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

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

Aims. Gap junction intercellular communication (GJIC) in osteocytes is impaired by oxidative stress, which is associated with age-related bone loss. Ageing is accompanied by the accumulation of advanced oxidation protein products (AOPPs). However, it is still unknown whether AOPP accumulation is involved in the impairment of osteocytes’ GJIC. This study aims to investigate the effect of AOPP accumulation on osteocytes’ GJIC in aged male mice and its mechanism. Methods. Changes in AOPP levels, expression of connexin43 (Cx43), osteocyte network, and bone mass were detected in 18-month-old and three-month-old male mice. Cx43 expression, GJIC function, mitochondria membrane potential, reactive oxygen species (ROS) levels, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation were detected in murine osteocyte-like cells (MLOY4 cells) treated with AOPPs. The Cx43 expression, osteocyte network, bone mass, and mechanical properties were detected in three-month-old mice treated with AOPPs for 12 weeks. Results. The AOPP levels were increased in aged mice and correlated with degeneration of osteocyte network, loss of bone mass, and decreased Cx43 expression. AOPP intervention induced NADPH oxidase activation and mitochondrial dysfunction, triggered ROS generation, reduced Cx43 expression, and ultimately impaired osteocytes’ GJIC, which were ameliorated by NADPH oxidase inhibitor apocynin, mitochondria-targeted superoxide dismutase mimetic (mito-TEMPO), and ROS scavenger N-acetyl cysteine. Chronic AOPP loading accelerated the degradation of osteocyte networks and decreased Cx43 expression, resulting in deterioration of bone mass and mechanical properties in vivo. Conclusion. Our study suggests that AOPP accumulation contributes to age-related impairment of GJIC in osteocytes of male mice, which may be part of the pathogenic mechanism responsible for bone loss during ageing. Cite this article: Bone Joint Res 2022;11(7):413–425

The rotator cuff tendinopathy is one of the most common shoulder problems leading to full-thickness rotator cuff tendon tear and, eventually, to degenerative arthritis. Recent research on rotator cuff tendon degeneration has focused on its relationship to cell death. The types of cell death known to be associated with rotator cuff tendon degeneration are apoptosis, necrosis, and autophagic cell death. The increased incidence of cell death in degenerative tendon tissue may affect the rates of collagen synthesis and repair, possibly weakening tendon tissue and increasing the risk of tendon rupture. The biomolecular mechanisms of the degenerative changes leading to apoptotic cell death in rotator cuff tenofibroblasts have been identified as oxidative-stress-related cascade mechanisms. Furthermore, apoptosis, necrosis, and autophagic cell death are all known to be mediated by oxidative stress, a condition in which ROS (reactive oxygen species) are overproduced. Lower levels of oxidative stress trigger apoptosis; higher levels mediate necrosis. Although the signaltransduction pathway leading to autophagy has not yet been fully established, ROS are known to be essential to autophagy. A neuronal theory regarding rotator cuff degeneration has been developed from the findings that glutamate, a neural transmitter, is present in increased concentrations in tendon tissues with tendinopathy and that it induces rat supraspinatus tendon cell death. Recent studies have reported that hypoxia involved in rotator cuff tendon degeneration. Because antioxidants are known to scavenge for intracellular ROS, some studies have been conducted to determine whether antioxidants can reduce cell death in rotator cuff tendon-origin fibroblasts. The first study reported that an antioxidant has the ability to reduce apoptosis in oxidative-stressed rotator cuff tenofibroblasts. The second study reported that antioxidants have both antiapoptotic effects and antinecrotic effects on rotator cuff tendon-origin fibroblasts exposed to an oxidative stimulus. The third study reported that an antioxidant has antiautophagic-cell-death effects on rotator cuff tendon-origin fibroblasts exposed to an oxidative stimulus. The fourth study reported that glutamate markedly increases cell death in rotator cuff tendonorigin fibroblasts. The glutamate-induced cytotoxic effects were reduced by an antioxidant, demonstrating its cytoprotective effects against glutamate-induced tenofibroblast cell death. The fifth study reported that hypoxia significantly increases intracellular ROS and apoptosis. The hypoxia-induced cytotoxic effects were markedly attenuated by antioxidants, demonstrating their cytoprotective effects against hypoxia-induced tenofibroblast cell death. In conclusion, antioxidants have cytoprotective effects on tenofibroblasts exposed in vitro to an oxidative stressor, a neurotransmitter, or hypoxia. These cytoprotective effects result from antiapoptotic, antinecrotic, and antiautophagic actions involving the inhibition of ROS formation. These findings suggest that antioxidants may have therapeutic potential for rotator cuff tendinopathy. Further studies must be conducted in order to apply these in vitro findings to clinical situations

Aims. The purpose of this study was to evaluate the in vitro effects of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and a downregulator of intracellular reactive oxygen species (ROS), on high glucose-induced oxidative stress on tenocytes. Methods. Tenocytes from normal Sprague-Dawley rats were cultured in both control and high-glucose conditions. Apocynin was added at cell seeding, dividing the tenocytes into four groups: the control group; regular glucose with apocynin (RG apo+); high glucose with apocynin (HG apo+); and high glucose without apocynin (HG apo–). Reactive oxygen species production, cell proliferation, apoptosis and messenger RNA (mRNA) expression of NOX1 and 4, and interleukin-6 (IL-6) were determined in vitro. Results. Expression of NOX1, NOX4, and IL-6 mRNA in the HG groups was significantly higher compared with that in the RG groups, and NOX1, NOX4, and IL-6 mRNA expression in the HG apo+ group was significantly lower compared with that in the HG apo– group. Cell proliferation in the RG apo+ group was significantly higher than in the control group and was also significantly higher in the HG apo+ group than in the HG apo– group. Both the ROS accumulation and the amounts of apoptotic cells in the HG groups were greater than those in the RG groups and were significantly less in the HG apo+ group than in the HG apo– group. Conclusion. Apocynin reduced ROS production and cell death via NOX inhibition in high-glucose conditions. Apocynin is therefore a potential prodrug in the treatment of diabetic tendinopathy. Cite this article:Bone Joint Res 2020;9(1):23–28

Invertebral disc degeneration (IDD) is a degenerative disease involving a variety of musculoskeletal and spinal disorders such as lower back pain (LBP). Secretome derived from mesenchymal stem cells (MSCs) have exerted beneficial effect on tissue regeneration. In this study, the goal was to investigate the paracrine and the anti-inflammatory effects of secretome from interleukin IL1β preconditioned Bone Marrow MSCs (BMSCs) on human nucleus pulposus cells (hNPCs) in a 3D in vitro model. Secretome was collected from BMSCs (BMSCs-sec) after preconditioning with 10 ng/mL IL1β. hNPCs were isolated from surgical specimens, culture expanded in vitro, encapsulated in alginate beads and treated with: growth medium; IL1β 10 ng/mL; IL1β 10 ng/mL for 24 hours and then BMSCs-sec. We examined: i) cell proliferation and viability (flow cytometry), ii) nitrite production (Griess assay) and ROS quantification (Immunofluorescence) iii) glycosaminoglycan (GAG) amount (DMBB) and iv) gene expression levels of extracellular matrix (ECM) components and inflammatory mediators (qPCR). One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D. In vitro tests showed an enhancement of hNPCs proliferation after treatment with BMSCs-sec (p ≤ 0.05) compared to IL1β group. After 24 hours, the percentage of dead cells was higher in IL1β treated hNPCs compared to control group and decreased significantly in combined IL1β and BMSCs-sec sample group (p ≤ 0.01). Nitrite and ROS production were significantly mitigated and GAGs content was improved by preconditioned BMSCs-sec (p ≤ 0.05). Furthermore, gene expression levels were modulated by BMSCs-sec treatment compared to controls. Our results supported the potential use of BMSCs' secretome as a cell-free strategy for IDD, overcoming the side effects of cell-therapy. Moreover, secretome derived from IL1β preconditioned BMSCs was able to reduce hNPCs death, attenuate ECM degradation and oxidative stress counteracting IDD progression. Acknowledgements: Financial support was received from the “iPSpine” and “RESPINE” Horizon 2020 projects

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

Introduction and Objective. Hyaluronic acid (HA) is an effective option for the treatment of osteoarthritis (OA) patients due to several properties such as normalization of the mechanical and rheological properties of the synovial fluid and amelioration of OA symptoms and joints function by promoting cartilage nutrition. Since OA progression is also significantly related to oxidative stress and reactive oxygen species (ROS), sodium succinate (SS) is envisioned as a promising compound for cartilage treatment by providing antioxidant defense able to normalize intracellular metabolism and tissue respiration via mitochondrial mechanism of action. The scope of this study was to investigate on an in vitro inflammatory model the efficacy of Diart. ®. product, a combination of HA and SS. Materials and Methods. Donor-matched chondrocytes and synoviocytes were obtained from KL 3–4 OA patients undergoing total knee replacement. At passage 4, inflammation was promoted with 1 ng/ml IL-1B for 48 hours in absence and presence of Diart. ®. at 1:3 dilution rate. Nitric oxide (NO) from cell culture supernatant was measured by Griess reaction. Mitochondrial and cytoplasmatic ROS evaluation was assessed by flow cytometry with MitoSox and dichlorodihydrofluorescein diacetate (DCFDA) assays. Gene expression of inflammation/oxidative stress-related transcripts (MMP1/MMP3/INOS/COX2) was evaluated by qRT-PCR using TBP as reference. Results. NO was detected only in inflamed chondrocytes and Diart® was able to abolish its levels. NO was not detected in synoviocytes in all conditions. IL-1B reduced both cytoplasmic (−66%) and mitochondrial (−68%) ROS in chondrocytes, with Diart® partially restoring (+40%) mitochondrial levels. In synoviocytes, IL-1B did not alter ROS, with Diart® modestly increasing (+27%) mitochondrial levels. Inflammation was able to increase transcript levels of all tested markers, with the exception of INOS in synoviocytes. In chondrocytes, Diart® significantly (p < 0.05) reduced COX2 (−75%) and MMP1 (−33%). In synoviocytes, Diart® significantly reduced COX2 (−77%) and MMP3 (−84%), with MMP1 53% decreased albeit without reaching statistical significance. Conclusions. Diart. ®. biochemical and physiologic properties in the tested in vitro model of inflammation on donor-matched chondrocytes and synoviocytes allowed reducing inflammation and oxidative stress-related markers, prompting the use of this combination as successful strategy to manage OA-related symptoms

Objectives. Recently, high failure rates of metal-on-metal (MOM) hip implants have raised concerns of cobalt toxicity. Adverse reactions occur to cobalt nanoparticles (CoNPs) and cobalt ions (Co. 2+. ) during wear of MOM hip implants, but the toxic mechanism is not clear. Methods. To evaluate the protective effect of zinc ions (Zn. 2+. ), Balb/3T3 mouse fibroblast cells were pretreated with 50 μM Zn. 2+. for four hours. The cells were then exposed to different concentrations of CoNPs and Co. 2+. for four hours, 24 hours and 48 hours. The cell viabilities, reactive oxygen species (ROS) levels, and inflammatory cytokines were measured. Results. CoNPs and Co. 2+. can induce the increase of ROS and inflammatory cytokines, such as tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). However, Zn pretreatment can significantly prevent cytotoxicity induced by CoNPs and Co. 2+. , decrease ROS production, and decrease levels of inflammatory cytokines in Balb/3T3 mouse fibroblast cells. Conclusion. These results suggest that Zn pretreatment can provide protection against inflammation and cytotoxicity induced by CoNPs and Co. 2+. in Balb/3T3 cells. Cite this article: Y. Liu, H. Zhu, H. Hong, W. Wang, F. Liu. Can zinc protect cells from the cytotoxic effects of cobalt ions and nanoparticles derived from metal-on-metal joint arthroplasties? Bone Joint Res 2017;6:649–655. DOI: 10.1302/2046-3758.612.BJR-2016-0137.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

Introduction. Particle-induced oxidative stress in cells is a unifying factor that determines toxicity and carcinogenicity potential in biomaterials. A previous study by Bladen et al. showed the production of significant levels of reactive oxygen species (ROS) following the stimulation of phagocytes by UHMWPE and CoCr wear debris [1]. Latest generation bearing materials such as silicon nitride also need to be tested for potential generation of ROS in phagocytic cells. This study aimed to investigate the production of reactive oxygen species in L929 fibroblasts stimulated with clinically relevant doses of nanoscale and micron-sized silicon nitride (Si. 3. N. 4. ) particles, silica nanoparticles, and CoCr wear debris. Silica nanoparticles were included as a comparison material for situations where the Si. 3. N. 4. particle's surface are oxidised to silicon dioxide [2]. Materials and Methods. Si. 3. N. 4. particles (<50 nm and <1 µm, Sigma), silica nanopowder (<100 nm, Sigma) and clinically relevant CoCr wear particles were heat-treated at 180°C for 4 h to remove endotoxin. Particles were then re-suspended in sterile water by sonication. L929 murine fibroblasts were cultured with low doses (0.5 µm. 3. /cell) and high doses (50 µm. 3. /cell) of Si. 3. N. 4. particles, and high doses (50 µm. 3. /cell) of silica nanoparticles and CoCr wear debris. Cells were incubated for three and six days at 37°C with 5% (v/v) CO. 2. tert-Butyl hydroperoxide (TBHP) was used as a positive control for the production of ROS in the cells. Intracellular ROS was measured using Image-IT LIVE kit (Invitrogen). This assay is based on carboxy-2',7'-dichlorodihydro-fluorescein diacetate (carboxy-H2DCFDA), which forms a non-fluorescent derivative by intracellular esterases and then reacts with intracellular ROS to form green fluoroscence producing derivative carboxy- dichlorodihydro-fluorescein. Images were captured using a confocal microscope and analysed using ImageJ for corrected total cell fluorescence (CTCF). The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc tests. Results and Discussion. Si. 3. N. 4. nanoparticles significantly reduced the ROS levels in L929 fibroblasts at low doses (0.5 μm. 3. /cell) and high doses (50 μm. 3. /cell) over a period of six days; whereas no significant change in the levels of ROS was observed in cells treated with micron-sized Si. 3. N. 4. particles [Figure 1]. Only a few cells treated with high doses of CoCr wear particles (50 μm. 3. /cell) survived for up to six days and produced significantly higher levels of ROS [Figure 1, 2]. Interestingly, cells challenged with high doses (50 μm. 3. /cell) of Si. 3. N. 4. and silica nanoparticles produced statistically similar levels of ROS in cells [Figure 1]. This might be due to the potential surface oxidation of Si. 3. N. 4. nanoparticles, which makes their surface chemistry and biological identity similar to silica nanoparticles. Conclusion. Unlike existing implant materials such as UHMWPE and CoCr, silicon nitride has demonstrated the capacity to reduce or maintain normal levels of ROS in macrophages depending on the particle size and dose. Acknowledgements. The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. GA-310477 LifeLongJoints

Objectives. We have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells. Materials and Methods. We seeded mouse skeletal muscle cells C2C12 on the hydroxyapatite/calcium sulphate biomaterial and the phenotype of the cells was analysed. To mimic surgical conditions with leakage of extra cellular matrix (ECM) proteins and growth factors, we cultured rat bone cells ROS 17/2.8 in a bioreactor and harvested the secreted proteins. The secretome was added to rat muscle cells L6. The phenotype of the muscle cells after treatment with the media was assessed using immunostaining and light microscopy. Results. C2C12 cells differentiated into osteoblast-like cells expressing prominent bone markers after seeding on the biomaterial. The conditioned media of the ROS 17/2.8 contained bone morphogenetic protein-2 (BMP-2 8.4 ng/mg, standard deviation (. sd. ) 0.8) and BMP-7 (50.6 ng/mg, . sd. 2.2). In vitro, this secretome induced differentiation of skeletal muscle cells L6 towards an osteogenic lineage. Conclusion. Extra cellular matrix proteins and growth factors leaking from a bone cavity, along with a ceramic biomaterial, can synergistically enhance the process of ectopic ossification. The overlaying muscle acts as an osteoinductive niche, and provides the required cells for bone formation. Cite this article: D. B. Raina, A. Gupta, M. M. Petersen, W. Hettwer, M. McNally, M. Tägil, M-H. Zheng, A. Kumar, L. Lidgren. Muscle as an osteoinductive niche for local bone formation with the use of a biphasic calcium sulphate/hydroxyapatite biomaterial. Bone Joint Res 2016;5:500–511. DOI: 10.1302/2046-3758.510.BJR-2016-0133.R1

The use of mesenchymal stromal cells (MSCs) in regenerative medicine and tissue engineering is well established, given their properties of self-renewal and differentiation. However, several studies have shown that these properties diminish with age, and understanding the pathways involved are important to provide regenerative therapies in an ageing population. In this PRISMA systematic review, we investigated the effects of chronological donor ageing on the senescence of MSCs. We identified 3023 studies after searching four databases including PubMed, Web of Science, Cochrane, and Medline. Nine studies met the inclusion and exclusion criteria and were included in the final analyses. These studies showed an increase in the expression of p21, p53, p16, ROS, and NF- B with chronological age. This implies an activated DNA damage response (DDR), as well as increased levels of stress and inflammation in the MSCs of older donors. Additionally, highlighting the effects of an activated DDR in cells from older donors, a decrease in the expression of proliferative markers including Ki67, MAPK pathway elements, and Wnt/ -catenin pathway elements was observed. Furthermore, we found an increase in the levels of SA- -galactosidase, a specific marker of cellular senescence. Together, these findings support an association between chronological age and MSC senescence. The precise threshold for chronological age where the reported changes become significant is yet to be defined and should form the basis for further scientific investigations. The outcomes of this review should direct further investigations into reversing the biological effects of chronological age on the MSC senescence phenotype

Stem cells are known to have low levels of intracellular reactive oxygen species (ROS) and high levels of glutathione. ROS are thought to interact with several pathways that affect the transcription machinery required for stem cell differentiation, and are critical for maintaining stem cell function. In this study, we are developing a new fluorescent probe that rapidly and reversibly reacts with glutathione (GSH), the most abundant non-protein thiol in living cells that acts as an antioxidant and redox regulator. Multipotent perivascular progenitor cells derived from human ESCs (hESC-PVPCs): Differentiated ESCs as embryoid bodies in the presence of BMP4 to induce mesoderm differentiation followed by a simple cell selection strategy using attachment of single cells onto collagen-coated dishes. Differential gene expression profiling was performed among H9 hESCs, EBs induced by BMP4 and naturally selected CD140B+CD44+ population at Day 7 (PVPCs). Colony-forming assay: GSHhigh and GSHlow PVPCs were plated on 10-cm tissue culture-treated polystyrene dishes in triplicate in growth medium and cultured for 14 days. Transwell migration assay: GSHhigh and GSHlow PVPCs at passage 4 were resuspended at 1 × 10. 6. /mL in the migration medium and seeded in the upper chamber. The following human recombinant SDF-1 and PDGF-AA proteins were used as chemoattractants in the lower compartment. Probe-GSH conjugate shows shifts in fluorescence excitation and emission spectra that enables ratiometric measurement of GSH levels. Using these properties, stem cells can be purified by FACS-based technology according to intracellular GSH level. We are developing a protocol both for comparing GSH level in stem cell from different culture conditions and for preparing stem cells with high-GSH level . Our results reveal that GSHhigh PVPC purified by FACS show increased colony forming ability compared with that GSHlow PVPC, indicating that intracellular GSH contributes to the maintenance of stemness. Moreover, transplantation of GSHlow PVPC is more effective than that of GSHlow PVPC for cartilage regeneration in osteochondral defect. This technique enable FACS-based sorting of stem cells according to intracellular GSH levels and thus investigation of functional role of GSH (high antioxidant capacity) in the stem cell maintenance and chondrogenic differentiation

Summary Statement. A novel transcutaneous CO. 2. therapy significantly enhanced the antitumor effectiveness of X-ray irradiation in human MFH xenografts The results strongly suggest that transcutaneous CO. 2. therapy could be a novel therapeutic tool for overcoming radioresistance in human malignancies. Introduction. Hypoxia contributes to tumor radioresistance. In the presence of oxygen, reactive oxygen species (ROS) play crucial roles in cellular apoptosis to irradiation. We previously showed that a novel transcutaneous application of CO. 2. can improve hypoxia and that it induces apoptosis and decreases the expression of HIF-1α in sarcoma. Therefore, we hypothesised that a transcutaneous application of CO. 2. may increase radiosensitivity in sarcoma by improvement of hypoxic condition and increasing ROS production in tumors. The purpose of this study is to examine the effect of transcutaneous application of CO. 2. on radiosensitivity in human malignant fibrous histiocytoma (MFH) cells. Methods. Cells. We used a human MFH cell line, Nara-H in this study. X-ray irradiation. X-ray irradiation was performed at a dose rate of 0.64 to 0.66 Gy/min. Colony formation assay. In vitro cell viability after X-ray irradiation was assessed by colony formation assay. In vivo studies. Nara-H cells were subcutaneously implanted to 24 nude mice which were randomly divided into 4 groups; CO. 2. group, X-ray group, Combination group and Control group. CO. 2. therapy was performed as we previously reported (1, 2). In combination group, mice were treated twice a week by X-ray at 3.2 Gy shortly after CO. 2. therapy for 2 weeks. The changes in body weight and tumor volume were monitored for 14 days in all 4 groups. The implanted tumors were excised at the end of experiment. We also excised tumors on the first day of each treatment in all 4 groups, and examined apoptosis and ROS expression by FACS analysis. The animal experiments were approved by the Animal Committee in our institute. Immunoblot analysis. The protein expression of HIF-1α, ROS-related proteins (p38 and JNK/SAPK), and apoptosis-related proteins (caspase-3 and PARP) were assessed by immunoblot analysis. FACS analysis. DNA fragmentation and ROS production in tumors were assessed by FACS analysis. Statistical analysis. ANOVA with post hoc test to compare for continuous values. All tests were considered significant at p<0.05. Results. Approximately 50% of Nara-H cells survived after a total of 3.2 Gy X-ray irradiation. Tumor volume in combination group was significantly reduced at the end of experiment (47% of that in X-ray group and 28% of that in control group). In Combination group, apoptosis with ROS production markedly increased when compared with those in Control, CO. 2. or X-ray group at 24 hours after treatment. Immunoblot analysis showed that, in combination group, the expression of phospho-p38, phospho-JNK/SPAK, and cleavage of both caspase-3 and PARP were increased compared with other groups, conversely, the expression of HIF-1α was decreased. Discussion/Conclusion. In this study, we demonstrated that the combination therapy showed more significant effects on apoptosis and ROS production through improving hypoxia in MFH cells in vivo. Our findings strongly suggest that the combination therapy of CO. 2. and X-ray could be a novel therapeutic strategy for the treatment of human MFH, and that transcutaneous application of CO. 2. may be one of the best radiosensitisers

Implant-related infection (IRI) is closely related to the local immunity of peri-implant tissues. The generation of reactive oxygen species (ROS) in activated macrophages plays a prominent role in the innate immune response. In previous studies, we indicated that implant wear particles promote endotoxin tolerance by decreasing the release of proinflammatory cytokines. However, it is unclear whether ROS are involved in the damage of the local immunity of peri-implant tissues. In the present study, we assessed the mechanism of local immunosuppression using titanium (Ti) particles and/or lipopolysaccharide (LPS) to stimulate RAW 264.7 cells. The results indicate that the Ti particles induced the generation of a moderate amount of ROS through nicotinamide adenine dinucleotide phosphate oxidase-1 (NOX-1), but not through catalase. Pre-exposure to Ti particles inhibited ROS generation and extracellular regulated protein kinase (ERK) activation in LPS-stimulated macrophages. These findings indicate that chronic stimulation by Ti particles may lead to a state of oxidative stress and persistent inflammation, which may result in the attenuation of the immune response of macrophages to bacterial components such as LPS. Eventually, immunosuppression develops in peri-implant tissues, which may be a risk factor for IRI

Free radicals, such as reactive oxygen species (ROS) which are released abruptly after deflation of an ischaemic tourniquet, cause reperfusion injuries. Ischaemic precondition (IPC), however, can reduce the injury. In clinical practice, the sequential application and release of tourniquets is often used in bilateral total knee replacement (TKR) to obtain a clearer operative field, but the effects on the production of free radicals and lipid peroxidation have not been studied. In this study, we have observed the production of free radicals and the subsequent lipid peroxidation in bilateral TKR with sequential application of a tourniquet to examine the effect of IPC. Patients undergoing elective TKR under intrathecal anaesthesia were studied. Blood samples were obtained after spinal anaesthesia, one minute before and five and 20 minutes after release of each tourniquet. We used the lucigenin chemiluminescence analysis and the phosphatidylcholine hydroperoxide (PCOOH) assay to measure the production of ROS and lipid peroxidation. Our results showed that production of ROS significantly increased at five and 20 minutes after release of the first tourniquet and at five minutes after release of the second tourniquet, but returned to normal at 20 minutes after the second reperfusion. The peak production of ROS was at 20 minutes after the first reperfusion; lipid peroxidation did not change significantly. We conclude that in spite of significant production of ROS after the release of tourniquet, the IPC phenomenon occurs during bilateral TKR with sequential application of a tourniquet

Introduction: Oxidative stress occurs when reactive oxygen species (ROS) are produced faster than they can be removed by cellular defence mechanisms contributing to ageing, many chronic diseases, such as atherosclerosis, RA, Parkinson and Alzheimer’s disease and skeletal pathologies. Here we address the impact of ROS on the viability of early osteogenic precursors in the bone marrow and study the influence of estrogen on this interaction. Cells have a number of mechanisms to protect themselves from ROS, which are constantly being formed in the cell through normal metabolic pathways, such as Vitamin E, C and estrogen. Estrogen has been shown to prevent intracellular accumulation of peroxide and to attenuate oxidant-induced death of neuronal and endothelial cells. In addition, it contributes significantly to bone turnover and relieves postmenopausal symptoms. This study has focused on the potential anti-oxidant properties of estrogen against oxidative on bone marrow stromal cells. stress induced by H. 2. O. 2. Methods: Primary bone marrow stromal cells were pre-treated with several different doses between 10. −6. M – 10. −8. M of estrogen prior to H. 2. O. 2. administration at 0.08–0.4 mM 30% (v/v) for 2–24h. The cellular production of ROS was determined by using the free radical indicator DCFH-DA. Apoptosis was determined by morphological criteria. Results: H. 2. O. 2. induced an increase in apoptosis of osteoprogenitor cells (p< 0.05). Determination of apoptosis and cell number by nuclear staining, indicated that pre-treatment of bone marrow stromal cells with 17-beta estradiol reduced the apoptotic response induced by H. 2. O. 2. (p< 0.05) and restored cell number to control levels. In order to test the anti-oxidant activity of estrogen, the dye DCFH-DA was introduced in a cell free system in the presence or absence of 17-beta estradiol and H. 2. O. 2. The same experiment was repeated in the presence of bone marrow stromal cells. H. 2. O. 2. increased both intracellularly and extracellularly oxidant activity and estradiol has the capacity of modifying this activity both inside and outside the cell. Discussion: These data demonstrate the ability of estrogen, used at physiological doses, to block oxidant-induced apoptosis of osteoprogenitor cells. Estrogen appears to reduce the generation of ROS in these cells. These data could have important implications on the maintenance of osteogenic stem cells during fractures, ageing and disease

Aim. This aim of this study was to investigate apoptosis, reactive oxygen species (ROS), and their upstream markers in Anteromedial Gonarthrosis (AMG). Methods. Ten resection specimens, from patients undergoing unicompartmental knee replacement for AMG, and ten control specimens, collected from vascular disease patients undergoing above knee amputation, were used. Routine histology and immunohistochemical studies were conducted for Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Active Caspase 3, Cytochrome C, Active Bax, Bim, 3-Nitrotyrosine and Forkhead box O3A (FOXO 3A). Results. Cell death was shown predominantly in the surface layer of chondrocytes of damaged cartilage (p<0.001). There was a significant difference in TUNEL staining between regions (p=0.001). This ranged from 26% (most damaged) to 4% (undamaged) and was significantly higher (p<0.001) in AMG compared to the control samples which showed an average of 2% TUNEL overall. Upstream markers of apoptosis (Active Caspase 3, Cytochrome C, Active Bax), assessed qualitatively, were present in a similar distribution to that of TUNEL staining. 3-Nitrotyrosine, an indicator of ROS mediated damage, was also shown to be a predominantly surface phenomenon. There was a significant difference (p<0.001) between regions, ranging from 58% (most damaged) to 10% (undamaged). Again, this was significantly higher that the control samples (p<0.001). In line with indicators of ROS mediated damage, Bim and FOXO3A were also detected. Discussion. In AMG, apoptosis and ROS appear to be a part of the biological process leading to cartilage degeneration. Such cellular responses in ‘stressed’ chondrocytes provide possible targets for disease modification, thus delaying or preventing the need for joint arthroplasty. Further work is required to demonstrate these pathways and the effects of intervention