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

Introduction

Mechanical loading has been hypothesized to play an important role in the development, remodeling and in diseases of many skeletal tissues, including cartilage. In order to study the metabolic response of cartilage to physical forces, in vitro systems have often been used because of the precise control with which mechanical loads can be applied. We developed a new mechanical loading system, in which we were able to load the intact femoral condyle in order to preserve the native cartilage/subchondral bone structure. This system represents a more ‚in vivo‘ situation than cartilage explants or chondrocyte cell culture systems.

Our approach focused on changes in mRNA expression of type II collagen, type VI collagen, and aggrecan in loaded versus adjacent unloaded cartilage in order to analyse the early response of chondrocytes to well-defined mechanical stresses.

Methods

Femoral condyles were obtained from two-year-old cows. The integrity of the cartilage surface was controlled by staining with safranin O. The femoral condyles were compressed in an Instron 8501 material testing machine. Cyclic compression pressure was applied for 2000 cycles in a sinusoidal waveform of 0. 5 Hz-frequency with a peak stress of 0. 2 to12. 5 MPa. Following loading, full depth cartilage sections were cut out and one half immediately frozen in liquid nitrogen for RNA isolation and the other half soaked in 4% paraformaldehyde for paraffin embedding. As control, the adjacent unloaded cartilage was collected and treated in the same way. Total RNA was isolated and changes in mRNA expression were quantitated by competitive quantitative PCR, using an internal standard of a C-terminal truncated version of the corresponding genes. The PCR-reactions were separated by agarose gel electrophoresis and amplified fragments quantified using video-densitometry analysis. The results were expressed as the ratio of mRNA from loaded to unloaded cartilage

Results

Cyclic compression with peak stresses of 12. 5, 6. 3, 2. 5 and 0. 6 MPa lead to a two-fold decrease in the mRNA expression of type II collagen and aggrecan and a threefold decrease of type VI collagen, in consideration of the intra-assay variability of about 30%. Compression with peak stresses of 0. 3 and 0. 2 MPa lead to a three-fold increase of the mRNA expression of type II collagen, a four-fold increase of aggrecan and a slight decrease of type VI collagen.

Low compression strength leads to an increase of the mRNA expression of the major components of cartilage, type II collagen and aggrecan, whereas high loading leads to a decrease of the mRNA expression.

Conclusion

The results show that our system can be used to analyze early responses of chondrocytes to well-defined mechanical stresses in an intact cartilage/bone-system and therefore will enable us to investigate the role of physiological and non-physiological high loading on the induction of cartilage degradation and regeneration in joint trauma and osteoarthritis. Since the cartilage/bone samples are incubated in medium during the experiment, this system will also offer us the opportunity to investigate additives to the medium as potential pharmacological therapeutics in osteoarthritis.

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

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 and Aims: Assessment of the metabolic state of articular cartilage (AC) is important in understanding the initiation and progression of osteoarthritis (OA). The purpose of this study was to evaluate changes in gene expression of the major AC extracellular matrix (ECM) components, in addition to a number of molecules involved in OA, including the novel glycoprotein lubricin, following lateral meniscectomy in a sheep model of OA. Method: AC tissue from both medial (MTP) and lateral (LTP) tibial plateaux were collected from six non-operated control (NOC) and six lateral meniscectomised (MEN) pure-bred Merino sheep six months post-surgery for semi-quantitative RT-PCR to assess patterns of mRNA expression (relative to GAPDH). Histological evaluation using a modified Mankin score was undertaken in the same sheep to grade the AC and immunohistochemical localisation of gene products was performed. Results: Cartilage degeneration was evident both macroscopically and histologically in the LTP following MEN, with less marked changes appearing in the MTP. The mean total tissue RNA increased greater than five-fold in the LTP following MEN (p< 0.01). Expression of aggrecan (p< 0.01) and collagen type II (p< 0.01) were found to be significantly elevated in LTP AC following MEN. Increased expression of biglycan (p< 0.01) was observed in LTP AC following MEN, whereas conversely, there was a decreased expression of decorin (p< 0.01), the other fibril associated small leucine rich proteoglycan. Expression of both lubricin (p< 0.01) and connective tissue growth factor (CTGF) (p< 0.05) were also found to decrease following MEN in LTP AC. TGFβ demonstrated no change in expression following MEN. Significant changes in gene expression were generally not seen in the MTP following MEN; however trends were observed reflecting similar gene profile changes to those occurring in the LTP. Conclusion: Strong up-regulation in gene expression of the major cartilage ECM components was found, reflecting an anabolic response and attempted tissue repair. Significant changes were also observed for other ECM macromolecules thought to be involved in degenerative joint disease, contributing to alterations in the gene expression profile associated with OA

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 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

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

Articular cartilage (AC) has a poor innate healing capacity following significant injury. Autologous chondrocyte implantation is a repair technique which utilises in vitro-expanded chondrocytes combined with a periosteal patch. The chondrocytes are enzymatically digested from arthroscopically harvested tissue at an initial surgery and expanded in monolayer culture prior to implantation at a second procedure. Unfortunately, in vitro expanded chondrocytes appear unable to retain their fundamental phenotype resulting in dedifferentiated cells which produce a matrix of inferior quality. This study compares the matrix-component gene expression profiles of chondrocytes in their native chondrons and through multiple divisions in monolayer culture. We hypothesised that there would be a rapid decline of matrix-component gene expression within a few cell replications in monolayer culture. The goal is to understand more fully the process of chondrocyte dedifferentiation and to compare matrix-component gene expression during cellular expansion in vitro. Human AC was obtained from tissue donors and operative patients. A portion of the AC was stored at −80°C for use as a control while the remainder was homogenised and enzymatically digested with collagenase. The released cells were plated in monolayer culture and passaged (2:1) when they approached confluence. RNA was extracted from the frozen cartilage control and the passaged chondrogenic cell lines from which cDNA was generated. Real time PCR was performed with primers specific for collagen I, collagen II, aggrecan, and GAPDH. Gene expression was quantified and profiles from the cells in their native chondron and passaged cells (p0-p9) were compared. Cells, when removed from the extra-cellular matrix and plated in monolayer, experienced an immediate upregulation of collagen I which persisted throughout all passages. In contrast, there was a stepwise decrease in collagen II with each successive passage until p8-p9 when the expression became undetectable. Aggrecan expression only decreased minimally as the cells were passaged. Rapid dedifferentiation of monolayer cultured chondrocytes is a persistent barrier to AC tissue engineering including ACI. This study quantified the expression of relevant genes relating to AC generation and is an important first step to understanding cellular events, as alternative expansion techniques and cellular alternatives are sought

Aim: To determine the pattern of gene expression induced in cultured human chondrocytes in response to compressive mechanical loads. Methods: Chondrocytes were obtained from tissue discarded at the time of a number of total knee replacements and where established in primary cell culture. The cultured chondrocytes were then subjected to compressive and tensile loads using a Flexcell machine. The RNA was subsequently extracted from these chondrocytes and the alterations in gene expression determined using the Affymetrix Gene Array machine. Results: Intended as an in vitro model for Osteoarthritis, it was found that mechanical stimulation of human chondrocytes caused a significant alteration in the expression of a number of classes of compounds. These included enzymes, inflammatory mediators and structural proteins. Conclusions: This study identified several interesting candidate genes whose expression was significantly altered after being exposed to a laboratory model for osteoarthrosis. Further study of these genes and their expression may lead to important clinical applications

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: Establishing pathogenic mechanisms that are important for OA progression would support development of therapies to delay arthoplasty and extend the life of the joint. The aim of this study was to define a human model system for comparing minimal and advanced OA cartilage at the tissue, cellular, and molecular level. Method: Cartilage was isolated from femoral condyles of patients undergoing knee arthroplasty, with advanced OA cartilage obtained from areas within 1cm of overt lesions, and minimal OA cartilage taken from areas with no obvious surface defects. Representative histological sections were scored for disease severity based on four categories: fibrillation, chondrocyte cloning, matrix depletion and cellularity using Bioquant Nova v5.00.8 software. The proteoglycan and hydroxyproline content of the cartilage was determined by biochemical analysis. Following RNA isolation and reverse transcription, the cDNA was analysed for relative gene expression using real-time PCR. Gene expression patterns were compared on a patient-matched basis. Results: Histological analysis showed that the advanced OA cartilage differed from the minimal cartilage with regard to cloning (p< 0.001), fibrillation (p< 0.001), and proteoglycan depletion (p< 0.001). There was no difference in overall cellularity. The advanced OA cartilage had significantly less proteoglycan content than the minimal tissue, with no difference found in hydroxyproline content. The following changes were observed in the relative expression level of specific genes: 1) the steady state level of osteopontin mRNA showed an overall 3.5-fold increase in advanced OA cartilage compared to minimal (p< 0.01); 2) The mRNA coding for aggre-can was down-regulated in advanced disease tissue to less than 50% the level found in minimal tissue in nine out of 11 patients; 3) the expression of mRNA coding for link protein was also significantly decreased in advanced OA cartilage compared to minimal in nine out of 11 patients; and 4) the mRNA level coding for collagen II did not show an obvious pattern of expression in the minimal versus advanced cartilage. The expression of mRNA coding for MMPs was variable with regard to disease state with the majority of patients showing decreased MMP3, MMP9, and MMP13 mRNA expression in advanced OA tissue compared to minimal. Conclusions: This study clearly demonstrates that patient-matched minimal and advanced OA cartilage show significant differences in cell and matrix characteristics. In addition, differential patterns of gene expression are observed in this model that may relate to the pathogenic mechanism operating during progression of OA

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

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

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

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

Aim: Collagen meniscus implant (CMI) is a tissue engineering technique for the management of irreparable meniscal lesions. In this study we evaluate morphological and biochemical changes occurring in CMI after implantation, in order to better define tissue ingrowth inside the scaffold. Gene expression technique was also adopted to characterize the phenotype of the invading cells. Methods and materials: Morphological analysis was performed by light microscopy, immunohistochemistry (type I and II collagen), SEM and TEM on 5 biopsy specimens, harvested from 5 different patients (range, 6 to 16 months after surgery). Biochemical evaluation was carried out using Flurophore Assisted Carbohydrate Electrophoresis (FACE): this assay allowed to measure glycosaminoglycans (GAG) production in extracellular matrix of 2 biopsy specimens, harvested respectively 6 and 16 months after implantation. Real Time PCR was performed on the same 2 biopsy samples for detecting tissue-specific gene expression (collagen); RNAaseP gene expression was used as housekeeping gene. All these investigations were also applied on non implanted scaffolds for comparison. Results: Scaffold sections appeared composed by parallel connective laminae of 10-30B5m, connected by smaller (5-10B5m) connective bundles, surrounding elongated lacunae of 40-60B5m in diameter. In the biopsies specimens, the lacunae were filled by connective tissue with newly formed vessels and fibroblast-like cells. In the extracellular matrix, the collagen fibrils showed uniform diameters. The original structure of CMI was still recognizable and no inflammatory cells were detected inside the implant. A more organized architecture of the fibrillar network was evident in specimens with longer follow-up. Immunohistochemistry revealed exclusively type I collagen in the scaffold, while type II collagen appeared and was predominant in the biopsies specimens. FACE analysis carried out in the scaffold did not detect any GAG disaccharides. Conversely, high amount of disaccharides (unsulphated chondroitin, 4 and 6 sulphated chondroitin) were detected, together with hyaluronan, in the implants. Real Time PCR showed signal for Collagen type I alpha 1 and no signal for Collagen type II alpha 1. In the scaffolds used for comparison, no gene expression was recorded. Conclusions: The morphological findings of this study demonstrate that CMI acts as a biocompatible scaffold which provide a three-dimensional structure available for colonization by connective cells and vessels. Biochemical data are consistent with an active and specific production of extracellular matrix in the scaffold after implantation. The absence of signal for type II collagen gene in biopsies specimens can be attributed to different maturation stages of the ingrowing tissue

Destruction of articular cartilage in osteoarthritis (OA) is mediated by proteases and cytokines, which are silenced by epigenetic mechanisms in normal chondrocytes, but aberrantly expressed in OA. This is associated with DNA de-methylation of specific CpGs in the promoter regions (. Arthritis Rheum. , . 2005. ; . 52. :. 3110. –24. ). A widely used in vitro model to study the transcriptional regulation in OA is treating monolayer cultures of normal articular chondrocytes with inflammatory cytokines (IL-1b, TNFa or oncostatin M (OSM)) and investigating gene expression after 8–24 hours. The cytokines up-regulate catabolic, but down-regulate chondrocytic genes. However, whether this up- or down regulation is maintained after cytokine withdrawal is rarely investigated. In OA, the expression of catabolic genes is maintained in absence of cytokines and is transmitted to daughter cells, suggesting that epigenetic changes have resulted in permanent up-regulation. We asked whether it is possible to reproduce the epigenetic changes in vitro. Hence we compared gene expression and DNA methylation status in short-term (24h) versus long-term (2–3 weeks) cultures and, in particular, investigated the effects of cytokine withdrawal on these parameters. Healthy chondrocytes, harvested from human femoral heads after hemiarthroplasty, were cultured in monolayer and passaged once (P1). For short-term culture, the P1 chondrocytes were divided into control culture or cultures with one-shot of IL-1b/OSM, harvested after 24h and 72h. For long-term culture, the cells were cultured with or without IL-1b/OSM, the latter added twice a week. Half the cells were harvested at confluence (3 weeks) and the other halves were passaged again and cultured without cytokines until confluence (2–3 weeks). RNA and genomic DNA were extracted from the same sample. IL-1b, MMP-3, MMP-13 and COL2A1 expression was quantified by real-time PCR. The percentage of cells with DNA methylation at the CpG site at −299bp of IL-1b promoter (a key CpG site) was quantified by a method we reported previously (. Epigenetics. , . 2007. ; . 2. : . 86. –95. ). As expected, expression of IL-1b MMP-3, MMP-13 had increased 100–4500-fold 24h after IL-1b/OSM treatment, but decreased considerably after cytokine withdrawal. COL2A1 expression was virtually abolished by IL-1b/OSM and not regained after 72h. The % DNA methylation did not change during the 72h. Repeated treatment with IL-1b/OSM in long-term culture also increased expression of IL-1b and the MMPs. However, this time expression was maintained or even increased after cytokine withdrawal and passaging. Expression inversely correlated with DNA methylation, which dropped from 59% to 35%. This de-methylation was preserved after passaging and cytokine withdrawal. Conclusion: The widely used short-term cytokine-treated monolayer cultures of articular chondrocytes do not approximate the in vivo situation, where long-term aberrant expression correlates with DNA de-methylation. However, long-term treatment can mimic the loss of DNA methylation, which results in increased gene expression that is maintained after cytokine withdrawal. This model will facilitate studies on the mechanisms of DNA de-methylation, which might ultimately lead to novel therapeutic approaches for the treatment of OA

Joint instability was induced by posterior cruciate ligament (PCL) transection. This resulted in significant changes in medial collateral ligament (MCL) gene expression as early as three days after injury that persisted as long as 6 weeks. We noted substantial changes in expression of matrix-metalloproteinases (MMPs) −1, −3 and -13, with reciprocal effects on their specific inhibitors TIMP-1 and −3. Sustained changes in expression of these angiogenesis-associated matrix-degrading enzymes likely account for the observed degradation of the mechanical properties of secondary stabilizers in chronically unstable joints. To determine changes in gene-expression induced by traumatic instability. Instability activates aberrant expression of angiogenesis-associated matrix metalloproteinases. PCL transection induces a significant increase in the expression of MMP-3 and decrease in its specific inhibitor TIMP-3 with opposite effects on MMP-1 and TIMP-1 as early as three days after injury. Understanding the changes in gene expression induced by instability may lead to specific treatments that could prevent the “collateral damage” to secondary stabilizing structures. Under anaesthesia, four cohorts of six adult rabbits underwent surgical transection of the PCL. Three days, and two, six and sixteen weeks later, the MCL was harvested and the relative expression of TGF-β, MMP-1, -3, and −13, and their tissues inhibitors, and urokinase-type plasminogen activator (uPA) was measured using semi-quantitative RT-PCR. Previous work revealed increased in blood flow by two weeks and increased vascular volume by six weeks in the MCL of PCL-deficient joints. These changes are preceded by substantial changes in expression of mRNA for matrix degradation enzymes involved in the early stages of angiogenesis. This aberrant expression of matrix metalloproteinases likely accounts for the progressive degradation of the mechanical properties of secondary stabilizing structures seen in chronic instability. Funding: This work was supported by funding from the CIHR and the Alberta Heritage Foundation for Medical Research. Please contact author for figures and/or graphs