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

In this edition of Cochrane Corner, two updated reviews have been published in the Cochrane database. The first is a very broad review of rehabilitation strategies for patients with ankle fractures and includes early mobilization, the use of removable splints, and physical therapy techniques for patients treated with and without surgery. The second evaluates interventions for fixing tibial plateau fractures, including fixation techniques and bone graft substitutes.

The Cochrane Collaboration has produced five new reviews relevant to bone and joint surgery since the publication of the last Cochrane Corner These reviews are relevant to a wide range of musculoskeletal specialists, and include reviews in Morton’s neuroma, scoliosis, vertebral fractures, carpal tunnel syndrome, and lower limb arthroplasty.

This edition of the Cochrane Corner looks at the three reviews that were published in the second half of 2023: surgical versus non-surgical interventions for displaced intra-articular calcaneal fractures; cryotherapy following total knee arthroplasty; and physical activity and education about physical activity for chronic musculoskeletal pain in children and adolescents.

Objectives. To explore the therapeutic potential of combining bone marrow-derived mesenchymal stem cells (BM-MSCs) and hydroxyapatite (HA) granules to treat nonunion of the long bone. Methods. Ten patients with an atrophic nonunion of a long bone fracture were selectively divided into two groups. Five subjects in the treatment group were treated with the combination of 15 million autologous BM-MSCs, 5g/cm. 3. (HA) granules and internal fixation. Control subjects were treated with iliac crest autograft, 5g/cm. 3. HA granules and internal fixation. The outcomes measured were post-operative pain (visual analogue scale), level of functionality (LEFS and DASH), and radiograph assessment. Results. Post-operative pain evaluation showed no significant differences between the two groups. The treatment group demonstrated faster initial radiographic and functional improvements. Statistically significant differences in functional scores were present during the first (p = 0.002), second (p = 0.005) and third (p = 0.01) month. Both groups achieved similar outcomes by the end of one-year follow-up. No immunologic or neoplastic side effects were reported. Conclusions. All cases of nonunion of a long bone presented in this study were successfully treated using autologous BM-MSCs. The combination of autologous BM-MSCs and HA granules is a safe method for treating nonunion. Patients treated with BM-MSCs had faster initial radiographic and functional improvements. By the end of 12 months, both groups had similar outcomes. Cite this article: H.D. Ismail, P. Phedy, E. Kholinne, Y. P. Djaja, Y. Kusnadi, M. Merlina, N. D. Yulisa. Mesenchymal stem cell implantation in atrophic nonunion of the long bones: A translational study. Bone Joint Res 2016;5:287–293. DOI: 10.1302/2046-3758.57.2000587

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 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

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

Objectives. This study aimed to investigate the functional effects of microRNA (miR)-214-5p on osteoblastic cells, which might provide a potential role of miR-214-5p in bone fracture healing. Methods. Blood samples were obtained from patients with hand fracture or intra-articular calcaneal fracture and from healthy controls (HCs). Expression of miR-214-5p was monitored by qRT-PCR at day 7, 14 and 21 post-surgery. Mouse osteoblastic MC3T3-E1 cells were transfected with antisense oligonucleotides (ASO)-miR-214-5p, collagen type IV alpha 1 (COL4A1) vector or their controls; thereafter, cell viability, apoptotic rate, and the expression of collagen type I alpha 1 (COL1A1), type II collagen (COL-II), and type X collagen (COL-X) were determined. Luciferase reporter assay, qRT-PCR, and Western blot were performed to ascertain whether COL4A1 was a target of miR-214-5p. Results. Plasma miR-214-5p was highly expressed in patients with bone fracture compared with HCs after fracture (p < 0.05 or p < 0.01). Inhibition of miR-214-5p increased the viability of MC3T3-E1 cells and the expressions of COL1A1 and COL-X, but decreased the apoptotic rate and COL-II expression (p < 0.05 or p < 0.01). COL4A1 was a target of miR-214-5p, and was negatively regulated by miR-214-5p (p < 0.05 or p < 0.01). Overexpression of COL4A1 showed a similar impact on cell viability, apoptotic rate, and COL1A1, COL-II, and COL-X expressions inhibiting miR-214-5p (p < 0.01). Conclusion. Inhibition of miR-214-5p promotes cell survival and extracellular matrix (ECM) formation of osteoblastic MC3T3-E1 cells by targeting COL4A1. Cite this article: Q. S. Li, F. Y. Meng, Y. H. Zhao, C. L. Jin, J. Tian, X. J. Yi. Inhibition of microRNA-214-5p promotes cell survival and extracellular matrix formation by targeting collagen type IV alpha 1 in osteoblastic MC3T3-E1 cells. Bone Joint Res 2017;6:464–471. DOI: 10.1302/2046-3758.68.BJR-2016-0208.R2

We used demineralised bone matrix (DBM) to augment re-attachment of tendon to a metal prosthesis in an in vivo ovine model of reconstruction of the extensor mechanism at the knee. We hypothesised that augmentation of the tendon-implant interface with DBM would enhance the functional and histological outcomes as compared with previously reported control reconstructions without DBM. Function was assessed at six and 12 weeks postoperatively, and histological examination was undertaken at 12 weeks. A significant increase of 23.5% was observed in functional weight-bearing at six weeks in the DBM-augmented group compared with non-augmented controls (p = 0.004). By 12 weeks augmentation with DBM resulted in regeneration of a more direct-type enthesis, with regions of fibrocartilage, mineralised fibrocartilage and bone. In the controls the interface was predominantly indirect, with the tendon attached to the bone graft-hydroxyapatite base plate by perforating collagen fibres

Objectives. Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage. Materials and Methods. Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1. Results. MiR-138-5p was significantly increased in OA cartilage and in chondrocytes in response to IL-1β-stimulation. Overexpression of miR-138-5p significantly increased the IL-1β-induced downregulation of COL2A1, ACAN, and GAGs, and increased the IL-1β-induced over expression of MMP-13.We found that FOXC1 is directly regulated by miR-138-5p. Additionally, co-transfection with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 resulted in higher levels of COL2A1, ACAN, and GAGs, but lower levels of MMP-13. Conclusion. miR-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes, possibly by targeting FOXC1. Cite this article: Y. Yuan, G. Q. Zhang, W. Chai,M. Ni, C. Xu, J. Y. Chen. Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation. Bone Joint Res 2016;5:523–530. DOI: 10.1302/2046-3758.510.BJR-2016-0074.R2

We used an in vivo model to assess the use of an autogenous cancellous bone block and marrow graft for augmenting tendon reattachment to metallic implants. We hypothesised that augmentation of the tendon-implant interface with a bone block would enable retention of the graft on the implant surface, enhance biological integration, and result in more consistent functional outcomes compared with previously reported morcellised graft augmentation techniques. A significant improvement in functional weight-bearing was observed between six and 12 weeks. The significant increase in ground reaction force through the operated limb between six and 12 weeks was greater than that reported previously with morcellised graft augmented reconstructions. Histological appearance and collagen fibre orientation with bone block augmentation more closely resembled that of an intact enthesis compared with the morcellised grafting technique. Bone block augmentation of tendon-implant interfaces results in more reliable functional and histological outcomes, with a return to pre-operative levels of weight-bearing by 24 weeks

Objectives. There are various pin-in-plaster methods for treating fractures of the distal radius. The purpose of this study is to introduce a modified technique of ‘pin in plaster’. Methods. Fifty-four patients with fractures of the distal radius were followed for one year post-operatively. Patients were excluded if they had type B fractures according to AO classification, multiple injuries or pathological fractures, and were treated more than seven days after injury. Range of movement and functional results were evaluated at three and six months and one and two years post-operatively. Radiographic parameters including radial inclination, tilt, and height, were measured pre- and post-operatively. Results. The average radial tilt was 10.6° of volar flexion and radial height was 10.2 mm at the sixth month post-operatively. Three cases of pin tract infection were recorded, all of which were treated successfully with oral antibiotics. There were no cases of pin loosening. A total of 73 patients underwent surgery, and three cases of radial nerve irritation were recorded at the time of cast removal. All radial nerve palsies resolved at the six-month follow-up. There were no cases of median nerve compression or carpal tunnel syndrome, and no cases of tendon injury. Conclusion. Our modified technique is effective to restore anatomic congruity and maintain reduction in fractures of the distal radius. Cite this article: Bone Joint Res 2015;4:176–180

We developed an in vivo model of the attachment of a patellar tendon to a metal implant to simulate the reconstruction of an extensor mechanism after replacement of the proximal tibia. In 24 ewes, the patellar tendon was attached to a hydroxyapatite (HA)-coated titanium prosthesis. In 12, the interface was augmented with autograft containing cancellous bone and marrow. In the remaining ewes, the interface was not grafted. Kinematic gait analysis showed nearly normal function of the joint by 12 weeks. Force-plate assessment showed a significant increase in functional weight-bearing in the grafted animals (p = 0.043). The tendon-implant interface showed that without graft, encapsulation of fibrous tissue occurred. With autograft, a developing tendon-bone-HA-implant interface was observed at six weeks and by 12 weeks a layered tendon-fibrocartilage-bone interface was seen which was similar to a direct-type enthesis. With stable mechanical fixation, an appropriate bioactive surface and biological augmentation the development of a functional tendon-implant interface can be achieved

The meniscofemoral ligaments (MFL) of the knee have both functional and clinical importance, but have been poorly described. We examined 42 human cadaver knees: there was at least one MFL in every joint and both ligaments were present in 27. The anterior MFL was present in the knees in all 18 males and in 17 of the 24 females. The posterior MFL was present in 16 males and 22 females. Measurement of the ligaments showed that they were of significant size. The mean midpoint width for the anterior MFL was 5.09 ± 1.41 mm in males and 2.99 ± 1.29 mm in females. The mean width of the posterior MFL was 5.48 ± 2.13 mm in males and 3.79 ± 2.56 mm in females. The average length of the anterior MFL was 27.09 ± 2.15 mm in males and 24.38 ± 3.39 mm in females, and the posterior MFL was 31.13 ± 2.54 mm and 27.59 ± 3.74 mm, respectively. There were anatomical variations in 16 (38%) knees (62.5% female, 37.5% male), more commonly in the posterior ligament. We conclude that the meniscofemoral ligaments are anatomically and probably functionally important structures in the human knee

An understanding of the remodelling of tendon is crucial for the development of scientific methods of treatment and rehabilitation. This study tested the hypothesis that tendon adapts structurally in response to changes in functional loading. A novel model allowed manipulation of the mechanical environment of the patellar tendon in the presence of normal joint movement via the application of an adjustable external fixator mechanism between the patella and the tibia in sheep, while avoiding exposure of the patellar tendon itself. Stress shielding caused a significant reduction in the structural and material properties of stiffness (79%), ultimate load (69%), energy absorbed (61%), elastic modulus (76%) and ultimate stress (72%) of the tendon compared with controls. Compared with the material properties the structural properties exhibited better recovery after re-stressing with stiffness 97%, ultimate load 92%, energy absorbed 96%, elastic modulus 79% and ultimate stress 80%. The cross-sectional area of the re-stressed tendons was significantly greater than that of stress-shielded tendons. The remodelling phenomena exhibited in this study are consistent with a putative feedback mechanism under strain control. This study provides a basis from which to explore the interactions of tendon remodelling and mechanical environment

This study investigates the use of porous biphasic ceramics as graft extenders in impaction grafting of the femur during revision hip surgery. Impaction grafting of the femur was performed in four groups of sheep. Group one received pure allograft, group two 50% allograft and 50% BoneSave, group three 50% allograft and 50% BoneSave type 2 and group four 10% allograft and 90% BoneSave as the graft material. Function was assessed using an index of pre- and post-operative peak vertical ground reaction force ratios. Changes in bone mineral density were measured by dual energy X ray absorptiometry (DEXA) scanning. Loosening and subsidence were assessed radiographically and by histological examination of the explanted specimens. There was no statistically significant difference between the four groups after 18 months of unrestricted functional loading for all outcome measures