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

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

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

Objectives. The molecular mechanism of rheumatoid arthritis (RA) remains elusive. We conducted a protein-protein interaction network-based integrative analysis of genome-wide association studies (GWAS) and gene expression profiles of RA. Methods. We first performed a dense search of RA-associated gene modules by integrating a large GWAS meta-analysis dataset (containing 5539 RA patients and 20 169 healthy controls), protein interaction network and gene expression profiles of RA synovium and peripheral blood mononuclear cells (PBMCs). Gene ontology (GO) enrichment analysis was conducted by DAVID. The protein association networks of gene modules were generated by STRING. Results. For RA synovium, the top-ranked gene module is HLA-A, containing TAP2, HLA-A, HLA-C, TAPBP and LILRB1 genes. For RA PBMCs, the top-ranked gene module is GRB7, consisting of HLA-DRB5, HLA-DRA, GRB7, CD63 and KIT genes. Functional enrichment analysis identified three significant GO terms for RA synovium, including antigen processing and presentation of peptide antigen via major histocompatibility complex class I (false discovery rate (FDR) = 4.86 × 10 – 4), antigen processing and presentation of peptide antigen (FDR = 2.33 × 10 – 3) and eukaryotic translation initiation factor 4F complex (FDR = 2.52 × 10 – 2). Conclusion. This study reported several RA-associated gene modules and their functional association networks. Cite this article: X. Xiao, J. Hao, Y. Wen, W. Wang, X. Guo, F. Zhang. Genome-wide association studies and gene expression profiles of rheumatoid arthritis: an analysis. Bone Joint Res 2016;5:314–319. DOI: 10.1302/2046-3758.57.2000502

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

Objectives. Adult mice lacking the transcription factor NFAT1 exhibit osteoarthritis (OA). The precise molecular mechanism for NFAT1 deficiency-induced osteoarthritic cartilage degradation remains to be clarified. This study aimed to investigate if NFAT1 protects articular cartilage (AC) against OA by directly regulating the transcription of specific catabolic and anabolic genes in articular chondrocytes. Methods. Through a combined approach of gene expression analysis and web-based searching of NFAT1 binding sequences, 25 candidate target genes that displayed aberrant expression in Nfat1. -/-. AC at the initiation stage of OA, and possessed at least four NFAT1 binding sites in the promoter of each gene, were selected and tested for NFAT1 transcriptional activities by chromatin immunoprecipitation (ChIP) and promoter luciferase reporter assays using chondrocytes isolated from the AC of three- to four-month-old wild-type mice or Nfat1. -/-. mice with early OA phenotype. Results. Chromatin immunoprecipitation assays revealed that NFAT1 bound directly to the promoter of 21 of the 25 tested genes encoding cartilage-matrix proteins, growth factors, inflammatory cytokines, matrix-degrading proteinases, and specific transcription factors. Promoter luciferase reporter assays of representative anabolic and catabolic genes demonstrated that NFAT1-DNA binding functionally regulated the luciferase activity of specific target genes in wild-type chondrocytes, but not in Nfat1. -/-. chondrocytes or in wild-type chondrocytes transfected with plasmids containing mutated NFAT1 binding sequences. Conclusion. NFAT1 protects AC against degradation by directly regulating the transcription of target genes in articular chondrocytes. NFAT1 deficiency causes defective transcription of specific anabolic and catabolic genes in articular chondrocytes, leading to increased matrix catabolism and osteoarthritic cartilage degradation. Cite this article: M. Zhang, Q. Lu, T. Budden, J. Wang. NFAT1 protects articular cartilage against osteoarthritic degradation by directly regulating transcription of specific anabolic and catabolic genes. Bone Joint Res 2019;8:90–100. DOI: 10.1302/2046-3758.82.BJR-2018-0114.R1

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

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

Objectives. Several genome-wide association studies (GWAS) of bone mineral density (BMD) have successfully identified multiple susceptibility genes, yet isolated susceptibility genes are often difficult to interpret biologically. The aim of this study was to unravel the genetic background of BMD at pathway level, by integrating BMD GWAS data with genome-wide expression quantitative trait loci (eQTLs) and methylation quantitative trait loci (meQTLs) data. Method. We employed the GWAS datasets of BMD from the Genetic Factors for Osteoporosis Consortium (GEFOS), analysing patients’ BMD. The areas studied included 32 735 femoral necks, 28 498 lumbar spines, and 8143 forearms. Genome-wide eQTLs (containing 923 021 eQTLs) and meQTLs (containing 683 152 unique methylation sites with local meQTLs) data sets were collected from recently published studies. Gene scores were first calculated by summary data-based Mendelian randomisation (SMR) software and meQTL-aligned GWAS results. Gene set enrichment analysis (GSEA) was then applied to identify BMD-associated gene sets with a predefined significance level of 0.05. Results. We identified multiple gene sets associated with BMD in one or more regions, including relevant known biological gene sets such as the Reactome Circadian Clock (GSEA p-value = 1.0 × 10. -4. for LS and 2.7 × 10. -2. for femoral necks BMD in eQTLs-based GSEA) and insulin-like growth factor receptor binding (GSEA p-value = 5.0 × 10. -4. for femoral necks and 2.6 × 10. -2. for lumbar spines BMD in meQTLs-based GSEA). Conclusion. Our results provided novel clues for subsequent functional analysis of bone metabolism, and illustrated the benefit of integrating eQTLs and meQTLs data into pathway association analysis for genetic studies of complex human diseases. Cite this article: W. Wang, S. Huang, W. Hou, Y. Liu, Q. Fan, A. He, Y. Wen, J. Hao, X. Guo, F. Zhang. Integrative analysis of GWAS, eQTLs and meQTLs data suggests that multiple gene sets are associated with bone mineral density. Bone Joint Res 2017;6:572–576

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

Objectives. Effects of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression of genes involved in the proliferation and differentiation of osteoblasts in culture were analysed. The best sequence of growth factor addition that induces expansion of cells before their differentiation was sought. Methods. Primary human osteoblasts in in vitro culture were treated with IGF1, BMP2 or FGF2 (10 ng/ml) for 24 hours (IGF1) or 48 hours (BMP2 and FGF2). Experiments were performed during the exponential growth phase with approximately 1e7 cells per 75 cm. 2. flask. mRNA was reverse transcribed directly and analysed using RT-PCR Taqman assays. Expression levels of key genes involved in cell growth and differentiation (CDH11, TNFRSF11B, RUNX2, POSTN, ALP, WNT5A, LEF1, HSPA5, FOS, p21) were monitored using RT-PCR with gene-specific Taqman probes. . Results. Autocrine expression of BMP2 is stimulated by FGF2 and BMP2 itself. BMP2 and FGF2 act as proliferative factors as indicated by reduced expression of ALP and POSTN, whereas IGF1 exhibits a more subtle picture: the Wingless und Int-1 (Wnt) signalling pathway and the Smad pathway, but not p38 mitogen-activated protein (MAP) kinase signalling, were shown to be activated by IGF1, leading to proliferation and differentiation of the cells. . Conclusions. For future use of autologous bone cells in the management of bony defects, new treatment options take advantage of growth factors and differentiation factors. Thus, our results might help to guide the timely application of these factors for the expansion and subsequent differentiation of osteoblastic cells in culture. Cite this article: Bone Joint Res 2014;3:236–40

We stably transfected early passage chondrocytes with an anti-apoptotic Bcl-2 gene in vitro using a retrovirus vector. Samples of articular cartilage were obtained from 11 patients with a mean age of 69 years (61 to 75) who were undergoing total knee replacement for osteoarthritis. The Bcl-2-gene-transfected chondrocytes were compared with non-transfected and lac-Z-gene-transfected chondrocytes, both of which were used as controls. All three groups of cultured chondrocytes were incubated with nitric oxide (NO) for ten days. Using the Trypan Blue exclusion assay, an enzyme-linked immunosorbent assay and flow cytometric analysis, we found that the number of apoptotic chondrocytes was significantly higher in the non-transfected and lac-Z-transfected groups than in the Bcl-2-transfected group (p < 0.05). The Bcl-2-transfected chondrocytes were protected from NO-induced impairment of proteoglycan synthesis. We conclude that NO-induced chondrocyte death involves a mechanism which appears to be subject to regulation by an anti-apoptotic Bcl-2 gene. Therefore, Bcl-2 gene therapy may prove to be of therapeutic value in protecting human articular chondrocytes

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

Summary Statement. Differential expression of canonical and noncanonical Wnt signalling along cartilage canals and osteochondral junctions is dependent on age. Increased gene expression of PTHrP along cartilage canals and Ihh along osteochondral junctions suggests paracrine feedback in articular-epiphyseal cartilage. Introduction. Wnt signaling has been shown to regulate chondrocyte differentiation during pre-/post-natal cartilage development. In addition, parathyroid-related peptide(PTHrP) and Indian hedgehog(Ihh) create a negative feedback loop in growth cartilage, but less is known in articular cartilage. The objective of this study was to elucidate expression of regulatory molecules in chondrocytes surrounding cartilage canals and osteochondral junctions during neonatal and pre-adolescent development. We hypothesised there would be increased expression of canonical Wnt signalling molecules and Ihh in osteochondral junction chondrocytes compared to cartilage canal chondrocytes. In addition, we hypothesised that Wnt signaling and PTHrP expression would be greater in neonates than pre-adolescents. Patients & Methods. Osteochondral samples were obtained(IACUC-approved) from normal femoropatellar joints of 14 euthanised immature horses(6 neonates, 8 pre-adolescents). Samples were frozen in OCT for laser capture microdissection(LCM) or fixed in 4% paraformaldehyde and paraffin-embedded for immunohistochemistry. Chondrocytes surrounding cartilage canals and osteochondral junctions were captured using LCM. Following RNA isolation, equine-specific β-catenin, Wnt-4, Wnt-5b, Wnt-11, Dickkopf-1(Dkk-1), low-density lipoprotein receptor-related protein-4,-6(Lrp4, Lrp6), Axin1, Wnt inhibitory factor-1(WIF)-1, secreted Frizzled-related protein-1,-3,-5(sFRP), retinoic acid receptor gamma(RARG), RAR-inducible serine carboxypeptidase(SC-PEP), Ihh, PTHrP, VEGF, PDGF, MMP-13, and 18S mRNA expression levels were evaluated by two-step real-time qPCR. Following immunohistochemistry using rabbit polyclonal or mouse monoclonal primary antibodies (confirmed by Western blot), spatial tissue protein expression was scored (0–3). Statistical analysis included Wilcoxon signed rank test(paired samples) or rank sum test(unpaired samples)(P<0.05). Results. Gene expression in chondrocytes along cartilage canals was significantly higher for PTHrP, β-catenin, Lrp6, Axin1, sFRP5, RARgamma, and SC-PEP than osteochondral junctions. Conversely, gene expression of Ihh, Wnt4, Wnt11, sFRP3, and VEGF were higher in osteochondral junction chondrocytes than cartilage canals. There was higher protein expression of β-catenin, PDGF, VEGF, and MMP-13 along osteochondral junctions than cartilage canals of pre-adolescents. Neonates had higher gene expression of PTHrP, Wnt-5b, sFRP3, Lrp6, and RARG in cartilage canal chondrocytes than pre-adolescents, while Ihh, Wnt-11, Lrp4, and Dkk1 were significantly higher in pre-adolescents. Immunostaining was higher for β-catenin and Wnt-11 in pre-adolescent osteochondral junction cartilage than neonates. No differences were found between age groups for Wnt-4 immunostaining. Dkk1 protein expression was significantly higher in the middle cartilage layer of pre-adolescents than neonates. Immunostaining was greater for Ihh and PTHrP in the deep cartilage layer of pre-adolescents than neonates. PDGF, VEGF, and MMP13 protein expression was higher in the superficial cartilage layer of pre-adolescents than neonates. Discussion. Wnt/β-catenin and Ihh/PTHrP signaling regulate cartilage differentiation during development and are important in endochondral ossification. This study revealed cell-specific, age-related differences in gene/protein expression of both regulatory pathways. Cells surrounding cartilage canals typically appeared small/rounded compared to larger chondrocytes along osteochondral junctions, likely due to different developmental stages. Higher PTHrP gene expression along cartilage canals and Ihh expression along osteochondral junctions may reflect these stages, suggesting paracrine feedback in articular-epiphyseal cartilage. β-catenin signaling may induce chondrocyte hypertrophy, potentially by enhancing Ihh and MMP-13 expression. Differential expression of canonical(β-catenin, Wnt-4, Lrp4, Lrp6) and noncanonical Wnt signalling(Wnt-5b, Wnt-11) and Wnt inhibitors (Dkk1, Axin1, sFRP3, sFRP5, Wif-1) surrounding cartilage canals and osteochondral junctions provides evidence of age-related interactions during postnatal development

Summary. Shear stress and hydrostatic effects on the hMSCs early mechano gene response were similar. For the same magnitude gene response, the hydrostatic compression (1.5×10. 5. Pascal) is a 200000 times greater than the force exerted by shear stress (0.7 Pascal). Introduction. In the lab, a perfusion bioreactor designed to automate the production of bone constructs was developed. The proof of concept was established in a large animal model of clinical relevance. The cells perfused in the bioreactor are likely to perceive 2 types of stresses: shear stress and hydrostatic pressure. Optimization of this bioreactor implies a better understanding of the effects of these forces on the cells in order to have better proliferation and differentiation. An understanding of the response of one cell layer submit to various strength is relevant. The primary objective of this study was to test the hypothesis that hMSCs have the fundamental ability to distinguish between different types of mechanical signals as evidenced by distinct gene expression. The effect of shear stress on one cell layer cultures of hMSCs will be evaluated using a commercially available system called Ibidi. For the hydrostatic pressure as there is no commercial device available, our group has developed a prototype capable of delivering a well-defined mechanical loading to cells in culture. Validation of the techniques: In order to validate the systems (shear stress and cyclic pressure apparatus) used in this study, we have used an osteocytes-like cell line, MLO-Y4. When stimulated by a 30 minutes PFF at 7 dyn/cm. 2. or hydrostatic compression at 1.5 bar, cells responded by producing NO in the culture media. NO release after mechanical stimulation of hMSCs: hMSCs were subjected to increased PFF (7 to 42 dyn/cm. 2. ) for 30 minutes. This stimulation resulted in an increased release of NO in the media compared to non-stimulated cells (p<0.05). Interestingly the level of NO was maximal at 7 dyn/cm. 2. and decreased with higher flow rate. Similar observation was made after hMSCs stimulation by hydrostatic pressure for 30 minutes: a peak of NO release at 1.5 bar was observed. Early gene expression of known mechano-sensitive genes: Gene expression analysis immediately after stimulation (PFF or hydrostatic compression) was performed on a range of known mechano-sensitive genes: NOS2, PTGS2, PTGES, IER3 and EGR1. Immediately after stimulation by PFF at 7 dyn/cm. 2. or hydrostatic pressure at 1.5 bars, the expression of all the genes of interest appear to be up regulated in stimulated cells. Conclusions. In the present study, hMSCs cells responses to two different treatments, shear stress and hydrostatic pressure, were monitored in parallel to study the difference in sensitivity to both mechanical stresses. Both systems used ensure a stable and reproducible strain condition in a well-controlled environment. We demonstrated that the shear stress and the hydrostatic pressure effects on the hMSCs were similar. We note that, for the same magnitude response, force exerted by the hydrostatic compression (1.5×10. 5. Pascal) on the cells is a 200000 times greater than the force exerted by shear stress (0.7 Pascal). This shows that the direct effect of hydrostatic compression on the hMSCs is negligible compared to the shear stress

Objectives. Degenerative disc disease (DDD) and osteoarthritis (OA) are relatively frequent causes of disability amongst the elderly; they constitute serious socioeconomic costs and significantly impair quality of life. Previous studies to date have found that aggrecan variable number of tandem repeats (VNTR) contributes both to DDD and OA. However, current data are not consistent across studies. The purpose of this study was to evaluate systematically the relationship between aggrecan VNTR, and DDD and/or OA. Methods. This study used a highly sensitive search strategy to identify all published studies related to the relationship between aggrecan VNTR and both DDD and OA in multiple databases from January 1996 to December 2016. All identified studies were systematically evaluated using specific inclusion and exclusion criteria. Cochrane methodology was also applied to the results of this study. Results. The final selection of seven studies was comprehensively evaluated and includes results for 2928 alleles. The most frequent allele among all the studies was allele 27. After comparing the distributions of each allele with others, statistically significant differences have been found in the distribution of the alleles by the two groups, with an over-representation of allele (A)21 (disease: 3.22%, control: 0.44%). Thus, carrying A21 increased the risk of DDD. Such an association was not found to be statistically significant when considering the risk of OA. Conclusions. The findings suggest that VNTR A21 seems to be associated with higher risk to DDD, however, such an association may not be statistically significant regarding the risk of OA. Cite this article: L. Cong, G. Tu, D. Liang. A systematic review of the relationship between the distributions of aggrecan gene VNTR polymorphism and degenerative disc disease/osteoarthritis. Bone Joint Res 2018;7:308–317. DOI: 10.1302/2046-3758.74.BJR-2017-0207.R1

The most common reasons for total joint arthroplasty (TJA) failure are aseptic loosening (AL) and prosthetic joint infection (PJI). There is a big clinical challenge to identify the patients with high risk of AL/PJI before the TJA surgery. Although there is evidence that genetic factors contribute to the individual susceptibility to AL/PJI, a predictive model for identification of patients with a high genetic risk of TJA failure has not been developed yet. We aimed to develop a risk evaluation tool utilising the AL/PJI-associated polymorphisms for identification of patients with high genetic risk of TJA failure based on inflammation-gene polymorphism panel. Based on allele and genotype frequencies of twenty-five single nucleotide polymorphisms (SNPs) in TNF, IL2, IL6, IL10, IL1b, IL-1Ra, MBL2, MMP1, FTO genes and those influencing the serum levels of biomarkers of TJA outcomes (IL6, CCL2/MCP-1, CRP, ESR) in peripheral blood obtained from patients with TJA (AL, n=110; PJI, n=93; no complications, n=123), we calculated a hazard ratio and a relative entropy of alleles and genotypes associated with AL and PJI and their combinations in patient subgroups. We conducted a risk evaluation tool based on the presence of risk alleles and genotypes in TNF (rs361525, rs1800629), DARC (rs12075), MBL2 (rs11003125) and FTO (rs9939609, rs9930506) genes associated with implant failure (AL/PJI). Of these, FTO gene variations (rs9939609, rs9930506) were associated mainly with PJI (P=0.001, OR=2.04, 95%CI=1.132–2.603; P=0.011, OR=1.72, 95%CI=1.338–3.096) and DARC (rs12075) with AL (P=0.005, OR=1.79, 95%CI=1.193–2.696). This tool calculates a hazard ratio of a combination of SNPs associated with AL and PJI for identification of patients with high and low risk of AL/PJI TJA failure. We proposed a risk evaluation tool for stratification of patients before the TJA surgery based on the genetic risk of AL/PJI development. The effect size for each genotype combination described in the study is small. Further multiparametric data analysis and studies on an extended patient cohort and other non-genetic and genetic parameters are ongoing. Grant support: AZV MZ CR VES16-131852A, VES15-27726A, IGA LF UP_2016_011

Introduction. Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily of growth factors and are known to regulate proliferation and expression of the differentiated phenotype of chondrocytes, osteoblasts, and osteoclasts. To investigate the osteoblastic differentiation gene expressions that contribute to BMP-7 dependent ostogenesis, we performed gene expression profiling of BMP-7-treated mouse bone marrow stromal cells. Methods. D1 cells (mouse bone marrow stromal cells) were cultured in osteogenic differentiation medium (ODM) for 3 days, and then treated with BMP-7 for 24 hr. Total RNA was extracted using Trizol, purified using RNeasy columns. Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit to yield biotinylated cRNA. The data analysis up- and down-regulation developmental processes (anterior/posterior patterning, ectoderm development, embryogenesis, gametogenesis, mesoderm development, other development process, and segment specification) genes expression fold. Results. We detected 18 mRNAs (Id2, Igf2, Pparg, S100a10, Foxn3, Tulp3, Mycbp2, Notch3, Ptk7, Lrp4, Tnfrsf11b, Ogn, Cyr61, Mglap, Akp2, Ltbp4, Ibsp, and Thbs1) that were differentially up-regulated after BMP-7 stimulation. 3 mRNAs (Wars, Adss and Trim35) were differentially down-regulated after BMP-7 stimulation. Conclusion. The data indicate that BMP-7 regulate various developmental processes genes expression during osteoblastic differentiation. Though further studies are needed in relation to each expression gene profiles and osteoblastic differentiation, this information may serve as a point of comparison for osteoblastic differentiation of BMP-7. Furthermore, the data should facilitate the informed use of BMP-7 as a therapeutic agent and tissue engineering tool. Acknowledgement. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R01-2008-000-10089-0)

We have examined whether primary human muscle-derived cells can be used in ex vivo gene therapy to deliver BMP-2 and to produce bone in vivo. Two in vitro experiments and one in vivo experiment were used to determine the osteocompetence and BMP-2 secretion capacity of cells isolated from human skeletal muscle. We isolated five different populations of primary muscle cells from human skeletal muscle in three patients. In the first in vitro experiment, production of alkaline phosphatase by the cells in response to stimulation by rhBMP-2 was measured and used as an indicator of cellular osteocompetence. In the second, secretion of BMP-2 was measured after the cell populations had been transduced by an adenovirus encoding for BMP-2. In the in vivo experiment, the cells were cotransduced with a retrovirus encoding for a nuclear localised β-galactosidase gene and an adenovirus encoding for BMP-2. The cotransduced cells were then injected into the hind limbs of severe combined immune-deficient (SCID) mice and analysed radiographically and histologically. The nuclear localised β-galactosidase gene allowed identification of the injected cells in histological specimens. In the first in vitro experiment, the five different cell populations all responded to in vitro stimulation of rhBMP-2 by producing higher levels of alkaline phosphatase when compared with non-stimulated cells. In the second, the five different cell populations were all successfully transduced by an adenovirus to express and secrete BMP-2. The cells secreted between 444 and 2551 ng of BMP-2 over three days. In the in vivo experiment, injection of the transduced cells into the hind-limb musculature of SCID mice resulted in the formation of ectopic bone at 1, 2, 3 and 4 weeks after injection. Retroviral labelling of the cell nuclei showed labelled human muscle-derived cells occupying locations of osteoblasts in the ectopic bone, further supporting their osteocompetence. Cells from human skeletal muscle, because of their availability to orthopaedic surgeons, their osteocompetence, and their ability to express BMP-2 after genetic engineering, are an attractive cell population for use in BMP-2 gene therapy approaches