Aims. Pellino1 (Peli1) has been reported to regulate various inflammatory diseases. This study aims to explore the role of Peli1 in the occurrence and development of osteoarthritis (OA), so as to find new targets for the treatment of OA. Methods. After inhibiting Peli1 expression in chondrocytes with small interfering RNA (siRNA), interleukin (IL)-1β was used to simulate inflammation, and OA-related indicators such as synthesis, decomposition, inflammation, and apoptosis were detected. Toll-like receptor (TLR) and nuclear factor-kappa B (NF-κB) signalling pathway were detected. After inhibiting the expression of Peli1 in macrophages Raw 264.7 with siRNA and intervening with lipopolysaccharide (LPS), the polarization index of macrophages was detected, and the supernatant of macrophage medium was extracted as conditioned medium to act on chondrocytes and detect the apoptosis index. The OA model of mice was established by destabilized medial meniscus (DMM) surgery, and adenovirus was injected into the knee cavity to reduce the expression of Peli1. The degree of cartilage destruction and synovitis were evaluated by haematoxylin and eosin (H&E) staining, Safranin O/Fast Green staining, and immunohistochemistry. Results. In chondrocytes, knockdown of Peli1 produced anti-inflammatory and anti-apoptotic effects by targeting the TLR and NF-κB signalling pathways. We found that in macrophages, knockdown of Peli1 can inhibit M1-type polarization of macrophages. In addition, the corresponding conditioned culture medium of macrophages applied to chondrocytes can also produce an anti-apoptotic effect. During in vivo experiments, the results have also shown that knockdown Peli1 reduces cartilage destruction and synovial inflammation. Conclusion. Knockdown of Peli1 has a therapeutic effect on OA, which therefore makes it a potential therapeutic target for OA. Cite this article: Bone Joint Res 2023;12(2):121–132

Aims. Tert-butylhydroquinone (tBHQ) has been identified as an inhibitor of oxidative stress-induced injury and apoptosis in human neural stem cells. However, the role of tBHQ in osteoarthritis (OA) is unclear. This study was carried out to investigate the role of tBHQ in OA. Methods. OA animal model was induced by destabilization of the medial meniscus (DMM). Different concentrations of tBHQ (25 and 50 mg/kg) were intraperitoneally injected in ten-week-old female mice. Chondrocytes were isolated from articular cartilage of mice and treated with 5 ng/ml lipopolysaccharide (LPS) or 10 ng/ml interleukin 1 beta (IL-1β) for 24 hours, and then treated with different concentrations of tBHQ (10, 20, and 40 μM) for 12 hours. The expression levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were measured. The expression levels of interleukin 6 (IL-6), IL-1β, and tumour necrosis factor alpha (TNF-α) leptin in plasma were measured using enzyme-linked immunoabsorbent assay (ELISA) kits. The expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signalling pathway proteins, and macrophage repolarization-related markers, were detected by western blot. Results. Tert-butylhydroquinone significantly attenuated cartilage destruction in DMM-induced mice in vivo. It demonstrated clear evidence of inhibiting IL-1β-induced chondrocyte apoptosis, inflammation, and differentiation defect in vitro. Meanwhile, tBHQ inhibited LPS-induced activation of NF-κB and MAPK signalling pathways, and also inhibited LPS-induced reactive oxygen species production and macrophages repolarization in vitro. Conclusion. Taken together, tBHQ might be a potential therapeutic strategy for protecting against OA development. Cite this article: Bone Joint Res 2021;10(11):704–713

Aims. It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth. Methods. C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT-PCR. ELISA and qRT-PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs. Results. Mechanical stimulation promoted macrophage M2 polarization in vivo and in vitro. The conditioned media from mechanically stimulated BMDMs (MS-CM) enhanced MSC chondrogenic differentiation, and mechanically stimulated BMDMs generated more TGF-β1. Further, neutralizing TGF-β1 in MS-CM can attenuate its pro-chondrogenic effect. In vivo, mechanical stimulation promoted TGF-β1 generation, MSC chondrogenesis, and T-B healing, which were abolished following macrophage depletion. Conclusion. Macrophages subjected to appropriate mechanical stimulation could polarize toward the M2 phenotype and secrete TGF-β1 to promote MSC chondrogenesis, which subsequently augments T-B healing. Cite this article: Bone Joint Res 2023;12(3):219–230

Aims. The use of 3D-printed titanium implant (DT) can effectively guide bone regeneration. DT triggers a continuous host immune reaction, including macrophage type 1 polarization, that resists osseointegration. Interleukin 4 (IL4) is a specific cytokine modulating osteogenic capability that switches macrophage polarization type 1 to type 2, and this switch favours bone regeneration. Methods. IL4 at concentrations of 0, 30, and 100 ng/ml was used at day 3 to create a biomimetic environment for bone marrow mesenchymal stromal cell (BMMSC) osteogenesis and macrophage polarization on the DT. The osteogenic and immune responses of BMMSCs and macrophages were evaluated respectively. Results. DT plus 30 ng/ml of IL4 (DT + 30 IL4) from day 3 to day 7 significantly (p < 0.01) enhanced macrophage type 2 polarization and BMMSC osteogenesis compared with the other groups. Local injection of IL4 enhanced new bone formation surrounding the DT. Conclusion. DT + 30 IL4 may switch macrophage polarization at the appropriate timepoints to promote bone regeneration. Cite this article: Bone Joint Res 2021;10(7):411–424

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. Up to 10% of fractures result in undesirable outcomes, for which female sex is a risk factor. Cellular sex differences have been implicated in these different healing processes. Better understanding of the mechanisms underlying bone healing and sex differences in this process is key to improved clinical outcomes. This study utilized a macrophage–mesenchymal stem cell (MSC) coculture system to determine: 1) the precise timing of proinflammatory (M1) to anti-inflammatory (M2) macrophage transition for optimal bone formation; and 2) how such immunomodulation was affected by male versus female cocultures. Methods. A primary murine macrophage-MSC coculture system was used to demonstrate the optimal transition time from M1 to M2 (polarized from M1 with interleukin (IL)-4) macrophages to maximize matrix mineralization in male and female MSCs. Outcome variables included Alizarin Red staining, alkaline phosphatase (ALP) activity, and osteocalcin protein secretion. Results. We found that 96 hours of M1 phenotype in male cocultures allowed for maximum matrix mineralization versus 72 hours in female cocultures. ALP activity and osteocalcin secretion were also enhanced with the addition of IL-4 later in male versus female groups. The sex of the cells had a statistically significant effect on the optimal IL-4 addition time to maximize osteogenesis. Conclusion. These results suggest that: 1) a 72- to 96-hour proinflammatory environment is critical for optimal matrix mineralization; and 2) there are immunological differences in this coculture environment due to sex. Optimizing immunomodulation during fracture healing may enhance and expedite the bone regeneration response. These findings provide insight into precise immunomodulation for enhanced bone healing that is sex-specific. Cite this article: K. Nathan, L. Y. Lu, T. Lin, J. Pajarinen, E. Jämsen, J-F. Huang, M. Romero-Lopez, M. Maruyama, Y. Kohno, Z. Yao, S. B. Goodman. Precise immunomodulation of the M1 to M2 macrophage transition enhances mesenchymal stem cell osteogenesis and differs by sex. Bone Joint Res 2019;8:481–488. DOI: 10.1302/2046-3758.810.BJR-2018-0231.R2

Aims. MicroRNA-183 (miR-183) is known to play important roles in osteoarthritis (OA) pain. The aims of this study were to explore the specific functions of miR-183 in OA pain and to investigate the underlying mechanisms. Methods. Clinical samples were collected from patients with OA, and a mouse model of OA pain was constructed by surgically induced destabilization of the medial meniscus (DMM). Reverse transcription quantitative polymerase chain reaction was employed to measure the expression of miR-183, transforming growth factor α (TGFα), C-C motif chemokine ligand 2 (CCL2), proinflammatory cytokines (interleukin (IL)-6, IL-1β, and tumour necrosis factor-α (TNF-α)), and pain-related factors (transient receptor potential vanilloid subtype-1 (TRPV1), voltage-gated sodium 1.3, 1.7, and 1.8 (Nav1.3, Nav1.7, and Nav1.8)). Expression of miR-183 in the dorsal root ganglia (DRG) of mice was evaluated by in situ hybridization. TGFα, CCL2, and C-C chemokine receptor type 2 (CCR2) levels were examined by immunoblot analysis and interaction between miR-183 and TGFα, determined by luciferase reporter assay. The extent of pain in mice was measured using a behavioural assay, and OA severity assessed by Safranin O and Fast Green staining. Immunofluorescent staining was conducted to examine the infiltration of macrophages in mouse DRG. Results. miR-183 was downregulated in tissue samples from patients and mice with OA. In DMM mice, overexpression of miR-183 inhibited the expression of proinflammatory cytokines (IL-6, IL-1β, TNF-α) and pain-related factors (TRPV1, Nav1.3, Nav1.7, Nav1.8) in DRG. OA pain was relieved by miR-183-mediated inhibition of macrophage infiltration, and dual luciferase reporter assay demonstrated that miR-183 directly targeted TGFα. Conclusion. Our data demonstrate that miR-183 can ameliorate OA pain by inhibiting the TGFα-CCL2/CCR2 signalling axis, providing an excellent therapeutic target for OA treatment. Cite this article: Bone Joint Res 2021;10(8):548–557

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

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

Aims. Treatment for delayed wound healing resulting from peripheral vascular diseases and diabetic foot ulcers remains a challenge. A novel surgical technique named ‘tibial cortex transverse transport’ (TTT) has been developed for treating peripheral ischaemia, with encouraging clinical effects. However, its underlying mechanisms remain unclear. In the present study, we explored the potential biological mechanisms of TTT surgery using various techniques in a rat TTT animal model. Methods. A novel rat model of TTT was established with a designed external fixator, and effects on wound healing were investigated. Laser speckle perfusion imaging, vessel perfusion, histology, and immunohistochemistry were used to evaluate the wound healing processes. Results. Gross and histological examinations showed that TTT technique accelerated wound closure and enhanced the quality of the newly formed skin tissues. In the TTT group, haematoxylin and eosin (H&E) staining demonstrated a better epidermis and dermis recovery, while immunohistochemical staining showed that TTT technique promoted local collagen deposition. The TTT technique also benefited to angiogenesis and immunomodulation. In the TTT group, blood flow in the wound area was higher than that of other groups according to laser speckle imaging with more blood vessels observed. Enhanced neovascularization was seen in the TTT group with double immune-labelling of CD31 and α-Smooth Muscle Actin (α-SMA). The number of M2 macrophages at the wound site in the TTT group was also increased. Conclusion. The TTT technique accelerated wound healing through enhanced angiogenesis and immunomodulation. Cite this article: Bone Joint Res 2022;11(4):189–199

Aims. The aim of this study was to determine the fracture haematoma (fxH) proteome after multiple trauma using label-free proteomics, comparing two different fracture treatment strategies. Methods. A porcine multiple trauma model was used in which two fracture treatment strategies were compared: early total care (ETC) and damage control orthopaedics (DCO). fxH was harvested and analyzed using liquid chromatography-tandem mass spectrometry. Per group, discriminating proteins were identified and protein interaction analyses were performed to further elucidate key biomolecular pathways in the early fracture healing phase. Results. The early fxH proteome was characterized by immunomodulatory and osteogenic proteins, and proteins involved in the coagulation cascade. Treatment-specific proteome alterations were observed. The fxH proteome of the ETC group showed increased expression of pro-inflammatory proteins related to, among others, activation of the complement system, neutrophil functioning, and macrophage activation, while showing decreased expression of proteins related to osteogenesis and tissue remodelling. Conversely, the fxH proteome of the DCO group contained various upregulated or exclusively detected proteins related to tissue regeneration and remodelling, and proteins related to anti-inflammatory and osteogenic processes. Conclusion. The early fxH proteome of the ETC group was characterized by the expression of immunomodulatory, mainly pro-inflammatory, proteins, whereas the early fxH proteome of the DCO group was more regenerative and osteogenic in nature. These findings match clinical observations, in which enhanced surgical trauma after multiple trauma causes dysbalanced inflammation, potentially leading to reduced tissue regeneration, and gained insights into regulatory mechanisms of fracture healing after severe trauma. Cite this article: Bone Joint Res 2024;13(5):214–225

Aims. The role of N,N-dimethylformamide (DMF) in diabetes-induced osteoporosis (DM-OS) progression remains unclear. Here, we aimed to explore the effect of DMF on DM-OS development. Methods. Diabetic models of mice, RAW 264.7 cells, and bone marrow macrophages (BMMs) were established by streptozotocin stimulation, high glucose treatment, and receptor activator of nuclear factor-κB ligand (RANKL) treatment, respectively. The effects of DMF on DM-OS development in these models were examined by micro-CT analysis, haematoxylin and eosin (H&E) staining, osteoclast differentiation of RAW 264.7 cells and BMMs, H&E and tartrate-resistant acid phosphatase (TRAP) staining, enzyme-linked immunosorbent assay (ELISA) of TRAP5b and c-terminal telopeptides of type 1 (CTX1) analyses, reactive oxygen species (ROS) analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), Cell Counting Kit-8 (CCK-8) assay, and Western blot. Results. The established diabetic mice were more sensitive to ovariectomy (OVX)-induced osteoporosis, and DMF treatment inhibited the sensitivity. OVX-treated diabetic mice exhibited higher TRAP5b and c-terminal telopeptides of type 1 (CTX1) levels, and DMF treatment inhibited the enhancement. DMF reduced RAW 264.7 cell viability. Glucose treatment enhanced the levels of TRAP5b, cathepsin K, Atp6v0d2, and H. +. -ATPase, ROS, while DMF reversed this phenotype. The glucose-increased protein levels were inhibited by DMF in cells treated with RANKL. The expression levels of antioxidant enzymes Gclc, Gclm, Ho-1, and Nqo1 were upregulated by DMF. DMF attenuated high glucose-caused osteoclast differentiation by targeting mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signalling in BMMs. Conclusion. DMF inhibits high glucose-induced osteoporosis by targeting MAPK and NF-κB signalling. Cite this article: Bone Joint Res 2022;11(4):200–209

Objectives. The aim of this study was to examine whether asymmetric loading influences macrophage elastase (MMP12) expression in different parts of a rat tail intervertebral disc and growth plate and if MMP12 expression is correlated with the severity of the deformity. Methods. A wedge deformity between the ninth and tenth tail vertebrae was produced with an Ilizarov-type mini external fixator in 45 female Wistar rats, matched for their age and weight. Three groups were created according to the degree of deformity (10°, 30° and 50°). A total of 30 discs and vertebrae were evaluated immunohistochemically for immunolocalisation of MMP12 expression, and 15 discs were analysed by western blot and zymography in order to detect pro- and active MMP12. Results. No MMP12 expression was detected in the nucleus pulposus. Expression of MMP12 in the annulus progressively increased from group I to groups II and III, mainly at the concave side. Many growth plate chondrocytes expressed MMP12 in the control group, less in group I and rare in groups II and III. Changes in cell phenotype and reduction of cell number were observed, together with disorganisation of matrix microstructure similar to disc degeneration. ProMMP12 was detected at the area of 54 kDa and active MMP12 at 22 kDa. Conclusions. Expression of MMP12 after application of asymmetric loading in a rat tail increased in the intervertebral disc but decreased in the growth plate and correlated with the degree of the deformity and the side of the wedged disc. Cite this article: Bone Joint Res 2014;3:273–9

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We aimed to develop a gene signature that predicts the occurrence of postmenopausal osteoporosis (PMOP) by studying its genetic mechanism.

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Despite the interest in the association of gut microbiota with bone health, limited population-based studies of gut microbiota and bone mineral density (BMD) have been made. Our aim is to explore the possible association between gut microbiota and BMD.

Aims. Osteoporosis is characterized by decreased trabecular bone volume, and microarchitectural deterioration in the medullary cavity. Interleukin-19 (IL-19), a member of the IL-10 family, is an anti-inflammatory cytokine produced primarily by macrophages. The aim of our study was to investigate the effect of IL-19 on osteoporosis. Methods. Blood and femoral bone marrow suspension IL-19 levels were first measured in the lipopolysaccharide (LPS)-induced bone loss model. Small interfering RNA (siRNA) was applied to knock down IL-19 for further validation. Thereafter, osteoclast production was stimulated with IL-19 in combination with mouse macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). The effect of IL-19 was subsequently evaluated using tartrate-resistant acid phosphatase (TRAP) staining and quantitative real-time polymerase chain reaction (RT-qPCR). The effect of IL-19 on osteoprotegerin (OPG) was then assessed using in vitro recombinant IL-19 treatment of primary osteoblasts and MLO-Y4 osteoblast cell line. Finally, transient transfection experiments and chromatin immunoprecipitation (ChIP) experiments were used to examine the exact mechanism of action. Results. In the LPS-induced bone loss mouse model, the levels of IL-19 in peripheral blood serum and femoral bone marrow suspension were significantly increased. The in vivo results indicated that global IL-19 deletion had no significant effect on RANKL content in the serum and bone marrow, but could increase the content of OPG in serum and femoral bone marrow, suggesting that IL-19 inhibits OPG expression in bone marrow mesenchymal stem cells (BMSCs) and thus increases bone resorption. Conclusion. IL-19 promotes bone resorption by suppressing OPG expression in BMSCs in a LPS-induced bone loss mouse model, which highlights the potential benefits and side effects of IL-19 for future clinical applications. Cite this article: Bone Joint Res 2023;12(11):691–701

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

Aims. Currently, the effect of drug treatment for osteoporosis is relatively poor, and the side effects are numerous and serious. Melatonin is a potential drug to improve bone mass in postmenopausal women. Unfortunately, the mechanism by which melatonin improves bone metabolism remains unclear. The aim of this study was to further investigate the potential mechanism of melatonin in the treatment of osteoporosis. Methods. The effects of melatonin on mitochondrial apoptosis protein, bmal1 gene, and related pathway proteins of RAW264.7 (mouse mononuclear macrophage leukaemia cells) were analyzed by western blot. Cell Counting Kit-8 was used to evaluate the effect of melatonin on cell viability. Flow cytometry was used to evaluate the effect of melatonin on the apoptosis of RAW264.7 cells and mitochondrial membrane potential. A reactive oxygen species (ROS) detection kit was used to evaluate the level of ROS in osteoclast precursors. We used bmal1-small interfering RNAs (siRNAs) to downregulate the Bmal1 gene. We established a postmenopausal mouse model and verified the effect of melatonin on the bone mass of postmenopausal osteoporosis in mice via micro-CT. Bmal1 lentiviral activation particles were used to establish an in vitro model of overexpression of the bmal1 gene. Results. Melatonin promoted apoptosis of RAW264.7 cells and increased the expression of BMAL1 to inhibit the activation of ROS and phosphorylation of mitogen-activated protein kinase (MAPK)-p38. Silencing the bmal1 gene weakened the above effects of melatonin. After that, we used dehydrocorydaline (DHC) to enhance the activation of MAPK-p38, and the effects of melatonin on reducing ROS levels and promoting apoptosis of RAW264.7 cells were also blocked. Then, we constructed a mouse model of postmenopausal osteoporosis and administered melatonin. The results showed that melatonin improves bone loss in ovariectomized mice. Finally, we established a model of overexpression of the bmal1 gene, and these results suggest that the bmal1 gene can regulate ROS activity and change the level of the MAPK-p38 signalling pathway. Conclusion. Our study confirmed that melatonin promotes the apoptosis of RAW264.7 cells through BMAL1/ROS/MAPK-p38, and revealed the therapeutic effect and mechanism of melatonin in postmenopausal osteoporosis. This finding enriches BMAL1 as a potential target for the treatment of osteoporosis and the pathogenesis of postmenopausal osteoporosis. Cite this article: Bone Joint Res 2023;12(11):677–690