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

Aims. This study intended to investigate the effect of vericiguat (VIT) on titanium rod osseointegration in aged rats with iron overload, and also explore the role of VIT in osteoblast and osteoclast differentiation. Methods. In this study, 60 rats were included in a titanium rod implantation model and underwent subsequent guanylate cyclase treatment. Imaging, histology, and biomechanics were used to evaluate the osseointegration of rats in each group. First, the impact of VIT on bone integration in aged rats with iron overload was investigated. Subsequently, VIT was employed to modulate the differentiation of MC3T3-E1 cells and RAW264.7 cells under conditions of iron overload. Results. Utilizing an OVX rat model, we observed significant alterations in bone mass and osseointegration due to VIT administration in aged rats with iron overload. The observed effects were concomitant with reductions in bone metabolism, oxidative stress, and inflammation. To elucidate whether these effects are associated with osteoclast and osteoblast activity, we conducted in vitro experiments using MC3T3-E1 cells and RAW264.7 cells. Our findings indicate that iron accumulation suppressed the activity of MC3T3-E1 while enhancing RAW264.7 function. Furthermore, iron overload significantly decreased oxidative stress levels; however, these detrimental effects can be mitigated by VIT treatment. Conclusion. Collectively, our data provide compelling evidence that VIT has the potential to reverse the deleterious consequences of iron overload on osseointegration and bone mass during ageing. Cite this article: Bone Joint Res 2024;13(9):427–440

Aims. This study examined whether systemic administration of melatonin would have different effects on osseointegration in ovariectomized (OVX) rats, depending on whether this was administered during the day or night. Methods. In this study, a titanium rod was implanted in the medullary cavity of one femoral metaphysis in OVX rats, and then the rats were randomly divided into four groups: Sham group (Sham, n = 10), OVX rat group (OVX, n = 10), melatonin day treatment group (OVX + MD, n = 10), and melatonin night treatment group (OVX + MN, n = 10). The OVX + MD and OVX + MN rats were treated with 30 mg/kg/day melatonin at 9 am and 9 pm, respectively, for 12 weeks. At the end of the research, the rats were killed to obtain bilateral femora and blood samples for evaluation. Results. Micro-CT and histological evaluation showed that the bone microscopic parameters of femoral metaphysis trabecular bone and bone tissue around the titanium rod in the OVX + MD group demonstrated higher bone mineral density, bone volume fraction, trabecular number, connective density, trabecular thickness, and lower trabecular speculation (p = 0.004) than the OVX + MN group. Moreover, the biomechanical parameters of the OVX + MD group showed higher pull-out test and three-point bending test values, including fixation strength, interface stiffness, energy to failure, energy at break, ultimate load, and elastic modulus (p = 0.012) than the OVX + MN group. In addition, the bone metabolism index and oxidative stress indicators of the OVX + MD group show lower values of Type I collagen cross-linked C-telopeptide, procollagen type 1 N propeptide, and malondialdehyde (p = 0.013), and higher values of TAC and SOD (p = 0.002) compared with the OVX + MN group. Conclusion. The results of our study suggest that systemic administration with melatonin at 9 am may improve the initial osseointegration of titanium rods under osteoporotic conditions more effectively than administration at 9 pm. Cite this article: Bone Joint Res 2022;11(11):751–762

Aims. Exosomes (exo) are involved in the progression of osteoarthritis (OA). This study aimed to investigate the function of dysfunctional chondrocyte-derived exo (DC-exo) on OA in rats and rat macrophages. Methods. Rat-derived chondrocytes were isolated, and DCs induced with interleukin (IL)-1β were used for exo isolation. Rats with OA (n = 36) or macrophages were treated with DC-exo or phosphate-buffered saline (PBS). Macrophage polarization and autophagy, and degradation and chondrocyte activity of cartilage tissues, were examined. RNA sequencing was used to detect genes differentially expressed in DC-exo, followed by RNA pull-down and ribonucleoprotein immunoprecipitation (RIP). Long non-coding RNA osteoarthritis non-coding transcript (OANCT) and phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5) were depleted in DC-exo-treated macrophages and OA rats, in order to observe macrophage polarization and cartilage degradation. The PI3K/AKT/mammalian target of rapamycin (mTOR) pathway activity in cells and tissues was measured using western blot. Results. DC-exo inhibited macrophage autophagy (p = 0.002) and promoted M1 macrophage polarization (p = 0.002). DC-exo at 20 μg/ml induced collagen degradation (p < 0.001) and inflammatory cell infiltration (p = 0.023) in rats. OANCT was elevated in DC (p < 0.001) and in cartilage tissues of OA patients (p < 0.001), and positively correlated with patients’ Kellgren-Lawrence grade (p < 0.001). PIK3R5 was increased in DC-exo-treated cartilage tissues (p < 0.001), and OANCT bound to fat mass and obesity-associated protein (FTO) (p < 0.001). FTO bound to PIK3R5 (p < 0.001) to inhibit the stability of PIK3R5 messenger RNA (mRNA) (p < 0.001) and disrupt the PI3K/AKT/mTOR pathway (p < 0.001). Conclusion. Exosomal OANCT from DC could bind to FTO protein, thereby maintaining the mRNA stability of PIK3R5, further activating the PI3K/AKT/mTOR pathway to exacerbate OA. Cite this article: Bone Joint Res 2022;11(9):652–668

Objectives. This study aimed to assess the effect of age and osteoporosis on the proliferative and differentiating capacity of bone-marrow-derived mesenchymal stem cells (MSCs) in female rats. We also discuss the role of these factors on expression and migration of cells along the C-X-C chemokine receptor type 4 (CXCR-4) / stromal derived factor 1 (SDF-1) axis. Methods. Mesenchymal stem cells were harvested from the femora of young, adult, and osteopenic Wistar rats. Cluster of differentiation (CD) marker and CXCR-4 expression was measured using flow cytometry. Cellular proliferation was measured using Alamar Blue, osteogenic differentiation was measured using alkaline phosphatase expression and alizarin red production, and adipogenic differentiation was measured using Oil red O. Cells were incubated in Boyden chambers to quantify their migration towards SDF-1. Data was analyzed using a Student’s t-test, where p-values < 0.05 were considered significant. Results. CD marker expression and proliferation of the MSCs from the three groups was not significantly different. The young MSCs demonstrated significantly increased differentiation into bone and fat and superior migration towards SDF-1. The migration of SDF-1 doubled with young rats compared with the adult rats (p = 0.023) and it was four times higher when compared with cells isolated from ovariectomized (OVX) osteopenic rats (p = 0.013). Conclusion. Young rat MSCs are significantly more responsive to osteogenic differentiation, and, contrary to other studies, also demonstrated increased adipogenic differentiation compared with cells from adult and ostopenic rats. Young-rat-derived cells also showed superior migration towards SDF-1 compared with MSCs from OVX and adult control rats. Cite this article: A. Sanghani-Kerai, L. Osagie-Clouard, G. Blunn, M. Coathup. The influence of age and osteoporosis on bone marrow stem cells from rats. Bone Joint Res 2018;7:289–297. DOI: 10.1302/2046-3758.74.BJR-2017-0302.R1

Aims. Ultrasound-guided injection techniques are expected to enhance therapeutic efficacy for skeletal muscle injuries and disorders, but basic knowledge is lacking. The purpose of this study was to examine the diagnostic accuracy of ultrasound for abnormal skeletal muscle lesions, and to examine the distribution patterns of solution and cells injected into abnormal muscle lesions under ultrasound guidance. Methods. A cardiotoxin (CTX)-induced muscle injury model was used. Briefly, CTX was injected into tibialis anterior muscle in rats under ultrasound observation. First, the diagnostic accuracy of abnormal muscle lesions on ultrasound was examined by comparing ultrasound findings and histology. Next, Fast Green solution and green fluorescent protein (GFP)-labelled cells were simultaneously injected into the abnormal muscle lesions under ultrasound guidance, and their distribution was evaluated. Results. Evaluation of short-axis ultrasound images and cross-sectional histological staining showed a strong correlation (r = 0.927; p < 0.001) between the maximum muscle damage area in ultrasound and haematoxylin and eosin (H&E) staining evaluations. Histological analysis showed that ultrasound-guided injection could successfully deliver Fast Green solution around the myofibres at the site of injury. In contrast, the distribution of injected cells was very localized compared to the area stained with Fast Green. Conclusion. This experimental animal study demonstrated the potential of ultrasound to quantitatively visualize abnormalities of skeletal muscle. It also showed that ultrasound-guided injections allowed for highly accurate distribution of solution and cells in abnormal muscle tissue, but the patterns of solution and cell distribution were markedly different. Although future studies using a more clinically relevant model are necessary, these results are important findings when considering biological therapies for skeletal muscle injuries and disorders. Cite this article: Bone Joint Res 2025;14(1):33–41

Objectives. Diabetes mellitus (DM) is known to impair fracture healing. Increasing evidence suggests that some microRNA (miRNA) is involved in the pathophysiology of diabetes and its complications. We hypothesized that the functions of miRNA and changes to their patterns of expression may be implicated in the pathogenesis of impaired fracture healing in DM. Methods. Closed transverse fractures were created in the femurs of 116 rats, with half assigned to the DM group and half assigned to the control group. Rats with DM were induced by a single intraperitoneal injection of streptozotocin. At post-fracture days five, seven, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture days five and 11. For further analysis, real-time polymerase chain reaction (PCR) analysis was performed at each timepoint. Results. Microarray analysis showed that there were 14 miRNAs at day five and 17 miRNAs at day 11, with a greater than twofold change in the DM group compared with the control group. Among these types of miRNA, five were selected based on a comparative and extended literature review. Real-time PCR analysis revealed that five types of miRNA (miR-140-3p, miR-140-5p, miR-181a-1-3p, miR-210-3p, and miR-222-3p) were differentially expressed with changing patterns of expression during fracture healing in diabetic rats compared with controls. Conclusions. Our findings provide information to further understand the pathology of impaired fracture healing in a diabetic rat model. These results may allow the potential development of molecular therapy using miRNA for the treatment of impaired fracture healing in patients with DM. Cite this article: S. Takahara, S. Y. Lee, T. Iwakura, K. Oe, T. Fukui, E. Okumachi, T. Waki, M. Arakura, Y. Sakai, K. Nishida, R. Kuroda, T. Niikura. Altered expression of microRNA during fracture healing in diabetic rats. Bone Joint Res 2018;7:139–147. DOI: 10.1302/2046-3758.72.BJR-2017-0082.R1

Aims. Post-traumatic osteoarthritis (PTOA) is a subset of osteoarthritis (OA). The gut microbiome is shown to be involved in OA. However, the effect of exercise on gut microbiome in PTOA remains elusive. Methods. A total of 18 eight-week Sprague-Dawley rats were assigned into three groups: Sham/sedentary (Sham/Sed), PTOA/sedentary (PTOA/Sed), and PTOA/treadmill-walking (PTOA/TW). PTOA model was induced by transection of the anterior cruciate ligament (ACLT) and the destabilization of the medial meniscus (DMM). Treadmill-walking (15 m/min, 30 min/d, five days/week for eight weeks) was employed in the PTOA/TW group. The response of cartilage, subchondral bone, serology, and gut microbiome and their correlations were assessed. Results. Eight-week treadmill-walking was effective at maintaining the integrity of cartilage-subchondral bone unit and reducing the elevated systematic inflammation factors and microbiome-derived metabolites. Furthermore, 16S ribosomal ribonucleic acid (rRNA) sequencing showed disease-relevant microbial shifts in PTOA animals, characterized by the decreased abundance of phylum TM7 and the increase of phylum Fusobacteria. At the genus level, the abundance of Lactobacillus, Turicibacter, Adlercreutzia, and Cetobacterium were increased in the PTOA animals, while the increase of Adlercreutzia and Cetobacterium was weakened as a response to exercise. The correlation analysis showed that genus Lactobacillus and Adlercreutzia were correlated to the structural OA phenotypes, while phylum Fusobacteria and genus Cetobacterium may contribute to the effects of exercise on the diminishment of serological inflammatory factors. Conclusion. Exercise is effective at maintaining the integrity of cartilage-subchondral bone unit, and the exercise-induced modification of disease-relevant microbial shifts is potentially involved in the mechanisms of exercise-induced amelioration of PTOA. Cite this article: Bone Joint Res 2022;11(4):214–225

Aims. To fully verify the reliability and reproducibility of an experimental method in generating standardized micromotion for the rat femur fracture model. Methods. A modularized experimental device has been developed that allows rat models to be used instead of large animal models, with the aim of reducing systematic errors and time and money constraints on grouping. The bench test was used to determine the difference between the measured and set values of the micromotion produced by this device under different simulated loading weights. The displacement of the fixator under different loading conditions was measured by compression tests, which was used to simulate the unexpected micromotion caused by the rat’s ambulation. In vivo preliminary experiments with a small sample size were used to test the feasibility and effectiveness of the whole experimental scheme and surgical scheme. Results. The bench test showed that a weight loading < 500 g did not affect the operation of experimental device. The compression test demonstrated that the stiffness of the device was sufficient to keep the uncontrollable motion between fracture ends, resulting from the rat’s daily activities, within 1% strain. In vivo results on 15 rats prove that the device works reliably, without overburdening the experimental animals, and provides standardized micromotion reproductively at the fracture site according to the set parameters. Conclusion. Our device was able to investigate the effect of micromotion parameters on fracture healing by generating standardized micromotion to small animal models. Cite this article: Bone Joint Res 2021;10(11):714–722

Aims. Abnormal lipid metabolism is involved in the development of osteoarthritis (OA). Growth differentiation factor 11 (GDF11) is crucial in inhibiting the differentiation of bone marrow mesenchymal stem cells into adipocytes. However, whether GDF11 participates in the abnormal adipogenesis of chondrocytes in OA cartilage is still unclear. Methods. Six-week-old female mice were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical staining (IHC), and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. Primary condylar chondrocytes of rats were stimulated with fluid flow shear stress (FFSS) and collected for oil red staining, immunofluorescence staining, qRT-PCR, and immunoprecipitation analysis. Results. Abnormal adipogenesis, characterized by increased expression of CCAAT/enhancer-binding protein α (CEBPα), fatty acid binding protein 4 (FABP4), Perilipin1, Adiponectin (AdipoQ), and peroxisome proliferator-activated receptor γ (PPARγ), was enhanced in the degenerative cartilage of TMJ OA in UAC mice, accompanied by decreased expression of GDF11. After FFSS stimulation, there were fat droplets in the cytoplasm of cultured cells with increased expression of PPARγ, CEBPα, FABP4, Perilipin1, and AdipoQ and decreased expression of GDF11. Exogenous GDF11 inhibited increased lipid droplets and expression of AdipoQ, CEBPα, and FABP4 induced by FFSS stimulation. GDF11 did not affect the change in PPARγ expression under FFSS, but promoted its post-translational modification by small ubiquitin-related modifier (SUMOylation). Local injection of GDF11 alleviated TMJ OA-related cartilage degeneration and abnormal adipogenesis in UAC mice. Conclusion. Abnormal adipogenesis of chondrocytes and decreased GDF11 expression were observed in degenerative cartilage of TMJ OA. GDF11 supplementation effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration. GDF11 may inhibit the abnormal adipogenesis of chondrocytes by affecting the SUMOylation of PPARγ. Cite this article: Bone Joint Res 2022;11(7):453–464

Objectives. This study aims to evaluate if micro-CT can work as a method for the 3D assessment and analysis of cancellous bone by comparing micro-CT with undecalcified histological sections in OVX rats. Methods. The mandible and tibia of sham, ovariectomised (OVX) and zoledronate-injected ovariectomised (OVX-ZOL) rats were assessed morphometrically. Specimens were scanned by micro-CT. Undecalcified histological sections were manufactured from the specimen scanned by micro-CT and stained with haematoxylin and eosin. Bivariate linear regressions and one-way analysis of variance were undertaken for statistics using SPSS 16.0.1 software. Results. There were highly significant correlations between undecalcified histological sections and micro-CT for all parameters (bone volume density (BV/TV), bone surface density (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp))in the mandible and tibia. Bone histomorphometric parameters analysed by both methods exhibited significant differences among sham, OVX, and OVX-ZOL groups. There were significant correlations between mandible and tibia in BV/TV, BS/BV, and Tb.Sp. Conclusions. Micro-CT is a complementary tool to histological sections in basic research that could improve our understanding of bone histomorphometry. The mandible can be used as an effective site to assess bone morphometry of OVX or metabolic bone disease rat models. Cite this article: H. Liu, W. Li, Y. S. Liu, Y. S. Zhou. Bone micro-architectural analysis of mandible and tibia in ovariectomised rats: A quantitative structural comparison between undecalcified histological sections and micro-CT. Bone Joint Res 2016;5:253–262

Aims. A growing number of fractures progress to delayed or nonunion, causing significant morbidity and socioeconomic impact. Localized delivery of stem cells and subcutaneous parathyroid hormone (PTH) has been shown individually to accelerate bony regeneration. This study aimed to combine the therapies with the aim of upregulating fracture healing. Methods. A 1.5 mm femoral osteotomy (delayed union model) was created in 48 female juvenile Wistar rats, aged six to nine months, and stabilized using an external fixator. At day 0, animals were treated with intrafracture injections of 1 × 10. 6. cells/kg bone marrow mesenchymal stem cells (MSCs) suspended in fibrin, daily subcutaneous injections of high (100 μg/kg) or low (25 μg/kg) dose PTH 1-34, or a combination of PTH and MSCs. A group with an empty gap served as a control. Five weeks post-surgery, the femur was excised for radiological, histomorphometric, micro-CT, and mechanical analysis. Results. Combination therapy treatment led to increased callus formation compared to controls. In the high-dose combination group there was significantly greater mineralized tissue volume and trabecular parameters compared to controls (p = 0.039). This translated to significantly improved stiffness (and ultimate load to failure (p = 0.049). The high-dose combination therapy group had the most significant improvement in mean modified Radiographic Union Score for Tibia fractures (RUST) compared to controls (13.8 (SD 1.3) vs 5.8 (SD 0.5)). All groups demonstrated significant increases in the radiological scores – RUST and Allen score – histologically compared to controls. Conclusion. We demonstrate the beneficial effect of localized MSC injections on fracture healing combined with low- or high-dose teriparatide, with efficacy dependent on PTH dose. Cite this article: Bone Joint Res 2021;10(10):659–667

Objectives. Mesenchymal stem cells (MSCs) are of growing interest in terms of bone regeneration. Most preclinical trials utilize bone-marrow-derived mesenchymal stem cells (bMSCs), although this is not without isolation and expansion difficulties. The aim of this study was: to compare the characteristics of bMSCs and adipose-derived mesenchymal stem cells (AdMSCs) from juvenile, adult, and ovarectomized (OVX) rats; and to assess the effect of human parathyroid hormone (hPTH) 1-34 on their osteogenic potential and migration to stromal cell-derived factor-1 (SDF-1). Methods. Cells were isolated from the adipose and bone marrow of juvenile, adult, and previously OVX Wistar rats, and were characterized with flow cytometry, proliferation assays, osteogenic and adipogenic differentiation, and migration to SDF-1. Experiments were repeated with and without intermittent hPTH 1-34. Results. Juvenile and adult MSCs demonstrated significantly increased osteogenic and adipogenic differentiation and superior migration towards SDF-1 compared with OVX groups; this was the case for AdMSCs and bMSCs equally. Parathyroid hormone (PTH) increased parameters of osteogenic differentiation and migration to SDF-1. This was significant for all cell types, although it had the most significant effect on cells derived from OVX animals. bMSCs from all groups showed increased mineralization and migration to SDF-1 compared with AdMSCs. Conclusion. Juvenile MSCs showed significantly greater migration to SDF-1 and significantly greater osteogenic and adipogenic differentiation compared with cells from osteopenic rats; this was true for bMSCs and AdMSCs. The addition of PTH increased these characteristics, with the most significant effect on cells derived from OVX animals, further illustrating possible clinical application of both PTH and MSCs in bone regenerative therapies. Cite this article:L. Osagie-Clouard, A. Sanghani-Kerai, M. Coathup, R. Meeson, T. Briggs, G. Blunn. The influence of parathyroid hormone 1-34 on the osteogenic characteristics of adipose- and bone-marrow-derived mesenchymal stem cells from juvenile and ovarectomized rats. Bone Joint Res 2019;8:397–404. DOI: 10.1302/2046-3758.88.BJR-2019-0018.R1

Objectives. Osteoarthritis (OA) is the most common form of arthritis, affecting approximately 15% of the human population. Recently, increased concentration of nitric oxide in serum and synovial fluid in patients with OA has been observed. However, the exact role of nitric oxide in the initiation of OA has not been elucidated. The aim of the present study was to investigate the role of nitric oxide in innate immune regulation during OA initiation in rats. Methods. Rat OA was induced by performing meniscectomy surgery while cartilage samples were collected 0, 7, and 14 days after surgery. Cartilage cytokine levels were determined by using enzyme-linked immunosorbent assay, while other proteins were assessed by using Western blot. Results. In the time course of the study, nitric oxide was increased seven and 14 days after OA induction. Pro-inflammatory cytokines including tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were decreased. L-NG-Nitroarginine methyl ester (L-NAME, a non-specific nitric oxide synthase inhibitor) significantly decreased cartilage nitric oxide and blocked immune suppression. Further, L-NAME decreased Matrix metalloproteinase (MMPs) and increased tissue inhibitor of metalloproteinase (TIMP) expression in meniscectomised rats. Conclusion. Nitric oxide-dependent innate immune suppression protects cartilage from damage in the early stages of OA initiation in rats. Cite this article: C-C. Hsu, C-L. Lin, I-M. Jou, P-H. Wang, J-S. Lee. The protective role of nitric oxide-dependent innate immunosuppression in the early stage of cartilage damage in rats: Role of nitric oxide in ca rtilage da mage. Bone Joint Res 2017;6:253–258. DOI: 10.1302/2046-3758.64.BJJ-2016-0161.R1

Objectives. Electromagnetic fields (EMF) are widely used in musculoskeletal disorders. There are indications that EMF might also be effective in the treatment of osteoporosis. To justify clinical follow-up experiments, we examined the effects of EMF on bone micro-architectural changes in osteoporotic and healthy rats. Moreover, we tested the effects of EMF on fracture healing. Methods. EMF (20 Gauss) was examined in rats (aged 20 weeks), which underwent an ovariectomy (OVX; n = 8) or sham-ovariectomy (sham-OVX; n = 8). As a putative positive control, all rats received bilateral fibular osteotomies to examine the effects on fracture healing. Treatment was applied to one proximal lower leg (three hours a day, five days a week); the lower leg was not treated and served as a control. Bone architectural changes of the proximal tibia and bone formation around the osteotomy were evaluated using in vivo microCT scans at start of treatment and after three and six weeks. Results. In both OVX and sham-OVX groups, EMF did not result in cancellous or cortical bone changes during follow-up. Moreover, EMF did not affect the amount of mineralised callus volume around the fibular osteotomy. Conclusions. In this study we were unable to reproduce the strong beneficial findings reported by others. This might indicate that EMF treatment is very sensitive to the specific set-up, which would be a serious hindrance for clinical use. No evidence was found that EMF treatment can influence bone mass for the benefit of osteoporotic patients. Cite this article: Bone Joint Res 2014;3:230–5

In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine. Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610–618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2

Objectives. Insufficient protein ingestion may affect muscle and bone mass, increasing the risk of osteoporotic fractures in the elderly, and especially in postmenopausal women. We evaluated how a low-protein diet affects bone parameters under gonadal hormone deficiency and the improvement led by hormone replacement therapy (HRT) with 17β-oestradiol. Methods. Female Wistar rats were divided into control (C), ovariectomized (OVX), and 17β-oestradiol-treated ovariectomized (OVX-HRT) groups, which were fed a control or an isocaloric low-protein diet (LP; 6.6% protein; seven animals per group). Morphometric, serum, and body composition parameters were assessed, as well as bone parameters, mechanical resistance, and mineralogy. Results. The results showed that protein restriction negatively affected body chemical composition and bone metabolism by the sex hormone deficiency condition in the OVX group. The association between undernutrition and hormone deficiency led to bone and muscle mass loss and increased the fragility of the bone (as well as decreasing relative femoral weight, bone mineral density, femoral elasticity, peak stress, and stress at offset yield). Although protein restriction induced more severe adverse effects compared with the controls, the combination with HRT showed an improvement in minimizing these damaging effects, as it was seen that HRT had some efficacy in maintaining muscle and bone mass, preserving the bone resistance and minimizing some deleterious processes during the menopause. Conclusion. Protein restriction has adverse effects on metabolism, leading to more severe menopausal symptoms, and HRT could minimize these effects. Therefore, special attention should be given to a balanced diet during menopause and HRT. Cite this article: Bone Joint Res 2019;8:573–581

Aims. This study aimed to investigate whether human umbilical cord mesenchymal stem cells (UC-MSCs) can prevent articular cartilage degradation and explore the underlying mechanisms in a rat osteoarthritis (OA) model induced by monosodium iodoacetate (MIA). Methods. Human UC-MSCs were characterized by their phenotype and multilineage differentiation potential. Two weeks after MIA induction in rats, human UC-MSCs were intra-articularly injected once a week for three weeks. The therapeutic effect of human UC-MSCs was evaluated by haematoxylin and eosin, toluidine blue, Safranin-O/Fast green staining, and Mankin scores. Markers of joint cartilage injury and pro- and anti-inflammatory markers were detected by immunohistochemistry. Results. Histopathological analysis showed that intra-articular injection of human UC-MSCs significantly inhibited the progression of OA, as demonstrated by reduced cartilage degradation, increased Safranin-O staining, and lower Mankin scores. Immunohistochemistry showed that human UC-MSC treatment down-regulated the expression of matrix metalloproteinase-13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), and enhanced the expression of type II collagen and ki67 in the articular cartilage. Furthermore, human UC-MSCs significantly decreased the expression of interleukin (IL)-1β and tumour necrosis factor-α (TNF-α), while increasing TNF-α-induced protein 6 and IL-1 receptor antagonist. Conclusion. Our results demonstrated that human UC-MSCs ameliorate MIA-induced OA by preventing cartilage degradation, restoring the proliferation of chondrocytes, and inhibiting the inflammatory response, which implies that human UC-MSCs may be a promising strategy for the treatment of OA. Cite this article: Bone Joint Res 2021;10(3):226–236

Aims. The involvement of cyclin D1 in the proliferation of microglia, and the generation and maintenance of bone cancer pain (BCP), have not yet been clarified. We investigated the expression of microglia and cyclin D1, and the influences of cyclin D1 on pain threshold. Methods. Female Sprague Dawley (SD) rats were used to establish a rat model of BCP, and the messenger RNA (mRNA) and protein expression of ionized calcium binding adaptor molecule 1 (IBA1) and cyclin D1 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. The proliferation of spinal microglia was detected by immunohistochemistry. The pain behaviour test was assessed by quantification of spontaneous flinches, limb use, and guarding during forced ambulation, mechanical paw withdrawal threshold, and thermal paw withdrawal latency. Results. IBA1 and cyclin D1 in the ipsilateral spinal horn increased in a time-dependent fashion. Spinal microglia proliferated in BCP rats. The microglia inhibitor minocycline attenuated the pain behaviour in BCP rats. The cyclin-dependent kinase inhibitor flavopiridol inhibited the proliferation of spinal microglia, and was associated with an improvement in pain behaviour in BCP rats. Conclusion. Our results revealed that the inhibition of spinal microglial proliferation was associated with a decrease in pain behaviour in a rat model of BCP. Cyclin D1 acts as a key regulator of the proliferation of spinal microglia in a rat model of BCP. Disruption of cyclin D1, the restriction-point control of cell cycle, inhibited the proliferation of microglia and attenuated the pain behaviours in BCP rats. Cyclin D1 and the proliferation of spinal microglia may be potential targets for the clinical treatment of BCP. Cite this article: Bone Joint Res 2022;11(11):803–813

Aims. To investigate the efficacy of ethylenediaminetetraacetic acid-normal saline (EDTA-NS) in dispersing biofilms and reducing bacterial infections. Methods. EDTA-NS solutions were irrigated at different durations (1, 5, 10, and 30 minutes) and concentrations (1, 2, 5, 10, and 50 mM) to disrupt Staphylococcus aureus biofilms on Matrigel-coated glass and two materials widely used in orthopaedic implants (Ti-6Al-4V and highly cross-linked polyethylene (HXLPE)). To assess the efficacy of biofilm dispersion, crystal violet staining biofilm assay and colony counting after sonification and culturing were performed. The results were further confirmed and visualized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). We then investigated the efficacies of EDTA-NS irrigation in vivo in rat and pig models of biofilm-associated infection. Results. When 10 mM or higher EDTA-NS concentrations were used for ten minutes, over 99% of S. aureus biofilm formed on all three types of materials was eradicated in terms of absorbance measured at 595 nm and colony-forming units (CFUs) after culturing. Consistently, SEM and CSLM scanning demonstrated that less adherence of S. aureus could be observed on all three types of materials after 10 mM EDTA-NS irrigation for ten minutes. In the rat model, compared with NS irrigation combined with rifampin (Ti-6Al-4V wire-implanted rats: 60% bacteria survived; HXLPE particle-implanted rats: 63.3% bacteria survived), EDTA-NS irrigation combined with rifampin produced the highest removal rate (Ti-6Al-4V wire-implanted rats: 3.33% bacteria survived; HXLPE particle-implanted rats: 6.67% bacteria survived). In the pig model, compared with NS irrigation combined with rifampin (Ti-6Al-4V plates: 75% bacteria survived; HXLPE bearings: 87.5% bacteria survived), we observed a similar level of biofilm disruption on Ti-6Al-4V plates (25% bacteria survived) and HXLPE bearings (37.5% bacteria survived) after EDTA-NS irrigation combined with rifampin. The in vivo study revealed that the biomass of S. aureus biofilm was significantly reduced when treated with rifampin following irrigation and debridement, as indicated by both the biofilm bacterial burden and crystal violet staining. EDTA-NS irrigation (10 mM/10 min) combined with rifampin effectively removes S. aureus biofilm-associated infections both in vitro and in vivo. Conclusion. EDTA-NS irrigation with or without antibiotics is effective in eradicating S. aureus biofilm-associated infection both ex and in vivo. Cite this article: Bone Joint Res 2024;13(1):40–51

Aims. A number of anti-retroviral therapies (ART) have been implicated in potentially contributing to HIV-associated bone disease. The aim of this study was to evaluate the effect of combination ART on the fracture healing process. Methods. A total of 16 adult male Wistar rats were randomly divided into two groups (n = eight each): Group 1 was given a combination of Tenfovir 30 mg, Lamivudine 30 mg, and Efavirenz 60 mg per day orally, whereas Group 2 was used as a control. After one week of medication preload, all rats underwent a standardized surgical procedure of mid-shaft tibial osteotomy fixed by intramedullary nail with no gap at the fracture site. Progress in fracture healing was monitored regularly for eight weeks. Further evaluations were carried out after euthanasia by micro-CT, mechanically and histologically. Two blinded orthopaedic surgeons used the Radiological Union Scoring system for the Tibia (RUST) to determine fracture healing. Results. The fracture healing process was different between the two groups at week 4 after surgery; only two out of eight rats showed full healing in Group 1 (ART-treated), while seven out of eight rats had bone union in Group 2 (control) (p = 0.040). However, at week eight postoperatively, there was no statistical difference in bone healing; seven out of eight progressed to full union in both groups. Conclusion. This study demonstrated that combination ART resulted in delayed fracture healing at week 4 after surgery in rats, but did not result in the development of nonunion. Cite this article: Bone Joint Res 2022;11(8):585–593

Aims. Although low-intensity pulsed ultrasound (LIPUS) combined with disinfectants has been shown to effectively eliminate portions of biofilm in vitro, its efficacy in vivo remains uncertain. Our objective was to assess the antibiofilm potential and safety of LIPUS combined with 0.35% povidone-iodine (PI) in a rat debridement, antibiotics, and implant retention (DAIR) model of periprosthetic joint infection (PJI). Methods. A total of 56 male Sprague-Dawley rats were established in acute PJI models by intra-articular injection of bacteria. The rats were divided into four groups: a Control group, a 0.35% PI group, a LIPUS and saline group, and a LIPUS and 0.35% PI group. All rats underwent DAIR, except for Control, which underwent a sham procedure. General status, serum biochemical markers, weightbearing analysis, radiographs, micro-CT analysis, scanning electron microscopy of the prostheses, microbiological analysis, macroscope, and histopathology evaluation were performed 14 days after DAIR. Results. The group with LIPUS and 0.35% PI exhibited decreased levels of serum biochemical markers, improved weightbearing scores, reduced reactive bone changes, absence of viable bacteria, and decreased inflammation compared to the Control group. Despite the greater antibiofilm activity observed in the PI group compared to the LIPUS and saline group, none of the monotherapies were successful in preventing reactive bone changes or eliminating the infection. Conclusion. In the rat model of PJI treated with DAIR, LIPUS combined with 0.35% PI demonstrated stronger antibiofilm potential than monotherapy, without impairing any local soft-tissue. Cite this article: Bone Joint Res 2024;13(7):332–341

Aims. Mesenchymal stem cells (MSCs) are usually cultured in a normoxic atmosphere (21%) in vitro, while the oxygen concentrations in human tissues and organs are 1% to 10% when the cells are transplanted in vivo. However, the impact of hypoxia on MSCs has not been deeply studied, especially its translational application. Methods. In the present study, we investigated the characterizations of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in hypoxic (1%) and normoxic (21%) atmospheres with a long-term culture from primary to 30 generations, respectively. The comparison between both atmospheres systematically analyzed the biological functions of MSCs, mainly including stemness maintenance, immune regulation, and resistance to chondrocyte apoptosis, and studied their joint function and anti-inflammatory effects in osteoarthritis (OA) rats constructed by collagenase II. Results. We observed that long-term hypoxic culture surpassed normoxic atmosphere during hUC-MSCs culture in respect of promoting proliferation, anti-tumorigenicity, maintaining normal karyotype and stemness, inhibiting senescence, and improving immunoregulatory function and the role of anti-apoptosis in chondrocytes. Furthermore, we demonstrated that the transplantation of long-term hypoxic hUC-MSCs (Hy-MSCs) had a better therapeutic effect on OA rats compared with the hUC-MSCs cultured in the normoxic atmosphere (No-MSCs) in terms of the improved function and swelling recovery in the joints, and substantially inhibited the secretion of pro-inflammatory factors, which effectively alleviated cartilage damage by reducing the expression of matrix metallopeptidase 13 (MMP-13). Conclusion. Our results demonstrate that Hy-MSCs possess immense potential for clinical applications via promoting stemness maintenance and enhancing immunoregulatory function. Cite this article: Bone Joint Res 2024;13(12):763–777

Aims. 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. Methods. An in vitro model for oxidative stress-induced injury in NPCs was developed to elucidate the mechanisms underlying the upregulation of DDIT4 expression, activation of the reactive oxygen species (ROS)-thioredoxin-interacting protein (TXNIP)-NLRP3 signalling pathway, and nucleus pulposus pyroptosis. Furthermore, the mechanism of action of small interfering DDIT4 (siDDIT4) on NPCs in vitro was validated. A triplex hydrogel named siDDIT4@G5-P-HA was created by adsorbing siDDIT4 onto fifth-generation polyamidoamine (PAMAM) dendrimer using van der Waals interactions, and then coating it with hyaluronic acid (HA). In addition, we established a rat puncture IVDD model to decipher the hydrogel’s mechanism in IVDD. Results. A correlation between DDIT4 expression levels and disc degeneration was shown with human nucleus pulposus and needle-punctured rat disc specimens. We confirmed that DDIT4 was responsible for activating the ROS-TXNIP-NLRP3 axis during oxidative stress-induced pyroptosis in rat nucleus pulposus in vitro. Mitochondria were damaged during oxidative stress, and DDIT4 contributed to mitochondrial damage and ROS production. In addition, siDDIT4@G5-P-HA hydrogels showed good delivery activity of siDDIT4 to NPCs. In vitro studies illustrated the potential of the siDDIT4@G5-P-HA hydrogel for alleviating IVDD in rats. Conclusion. DDIT4 is a key player in mediating pyroptosis and IVDD in NPCs through the ROS-TXNIP-NLRP3 axis. Additionally, siDDIT4@G5-P-HA hydrogel has been found to relieve IVDD in rats. Our research offers an innovative treatment option for IVDD. Cite this article: Bone Joint Res 2024;13(5):247–260

Aims. The optimum type of antibiotics and their administration route for treating Gram-negative (GN) periprosthetic joint infection (PJI) remain controversial. This study aimed to determine the GN bacterial species and antibacterial resistance rates related to clinical GN-PJI, and to determine the efficacy and safety of intra-articular (IA) antibiotic injection after one-stage revision in a GN pathogen-induced PJI rat model of total knee arthroplasty. Methods. A total of 36 consecutive PJI patients who had been infected with GN bacteria between February 2015 and December 2021 were retrospectively recruited in order to analyze the GN bacterial species involvement and antibacterial resistance rates. Antibiotic susceptibility assays of the GN bacterial species were performed to screen for the most sensitive antibiotic, which was then used to treat the most common GN pathogen-induced PJI rat model. The rats were randomized either to a PJI control group or to three meropenem groups (intraperitoneal (IP), IA, and IP + IA groups). After two weeks of treatment, infection control level, the side effects, and the volume of antibiotic use were evaluated. Results. Escherichia coli was the most common pathogen in GN-PJI, and meropenem was the most sensitive antibiotic. Serum inflammatory markers, weightbearing activity, and Rissing score were significantly improved by meropenem, especially in the IA and IP + IA groups ( p < 0.05). Meropenem in the IA group eradicated E. coli from soft-tissue, bone, and prosthetic surfaces, with the same effect as in the IP + IA group. Radiological results revealed that IA and IP + IA meropenem were effective at relieving bone damage. Haematoxylin and eosin staining also showed that IA and IP + IA meropenem improved synovial inflammation and bone destruction. No pathological changes in the main organs or abnormal serum markers were observed in any of the meropenem-treated rats. The IA group required the lowest amount of meropenem, followed by the IP and IP + IA groups. Conclusion. IA-only meropenem with a two-week treatment course was effective and safe for PJI control following one-stage revision in a rat model, with less meropenem use. Cite this article: Bone Joint Res 2024;13(10):546–558

Aims. Methicillin-resistant Staphylococcus aureus (MRSA) can cause wound infections via a ‘Trojan Horse’ mechanism, in which neutrophils engulf intestinal MRSA and travel to the wound, releasing MRSA after apoptosis. The possible role of intestinal MRSA in prosthetic joint infection (PJI) is unknown. Methods. Rats underwent intestinal colonization with green fluorescent protein (GFP)-tagged MRSA by gavage and an intra-articular wire was then surgically implanted. After ten days, the presence of PJI was determined by bacterial cultures of the distal femur, joint capsule, and implant. We excluded several other possibilities for PJI development. Intraoperative contamination was excluded by culturing the specimen obtained from surgical site. Extracellular bacteraemia-associated PJI was excluded by comparing with the infection rate after intravenous injection of MRSA or MRSA-carrying neutrophils. To further support this theory, we tested the efficacy of prophylactic membrane-permeable and non-membrane-permeable antibiotics in this model. Results. After undergoing knee surgery eight or 72 hours after colonization, five out of 20 rats (25.0%) and two out of 20 rats (10.0%) developed PJI, respectively. Strikingly, 11 out of 20 rats (55.0%) developed PJI after intravenous injection of MRSA-carrying neutrophils that were isolated from rats with intestinal MRSA colonization. None of the rats receiving intravenous injections of MRSA developed PJI. These results suggest that intestinal MRSA carried by neutrophils could cause PJI in our rat model. Ten out of 20 (50.0%) rats treated with non-membrane-permeable gentamicin developed PJI, whereas only one out of 20 (5.0%) rats treated with membrane-permeable linezolid developed PJI. Conclusion. Neutrophils as carriers of intestinal MRSA may play an important role in PJI development. Cite this article:Bone Joint Res. 2020;9(4):152–161

Aims. Osteoarthritis (OA) is a disabling joint disorder and mechanical loading is an important pathogenesis. This study aims to investigate the benefits of less mechanical loading created by intermittent tail suspension for knee OA. Methods. A post-traumatic OA model was established in 20 rats (12 weeks old, male). Ten rats were treated with less mechanical loading through intermittent tail suspension, while another ten rats were treated with normal mechanical loading. Cartilage damage was determined by gross appearance, Safranin O/Fast Green staining, and immunohistochemistry examinations. Subchondral bone changes were analyzed by micro-CT and tartrate-resistant acid phosphatase (TRAP) staining, and serum inflammatory cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA). Results. Our radiographs showed that joint space was significantly enlarged in rats with less mechanical loading. Moreover, cartilage destruction was attenuated in the less mechanical loading group with lower histological damage scores, and lower expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5, matrix metalloproteinase (MMP)-3, and MMP-13. In addition, subchondral bone abnormal changes were ameliorated in OA rats with less mechanical loading, as reduced bone mineral density (BMD), bone volume/tissue volume (BV/TV), and number of osteophytes and osteoclasts in the subchondral bone were observed. Finally, the level of serum inflammatory cytokines was significantly downregulated in the less mechanical loading group compared with the normal mechanical loading group, as well as the expression of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), caspase-1, and interleukin 1β (IL-1β) in the cartilage. Conclusion. Less mechanical loading alleviates cartilage destruction, subchondral bone changes, and secondary inflammation in OA joints. This study provides fundamental insights into the benefit of non-weight loading rest for patients with OA. Cite this article: Bone Joint Res 2020;9(10):731–741

Aims. Treatment outcomes for methicillin-resistant Staphylococcus aureus (MRSA) periprosthetic joint infection (PJI) using systemic vancomycin and antibacterial cement spacers during two-stage revision arthroplasty remain unsatisfactory. This study explored the efficacy and safety of intra-articular vancomycin injections for PJI control after debridement and cement spacer implantation in a rat model. Methods. Total knee arthroplasty (TKA), MRSA inoculation, debridement, and vancomycin-spacer implantation were performed successively in rats to mimic first-stage PJI during the two-stage revision arthroplasty procedure. Vancomycin was administered intraperitoneally or intra-articularly for two weeks to control the infection after debridement and spacer implantation. Results. Rats receiving intra-articular vancomycin showed the best outcomes among the four treatment groups, with negative bacterial cultures, increased weight gain, increased capacity for weightbearing activities, increased residual bone volume preservation, and reduced inflammatory reactions in the joint tissues, indicating MRSA eradication in the knee. The vancomycin-spacer and/or systemic vancomycin failed to eliminate the MRSA infections following a two-week antibiotic course. Serum vancomycin levels did not reach nephrotoxic levels in any group. Mild renal histopathological changes, without changes in serum creatinine levels, were observed in the intraperitoneal vancomycin group compared with the intra-articular vancomycin group, but no changes in hepatic structure or serum alanine aminotransferase or aspartate aminotransferase levels were observed. No local complications were observed, such as sinus tract or non-healing surgical incisions. Conclusion. Intra-articular vancomycin injection was effective and safe for PJI control following debridement and spacer implantation in a rat model during two-stage revision arthroplasties, with better outcomes than systemic vancomycin administration. Cite this article: Bone Joint Res 2022;11(6):371–385

Aims. There is an increasing concern of osteoporotic fractures in the ageing population. Low-magnitude high-frequency vibration (LMHFV) was shown to significantly enhance osteoporotic fracture healing through alteration of osteocyte lacuno-canalicular network (LCN). Dentin matrix protein 1 (DMP1) in osteocytes is known to be responsible for maintaining the LCN and mineralization. This study aimed to investigate the role of osteocyte-specific DMP1 during osteoporotic fracture healing augmented by LMHFV. Methods. A metaphyseal fracture was created in the distal femur of ovariectomy-induced osteoporotic Sprague Dawley rats. Rats were randomized to five different groups: 1) DMP1 knockdown (KD), 2) DMP1 KD + vibration (VT), 3) Scramble + VT, 4) VT, and 5) control (CT), where KD was performed by injection of short hairpin RNA (shRNA) into marrow cavity; vibration treatment was conducted at 35 Hz, 0.3 g; 20 minutes/day, five days/week). Assessments included radiography, micro-CT, dynamic histomorphometry and immunohistochemistry on DMP1, sclerostin, E11, and fibroblast growth factor 23 (FGF23). In vitro, murine long bone osteocyte-Y4 (MLO-Y4) osteocyte-like cells were randomized as in vivo groupings. DMP1 KD was performed by transfecting cells with shRNA plasmid. Assessments included immunocytochemistry on osteocyte-specific markers as above, and mineralized nodule staining. Results. Healing capacities in DMP1 KD groups were impaired. Results showed that DMP1 KD significantly abolished vibration-enhanced fracture healing at week 6. DMP1 KD significantly altered the expression of osteocyte-specific markers. The lower mineralization rate in DMP1 KD groups indicated that DMP1 knockdown was associated with poor fracture healing process. Conclusion. The blockage of DMP1 would impair healing outcomes and negate LMHFV-induced enhancement on fracture healing. These findings reveal the importance of DMP1 in response to the mechanical signal during osteoporotic fracture healing. Cite this article: Bone Joint Res 2022;11(7):465–476

Aims. With the ageing population, fragility fractures have become one of the most common conditions. The objective of this study was to investigate whether microbiological outcomes and fracture-healing in osteoporotic bone is worse than normal bone with fracture-related infection (FRI). Methods. A total of 120 six-month-old Sprague-Dawley (SD) rats were randomized to six groups: Sham, sham + infection (Sham-Inf), sham with infection + antibiotics (Sham-Inf-A), ovariectomized (OVX), OVX + infection (OVX-Inf), and OVX + infection + antibiotics (OVX-Inf-A). Open femoral diaphysis fractures with Kirschner wire fixation were performed. Staphylococcus aureus at 4 × 10. 4. colony-forming units (CFU)/ml was inoculated. Rats were euthanized at four and eight weeks post-surgery. Radiography, micro-CT, haematoxylin-eosin, mechanical testing, immunohistochemistry (IHC), gram staining, agar plating, crystal violet staining, and scanning electron microscopy were performed. Results. Agar plating analysis revealed a higher bacterial load in bone (p = 0.002), and gram staining showed higher cortical bone colonization (p = 0.039) in OVX-Inf compared to Sham-Inf. OVX-Inf showed significantly increased callus area (p = 0.013), but decreased high-density bone volume (p = 0.023) compared to Sham-Inf. IHC staining showed a significantly increased expression of TNF-α in OVX-Inf compared to OVX (p = 0.049). Significantly reduced bacterial load on bone (p = 0.001), enhanced ultimate load (p = 0.001), and energy to failure were observed in Sham-Inf-A compared to Sham-Inf (p = 0.028), but not in OVX-Inf-A compared to OVX-Inf. Conclusion. In osteoporotic bone with FRI, infection was more severe with more bone lysis and higher bacterial load, and fracture-healing was further delayed. Systemic antibiotics significantly reduced bacterial load and enhanced callus quality and strength in normal bone with FRI, but not in osteoporotic bone. Cite this article: Bone Joint Res 2022;11(2):49–60

Aims. Distraction osteogenesis (DO) is a useful orthopaedic procedure employed to lengthen and reshape bones by stimulating bone formation through controlled slow stretching force. Despite its promising applications, difficulties are still encountered. Our previous study demonstrated that pulsed electromagnetic field (PEMF) treatment significantly enhances bone mineralization and neovascularization, suggesting its potential application. The current study compared a new, high slew rate (HSR) PEMF signal, with different treatment durations, with the standard Food and Drug Administration (FDA)-approved signal, to determine if HSR PEMF is a better alternative for bone formation augmentation. Methods. The effects of a HSR PEMF signal with three daily treatment durations (0.5, one, and three hours/day) were investigated in an established rat DO model with comparison of an FDA-approved classic signal (three hrs/day). PEMF treatments were applied to the rats daily for 35 days, starting from the distraction phase until termination. Radiography, micro-CT (μCT), biomechanical tests, and histological examinations were employed to evaluate the quality of bone formation. Results. All rats tolerated the treatment well and no obvious adverse effects were found. By comparison, the HSR signal (three hrs/day) treatment group achieved the best healing outcome, in that endochondral ossification and bone consolidation were enhanced. In addition, HSR signal treatment (one one hr/day) had similar effects to treatment using the classic signal (three three hrs/day), indicating that treatment duration could be significantly shortened with the HSR signal. Conclusion. HSR signal may significantly enhance bone formation and shorten daily treatment duration in DO, making it a potential candidate for a new clinical protocol for patients undergoing DO treatments. Cite this article: Bone Joint Res 2021;10(12):767–779

Aims. Pigment epithelium-derived factor (PEDF) is known to induce several types of tissue regeneration by activating tissue-specific stem cells. Here, we investigated the therapeutic potential of PEDF 29-mer peptide in the damaged articular cartilage (AC) in rat osteoarthritis (OA). Methods. Mesenchymal stem/stromal cells (MSCs) were isolated from rat bone marrow (BM) and used to evaluate the impact of 29-mer on chondrogenic differentiation of BM-MSCs in culture. Knee OA was induced in rats by a single intra-articular injection of monosodium iodoacetate (MIA) in the right knees (set to day 0). The 29-mer dissolved in 5% hyaluronic acid (HA) was intra-articularly injected into right knees at day 8 and 12 after MIA injection. Subsequently, the therapeutic effect of the 29-mer/HA on OA was evaluated by the Osteoarthritis Research Society International (OARSI) histopathological scoring system and changes in hind paw weight distribution, respectively. The regeneration of chondrocytes in damaged AC was detected by dual-immunostaining of 5-bromo-2'-deoxyuridine (BrdU) and chondrogenic markers. Results. The 29-mer promoted expansion and chondrogenic differentiation of BM-MSCs cultured in different defined media. MIA injection caused chondrocyte death throughout the AC, with cartilage degeneration thereafter. The 29-mer/HA treatment induced extensive chondrocyte regeneration in the damaged AC and suppressed MIA-induced synovitis, accompanied by the recovery of cartilage matrix. Pharmacological inhibitors of PEDF receptor (PEDFR) and signal transducer and activator of transcription 3 (STAT3) signalling substantially blocked the chondrogenic promoting activity of 29-mer on the cultured BM-MSCs and injured AC. Conclusion. The 29-mer/HA formulation effectively induces chondrocyte regeneration and formation of cartilage matrix in the damaged AC. Cite this article: Bone Joint Res 2024;13(4):137–148

Aims. Poly (ADP-ribose) polymerase (PARP) inhibitor has been reported to attenuate inflammatory response in rat models of inflammation. This study was designed to investigate the effect of PARP signalling in osteoarthritis (OA) cartilage inflammatory response in an OA rat model. Methods. The OA model was established by anterior cruciate ligament transection with medial meniscectomy in Wistar rats. The poly (ADP-ribose) polymerase 1 (PARP-1) shRNA (short hairpin (sh)-PARP-1) and negative control shRNA (sh-NC) were delivered using a lentiviral vector and were intra-articularly injected into rats after surgery. The weight-bearing distribution of the hind limbs and the knee joint width were measured every two weeks. The expression levels of PARP-1, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in cartilage were determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The serum concentrations of inflammatory cytokines were detected using enzyme-linked immunosorbent assay (ELISA). Results. PARP-1 expression level significantly increased in the cartilage of the established OA rat model. sh-PARP-1 treatment suppressed PARP-1 levels, decreased the Δ Force (the difference between the weight on ipsilateral limb and contralateral limb) and the knee joint width, inhibited cartilage matrix catabolic enzymes, and ameliorated OA cartilage degradation and attenuated inflammatory response. Conclusion. PARP-1 inhibition attenuates OA cartilage inflammatory response in the OA rat model. Cite this article: Bone Joint Res 2021;10(7):401–410

Aims. Cartilage injuries rarely heal spontaneously and often require surgical intervention, leading to the formation of biomechanically inferior fibrous tissue. This study aimed to evaluate the possible effect of amelogenin on the healing process of a large osteochondral injury (OCI) in a rat model. Methods. A reproducible large OCI was created in the right leg femoral trochlea of 93 rats. The OCIs were treated with 0.1, 0.5, 1.0, 2.5, or 5.0 μg/μl recombinant human amelogenin protein (rHAM. +. ) dissolved in propylene glycol alginate (PGA) carrier, or with PGA carrier alone. The degree of healing was evaluated 12 weeks after treatment by morphometric analysis and histological evaluation. Cell recruitment to the site of injury as well as the origin of the migrating cells were assessed four days after treatment with 0.5 μg/μl rHAM. +. using immunohistochemistry and immunofluorescence. Results. A total of 12 weeks after treatment, 0.5 μg/μl rHAM. +. brought about significant repair of the subchondral bone and cartilage. Increased expression of proteoglycan and type II collagen and decreased expression of type I collagen were revealed at the surface of the defect, and an elevated level of type X collagen at the newly developed tide mark region. Conversely, the control group showed osteoarthritic alterations. Recruitment of cells expressing the mesenchymal stem cell (MSC) markers CD105 and STRO-1, from adjacent bone marrow toward the OCI, was noted four days after treatment. Conclusion. We found that 0.5 μg/μl rHAM. +. induced in vivo healing of injured articular cartilage and subchondral bone in a rat model, preventing the destructive post-traumatic osteoarthritic changes seen in control OCIs, through paracrine recruitment of cells a few days after treatment. Cite this article: Bone Joint Res 2023;12(10):615–623

Aims. To explore the efficacy of extracorporeal shockwave therapy (ESWT) in the treatment of osteochondral defect (OCD), and its effects on the levels of transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, -3, -4, -5, and -7 in terms of cartilage and bone regeneration. Methods. The OCD lesion was created on the trochlear groove of left articular cartilage of femur per rat (40 rats in total). The experimental groups were Sham, OCD, and ESWT (0.25 mJ/mm. 2. , 800 impulses, 4 Hz). The animals were euthanized at 2, 4, 8, and 12 weeks post-treatment, and histopathological analysis, micro-CT scanning, and immunohistochemical staining were performed for the specimens. Results. In the histopathological analysis, the macro-morphological grading scale showed a significant increase, while the histological score and cartilage repair scale of ESWT exhibited a significant decrease compared to OCD at the 8- and 12-week timepoints. At the 12-week follow-up, ESWT exhibited a significant improvement in the volume of damaged bone compared to OCD. Furthermore, immunohistochemistry analysis revealed a significant decrease in type I collagen and a significant increase in type II collagen within the newly formed hyaline cartilage following ESWT, compared to OCD. Finally, SRY-box transcription factor 9 (SOX9), aggrecan, and TGF-β, BMP-2, -3, -4, -5, and -7 were significantly higher in ESWT than in OCD at 12 weeks. Conclusion. ESWT promoted the effect of TGF-β/BMPs, thereby modulating the production of extracellular matrix proteins and transcription factor involved in the regeneration of articular cartilage and subchondral bone in an OCD rat model. Cite this article: Bone Joint Res 2024;13(7):342–352

Aims. The purpose of this study was to determine whether intracellular Staphylococcus aureus is associated with recurrent infection in a rat model of open fracture. Methods. After stabilizing with Kirschner wire, we created a midshaft femur fracture in Sprague-Dawley rats and infected the wound with green fluorescent protein (GFP)-tagged S. aureus. After repeated debridement and negative swab culture was achieved, the isolation of GFP-containing cells from skin, bone marrow, and muscle was then performed. The composition and viability of intracellular S. aureus in isolated GFP-positive cells was assessed. We suppressed the host immune system and observed whether recurrent infection would occur. Finally, rats were assigned to one of six treatment groups (a combination of antibiotic treatment and implant removal/retention). The proportion of successful eradication was determined. Results. Green fluorescent protein-containing cells were successfully isolated after the swab culture was negative from skin (n = 0, 0%), muscle (n = 10, 100%), and bone marrow (n = 10, 100%) of a total of ten rats. The phagocytes were predominant in GFP-positive cells from muscle (73%) and bone marrow (81%) with a significantly higher viability of intracellular S. aureus (all p-values < 0.001). The recurrent infection occurred in up to 75% of rats after the immunosuppression. The proportion of successful eradication was not associated with implant retention or removal, and the efficacy of linezolid in eradicating intracellular S. aureus is significantly higher than that of vancomycin. Conclusion. Intracellular S. aureus is associated with recurrent infection in the rat model of open fracture. Usage of linezolid, a membrane-permeable antibiotic, is an effective strategy against intracellular S. aureus. Cite this article:Bone Joint Res. 2020;9(2):71–76

Aims. In recent conflicts, most injuries to the limbs are due to blasts resulting in a large number of lower limb amputations. These lead to heterotopic ossification (HO), phantom limb pain (PLP), and functional deficit. The mechanism of blast loading produces a combined fracture and amputation. Therefore, to study these conditions, in vivo models that replicate this combined effect are required. The aim of this study is to develop a preclinical model of blast-induced lower limb amputation. Methods. Cadaveric Sprague-Dawley rats’ left hindlimbs were exposed to blast waves of 7 to 13 bar burst pressures and 7.76 ms to 12.68 ms positive duration using a shock tube. Radiographs and dissection were used to identify the injuries. Results. Higher burst pressures of 13 and 12 bar caused multiple fractures at the hip, and the right and left limbs. Lowering the pressure to 10 bar eliminated hip fractures; however, the remaining fractures were not isolated to the left limb. Further reducing the pressure to 9 bar resulted in the desired isolated fracture of the left tibia with a dramatic reduction in the fractures to other sites. Conclusion. In this paper, a rodent blast injury model has been developed in the hindlimb of cadaveric rats that combines the blast and fracture in one insult, necessitating amputation. Experimental setup with 9 bar burst pressure and 9.13 ms positive duration created a fracture at the tibia with total reduction in non-targeted fractures, rendering 9 bar burst pressure suitable for translation to a survivable model to investigate blast injury-associated diseases. Cite this article: Bone Joint Res 2021;10(3):166–173

Aims. Fibrinolysis plays a key transition step from haematoma formation to angiogenesis and fracture healing. Low-magnitude high-frequency vibration (LMHFV) is a non-invasive biophysical modality proven to enhance fibrinolytic factors. This study investigates the effect of LMHFV on fibrinolysis in a clinically relevant animal model to accelerate osteoporotic fracture healing. Methods. A total of 144 rats were randomized to four groups: sham control; sham and LMHFV; ovariectomized (OVX); and ovariectomized and LMHFV (OVX-VT). Fibrinolytic potential was evaluated by quantifying fibrin, tissue plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1) along with healing outcomes at three days, one week, two weeks, and six weeks post-fracture. Results. All rats achieved healing, and x-ray relative radiopacity for OVX-VT was significantly higher compared to OVX at week 2. Martius Scarlet Blue (MSB) staining revealed a significant decrease of fibrin content in the callus in OVX-VT compared with OVX on day 3 (p = 0.020). Mean tPA from muscle was significantly higher for OVX-VT compared to OVX (p = 0.020) on day 3. Mechanical testing revealed the mean energy to failure was significantly higher for OVX-VT at 37.6 N mm (SD 8.4) and 71.9 N mm (SD 30.7) compared with OVX at 5.76 N mm (SD 7.1) (p = 0.010) and 17.7 N mm (SD 11.5) (p = 0.030) at week 2 and week 6, respectively. Conclusion. Metaphyseal fracture healing is enhanced by LMHFV, and one of the important molecular pathways it acts on is fibrinolysis. LMHFV is a promising intervention for osteoporotic metaphyseal fracture healing. The improved mechanical properties, acceleration of fracture healing, and safety justify its role into translation to future clinical studies. Cite this article: Bone Joint Res 2021;10(1):41–50

Aims. Adenosine, lidocaine, and Mg. 2+. (ALM) therapy exerts differential immuno-inflammatory responses in males and females early after anterior cruciate ligament (ACL) reconstruction (ACLR). Our aim was to investigate sex-specific effects of ALM therapy on joint tissue repair and recovery 28 days after surgery. Methods. Male (n = 21) and female (n = 21) adult Sprague-Dawley rats were randomly divided into ALM or Saline control treatment groups. Three days after ACL rupture, animals underwent ACLR. An ALM or saline intravenous infusion was commenced prior to skin incision, and continued for one hour. An intra-articular bolus of ALM or saline was also administered prior to skin closure. Animals were monitored to 28 days, and joint function, pain, inflammatory markers, histopathology, and tissue repair markers were assessed. Results. Despite comparable knee function, ALM-treated males had reduced systemic inflammation, synovial fluid angiogenic and pro-inflammatory mediators, synovitis, and fat pad fibrotic changes, compared to controls. Within the ACL graft, ALM-treated males had increased expression of tissue repair markers, decreased inflammation, increased collagen organization, and improved graft-bone healing. In contrast to males, females had no evidence of persistent systemic inflammation. Compared to controls, ALM-treated females had improved knee extension, gait biomechanics, and elevated synovial macrophage inflammatory protein-1 alpha (MIP-1α). Within the ACL graft, ALM-treated females had decreased inflammation, increased collagen organization, and improved graft-bone healing. In articular cartilage of ALM-treated animals, matrix metalloproteinase (MMP)-13 expression was blunted in males, while in females repair markers were increased. Conclusion. At 28 days, ALM therapy reduces inflammation, augments tissue repair patterns, and improves joint function in a sex-specific manner. The study supports transition to human safety trials. Cite this article: Bone Joint Res 2024;13(6):279–293

Objectives. Cellular movement and relocalisation are important for many physiologic properties. Local mesenchymal stem cells (MSCs) from injured tissues and circulating MSCs aid in fracture healing. Cytokines and chemokines such as Stromal cell-derived factor 1(SDF-1) and its receptor chemokine receptor type 4 (CXCR4) play important roles in maintaining mobilisation, trafficking and homing of stem cells from bone marrow to the site of injury. We investigated the differences in migration of MSCs from the femurs of young, adult and ovariectomised (OVX) rats and the effect of CXCR4 over-expression on their migration. Methods. MSCs from young, adult and OVX rats were put in a Boyden chamber to establish their migration towards SDF-1. This was compared with MSCs transfected with CXCR4, as well as MSCs differentiated to osteoblasts. Results. MSCs from OVX rats migrate significantly (p < 0.05) less towards SDF-1 (9%, . sd. 5%) compared with MSCs from adult (15%, . sd. 3%) and young rats (25%, . sd. 4%). Cells transfected with CXCR4 migrated significantly more towards SDF-1 compared with non-transfected cells, irrespective of whether these cells were from OVX (26.5%, . sd. 4%), young (47%, . sd. 17%) or adult (21%, . sd. 4%) rats. Transfected MSCs differentiated to osteoblasts express CXCR4 but do not migrate towards SDF-1. Conclusions. MSC migration is impaired by age and osteoporosis in rats, and this may be associated with a significant reduction in bone formation in osteoporotic patients. The migration of stem cells can be ameliorated by upregulating CXCR4 levels which could possibly enhance fracture healing in osteoporotic patients. Cite this article: A. Sanghani-Kerai, M. Coathup, S. Samazideh, P. Kalia, L. Di Silvio, B. Idowu, G. Blunn. Osteoporosis and ageing affects the migration of stem cells and this is ameliorated by transfection with CXCR4. Bone Joint Res 2017;6:–365. DOI: 10.1302/2046-3758.66.BJR-2016-0259.R1

Aims. Periprosthetic joint infections (PJIs) are rare, but represent a great burden for the patient. In addition, the incidence of methicillin-resistant Staphylococcus aureus (MRSA) is increasing. The aim of this rat experiment was therefore to compare the antibiotics commonly used in the treatment of PJIs caused by MRSA. Methods. For this purpose, sterilized steel implants were implanted into the femur of 77 rats. The metal devices were inoculated with suspensions of two different MRSA strains. The animals were divided into groups and treated with vancomycin, linezolid, cotrimoxazole, or rifampin as monotherapy, or with combination of antibiotics over a period of 14 days. After a two-day antibiotic-free interval, the implant was explanted, and bone, muscle, and periarticular tissue were microbiologically analyzed. Results. Vancomycin and linezolid were able to significantly (p < 0.05) reduce the MRSA bacterial count at implants. No significant effect was found at the bone. Rifampin was the only monotherapy that significantly reduced the bacterial count on implant and bone. The combination with vancomycin or linezolid showed significant efficacy. Treatment with cotrimoxazole alone did not achieve a significant bacterial count reduction. The combination of linezolid plus rifampin was significantly more effective on implant and bone than the control group in both trials. Conclusion. Although rifampicin is effective as a monotherapy, it should not be used because of the high rate of resistance development. Our animal experiments showed the great importance of combination antibiotic therapies. In the future, investigations with higher case numbers, varied bacterial concentrations, and changes in individual drug dosages will be necessary to be able to draw an exact comparison, possibly within a clinical trial. Cite this article: Bone Joint Res 2022;11(3):143–151

Objectives. Our objective in this article is to test the hypothesis that type 2 diabetes mellitus (T2DM) is a factor in the onset and progression of osteoarthritis, and to characterise the quality of the articular cartilage in an appropriate rat model. Methods. T2DM rats were obtained from the UC Davis group and compared with control Lewis rats. The diabetic rats were sacrificed at ages from six to 12 months, while control rats were sacrificed at six months only. Osteoarthritis severity was determined via histology in four knee quadrants using the OARSI scoring guide. Immunohistochemical staining was also performed as a secondary form of osteoarthritic analysis. Results. T2DM rats had higher mean osteoarthritis scores than the control rats in each of the four areas that were analysed. However, only the results at the medial and lateral femur and medial tibia were significant. Cysts were also found in T2DM rats at the junction of the articular cartilage and subchondral bone. Immunohistochemical analysis does not show an increase in collagen II between control and T2DM rats. Mass comparisons also showed a significant relationship between mass and osteoarthritis score. Conclusions. T2DM was found to cause global degeneration in the UCD rat knee joints, suggesting that diabetes itself is a factor in the onset and progression of osteoarthritis. The immunohistochemistry stains showed little to no change in collagen II degeneration between T2DM and control rats. Overall, it seems that the animal model used is pertinent to future studies of T2DM in the development and progression of osteoarthritis. Cite this article: Bone Joint Res 2014;3:203–11

Objectives. To elucidate the effects of age on the expression levels of the receptor activator of the nuclear factor-κB ligand (RANKL) and osteoclasts in the periodontal ligament during orthodontic mechanical loading and post-orthodontic retention. Materials and Methods. The study included 20 male Sprague-Dawley rats, ten in the young group (aged four to five weeks) and ten in the adult group (aged 18 to 20 weeks). In each rat, the upper-left first molar was subjected to a seven-day orthodontic force loading followed by a seven-day retention period. The upper-right first molar served as a control. The amount of orthodontic tooth movement was measured after seven-day force application and seven-day post-orthodontic retention. The expression levels of RANKL and the tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were evaluated on day 7 (end of mechanical force loading) and day 14 (after seven days of post-orthodontic retention). Statistical analysis was performed using the t-test, and significance was set at p < 0.05. Results. There was no significant difference between the amount of tooth movement in the young group (0.96, standard deviation (. sd. ) 0.30mm) and that in the adult group (0.80mm, . sd. 0.28) (p > 0.05) after the seven-day force application. On the compression side, the expression of RANKL and TRAP-positive osteoclasts in both the young and the adult groups increased after the application of force for seven days, and then decreased at the end of the seven-day retention period. However, by the end of the period, the expression of RANKL on the compression side dropped to the control level in the young group (p > 0.05), while it was still higher than that on the control side in the adult group (p < 0.05). The expression of RANKL on the compression side did not show significant difference between the young and the adult groups after seven-day force application (p > 0.05), but it was significantly higher in the adult group than that in the young group after seven-day post-orthodontic retention (p < 0.05). Similarly, the decreasing trend of TRAP-positive osteoclasts during the retention period in the adult group was less obvious than that in the young group. Conclusions. The bone-resorptive activity in the young rats was more dynamic than that in the adult rats. The expression of RANKL and the number of osteoclasts in adult rats did not drop to the control level during the post-orthodontic retention period while RANKL expression and the number of osteoclasts in young rats had returned to the baseline. Cite this article: X. Li, M. Li, J. Lu, Y. Hu, L. Cui, D. Zhang, Y. Yang. Age-related effects on osteoclastic activities after orthodontic tooth movement. Bone Joint Res 2016;5:492–499. DOI: 10.1302/2046-3758.510.BJR-2016-0004.R2

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

Objectives. Experimental studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) may have negative effects on fracture healing. This study aimed to assess the effect of immediate and delayed short-term administration of clinically relevant parecoxib doses and timing on fracture healing using an established animal fracture model. Methods. A standardized closed tibia shaft fracture was induced and stabilized by reamed intramedullary nailing in 66 Wistar rats. A ‘parecoxib immediate’ (Pi) group received parecoxib (3.2 mg/kg bodyweight twice per day) on days 0, 1, and 2. A ‘parecoxib delayed’ (Pd) group received the same dose of parecoxib on days 3, 4, and 5. A control group received saline only. Fracture healing was evaluated by biomechanical tests, histomorphometry, and dual-energy x-ray absorptiometry (DXA) at four weeks. Results. For ultimate bending moment, the median ratio between fractured and non-fractured tibia was 0.61 (interquartile range (IQR) 0.45 to 0.82) in the Pi group, 0.44 (IQR 0.42 to 0.52) in the Pd group, and 0.50 (IQR 0.41 to 0.75) in the control group (n = 44; p = 0.068). There were no differences between the groups for stiffness, energy, deflection, callus diameter, DXA measurements (n = 64), histomorphometrically osteoid/bone ratio, or callus area (n = 20). Conclusion. This study demonstrates no negative effect of immediate or delayed short-term administration of parecoxib on diaphyseal fracture healing in rats. Cite this article: G. A. Hjorthaug, E. Søreide, L. Nordsletten, J. E. Madsen, F. P. Reinholt, S. Niratisairak, S. Dimmen. Short-term perioperative parecoxib is not detrimental to shaft fracture healing in a rat model. Bone Joint Res 2019;8:472–480. DOI: 10.1302/2046-3758.810.BJR-2018-0341.R1

Objectives . Rotator cuff tears are among the most common and debilitating upper extremity injuries. Chronic cuff tears result in atrophy and an infiltration of fat into the muscle, a condition commonly referred to as ‘fatty degeneration’. While stem cell therapies hold promise for the treatment of cuff tears, a suitable immunodeficient animal model that could be used to study human or other xenograft-based therapies for the treatment of rotator cuff injuries had not previously been identified. Methods . A full-thickness, massive supraspinatus and infraspinatus tear was induced in adult T-cell deficient rats. We hypothesised that, compared with controls, 28 days after inducing a tear we would observe a decrease in muscle force production, an accumulation of type IIB fibres, and an upregulation in the expression of genes involved with muscle atrophy, fibrosis and inflammation. Results . Chronic cuff tears in nude rats resulted in a 30% to 40% decrease in muscle mass, a 23% reduction in production of muscle force, and an induction of genes that regulate atrophy, fibrosis, lipid accumulation, inflammation and macrophage recruitment. Marked large lipid droplet accumulation was also present. Conclusions . The extent of degenerative changes in nude rats was similar to what was observed in T-cell competent rats. T cells may not play an important role in regulating muscle degeneration following chronic muscle unloading. The general similarities between nude and T-cell competent rats suggest the nude rat is likely an appropriate preclinical model for the study of xenografts that have the potential to enhance the treatment of chronically torn rotator cuff muscles. Cite this article: Bone Joint Res 2014;3:262–72

Objectives. Metabolic syndrome and low-grade systemic inflammation are associated with knee osteoarthritis (OA), but the relationships between these factors and OA in other synovial joints are unclear. The aim of this study was to determine if a high-fat/high-sucrose (HFS) diet results in OA-like joint damage in the shoulders, knees, and hips of rats after induction of obesity, and to identify potential joint-specific risks for OA-like changes. Methods. A total of 16 male Sprague-Dawley rats were allocated to either the diet-induced obesity group (DIO, 40% fat, 45% sucrose, n = 9) or a chow control diet (n = 7) for 12 weeks. At sacrifice, histological assessments of the shoulder, hip, and knee joints were performed. Serum inflammatory mediators and body composition were also evaluated. The total Mankin score for each animal was assessed by adding together the individual Modified Mankin scores across all three joints. Linear regression modelling was conducted to evaluate predictive relationships between serum mediators and total joint damage. Results. The HFS diet, in the absence of trauma, resulted in increased joint damage in the shoulder and knee joints of rats. Hip joint damage, however, was not significantly affected by DIO, consistent with findings in human studies. The total Mankin score was increased in DIO animals compared with the chow group, and was associated with percentage of body fat. Positive significant predictive relationships for total Mankin score were found between body fat and two serum mediators (interleukin 1 alpha (IL-1α) and vascular endothelial growth factor (VEGF)). Conclusion. Systemic inflammatory alterations from DIO in this model system may result in a higher risk for development of knee, shoulder, and multi-joint damage with a HFS diet. Cite this article: K. H. Collins, D. A. Hart, R. A. Seerattan, R. A. Reimer, W. Herzog. High-fat/high-sucrose diet-induced obesity results in joint-specific development of osteoarthritis-like degeneration in a rat model. Bone Joint Res 2018;7:274–281. DOI: 10.1302/2046-3758.74.BJR-2017-0201.R2

Aims. Rotator cuff (RC) tears are common musculoskeletal injuries which often require surgical intervention. Noninvasive pulsed electromagnetic field (PEMF) devices have been approved for treatment of long-bone fracture nonunions and as an adjunct to lumbar and cervical spine fusion surgery. This study aimed to assess the effect of continuous PEMF on postoperative RC healing in a rat RC repair model. Methods. A total of 30 Wistar rats underwent acute bilateral supraspinatus tear and repair. A miniaturized electromagnetic device (MED) was implanted at the right shoulder and generated focused PEMF therapy. The animals’ left shoulders served as controls. Biomechanical, histological, and bone properties were assessed at three and six weeks. Results. Extension of the tendon from preload to the maximum load to failure was significantly better in the PEMF-treated shoulders at three weeks compared to controls (p = 0.038). The percentage strain was significantly higher in the PEMF group at both timepoints (p = 0.037). Collagen organization was significantly better (p = 0.034) as was tissue mineral density in the PEMF-treated group at three weeks (p = 0.028). Tendon immunohistochemistry revealed a prominent increase in type I collagen at the repair site at three weeks following continuous PEMF treatment compared with controls. None of the other tested parameters differed between the groups. Conclusion. MED-generated PEMF may enhance early postoperative tendon-to-bone healing in an acute rat supraspinatus detachment and repair model. Superior biomechanical elasticity parameters together with better collagen organization suggest improved RC healing. Cite this article: Bone Joint Res 2021;10(5):298–306

Aims. The effect of the gut microbiota (GM) and its metabolite on bone health is termed the gut-bone axis. Multiple studies have elucidated the mechanisms but findings vary greatly. A systematic review was performed to analyze current animal models and explore the effect of GM on bone. Methods. Literature search was performed on PubMed and Embase databases. Information on the types and strains of animals, induction of osteoporosis, intervention strategies, determination of GM, assessment on bone mineral density (BMD) and bone quality, and key findings were extracted. Results. A total of 30 studies were included, of which six studies used rats and 24 studies used mice. Osteoporosis or bone loss was induced in 14 studies. Interventions included ten with probiotics, three with prebiotics, nine with antibiotics, two with short-chain fatty acid (SCFA), six with vitamins and proteins, two with traditional Chinese medicine (TCM), and one with neuropeptide Y1R antagonist. In general, probiotics, prebiotics, nutritional interventions, and TCM were found to reverse the GM dysbiosis and rescue bone loss. Conclusion. Despite the positive therapeutic effect of probiotics, prebiotics, and nutritional or pharmaceutical interventions on osteoporosis, there is still a critical knowledge gap regarding the role of GM in rescuing bone loss and its related pathways. Cite this article: Bone Joint Res 2021;10(1):51–59

Objectives. The purpose of this study was to evaluate in vivo biocompatibility of novel single-walled carbon nanotubes (SWCNT)/poly(lactic-co-glycolic acid) (PLAGA) composites for applications in bone and tissue regeneration. Methods. A total of 60 Sprague-Dawley rats (125 g to 149 g) were implanted subcutaneously with SWCNT/PLAGA composites (10 mg SWCNT and 1gm PLAGA 12 mm diameter two-dimensional disks), and at two, four, eight and 12 weeks post-implantation were compared with control (Sham) and PLAGA (five rats per group/point in time). Rats were observed for signs of morbidity, overt toxicity, weight gain and food consumption, while haematology, urinalysis and histopathology were completed when the animals were killed. Results. No mortality and clinical signs were observed. All groups showed consistent weight gain, and the rate of gain for each group was similar. All groups exhibited a similar pattern for food consumption. No difference in urinalysis, haematology, and absolute and relative organ weight was observed. A mild to moderate increase in the summary toxicity (sumtox) score was observed for PLAGA and SWCNT/PLAGA implanted animals, whereas the control animals did not show any response. Both PLAGA and SWCNT/PLAGA showed a significantly higher sumtox score compared with the control group at all time intervals. However, there was no significant difference between PLAGA and SWCNT/PLAGA groups. Conclusions. Our results demonstrate that SWCNT/PLAGA composites exhibited in vivo biocompatibility similar to the Food and Drug Administration approved biocompatible polymer, PLAGA, over a period of 12 weeks. These results showed potential of SWCNT/PLAGA composites for bone regeneration as the low percentage of SWCNT did not elicit a localised or general overt toxicity. Following the 12-week exposure, the material was considered to have an acceptable biocompatibility to warrant further long-term and more invasive in vivo studies. Cite this article: Bone Joint Res 2015;4:70–7