Aims. Cell-free DNA (cfDNA) and circulating tumour DNA (ctDNA) are used for prognostication and monitoring in patients with carcinomas, but their utility is unclear in sarcomas. The objectives of this pilot study were to explore the prognostic significance of cfDNA and investigate whether tumour-specific alterations can be detected in the circulation of sarcoma patients. Methods. Matched tumour and blood were collected from 64 sarcoma patients (n = 70 samples) prior to resection of the primary tumour (n = 57) or disease recurrence (n = 7). DNA was isolated from plasma, quantified, and analyzed for cfDNA. A subset of cases (n = 6) underwent whole exome sequencing to identify tumour-specific alterations used to detect ctDNA using digital droplet polymerase chain reaction (ddPCR). Results. Cell-free was present in 69 of 70 samples above 0.5 ng/ml. Improved disease-free survival was found for patients with lower cfDNA levels (90% vs 48% at one-year for ≤ 6 ng/ml and > 6 ng/ml, respectively; p = 0.005). Digital droplet PCR was performed as a pilot study and mutant alleles were detectable at 0.5% to 2.5% of the wild type genome, and at a level of 0.25 ng tumour DNA. Tumour-specific alterations (ctDNA) were found in five of six cases. Conclusion. This work demonstrates the feasibility and potential utility of cfDNA and ctDNA as biomarkers for bone and soft-tissue sarcomas, despite the lack of recurrent genomic alterations. A larger study is required to validate these findings. Cite this article: Bone Joint Res 2021;10(9):602–610

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

Previous research has shown an increase in chromosomal aberrations in patients with worn implants. The type of aberration depended on the type of metal alloy in the prosthesis. We have investigated the metal-specific difference in the level of DNA damage (DNA stand breaks and alkali labile sites) induced by culturing human fibroblasts in synovial fluid retrieved at revision arthroplasty. All six samples from revision cobalt-chromium metal-on-metal and four of six samples from cobalt-chromium metal-on-polyethylene prostheses caused DNA damage. By contrast, none of six samples from revision stainless-steel metal-on-polyethylene prostheses caused significant damage. Samples of cobalt-chromium alloy left to corrode in phosphate-buffered saline also caused DNA damage and this depended on a synergistic effect between the cobalt and chromium ions. Our results further emphasise that epidemiological studies of orthopaedic implants should take account of the type of metal alloy used

Aims. Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants. Methods. pEVs and cEVs were isolated by size exclusion chromatography (SEC) and physically characterized by nanoparticle tracking analysis (NTA), protein content, and purity. OA conditions were induced in human cartilage explants (10 ng/ml oncostatin M and 2 ng/ml tumour necrosis factor alpha (TNFα)) and treated with 1 × 10. 9. particles of pEVs or cEVs for 14 days. Then, DNA, glycosaminoglycans (GAG), and collagen content were quantified, and a histological study was performed. EV uptake was monitored using PKH26 labelled EVs. Results. Significantly higher content of DNA and collagen was observed for the pEV-treated group compared to control and cEV groups. No differences were found in GAG quantification nor in EVs uptake within any treated group. Conclusion. In conclusion, pEVs showed better performance than cEVs in our in vitro OA model. Although further studies are needed, pEVs are shown as a potential alternative to cEVs for cell-free regenerative medicine. Cite this article: Bone Joint Res 2023;12(10):667–676

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

The DNA content of the tumour cells in 10 patients with primary renal cell carcinomas was analysed; from six of the patients skeletal metastases were also studied. Four patients had homogenously diploid primary tumours, with solitary metastases. Six patients had aneuploid primary tumours, three with solitary and three with multiple metastases. In two patients radical excision of diploid metastases resulted in long disease-free intervals. Patients with diploid tumours survived significantly longer than patients with aneuploid tumours. These results indicate that tumour DNA content might be a useful prognostic indicator. The measurement of DNA content may be a suitable method of identifying those patients likely to survive long enough to benefit from major surgical resection and reconstruction

We investigated 21 pairs of twins for zygosity and idiopathic scoliosis. DNA fingerprinting confirmed that 13 pairs were monozygotic and eight were dizygotic. There was concordance for idiopathic scoliosis in 92.3% of monozygotic and 62.5% of dizygotic twins. Of the 12 pairs of monozygotic twins concordant for idiopathic scoliosis, six showed discordant curve patterns but eight had differences in Cobb angle of less than 10°. Seven of the ten pairs of monozygotic twins had similar back shapes. Our findings suggest that there is a genetic factor in the aetiology of idiopathic scoliosis; they also indicate that there is a genetic factor in both the severity of the curve and the general shape of the back

Aims

Bacterial infection activates neutrophils to release neutrophil extracellular traps (NETs) in bacterial biofilms of periprosthetic joint infections (PJIs). The aim of this study was to evaluate the increase in NET activation and release (NETosis) and haemostasis markers in the plasma of patients with PJI, to evaluate whether such plasma induces the activation of neutrophils, to ascertain whether increased NETosis is also mediated by reduced DNaseI activity, to explore novel therapeutic interventions for NETosis in PJI in vitro, and to evaluate the potential diagnostic use of these markers.

Adolescent idiopathic scoliosis (AIS), defined by an age at presentation of 11 to 18 years, has a prevalence of 0.47% and accounts for approximately 90% of all cases of idiopathic scoliosis. Despite decades of research, the exact aetiology of AIS remains unknown. It is becoming evident that it is the result of a complex interplay of genetic, internal, and environmental factors. It has been hypothesized that genetic variants act as the initial trigger that allow epigenetic factors to propagate AIS, which could also explain the wide phenotypic variation in the presentation of the disorder. A better understanding of the underlying aetiological mechanisms could help to establish the diagnosis earlier and allow a more accurate prediction of deformity progression. This, in turn, would prompt imaging and therapeutic intervention at the appropriate time, thereby achieving the best clinical outcome for this group of patients.

Cite this article: Bone Joint J 2022;104-B(8):915–921.

Aims

Psychoeducative prehabilitation to optimize surgical outcomes is relatively novel in spinal fusion surgery and, like most rehabilitation treatments, they are rarely well specified. Spinal fusion patients experience anxieties perioperatively about pain and immobility, which might prolong hospital length of stay (LOS). The aim of this prospective cohort study was to determine if a Preoperative Spinal Education (POSE) programme, specified using the Rehabilitation Treatment Specification System (RTSS) and designed to normalize expectations and reduce anxieties, was safe and reduced LOS.

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

Aims. Aseptic loosening is a leading cause of uncemented arthroplasty failure, often accompanied by fibrotic tissue at the bone-implant interface. A biological target, neutrophil extracellular traps (NETs), was investigated as a crucial connection between the innate immune system’s response to injury, fibrotic tissue development, and proper bone healing. Prevalence of NETs in peri-implant fibrotic tissue from aseptic loosening patients was assessed. A murine model of osseointegration failure was used to test the hypothesis that inhibition (through Pad4-/- mice that display defects in peptidyl arginine deiminase 4 (PAD4), an essential protein required for NETs) or resolution (via DNase 1 treatment, an enzyme that degrades the cytotoxic DNA matrix) of NETs can prevent osseointegration failure and formation of peri-implant fibrotic tissue. Methods. Patient peri-implant fibrotic tissue was analyzed for NETs biomarkers. To enhance osseointegration in loose implant conditions, an innate immune system pathway (NETs) was either inhibited (Pad4-/- mice) or resolved with a pharmacological agent (DNase 1) in a murine model of osseointegration failure. Results. NETs biomarkers were identified in peri-implant fibrotic tissue collected from aseptic loosening patients and at the bone-implant interface in a murine model of osseointegration failure. Inhibition (Pad4-/-) or resolution (DNase 1) of NETs improved osseointegration and reduced fibrotic tissue despite loose implant conditions in mice. Conclusion. This study identifies a biological target (NETs) for potential noninvasive treatments of aseptic loosening by discovering a novel connection between the innate immune system and post-injury bone remodelling caused by implant loosening. By inhibiting or resolving NETs in an osseointegration failure murine model, fibrotic tissue encapsulation around an implant is reduced and osseointegration is enhanced, despite loose implant conditions. Cite this article: Bone Joint J 2021;103-B(7 Supple B):135–144

Objectives. The cytotoxicity induced by cobalt ions (Co. 2+. ) and cobalt nanoparticles (Co-NPs) which released following the insertion of a total hip prosthesis, has been reported. However, little is known about the underlying mechanisms. In this study, we investigate the toxic effect of Co. 2+. and Co-NPs on liver cells, and explain further the potential mechanisms. Methods. Co-NPs were characterised for size, shape, elemental analysis, and hydrodynamic diameter, and were assessed by Transmission Electron Microscope, Scanning Electron Microscope, Energy Dispersive X-ray Spectroscopy and Dynamic Light Scattering. BRL-3A cells were used in this study. Cytotoxicity was evaluated by MTT and lactate dehydrogenase release assay. In order to clarify the potential mechanisms, reactive oxygen species, Bax/Bcl-2 mRNA expression, IL-8 mRNA expression and DNA damage were assessed on BRL-3A cells after Co. 2+. or Co-NPs treatment. Results. Results showed cytotoxic effects of Co. 2+. and Co-NPs were dependent upon time and dosage, and the cytotoxicity of Co-NPs was greater than that of Co. 2+. In addition, Co-NPs elicited a significant (p < 0.05) reduction in cell viability with a concomitant increase in lactic dehydrogenase release, reactive oxygen species generation, IL-8 mRNA expression, Bax/Bcl-2 mRNA expression and DNA damage after 24 hours of exposure. Conclusion. Co-NPs induced greater cytotoxicity and genotoxicity in BRL-3A cells than Co. 2+. Cell membrane damage, oxidative stress, immune inflammation and DNA damage may play an important role in the effects of Co-NPs on liver cells. Cite this article: Y. K. Liu, X. X. Deng, H.L. Yang. Cytotoxicity and genotoxicity in liver cells induced by cobalt nanoparticles and ions. Bone Joint Res 2016;5:461–469. DOI: 10.1302/2046-3758.510.BJR-2016-0016.R1

The ability to edit DNA at the nucleotide level using clustered regularly interspaced short palindromic repeats (CRISPR) systems is a relatively new investigative tool that is revolutionizing the analysis of many aspects of human health and disease, including orthopaedic disease. CRISPR, adapted for mammalian cell genome editing from a bacterial defence system, has been shown to be a flexible, programmable, scalable, and easy-to-use gene editing tool. Recent improvements increase the functionality of CRISPR through the engineering of specific elements of CRISPR systems, the discovery of new, naturally occurring CRISPR molecules, and modifications that take CRISPR beyond gene editing to the regulation of gene transcription and the manipulation of RNA. Here, the basics of CRISPR genome editing will be reviewed, including a description of how it has transformed some aspects of molecular musculoskeletal research, and will conclude by speculating what the future holds for the use of CRISPR-related treatments and therapies in clinical orthopaedic practice. Cite this article: Bone Joint Res 2020;9(7):351–359

Aims. Extracellular matrix (ECM) and its architecture have a vital role in articular cartilage (AC) structure and function. We hypothesized that a multi-layered chitosan-gelatin (CG) scaffold that resembles ECM, as well as native collagen architecture of AC, will achieve superior chondrogenesis and AC regeneration. We also compared its in vitro and in vivo outcomes with randomly aligned CG scaffold. Methods. Rabbit bone marrow mesenchymal stem cells (MSCs) were differentiated into the chondrogenic lineage on scaffolds. Quality of in vitro regenerated cartilage was assessed by cell viability, growth, matrix synthesis, and differentiation. Bilateral osteochondral defects were created in 15 four-month-old male New Zealand white rabbits and segregated into three treatment groups with five in each. The groups were: 1) untreated and allogeneic chondrocytes; 2) multi-layered scaffold with and without cells; and 3) randomly aligned scaffold with and without cells. After four months of follow-up, the outcome was assessed using histology and immunostaining. Results. In vitro testing showed that the secreted ECM oriented itself along the fibre in multi-layered scaffolds. Both types of CG scaffolds supported cell viability, growth, and matrix synthesis. In vitro chondrogenesis on scaffold showed an around 400-fold increase in collagen type 2 (COL2A1) expression in both CG scaffolds, but the total glycosaminoglycan (GAG)/DNA deposition was 1.39-fold higher in the multi-layered scaffold than the randomly aligned scaffold. In vivo cartilage formation occurred in both multi-layered and randomly aligned scaffolds treated with and without cells, and was shown to be of hyaline phenotype on immunostaining. The defects treated with multi-layered + cells, however, showed significantly thicker cartilage formation than the randomly aligned scaffold. Conclusion. We demonstrated that MSCs loaded CG scaffold with multi-layered zonal architecture promoted superior hyaline AC regeneration. Cite this article: Bone Joint Res 2020;9(9):601–612

It has been proposed that intervertebral disc degeneration might be caused by low-grade infection. The purpose of the present study was to assess the incidence of herpes viruses in intervertebral disc specimens from patients with lumbar disc herniation. A polymerase chain reaction based assay was applied to screen for the DNA of eight different herpes viruses in 16 patients and two controls. DNA of at least one herpes virus was detected in 13 specimens (81.25%). Herpes Simplex Virus type-1 (HSV-1) was the most frequently detected virus (56.25%), followed by Cytomegalovirus (CMV) (37.5%). In two patients, co-infection by both HSV-1 and CMV was detected. All samples, including the control specimens, were negative for Herpes Simplex Virus type-2, Varicella Zoster Virus, Epstein Barr Virus, Human Herpes Viruses 6, 7 and 8. The absence of an acute infection was confirmed both at the serological and mRNA level. To our knowledge this is the first unequivocal evidence of the presence of herpes virus DNA in intervertebral disc specimens of patients with lumbar disc herniation suggesting the potential role of herpes viruses as a contributing factor to the pathogenesis of degenerative disc disease

Aims. Here we introduce a wide and complex study comparing effects of growth factors used alone and in combinations on human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation. Certain ways of cell behaviour can be triggered by specific peptides – growth factors, influencing cell fate through surface cellular receptors. Methods. In our study transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were used in order to induce osteogenesis and proliferation of hMSCs from bone marrow. These cells are naturally able to differentiate into various mesodermal cell lines. Effect of each factor itself is pretty well known. We designed experimental groups where two and more growth factors were combined. We supposed cumulative effect would appear when more growth factors with the same effect were combined. The cellular metabolism was evaluated using MTS assay and double-stranded DNA (dsDNA) amount using PicoGreen assay. Alkaline phosphatase (ALP) activity, as early osteogenesis marker, was observed. Phase contrast microscopy was used for cell morphology evaluation. Results. TGF-β and bFGF were shown to significantly enhance cell proliferation. VEGF and IGF-1 supported ALP activity. Light microscopy showed initial extracellular matrix mineralization after VEGF/IGF-1 supply. Conclusion. A combination of more than two growth factors did not support the cellular metabolism level and ALP activity even though the growth factor itself had a positive effect. This is probably caused by interplay of various messengers shared by more growth factor signalling cascades. Cite this article: Bone Joint Res 2020;9(7):412–420

The weight-bearing status of articular cartilage has been shown to affect its biochemical composition. We have investigated the topographical variation of sulphated glycosaminoglycan (GAG) relative to the DNA content of the chondrocyte in human distal femoral articular cartilage. Paired specimens of distal femoral articular cartilage, from weight-bearing and non-weight-bearing regions, were obtained from 13 patients undergoing above-knee amputation. After papain enzyme digestion, spectrophotometric GAG and fluorometric DNA assays assessed the biochemical composition of the samples. The results were analysed using a paired t-test. Although there were no significant differences in cell density between the regions, the weight-bearing areas showed a significantly higher concentration of GAG relative to DNA when compared with non-weight-bearing areas (p = 0.02). We conclude that chondrocytes are sensitive to their mechanical environment, and that local loading conditions influence the metabolism of the cells and hence the biochemical structure of the tissue

Various chemicals are commonly used as adjuvant treatment to surgery for giant-cell tumour (GCT) of bone. The comparative effect of these solutions on the cells of GCT is not known. In this study we evaluated the cytotoxic effect of sterile water, 95% ethanol, 5% phenol, 3% hydrogen peroxide (H. 2. O. 2. ) and 50% zinc chloride (ZnCI. 2. ) on GCT monolayer tumour cultures which were established from six patients. The DNA content, the metabolic activity and the viability of the cultured samples of tumour cells were assessed at various times up to 120 hours after their exposure to these solutions. Equal cytotoxicity to the GCT monolayer culture was observed for 95% ethanol, 5% phenol, 3% H. 2. O. 2. and 50% ZnCI. 2. The treated samples showed significant reductions in DNA content and metabolic activity 24 hours after treatment and this was sustained for up to 120 hours. The samples treated with sterile water showed an initial decline in DNA content and viability 24 hours after treatment, but the surviving cells were viable and had proliferated. No multinucleated cell formation was seen in these cultures. These results suggest that the use of chemical adjuvants other than water could help improve local control in the treatment of GCT of bone

Aims. The purpose of this study was to use pharmacogenetics to determine the frequency of genetic variants in our total knee arthroplasty (TKA) patients that could affect postoperative pain medications. Pharmacogenetic testing evaluates patient DNA to determine if a drug is expected to have a normal clinical effect, heightened effect, or no effect at all on the patient. It also predicts whether patients are likely to experience side effects from medicine. We further sought to determine if changing the multimodal programme based on these results would improve pain control or reduce side effects. Methods. In this pilot study, buccal samples were collected from 31 primary TKA patients. Pharmacogenetics testing examined genetic variants in genes OPRM1, CYP1A2, CYP2B6, CYP2C19, CYP3A4, CYP2C9, and CYP2D6. These genes affect the pharmacodynamics and pharmacokinetics of non-steroidal anti-inflammatory drugs and opioids. We examined the frequency of genetic variants to any of the medications we prescribed including celecoxib, hydrocodone, and tramadol. Patients were randomized to one of two groups: the control group received the standard postoperative pain regimen, and the study group received a customized regimen based on the pharmacogenetic results. For the first ten postoperative days, patients recorded pain scores, medication, and side effects. Results. Genetic variants involving one or more medications in the multimodal pain protocol occurred in 13 of the 31 patients (42%). In total, eight patients (26%) had variants affecting more than one of the medications. For the 25 patients who recorded pain and medication logs, the mean pain levels and morphine equivalents (MEQs) consumed in the first ten days were higher in the control group than in the custom-guided group (p = 0.019 for pain and p = 0.655 for MEQ). Conclusion. Overall, 42% of patients had a variant involving one of the pain medications prescribed in our perioperative pain program for TKA. Ongoing research will help determine if using these data to modify a patient’s medication will improve outcomes. Cite this article: Bone Joint J 2020;102-B(6 Supple A):73–78

Objectives. Platelet-rich plasma (PRP) is being used increasingly often in the clinical setting to treat tendon-related pathologies. Yet the optimal PRP preparations to promote tendon healing in different patient populations are poorly defined. Here, we sought to determine whether increasing the concentration of platelet-derived proteins within a derivative of PRP, platelet lysate (PL), enhances tenocyte proliferation and migration in vitro, and whether the mitogenic properties of PL change with donor age. Methods. Concentrated PLs from both young (< 50 years) and aged (> 50 years) donors were prepared by exposing pooled PRP to a series of freeze-thaw cycles followed by dilution in plasma, and the levels of several platelet-derived proteins were measured using multiplex immunoassay technology. Human tenocytes were cultured with PLs to simulate a clinically relevant PRP treatment range, and cell growth and migration were assessed using DNA quantitation and gap closure assays, respectively. Results. Platelet-derived protein levels increased alongside higher PL concentrations, and PLs from both age groups improved tenocyte proliferation relative to control conditions. However, PLs from aged donors yielded a dose-response relationship in tenocyte behaviour, with higher PL concentrations resulting in increased tenocyte proliferation and migration. Conversely, no significant differences in tenocyte behaviour were detected when increasing the concentration of PLs from younger donors. Conclusion. Higher PL concentrations, when prepared from the PRP of aged but not young donors, were more effective than lower PL concentrations at promoting tenocyte proliferation and migration in vitro. Cite this article: D. R. Berger, C. J. Centeno, N. J. Steinmetz. Platelet lysates from aged donors promote human tenocyte proliferation and migration in a concentration-dependent manner. Bone Joint Res 2019;8:32–40. DOI: 10.1302/2046-3758.81.BJR-2018-0164.R1

A retrospective series of 45 cases of chronic osteomyelitis collected over a period of 14 years was histologically classified into tuberculous osteomyelitis (25) and chronic non-granulomatous osteomyelitis (20). The tuberculous osteomyelitis group was divided into three subgroups: a) typical granulomas (13 cases); b) ill-defined granulomas (seven cases), and c) suspected granulomas (five cases). An in-house polymerase chain reaction amplifying the 245 bp nucleotide sequence, and capable of detecting 10 fg of DNA of Mycobacterium tuberculosis, was used on the DNA extracted from the paraffin blocks. The polymerase chain reaction was positive in 72% of cases (18) of tuberculous osteomyelitis, but when typical cases of tuberculous osteomyelitis with confirmed granulomas were considered (13), this increased to 84.6% (11). The chronic non-granulomatous osteomyelitis group gave positive polymerase chain reaction results in 20% of the cases (4). Our preliminary study on tuberculous osteomyelitis shows that the polymerase chain reaction can be a very useful diagnostic tool, since a good correlation was seen between typical granulomas and polymerase chain reaction with a sensitivity of 84.6% and a specificity of 80%. In addition, our study shows that tuberculous osteomyelitis can be diagnosed in formalin-fixed paraffin-embedded tissues in the absence of typical granulomas

Objectives. The aims of this study were to determine whether the administration of anti-inflammatory and antifibrotic agents affect the proliferation, viability, and expression of markers involved in the fibrotic development of the fibroblasts obtained from arthrofibrotic tissue in vitro, and to evaluate the effect of the agents on arthrofibrosis prevention in vivo. Methods. Dexamethasone, diclofenac, and decorin, in different concentrations, were employed to treat fibroblasts from arthrofibrotic tissue (AFib). Cell proliferation was measured by DNA quantitation, and viability was analyzed by Live/Dead staining. The levels of procollagen type I N-terminal propeptide (PINP) and procollagen type III N-terminal propeptide (PIIINP) were evaluated with enzyme-linked immunosorbent assay (ELISA) kits. In addition, the expressions of fibrotic markers were detected by real-time polymerase chain reaction (PCR). Fibroblasts isolated from healthy tissue (Fib) served as control. Further, a rabbit model of joint contracture was used to evaluate the antifibrotic effect of the three different agents. Results. Dexamethasone maintained the viability and promoted the proliferation of AFib. Diclofenac decreased the viability and inhibited the cell proliferation during the first week of cultivation. However, decorin inhibited AFib proliferation and downregulated the expressions of fibrotic markers. Additionally, decorin could improve the flexion contracture angle and inhibit the deposition of interstitial matrix components in the rabbit joint model. Conclusion. Decorin decreased the expression of myofibroblast markers in AFib, inhibited the proliferation of AFib, and prevented the initial procedure of arthrofibrosis in vivo, suggesting that decorin could be a promising treatment to inhibit the development of arthrofibrosis. Cite this article: X. Tang, S. Teng, M. Petri, C. Krettek, C. Liu, M. Jagodzinski. The effect of anti-inflammatory and antifibrotic agents on fibroblasts obtained from arthrofibrotic tissue: An in vitro and in vivo study. Bone Joint Res 2018;7:213–222. DOI: 10.1302/2046-3758.73.BJR-2017-0219.R2

The role of heritable thrombophilic risk factors in the pathogenesis of the Perthes’ disease is controversial. The clinical and radiological findings of Perthes’ disease may be indistinguishable from those of Gaucher’s disease, and the most common Jewish N370S Gaucher mutation is threefold greater in patients with Perthes’ disease. Familial osteonecrosis of the femoral head is associated with variant mutations of collagen type II (COL2A1 mutations). We therefore studied the potential role of genetic thrombophilia and the Gaucher and COL2A1 mutations in children with Perthes’ disease. Genomic DNA of 119 children with radiologically-confirmed Perthes’ disease diagnosed between 1986 and 2005 was analysed for the thrombophilic polymorphisms Factor V Leiden, 677T-MTHFR and FIIG20210A. The results were compared with those of a group of 276 children without Perthes’ disease. DNA was also analysed for the Gaucher mutations N370S, G insertion (84GG), L444P, Intron 2 (IVS2+1G> A) and R496H. Enzymic assays confirmed the Gaucher disease status. Collagen (COL2A1) mutations of the 12q13 gene were also analysed. The prevalence of thrombophilic markers was similar among the 119 patients with Perthes’ disease and the 276 control subjects. The prevalence of the Gaucher mutation was consistent with Israeli population carriership data and did not confirm an earlier-claimed association with Perthes’ disease. All 199 patients were negative for the studied COL2A1 mutations. We found no genetic association between Perthes’ disease and either Gaucher’s disease or COL2A1 mutations or increased genetic thrombophilia among our patients compared with the control group. A systematic review of case-control studies suggested that there was a positive association between Perthes’ disease and Factor V Leiden. The impact of this association upon the disease, although not consistent across the studies, remains unclear

Objectives. X-linked hypophosphataemic rickets (XLHR) is a disease of impaired bone mineralization characterized by hypophosphataemia caused by renal phosphate wasting. The main clinical manifestations of the disorder are O-shaped legs, X-shaped legs, delayed growth, and bone pain. XLHR is the most common inheritable form of rickets, with an incidence of 1/20 000 in humans. It accounts for approximately 80% of familial cases of hypophosphataemia and serves as the prototype of defective tubular phosphate (PO4. 3+. ) transport, due to extra renal defects resulting in unregulated FGF23 activity. XLHR is caused by loss-of-function mutations in the PHEX gene. The aim of this research was to identify the genetic defect responsible for familial hypophosphataemic rickets in a four-generation Chinese Han pedigree and to analyze the function of this mutation. Methods. The genome DNA samples of all members in the pedigree were extracted from whole blood. We sequenced all exons of the PHEX and FGF23 genes, as well as the adjacent splice site sequence with Sanger sequencing. Next, we analyzed the de novo mutation c.1692 del A of the PHEX gene with an online digital service and investigated the mutant PHEX with SWISS-MODEL, immunofluorescence, and protein stability detection. Results. Through Sanger sequencing, we found a de novo mutation, c.1692 del A, in exon 16 of the PHEX gene in this pedigree. This mutation can make the PHEX protein become unstable and decay rapidly, which results in familial XLHR. Conclusion. We have found a de novo loss-of-function mutation, c.1692 del A, in exon 16 of the PHEX gene that can cause XLHR. Cite this article: J. Huang, X. Bao, W. Xia, L. Zhu, J. Zhang, J. Ma, N. Jiang, J. Yang, Q. Chen, T. Jing, J. Liu, D. Ma, G. Xu. Functional analysis of a de novo mutation c.1692 del A of the PHEX gene in a Chinese family with X-linked hypophosphataemic rickets. Bone Joint Res 2019;8:405–413. DOI: 10.1302/2046-3758.88.BJR-2018-0276.R1

Objectives. To determine the pattern of mutations of the WISP3 gene in clinically identified progressive pseudorheumatoid dysplasia (PPD) in an Indian population. Patients and Methods. A total of 15 patients with clinical features of PPD were enrolled in this study. Genomic DNA was isolated and polymerase chain reaction performed to amplify the WISP3 gene. Screening for mutations was done by conformation-sensitive gel electrophoresis, beginning with the fifth exon and subsequently proceeding to the remaining exons. Sanger sequencing was performed for both forward and reverse strands to confirm the mutations. Results. In all, two of the 15 patients had compound heterozygous mutations: one a nonsense mutation c.156C>A (p.C52*) in exon 2, and the other a missense mutation c.677G>T (p.G226V) in exon 4. All others were homozygous, with three bearing a nonsense mutation c.156C>A (p.C52*) in exon 2, three a missense mutation c.233G>A (p.C78Y) in exon 2, five a missense mutation c.1010G>A (p.C337Y) in exon 5, one a nonsense mutation c.348C>A (p.Y116*) in exon 3, and one with a novel deletion mutation c.593_597delATAGA (p.Y198*) in exon 4. Conclusion. We identified a novel mutation c.593_597delATAGA (p.Y198*) in the fourth exon of the WISP3 gene. We also confirmed c.1010G>A as one of the common mutations in an Indian population with progressive pseudorheumatoid dysplasia. Cite this article: V. Madhuri, M. Santhanam, K. Rajagopal, L. K. Sugumar, V. Balaji. WISP3 mutational analysis in Indian patients diagnosed with progressive pseudorheumatoid dysplasia and report of a novel mutation at p.Y198* Bone Joint Res 2016;5:301–306. DOI: 10.1302/2046-3758.57.2000520

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Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism.

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Circular RNA (circRNA) is involved in the regulation of articular cartilage degeneration induced by inflammatory factors or oxidative stress. In a previous study, we found that the expression of circStrn3 was significantly reduced in chondrocytes of osteoarthritis (OA) patients and OA mice. Therefore, the aim of this paper was to explore the role and mechanism of circStrn3 in osteoarthritis.

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

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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.

Infection of human cartilage with HIV in vivo has not previously been reported. Specimens of articular cartilage taken at postmortem from ten patients who were HIV-positive were examined. Two had AIDS and eight were believed to have stage-2 disease. The standard polymerase chain reaction (PCR) protocol was modified to allow semiquantitative analysis of the samples. Oligonucleotide primers labelled with . 32. P gamma-ATP were used to detect a segment of HIV DNA and a control DNA gene segment (HLA genome) to estimate the ratio of infected cells. The . 32. P-labelled PCR products were separated on acrylamide gels and visualised directly by autoradiography and computer densitometry. Infection of human cartilage in vivo was demonstrated in nine of the ten samples in which the PCR analysis was positive. The other did not react sufficiently to produce detectable radiolabelled PCR product despite repeated DNA digestion and extraction. Cartilage infected with HIV could be a potential source of HIV when used in operations

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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.

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Osteoarthritis (OA) is a common degenerative joint disease characterized by chronic inflammatory articular cartilage degradation. Long noncoding RNAs (lncRNAs) have been previously indicated to play an important role in inflammation-related diseases. Herein, the current study set out to explore the involvement of lncRNA H19 in OA.

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This study aimed to investigate the role and mechanism of meniscal cell lysate (MCL) in fibroblast-like synoviocytes (FLSs) and osteoarthritis (OA).

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After a few passages of in vitro culture, primary human articular chondrocytes undergo senescence and loss of their phenotype. Most of the available chondrocyte cell lines have been obtained from cartilage tissues different from diarthrodial joints, and their utility for osteoarthritis (OA) research is reduced. Thus, the goal of this research was the development of immortalized chondrocyte cell lines proceeded from the articular cartilage of patients with and without OA.

We analysed the effects of commonly used medications on human osteoblastic cell activity in vitro, specifically proliferation and tissue mineralisation. A list of medications was retrieved from the records of patients aged > 65 years filed in the database of the largest health maintenance organisation in our country (> two million members). Proliferation and mineralisation assays were performed on the following drugs: rosuvastatin (statin), metformin (antidiabetic), metoprolol (β-blocker), citalopram (selective serotonin reuptake inhibitor [SSRI]), and omeprazole (proton pump inhibitor (PPI)). All tested drugs significantly stimulated DNA synthesis to varying degrees, with rosuvastatin 5 µg/ml being the most effective among them (mean 225% (. sd. 20)), compared with metformin 10 µg/ml (185% (. sd.  10)), metoprolol 0.25 µg/ml (190% (. sd. 20)), citalopram 0.05 µg/ml (150% (. sd. 10)) and omeprazole 0.001 µg/ml (145% (. sd. 5)). Metformin and metoprolol (to a small extent) and rosuvastatin (to a much higher extent) inhibited cell mineralisation (85% (. sd. 5)). Our results indicate the need to evaluate the medications prescribed to patients in terms of their potential action on osteoblasts. Appropriate evaluation and prophylactic treatment (when necessary) might lower the incidence and costs associated with potential medication-induced osteoporosis. Cite this article: Bone Joint J 2013;95-B:1575–80

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In this investigation, we administered oxidative stress to nucleus pulposus cells (NPCs), recognized DNA-damage-inducible transcript 4 (DDIT4) as a component in intervertebral disc degeneration (IVDD), and devised a hydrogel capable of conveying small interfering RNA (siRNA) to IVDD.

Periprosthetic joint infection (PJI) remains an extremely challenging complication. We have focused on this issue more over the last decade than previously, but there are still many unanswered questions. We now have a workable definition that everyone should align to, but we need to continue to focus on identifying the organisms involved. Surgical strategies are evolving and care is becoming more patient-centred. There are some good studies under way. There are, however, still numerous problems to resolve, and the challenge of PJI remains a major one for the orthopaedic community. This annotation provides some up-to-date thoughts about where we are, and the way forward. There is still scope for plenty of research in this area.

Cite this article: Bone Joint J 2022;104-B(11):1193–1195.

Objectives. We sought to determine if a durable bilayer implant composed of trabecular metal with autologous periosteum on top would be suitable to reconstitute large osteochondral defects. This design would allow for secure implant fixation, subsequent integration and remodeling. Materials and Methods. Adult sheep were randomly assigned to one of three groups (n = 8/group): 1. trabecular metal/periosteal graft (TMPG), 2. trabecular metal (TM), 3. empty defect (ED). Cartilage and bone healing were assessed macroscopically, biochemically (type II collagen, sulfated glycosaminoglycan (sGAG) and double-stranded DNA (dsDNA) content) and histologically. Results. At 16 weeks post-operatively, histological scores amongst treatment groups were not statistically different (TMPG: overall 12.7, cartilage 8.6, bone 4.1; TM: overall 14.2, cartilage 9.5, bone 4.9; ED: overall 13.6, cartilage 9.1, bone 4.5). Metal scaffolds were incorporated into the surrounding bone, both in TM and TMPG. The sGAG yield was lower in the neo-cartilage regions compared with the articular cartilage (AC) controls (TMPG 20.8/AC 39.5, TM 25.6/AC 33.3, ED 32.2/AC 40.2 µg sGAG/1 mg respectively), with statistical significance being achieved for the TMPG group (p < 0.05). Hypercellularity of the neo-cartilage was found in TM and ED, as the dsDNA content was significantly higher (p < 0.05) compared with contralateral AC controls (TM 126.7/AC 71.1, ED 99.3/AC 62.8 ng dsDNA/1 mg). The highest type II collagen content was found in neo-cartilage after TM compared with TMPG and ED (TM 60%/TMPG 40%/ED 39%). Inter-treatment differences were not significant. Conclusions. TM is a highly suitable material for the reconstitution of osseous defects. TM enables excellent bony ingrowth and fast integration. However, combined with autologous periosteum, such a biocomposite failed to promote satisfactory neo-cartilage formation. Cite this article: E. H. Mrosek, H-W. Chung, J. S. Fitzsimmons, S. W. O’Driscoll, G. G. Reinholz, J. C. Schagemann. Porous tantalum biocomposites for osteochondral defect repair: A follow-up study in a sheep model. Bone Joint J 2016;5:403–411. DOI: 10.1302/2046-3758.59.BJR-2016-0070.R1

Tendon is a bradytrophic and hypovascular tissue, hence, healing remains a major challenge. The molecular key events involved in successful repair have to be unravelled to develop novel strategies that reduce the risk of unfavourable outcomes such as non-healing, adhesion formation, and scarring. This review will consider the diverse pathophysiological features of tendon-derived cells that lead to failed healing, including misrouted differentiation (e.g. de- or transdifferentiation) and premature cell senescence, as well as the loss of functional progenitors. Many of these features can be attributed to disturbed cell-extracellular matrix (ECM) or unbalanced soluble mediators involving not only resident tendon cells, but also the cross-talk with immigrating immune cell populations. Unrestrained post-traumatic inflammation could hinder successful healing. Pro-angiogenic mediators trigger hypervascularization and lead to persistence of an immature repair tissue, which does not provide sufficient mechano-competence. Tendon repair tissue needs to achieve an ECM composition, structure, strength, and stiffness that resembles the undamaged highly hierarchically ordered tendon ECM. Adequate mechano-sensation and -transduction by tendon cells orchestrate ECM synthesis, stabilization by cross-linking, and remodelling as a prerequisite for the adaptation to the increased mechanical challenges during healing. Lastly, this review will discuss, from the cell biological point of view, possible optimization strategies for augmenting Achilles tendon (AT) healing outcomes, including adapted mechanostimulation and novel approaches by restraining neoangiogenesis, modifying stem cell niche parameters, tissue engineering, the modulation of the inflammatory cells, and the application of stimulatory factors.

Cite this article: Bone Joint Res 2022;11(8):561–574.

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Osteoporosis (OP) is a metabolic bone disease, characterized by a decrease in bone mineral density (BMD). However, the research of regulatory variants has been limited for BMD. In this study, we aimed to explore novel regulatory genetic variants associated with BMD.

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Bacteriophages infect, replicate inside bacteria, and are released from the host through lysis. Here, we evaluate the effects of repetitive doses of the Staphylococcus aureus phage 191219 and gentamicin against haematogenous and early-stage biofilm implant-related infections in Galleria mellonella.

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The aim of this study was to identify factors associated with five-year cancer-related mortality in patients with limb and trunk soft-tissue sarcoma (STS) and develop and validate machine learning algorithms in order to predict five-year cancer-related mortality in these patients.

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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.

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It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth.

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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.

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Rheumatoid arthritis (RA) is a common chronic immune disease. Berberine, as its main active ingredient, was also contained in a variety of medicinal plants such as Berberaceae, Buttercup, and Rutaceae, which are widely used in digestive system diseases in traditional Chinese medicine with anti-inflammatory and antibacterial effects. The aims of this article were to explore the therapeutic effect and mechanism of berberine on rheumatoid arthritis.