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

Introduction: Infection constitutes a serious complication of joint arthroplasty, with an incidence of 1–2% after primary arthroplasty and even higher after revision procedures. Detection of low-grade infection in a prosthetic joint can often be very difficult, with huge implications on the subsequent treatment, cost and patient morbidity. Revision of an unrecognised infected arthroplasty may lead to less satisfactory results in a high proportion of cases. We utilized Polymerase Chain Reaction, a molecular biology technique to detect bacterial DNA from the synovial fluid of patients undergoing revision surgery, in comparison with conventional infection detection techniques. Methods: We prospectively assessed 81 patients undergoing revision arthroplasty (67 hips and 14 knees). Each patient was pre-operatively assessed clinically and radiologically. ESR and CRP results were noted. During revision, synovial fluid and tissue cultures were obtained and antibiotics were given only after the specimens were taken. Standard microbiology and histology study were done on tissue samples. In addition Polymerase Chain Reaction study was performed on the synovial fluid. In this method, DNA is extracted from the bacterial cell; it is polymerised and finally visualized by gel electrophoresis. Post-operatively patients were followed up at regular intervals. Diagnosis of infection included correlation between clinical, radiological and laboratory investigations along with intra-operative findings, tissue culture and histology results and a period of postoperative follow up of 12 to 36 months. Results: Eleven (13.58%) of the 81 cases that had revision arthroplasty were clinically infected. Polymerase chain reaction was positive in 30 cases, tissue cultures were positive in 8 cases and histology was positive in 10 cases for infection. PCR showed sensitivity and specificity of 0.92 and 0.72 respectively. Tissue culture showed sensitivity and specificity of 0.72 and 0.81 respectively. Histology showed sensitivity and specificity of 0.9 and 1 respectively. Discussion: Twenty out of 30 PCR positive cases did not show any clinical evidence of infection. It is unclear whether this represents contamination during surgery or in the PCR lab. Alternatively this may represent true positive PCR results in cases with low bacterial count or bacteria growing within a biofilm that can be detected only by ultrasonication of the implant and immunofluorescence methods. PCR could also be detecting non-culturable forms of bacteria or bacterial fragments. Conclusion: PCR has high sensitivity and low specificity for detection of bacterial DNA. The combination of tissue cultures and histology can still provide a reliable diagnosis of infection

The incidence of infection remains 1–2% after primary total joint arthroplasty and even higher after revision procedures in spite of advances in prophylactic antibiotics and clean air operating theatre environment. Detection of low-grade infection in a prosthetic joint can often be very difficult. None of the investigations available so far have 100% sensitivity and specificity. This has huge implications on the subsequent treatment, cost and patient morbidity. Revision of an unrecognized infected arthroplasty may lead to less satisfactory results in a high proportion of cases. We utilized Polymerase Chain Reaction, a molecular biology technique to detect bacterial DNA from the synovial fluid of patients undergoing revision surgery. We prospectively assessed 70 patients undergoing revision arthroplasty (57 hips and 13 knees). Each patient was pre operatively assessed clinically and radiologically. ESR and CRP results were noted. During revision, synovial fluid and tissue cultures from capsule, bone and bone-cement interface were obtained. None of the patients received pre or intra operative antibiotics till the specimens were taken. Standard microbiology and histology study were done on tissue samples. In addition Polymerase Chain Reaction study was done on the synovial fluid. In this method, DNA is extracted from the bacterial cell, it is polymerized and finally visualized by gel electrophoresis. Post operatively patients were followed up at regular intervals. Diagnosis of infection included correlation between clinical, radiological and laboratory investigations along with intraoperative findings, tissue culture and histology results and a period of post operative follow up (12 months to 36 months). Six (8%) of the 70 cases that had revision arthroplasty were clinically infected. Polymerase chain reaction was positive in 25 cases, tissue cultures were positive in 5 cases and histology was positive in 5 cases for infection. PCR showed sensitivity and specificity of 83% and 69% respectively. Tissue culture showed sensitivity and specificity of 83% and 100% respectively. Histology showed sensitivity and specificity of 83% and 100% respectively. 20 out of 25 PCR positive cases did not show any clinical evidence of infection. It is unclear whether this represents contamination during surgery or in the PCR lab. Alternatively this may represent true positive PCR results in cases with low bacterial count that can be detected by ultrasonication of implant and immunofluorescence methods. PCR is more sensitive in detection of bacterial DNA. However it has low specificity and combination of tissue cultures and histology can still provide a reliable diagnosis of infection

Aims. Preoperative diagnosis is important for revision surgery after prosthetic joint infection (PJI). The purpose of our study was to determine whether reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which is used to detect bacterial ribosomal RNA (rRNA) preoperatively, can reveal PJI in low volumes of aspirated fluid. Methods. We acquired joint fluid samples (JFSs) by preoperative aspiration from patients who were suspected of having a PJI and failed arthroplasty; patients with preoperative JFS volumes less than 5 ml were enrolled. RNA-based polymerase chain reaction (PCR) and bacterial culture were performed, and diagnostic efficiency was compared between the two methods.According to established Musculoskeletal Infection Society (MSIS) criteria, 21 of the 33 included patients were diagnosed with PJI. Results. RNA-based PCR exhibited 57.1% sensitivity, 91.7% specificity, 69.7% accuracy, 92.3% positive predictive value, and 55.0% negative predictive value. The corresponding values for culture were 28.6%, 83.3%, 48.5%, 75.0%, and 40.0%, respectively. A significantly higher sensitivity was thus obtained with the PCR method versus the culture method. Conclusion. In situations in which only a small JFS volume can be acquired, RNA-based PCR analysis increases the utility of preoperative puncture for patients who require revision surgery due to suspected PJI. Cite this article:Bone Joint Res. 2020;9(5):219–224

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. The preoperative diagnosis of periprosthetic joint infection (PJI) remains a challenge due to a lack of biomarkers that are both sensitive and specific. We investigated the performance characteristics of polymerase chain reaction (PCR), interleukin-6 (IL6), and calprotectin of synovial fluid in the diagnosis of PJI. Methods. We performed systematic search of PubMed, Embase, The Cochrane Library, Web of Science, and Science Direct from the date of inception of each database through to 31 May 2021. Studies which described the diagnostic accuracy of synovial fluid PCR, IL6, and calprotectin using the Musculoskeletal Infection Society criteria as the reference standard were identified. Results. Overall, 31 studies were identified: 20 described PCR, six described IL6, and five calprotectin. The sensitivity and specificity were 0.78 (95% confidence interval (CI) 0.67 to 0.86) and 0.97 (95% CI 0.94 to 0.99), respectively, for synovial PCR;, 0.86 (95% CI 0.74 to 0.92), and 0.94 (95% CI 0.90 to 0.96), respectively, for synovial IL6; and 0.94 (95% CI 0.82 to 0.98) and 0.93 (95% CI 0.85 to 0.97), respectively, for synovial calprotectin. Likelihood ratio scattergram analyses recommended clinical utility of synovial fluid PCR and IL6 as a confirmatory test only. Synovial calprotectin had utility in the exclusion and confirmation of PJI. Conclusion. Synovial fluid PCR and IL6 had low sensitivity and high specificity in the diagnosis of PJI, and is recommended to be used as confirmatory test. In contrast, synovial fluid calprotectin had both high sensitivity and specificity with utility in both the exclusion and confirmation of PJI. We recommend use of synovial fluid calprotectin studies in the preoperative workup of PJI. Cite this article: Bone Joint J 2022;104-B(3):311–320

Objectives. The objective of this study was to develop a test for the rapid (within 25 minutes) intraoperative detection of bacteria from synovial fluid to diagnose periprosthetic joint infection (PJI). Methods. The 16s rDNA test combines a polymerase chain reaction (PCR) for amplification of 16s rDNA with a lateral flow immunoassay in one fully automated system. The synovial fluid of 77 patients undergoing joint aspiration or primary or revision total hip or knee surgery was prospectively collected. The cohort was divided into a proof-of-principle cohort (n = 17) and a validation cohort (n = 60). Using the proof-of-principle cohort, an optimal cut-off for the discrimination between PJI and non-PJI samples was determined. PJI was defined as detection of the same bacterial species in a minimum of two microbiological samples, positive histology, and presence of a sinus tract or intra-articular pus. Results. The 16s rDNA test proved to be very robust and was able to provide a result in 97% of all samples within 25 minutes. The 16s rDNA test was able to diagnose PJI with a sensitivity of 87.5% and 82%, and a specificity of 100% and 89%, in the proof-of-principle and validation cohorts, respectively. The microbiological culture of synovial fluid achieved a sensitivity of 80% and a specificity of 93% in the validation cohort. Conclusion. The 16s rDNA test offers reliable intraoperative detection of all bacterial species within 25 minutes with a sensitivity and specificity comparable with those of conventional microbiological culture of synovial fluid for the detection of PJI. The 16s rDNA test performance is independent of possible blood contamination, culture time and bacterial species. Cite this article: V. Janz, J. Schoon, C. Morgenstern, B. Preininger, S. Reinke, G. Duda, A. Breitbach, C. F. Perka, S. Geissler. Rapid detection of periprosthetic joint infection using a combination of 16s rDNA polymerase chain reaction and lateral flow immunoassay: A Pilot Study. Bone Joint Res 2018;7:12–19. DOI: 10.1302/2046-3758.71.BJR-2017-0103.R2

Tuberculosis of the spine is very common and it is important to do confirmatory testing. This retrospective study involved 40 patients in whom tuberculosis of the spine was diagnosed after clinical examination and investigations. All underwent decompression of the spine for neurological fallout. Intra-operatively, histological tissue, MCS and polymerase chain reaction (PCR) were assessed. PCR was positive in only 50% of the patients, but was complementary to histology and MCS

Aims. This study aimed to assess the performance of an automated multiplex polymerase chain reaction (mPCR) technique for rapid diagnosis of native joint septic arthritis. Patients and Methods. Consecutive patients with suspected septic arthritis undergoing aseptic diagnostic joint aspiration were included. The aspirate was used for analysis by mPCR and conventional microbiological analysis. A joint was classed as septic according to modified Newman criteria. Based on receiver operating characteristic (ROC) analysis, the area under the ROC curve (AUC) values of the mPCR and the synovial fluid culture were compared using the z-test. A total of 72 out of 76 consecutive patients (33 women, 39 men; mean age 64 years (22 to 92)) with suspected septic arthritis were included in this study. Results. Of 72 patients, 42 (58%) were deemed to have septic joints. The sensitivity of mPCR and synovial fluid culture was 38% and 29%, respectively. No significant differences were found between the AUCs of both techniques (p = 0.138). A strong concordance of 89% (Cohen’s kappa: 0.65) was shown. The mPCR failed to detect Staphylococcus aureus (n = 1) and Streptococcus pneumoniae (n = 1; no primer included in the mPCR), whereas the synovial fluid culture missed six microorganisms (positive mPCR: S. aureus (n = 2), Cutibacterium acnes (n = 3), coagulase-negative staphylococci (n = 2)). Conclusion. The automated mPCR showed at least a similar performance to the synovial fluid culture (the current benchmark) in diagnosing septic arthritis, having the great advantage of a shorter turnaround time (within five hours). Cite this article: Bone Joint J 2019;101-B:288–296

Introduction:. Skeletal TB has a paucibacillary nature. It is often found in poorly accessible areas for biopsy purposes. Retrieved samples may have a poor representation of the underlying disease process. Additionally, patients have normally commenced anti-tubercular medication that further decreases the number of bacilli. This has resulted in poor sensitivity and specificity outcomes for the tests that are traditionally done. The polymerase chain reaction (PCR) has been proven to be a useful test for the demonstration of extrapulmonary TB. It has a high specificity and sensitivity. Objective:. The study measures the sensitivity and specificity of PCR done on fresh biopsies from patients suspected of a tuberculous spinal infection. Method and Results:. A retrospective review of results was done of spinal tissue biopsies. A total of 30 consecutive patients were identified. There were 15 males (mean age = 40,3 yrs) and 15 females (mean age = 45,8 yrs). 18 of the patients were HIV positive. Acid fast bacilli were demonstrated in 4 (13,33%) patients by staining. Culture was positive in 10 (33,33%), in which 2 had commenced antitubercular therapy. Histology reported features in keeping with tuberculosis in 9 (30%) patients. Furthermore, multiple myeloma, adenocarcinoma, osteomyelitis and thyroid carcinoma was reported. PCR was positive in 12 (40%) patients. Sensitivity of PCR was calculated as 85,7% whilst specificity was 72,7% (the amount correctly classified was 77,8%). Only 4 patients had positive tests for all 4 methods used for diagnosing tuberculosis. Conclusion:. Tuberculosis should always be considered in spinal lesions. PCR provides a quick and effective means of identifying it. This allows early commencement of anti-tubercular treatment

Introduction: Although there is evidence that laminar flow operating theatres (LFOTs) can reduce the incidence of wound infection over standard operating theatres (STOTs) when no routine peri-operative antibiotics were used, the evidence for the use with concurrent parenteral antibiotics is less compelling. A number of prior studies have compared the bacterial load observed in LFOTs and STOTs by wound culture and air sampling during surgery. However many organisms responsible for low grade infection after THR are not readily identified on routine culture and may be detectable only by more sensitive techniques such as the polymerase chain reaction (PCR), a molecular biology test for the presence of bacterial DNA. The purpose of this study was to compare the wound contamination rate during THRs performed in STOT with that in LFOTs using PCR. Method: Patients undergoing primary THR for osteoarthritis without a history of joint infection were recruited for the study. Surgery was performed in either STOTs or LFOTs, using identical skin preparation solutions, surgical drapes and operating attire. Specimens of the deep tissue, taken at the beginning and end of surgery, were each immediately separated into two sterile containers, one sent for culture (aerobic, anaerobic and enriched meat broth) and the other frozen at minus 80 degrees Celsius for PCR at a later date. Results: In each theatre type, 40 specimens from 20 THRs were analysed by both PCR and culture (80 specimens and 40 THRs in total). Using PCR, bacterial DNA was identified on 12 of 40 specimens (30%) from STOTs. Of these 12, three were taken at the start of surgery and nine at the end of the surgery, equivalent to a 45% wound contamination rate (9 of 20). Only two specimens (5%), both taken at the end of surgery, were positive on enriched culture. In LFOTs, bacterial DNA was identified by PCR on eight of 40 specimens (20%). Of these eight, two were taken at the start of surgery and six at the end of surgery, equivalent to a 30% wound contamination rate (6 of 20). None of the specimens were positive on enriched culture. Discussions: We concluded that wound contamination of primary THR occurs frequently in both STOTs and LFOTs. Although STOTs showed evidence of more frequent wound contamination than LFOTs, with the numbers available, no significant difference was detected. These data are important in that they confirm that continued vigilance to technique continue to be important as significant wound contamination can occur despite the use of ultra clean air operating theatres

Aim. We used a polymerase chain reaction (PCR) lateral flow assay1) to rapidly diagnose joint infection. We evaluated the usefulness of multiplex-PCR (PCR lateral flow assay: PCR-LF) using detailed clinical data. Method. A total of 35 synovial fluid samples were collected from 26 patients in whom bacterial infection was suspected, including 22 from knee joints, 11 from hip joints, and 2 from other joints. After purifying the DNA from the samples, multiplex PCR targeting two MRSA-associated genes (femA and mecA) and the bacterial 16S rRNA gene was performed. Amplified gene fragments were specifically detected with DNA probes immobilized on stick devices through DNA-DNA hybridization and visualization, enabling diagnosis of MRSA, MSSA, MRCNS, gram-positive, and/or gram-negative bacterial infection. Genetic identification of bacteria by determining the 16S rRNA gene sequence was also performed using multiplex PCR-positive samples. Finally, the usefulness of our PCR-LF method was evaluated using detailed clinical data. Results. The results of PCR-LF were 9 gram-positive and 1 gram-negative bacterial infections. Eleven bacterial species were identified based on 16S rRNA gene sequences. Ten (90.9%)of the eleven samples (bacterial species) were identified using our PCR-LF. Five samples were detected in bacterial cultures; two are MSSA, one is Streptococcus agalactiae, one is Escherichia coli, one is Prevotella oralis. We diagnosed 6 samples as clinical infections. Therefore, the sensitivity and specificity of the culture tests were 83% and 100%, respectively, while for PCR-LF, these values were 83% and 83%. Conclusions. PCR-LF is highly sensitive and effective for the rapid diagnosis of joint infection; however, dead bacteria may also be detected. Moreover, because the target bacterial species are limited, clinical diagnosis based on the results of multiple examinations is necessary

The lack of an accurate, rapid diagnostic test for mycobacterium tuberculosis (TB) is a major handicap in the management of spinal TB. GeneXpert, a new, rapid molecular diagnostic test is recommended as the first line investigation for suspected pulmonary TB in areas with a high prevalence of HIV or drug resistance, yet it has not been validated for the diagnosis of musculoskeletal TB.

The aim of this study was to assess the accuracy of GeneXpert in diagnosing spinal TB.

A prospective clinical study of 69 consecutive adults with suspected spinal TB was conducted at a tertiary hospital in an area with the highest incidence and prevalence of TB in the world. GeneXpert was used on tissue samples of the enrolled patients and its diagnostic accuracy compared with a reference standard of tissue in liquid culture. A total of 71 spine samples from 69 patients (two re-biopsies) were included in the study.

The GeneXpert test showed a sensitivity of 95.6% and specificity of 96.2% for spinal TB. The results of the GeneXpert test were available within 48 hours compared with a median of 35 days (IQR 15 to 43) for cultures. All cases of multi-drug resistant TB (MDR TB) were diagnosed accurately with the GeneXpert test. The MDR TB rate was 5.8%.

Cite this article: Bone Joint J 2014;96-B:1366–9.

Background: Chromosomal translocation are frequently observed in leukemias and sarcomas; these translocations break specific genes in the involved chromosomes and create novel chimeric genes that encode a fusion protein. Advances in these techniques have increased knowledge of the genes involved in tumoral development; molecular techniques have enabled more precise diagnosis as well as identification of new prognostic factors.

Aims: To explore the use of Reverse-Transcriptase Poly-merase Chain Reaction (RT-PCR) assay for detecting fusion transcripts in a series of Soft Tissue Sarcomas (STS) and compare the results with histopathologic diagnosis.

Material and methods: We studied 80 biopsies performed at Orthopaedic Oncology and Reconstructive Surgery Department, CTO-CFR-M.Adelaide Hospital Turin Italy, with clinical suspect of STS. Histological diagnosis was obtained contemporary to evaluation of chimeric transcripts detected by RT-PCR. cDNA were PCR amplified using primer specific for each sarcoma. Paraffin-embedded tissue samples were not used because the poor quality of the extracted RNA may give wrong positive results. Results Histology confirmed 21 Ewing Sarcoma (ES), 14 Synovial sarcoma (SS), 7 Mixoid liposarcoma (M-LPS), 4 Extraskeletal Myxoid Chondrosarcoma (E-MCDS), 4 Dermatofibrosarcoma protuberans (DFSP), 10 Rhabdo-myosarcoma, 10 Leiomyosarcoma. Of the 21 tumors diagnosed as ES, 21 (100%) expressed EWS-FLI1 chimeric transcripts. All 14 SS were positive for SYT-SSX fusion transcripts. Of the 7 cases with diagnosis of M-LPS, six were positive for EWS-CHOP transcripts; of the four cases of E-MCS 3 were positive for EWS-CHN fusion transcripts. All 4 DFSP were positive for COL1A1-PDGFB transcripts. Expression of Myo-D1, tested in ten cases of Rhabdomyosarcoma, was positive while in ten cases of Leiomyosarcoma no expression of Myo-D1 was detected by RT-PCR Ten cases were non sarcoma and negative for molecular biology.

Conclusion These results demonstrate a strong concordance between the standard histopathological diagnosis and molecular results. These techniques could be a useful method to increase the quality of histologic diagnosis in difficult cases.

We investigated the use of PCR (the Polymerase Chain Reaction) to detect the presence of infection in a group of patients undergoing revision arthroplasty for loose TJR (total joint replacement), compared to internationally agreed criteria used as the ‘gold standard’ for infection.

We prospectively tested samples taken from 108 patients undergoing revision arthroplasty (76 hips, 32 knees). Antibiotics were omitted prior to obtaining samples. DNA was extracted by 2 methods – a previously published technique (reference) and a commercial extraction kit (Qiagen^®). PCR involved amplification of an 882 base pair segment of the universal bacterial 16S RNA gene. During revision arthroplasty multiple specimens were taken from around the joint for microbiological and histological examination and the presence or absence of pus was noted. The patient was deemed to be infected if one of the following criteria was found: presence of a sinus pre-operatively; 2 or more intra-operative cultures positive for the same organism; an acute inflammatory response on histology; pus in the joint at revision.

Using the published DNA extraction technique PCR had a sensitivity of 50%, specificity of 93%, positive predictive value of 67% and negative predictive value of 88%. Using commercial extraction the sensitivity improved to 60%, specificity to 98%, positive predictive value to 90% and negative predictive value to 90%.

The previous report stated that PCR had a high sensitivity but a low specificity for detecting low grade infection. However, when using the published technique we found the opposite results – a moderate sensitivity and a high specificity. Introduction of a new DNA extraction technique improved the sensitivity. The refined PCR technique had a high accuracy, but further work is needed to improve sensitivity before we would recommend this method for routine clinical use.

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. 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. Astragalus polysaccharide (APS) participates in various processes, such as the enhancement of immunity and inhibition of tumours. APS can affect osteoporosis (OP) by regulating the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). This study was designed to elucidate the mechanism of APS in hBMSC proliferation and osteoblast differentiation. Methods. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the expression of microRNA (miR)-760 and ankyrin repeat and FYVE domain containing 1 (ANKFY1) in OP tissues and hBMSCs. Cell viability was measured using the Cell Counting Kit-8 assay. The expression of cyclin D1 and osteogenic marker genes (osteocalcin (OCN), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2)) was evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mineral deposits were detected through Alizarin Red S staining. In addition, Western blotting was performed to detect the ANKFY1 protein levels following the regulation of miR-760. The relationship between miR-760 and ANKFY1 was determined using a luciferase reporter assay. Results. The expression of miR-760 was upregulated in OP tissues, whereas ANKFY1 expression was downregulated. APS stimulated the differentiation and proliferation of hBMSCs by: increasing their viability; upregulating the expression levels of cyclin D1, ALP, OCN, and RUNX2; and inducing osteoblast mineralization. Moreover, APS downregulated the expression of miR-760. Overexpression of miR-760 was found to inhibit the promotive effect of APS on hBMSC differentiation and proliferation, while knockdown of miR-760 had the opposite effect. ANKFY1 was found to be the direct target of miR-760. Additionally, ANKFY1 participated in the APS-mediated regulation of miR-760 function in hBMSCs. Conclusion. APS promotes the osteogenic differentiation and proliferation of hBMSCs. Moreover, APS alleviates the effects of OP by downregulating miR-760 and upregulating ANKFY1 expression. Cite this article: Bone Joint Res 2023;12(8):476–485

Aims. 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. Methods. Porcine chondrocytes were treated with vehicle or various doses of suramin. The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN); COL1A1; COL10A1; SRY-box transcription factor 9 (SOX9); nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX); interleukin (IL)-1β; tumour necrosis factor alpha (TNFα); IL-8; and matrix metallopeptidase 13 (MMP-13) in chondrocytes at both messenger RNA (mRNA) and protein levels was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. In addition, the supplementation of suramin to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by biochemical analyses and immunofluorescence, as well as by immunohistochemistry. The expression of reactive oxygen species (ROS) and NOX activity were assessed by luciferase reporter gene assay, immunofluorescence analysis, and flow cytometry. Mutagenesis analysis, Alcian blue staining, reverse transcriptase polymerase chain reaction (RT-PCR), and western blot assay were used to determine whether p67. phox. was involved in suramin-enhanced chondrocyte phenotype maintenance. Results. Suramin enhanced the COL2A1 and ACAN expression and lowered COL1A1 synthesis. Also, in 3D pellet culture GAG and COL2A1 production was significantly higher in pellets consisting of chondrocytes expanded with suramin compared to controls. Surprisingly, suramin also increased ROS generation, which is largely caused by enhanced NOX (p67. phox. ) activity and membrane translocation. Overexpression of p67. phox. but not p67. phox. AD (deleting amino acid (a.a) 199 to 212) mutant, which does not support ROS production in chondrocytes, significantly enhanced chondrocyte phenotype maintenance, SOX9 expression, and AKT (S473) phosphorylation. Knockdown of p67. phox. with its specific short hairpin (sh) RNA (shRNA) abolished the suramin-induced effects. Moreover, when these cells were treated with the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor LY294002 or shRNA of AKT1, p67. phox. -induced COL2A1 and ACAN expression was significantly inhibited. Conclusion. Suramin could redifferentiate dedifferentiated chondrocytes dependent on p67. phox. activation, which is mediated by the PI3K/AKT/SOX9 signalling pathway. Cite this article: Bone Joint Res 2022;11(10):723–738

Aims. 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. Methods. Minus RNA sequencing, fluorescence in situ hybridization, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of circStrn3 in human and mouse OA cartilage tissues and chondrocytes. Chondrocytes were then stimulated to secrete exosomal miR-9-5p by cyclic tensile strain. Intra-articular injection of exosomal miR-9-5p into the model induced by destabilized medial meniscus (DMM) surgery was conducted to alleviate OA progression. Results. Tensile strain could decrease the expression of circStrn3 in chondrocytes. CircStrn3 expression was significantly decreased in human and mouse OA cartilage tissues and chondrocytes. CircStrn3 could inhibit matrix metabolism of chondrocytes through competitively ‘sponging’ miRNA-9-5p targeting Kruppel-like factor 5 (KLF5), indicating that the decrease in circStrn3 might be a protective factor in mechanical instability-induced OA. The tensile strain stimulated chondrocytes to secrete exosomal miR-9-5p. Exosomes with high miR-9-5p expression from chondrocytes could inhibit osteoblast differentiation by targeting KLF5. Intra-articular injection of exosomal miR-9-5p alleviated the progression of OA induced by destabilized medial meniscus surgery in mice. Conclusion. Taken together, these results demonstrate that reduction of circStrn3 causes an increase in miR-9-5p, which acts as a protective factor in mechanical instability-induced OA, and provides a novel mechanism of communication among joint components and a potential application for the treatment of OA. Cite this article: Bone Joint Res 2023;12(1):33–45