Aims. Knee osteoarthritis (OA) involves a variety of tissues in the joint. Gene expression profiles in different tissues are of great importance in order to understand OA. Methods. First, we obtained gene expression profiles of cartilage, synovium, subchondral bone, and meniscus from the Gene Expression Omnibus (GEO). Several datasets were standardized by merging and removing batch effects. Then, we used unsupervised clustering to divide OA into three subtypes. The gene ontology and pathway enrichment of three subtypes were analyzed. CIBERSORT was used to evaluate the infiltration of immune cells in different subtypes. Finally, OA-related genes were obtained from the Molecular Signatures Database for validation, and diagnostic markers were screened according to clinical characteristics. Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) was used to verify the effectiveness of markers. Results. C1 subtype is mainly concentrated in the development of skeletal muscle organs, C2 lies in metabolic process and immune response, and C3 in pyroptosis and cell death process. Therefore, we divided OA into three subtypes: bone remodelling subtype (C1), immune metabolism subtype (C2), and cartilage degradation subtype (C3). The number of macrophage M0 and activated mast cells of C2 subtype was significantly higher than those of the other two subtypes. COL2A1 has significant differences in different subtypes. The expression of COL2A1 is related to age, and trafficking protein particle complex subunit 2 is related to the sex of OA patients. Conclusion. This study linked different tissues with gene expression profiles, revealing different molecular subtypes of patients with knee OA. The relationship between clinical characteristics and OA-related genes was also studied, which provides a new concept for the diagnosis and treatment of OA. Cite this article: Bone Joint Res 2023;12(12):702–711

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

Aims. Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully understood. In this study, we aimed to characterize the local changes in gene expression (GE) associated with diabetic fracture. We used an unbiased approach to compare GE in the fracture callus of Zucker diabetic fatty (ZDF) rats relative to wild-type (WT) littermates at three weeks following femoral osteotomy. Methods. Zucker rats, WT and homozygous for leptin receptor mutation (ZDF), were fed a moderately high-fat diet to induce T2DM only in the ZDF animals. At ten weeks of age, open femoral fractures were simulated using a unilateral osteotomy stabilized with an external fixator. At three weeks post-surgery, the fractured femur from each animal was retrieved for analysis. Callus formation and the extent of healing were assessed by radiograph and histology. Bone tissue was processed for total RNA extraction and messenger RNA (mRNA) sequencing (mRNA-Seq). Results. Radiographs and histology demonstrated impaired fracture healing in ZDF rats with incomplete bony bridge formation and an influx of intramedullary inflammatory tissue. In comparison, near-complete bridging between cortices was observed in Sham WT animals. Of 13,160 genes, mRNA-Seq analysis identified 13 that were differentially expressed in ZDF rat callus, using a false discovery rate (FDR) threshold of 10%. Seven genes were upregulated with high confidence (FDR = 0.05) in ZDF fracture callus, most with known roles in inflammation. Conclusion. These findings suggest that elevated or prolonged inflammation contributes to delayed fracture healing in T2DM. The identified genes may be used as biomarkers to monitor and treat delayed fracture healing in diabetic patients. Cite this article: Bone Joint Res 2023;12(10):657–666

Objectives. This study aimed to investigate time-dependent gene expression of injured human anterior cruciate ligament (ACL), and to evaluate the histological changes of the ACL remnant in terms of cellular characterisation. Methods. Injured human ACL tissues were harvested from 105 patients undergoing primary ACL reconstruction and divided into four phases based on the period from injury to surgery. Phase I was < three weeks, phase II was three to eight weeks, phase III was eight to 20 weeks, and phase IV was ≥ 21 weeks. Gene expressions of these tissues were analysed in each phase by quantitative real-time polymerase chain reaction using selected markers (collagen types 1 and 3, biglycan, decorin, α-smooth muscle actin, IL-6, TGF-β1, MMP-1, MMP-2 and TIMP-1). Immunohistochemical staining was also performed using primary antibodies against CD68, CD55, Stat3 and phosphorylated-Stat3 (P-Stat3). . Results. Expression of IL-6 was mainly seen in phases I, II and III, collagen type 1 in phase II, MMP-1, 2 in phase III, and decorin, TGF-β1 and α-smooth muscle actin in phase IV. Histologically, degradation and scar formation were seen in the ACL remnant after phase III. The numbers of CD55 and P-Stat3 positive cells were elevated from phase II to phase III. . Conclusions. Elevated cell numbers including P-Stat3 positive cells were not related to collagens but to MMPs’ expressions

Aims. Degenerative cervical spondylosis (DCS) is a common musculoskeletal disease that encompasses a wide range of progressive degenerative changes and affects all components of the cervical spine. DCS imposes very large social and economic burdens. However, its genetic basis remains elusive. Methods. Predicted whole-blood and skeletal muscle gene expression and genome-wide association study (GWAS) data from a DCS database were integrated, and functional summary-based imputation (FUSION) software was used on the integrated data. A transcriptome-wide association study (TWAS) was conducted using FUSION software to assess the association between predicted gene expression and DCS risk. The TWAS-identified genes were verified via comparison with differentially expressed genes (DEGs) in DCS RNA expression profiles in the Gene Expression Omnibus (GEO) (Accession Number: GSE153761). The Functional Mapping and Annotation (FUMA) tool for genome-wide association studies and Meta tools were used for gene functional enrichment and annotation analysis. Results. The TWAS detected 420 DCS genes with p < 0.05 in skeletal muscle, such as ribosomal protein S15A (RPS15A) (PTWAS = 0.001), and 110 genes in whole blood, such as selectin L (SELL) (PTWAS = 0.001). Comparison with the DCS RNA expression profile identified 12 common genes, including Apelin Receptor (APLNR) (PTWAS = 0.001, PDEG = 0.025). In total, 148 DCS-enriched Gene Ontology (GO) terms were identified, such as mast cell degranulation (GO:0043303); 15 DCS-enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified, such as the sphingolipid signalling pathway (ko04071). Nine terms, such as degradation of the extracellular matrix (R-HSA-1474228), were common to the TWAS enrichment results and the RNA expression profile. Conclusion. Our results identify putative susceptibility genes; these findings provide new ideas for exploration of the genetic mechanism of DCS development and new targets for preclinical intervention and clinical treatment. Cite this article: Bone Joint Res 2023;12(1):80–90

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.

Aims. This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD). Methods. The gene expression profile of GSE23130 was downloaded from the Gene Expression Omnibus (GEO) database. Extracellular protein-differentially expressed genes (EP-DEGs) were screened by protein annotation databases, and we used Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) to analyze the functions and pathways of EP-DEGs. STRING and Cytoscape were used to construct protein-protein interaction (PPI) networks and identify hub EP-DEGs. NetworkAnalyst was used to analyze transcription factors (TFs) and microRNAs (miRNAs) that regulate hub EP-DEGs. A search of the Drug Signatures Database (DSigDB) for hub EP-DEGs revealed multiple drug molecules and drug-target interactions. Results. A total of 56 EP-DEGs were identified in the differential expression analysis. EP-DEGs were enriched in the extracellular structure organization, ageing, collagen-activated signalling pathway, PI3K-Akt signalling pathway, and AGE-RAGE signalling pathway. PPI network analysis showed that the top ten hub EP-DEGs are closely related to IDD. Correlation analysis also demonstrated a significant correlation between the ten hub EP-DEGs (p＜0.05), which were selected to construct TF–gene interaction and TF–miRNA coregulatory networks. In addition, ten candidate drugs were screened for the treatment of IDD. Conclusion. The findings clarify the roles of extracellular proteins in IDD and highlight their potential as promising novel therapeutic targets. Cite this article: Bone Joint Res 2023;12(9):522–535

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

Aims. This study aimed to explore the biological and clinical importance of dysregulated key genes in osteoarthritis (OA) patients at the cartilage level to find potential biomarkers and targets for diagnosing and treating OA. Methods. Six sets of gene expression profiles were obtained from the Gene Expression Omnibus database. Differential expression analysis, weighted gene coexpression network analysis (WGCNA), and multiple machine-learning algorithms were used to screen crucial genes in osteoarthritic cartilage, and genome enrichment and functional annotation analyses were used to decipher the related categories of gene function. Single-sample gene set enrichment analysis was performed to analyze immune cell infiltration. Correlation analysis was used to explore the relationship among the hub genes and immune cells, as well as markers related to articular cartilage degradation and bone mineralization. Results. A total of 46 genes were obtained from the intersection of significantly upregulated genes in osteoarthritic cartilage and the key module genes screened by WGCNA. Functional annotation analysis revealed that these genes were closely related to pathological responses associated with OA, such as inflammation and immunity. Four key dysregulated genes (cartilage acidic protein 1 (CRTAC1), iodothyronine deiodinase 2 (DIO2), angiopoietin-related protein 2 (ANGPTL2), and MAGE family member D1 (MAGED1)) were identified after using machine-learning algorithms. These genes had high diagnostic value in both the training cohort and external validation cohort (receiver operating characteristic > 0.8). The upregulated expression of these hub genes in osteoarthritic cartilage signified higher levels of immune infiltration as well as the expression of metalloproteinases and mineralization markers, suggesting harmful biological alterations and indicating that these hub genes play an important role in the pathogenesis of OA. A competing endogenous RNA network was constructed to reveal the underlying post-transcriptional regulatory mechanisms. Conclusion. The current study explores and validates a dysregulated key gene set in osteoarthritic cartilage that is capable of accurately diagnosing OA and characterizing the biological alterations in osteoarthritic cartilage; this may become a promising indicator in clinical decision-making. This study indicates that dysregulated key genes play an important role in the development and progression of OA, and may be potential therapeutic targets. Cite this article: Bone Joint Res 2024;13(2):66–82

Aims. This study aimed, through bioinformatics analysis, to identify the potential diagnostic markers of osteoarthritis, and analyze the role of immune infiltration in synovial tissue. Methods. The gene expression profiles were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified by R software. Functional enrichment analyses were performed and protein-protein interaction networks (PPI) were constructed. Then the hub genes were screened. Biomarkers with high value for the diagnosis of early osteoarthritis (OA) were validated by GEO datasets. Finally, the CIBERSORT algorithm was used to evaluate the immune infiltration between early-stage OA and end-stage OA, and the correlation between the diagnostic marker and infiltrating immune cells was analyzed. Results. A total of 88 DEGs were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that DEGs were significantly enriched in leucocyte migration and interleukin (IL)-17 signalling pathways. Disease ontology (DO) indicated that DEGs were mostly enriched in rheumatoid arthritis. Six hub genes including FosB proto-oncogene, AP-1 transcription factor subunit (FOSB); C-X-C motif chemokine ligand 2 (CXCL2); CXCL8; IL-6; Jun proto-oncogene, AP-1 transcription factor subunit (JUN); and Activating transcription factor 3 (ATF3) were identified and verified by GEO datasets. ATF3 (area under the curve = 0.975) turned out to be a potential biomarker for the diagnosis of early OA. Several infiltrating immune cells varied significantly between early-stage OA and end-stage OA, such as resting NK cells (p = 0.016), resting dendritic cells (p = 0.043), and plasma cells (p = 0.043). Additionally, ATF3 was significantly correlated with resting NK cells (p = 0.034), resting dendritic cells (p = 0.026), and regulatory T cells (Tregs, p = 0.018). Conclusion. ATF3 may be a potential diagnostic marker for early diagnosis and treatment of OA, and immune cell infiltration provides new perspectives for understanding the mechanism during OA progression. Cite this article: Bone Joint Res 2022;11(9):679–689

Aims. Lumbar spinal stenosis (LSS) is a common skeletal system disease that has been partly attributed to genetic variation. However, the correlation between genetic variation and pathological changes in LSS is insufficient, and it is difficult to provide a reference for the early diagnosis and treatment of the disease. Methods. We conducted a transcriptome-wide association study (TWAS) of spinal canal stenosis by integrating genome-wide association study summary statistics (including 661 cases and 178,065 controls) derived from Biobank Japan, and pre-computed gene expression weights of skeletal muscle and whole blood implemented in FUSION software. To verify the TWAS results, the candidate genes were furthered compared with messenger RNA (mRNA) expression profiles of LSS to screen for common genes. Finally, Metascape software was used to perform enrichment analysis of the candidate genes and common genes. Results. TWAS identified 295 genes with permutation p-values < 0.05 for skeletal muscle and 79 genes associated for the whole blood, such as RCHY1 (PTWAS = 0.001). Those genes were enriched in 112 gene ontology (GO) terms and five Kyoto Encyclopedia of Genes and Genomes pathways, such as ‘chemical carcinogenesis - reactive oxygen species’ (LogP value = −2.139). Further comparing the TWAS significant genes with the differentially expressed genes identified by mRNA expression profiles of LSS found 18 overlapped genes, such as interleukin 15 receptor subunit alpha (IL15RA) (PTWAS = 0.040, PmRNA = 0.010). Moreover, 71 common GO terms were detected for the enrichment results of TWAS and mRNA expression profiles, such as negative regulation of cell differentiation (LogP value = −2.811). Conclusion. This study revealed the genetic mechanism behind the pathological changes in LSS, and may provide novel insights for the early diagnosis and intervention of LSS. Cite this article: Bone Joint Res 2023;12(6):387–396

Aims. Osteoarthritis (OA) is a common degenerative joint disease worldwide, which is characterized by articular cartilage lesions. With more understanding of the disease, OA is considered to be a disorder of the whole joint. However, molecular communication within and between tissues during the disease process is still unclear. In this study, we used transcriptome data to reveal crosstalk between different tissues in OA. Methods. We used four groups of transcription profiles acquired from the Gene Expression Omnibus database, including articular cartilage, meniscus, synovium, and subchondral bone, to screen differentially expressed genes during OA. Potential crosstalk between tissues was depicted by ligand-receptor pairs. Results. During OA, there were 626, 97, 1,060, and 2,330 differentially expressed genes in articular cartilage, meniscus, synovium, and subchondral bone, respectively. Gene Ontology enrichment revealed that these genes were enriched in extracellular matrix and structure organization, ossification, neutrophil degranulation, and activation at different degrees. Through ligand-receptor pairing and proteome of OA synovial fluid, we predicted ligand-receptor interactions and constructed a crosstalk atlas of the whole joint. Several interactions were reproduced by transwell experiment in chondrocytes and synovial cells, including TNC-NT5E, TNC-SDC4, FN1-ITGA5, and FN1-NT5E. After lipopolysaccharide (LPS) or interleukin (IL)-1β stimulation, the ligand expression of chondrocytes and synovial cells was upregulated, and corresponding receptors of co-culture cells were also upregulated. Conclusion. Each tissue displayed a different expression pattern in transcriptome, demonstrating their specific roles in OA. We highlighted tissue molecular crosstalk through ligand-receptor pairs in OA pathophysiology, and generated a crosstalk atlas. Strategies to interfere with these candidate ligands and receptors may help to discover molecular targets for future OA therapy. Cite this article: Bone Joint Res 2022;11(12):862–872

Aims. The present study investigated receptor activator of nuclear factor kappa-Β ligand (RANKL), osteoprotegerin (OPG), and Runt-related transcription factor 2 (RUNX2) gene expressions in giant cell tumour of bone (GCTB) patients in relationship with tumour recurrence. We also aimed to investigate the influence of CpG methylation on the transcriptional levels of RANKL and OPG. Methods. A total of 32 GCTB tissue samples were analyzed, and the expression of RANKL, OPG, and RUNX2 was evaluated by quantitative polymerase chain reaction (qPCR). The methylation status of RANKL and OPG was also evaluated by quantitative methylation-specific polymerase chain reaction (qMSP). Results. We found that RANKL and RUNX2 gene expression was upregulated more in recurrent than in non-recurrent GCTB tissues, while OPG gene expression was downregulated more in recurrent than in non-recurrent GCTB tissues. Additionally, we proved that changes in DNA methylation contribute to upregulating the expression of RANKL and downregulating the expression of OPG, which are critical for bone homeostasis and GCTB development. Conclusion. Our results suggest that the overexpression of RANKL/RUNX2 and the lower expression of OPG are associated with recurrence in GCTB patients. Cite this article: Bone Joint Res 2024;13(2):84–91

Aims. To test the hypothesis that reseeded anterior cruciate ligament (ACL)-derived cells have a better ability to survive and integrate into tendon extracellular matrix (ECM) and accelerate the ligamentization process, compared to adipose-derived mesenchymal stem cells (ADMSCs). Methods. Acellularized tibialis allograft tendons were used. Tendons were randomly reseeded with ACL-derived cells or ADMSCs. ACL-derived cells were harvested and isolated from remnants of ruptured ACLs during reconstruction surgery and cultured at passage three. Cell suspensions (200 µl) containing 2 × 10. 6. ACL-derived cells or ADMSCs were prepared for the purpose of reseeding. At days 1, 3, and 7 post-reseeding, graft composites were assessed for repopulation with histological and immunohistochemical analysis. Matrix protein contents and gene expression levels were analyzed. Results. In the graft reseeded with ACL-derived cells, a large number of elongated cells that integrated into the matrix were evident at day 3 and day 7. However, in the graft reseeded with ADMSCs, only a small number of elongated cells were found integrated into the matrix. Immunofluorescence for Ki-67 and type I collagen confirmed the pronounced production of type I collagen by Ki-67-positive ACL-derived cells integrated into the ECM. A messenger RNA (mRNA) expression assay demonstrated significantly higher gene expression levels of types I (p = 0.013) and III (p = 0.050) collagen in the composites reseeded with ACL-derived cells than ADMSCs. Conclusion. ACL-derived cells, when reseeded to acellularized tendon graft, demonstrated earlier better survival and integration in the tendon ECM and resulted in higher gene expression levels of collagen, which may be essential to the normal ligamentization process compared to ADMSCs. Cite this article: Bone Joint Res 2022;11(11):777–786

Aims. Glucose-insulin-potassium (GIK) is protective following cardiac myocyte ischaemia-reperfusion (IR) injury, however the role of GIK in protecting skeletal muscle from IR injury has not been evaluated. Given the similar mechanisms by which cardiac and skeletal muscle sustain an IR injury, we hypothesized that GIK would similarly protect skeletal muscle viability. Methods. A total of 20 C57BL/6 male mice (10 control, 10 GIK) sustained a hindlimb IR injury using a 2.5-hour rubber band tourniquet. Immediately prior to tourniquet placement, a subcutaneous osmotic pump was placed which infused control mice with saline (0.9% sodium chloride) and treated mice with GIK (40% glucose, 50 U/l insulin, 80 mEq/L KCl, pH 4.5) at a rate of 16 µl/hr for 26.5 hours. At 24 hours following tourniquet removal, bilateral (tourniqueted and non-tourniqueted) gastrocnemius muscles were triphenyltetrazolium chloride (TTC)-stained to quantify percentage muscle viability. Bilateral peroneal muscles were used for gene expression analysis, serum creatinine and creatine kinase activity were measured, and a validated murine ethogram was used to quantify pain before euthanasia. Results. GIK treatment resulted in a significant protection of skeletal muscle with increased viability (GIK 22.07% (SD 15.48%)) compared to saline control (control 3.14% (SD 3.29%)) (p = 0.005). Additionally, GIK led to a statistically significant reduction in gene expression markers of cell death (CASP3, p < 0.001) and inflammation (NOS2, p < 0.001; IGF1, p = 0.007; IL-1β, p = 0.002; TNFα, p = 0.012), and a significant reduction in serum creatine kinase (p = 0.004) and creatinine (p < 0.001). GIK led to a significant reduction in IR-related pain (p = 0.030). Conclusion. Systemic GIK infusion during and after limb ischaemia protects murine skeletal muscle from cell death, kidneys from reperfusion metabolites, and reduces pain by reducing post-ischaemic inflammation. Cite this article: Bone Joint Res 2023;12(3):212–218

Aims. Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice. Methods. We used a murine model of chronic inflammation induced by continuous intramedullary delivery of lipopolysaccharide-contaminated polyethylene particles (cPE) using an osmotic pump. Specimens were evaluated using micro-CT and histomorphometric analyses. Sex-specific osteogenic and osteoclastic differentiation potentials were also investigated in vitro, including alkaline phosphatase, Alizarin Red, tartrate-resistant acid phosphatase staining, and gene expression using reverse transcription polymerase chain reaction (RT-PCR). Results. Local delivery of NF-κB decoy ODN in vivo increased osteogenesis in males, but not females, in the presence of chronic inflammation induced by cPE. Bone resorption activity was decreased in both sexes. In vitro osteogenic and osteoclastic differentiation assays during inflammatory conditions did not reveal differences among the groups. Receptor activator of nuclear factor kappa Β ligand (Rankl) gene expression by osteoblasts was significantly decreased only in males when treated with ODN. Conclusion. We demonstrated that NF-κB decoy ODN increased osteogenesis in male mice and decreased bone resorption activity in both sexes in preclinical models of chronic inflammation. NF-κB signalling could be a therapeutic target for chronic inflammatory diseases involving bone, especially in males. Cite this article: Bone Joint Res 2024;13(1):28–39

Objectives. Re-rupture is common after primary flexor tendon repair. Characterization of the biological changes in the ruptured tendon stumps would be helpful, not only to understand the biological responses to the failed tendon repair, but also to investigate if the tendon stumps could be used as a recycling biomaterial for tendon regeneration in the secondary grafting surgery. Methods. A canine flexor tendon repair and failure model was used. Following six weeks of repair failure, the tendon stumps were analyzed and characterized as isolated tendon-derived stem cells (TDSCs). Results. Failed-repair stump tissue showed cellular accumulation of crumpled and disoriented collagen fibres. Compared with normal tendon, stump tissue had significantly higher gene expression of collagens I and III, matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF). The stump TDSCs presented both mesenchymal stem and haematopoietic cell markers with significantly increased expression of CD34, CD44, and CD90 markers. Stump TDSCs exhibited similar migration but a lower proliferation rate, as well as similar osteogenic differentiation but a lower chondrogenic/adipogenic differentiation capability, compared with normal TDSCs. Stump TDSCs also showed increasing levels of SRY-box 2 (Sox2), octamer-binding transcription factor 4 (Oct4), tenomodulin (TNMD), and scleraxis (Scx) protein and gene expression. Conclusion. We found that a failed repair stump had increased cellularity that preserved both mesenchymal and haematopoietic stem cell characteristics, with higher collagen synthesis, MMP, and growth factor gene expression. This study provides evidence that tendon stump tissue has regenerative potential. Cite this article: C-C. Lu, T. Zhang, R. L. Reisdorf, P. C. Amadio, K-N. An, S. L. Moran, A. Gingery, C. Zhao. Biological analysis of flexor tendon repair-failure stump tissue: A potential recycling of tissue for tendon regeneration. Bone Joint Res 2019;8:232–245. DOI: 10.1302/2046-3758.86.BJR-2018-0239.R1

Aims. Proliferation, migration, and differentiation of anterior cruciate ligament (ACL) remnant and surrounding cells are fundamental processes for ACL reconstruction; however, the interaction between ACL remnant and surrounding cells is unclear. We hypothesized that ACL remnant cells preserve the capability to regulate the surrounding cells’ activity, collagen gene expression, and tenogenic differentiation. Moreover, extracorporeal shock wave (ESW) would not only promote activity of ACL remnant cells, but also enhance their paracrine regulation of surrounding cells. Methods. Cell viability, proliferation, migration, and expression levels of Collagen-I (COL-I) A1, transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF) were compared between ACL remnant cells untreated and treated with ESW (0.15 mJ/mm. 2. , 1,000 impulses, 4 Hz). To evaluate the subsequent effects on the surrounding cells, bone marrow stromal cells (BMSCs)’ viability, proliferation, migration, and levels of Type I Collagen, Type III Collagen, and tenogenic gene (Scx, TNC) expression were investigated using coculture system. Results. ESW-treated ACL remnant cells presented higher cell viability, proliferation, migration, and increased expression of COL-I A1, TGF-β, and VEGF. BMSC proliferation and migration rate significantly increased after coculture with ACL remnant cells with and without ESW stimulation compared to the BMSCs alone group. Furthermore, ESW significantly enhanced ACL remnant cells’ capability to upregulate the collagen gene expression and tenogenic differentiation of BMSCs, without affecting cell viability, TGF-β, and VEGF expression. Conclusion. ACL remnant cells modulated activity and differentiation of surrounding cells. The results indicated that ESW enhanced ACL remnant cells viability, proliferation, migration, and expression of collagen, TGF-β, VEGF, and paracrine regulation of BMSC proliferation, migration, collagen expression, and tenogenesis. Cite this article: Bone Joint Res 2020;9(8):457–467

Aims. The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction. Methods. The ACL remnant, bone marrow, and hamstring tendons were surgically harvested from rabbits. The apoptosis rate, cell proliferation, and expression of types I and III collagen, transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and tenogenic genes (scleraxis (SCX), tenascin C (TNC), and tenomodulin (TNMD)) of the hamstring tendons were compared between the co-culture medium (ACL remnant cells (ACLRCs) and BMSCs co-culture) and control medium (BMSCs-only culture). We also evaluated the apoptosis, cell proliferation, migration, and gene expression of hamstring tenocytes with exposure to co-culture and control media. Results. Compared to BMSCs-only culture medium, the co-culture medium showed substantially decreased early and late apoptosis rates, attenuation of intrinsic and extrinsic apoptotic pathways, and enhanced proliferation of the hamstring tendons and tenocytes. In addition, the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes in the hamstring tendons and tenocytes significantly increased in the co-culture medium compared to that in the control medium. Conclusion. In the presence of ACLRCs and BMSCs, the hamstring tendons and tenocytes significantly attenuated apoptosis and enhanced the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes. This in vitro study suggests that the ACLRCs mixed with BMSCs could aid regeneration of the hamstring tendon graft during ACL reconstruction. Cite this article: Bone Joint Res 2023;12(1):9–21

Aims. Osteoarthritis (OA) is a common degenerative joint disease. The osteocyte transcriptome is highly relevant to osteocyte biology. This study aimed to explore the osteocyte transcriptome in subchondral bone affected by OA. Methods. Gene expression profiles of OA subchondral bone were used to identify disease-relevant genes and signalling pathways. RNA-sequencing data of a bone loading model were used to identify the loading-responsive gene set. Weighted gene co-expression network analysis (WGCNA) was employed to develop the osteocyte mechanics-responsive gene signature. Results. A group of 77 persistent genes that are highly relevant to extracellular matrix (ECM) biology and bone remodelling signalling were identified in OA subchondral lesions. A loading responsive gene set, including 446 principal genes, was highly enriched in OA medial tibial plateaus compared to lateral tibial plateaus. Of this gene set, a total of 223 genes were identified as the main contributors that were strongly associated with osteocyte functions and signalling pathways, such as ECM modelling, axon guidance, Hippo, Wnt, and transforming growth factor beta (TGF-β) signalling pathways. We limited the loading-responsive genes obtained via the osteocyte transcriptome signature to identify a subgroup of genes that are highly relevant to osteocytes, as the mechanics-responsive osteocyte signature in OA. Based on WGCNA, we found that this signature was highly co-expressed and identified three clusters, including early, late, and persistently responsive genes. Conclusion. In this study, we identified the mechanics-responsive osteocyte signature in OA-lesioned subchondral bone. Cite this article: Bone Joint Res 2022;11(6):362–370

Aims. Arthrofibrosis is a relatively common complication after joint injuries and surgery, particularly in the knee. The present study used a previously described and validated rabbit model to assess the biomechanical, histopathological, and molecular effects of the mast cell stabilizer ketotifen on surgically induced knee joint contractures in female rabbits. Methods. A group of 12 skeletally mature rabbits were randomly divided into two groups. One group received subcutaneous (SQ) saline, and a second group received SQ ketotifen injections. Biomechanical data were collected at eight, ten, 16, and 24 weeks. At the time of necropsy, posterior capsule tissue was collected for histopathological and gene expression analyses (messenger RNA (mRNA) and protein). Results. At the 24-week timepoint, there was a statistically significant increase in passive extension among rabbits treated with ketotifen compared to those treated with saline (p = 0.03). However, no difference in capsular stiffness was detected. Histopathological data failed to demonstrate a decrease in the density of fibrous tissue or a decrease in α-smooth muscle actin (α-SMA) staining with ketotifen treatment. In contrast, tryptase and α-SMA protein expression in the ketotifen group were decreased when compared to saline controls (p = 0.007 and p = 0.01, respectively). Furthermore, there was a significant decrease in α-SMA (ACTA2) gene expression in the ketotifen group compared to the control group (p < 0.001). Conclusion. Collectively, these data suggest that ketotifen mitigates the severity of contracture formation in a rabbit model of arthrofibrosis. Cite this article: Bone Joint Res 2020;9(6):302–310

Aims. We aimed to develop a gene signature that predicts the occurrence of postmenopausal osteoporosis (PMOP) by studying its genetic mechanism. Methods. Five datasets were obtained from the Gene Expression Omnibus database. Unsupervised consensus cluster analysis was used to determine new PMOP subtypes. To determine the central genes and the core modules related to PMOP, the weighted gene co-expression network analysis (WCGNA) was applied. Gene Ontology enrichment analysis was used to explore the biological processes underlying key genes. Logistic regression univariate analysis was used to screen for statistically significant variables. Two algorithms were used to select important PMOP-related genes. A logistic regression model was used to construct the PMOP-related gene profile. The receiver operating characteristic area under the curve, Harrell’s concordance index, a calibration chart, and decision curve analysis were used to characterize PMOP-related genes. Then, quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression of the PMOP-related genes in the gene signature. Results. We identified three PMOP-related subtypes and four core modules. The muscle system process, muscle contraction, and actin filament-based movement were more active in the hub genes. We obtained five feature genes related to PMOP. Our analysis verified that the gene signature had good predictive power and applicability. The outcomes of the GSE56815 cohort were found to be consistent with the results of the earlier studies. qRT-PCR results showed that RAB2A and FYCO1 were amplified in clinical samples. Conclusion. The PMOP-related gene signature we developed and verified can accurately predict the risk of PMOP in patients. These results can elucidate the molecular mechanism of RAB2A and FYCO1 underlying PMOP, and yield new and improved treatment strategies, ultimately helping PMOP monitoring. Cite this article: Bone Joint Res 2022;11(8):548–560

Aims. To assess the alterations in cell-specific DNA methylation associated with chondroitin sulphate response using peripheral blood collected from Kashin-Beck disease (KBD) patients before initiation of chondroitin sulphate treatment. Methods. Peripheral blood samples were collected from KBD patients at baseline of chondroitin sulphate treatment. Methylation profiles were generated using reduced representation bisulphite sequencing (RRBS) from peripheral blood. Differentially methylated regions (DMRs) were identified using MethylKit, while DMR-related genes were defined as those annotated to the gene body or 2.2-kilobase upstream regions of DMRs. Selected DMR-related genes were further validated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to assess expression levels. Tensor composition analysis was performed to identify cell-specific differential DNA methylation from bulk tissue. Results. This study revealed 21,060 hypermethylated and 44,472 hypomethylated DMRs, and 13,194 hypermethylated and 22,448 hypomethylated CpG islands for differential global methylation for chondroitin sulphate treatment response. A total of 12,666 DMR-related genes containing DMRs were identified in their promoter regions, such as CHL1 (false discovery rate (FDR) = 2.11 × 10. -11. ), RIC8A (FDR = 7.05 × 10. -4. ), and SOX12 (FDR = 1.43 × 10. -3. ). Additionally, RIC8A and CHL1 were hypermethylated in responders, while SOX12 was hypomethylated in responders, all showing decreased gene expression. The patterns of cell-specific differential global methylation associated with chondroitin sulphate response were observed. Specifically, we found that DMRs located in TESPA1 and ATP11A exhibited differential DNA methylation between responders and non-responders in granulocytes, monocytes, and B cells. Conclusion. Our study identified cell-specific changes in DNA methylation associated with chondroitin sulphate response in KBD patients. Cite this article: Bone Joint Res 2024;13(5):237–246

Aims. To explore the novel molecular mechanisms of histone deacetylase 4 (HDAC4) in chondrocytes via RNA sequencing (RNA-seq) analysis. Methods. Empty adenovirus (EP) and a HDAC4 overexpression adenovirus were transfected into cultured human chondrocytes. The cell survival rate was examined by real-time cell analysis (RTCA) and EdU and flow cytometry assays. Cell biofunction was detected by Western blotting. The expression profiles of messenger RNAs (mRNAs) in the EP and HDAC4 transfection groups were assessed using whole-transcriptome sequencing (RNA-seq). Volcano plot, Gene Ontology, and pathway analyses were performed to identify differentially expressed genes (DEGs). For verification of the results, the A289E/S246/467/632 A sites of HDAC4 were mutated to enhance the function of HDAC4 by increasing HDAC4 expression in the nucleus. RNA-seq was performed to identify the molecular mechanism of HDAC4 in chondrocytes. Finally, the top ten DEGs associated with ribosomes were verified by quantitative polymerase chain reaction (QPCR) in chondrocytes, and the top gene was verified both in vitro and in vivo. Results. HDAC4 markedly improved the survival rate and biofunction of chondrocytes. RNA-seq analysis of the EP and HDAC4 groups showed that HDAC4 induced 2,668 significant gene expression changes in chondrocytes (1,483 genes upregulated and 1,185 genes downregulated, p < 0.05), and ribosomes exhibited especially large increases. The results were confirmed by RNA-seq of the EP versus mutated HDAC4 groups and the validations in vitro and in vivo. Conclusion. The enhanced ribosome pathway plays a key role in the mechanism by which HDAC4 improves the survival rate and biofunction of chondrocytes. Cite this article: Bone Joint Res 2023;12(7):433–446

Aims. This study aimed to explore the role of small colony variants (SCVs) of Staphylococcus aureus in intraosseous invasion and colonization in patients with periprosthetic joint infection (PJI). Methods. A PJI diagnosis was made according to the MusculoSkeletal Infection Society (MSIS) for PJI. Bone and tissue samples were collected intraoperatively and the intracellular invasion and intraosseous colonization were detected. Transcriptomics of PJI samples were analyzed and verified by polymerase chain reaction (PCR). Results. SCVs can be isolated from samples collected from chronic PJIs intraoperatively. Transmission electron microscopy (TEM) and immunofluorescence (IF) showed that there was more S. aureus in bone samples collected from chronic PJIs, but much less in bone samples from acute PJIs, providing a potential mechanism of PJI. Immunofluorescence results showed that SCVs of S. aureus were more likely to invade osteoblasts in vitro. Furthermore, TEM and IF also demonstrated that SCVs of S. aureus were more likely to invade and colonize in vivo. Cluster analysis and principal component analysis (PCA) showed that there were substantial differences in gene expression profiles between chronic and acute PJI. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these differentially expressed genes were enriched to chemokine-related signal pathways. PCR also verified these results. Conclusion. Our study has shown that the S. aureus SCVs have a greater ability to invade and colonize in bone, resulting in S. aureus remaining in bone tissues long-term, thus explaining the pathogenesis of chronic PJI. Cite this article: Bone Joint Res 2022;11(12):843–853

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

Aims. The antidiabetic agent metformin inhibits fibrosis in various organs. This study aims to elucidate the effects of hyperglycaemia and metformin on knee joint capsule fibrosis in mice. Methods. Eight-week-old wild-type (WT) and type 2 diabetic (db/db) mice were divided into four groups without or with metformin treatment (WT met(-/+), Db met(-/+)). Mice received daily intraperitoneal administration of metformin and were killed at 12 and 14 weeks of age. Fibrosis morphology and its related genes and proteins were evaluated. Fibroblasts were extracted from the capsules of 14-week-old mice, and the expression of fibrosis-related genes in response to glucose and metformin was evaluated in vitro. Results. The expression of all fibrosis-related genes was higher in Db met(-) than in WT met(-) and was suppressed by metformin. Increased levels of fibrosis-related genes, posterior capsule thickness, and collagen density were observed in the capsules of db/db mice compared with those in WT mice; these effects were suppressed by metformin. Glucose addition increased fibrosis-related gene expression in both groups of mice in vitro. When glucose was added, metformin inhibited the expression of fibrosis-related genes other than cellular communication network factor 2 (Ccn2) in WT mouse cells. Conclusion. Hyperglycaemia promotes fibrosis in the mouse knee joint capsule, which is inhibited by metformin. These findings can help inform the development of novel strategies for treating knee joint capsule fibrosis. Cite this article: Bone Joint Res 2024;13(7):321–331

Objectives. The biomembrane (induced membrane) formed around polymethylmethacrylate (PMMA) spacers has value in clinical applications for bone defect reconstruction. Few studies have evaluated its cellular, molecular or stem cell features. Our objective was to characterise induced membrane morphology, molecular features and osteogenic stem cell characteristics. Methods. Following Institutional Review Board approval, biomembrane specimens were obtained from 12 patient surgeries for management of segmental bony defects (mean patient age 40.7 years, standard deviation 14.4). Biomembranes from nine tibias and three femurs were processed for morphologic, molecular or stem cell analyses. Gene expression was determined using the Affymetrix GeneChip Operating Software (GCOS). Molecular analyses compared biomembrane gene expression patterns with a mineralising osteoblast culture, and gene expression in specimens with longer spacer duration (> 12 weeks) with specimens with shorter durations. Statistical analyses used the unpaired student t-test (two tailed; p < 0.05 was considered significant). Results. Average PMMA spacer in vivo time was 11.9 weeks (six to 18). Trabecular bone was present in 33.3% of the biomembrane specimens; bone presence did not correlate with spacer duration. Biomembrane morphology showed high vascularity and collagen content and positive staining for the key bone forming regulators, bone morphogenetic protein 2 (BMP2) and runt-related transcription factor 2 (RUNX2). Positive differentiation of cultured biomembrane cells for osteogenesis was found in cells from patients with PMMA present for six to 17 weeks. Stem cell differentiation showed greater variability in pluripotency for osteogenic potential (70.0%) compared with chondrogenic or adipogenic potentials (100% and 90.0%, respectively). Significant upregulation of BMP2 and 6, numerous collagens, and bone gla protein was present in biomembrane compared with the cultured cell line. Biomembranes with longer resident PMMA spacer duration (vs those with shorter residence) showed significant upregulation of bone-related, stem cell, and vascular-related genes. Conclusion. The biomembrane technique is gaining favour in the management of complicated bone defects. Novel data on biological mechanisms provide improved understanding of the biomembrane’s osteogenic potential and molecular properties. Cite this article: Dr H. E. Gruber. Osteogenic, stem cell and molecular characterisation of the human induced membrane from extremity bone defects. Bone Joint Res 2016;5:106–115. DOI: 10.1302/2046-3758.54.2000483

Aims. Minimally manipulated cells, such as autologous bone marrow concentrates (BMC), have been investigated in orthopaedics as both a primary therapeutic and augmentation to existing restoration procedures. However, the efficacy of BMC in combination with tissue engineering is still unclear. In this study, we aimed to determine whether the addition of BMC to an osteochondral scaffold is safe and can improve the repair of large osteochondral defects when compared to the scaffold alone. Methods. The ovine femoral condyle model was used. Bone marrow was aspirated, concentrated, and used intraoperatively with a collagen/hydroxyapatite scaffold to fill the osteochondral defects (n = 6). Tissue regeneration was then assessed versus the scaffold-only group (n = 6). Histological staining of cartilage with alcian blue and safranin-O, changes in chondrogenic gene expression, microCT, peripheral quantitative CT (pQCT), and force-plate gait analyses were performed. Lymph nodes and blood were analyzed for safety. Results. The results six months postoperatively showed that there were no significant differences in bone regrowth and mineral density between BMC-treated animals and controls. A significant upregulation of messenger RNA (mRNA) for types I and II collagens in the BMC group was observed, but there were no differences in the formation of hyaline-like cartilage between the groups. A trend towards reduced sulphated glycosaminoglycans (sGAG) breakdown was detected in the BMC group but this was not statistically significant. Functional weightbearing was not affected by the inclusion of BMC. Conclusion. Our results indicated that the addition of BMC to scaffold is safe and has some potentially beneficial effects on osteochondral-tissue regeneration, but not on the functional endpoint of orthopaedic interest. Cite this article: Bone Joint Res 2021;10(10):677–689

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. 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. Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Materials and Methods. Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. Results. No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. Conclusion. For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised. Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560–568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3

Aims. Developmental dysplasia of the hip (DDH) is a complex musculoskeletal disease that occurs mostly in children. This study aimed to investigate the molecular changes in the hip joint capsule of patients with DDH. Methods. High-throughput sequencing was used to identify genes that were differentially expressed in hip joint capsules between healthy controls and DDH patients. Biological assays including cell cycle, viability, apoptosis, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were performed to determine the roles of the differentially expressed genes in DDH pathology. Results. More than 1,000 genes were differentially expressed in hip joint capsules between healthy controls and DDH. Both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that extracellular matrix (ECM) modifications, muscle system processes, and cell proliferation were markedly influenced by the differentially expressed genes. Expression of Collagen Type I Alpha 1 Chain (COL1A1), COL3A1, matrix metalloproteinase-1 (MMP1), MMP3, MMP9, and MMP13 was downregulated in DDH, with the loss of collagen fibres in the joint capsule. Expression of transforming growth factor beta 1 (TGF-β1) was downregulated, while that of TGF-β2, Mothers against decapentaplegic homolog 3 (SMAD3), and WNT11 were upregulated in DDH, and alpha smooth muscle actin (αSMA), a key myofibroblast marker, showed marginal increase. In vitro studies showed that fibroblast proliferation was suppressed in DDH, which was associated with cell cycle arrest in G0/G1 and G2/M phases. Cell cycle regulators including Cyclin B1 (CCNB1), Cyclin E2 (CCNE2), Cyclin A2 (CCNA2), Cyclin-dependent kinase 1 (CDK1), E2F1, cell division cycle 6 (CDC6), and CDC7 were downregulated in DDH. Conclusion. DDH is associated with the loss of collagen fibres and fibroblasts, which may cause loose joint capsule formation. However, the degree of differentiation of fibroblasts to myofibroblasts needs further study. Cite this article: Bone Joint Res 2021;10(9):558–570

Aims. Osteoarthritis (OA) is characterized by persistent destruction of articular cartilage. It has been found that microRNAs (miRNAs) are closely related to the occurrence and development of OA. The purpose of the present study was to investigate the mechanism of miR-486 in the development and progression of OA. Methods. The expression levels of miR-486 in cartilage were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN), matrix metalloproteinase (MMP)-13, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) in SW1353 cells at both messenger RNA (mRNA) and protein levels was determined by qRT-PCR, western blot, and enzyme-linked immunosorbent assay (ELISA). Double luciferase reporter gene assay, qRT-PCR, and western blot assay were used to determine whether silencing information regulator 6 (SIRT6) was involved in miR-486 induction of chondrocyte-like cells to a more catabolic phenotype. Results. Compared with osteonecrosis, the expression of miR-486 was significantly upregulated in cartilage from subjects with severe OA. In addition, overexpressed miR-486 promoted a catabolic phenotype in SW1353 cells by upregulating the expressions of ADAMTS4 and MMP-13 and down-regulating the expressions of COL2A1 and ACAN. Conversely, inhibition of miR-486 had the opposite effect. Furthermore, overexpression of miR-486 significantly inhibited the expression of SIRT6, confirming that SIRT6 is a direct target of miR-486. Moreover, SW1353 cells were transfected with small interfering RNA (si)-SIRT6 and it was found that SIRT6 was involved in and inhibited miR-486-induced changes to SW1353 gene expression. Conclusion. Our results indicate that miR-486 promotes a catabolic phenotype in SW1353 cells in OA by targeting SIRT6. Our findings might provide a potential therapeutic target and theoretical basis for OA. Cite this article: Bone Joint Res 2021;10(7):459–466

Aims. Interleukin (IL)-1β is one of the major pathogenic regulators during the pathological development of intervertebral disc degeneration (IDD). However, effective treatment options for IDD are limited. Suramin is used to treat African sleeping sickness. This study aimed to investigate the pharmacological effects of suramin on mitigating IDD and to characterize the underlying mechanism. Methods. Porcine nucleus pulposus (NP) cells were treated with vehicle, 10 ng/ml IL-1β, 10 μM suramin, or 10 μM suramin plus IL-1β. The expression levels of catabolic and anabolic proteins, proinflammatory cytokines, mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB-related signalling molecules were assessed by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence analysis. Flow cytometry was applied to detect apoptotic cells. The ex vivo effects of suramin were examined using IDD organ culture and differentiation was analyzed by Safranin O-Fast green and Alcian blue staining. Results. Suramin inhibited IL-1β-induced apoptosis, downregulated matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5, and upregulated collagen 2A (Col2a1) and aggrecan in IL-1β-treated NP cells. IL-1β-induced inflammation, assessed by IL-1β, IL-8, and tumour necrosis factor α (TNF-α) upregulation, was alleviated by suramin treatment. Suramin suppressed IL-1β-mediated proteoglycan depletion and the induction of MMP-3, ADAMTS-4, and pro-inflammatory gene expression in ex vivo experiments. Conclusion. Suramin administration represents a novel and effectively therapeutic approach, which could potentially alleviate IDD by reducing extracellular matrix (ECM) deposition and inhibiting apoptosis and inflammatory responses in the NP cells. Cite this article: Bone Joint Res 2021;10(8):498–513

Objectives. The long head of the biceps (LHB) is often resected in shoulder surgery and could therefore serve as a cell source for tissue engineering approaches in the shoulder. However, whether it represents a suitable cell source for regenerative approaches, both in the inflamed and non-inflamed states, remains unclear. In the present study, inflamed and native human LHBs were comparatively characterized for features of regeneration. Methods. In total, 22 resected LHB tendons were classified into inflamed samples (n = 11) and non-inflamed samples (n = 11). Proliferation potential and specific marker gene expression of primary LHB-derived cell cultures were analyzed. Multipotentiality, including osteogenic, adipogenic, chondrogenic, and tenogenic differentiation potential of both groups were compared under respective lineage-specific culture conditions. Results. Inflammation does not seem to affect the proliferation rate of the isolated tendon-derived stem cells (TDSCs) and the tenogenic marker gene expression. Cells from both groups showed an equivalent osteogenic, adipogenic, chondrogenic and tenogenic differentiation potential in histology and real-time polymerase chain reaction (RT-PCR) analysis. Conclusion. These results suggest that the LHB tendon might be a suitable cell source for regenerative approaches, both in inflamed and non-inflamed states. The LHB with and without tendinitis has been characterized as a novel source of TDSCs, which might facilitate treatment of degeneration and induction of regeneration in shoulder surgery. Cite this article: J. Schmalzl, P. Plumhoff, F. Gilbert, F. Gohlke, C. Konrads, U. Brunner, F. Jakob, R. Ebert, A. F. Steinert. Tendon-derived stem cells from the long head of the biceps tendon: Inflammation does not affect the regenerative potential. Bone Joint Res 2019;8:414–424. DOI: 10.1302/2046-3758.89.BJR-2018-0214.R2

MicroRNAs (miRNAs) are a class of small non-coding RNAs that have emerged as potential predictive, prognostic, and therapeutic biomarkers, relevant to many pathophysiological conditions including limb immobilization, osteoarthritis, sarcopenia, and cachexia. Impaired musculoskeletal homeostasis leads to distinct muscle atrophies. Understanding miRNA involvement in the molecular mechanisms underpinning conditions such as muscle wasting may be critical to developing new strategies to improve patient management. MicroRNAs are powerful post-transcriptional regulators of gene expression in muscle and, importantly, are also detectable in the circulation. MicroRNAs are established modulators of muscle satellite stem cell activation, proliferation, and differentiation, however, there have been limited human studies that investigate miRNAs in muscle wasting. This narrative review summarizes the current knowledge as to the role of miRNAs in the skeletal muscle differentiation and atrophy, synthesizing the findings of published data. Cite this article: Bone Joint Res 2020;9(11):798–807

Osteoarthritis (OA), one of the most common motor system disorders, is a degenerative disease involving progressive joint destruction caused by a variety of factors. At present, OA has become the fourth most common cause of disability in the world. However, the pathogenesis of OA is complex and has not yet been clarified. Long non-coding RNA (lncRNA) refers to a group of RNAs more than 200 nucleotides in length with limited protein-coding potential, which have a wide range of biological functions including regulating transcriptional patterns and protein activity, as well as binding to form endogenous small interference RNAs (siRNAs) and natural microRNA (miRNA) molecular sponges. In recent years, a large number of lncRNAs have been found to be differentially expressed in a variety of pathological processes of OA, including extracellular matrix (ECM) degradation, synovial inflammation, chondrocyte apoptosis, and angiogenesis. Obviously, lncRNAs play important roles in regulating gene expression, maintaining the phenotype of cartilage and synovial cells, and the stability of the intra-articular environment. This article reviews the results of the latest research into the role of lncRNAs in a variety of pathological processes of OA, in order to provide a new direction for the study of OA pathogenesis and a new target for prevention and treatment. Cite this article: Bone Joint Res 2021;10(2):122–133

Objectives. The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy. Methods. Using tenocytes from normal Sprague-Dawley rats, cultured both in control and high glucose conditions, reactive oxygen species (ROS) production, cell proliferation, messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, interleukin-6 (IL-6), matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 and type I and III collagens were determined after 48 and 72 hours in vitro. In an in vivo study, using diabetic rats and controls, NOX1 and 4 expressions in Achilles tendon were also determined. Results. In tenocyte cultures grown under high glucose conditions, gene expressions of NOX1, MMP-2, TIMP-1 and -2 after 48 and 72 hours, NOX4 after 48 hours and IL-6, type III collagen and TIMP-2 after 72 hours were significantly higher than those in control cultures grown under control glucose conditions. Type I collagen expression was significantly lower after 72 hours. ROS accumulation was significantly higher after 48 hours, and cell proliferation after 48 and 72 hours was significantly lower in high glucose than in control glucose conditions. In the diabetic rat model, NOX1 expression within the Achilles tendon was also significantly increased. Conclusion. This study suggests that high glucose conditions upregulate the expression of mRNA for NOX1 and IL-6 and the production of ROS. Moreover, high glucose conditions induce an abnormal tendon matrix expression pattern of type I collagen and a decrease in the proliferation of rat tenocytes. Cite this article: Y. Ueda, A. Inui, Y. Mifune, R. Sakata, T. Muto, Y. Harada, F. Takase, T. Kataoka, T. Kokubu, R. Kuroda. The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo. Bone Joint Res 2018;7:362–372. DOI: 10.1302/2046-3758.75.BJR-2017-0126.R2

Aims. Tocilizumab, an interleukin-6 (IL-6) receptor (IL-6R) targeting antibody, enhances the anti-tumour effect of conventional chemotherapy in preclinical models of cancer. We investigated the anti-tumour effect of tocilizumab in osteosarcoma (OS) cell lines. Methods. We used the 143B, HOS, and Saos-2 human OS cell lines. We first analyzed the IL-6 gene expression and IL-6Rα protein expression in OS cells using reverse transcription real time quantitative-polymerase chain reaction (RT-qPCR) analysis and western blotting, respectively. We also assessed the effect of tocilizumab on OS cells using proliferation and invasion assay. Results. The OS cell lines 143B, HOS, and Saos-2 expressed IL-6R. Recombinant human IL-6 treatment increased proliferation of 143B and HOS cells. Tocilizumab treatment decreased proliferation and invasion of 143B, HOS, and Saos-2. Conclusion. In conclusion, we confirmed the production of IL-6 and the expression of IL-6R in OS cells and demonstrated that tocilizumab inhibits proliferation and invasion in OS cells. Cite this article: Bone Joint Res 2020;9(11):821–826

Aims. Osteoarthritis (OA) is the most prevalent joint disease. However, the specific and definitive genetic mechanisms of OA are still unclear. Methods. Tissue-related transcriptome-wide association studies (TWAS) of hip OA and knee OA were performed utilizing the genome-wide association study (GWAS) data of hip OA and knee OA (including 2,396 hospital-diagnosed hip OA patients versus 9,593 controls, and 4,462 hospital-diagnosed knee OA patients versus 17,885 controls) and gene expression reference to skeletal muscle and blood. The OA-associated genes identified by TWAS were further compared with the differentially expressed genes detected by the messenger RNA (mRNA) expression profiles of hip OA and knee OA. Functional enrichment and annotation analysis of identified genes was performed by the DAVID and FUMAGWAS tools. Results. We detected 33 common genes, eight common gene ontology (GO) terms, and one common pathway for hip OA, such as calcium and integrin-binding protein 1 (CIB1) (PTWAS = 0.025, FCmRNA = -1.575 for skeletal muscle), adrenomedullin (ADM) (PTWAS = 0.022, FCmRNA = -4.644 for blood), Golgi apparatus (PTWAS <0.001, PmRNA = 0.012 for blood), and phosphatidylinositol 3' -kinase-protein kinase B (PI3K-Akt) signalling pathway (PTWAS = 0.033, PmRNA = 0.005 for blood). For knee OA, we detected 24 common genes, eight common GO terms, and two common pathways, such as histocompatibility complex, class II, DR beta 1 (HLA-DRB1) (PTWAS = 0.040, FCmRNA = 4.062 for skeletal muscle), Follistatin-like 1 (FSTL1) (PTWAS = 0.048, FCmRNA = 3.000 for blood), cytoplasm (PTWAS < 0.001, PmRNA = 0.005 for blood), and complement and coagulation cascades (PTWAS = 0.017, PmRNA = 0.001 for skeletal muscle). Conclusion. We identified a group of OA-associated genes and pathways, providing novel clues for understanding the genetic mechanism of OA. Cite this article:Bone Joint Res. 2020;9(3):130–138

Background. Resveratrol is a polyphenolic compound commonly found in the skins of red grapes. Sirtuin 1 (SIRT1) is a human gene that is activated by resveratrol and has been shown to promote longevity and boost mitochondrial metabolism. We examined the effect of resveratrol on normal and osteoarthritic (OA) human chondrocytes. Methods. Normal and OA chondrocytes were incubated with various concentrations of resveratrol (1 µM, 10 µM, 25 µM and 50 µM) and cultured for 24, 48 or 72 hours or for six weeks. Cell proliferation, gene expression, and senescence were evaluated. Results. SIRT1 was significantly upregulated in normal chondrocytes with resveratrol concentrations of 25 µM and 50 µM on both two- (2D) (both p = 0.001) and three-dimensional (3D) cultures (p = 0.008 and 0.001, respectively). It was significantly upregulated in OA chondrocytes treated with 10 µM, 25 µM and 50 µM resveratrol on 2D cultures (p = 0.036, 0.002 and 0.001, respectively) and at 50 µM concentration on 3D cultures (p = 0.001). At 72 hours, the expression of collagen (COL)-10, aggrecan (AGG), and runt-related transcription factor 2 (RUNX2) was significantly greater in both 25 µM (p = 0.011, 0.006 and 0.015, respectively) and 50 µM (p = 0.019, 0.004 and 0.002, respectively) resveratrol-treated normal chondrocyte cultures. In OA chondrocytes, expression of COL10 and RUNX2 was significantly greater in 25 µM (p = 0.004 and 0.024) and 50 µM (p = 0.004 and 0.019) cultures at 72 hours on 3D cultures. Conclusions. At concentrations of 25 µM and/or 50 µM, resveratrol treatment significantly upregulates SIRT1 gene expression in normal and osteoarthritic chondrocytes. Resveratrol induces chondrocytes into a hypertrophic state through upregulation of COL1, COL10, and RUNX2. Cite this article: Bone Joint Res 2014;3:51–9

Aims. This study aimed to uncover the hub long non-coding RNAs (lncRNAs) differentially expressed in osteoarthritis (OA) cartilage using an integrated analysis of the competing endogenous RNA (ceRNA) network and co-expression network. Methods. Expression profiles data of ten OA and ten normal tissues of human knee cartilage were obtained from the Gene Expression Omnibus (GEO) database (GSE114007). The differentially expressed messenger RNAs (DEmRNAs) and lncRNAs (DElncRNAs) were identified using the edgeR package. We integrated human microRNA (miRNA)-lncRNA/mRNA interactions with DElncRNA/DEmRNA expression profiles to construct a ceRNA network. Likewise, lncRNA and mRNA expression profiles were used to build a co-expression network with the WGCNA package. Potential hub lncRNAs were identified based on an integrated analysis of the ceRNA network and co-expression network. StarBase and Multi Experiment Matrix databases were used to verify the lncRNAs. Results. We detected 1,212 DEmRNAs and 49 DElncRNAs in OA and normal knee cartilage. A total of 75 dysregulated lncRNA-miRNA interactions and 711 dysregulated miRNA-mRNA interactions were obtained in the ceRNA network, including ten DElncRNAs, 69 miRNAs, and 72 DEmRNAs. Similarly, 1,330 dysregulated lncRNA-mRNA interactions were used to construct the co-expression network, which included ten lncRNAs and 407 mRNAs. We finally identified seven hub lncRNAs, named MIR210HG, HCP5, LINC00313, LINC00654, LINC00839, TBC1D3P1-DHX40P1, and ISM1-AS1. Subsequent enrichment analysis elucidated that these lncRNAs regulated extracellular matrix organization and enriched in osteoclast differentiation, the FoxO signalling pathway, and the tumour necrosis factor (TNF) signalling pathway in the development of OA. Conclusion. The integrated analysis of the ceRNA network and co-expression network identified seven hub lncRNAs associated with OA. These lncRNAs may regulate extracellular matrix changes and chondrocyte homeostasis in OA progress. Cite this article:Bone Joint Res. 2020;9(3):90–98

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

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. The aim of this study was to identify key pathological genes in osteoarthritis (OA). Methods. We searched and downloaded mRNA expression data from the Gene Expression Omnibus database to identify differentially expressed genes (DEGs) of joint synovial tissues from OA and normal individuals. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analyses were used to assess the function of identified DEGs. The protein-protein interaction (PPI) network and transcriptional factors (TFs) regulatory network were used to further explore the function of identified DEGs. The quantitative real-time polymerase chain reaction (qRT-PCR) was applied to validate the result of bioinformatics analysis. Electronic validation was performed to verify the expression of selected DEGs. The diagnosis value of identified DEGs was accessed by receiver operating characteristic (ROC) analysis. Results. A total of 1085 DEGs were identified. KEGG pathway analysis displayed that Wnt was a significantly enriched signalling pathway. Some hub genes with high interactions such as USP46, CPVL, FKBP5, FOSL2, GADD45B, PTGS1, and ZNF423 were identified in the PPI and TFs network. The results of qRT-PCR showed that GADD45B, ADAMTS1, and TFAM were down-regulated in joint synovial tissues of OA, which was consistent with the bioinformatics analysis. The expression levels of USP46, CPVL, FOSL2, and PTGS1 in electronic validation were compatible with the bio-informatics result. CPVL and TFAM had a potential diagnostic value for OA based on the ROC analysis. Conclusion. The deregulated genes including USP46, CPVL, FKBP5, FOSL2, GADD45B, PTGS1, ZNF423, ADAMTS1, and TFAM might be involved in the pathology of OA. Cite this article: X. Zhang, Y. Bu, B. Zhu, Q. Zhao, Z. Lv, B. Li, J. Liu. Global transcriptome analysis to identify critical genes involved in the pathology of osteoarthritis. Bone Joint Res 2018;7:298–307. DOI: 10.1302/2046-3758.74.BJR-2017-0245.R1

Objectives. This study aimed to examine the effects of SRT1720, a potent SIRT1 activator, on osteoarthritis (OA) progression using an experimental OA model. Methods. Osteoarthritis was surgically induced by destabilization of the medial meniscus in eight-week-old C57BL/6 male mice. SRT1720 was administered intraperitoneally twice a week after surgery. Osteoarthritis progression was evaluated histologically using the Osteoarthritis Research Society International (OARSI) score at four, eight, 12 and 16 weeks. The expression of SIRT1, matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), cleaved caspase-3, PARP p85, and acetylated nuclear factor (NF)-κB p65 in cartilage was examined by immunohistochemistry. Synovitis was also evaluated histologically. Primary mouse epiphyseal chondrocytes were treated with SRT1720 in the presence or absence of interleukin 1 beta (IL-1β), and gene expression changes were examined by real-time polymerase chain reaction (PCR). Results. The OARSI score was significantly lower in mice treated with SRT1720 than in control mice at eight and 12 weeks associated with the decreased size of osteophytes at four and eight weeks. The delayed OA progression in the mice treated with SRT1720 was also associated with increased SIRT1-positive chondrocytes and decreased MMP-13-, ADAMTS-5-, cleaved caspase-3-, PARP p85-, and acetylated NF-κB p65-positive chondrocytes and decreased synovitis at four and eight weeks. SRT1720 treatment partially rescued the decreases in collagen type II alpha 1 (COL2A1) and aggrecan caused by IL-1β, while also reducing the induction of MMP-13 by IL-1β in vitro. Conclusion. The intraperitoneal injection of SRT1720 attenuated experimental OA progression in mice, indicating that SRT1720 could be a new therapeutic approach for OA. Cite this article: K. Nishida, T. Matsushita, K. Takayama, T. Tanaka, N. Miyaji, K. Ibaraki, D. Araki, N. Kanzaki, T. Matsumoto, R. Kuroda. Intraperitoneal injection of the SIRT1 activator SRT1720 attenuates the progression of experimental osteoarthritis in mice. Bone Joint Res 2018;7:252–262. DOI: 10.1302/2046-3758.73.BJR-2017-0227.R1

Objectives. The objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing. Methods. Human PBMNCs were cultured for seven days with the QQ-culture method using a serum-free medium containing five specific cytokines and growth factors. The QQ-cultured PBMNCs (QQMNCs) obtained were counted and characterised by flow cytometry and real-time polymerase chain reaction (RT-PCR). Angiogenic and osteo-inductive potentials were evaluated using tube formation assays and co-culture with mesenchymal stem cells with osteo-inductive medium in vitro. In order to evaluate the therapeutic potential of QQMNCs, cells were transplanted into an immunodeficient rat femur nonunion model. The rats were randomised into three groups: control; PBMNCs; and QQMNCs. The fracture healing was evaluated radiographically and histologically. Results. The total number of PBMNCs was decreased after QQ-culture, however, the number of CD34+ and CD206+ cells were found to have increased as assessed by flow cytometry analysis. In addition, gene expression of angiogenic factors was upregulated in QQMNCs. In the animal model, the rate of bone union was higher in the QQMNC group than in the other groups. Radiographic scores and bone volume were significantly associated with the enhancement of angiogenesis in the QQMNC group. Conclusion. We have demonstrated that QQMNCs have superior potential to accelerate fracture healing compared with PBMNCs. The QQMNCs could be a promising option for fracture nonunion. Cite this article: K. Mifuji, M. Ishikawa, N. Kamei, R. Tanaka, K. Arita, H. Mizuno, T. Asahara, N. Adachi, M. Ochi. Angiogenic conditioning of peripheral blood mononuclear cells promotes fracture healing. Bone Joint Res 2017;6: 489–498. DOI: 10.1302/2046-3758.68.BJR-2016-0338.R1

Objectives. Enhanced micromotions between the implant and surrounding bone can impair osseointegration, resulting in fibrous encapsulation and aseptic loosening of the implant. Since the effect of micromotions on human bone cells is sparsely investigated, an in vitro system, which allows application of micromotions on bone cells and subsequent investigation of bone cell activity, was developed. Methods. Micromotions ranging from 25 µm to 100 µm were applied as sine or triangle signal with 1 Hz frequency to human osteoblasts seeded on collagen scaffolds. Micromotions were applied for six hours per day over three days. During the micromotions, a static pressure of 527 Pa was exerted on the cells by Ti6Al4V cylinders. Osteoblasts loaded with Ti6Al4V cylinders and unloaded osteoblasts without micromotions served as controls. Subsequently, cell viability, expression of the osteogenic markers collagen type I, alkaline phosphatase, and osteocalcin, as well as gene expression of osteoprotegerin, receptor activator of NF-κB ligand, matrix metalloproteinase-1, and tissue inhibitor of metalloproteinase-1, were investigated. Results. Live and dead cell numbers were higher after 25 µm sine and 50 µm triangle micromotions compared with loaded controls. Collagen type I synthesis was downregulated in respective samples. The metabolic activity and osteocalcin expression level were higher in samples treated with 25 µm micromotions compared with the loaded controls. Furthermore, static loading and micromotions decreased the osteoprotegerin/receptor activator of NF-κB ligand ratio. Conclusion. Our system enables investigation of the behaviour of bone cells at the bone-implant interface under shear stress induced by micromotions. We could demonstrate that micromotions applied under static pressure conditions have a significant impact on the activity of osteoblasts seeded on collagen scaffolds. In future studies, higher mechanical stress will be applied and different implant surface structures will be considered. Cite this article: J. Ziebart, S. Fan, C. Schulze, P. W. Kämmerer, R. Bader, A. Jonitz-Heincke. Effects of interfacial micromotions on vitality and differentiation of human osteoblasts. Bone Joint Res 2018;7:187–195. DOI: 10.1302/2046-3758.72.BJR-2017-0228.R1

Objectives. The present study describes a novel technique for revitalising allogenic intrasynovial tendons by combining cell-based therapy and mechanical stimulation in an ex vivo canine model. Methods. Specifically, canine flexor digitorum profundus tendons were used for this study and were divided into the following groups: (1) untreated, unprocessed normal tendon; (2) decellularised tendon; (3) bone marrow stromal cell (BMSC)-seeded tendon; and (4) BMSC-seeded and cyclically stretched tendon. Lateral slits were introduced on the tendon to facilitate cell seeding. Tendons from all four study groups were distracted by a servohydraulic testing machine. Tensile force and displacement data were continuously recorded at a sample rate of 20 Hz until 200 Newton of force was reached. Before testing, the cross-sectional dimensions of each tendon were measured with a digital caliper. Young’s modulus was calculated from the slope of the linear region of the stress-strain curve. The BMSCs were labeled for histological and cell viability evaluation on the decellularized tendon scaffold under a confocal microscope. Gene expression levels of selected extracellular matrix tendon growth factor genes were measured. Results were reported as mean ± SD and data was analyzed with one-way ANOVAs followed by Tukey’s post hoc multiple-comparison test. Results. We observed no significant difference in cross-sectional area or in Young’s modulus among the four study groups. In addition, histological sections showed that the BMSCs were aligned well and viable on the tendon slices after two-week culture in groups three and four. Expression levels of several extracellular matrix tendon growth factors, including collagen type I, collagen type III, and matrix metalloproteinase were significantly higher in group four than in group three (p < 0.05). Conclusion. Lateral slits introduced into de-cellularised tendon is a promising method of delivery of BMSCs without compromising cell viability and tendon mechanical properties. In addition, mechanical stimulation of a cell-seeded tendon can promote cell proliferation and enhance expression of collagen types I and III in vitro. Cite this article: J. H. Wu, A. R. Thoreson, A. Gingery, K. N. An, S. L. Moran, P. C. Amadio, C. Zhao. The revitalisation of flexor tendon allografts with bone marrow stromal cells and mechanical stimulation: An ex vivo model revitalising flexor tendon allografts. Bone Joint Res 2017;6:179–185. DOI: 10.1302/2046-3758.63.BJR-2016-0207.R1