained with HLA-A*0201/peptide pentamers. Multimer-positive T cells were sorted and directly cloned by limiting dilution. Using this technique we have succeeded in establishing T cell clones directed against several HLA-A*0201-resticted peptides derived from Ewing Tumor (ET) specific antigens identified via previous DNA microarray analysis and supposed to play a central role in the pathogenesis of this tumor. These T cells not only specifically recognized peptide-pulsed target cells or antigen transfected cells in the context of HLA-A*0201 but also killed HLA-A*0201+ ET expressing the antigen while HLA-A*0201– ET were not affected. Allogeneic, tumor specific T cells can be easily isolated via Peptide/HLA-multimer technology and may benefit therapeutic strategies in allogeneic stem cell transplantation

Introduction: Despite a resurgence in cobalt-chromium metal-on-metal arthroplasty, the potential toxicity of metal ions in the periprosthetic area remains a cause for concern. Studies to date have assessed the acute effect of cobalt ions on osteoblasts over 48 hours. The aim of our study was to determine the response of osteoblasts to cobalt ions over a prolonged period of exposure. Methods. Primary human osteoblasts were cultured and treated with cobalt (10ppm) over 21 days. Osteoblast function was assessed via alkaline phosphatase activity and calcium deposition. ELISA were used to assess chemokine (IL-8, MCP-1 and TNF-α) secretion. Osteoblast gene expression was assessed using microarray analysis and real time PCR. Immunoflourescent cell staining of actin filaments was used to examine osteoblast morphology. Results: Chemokine (IL-8) secretion by osteoblasts was significantly increased after 10 days of stimulation with cobalt ions. In parallel with this, osteoblast function was also significantly inhibited as demonstrated by reduced alkaline phosphatase activity and calcium deposition. Regarding osteoblast phenotype, FSP-1, CTGF and TGF-β gene expression were upregulated indicating a transition in osteoblast phenotype. Immunoflourescent staining of actin filaments also showed a change in osteoblast morphology. Taken together, these data show cobalt ions induce a change in the osteoblast phenotype to that of a mesenchymal cell type. Conclusion: After 10 days of treatment with cobalt ions, osteoblasts no longer function as osteogenic cells. they undergo transition to a mesenchymal cell type. Furthermore, IL-8 secretion is increased which attracts neutrophils to the periprosthetic area thereby contributing to the inflammatory response that characterises osteolysis

Aims

Rheumatoid arthritis (RA) is a systematic autoimmune disorder, characterized by synovial inflammation, bone and cartilage destruction, and disease involvement in multiple organs. Although numerous drugs are employed in RA treatment, some respond little and suffer from severe side effects. This study aimed to screen the candidate therapeutic targets and promising drugs in a novel method.

Summary Statement. Dickkopf-3 is upregulated in OA cartilage and synovial tissue. In vitro studies show Dkk3 can prevent cartilage degradation and antagonise Wnt signaling. We hypothesis that Dkk3 can protect against OA-related cartilage destruction. Introduction. Our group has previously shown that Dkk3, a member of the Dkk family of Wnt antagonists, is upregulated in OA cartilage and synovium. Levels of Dkk3 in synovial fluid are also increased in individuals with tricompartmental OA and after arthroscopy. The role of Dkk3 in cartilage or the factors regulating its expression are not currently understood. Correct regulation of cell signalling pathways is integral to cartilage homeostasis and thus the prevention of OA pathogenesis. Dkk3 is a member of the Dkk family of Wnt antagonists and therefore may impact on chondrocyte biology through interaction with the Wnt pathway. Dkk3 has also been found to influence TGFβ signalling in other cell systems. Methods. Expression of Dkk3 was assessed in primary human articular chondrocytes (HAC) following treatment with interleukin-1,-6 (IL1, IL6), TNFα, FGF2 and oncostatin-M (OSM). Dkk3 expression was assessed following ex vivo injury of murine cartilage explants. The effect of Dkk3 on IL1/OSM-induced proteoglycan and collagen release from explants of bovine nasal (BNC)- and primary human-cartilage was assessed. SW1353 chondrosarcoma cells were treated with Dkk3+/−Wnt3a, TGFβ and Activin and TOPFlash and CAGA luciferase reporters used to measure Wnt and Smad signalling. RNA was extracted from primary HAC treated with Dkk3+/−TGFβ or Wnt3a. ADAM12 and TIMP3 expression were measured to assess TGFβ signalling and AXIN2 to assess Wnt signalling. Micromass HAC were treated with Wnt3a +/− Dkk3 and proteoglycan output assessed using alcian blue staining. β-catenin was silenced in primary HAC prior to TGFβ and Activin treatment. Dkk3 was silenced in primary HAC for microarray analysis. Results. Dkk3 expression was decreased in primary HAC following IL1/OSM treatment but increased by TNFα. Dkk3 expression was decreased immediately following injury to murine explants. In BNC explants, IL1/OSM-induced proteoglycan release was inhibited by Dkk3. Dkk3 antagonised chondrocyte Wnt signalling and Wnt3a-induced reductions in proteoglycan production in micromass cultures. Interestingly, Dkk3 enhanced TGFβ signalling, increasing TGFβ-induced TIMP3 and ADAM12 expression and TGFβ-induced luciferase from the CAGA-luc reporter. In contrast Dkk3 antagonised Activin-induced CAGA-luc activity, TIMP3 and ADAM12 expression. β-catenin knockdown did not significantly alter TGFβ- or Activin-induced expression of TIMP3 or ADAM12, suggesting that Dkk3-effects on these pathways is not mediated solely by inhibition of Wnt signalling. Conclusions. Dkk3 expression is increased in OA and can be regulated injury and inflammatory cytokines. This suggests a balance of Dkk3 effects depending upon the biological stimuli within the cartilage. Dkk3 may act in a protective role in the presence of inflammatory cytokines as exemplified by its ability to inhibit matrix loss. Dkk3 knockdown decreases DICER expression and thus changes in Dkk3 expression in OA may alter chondrocyte phenotype through alterations in miRNA activity. The ability of Dkk3 to antagonise Wnt, enhance TGFβ and antagonise Activin signalling would have multiple effects on chondrocyte activity. These results imply that Dkk3 could influence multiple OA-relevant processes, protect cartilage from degradation and be important in cartilage development and homeostasis

INTRODUCTION. There is increasing evidence for a multi-stage model of rotator cuff (RC) tendon tears, wherein healing is affected by tear size. The underlying pathophysiology however is not fully understood. Changes in the production and remodeling of the RC extracellular matrix (ECM) are likely to be important determinants of RC tendinopathy as they affect healing and the ability to bear loads. This study aimed to gain greater insight into size related tear pathogenesis by analyzing gene expression profiles from normal, small and massive RC tears. METHODS. The genetic profiles of 28 human RC tendons were analyzed using microarrays representing the entire genome. 11 massive and 5 small torn RC tendon specimens were obtained from tear edges intraoperatively, and compared to 12 age matched normal controls. Semiquantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemistry were performed for validation. RESULTS. Numerous insightful gene changes were detected. Key changes included upregulation of aggrecan in massive tendon tears compared to normal controls, but not in small tears (p < 0.05 and > 2-fold change). Matrix metallopeptidases (MMP)-3,-10,-12,-13,-15,-21,-25 and a disintegrin and metallopeptidase (ADAMs)-12,-15,-22 were significantly upregulated in tears. Aggrecan was upregulated in massive tendon tears but not in small tears. Amyloid was downregulated in the small and massive tear groups when compared to normals. BMP-5 was upregulated in small tears only when compared to normals. As part of the chemotaxis pathway, IL-3,-10,-13,-15,-18 were upregulated in tears, whereas downregulation of IL-1,-8,-11,-27, was seen. RT-PCR and immunohistochemistry confirmed altered gene expression. CONCLUSION. The gene profiles of normal, small and massive RC tear groups suggested they are biologically distinct groups. In addition to confirming altered gene expression in pathways reported in previous studies, this study has identified a number of novel pathways which are affected between the different tendon tear and normal groups. This study identified that RC tear pathogenesis is contributed to by ECM remodeling genes, chemotaxis genes, aggrecan and amyloid. Further investigation is required to determine whether some of these genes may potentially have a role as biomarkers of failure. Modulating these ECM pathways may be a useful treatment strategy for improving clinical outcomes

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. Gene expression profiles were assessed on a customised cDNA array including 230 cartilage- and stem cell-relevant genes. Data were analysed by hierarchical clustering and significance analysis of microarrays. Results were confirmed by real-time RT-PCR. Gene expression profiles clearly discriminated between normal and neoplastic cartilage. Between them, 73 differentially expressed genes were identified. The genes higher expressed in cartilage included several genes encoding matrix proteins. Among the genes higher expressed in chondrosarcoma, molecules involved in PTH and BMP signalling were found. Genes differentially expressed between tumours of different grade were identified. Among others, galectin 1 was significantly higher expressed in highly malignant tumours compared to grade I tumours. This correlation could be confirmed at protein level by immunohistological analysis. The comparative analysis of normal cartilage and chondrosarcoma gene expression showed that there are important molecular differences between the matrix of normal and neoplastic cartilage. Our results furthermore confirm that genes implicated in the regulation of the growth plate were expressed in chondrosarcoma. Remarkably, we identified galectin 1 as a marker correlating to malignancy on the level of gene and protein expression. More extended studies on this functionally polyvalent molecule would be necessary to establish it as a marker for malignancy in chondrosarcoma

Summary. We compare the difference in expression profiles of miRNAs during fracture healing between adult and aged female mice. This study reveals the possibility to improve impaired fracture healing in aged females by regulating key miRNAs at early stage. Introduction. Impaired fracture healing in aged female skeleton is still a clinical challenge (Holroyd et al., Best Pract Res Clin Endocrinol Metab, 2008, Virk, Lieberman, Arthritis Res Ther, 2012). Angiogenesis and osteogenesis are the two key stages during fracture healing, which are impaired in aged female (Naik et al., J Bone Miner Res, 2009). MicroRNAs (miRNAs) are key post-transcriptional non-coding regulators of gene expression, which has demonstrated important roles in angiogenesis and osteogenesis (Bae et al., Hum Mol Genet, 2012, Plummer et al., Cancer Res, 2013). Understanding how non-coding regulatory RNA in fracture healing changes with age will help identifying novel therapeutic targets that can be exploited to improve fracture healing in the aged females. Materials and methods. Bilateral femur transverse fractures were created in 9 female 12-month-old mice (Aged Group) and 9 female 12-week-old mice (Adult Group). Three mice in each group were sacrificed at 0, 2 and 4 weeks post fracture, respectively. Total RNA was extracted and hybridised on Agilent 8×60K Mouse miRNA Microarray. Then, differentially expressed miRNAs were identified in adult and aged female fracture mice, respectively (2-vs-0 weeks, 4-vs-0 weeks, P-value <= 0.05 & Fold change >=2.0). With the experimentally validated interactions among miRNAs and their targets, we constructed fracture-healing-related molecular network. Thereafter, we performed topological and dynamic network analysis to find key hub miRNAs in female fracture healing. Person correlation coefficient (r) analysis was performed on the expression data of the miRNAs in all the 18 mice to identify co-expression modules in the female fracture healing progress. Meanwhile, in order to analyze the angiogenesis in the early stage and osteogenesis in the later stage of female fracture healing, we performed microCT-based angiography at 2 weeks post fracture and micro-CT examination at 4 weeks post fracture on the right femur callus samples. Results & Discussion. Angiography showed smaller blood vessel volume in aged mice at early stage when compared to that in the adult mice. Reconstructed calluses showed lower bridging mineralization tissues within the gap in aged mice than that in the adult mice at the later stage. We found that the top hub miRNAs were differentially expressed in adult female mice but not in aged ones during fracture healing. Moreover, the differential expression of the top hub miRNAs was only observed at early stage (2 weeks) during fracture healing in adult female mice. This may help explain the difference of fracture healing between adult and aged female mice. It also indicated the molecular events controlled by the hub miRNAs in early stage could lead to the following differences between the adult and aged female mice at 4 weeks. The person correlation coefficient analysis revealed that there were five co-expression miRNA modules (r>0.8) participated in female fracture healing. The top hub miRNAs in fracture-healing-related molecular network were all included in the two largest modules. These results implied the possibility to improve the aged female fracture healing by regulating key miRNAs at early stage

Introduction: MSCs were demonstrated to exist within peripheral blood (PB) of several mammalian species including human, guinea pig, mice, rat, and rabbit. We have found increased numbers of circulating MSCs in human peripheral blood after fracture and in patients with cancers. We have also compared the difference between circulating MSCs and bone marrow MSCs and evaluated their potential clinical applications in tissue engineering and cell therapy. Methods and findings: Using culture conditions similar to those defined for bone marrow derived mesenchymal stromal cells (BMMSCs), we have isolated and expanded multi-colony and single colony derived PBMSCs strains from the GFP transgenic rats. Aspects of molecular, cellular and developmental properties of this poorly characterized peripheral blood subpopulation were examined. PBMSCs share some common phenotypic characteristics with BMMSCs, but are distinguishable in gene expression profile by cDNA microarray analysis, with 84 up-regulated and 83 down-regulated genes (> 2 fold, E-B/B-E> 100, P< 0.05). Most of these genes are related to cell proliferation, differentiation, cyto-skeleton, and calcium/iron homeostasis. Differentially expressed genes with fold change ≥10 were further confirmed with quantitative real time RT-PCR, and these genes are: retinol-binding protein 1 (CRBP1), cadherin 2, bone morphogenetic protein 6 (BMP6), SRY-box containing gene 11 (Sox11), the aquaporin 1 (AQP1), and so on, and they can be potential targets for further investigations. We have demonstrated that single colony derived PBMSCs strains possess extensive proliferation and multipotent differentiation potentials into osteoblasts, adipocytes, chondrocytes, endothelial cells and neuronal cells. In terms of potential clinical implications of PBMSCs, we have demonstrated that allogenic PB-MSCs enhance bone regeneration in rabbit ulna critical-sized bone defect model. We also demonstrated that BM-MSCs can be recruited towards to the sites of bone fracture and participate fracture healing. We are now working on using MSCs as a gene delivery vehicle for management of would healing or cancer therapy, and ways of enhancing the homing and recruitment MSCs towards to specific sites after their systemic delivery. Conclusion: Taken the above data together, PB-MSCs may be a new cell source for cell therapy, tissue engineering and gene therapy strategies

Wear debris from worn cobalt chrome joint replacements causes an increase in chromosomal translocations and aneuploidy. In this study the relationship between the amount of DNA damage and the changes in gene expression was investigated in human fibroblasts after exposure to artificial cobalt chrome particles. The comparison was made with different doses of particles, at different time intervals and in fibroblasts of different ages, those that had completed 10 population doublings (10 PD fibroblasts) and those that had completed 35 population doublings (35 PD fibroblasts). The genes (TGF-©¬2, p38 MAPK, Integrin ¥â1, SOD1, Caspase 10, PURA, FRA-1 and VNR) were chosen after a previous screen with cDNA microarrays. The percentage of senescent cells was evaluated using an immunohistochemical assay for ¥â-galactosidase activity. The 35 PD fibroblasts showed significantly more ¥â-galactosidase activity than the 10 PD fibroblasts. The level of DNA damage, as detected with the alkaline comet assay, was greater at higher doses, at longer exposures (up to 24 hours) and in 10 PD fibroblasts. The expression of all the genes listed above was generally lower after exposure to cobalt chrome particles using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The reduction in gene expression, like the increase in DNA damage was greater at higher doses and at longer exposure times. After 24hr exposure the reduction in gene expression was greater in 10 PD fibroblasts compared to 35 PD fibroblasts. After 6hr exposure this was only true at higher doses of particles and the opposite was seen after a lower dose of particles. These results show that levels of gene expression of TGF-©¬2, p38 MAPK, Integrin ¥â1, SOD1, Caspase10, PURA, FRA-1 and VNR may be correlated with the level of DNA damage and that this depends on the dose and length of exposure and the age of the cells. This highlights the potential importance of these genes in the mutagenicity of cobalt chrome particles in human fibroblasts

Purpose: In attempting to unravel the complex cellular responses leading to prosthetic loosening investigators have been limited to studying gene expression of extracellular molecules about which most is known whereas new microarray technology allows simultaneous expression profiling of thousands of genes from a complex sample such as the membrane formed around loosened hip prostheses. Methods: Two groups of 8 patients were recruited who have undergone primary total hip arthroplasty for osteoarthritis and subsequently developed either septic or aseptic loosening +/− osteolysis. The control group consisted of one group of 5 patients with the same initial diagnosis who had undergone identical procedures, developed no clinical or radiological signs of aseptic or septic loosening, but had come to revision surgery for other complications as defined by the Swedish Hip register: fracture without previous osteolysis, dislocation, technical error, implant fracture, polyethylene wear or pain. Peri-prosthetic membrane was harvested at the time of revision surgery and subjected to RNA extraction. cDNA was then synthesized and hybridised to a Human Genome u95 Genechip ® array which contains a complete set of known human genes. Data normalisation, data filtering and pattern identification was performed using Genechip®3.1 software (Affymetrix, Santa Clara, CA). Results: This has revealed the involvement of a large number of genes coding for transcriptional regulators upstream from the extracellular and cell-cell signalling molecules already known to be involved in osteolysis and deep infection and which may ultimately control the responses to wear particles and bacterial challenge. Differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation and immune response was found. Most interestingly pathways for control of local bone resorption and inflammatory response have been shown to be highly activated. Conclusions: The identification of these new pathogenetic mechanisms of total hip replacement failure make new indicators of disease susceptibility and prognosis plus new drug targets direct possibilities

Introduction: The existence of peripheral blood (PB) derived mesenchynal stem cells (PBMSCs) have been documented in several species including human. The circulating skeletal stem cells may provide a new source of stem cells that may be used for skeletal and other tissue engineering applications. The objective of this study is to further investigate and compare the biological characteristics of the PBMSCs with bone marrow derived MSCs in the GFP rats. Methods: The peripheral blood (PB) from the GFP rats was harvested by cardiac puncture using syringes containing sodium heparin. Mononuclear cells were isolated by density gradient centrifugation method and plated at a density of 1–3~105/cm2 in flasks with D-MEM medium containing 15% FCS. The bone marrow (BM) was also collected for obtaining BMMSCs, the bone chips for osteoblastic cells, and the skin for skin fibroblasts. The phenotypes of the cells were characterized by immunocytochemistry (ICC), and flow cytometry methods. Gene expression profiles of 3-paired PBMSCs and BMMSCs cDNA samples were examined by Affymetrix gene chips microarray analysis. The multipotent differentiation potentials of PBMSCs into osteoblasts, chondrocytes, and adipocytes were examined under specific inductive conditions and checked with lineage specific markers. Finally, the osteogenic potential of the PBMSCs was examined by an in vivo implantation model in which the PBMSCs were seeded with HA-TCP powder complexes, and implanted subcutaneously in the severe compromised immunodeficiency (SCID) mice for 12 weeks, whereas the bone-derived osteoblasts and skin fibroblasts were used as controls. Results: Compared with the BMMSCs, the PBMSCs shared some but not all common surface markers as demonstrated by (ICC) and flow cytometry examinations. The osteogenic differentiation of PBMSCs was defined with positive staining of type I collagen and osteocalcin; positive staining for alkaline phosphatase and Von Kossa staining for mineralized bone nodules. Adipogenic differentiation was evidenced by positive Oil red-O staining for accumulated lipids, and chondrogenic differentiation by positive type II collagen and Saferinin O positive staining. For gene expression profiles, in the Affymetrix chip general analysis, 83 genes were up regulated and 84 genes down regulated in the PBMSCs (vs BMMSCs, > 2 fold, E-B/B-E> 100, p< 0.05). Most of which genes are related to cell proliferation, differentiation, cytoskeleton, and calcium/iron homeostasis. After 12 weeks implantation in SCID mice, newly formed lamellar bone was clearly evident in the groups with PBMSCs implants, so as in the groups with osteoblasts implants, but only fibrous tissue was found in the group implanted with skin fibroblasts. Discussion: This study demonstrated that the multi-potent PBMSCs in the GFP rats resemble BMMSCs in many aspects, but they are distinguishable from the BMMSCs in some biological characteristics and gene profiles. Our study has confirmed that these PBMSCs possess osteogenic potential in vitro and in vivo, suggesting that these circulating stem cells could serve as an alternative source as bone marrow derived MSCs for tissue engineering purposes

Introduction and Aims: Although skeletal muscles have remarkable potential for adaptation, the amount of muscle length increase during gradual limb lengthening is always less than the amount of bone lengthening. The purpose of this study was to analyse gene expression in skeletal muscle undergoing adaptation to limb lengthening. Method: Ten adult goats were randomly divided into two groups of five animals. Group 1 underwent 20% (43–47mm) standard Ilizarov tibial lengthening and group 2 served as un-operated control. Muscle tissues from proximal myotendenous junctions of Peroneus Longus were harvested from the lengthened limb in the distraction group and corresponding limb in the control group and immediately snap frozen in liquid nitrogen. To screen for genes potentially associated with sarcomerogenesis, microarray technology was employed. Biotin labeled cRNA was hybridised to Affymetrix HU133A GeneChips, containing 22,284 gene transcripts. All created data files were analysed using computer software GeneSpring 5.0. Results: In both groups, 5092 (23%) gene transcripts flagged present. Thirty-two of these transcripts were differentially expressed between distracted and control groups (p < 0.05). Represented by these transcripts were 12 known and three unknown genes, which were up-regulated in lengthened muscles by more than 2.0 fold. The substantially up-regulated genes identified were MYOZ2 (myozenin 2), MYL4 (embryonic myosin alkali light chain), MYL6 (myosin light polypeptide 6), CRYAB (crystalline), PFN2 (profiling 2), ARPP-19 (cyclic AMP phosphorprotein), TUBB2 (tubulin beta 2), PPP1R12 (protein phosphatase 1), RCOR (REST corepressor), LIM (LIM protein), FN1 (fibronectin 1), ACTC (alpha-actin), and hypothetical protein FLJ10111. Among the genes found to be up-regulated are genes involved in the myogenesis pathway. Myozenin 2 gene is associated with the signalling and activity of Calcineurin/Calsarcin that plays a significant role in muscle cell proliferation and myofiber type differentiation. Crystallin gene may be involved in promoting muscle survival during differentiation. The functionality of the remaining genes range from cytoskeletal organisation (TUBB2), cyto-skeletal structure (PFN2, MYL4, MYL6), cell adhesion and motility (FN1), muscle development and differentiation (FHL1 and LIM), intercellular adhesion and intermediate filament organisation (PNN), muscle contraction and relaxation (PPP1R12A), neuronal-specific gene silencing (RCOR), and PKA-dependent intracellular messaging (ARPP-19). Conclusion: The findings suggest that tension stress observed during gradual limb lengthening using standard Ilizarov distraction protocol activates expression of genes involved in skeletal muscle growth, differentiation, and neogenesis. On-going studies involving immunohistochemistry, RT-PCR, and in situ hybridisation to confirm cellular localisation of up-regulated genes are underway

Objectives

The aim of this study was to provide a comprehensive understanding of alterations in messenger RNAs (mRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs) in cartilage affected by osteoarthritis (OA).

MicroRNAs (miRNAs) are a class of small non-coding RNAs that have emerged as potential predictive, prognostic, and therapeutic biomarkers, relevant to many pathophysiological conditions including limb immobilization, osteoarthritis, sarcopenia, and cachexia. Impaired musculoskeletal homeostasis leads to distinct muscle atrophies. Understanding miRNA involvement in the molecular mechanisms underpinning conditions such as muscle wasting may be critical to developing new strategies to improve patient management. MicroRNAs are powerful post-transcriptional regulators of gene expression in muscle and, importantly, are also detectable in the circulation. MicroRNAs are established modulators of muscle satellite stem cell activation, proliferation, and differentiation, however, there have been limited human studies that investigate miRNAs in muscle wasting. This narrative review summarizes the current knowledge as to the role of miRNAs in the skeletal muscle differentiation and atrophy, synthesizing the findings of published data.

Cite this article: Bone Joint Res 2020;9(11):798–807.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with limited coding potential, which have emerged as novel regulators in many biological and pathological processes, including growth, development, and oncogenesis. Accumulating evidence suggests that lncRNAs have a special role in the osteogenic differentiation of various types of cell, including stem cells from different sources such as embryo, bone marrow, adipose tissue and periodontal ligaments, and induced pluripotent stem cells. Involved in complex mechanisms, lncRNAs regulate osteogenic markers and key regulators and pathways in osteogenic differentiation. In this review, we provide insights into the functions and molecular mechanisms of lncRNAs in osteogenesis and highlight their emerging roles and clinical value in regenerative medicine and osteogenesis-related diseases.

Cite this article: J. Zhang, X. Hao, M. Yin, T. Xu, F. Guo. Long non-coding RNA in osteogenesis: A new world to be explored. Bone Joint Res 2019;8:73–80. DOI: 10.1302/2046-3758.82.BJR-2018-0074.R1.

The world-wide experience with metallic implants provides the evidence for biocompatibility of modern cobalt- and titanium-based alloys. However, a corrosion process leading to release of ions has to be taken into account, and controversial data are available about the ‘endogenous’ metal exposure resulting from implants. Adverse tissue reactions, cytotoxicity and toxic/ sensitizing effects of corrosion products on the immune system, as well as an involvement of metal ions in the pathogenesis of prosthesis loosening, are the main undesired effects. Moreover, metal ions could be responsible of long-term toxic effects, such as genotoxicity and carcinogenicity. The aim of our study was to analyze the levels of serum metal ions in a large series of patients who underwent total hip and knee replacement (THR and TKR, respectively); both stable and loosened implants were considered. Furthermore, a group of subjects with fracture fixation devices was evaluated. A consecutive series of 471 individuals (193 M; 278 F) was enrolled in the study, including 151 with hip prosthesis loosening (Group A: 52 M; 99 F; median age 67; median follow up 90 months) and 100 patients with stable hip prosthesis (Group B: 44 M; 56 F; median age 59; median follow up 34 months). Group A and B comprised implants with different coupling, i.e. ceramic-on-ceramic (alumina), metal-on-metal and metal-on-polyethylene, and different stem alloy (TiAlV-THR and CoCrMo-THR). 20 patients with knee prosthesis loosening (Group C: 3 M; 17 F; median age 67; median follow up 25 months), and 12 subjects with failed fracture fixation devices (Group D: 6 M; 6 F; median age 35; median follow up 12 months) were enrolled, too. Osteoarthritis was the most frequent disease that led to joint replacement (59%), followed by hip dysplasia (19%), and trauma (13%). Ion reference ranges were obtained from 188 subjects (88 M; 100 F; median age 52), including 56 healthy subjects and 132 candidates to primary THR or TKR. Serum samples were analyzed for chromium (Cr), cobalt (Co), molybdenum (Mo), nickel (Ni), aluminum (Al), titanium (Ti) and vanadium (V) content, in relation with the presence in the implant alloy, using a graphite furnace atomic absorption spectrometer (GFAAS), equipped with double background correction Deuterium/ Zeeman, autosampler and pyrolytic carbon-coated graphite tubes (Unica Solaar 939 QZ, Cambridge, UK). The highest value found for subjects with no implant was considered the upper reference limit for each element. Group A (loosened THR) and B (well-fixed THR) patients, compared with controls, showed different behaviour depending on the prosthesis coupling and stem composition. Metal-on-PE coupling and CoCrMo/Ni stem: a significant increase of Cr and Co serum values was observed, in both groups, even if the increase in loosened implants was higher than in the stable ones. Mo and Ni concentration did not show a significant variation. Ceramic-on-ceramic coupling: a high Cr release, and a high Al, Ti release was shown in loosened implants with CoCrMo stem, and TiAlV stem, respectively, whereas normal values were measured in patients with stable prostheses. Metal-on-metal coupling and CoCrMo stem: a significantly higher Co and Cr release, both in group A and B patients. The conspicuous corrosion rate in metal-on-metal implants, even if stable, was confirmed in the same population in the short- and medium-term. A serum Co, Cr increase was demonstrated in Group C patients, too, who had a failed knee prosthesis, and a Cr, Ni increase was observed in Group D patients with stainless steel fracture fixation devices. Metal corrosion leading to ion release needs further consideration, because it may enhance the inflammatory reaction, depress the immune system, and facilitate peri-prosthetic bacterial growth. Especially metal-on-metal coupling introduces additional biologic risks associated with increased degradation products of prosthetic materials. Otherwise, histiocytic/giant-cell reaction and peri-prosthetic osteolysis induced by polyethylene particles represent the main problem in knee and hip joint implants with metal/PE coupling, and the systemic effects due to the ion release become negligible. On the contrary, ceramic-on-ceramic coupling represents a good alternative, as demonstrated by the absence of metal release in stable implants, even if, in case of loosening, a serum ion increase is observed, probably due to the fretting at the head/neck connection. Also, metal corrosion of fixation devices is a concern; consequently, it could be appropriate to remove the implants, as early as their function is accomplished. In conclusion, ion profile should be carefully monitored and the epidemiological survey implemented, in order to establish the tolerance values in patients with implants, especially in young patients, where a long-term ‘endogenous’ exposure has to be faced. In particular, concerning systemic toxic effects, the ‘gene expression profiling’, through the use of microarray technology, could contribute to an improved understanding of the biological responses to metal ions released from orthopedic implants. In such a way a real risk-to-benefit ratio for the patient could be established. Finally, ion monitoring, as demonstrated by the different serum ion levels in stable and loosened implants, could allow to detect early signs of failure, when radiographic and clinical data are unclear, and to avoid the need of revision using a proper treatment

Objectives

Eukaryotic translation initiation factor 3 (eIF3) is a multi-subunit complex that plays a critical role in translation initiation. Expression levels of eIF3 subunits are elevated or decreased in various cancers, suggesting a role for eIF3 in tumorigenesis. Recent studies have shown that the expression of the eIF3b subunit is elevated in bladder and prostate cancer, and eIF3b silencing inhibited glioblastoma growth and induced cellular apoptosis. In this study, we investigated the role of eIF3b in the survival of osteosarcoma cells.

Objectives

Prosthetic joint infection (PJI) diagnosis is a major challenge in orthopaedics, and no reliable parameters have been established for accurate, preoperative predictions in the differential diagnosis of aseptic loosening or PJI. This study surveyed factors in synovial fluid (SF) for improving PJI diagnosis.

Objectives

The biomembrane (induced membrane) formed around polymethylmethacrylate (PMMA) spacers has value in clinical applications for bone defect reconstruction. Few studies have evaluated its cellular, molecular or stem cell features. Our objective was to characterise induced membrane morphology, molecular features and osteogenic stem cell characteristics.

This article provides an overview of the role of genomics in sarcomas and describes how new methods of analysis and comparative screening have provided the potential to progress understanding and treatment of sarcoma. This article reviews genomic techniques, the evolution of the use of genomics in cancer, the current state of genomic analysis, and also provides an overview of the medical, social and economic implications of recent genomic advances.

Objectives

Several genome-wide association studies (GWAS) of bone mineral density (BMD) have successfully identified multiple susceptibility genes, yet isolated susceptibility genes are often difficult to interpret biologically. The aim of this study was to unravel the genetic background of BMD at pathway level, by integrating BMD GWAS data with genome-wide expression quantitative trait loci (eQTLs) and methylation quantitative trait loci (meQTLs) data

Objectives

Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues.

Mesenchymal stem-cell based therapies have been proposed as novel treatments for intervertebral disc degeneration, a prevalent and disabling condition associated with back pain. The development of these treatment strategies, however, has been hindered by the incomplete understanding of the human nucleus pulposus phenotype and by an inaccurate interpretation and translation of animal to human research. This review summarises recent work characterising the nucleus pulposus phenotype in different animal models and in humans and integrates their findings with the anatomical and physiological differences between these species. Understanding this phenotype is paramount to guarantee that implanted cells restore the native functions of the intervertebral disc.

Cite this article: Bone Joint Res 2013;2:169–78.

The April 2015 Knee Roundup360 looks at: Genetic determinants of ACL strength; TKA outcomes influenced by prosthesis; Single- or two-stage revision for infected TKA?; Arthroscopic meniscectomy: a problem that just won’t go away!; Failure in arthroscopic ACL reconstruction; ACL reconstruction in the over 50s?; Knee arthroplasty for early osteoarthritis; All inside meniscal repair; Steroids, thrombogenic markers and TKA

Objectives

Sustained intra-articular delivery of pharmacological agents is an attractive modality but requires use of a safe carrier that would not induce cartilage damage or fibrosis. Collagen scaffolds are widely available and could be used intra-articularly, but no investigation has looked at the safety of collagen scaffolds within synovial joints. The aim of this study was to determine the safety of collagen scaffold implantation in a validated in vivo animal model of knee arthrofibrosis.

The February 2013 Children’s orthopaedics Roundup³⁶⁰ looks at: the human genome; new RNA; cells, matrix and gene enhancement; the histology of x-rays; THR and VTE in the Danish population; potential therapeutic targets for GCT; optimising vancomycin elution from cement; and how much sleep is enough.