The aetiology and pathophysiology of non-union is still unclear, but in this condition there is an abnormal bone metabolism. The paracrine matrix RAS has been implicated in the regulation of bone remodeling and injury responses, possibly via its effects on kinins. The influence of the local RAS or the genetic influence of the ACE/ BK2R genes to bone remodelling may thus be central to the disorder, or augmented in these conditions. We thus compared the distribution of the ACE I/D and BK2R “+9/-9” functional polymorphisms in patients with non-union and compared them to appropriate control. Gene analysis was performed on buccal cells collected from all subjects and the data was analysed for 59 patients (46 males, 13 females; mean age 40.1±15.7 years) with non-union and 81 control subjects (49 males, 32 females; mean age 51.4±22.81 years. The overall genotype distribution was consistent with Hardy-Wein-berg equilibrium for the overall and individual groups for ACE (p0.16), B1BKR (p0.68) and B2BKR genotypes (p0.12). As the -9 allele is associated with greater gene transcription and higher mRNA expression of the receptor we combined the -9/-9 homozygous and -9/+9 heterozygous groups and compared them with the homozygous +9/+9 groups. This showed a significant difference between the non-union and control groups, with the +9/+9 homozygous being less prominent in the former (p=0.03). The B2BKR -9 allele is associated with the incidence of non-union in fracture healing, in this first study to address this question. We found no association with either the ACE I/D or B1BKR genotypes. In conclusion, with previous findings that the absence of the -9 allele of the B2BKR +9/-9 polymorphism is associated with greater gene transcription and higher mRNA expression of the receptor our findings are suggestive that increased BK activity via the B2BKR may predispose to the development of non-union

Intra-articular injection is a common way to deliver biologics to joints, but their effectiveness is limited by rapid clearance from the joint space. This barrier can be overcome by genetically modifying cells within the joint such that they produce anti-arthritic gene products endogenously, thereby achieving sustained, therapeutic, intra-articular concentrations of the transgene products without re-dosing. A variety of non-viral and viral vectors have been subjected to preclinical testing to evaluate their suitability for delivering genes to joints. The first transfer of a gene to a human joint used an ex vivo protocol involving retrovirally transduced, autologous, synovial fibroblasts. Recent advances in vector technology allow in vivo delivery using adeno-associated virus (AAV). We have developed an AAV vector encoding the interleukin-1 receptor antagonist (AAV.IL-1Ra) for injection into joints with osteoarthritis (OA). It showed efficacy and safety in equine and rat models of OA, leading to a recently-completed, investigator-initiated, Phase I, dose-escalation clinical trial in 9 subjects with mid-stage OA of the knee (. ClinicalTrials.gov. Identifier: NCT02790723). Three cohorts of three subjects with mild to moderate OA in the index knee were injected intra-articularly under ultrasound guidance with a low (10e11 viral genomes) medium (10e12 viral genomes) or high (10e13 viral genomes) dose of AAV.IL-1Ra and followed for one year. The data confirm safety, with evidence of sustained intra-articular expression of IL-1Ra and a clinical response in certain subjects. Funding for a subsequent Phase Ib trial involving 50 subjects (. ClinicalTrials.gov. Identifier: NCT05835895), expected to start later this year, has been acquired. Progress in this area has stimulated commercial activity and there are now at least seven different companies developing gene therapies for OA and a number of clinical trials are in progress. Acknowledgement: Clinical trial funded by US Department of Defense Clinical Trial Award W81XWH-16-1-0540

Low back pain affects 80% of the population with half of cases attributed to intervertebral disc (IVD) degeneration. However, the majority of treatments focus on pain management, with none targeting the underlying pathophysiological causes. PCRX-201 presents a novel gene therapy approach that addresses this issue. PCRX-201 codes for interleukin-1 receptor antagonist (IL-1Ra), the natural inhibitor of the pro-inflammatory cytokine IL-1, which orchestrates the catabolic degeneration of the IVD. Our objective here is to determine the ability of PCRX-201 to infect human nucleus pulposus (NP) cells and tissue to increase the production of IL-1Ra and assess downstream effects on catabolic protein production. Degenerate human NP cells and tissue explants were infected with PCRX-201 at 0 or 3000 multiplicities of infection (MOI) and subsequently cultured for 5 days in monolayer (n=7), 21 days in alginate beads (n=6) and 14 days in tissue explants (n=5). Cell culture supernatant was collected throughout culture duration and downstream targets associated with pain and degeneration were assessed using ELISA. IL-1Ra production was increased in NP cells and tissue infected with PCRX-201. The production of downstream catabolic proteins such as IL-1β, IL-6, MMP3, ADAMTS4 and VEGF was decreased in both 3D-cultured NP cells and tissue explants. Here, we have demonstrated that a novel gene therapy, PCRX-201, is able to infect and increase the production of IL-1Ra in degenerate NP cells and tissue in vitro. The increase of IL-1Ra also resulted in a decrease in the production of a number of pro-inflammatory and catabolic proteins, suggesting PCRX-201 enables the inhibition of IL-1-driven IVD degeneration. At present, no treatments for IVD degeneration target the underlying pathology. The ability of FX201 to elicit anti-catabolic responses is promising and warrants further investigation in vitro and in vivo, to determine the efficacy of this exciting, novel gene therapy

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

Objective To decide whether recombined rat transforming growth factor beta-1 gene and insulin-like growth factor-1 gene have positive influences on ACLT-induced osteoarthritis-like changes in NZW rabbit articular cartilage. Methods Twenty-four NZW rabbits, with osteoarthritis caused by anterior cruciate ligament transection£. . ACLT£©, were distributed to 4 groups randomly and another six rabbits were taken as normal control group (group 1). Chondrocytes which had been transfected with TGF-¦Â1 gene, IGF-1 gene (group 3–5) were injected into the knee of these NZW rabbits. Experimental control group (group 2) was only suffered ACLT but nothing injected. After 4, 8 weeks, rabbits were sacrificed and evaluated by morphological grades, histological examination, examination of in situ hybridization, immunohistochemistry, and transmission electron microscopy (TEM). Results The data of morphological grades showed that the normal control showed a significant difference compared with experimental control group (P< 0.01). The groups with injected chondrocytes carring TGF-¦Â1 gene and double genes (group 3,5) had a significant difference compared with experimental control group (P< 0.05). The in situ hybridization and immunohis-tochemistry examination showed the same results as above, and the group carring double genes (group 5) had a significant difference with that single gene (group 3,4) (P< 0.05). After 8 weeks, the examination data showed that all groups lower than the data of 4 weeks except the normal control group and experimental control group (P< 0.05). Ultrastructural examination indicated that the ultrastructure of experimental control group was more turbulent than that of normal control group. The ultra-structure of the gene therapy groups was more normal than that of experimental control group after gene therapy, but it turned to be turbulent again after 8 weeks. Conclusion It is effectual on osteoarthritis to inject chondrocytes carring recombined TGF-¦Â1,IGF-1 genes into NZW rabbits knee joints. It was obvious that the therapy effect of double genes was better than single gene. The fact that gene expression was decreased gradually after 4 weeks makes out that gene therapy is limited by time. These results suggest that therapeutic TGF-¦Â1 and IGF-1 gene transfer may be applicable for the treatment of OA

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

Although 80% of fractures typically heal without any problems, there is a small proportion (<20%) that suffer complications such as delayed healing and potential progression to non-union. In patients with healing complications, the coordinated regulation between pro- and anti-inflammatory cytokines, such as interleukin-1β (IL-1β) and interleukin-1 receptor antagonist (IL-1Ra) respectively, is often dysregulated. The aim of this study is to develop a therapeutic strategy based on the local delivery of genes to reparative mesenchymal stromal cells (MSCs) migrating into the local fracture microenvironment, thereby promoting a more favourable healing environment to enhance fracture repair. Our approach involves the local delivery of nanoparticles complexing the non-viral vector polyethyleneimine (PEI) with therapeutic plasmid DNA (pDNA) encoding for IL-1Ra. pDNA encoding green fluorescent protein and Gaussia luciferase were used as reporter genes to determine the transfection efficiency of both rat and human MSCs using flow cytometry and to assess the transgene expression profile using a luciferase expression assay. The effect of transfection with PEI on the viability of MSCs was assessed using the metabolic assay Cell Titer Blue and dsDNA quantification. Levels of IL-1Ra produced by cells following transfection with nanoparticles encoding IL-1Ra was assessed using enzyme-linked immunosorbent assays (ELISA). HEK-Blue IL-1β reporter cells, which secrete alkaline phosphatase in response to IL-1β stimulation, were used to confirm that the IL-1Ra produced by transfected cells is functionally active, i.e. the successful antagonism of IL-1β bioactivity. We have determined that using PEI-based nanoparticles we can achieve a transfection efficiency of 14.8 + 1.8% in rat MSCs. Transgene expression was found to be transient, with a peak in expression at 7 days post-transfection and a gradual decrease over time, which was maintained for up to 4 weeks. Using an optimized concentration of PEI, the impact of the nanoparticles on MSC viability was limited, with no significant difference in cellular metabolic activity compared to non-transfected cells at 10 days post-transfection. We have additionally demonstrated the capacity to successfully transfect both rat and human MSCs with pDNA encoding for IL-1Ra, resulting in enhanced levels of IL-1Ra, which is functionally active. The use of non-viral gene therapy to locally deliver immunomodulatory genes, such as IL-1Ra, to MSCs presents a promising strategy to enhance bone healing. Specifically, the transgene expression levels achieved with such an approach can remain therapeutically effective and are transient in nature, presenting an advantage over other methods such as recombinant protein delivery and viral-based gene delivery methodologies

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

Treatment of segmental bone defects remains a major clinical problem, and innovative strategies are often necessary to successfully reconstruct large volumes of bone. When fractures occur, the resulting hematoma serves as a reservoir for growth factors and a space for cell infiltration, both crucial to the initiation of bone healing. Our previous studies have demonstrated very clear ultrastructural differences between fracture hematomas formed in normally healing fractures and those formed in segmental bone defects. However, there is little information available regarding potential differences in the underlying gene expression between hematomas formed in normal fractures, which usually heal by themselves, and segmental bone defects, which do not. Therefore, the aim of this study was to identify differences in gene expression within hematomas collected from 0.5 mm (normal fracture) and 5 mm (segmental bone defect) fracture sites during the earliest stages of bone healing. Osteotomies of 0.5 and 5 mm in the femur of Fisher 344 rats were stabilized with external fixators (RISystem AG). After 3 days the rats were sacrificed, and the fracture hematomas were collected for RNA-sequencing. Ingenuity pathway analysis (IPA) was used to identify upstream regulators and biological functions that were significantly enriched with differentially expressed genes from the RNA-sequencing analysis. Animal procedures were conducted following the IACUC protocol of the UT Health Science Center San Antonio. Key upstream regulators of bone formation were less active (e.g. TGFB1, FGF2, SMAD3) or even inhibited (e.g. WNT3A, RUNX2, BMP2) in non-healing defects when compared to normally healing fractures. Many upstream regulators that were uniquely enriched in healing defects were molecules recently discovered to have osteogenic effects during fracture healing (e.g. GLI1, EZH2). Upstream regulators uniquely enriched in non-healing defects were mainly involved in an abnormal modulation of hematopoiesis, revealing evidence of impaired maturation of functional macrophages and cytokines (e.g. IL3, CEBPE), both essential for successful bone healing. In addition, the enrichment pattern suggested a dysregulation of megakaryopoiesis (e.g. MRTFA, MRTFB, GATA2), which directly affects platelet production, and therefore fracture hematoma formation. Remarkably, the organization of the ECM was the most significantly enriched biological function in the normally healing fractures, and implies that the defect size directly affected the structural properties within the fracture hematoma. Conversely, genes encoding important ECM components (e.g. BGN, various collagens, IBSP, TNC), cell adhesion molecules, MMPs (MMP2), and TIMPs were all significantly downregulated in non-healing defects. Our most recent findings reveal new important key molecules that regulate defect size-dependent fracture healing. Combined with our previous results, which identified structural differences in fracture hematomas from both types of defects, current findings indicate that differential expression of genes is dictated by the structural properties of the hematomas formed during early fracture healing. Consequently, creating a bioscaffold that mimics the structure of normal fracture hematomas could be the first step towards developing new orthoregenerative treatment strategies that potentiate healing of large bone defects and non-healing fractures

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

Introduction. Within the intervertebral disc (IVD), nucleus pulposus (NP) cells reside within a unique microenvironment. Factors such as hypoxia, osmolality, pH and the presence of cytokines all dictate the function of NP cells and as such the cells must adapt to their environment to survive. Previously we have identified the expression of aquaporins (AQP) within human IVD tissue. AQPs allow the movement of water across the cell membrane and are important in cellular homeostasis. Here we investigated how AQP gene expression was regulated by the microenvironment of the IVD. Methods. Human NP cells were cultured in alginate beads prior to cytokine, osmolality, pH and hypoxia treatments and subsequent RT-qPCR to assess regulation of AQP gene expression. Results. Physiological conditions observed within the native IVD regulated AQP gene expression in human NP cells. Hyperosmotic treatment up-regulated the expression of AQP1 and 5 during hypoxic conditions, whereas AQP4 expression was down-regulated. During hypoxia and physiological pH treatments AQP5 expression was increased. Pro-inflammatory cytokines, increased during IVD degeneration, also altered AQP gene expression. Interleukin-1β (IL-1β) decreased expression of AQP1 and 3 yet up-regulated AQP9, interleukin-6 (IL-6) increased expression of AQP1, 3, and 9 and tumour necrosis factor α (TNFα) upregulated the gene expression of both AQP2 and 9. Conclusion. The microenvironment in which NP cells reside in vivo directly contributes to their correct function and survival. AQP gene expression was differentially regulated under healthy compared to degenerate conditions; this potentially highlights that during IVD degeneration NP cells differentially express AQPs. No conflicts of interest. Funded by BMRC, Sheffield Hallam University

Total hip arthroplasty (THA) wear debris induced macrophage expression of pro-inflammatory cytokines has been associated with osteolysis both in vitro and in animal and human subjects. Interleukin-1 receptor antagonist (IL-1RA) is an anti-inflammatory cytokine which may limit bone destruction. Polymorphisms (SNPs) within the IL-1RN gene are associated with differences in susceptibility to infectious and inflammatory conditions and disorders of bone remodelling. This study investigated the association between the IL-1RA+2018T/C SNP (rs419598) and osteolysis after THA, and with mRNA and protein expression in an in-vitro wear debris-macrophage stimulation assay. 611 North European Caucasians who had received a cemented THA for primary osteoarthritis were genotyped for the IL-1RN+2018 SNP using Taqman methods. 62 subjects with a Charnley THA were selected from the genotyping population. Control subjects had no radiographic osteolysis and the osteolysis group had previously undergone revision surgery for aseptic loosening. Peripheral blood mononuclear cells were extracted and stimulated with endotoxin-stripped titanium particles (TiCL, endotoxin level 0 Eu/ml) and endotoxin-stripped particles with adherent LPS added back (TiAB, endotoxin level 140 Eu/ml); non-stimulated and LPS-stimulated cells were used as negative and positive controls. Cell lysate IL-1RA mRNA levels were assessed by rqRT-PCT following a 3-hour stimulation. Cell supernatant IL-1RA protein levels were assayed after 24 hours stimulation using a multiplex method. The IL-1RN+2018C allele was underrepresented in patients with osteolysis after THA versus control THA subjects (chi-squared test 5.96, P=0.015). After correction for other risk factors for osteolysis, the adjusted odds ratio for osteolysis associated with carriage of the IL-1RN+2018C SNP was 0.69 (0.48 to 0.99, p=0.048). IL-1RA mRNA expression increased linearly with IL-1RN+2018C allele copy number in gene-dose dependent manner (ANOVA p=0.013). The IL-1RA+2018C allele did not significantly affect IL-1RA protein expression (ANOVA p> 0.05), however a similar trend towards increased levels with increased C allele copy number was observed. Carriage of the IL-1RA+2018C allele is associated with both a decreased risk of osteolysis after THA and increased IL-1RA mRNA expression in-vitro. The mechanism for this functional effect remains unclear, however these findings support the importance of the IL-1RA in osteolysis and aseptic loosening

Aim. The aim of this study was to gain insight into the in vivo expression of virulence and metabolic genes of Staphylococcus aureus in a prosthetic joint infection in a human subject. Method. Deep RNA sequencing (RNA-seq) was used for transcriptome profile of joint fluid obtained from a patient undergoing surgery due to acute S. aureus prosthetic joint infection. The S. aureus gene expression in the infection was compared with exponential culture of a S. aureus isolate obtained from the same sample using EdgeR. In addition, the genome of the isolate was sequenced on Miseq, assembled in CLC genomics workbench and annotated by MaGe. Moreover, using nuclear magnetic resonance (NMR) spectroscopy we analysed the metabolites in the joint fluid and in the culture supernatants to determine the biochemical composition of the environments. Results. Antibiotic susceptibility testing by disk diffusion (EUCAST) demonstrated that the strain was susceptible to β-lactams (penicillin and cefoxitin) and macrolides (erythromycin and roxitromycin). This was indirectly confirmed by the annotated genome, because of absence of known resistant genes. The patient showed no signs of improvement during 2-days treatment with antibiotics (different β-lactams and gentamicin) prior to the surgery. The RNA-seq data indicated that the strategy employed by S. aureus to survive and proliferate in the host during antibiotic treatment involved overexpression of various enzymes related to cell-wall synthesis and multidrug efflux pumps. Interestingly, these efflux pumps are only known to be related to fluoroquinolone resistance. Many of the genes encoding virulence factors were upregulated, including toxins and superantigen-like proteins, hemolysins, and immune evasion proteins. A number of chaperones and stress related genes were overexpressed indicating a stress response. Furthermore, the RNA-seq data provided clues of the potential major nutrient sources for the pathogen in vivo. Several amino acid degradation pathways were highly upregulated, e.g. arginine, histidine. Additional carbon sources included N-acetylneuraminate and purine/pyrimidine deoxyribonucleosides as indicated by the upregulation of the genes involved in the degradation pathways of these compounds and higher concentration of these substances in the joint fluid compared to culture supernatants. Conclusions. Our results show that the gene expression pattern of S. aureusin vivo is vastly different from that of an in vitro grown exponential culture, indicating that the pathogen adapts to host environmental conditions by altering gene expression. Finally our study emphasizes the importance of in vivo study in elucidating pathogenesis of S. aureus in prosthetic joint infections

Introduction and Aims: Aberrations in the balance of chondrocyte metabolism play an integral role in the degeneration of articular cartilage and subsequent osteoarthritis. Gene expression profiling allows a comparison of levels of mRNA expression in large numbers of genes simultaneously. This study compares the mRNA expression from osteoarthritic cartilage in knees and hips with that of normal cartilage. Method: Human cartilage samples were obtained from osteoarthritic knees and hips at the time of joint arthroplasty surgery. ‘Normal’ cartilage was obtained from femoral heads after fracture or from radial heads after trauma. Cartilage samples were either snap frozen in liquid nitrogen or enzymatically digested and established in primary cell culture prior to RNA isolation. The RNA was reverse-transcribed to cDNA, labelled with a fluorochrome and then hybridised to gene chips. Results: In addition to confirming that cells raised in primary cell culture dedifferentiate to a fibroblast-like state and cease to synthesise normal products of cartilage matrix we have also developed a reproducible method of processing snap frozen cartilage samples in order to produce a sufficiently pure quantity of mRNA to be used in gene chip technology. We now have gene chips completed for a ‘normal’ control, a standard osteo-arthritic knee and an osteoarthritic hip with a significant genetic history of early onset osteoarthritis. Early analysis and comparison of the data from these chips identifies some potential candidate genes for further analysis. Conclusion: Human articular cartilage lends itself to gene profiling using cDNA arrays as it contains only one cell type. Thus any changes in gene expression levels can be directly attributable to the chondrocyte. This early data analysis opens the door to a new search for the ‘arthritis gene’. For the data to be meaningful we will need to process gene chips on several more samples of arthritic and ‘normal’ cartilage

The superficial zone (SFZ) of articular cartilage has unique structural and biomechanical features, and is important for joint long-term function. Previous studies have shown that TGF-β/Alk5 signaling upregulating PRG4 expression maintains articular cartilage homeostasis. However, the exact role and molecular mechanism of TGF-β signaling in SFZ of articular cartilage homeostasis are still lacking. In this study, a combination of in vitro and in vivo approaches were used to elucidate the role of Alk5 signaling in maintaining the SFZ of articular cartilage and preventing osteoarthritis initiation. Mice with inducible cartilage SFZ-specific deletion of Alk5 were generated to assess the role of Alk5 in OA development. Alterations in cartilage structure were evaluated histologically. The chondrocyte apoptosis and cell cycle were detected by TUNEL and Edu staining, respectively. Isolation, culture and treatment of SFZ cells, the expressions of genes associated with articular cartilage homeostasis and TGF-β signaling were analyzed by qRT-PCR. The effects of TGF-β/Alk5 signaling on proliferation and differentiation of SFZ cells were explored by cells count and alcian blue staining. In addition, SFZ cells isolated from C57 mice were cultured in presence of TGF-β1 or SB505124 for 7 days and transplanted subcutaneously in athymic mice. Postnatal cartilage SFZ-specific deletion of Alk5 induced an OA-like phenotype with degradation of articular cartilage, synovial hyperplasia as well as enhanced chondrocyte apoptosis, overproduction of catabolic factors, and decreased expressions of anabolic factors in chondrocytes. qRT-PCR and IHC results confirmed that Alk5 gene was effectively deleted in articular cartilage SFZ cells. Next, the PRG4-positive cells in articular cartilage SFZ were significantly decreased in Alk5 cKO mice compared with those in Cre-negative control mice. The mRNA expression of Aggrecan and Col2 were decreased, meanwhile, expression of Mmp13 and Adamts5 were significantly increased in articular cartilage SFZ cells of Alk5 cKO mice. In addition, Edu and TUNEL staining results revealed that slow-cell cycle cell number and increase the apoptosis positive cell in articular cartilage SFZ of Alk5 cKO mice compared with Cre-negative mice, respectively. Furthermore, all groups of SFZ cells formed ectopic solid tissue masses 1 week after transplantation. Histological examination revealed that the TGF-β1-pretreated tissues was composed of small and round cells and was positive for alcian blue staining, while the SB505124-pretreated tissue contained more hypertrophic cells though it did stain with alcian blue. TGF-β/alk5 signaling is an essential regulator of the superficial layer of articular cartilage by maintaining chondrocyte number, its differentiation properties, and lubrication function. Furthermore, it plays a critical role in protecting cartilage from OA initiation

Introduction Aberrations in the balance of chondrocyte metabolism play an integral role in the degeneration of articular cartilage and subsequent arthritis. Gene expression profiling is a powerful tool which allows identification of differences in levels of mRNA expression of large numbers of genes simultaneously. The objective of this study was to compare mRNA expression from osteoarthritic cartilage with that of normal cartilage and by use of the Affymetrix system, identify target genes for further investigation. Methods Human cartilage samples were obtained from osteoarthritic knees and hips at the time of joint replacement surgery. Non-arthritic cartilage samples were obtained from notchplasty at time of cruciate ligament replacement surgery or from trauma surgery. Cartilage samples were either snap frozen in liquid nitrogen and RNA directly isolated from the frozen tissue or enzymatically digested and established in primary culture prior to RNA isolation. The RNA was reverse transcribed to cDNA, labelled with a fluorochrome and then hybridised to gene chips. This will allow us to: 1. Compare whether RNA expression in cell culture accurately reflects that in the tissue itself. 2. Determine whether there are differences between the gene profiles of knee and hip osteoarthritis. 3. Select candidate genes for further analysis. Results At present primary cell culture lines have been successfully established and are ready for RNA isolation. Frozen cartilage samples have undergone RNA isolation. Currently techniques are underway to maximise RNA extraction and sufficiently purify it to process a gene chip. Once the gene chip is made a list of up or down-regulated genes will be available for analysis. Human articular cartilage lends itself to gene profiling using cDNA arrays as it contains only one cell type. Thus any changes in gene expression levels can be directly attributed to the chondrocyte. Conclusions This technology opens the door to a new search for the ‘arthritis gene’. In relation to the conduct of this study, one or more of the authors is in receipt of a research grant from a non-commercial source

Paget’s disease of bone is a common disorder characterised by focal areas of increased bone resorption coupled to increased and disorganised bone formation. Pagetic osteoclasts have been studied extensively, however, due to the integral cross-talk between osteoclasts and osteoblasts, we propose that pagetic osteoblasts may also play a key role in the pathogenesis of Paget’s disease. Any phenotypic changes in the diseased osteoblasts are likely to result from alterations in the expression levels of specific genes. To determine any differences in expression between pagetic and non-pagetic osteoblasts and their precursors the gene expression profiles of RANK, RANKL, OPG, VEGF, IL-1beta, IL-6, MIP-1, TNF and M-CSF were investigated in primary cultures of human osteoblasts and in the osteoblast precursor population of bone marrow stromal cells. We present preliminary data of this study. Trabecular bone explants were finely chopped, washed free of marrow and cellular debris then either snap frozen in liquid nitrogen or placed in flasks to culture outgrowth osteoblast-like cells. Mononuclear stromal cells from bone marrow were isolated and grown in culture flasks. RNA and conditioned media were collected from cultured osteoblasts and stromal cells at confluency. The innovative method of Real-Time PCR, the most accurate technique available at present to quantitatively measure gene expression, was used for the comparison of gene expression levels in our samples. 18S ribosomal RNA was used as an endogenous control to normalise the expression in the various samples. RANK, MIP-1 and TNF were only detected in stromal cells whereas RANKL, OPG, VEGF, IL-1beta, IL-6 and M-CSF were detected in both osteoblasts and stromal cells. OPG displayed higher expression in osteoblasts while IL-1beta showed higher expression in stromal cells. To date we have not seen any significant differences in gene expression between pagetic and non-pagetic subjects when comparing a small number of samples. A larger cohort is currently being investigated. We are also comparing levels of secreted proteins in the conditioned media from pagetic and non-pagetic cell cultures. This may lead to further candidate genes involved in the pathology of the pagetic lesion

Background. Hyaluronan (HA) promotes extracellular matrix (ECM) production and inhibits the activity of matrix degrading enzymes in chondrocytes. The meniscus is composed of the avascular inner and vascular outer regions. Inner meniscus cells have a chondrocytic phenotype compared with outer meniscus cells. In this study, we examined the effect of HA on chondrocytic gene expression in human meniscus cells. Methods. Human meniscus cells were prepared from macroscopically intact lateral meniscus. Inner and outer meniscus cells were obtained from the inner and outer halves of the meniscus. The proliferative activity of meniscus cells was evaluated by WST-1 assay in the presence or absence of HA (MW = 600–1200 kDa; Seikagaku). Gene expression of SOX9, COL2A1, and COL1A1 was assessed by a quantitative real-time PCR analysis. The effect of HA on the gene expression and cellular proliferation was investigated under the treatment of interleukin (IL)-1α. Meniscal samples perforated by a 2-mm-diameter punch were maintained for 3 weeks in HA-supplemented media. Cultured meniscal samples were evaluated by histological analyses. Results. HA treatments stimulated cellular proliferation in both inner and outer meniscus cells. HA also increased COL2A1 expression in inner meniscus cells. On the other hand, HA did not induce COL2A1 expression in outer meniscus cells. Although IL-1α treatment decreased COL2A1 expression in inner meniscus cells, the decrease of COL2A1 expression was prevented by HA treatments. In addition, HA treatments increased cellular counts along the perforated surface of organ-cultured meniscal samples. Conclusion. The present study demonstrated that HA activated the proliferation and chondrocytic gene expression of inner meniscus cells. In addition, IL-1α-dependent decrease of COL2A1 expression was prevented by HA treatment. Our results suggest that intra-articular HA injection may be useful in the treatment of inner meniscal injury. Level of evidence. in vitro study, level IV. Disclosure. The authors have no conflicts of interest

Introduction Paget’s disease of bone is a common disorder characterised by focal areas of increased bone resorption by osteoclasts and disorganised bone formation by osteoblasts. Because there is integral cross-talk between osteoclasts and osteoblasts during normal bone remodelling, we propose that Pagetic osteoblasts may also play a key role in the pathogenesis of Paget’s disease. Any phenotypic changes in the diseased osteoblasts are likely to result from alterations in the expression levels of specific genes. Methods To determine any differences in expression between Pagetic and non-Pagetic osteoblasts and their precursors the gene expression profiles of RANK, RANKL, OPG, VEGF, IL-1beta, IL-6, MIP-1, TNF and M-CSF were investigated in primary cell cultures of human osteoblasts and in the osteoblast precursor population of bone marrow stromal cells. Trabecular bone explants were finely chopped, washed free of marrow and cellular debris then either snap frozen in liquid nitrogen or placed in flasks to culture outgrowth osteoblast-like cells. Mononuclear stromal cells from bone marrow were isolated and grown in culture flasks. RNA and conditioned media were collected from cultured osteoblasts and stromal cells at confluency. Real-Time PCR was used for the comparison of gene expression. 18S ribosomal RNA was used as an endogenous control to normalise the expression in the various samples. Results RANK, MIP-1 and TNF were only detected in stromal cells whereas RANKL, OPG, VEGF, IL-1beta, IL-6 and M-CSF were detected in both osteoblasts and stromal cells. OPG displayed higher expression in osteoblasts while IL-1beta showed higher expression in stromal cells. To-date we have not seen any significant differences in gene expression between pagetic and non-pagetic subjects when comparing a small number of samples. A larger cohort is currently being investigated. Mutations in the sequestosome 1 gene have been showed to be associated with Paget’s disease. When a small number of Pagetic samples were sequenced for these mutations we found one out of seven patients (14%) to possess a known transition mutation at position 1215 in this gene. Conclusions These results may further our understanding of the pathology of Paget’s disease

Aim. Previous studies have indicated that TNF-α and lymphotoxin-α (LTA) gene polymorphisms associate with the development of several different inflammatory diseases. However, potential associations of such gene polymorphisms with the susceptibility to extremity chronic osteomyelitis remain unknown. This study aimed to investigate potential links between TNF-α gene polymorphisms (rs1800629, rs361525, rs1799964, rs1800630, rs1799724 and rs1800750) and LTA gene polymorphism (rs909253) and the risk of developing extremity chronic osteomyelitis in Chinese population. Method. A total of 233 patients with extremity chronic osteomyelitis and 200 healthy controls were genotyped for the above 7 polymorphisms of TNF-α and LTA genes using the genotyping method*. Results. Significant difference was found regarding the genotype distribution of rs909253 between patients and healthy controls (P = 0.002). The mutant allele C frequency of rs909253 in patient group was significantly higher than that in control group (P = 0.001). Significant associations were identified between rs909253 and the risk of developing chronic osteomyelitis by dominant model (P = 0.040), recessive model (P = 0.002) and homozygous model (P = 0.001). Additionally, the mutant allele T frequency in rs1799964 in patient group was significantly higher than that in control group (P = 0.035). Significant link was found between rs1799964 and susceptibility to chronic osteomyelitis by recessive model (P = 0.048). However, no significant outcomes were identified regarding other TNF-α gene polymorphisms between the two groups. Conclusions. The present study demonstrated that rs909253 and rs1799964 polymorphisms may associate with the risk of developing chronic osteomyelitis in Chinese population. *SNaPshot