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Oral diseases, particularly dental caries and periodontal disease, pose significant global health challenges. The imbalance of the oral microbiota plays a key role in the occurrence of these diseases, prompting researchers to seek new strategies to restore oral ecological balance. Lactobacillus reuteri is a Gram-positive rod-shaped bacterium that exists in various body parts of humans, including the gastrointestinal tract, urinary tract, skin, and so on. This species has a potentially positive impact on oral health and plays an important role in maintaining systemic health. Recent studies have explored the application of Lactobacillus reuteri in the prevention and treatment of oral diseases, and its impact on systemic health has also been preliminarily revealed. The current review summarizes the role of Lactobacillus reuteri in oral health and systemic health and outlines its potential applications in the future. Lactobacillus reuteri has shown promising prospects in treating non-communicable biofilm-dependent oral diseases, but its mechanism of action and efficacy still need further research. In addition, Lactobacillus reuteri has also displayed some potential benefits in promoting overall health. Future research should focus on revealing the specific pathways of action of Lactobacillus reuteri, screening for the most beneficial strains, determining the most effective drug delivery strategies, developing oral and systemic health products based on Lactobacillus reuteri, and ensuring their safety in clinical applications. [ABSTRACT FROM AUTHOR]

Oxidative stress has been considered to be closely related to spaceflight‐induced bone loss; however, mechanism is elusive and there are no effective countermeasures. Using cultured rat calvarial osteoblasts exposed to microgravity simulated by a random positioning machine, this study addressed the hypotheses that microgravity‐induced shortening of primary cilia leads to oxidative stress and that primary cilium protection prevents oxidative stress and osteogenesis loss. Microgravity was found to induce oxidative stress (as represented by increased levels of reactive oxygen species (ROS) and malondialdehyde production, and decreased activities of antioxidant enzymes), which was perfectly replicated in osteoblasts growing in NG with abrogated primary cilia (created by transfection of an interfering RNA), suggesting the possibility that shortening of primary cilia leads to oxidative stress. Oxidative stress was accompanied by mitochondrial dysfunction (represented by increased mitochondrial ROS and decreased mitochondrial membrane potential) and intracellular Ca2+ overload, and the latter was found to be caused by increased activity of Ca2+ channel transient receptor potential vanilloid 4 (TRPV4), as also evidenced by TRPV4 agonist GSK1016790A‐elicited Ca2+ influx. Supplementation of HC‐067047, a specific antagonist of TRPV4, attenuated microgravity‐induced mitochondrial dysfunction, oxidative stress, and osteogenesis loss. Although TRPV4 was found localized in primary cilia and expressed at low levels in NG, microgravity‐induced shortening of primary cilia led to increased TRPV4 levels and Ca2+ influx. When primary cilia were protected by miR‐129‐3p overexpression or supplementation with a natural flavonoid moslosooflavone, microgravity‐induced increased TRPV4 expression, mitochondrial dysfunction, oxidative stress, and osteogenesis loss were all prevented. Our data revealed a new mechanism that primary cilia function as a controller for TRPV4 expression. Microgravity‐induced injury on primary cilia leads to increased expression and overactive channel of TRPV4, causing intracellular Ca2+ overload and oxidative stress, and primary cilium protection could be an effective countermeasure against microgravity‐induced oxidative stress and loss of osteogenic potential of osteoblasts. [ABSTRACT FROM AUTHOR]

Simple Summary: Vitamin D deficiency or insufficiency is prevalent in childhood cancer patients and survivors; however, the underlying etiology and effectiveness of vitamin D supplementation in preventing chemotherapy-induced bone loss are unclear. This study aimed to use a rat model to investigate whether methotrexate chemotherapy-induced vitamin D deficiency is related to intestinal damage and whether vitamin D supplementation can attenuate the resultant bone loss. We found that methotrexate chemotherapy causes vitamin D deficiency, bone growth impairments, bone loss, and altered intestinal vitamin D metabolism, which are associated with intestinal damage, and vitamin D supplementation inhibits methotrexate-induced bone loss due to its effect in suppressing methotrexate-induced bone lysis. This study has provided a mechanistic insight into vitamin D deficiency caused by methotrexate chemotherapy (which is commonly used in childhood oncology), and it has produced experimental evidence for the effectiveness of vitamin D supplementation in preventing chemotherapy-induced bone loss (commonly found in cancer patients and survivors). Vitamin D deficiency or insufficiency is prevalent in childhood cancer patients and survivors after chemotherapy; further studies are needed to investigate the underlying aetiology and effectiveness of vitamin D supplementation in preventing chemotherapy-induced bone loss. This study used a rat model of treatment with antimetabolite methotrexate to investigate whether methotrexate chemotherapy causes vitamin D deficiency and if vitamin D supplementation attenuates the resultant bone loss. Methotrexate treatment (five daily injections) decreased serum vitamin D levels (from 52 to <30 ng/mL), reduced body and bone lengthening and tibial trabecular bone volume, and altered intestinal vitamin D metabolism, which was associated with intestinal mucosal damage known to cause malabsorption of nutrients, including dietary vitamin D and calcium. During the early stage after chemotherapy, mRNA expression increased for vitamin D activation enzyme CYP27B1 and for calcium-binding protein TRPV6 in the intestine. During the intestinal healing stage, expression of vitamin D catabolism enzyme CYP24 increased, and that of TRPV6 was normalised. Furthermore, subcutaneous calcitriol supplementation diminished methotrexate-induced bone loss due to its effect suppressing methotrexate-induced increased bone resorption. Thus, in young rats, methotrexate chemotherapy causes vitamin D deficiency, growth impairments, bone loss, and altered intestinal vitamin D metabolism, which are associated with intestinal damage, and vitamin D supplementation inhibits methotrexate-induced bone loss. [ABSTRACT FROM AUTHOR]

Due to their superior antibacterial properties, biocompatibility and high conductivity, nanomaterials have shown a broad prospect in the biomedical field and have been widely used in the prevention and treatment of oral diseases. Also due to their small particle sizes and biodegradability, nanomaterials can provide solutions for tissue engineering, especially for oral tissue rehabilitation and regeneration. At present, research on nanomaterials in the field of dentistry focuses on the biological effects of various types of nanomaterials on different oral diseases and tissue engineering applications. In the current review, we have summarized the biological effects of nanoparticles on oral diseases, their potential action mechanisms and influencing factors. We have focused on the opportunities and challenges to various nanomaterial therapy strategies, with specific emphasis on overcoming the challenges through the development of biocompatible and smart nanomaterials. This review will provide references for potential clinical applications of novel nanomaterials in the field of oral medicine for the prevention, diagnosis and treatment of oral diseases. [ABSTRACT FROM AUTHOR]

The molecular mechanism for the microgravity‐induced decrease in bone formation remains unclear and there is a lack of effective specific preventative therapies. We recently reported that primary cilia of osteoblasts became shorter and even disappeared when the cells were exposed to random positioning machine (RPM)‐simulated microgravity and that the microgravity‐induced loss of osteogenic potential of osteoblasts could be attenuated when the resorption of primary cilia was prevented by treatment with 0.1 μM cytochalasin D. In the current study, it was further found that the loss of the osteogenic capacity of rat calvarial osteoblasts (ROBs) was associated with the inhibition of the BMP‐2/Smad1/5/8 signalling pathway, of which most of the signalling proteins including BMP‐2, BMPRII, Smad1/5/8 and p‐Smad1/5/8 were found localized to primary cilia. Accompanying the resorption of primary cilia following the cells being exposed to simulated microgravity, the expression levels of these signalling proteins were reduced significantly. Furthermore, the expression of miRNA‐129‐3p, a microRNA previously reported to control cilium biogenesis, was found to be reduced quickly and changed in a similar tendency with the length of primary cilia. Moreover, overexpression of miRNA‐129‐3p in ROBs significantly attenuated microgravity‐induced inhibition of BMP‐2 signalling and loss of osteogenic differentiation and mineralization. These results indicated the important role of miRNA‐129‐3p in microgravity‐induced resorption of primary cilia of osteoblasts and the potential of replenishing the miRNA‐129‐3p as an effective countermeasure against microgravity‐induced loss of primary cilia and impairment of osteoblast function. [ABSTRACT FROM AUTHOR]

Methotrexate (MTX) is a commonly used antimetabolite in cancer treatment. Its intensive use is linked with skeletal adverse effects such as reduced bone formation and bone loss, and yet little information is available on molecular mechanisms underlying MTX‐induced impaired bone formation. This study investigated the effects of MTX treatment at a clinical chemotherapy relevant dose on osteogenic differentiation in MC3T3E1 osteoblastic cells. To investigate the potential mechanisms, the expression of 87 genes regulating osteoblast differentiation and bone homeostasis was screened in MTX‐treated versus untreated cells by polymerase chain reaction (PCR) arrays and results illustrated significant upregulation of Notch2 and Notch target genes at both early and late stages of MC3T3E1 differentiation following MTX treatment. To confirm the roles of Notch2 pathway and its potential action mechanisms, MC3T3E1 cells were treated with MTX with an anti‐Notch2 neutralizing antibody or control IgG and effects were examined on osteogenesis and activation of the Wnt/β‐catenin pathway. Our results demonstrated that induction of Notch2 activity is associated with MTX adverse effects on osteogenic differentiation and blocking Notch2 rescues osteoblast differentiation by preserving activation of the Wnt/β‐catenin pathway. [ABSTRACT FROM AUTHOR]

Intensive cancer chemotherapy is well known to cause bone vasculature disfunction and damage, but the mechanism is poorly understood and there is a lack of treatment. Using a rat model of methotrexate (MTX) chemotherapy (five once-daily dosses at 0.75 mg/kg), this study investigated the roles of the Notch2 signalling pathway in MTX chemotherapy-induced bone micro-vasculature impairment. Gene expression, histological and micro-computed tomography (micro-CT) analyses revealed that MTX-induced micro-vasculature dilation and regression is associated with the induction of Notch2 activity in endothelial cells and increased production of inflammatory cytokine tumour necrosis factor alpha (TNFα) from osteoblasts (bone forming cells) and bone marrow cells. Blockade of Notch2 by a neutralising antibody ameliorated MTX adverse effects on bone micro-vasculature, both directly by supressing Notch2 signalling in endothelial cells and indirectly via reducing TNFα production. Furthermore, in vitro studies using rat bone marrow-derived endothelial cell revealed that MTX treatment induces Notch2/Hey1 pathway and negatively affects their ability in migration and tube formation, and Notch2 blockade can partially protect endothelial cell functions from MTX damage. [ABSTRACT FROM AUTHOR]

Childhood cancer methotrexate (MTX) chemotherapy often causes bone growth impairments, bone loss, and increased risks of fractures during or after treatment, for which the pathobiology is unclear and there is a lack of specific treatment. Our time course analyses of long bones from rats receiving intensive MTX treatment (mimicking a clinical protocol) found decreased trabecular bone volume, increased osteoclast formation and activity, increased adipogenesis in the expense of osteogenesis from the bone marrow stromal cells at days 6 and 9 following the first of five daily MTX doses. For exploring potential mechanisms, PCR array expression of 91 key factors regulating bone homeostasis was screened with the bone samples, which revealed MTX treatment-induced upregulation of Notch receptor NOTCH2, activation of which is known to be critical in skeletal development and bone homeostasis. Consistently, increased Notch2 activation in bones of MTX-treated rats was confirmed, accompanied by increased expression of Notch2 intracellular domain protein and Notch target genes HEY1, HES1 and HEYL. To confirm the roles of Notch2 signalling, a neutralising anti-Notch2 antibody or a control IgG was administered to rats during MTX treatment. Microcomputed tomography analyses demonstrated that trabecular bone volume was preserved by MTX+anti-Notch2 antibody treatment. Anti-Notch2 antibody treatment ameliorated MTX treatment-induced increases in osteoclast density and NFATc1 and RANKL expression, and attenuated MTX-induced bone marrow adiposity via regulating Wnt/β-catenin signalling and PPARγ expression. Thus, Notch2 signalling plays an important role in mediating MTX treatment-induced bone loss and bone marrow adiposity, and targeting Notch2 could be a potential therapeutic option. [ABSTRACT FROM AUTHOR]

Previous studies have shown that administration of antimetabolite methotrexate (MTX) caused a reduced trabecular bone volume and increased marrow adiposity (bone/fat switch), for which the underlying molecular mechanisms and recovery potential are unclear. Altered expression of microRNAs (miRNAs) has been shown to be associated with dysregulation of osteogenic and/or adipogenic differentiation by disrupting target gene expression. First, the current study confirmed the bone/fat switch following MTX treatment in precursor cell culture models in vitro. Then, using a rat intensive 5‐once daily MTX treatment model, this study aimed to identify miRNAs associated with bone damage and recovery (in a time course over Days 3, 6, 9, and 14 after the first MTX treatment). RNA isolated from bone samples of treated and control rats were subjected to miRNA array and reverse transcription‐polymerase chain reaction validation, which identified five upregulated miRNA candidates, namely, miR‐155‐5p, miR‐154‐5p, miR‐344g, miR‐6215, and miR‐6315. Target genes of these miRNAs were predicted using TargetScan and miRDB. Then, the protein‐protein network was established via STRING database, after which the miRNA‐key messenger RNA (mRNA) network was constructed by Cytoscape. Functional annotation and pathway enrichment analyses for miR‐6315 were performed by DAVID database. We found that TGF‐β signaling was the most significantly enriched pathway and subsequent dual‐luciferase assays suggested that Smad2 was the direct target of miR‐6315. Our current study showed that miR‐6315 might be a vital regulator involved in bone and marrow fat formation. Also, this study constructed a comprehensive miRNA–mRNA regulatory network, which may contribute to the pathogenesis/prognosis of MTX‐associated bone loss and bone marrow adiposity. [ABSTRACT FROM AUTHOR]

Osteoporosis is characterized by increased bone fragility, and the drugs used at present to treat osteoporosis can cause adverse reactions. Gentiopicroside (GEN), a class of natural compounds with numerous biological activities such as anti‐resorptive properties and protective effects against bone loss. Therefore, the aim of this work was to explore the effect of GEN on bone mesenchymal stem cells (BMSCs) osteogenesis for a potential osteoporosis therapy. In vitro, BMSCs were exposed to GEN at different doses for 2 weeks, whereas in vivo, ovariectomized osteoporosis was established in mice and the therapeutic effect of GEN was evaluated for 3 months. Our results in vitro showed that GEN promoted the activity of alkaline phosphatase, increased the calcified nodules in BMSCs and up‐regulated the osteogenic factors (Runx2, OSX, OCN, OPN and BMP2). In vivo, GEN promoted the expression of Runx2, OCN and BMP2, increased the level of osteogenic parameters, and accelerated the osteogenesis of BMSCs by activating the BMP pathway and Wnt/β‐catenin pathway, effect that was inhibited using the BMP inhibitor Noggin and Wnt/β‐catenin inhibitor DKK1. Silencing the β‐catenin gene and BMP2 gene blocked the osteogenic differentiation induced by GEN in BMSCs. This block was also observed when only β‐catenin was silenced, although the knockout of BMP2 did not affect β‐catenin expression induced by GEN. Therefore, GEN promotes BMSC osteogenesis by regulating β‐catenin‐BMP signalling, providing a novel strategy in the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

Electromagnetic fields (EMFs) have emerged as a versatile means for osteoporosis treatment and prevention. However, its optimal application parameters are still elusive. Here, we optimized the frequency parameter first by cell culture screening and then by animal experiment validation. Osteoblasts isolated from newborn rats (ROBs) were exposed 90 min/day to 1.8 mT SEMFs at different frequencies (ranging from 10 to 100 Hz, interval of 10 Hz). SEMFs of 1.8 mT inhibited ROB proliferation at 30, 40, 50, 60 Hz, but increased proliferation at 10, 70, 80 Hz. SEMFs of 10, 50, and 70 Hz promoted ROB osteogenic differentiation and mineralization as shown by alkaline phosphatase (ALP) activity, calcium content, and osteogenesis‐related molecule expression analyses, with 50 Hz showing greater effects than 10 and 70 Hz. Treatment of young rats with 1.8 mT SEMFs at 10, 50, or 100 Hz for 2 months significantly increased whole‐body bone mineral density (BMD) and femur microarchitecture, with the 50 Hz group showing the greatest effect. Furthermore, 1.8 mT SEMFs extended primary cilia lengths of ROBs and increased protein kinase A (PKA) activation also in a frequency‐dependent manner, again with 50 Hz SEMFs showing the greatest effect. Pretreatment of ROBs with the PKA inhibitor KT5720 abolished the effects of SEMFs to increase primary cilia length and promote osteogenic differentiation/mineralization. These results indicate that 1.8 mT SEMFs have a frequency window effect in promoting osteogenic differentiation/mineralization in ROBs and bone formation in growing rats, which involve osteoblast primary cilia length extension and PKA activation. [ABSTRACT FROM AUTHOR]

Intensive use of methotrexate (MTX) and/or dexamethasone (DEX) for treating childhood malignancies is known to cause chondrocyte apoptosis and growth plate dysfunction leading to bone growth impairments. However, mechanisms remain vague and it is unclear whether MTX and DEX combination treatment could have additive effects in the growth plate defects. In this study, significant cell apoptosis was induced in mature ATDC5 chondrocytes after treatment for 48 h with 10−5 M MTX and/or 10−6 M DEX treatment. PCR array assays with treated cells plus messenger RNA and protein expression confirmation analyses identified chemokine CXCL12 having the most prominent induction in each treatment group. Conditioned medium from treated chondrocytes stimulated migration of RAW264.7 osteoclast precursor cells and formation of osteoclasts, and these stimulating effects were inhibited by the neutralizing antibody for CXCL12. Additionally, while MTX and DEX combination treatment showed some additive effects on apoptosis induction, it did not have additive or counteractive effects on CXCL12 expression and its functions in enhancing osteoclastic recruitment and formation. In young rats treated acutely with MTX, there was increased expression of CXCL12 in the tibial growth plate, and more resorbing chondroclasts were found present at the border between the hypertrophic growth plate and metaphysis bone. Thus, the present study showed an association between induced chondrocyte apoptosis and stimulated osteoclastic migration and formation following MTX and/or DEX treatment, which could be potentially or at least partially linked molecularly by CXCL12 induction. This finding may contribute to an enhanced mechanistic understanding of bone growth impairments following MTX and/or DEX therapy. [ABSTRACT FROM AUTHOR]

Cancer chemotherapy can significantly impair the bone formation and cause myelosuppression; however, their recovery potentials and mechanisms remain unclear. This study investigated the roles of the β‐catenin signaling pathway in bone and bone marrow recovery potentials in rats treated with antimetabolite methotrexate (MTX) (five once‐daily injections, 0.75 mg/kg) with/without β‐catenin inhibitor indocyanine green (ICG)‐001 (oral, 200 mg/kg/day). ICG alone reduced trabecular bone volume and bone marrow cellularity. In MTX‐treated rats, ICG suppressed bone volume recovery on Day 11 after the first MTX injection. ICG exacerbated MTX‐induced decreases on Day 9 osteoblast numbers on bone surfaces, their formation in vitro from bone marrow stromal cells (osteogenic differentiation/mineralization), as well as expression of osteogenesis‐related markers Runx2, Osx, and OCN in bone, and it suppressed their subsequent recoveries on Day 11. On the other hand, ICG did not affect MTX‐induced increased osteoclast density and the level of the osteoclastogenic signal (RANKL/OPG expression ratio) in bone, suggesting that ICG inhibition of β‐catenin does nothing to abate the increased bone resorption induced by MTX. ICG also attenuated bone marrow cellularity recovery on Day 11, which was associated with the suppressed recovery of CD34+ or c‐Kit+ hematopoietic progenitor cell contents. Thus, β‐catenin signaling is important for osteogenesis and hematopoiesis recoveries following MTX chemotherapy. [ABSTRACT FROM AUTHOR]

pH‐magnetic dual‐responsive nanocomposites have been widely used in drug delivery and gene therapy. Recently, a polypseudorotaxane functionalized magnetic nanoparticle (MNP) was developed by synthesizing the magnetic nanoparticles with cyclodextrin (CD) molecules (CDMNP) via polyethylene glycol (PEG) (CDMNP‐PEG‐CD). The purpose of this study was to explore the antigenicity and immunogenicity of the nanoparticles in vivo prior to their further application explorations. Here, nanoparticles were assessed in vivo for retention, bio‐distribution and immuno‐reactivity. The results showed that, once administered intravenously, CDMNP‐PEG‐CD induced a temporary blood monocyte response and was cleared effectively from the body through the urine system in mice. The introduction of β‐CD and PEG/β‐CD polypseudorotaxane on SiO2 magnetic nanoparticles (SOMNP) limited particle intramuscular dispersion after being injected into mouse gastrocnemius muscle (GN), which led to the prolonged local inflammation and muscle toxicity by CDMNP and CDMNP‐PEG‐CD. In addition, T cells were found to be more susceptible for β‐CD–modified CDMNP; however, polypseudorotaxane modification partially attenuated β‐CD–induced T cell response in the implanted muscle. Our results suggested that CDMNP‐PEG‐CD nanoparticles or the decomposition components have potential to prime antigen‐presenting cells and to break the muscle autoimmune tolerance. [ABSTRACT FROM AUTHOR]

Resveratrol, a naturally occurring polyphenol in red grapes and berries, can act as a phytoestrogen. It has been shown to improve both systemic and cerebral circulatory functions, possibly through activation of endothelial estrogen receptors. in vitro and in vivo studies in rodent models also indicate a bone‐protective role for resveratrol, particularly in ovariectomized rat models that mimic postmenopausal osteoporosis caused by estrogen deficiency. Hypothesizing a circulatory benefit of resveratrol in bone tissue, we investigated whether resveratrol supplementation could improve bone health in postmenopausal women. The Resveratrol for Healthy Aging in Women (RESHAW) trial was a 24‐month randomized, double‐blind, placebo‐controlled, two‐period crossover intervention conducted to evaluate the effects of resveratrol (75 mg twice daily) on cognition, cerebrovascular function, bone health, cardiometabolic markers, and well‐being in postmenopausal women. After 12 months of supplementation with resveratrol versus placebo, there were positive effects on bone density in the lumbar spine (+0.016 ± 0.003 g/cm2) and neck of femur (+0.005 ± 0.002 g/cm2), which were accompanied by a 7.24% reduction in C‐terminal telopeptide type‐1 collagen levels, a bone resorption marker, compared with placebo. The increase in bone mineral density in the femoral neck resulted in an improvement in T‐score (+0.070 ± 0.018) and a reduction in the 10‐year probability of major and hip fracture risk. The magnitude of improvement was higher in women with poor bone health biomarker status. Importantly, the improvement in femoral neck T‐score with resveratrol correlated with improvement in perfusion. Our subanalysis also revealed that the bone‐protective benefit of resveratrol was greater in participants who supplemented with vitamin D plus calcium. Regular supplementation with 75 mg of resveratrol twice daily has the potential to slow bone loss in the lumbar spine and femoral neck, common fracture sites in postmenopausal women without overt osteoporosis. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

The transcriptional repressor Bmi‐1 is involved in cell‐cycle regulation and cell senescence, the deficiency of which has been shown to cause oxidative stress. This study investigated whether Bmi‐1 deficiency plays a role in promoting disc degeneration and the effect of treatment with antioxidant N‐acetylcysteine (NAC) on intervertebral disc degeneration. Bmi‐1−/− mice were treated with the antioxidant NAC, supplied in drinking water (Bmi‐1−/−+NAC). For in vitro experiments, mouse intervertebral discs were cultured under low oxygen tension and serum‐limiting conditions in the presence of tumour necrosis factor α and interleukin 1β in order to mimic degenerative insult. Disc metabolism parameters in these in vitro and in vivo studies were evaluated by histopathological, immunohistochemical and molecular methods. Bmi‐1−/− mice showed lower collagen Ⅱ and aggrecan levels and higher collagen Ⅹ levels than wild‐type and Bmi‐1−/−+NAC mice. Bmi‐1−/− mice showed significantly lower superoxide dismutase (SOD)‐1, SOD‐2, glutathione peroxidase (GPX)‐1 and GPX‐3 levels than their wild‐type littermates and Bmi‐1−/−+ NAC mice. Relative to Bmi‐1−/− mice, the control and Bmi‐1−/−+NAC mice showed significantly lower p16, p21, and p53 levels. These results demonstrate that Bmi‐1 plays an important role in attenuating intervertebral disc degeneration in mice by inhibiting oxidative stress and cell apoptosis. [ABSTRACT FROM AUTHOR]

The aim of this study is to develop a hybrid 3D printing platform integrating thermal-extrusion and electrospinning methods to fabricate bone scaffolds. The scaffolds made by mPEG–PCL–mPEG/HA biocomposite and their surface were enhanced by adding an electrospun fibre layer which improved adhesion and viability of osteoblastic cells. The scaffolds were evaluated by mechanical testing; biochemical analyses, SEM observation and their capabilities for supporting growth and adhesion of osteoblastic cells were also assessed in vitro. The 3D printing platform can manufacture the controllable and complicated shapes of bone scaffolds by controlling the thermal-extrusion and electrospinning equipment. It also can control the pore size, porosity and pore interconnectivity, and stack the scaffold structure by using different materials and different ways. Analyses showed that the bone scaffolds have a good mechanical strength and the scaffolds are suitable to support growth of MC3T3-E1 osteoblastic cells; and the electrospun fibres may increase the surface area of the fabricated scaffolds for the future application in modulating osteoblast response. Thus, it is feasible to produce bone tissue engineering scaffolds integrating thermal-extrusion and electrospinning techniques using our 3D printing platform. [ABSTRACT FROM AUTHOR]

Key points: There is a close relationship between skeletal muscle physiology and Ca2+/calmodulin (CaM) signalling.Despite the effects of Ca2+/CaM signalling on immune and inflammatory responses having been extensively explored, few studies have investigated the role of CaM pathway activation on the post‐injury muscle inflammatory response.In this study, we investigated the role of CaM‐dependent signalling in muscle inflammation in cardiotoxin induced myoinjuries in mice.The Ca2+/calmodulin‐dependent protein kinase II (CaMII), Ca2+/calmodulin‐dependent protein kinase IV (CaMKIV), and nuclear factor of activated T cells (NFAT) pathways are likely to be simultaneously activated in muscle cells and in infiltrating lymphocytes and to regulate the immune behaviours of myofibres in an inflammatory environment, and these pathways ultimately affect the outcome of muscle inflammation. Calcium/calmodulin (Ca2+/CaM) signalling is essential for immune and inflammatory responses in tissues. However, it is unclear if Ca2+/CaM signalling interferes with muscle inflammation. Here we investigated the roles of CaM‐dependent signalling in muscle inflammation in mice that had acute myoinjuries in the tibialis anterior muscle induced by intramuscular cardiotoxin (CTX) injections and received intraperitoneal injections of either the CaM inhibitor calmidazolium chloride (CCL) or CaM agonist calcium‐like peptide 1 (CALP1). Multiple inflammatory parameters, including muscle autoantigens and toll‐like receptors, mononuclear cell infiltration, cytokines and chemokines associated with peripheral muscle inflammation, were examined after the injury and treatment. CALP1 treatment enhanced intramuscular infiltration of monocytes/macrophages into the damaged tibialis anterior muscle and up‐regulated mRNA and protein levels of muscle autoantigens (Mi‐2, HARS and Ku70) and Toll‐like receptor 3 (TLR3), and mRNA levels of tumor necrosis factor α (TNF‐α), interleukin‐6 (IL‐6), Monocyte chemoattractant protein‐1 (MCP1), Monocyte chemoattractant protein‐3 (MCP3) and Macrophage inflammatory protein‐1(MIP‐1α) in damaged muscle. In contrast, CCL treatment decreased the intramuscular cell infiltration and mRNA levels of the inflammatory mediators. After CALP1 treatment, a substantial up‐regulation in Ca2+/calmodulin‐dependent protein kinase II (CaMKII), Ca2+/calmodulin‐dependent protein kinase IV (CaMKIV) and nuclear factor of activated T cells (NFAT) activity was detected in CD45+ cells isolated from the damaged muscle. More pro‐inflammatory F4/80+Ly‐6C+ cells were detected in CD45‐gated cells after CALP1 treatment than in those after CCL treatment or no treatment. Consistently, in interferon‐γ‐stimulated cultured myoblasts and myotubes, CALP1 treatment up‐regulated the activities of CaMKII, CaMKIV and NFAT, and levels of class I/II major histocompatibility complexes (MHC‐I/II) and TLR3. Our findings demonstrated that CaM‐dependent signalling pathways mediate the injury‐induced acute muscle inflammatory response. Key points: There is a close relationship between skeletal muscle physiology and Ca2+/calmodulin (CaM) signalling.Despite the effects of Ca2+/CaM signalling on immune and inflammatory responses having been extensively explored, few studies have investigated the role of CaM pathway activation on the post‐injury muscle inflammatory response.In this study, we investigated the role of CaM‐dependent signalling in muscle inflammation in cardiotoxin induced myoinjuries in mice.The Ca2+/calmodulin‐dependent protein kinase II (CaMII), Ca2+/calmodulin‐dependent protein kinase IV (CaMKIV), and nuclear factor of activated T cells (NFAT) pathways are likely to be simultaneously activated in muscle cells and in infiltrating lymphocytes and to regulate the immune behaviours of myofibres in an inflammatory environment, and these pathways ultimately affect the outcome of muscle inflammation. [ABSTRACT FROM AUTHOR]

Background: Inflammation is implicated in both hepatic cirrhosis development and hepatocellular carcinogenesis, and treatment with long-acting glucocorticoid dexamethasone prevented liver carcinogenesis in mice. However, it is unclear whether glucocorticoids have anti-inflammatory effect on hepatocellular carcinoma (HCC) and if short-acting glucocorticoids (with fewer adverse effects) inhibit paraneoplastic inflammation and HCC progression. Methods: To investigate whether different types of anti-inflammatory agents attenuate HCC progression, the current study compared effects of treatments with hydrocortisone (a short-acting glucocorticoid) or aspirin on HCC progression. HCC was induced in diethylnitrosamine-treated rats which were randomly divided into 4 groups (n=8), respectively receiving orally once daily vehicle, glucuronolactone, glucuronolactone+hydrocortisone, and glucuronolactone+aspirin. Diethylnitrosamine (DEN) was given to rats in drinking water (100mg/L) to induce HCC. At weeks 12 and 16 post-induction, effects were compared on HCC nodule formation, microvessel density, and macrophage infiltration, and levels of paraneoplastic protein expression of tumor necrosis factor (TNF)-α, p38 mitogen-activated protein kinase (p38), phosphorylated p38 (p-p38), nuclear factor (NF)-κB, interleukin (IL)-10, hepatocyte growth factor (HGF), transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF). Results: Compared to the model and glucuronolactone alone groups, HCC nodule number and microvessel density in the glucuronolactone+hydrocortisone group were significantly lower at week 12. At week 12 but not week 16, significantly lower levels of macrophages, TNF-α, p-p38, NF-κB, IL-10, HGF, TGF-β1 and VEGF were observed in the paraneoplastic tissue of the glucuronolactone+hydrocortisone group when compared with the control and glucuronolactone groups. Conclusion: The results suggest that hydrocortisone treatment reduces macrophage polarization, expression of inflammatory and anti-inflammatory cytokines, and angiogenesis in paraneoplastic tissue, and attenuates early HCC progression. Although hydrocortisone did not have attenuation effect on advanced solid tumor, the current study shows the potential benefits and supports potential clinical use of hydrocortisone in attenuating early progression of HCC, which is through suppressing paraneoplastic inflammation and angiogenesis. [ABSTRACT FROM AUTHOR]

Methotrexate (MTX), a widely used antimetabolite in paediatric cancer to treatment, has been widely reported to cause bone loss and bone marrow (BM) microvascular (particularly sinusoids) damage. Investigations must now investigate how MTX‐induced bone loss and microvasculature damage can be attenuated/prevented. In the present study, we examined the potency of icariin, an herbal flavonoid, in reducing bone loss and the dilation/damage of BM sinusoids in rats caused by MTX treatment. Groups of young rats were treated with five daily MTX injections (0.75 mg/kg) with and without icariin oral supplementation until Day 9 after the first MTX injection. Histological analyses showed a significant reduction in the bone volume/tissue volume (BV/TV) fraction (%) and trabecular number in the metaphysis trabecular bone of MTX‐treated rats, but no significant changes in trabecular thickness and trabecular spacing. However, the BV/TV (%) and trabecular number were found to be significantly higher in MTX + icariin‐treated rats than those of MTX alone‐treated rats. Gene expression analyses showed that icariin treatment maintained expression of osteogenesis‐related genes but suppressed the induction of adipogenesis‐related genes in bones of MTX‐treated rats. In addition, icariin treatment attenuated MTX‐induced dilation of BM sinusoids and upregulated expression of endothelial cell marker CD31 in the metaphysis bone of icariin + MTX‐treated rats. Furthermore, in vitro studies suggest that icariin treatment can potentially enhance the survival of cultured rat sinusoidal endothelial cells against cytotoxic effect of MTX and promote their migration and tube formation abilities, which is associated with enhanced production of nitric oxide. [ABSTRACT FROM AUTHOR]

Critical limb ischemia (CLI) is the advanced stage of peripheral artery disease spectrum and is defined by limb pain or impending limb loss because of compromised blood flow to the affected extremity. Current conventional therapies for CLI include amputation, bypass surgery, endovascular therapy, and pharmacological approaches. Although these conventional therapeutic strategies still remain as the mainstay of treatments for CLI, novel and promising therapeutic approaches such as proangiogenic gene/protein therapies and stem cell‐based therapies have emerged to overcome, at least partially, the limitations and disadvantages of current conventional therapeutic approaches. Such novel CLI treatment options may become even more effective when other complementary approaches such as utilizing proper bioscaffolds are used to increase the survival and engraftment of delivered genes and stem cells. Therefore, herein, we address the benefits and disadvantages of current therapeutic strategies for CLI treatment and summarize the novel and promising therapeutic approaches for CLI treatment. Our analyses also suggest that these novel CLI therapeutic strategies show considerable advantages to be used when current conventional methods have failed for CLI treatment. [ABSTRACT FROM AUTHOR]

Cancer chemotherapy can cause significant damage to the bone marrow (BM) microvascular (sinusoidal) system. Investigations must now address whether and how BM sinusoidal endothelial cells (SECs) can be protected during chemotherapy. Herein we examined the potential protective effects of genistein, a soy‐derived flavonoid, against BM sinusoidal damage caused by treatment with methotrexate (MTX). The groups of young adult rats were gavaged daily with genistein (20 mg/kg) or placebo. After 1 week, rats also received daily injections of MTX (0.75 mg/kg) or saline for 5 days and were killed after a further 4 days. Histological analyses showed that BM sinusoids were markedly dilated (p < 0.001) in the MTX‐alone group but were unaffected or less dilated in the genistein+MTX group. In control rats, genistein significantly enhanced expression of vascular endothelial growth factor (VEGF; p < 0.01), particularly in osteoblasts, and angiogenesis marker CD31 (p < 0.001) in bone. In MTX‐treated rats, genistein suppressed MTX‐induced apoptosis of BM SECs (p < 0.001 vs MTX alone group) and tended to increase expression of CD31 and VEGF (p < 0.05). Our in vitro studies showed that genistein in certain concentrations protected cultured SECs from MTX cytotoxic effects. Genistein enhanced tube formation of cultured SECs, which is associated with its ability to induce expression of endothelial nitric oxide synthase and production of nitric oxide. These data suggest that genistein can protect BM sinusoids during MTX therapy, which is associated, at least partially, with its indirect effect of promoting VEGF expression in osteoblasts and its direct effect of enhancing nitric oxide production in SECs. Flavonoid genistein may have a potency to protect bone marrow sinusoids during methotrexate therapy, which is associated, at least partially, with its indirect effect of promoting angiogenic factor vascular endothelial growth factor expression in osteoblasts and its direct effect of enhancing nitric oxide production in sinusoidal endothelial cells. [ABSTRACT FROM AUTHOR]

The purpose of this study was to develop a novel approach to treat bone osteosarcoma using a multipurpose scaffold aiming for local drug delivery. The slowly releasing microspheres was designed to deliver the chemotherapy drug adriamycin (ADM) and a decellularized (D) periosteum scaffold (which is known to be able to promote bone regeneration) was used to carry these microspheres. D‐periosteum was obtained by physical and chemical decellularization. Histological results showed that the cellular components were effectively removed. The D‐periosteum showed an excellent cytocompatibility and the ability to promote adhesion and growth of fibroblasts. Two kinds of slowly releasing microspheres, adriamycin gelatin microspheres (ADM‐GMS) and adriamycin poly (dl‐lactide‐co‐glycolide) gelatin microspheres (ADM‐PLGA‐GMS), were prepared and anchored to D‐periosteum, resulting in two types of drug‐releasing regenerative scaffolds. The effectiveness of these two scaffolds in killing human osteosarcoma cells was tested by evaluating cell viability overtime of the cancer cells cultured with the scaffolds. In summary, a gelatin/decellularized periosteum‐based biologic scaffold material was designed aiming for local delivery of chemotherapy drugs for osteosarcoma, with the results showing ability of the scaffolds in sustaining release of the cancer drug and in suppressing growth of the cancer cells in vitro. The effectiveness of these two scaffolds in killing human osteosarcoma cells was tested by evaluating cell viability overtime of the cancer cells cultured with the scaffolds. In summary, a gelatin/decellularized periosteum‐based biologic scaffold material was designed aiming for local delivery of chemotherapy drugs for osteosarcoma, with the results showing ability of the scaffolds in sustaining release of the cancer drug and in suppressing growth of the cancer cells in vitro [ABSTRACT FROM AUTHOR]

Extremely low‐frequency electromagnetic fields have been considered a potential candidate for the prevention and treatment of osteoporosis; however, their action mechanism and optimal magnetic flux density (intensity) parameter are still elusive. The present study found that 50‐Hz sinusoidal electromagnetic fields (SEMFs) at 1.8 mT increased the peak bone mass of young rats by increasing bone formation. Gene array expression studies with femoral bone samples showed that SEMFs increased the expression levels of collagen‐1α1 and Wnt10b, a critical ligand of the osteogenic Wnt/β‐catenin pathway. Consistently, SEMFs promoted osteogenic differentiation and maturation of rat calvarial osteoblasts (ROBs) in vitro through activating the Wnt10b/β‐catenin pathway. This osteogenesis‐promoting effect of SEMFs via Wnt10b/β‐catenin signaling was found to depend on the functional integrity of primary cilia in osteoblasts. When the primary cilia were abrogated by small interfering RNA (siRNA) targeting IFT88, the ability of SEMFs to promote the osteogenic differentiation of ROBs through activating Wnt10b/β‐catenin signaling was blocked. Although the knockdown of Wnt10b expression with RNA interference had no effect on primary cilia, it significantly suppressed the promoting effect of SEMFs on osteoblastic differentiation/maturation. Wnt10b was normally localized at the bases of primary cilia, but it disappeared (or was released) from the cilia upon SEMF treatment. Interestingly, primary cilia were elongated to different degrees by different intensities of 50‐Hz SEMFs, with the window effect observed at 1.8 mT, and the expression level of Wnt10b increased in accord with the lengths of primary cilia. These results indicate that 50‐Hz 1.8‐mT SEMFs increase the peak bone mass of growing rats by promoting osteogenic differentiation/maturation of osteoblasts, which is mediated, at least in part, by Wnt10b at the primary cilia and the subsequent activation of Wnt/β‐catenin signaling. © 2019 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Purpose: To assess the association between the fracture line distribution and the location of comminution in intra-articular distal radius fractures by building fracture mapping. Patients/methods: Forty cases with intra-articular fractures of distal radius were enrolled in the current prospective clinical study. Fracture lines and comminution zones were identified by reduced three-dimensional computed tomography reconstructions and then graphically superimposed onto a standard template to create two-dimensional fracture maps, followed by the conversion into heating maps. Based on qualitative descriptive fracture mapping analyses, the patterns of intra-articular distal radius fractures were determined. Results: It was observed that the highest fracture line intensity was located as an inverted "T" shape zone in the dorsal aspect of the joint with high incidence of fractures and the prominently intense color in heat mapping. The keystone projected area, the radial styloid process and the metacarpal radial side articular surface were found to be the least involved parts of the fracture. According to the mapping of the number and distribution of fracture lines, a new classification method for intra-articular fractures of the distal radius was redefined. Different surgical approaches and internal fixation techniques were proposed for different types. In this paper, we retrospectively compared the preoperative X-ray findings between different types. Based on the preoperative X-ray prediction, the distal intra-articular radius fractures were classified, so as to develop effective surgical strategies. In this study, a new surgical approach was attempted, but due to the lack of evidence-based evidence, long-term postoperative complications and hand function should be further evaluated. Conclusion: This study redefines a new method for the classification of intra-articular fractures of the distal radius, which allows doctors to have a clearer understanding of the characteristics of distal radius fractures. Moreover, the application value in fracture diagnosis is more significant, and the best surgical approach is selected for different types. [ABSTRACT FROM AUTHOR]

Wound healing is a complex but a fine‐tuned biological process in which human skin has the ability to regenerate itself following damage. However, in particular conditions such as deep burn or diabetes the process of wound healing is compromised. Despite investigations on the potency of a wide variety of stem cells for wound healing, adipose‐derived stem cells (ASCs) seem to possess the least limitations for clinical applications, and literature showed that ASCs can improve the process of wound healing very likely by promoting angiogenesis and/or vascularisation, modulating immune response, and inducing epithelialization in the wound. In the present review, advantages and disadvantages of various stem cells which can be used for promoting wound healing are discussed. In addition, potential mechanisms of action by which ASCs may accelerate wound healing are summarised. Finally, clinical studies applying ASCs for wound healing and the associated limitations are reviewed. Despite investigations on the potency of a wide variety of stem cells for wound healing, adipose‐derived stem cells (ASCs) seem to possess the least clinical limitations for clinical applications, and our review showed that ASCs can improve the process of wound healing very likely by promoting angiogenesis and/or vascularisation, modulating immune response, and inducing epithelialization in the wound. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) play a critical role in the initiation and progression of colorectal cancer (CRC), but little is known about the function of lncRNAs in the colorectal liver metastasis (CLM). This study was designed to identify specific lncRNAs correlating to liver metastasis of CRC, and to further assess their clinical value. Seventeen patients with primary CRC lesions, adjacent normal mucosa, and synchronous liver metastases lesions were divided into discovery set (six patients) and test set (11 patients). Transcriptome sequencing (RNAseq) was used to screen differential expression of lncRNAs in the discovery set. Based on bioinformatics data, quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR) was used to verify the target lncRNA in test set. The relationships between target lncRNA and clinical values were analysed in an expanded validation set of additional 91 patients. 23 upregulated and 14 downregulated lncRNAs were detected for distinguishing synchronous liver metastases, primary CRC lesions from adjacent normal mucosa in the RNAseq set. The expression levels of four lncRNAs in the 37 lncRNA signature were verified by qRT‐PCR in the test set. Compared with the paired normal mucosa, high expression levels of lnc‐small‐nucleolar RNA host gene 15 (SNHG15) were detected not only in primary CRC lesions but also in liver metastases lesions in the test set. Furthermore, in the expanded validation set, high expression of lnc‐SNHG15 was significantly associated with lymph‐node metastasis and liver metastasis (p < 0.05), and patients displaying high lncRNA‐SNHG15 expression exhibited a shorter median overall survival duration than those displaying low expression (30.7 vs. 35.2 months; p = 0.003). Multivariate analyses demonstrated that lncRNA‐SNHG15 overexpression may serve as a poor prognostic biomarker for CRC patients (p = 0.049; Cox's regression: 2.731). Lnc‐SNHG15 overexpression was significantly associated with CLM and high‐expression of lnc‐SNHG15 in CRC was an independent predictor of poor survival. Long non coding RNAs‐small‐nucleolar RNA host gene 15 (lncRNAs‐SNHG15) overexpression may serve as a poor prognostic biomarker for colorectal cancer (CRC) patients (p = 0.049; Cox Regression: 2.731). Lnc‐SNHG15 overexpression was significantly associated with colorectal liver metastasis and high expression of lnc‐SNHG15 in CRC was an independent predictor of poor survival. [ABSTRACT FROM AUTHOR]

The application of pulsed electromagnetic fields (PEMFs) in the prevention and treatment of osteoporosis has long been an area of interest. However, the clinical application of PEMFs remains limited because of the poor understanding of the PEMF action mechanism. Here, we report that PEMFs promote bone formation by activating soluble adenylyl cyclase (sAC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element‐binding protein (CREB) signaling pathways. First, it was found that 50 Hz 0.6 millitesla (mT) PEMFs promoted osteogenic differentiation of rat calvarial osteoblasts (ROBs), and that PEMFs activated cAMP–PKA–CREB signaling by increasing intracellular cAMP levels, facilitating phosphorylation of PKA and CREB, and inducing nuclear translocation of phosphorylated (p)‐CREB. Blocking the signaling by adenylate cyclase (AC) and PKA inhibitors both abolished the osteogenic effect of PEMFs. Second, expression of sAC isoform was found to be increased significantly by PEMF treatment. Blocking sAC using sAC‐specific inhibitor KH7 dramatically inhibited the osteogenic differentiation of ROBs. Finally, the peak bone mass of growing rats was significantly increased after 2 months of PEMF treatment with 90 min/day. The serum cAMP content, p‐PKA, and p‐CREB as well as the sAC protein expression levels were all increased significantly in femurs of treated rats. The current study indicated that PEMFs promote bone formation in vitro and in vivo by activating sAC–cAMP–PKA–CREB signaling pathway of osteoblasts directly or indirectly. Our results indicated that 50 Hz 0.6 millitesla pulsed electromagnetic fields (PEMFs) promote osteogenic differentiation and mineralization of osteoblasts and augment peak bone mass attainment in young rats, and that PEMFs stimulate osteogenic differentiation and mineralization of osteoblasts by activating soluble adenylyl cyclase–cyclic adenosine monophosphate–protein kinase A–cAMP response element‐binding protein signaling pathway. [ABSTRACT FROM AUTHOR]

Dexamethasone (Dex) is known to cause significant bone growth impairment in childhood. Although previous studies have suggested roles of osteocyte apoptosis in the enhanced osteoclastic recruitment and local bone loss, whether it is so in the growing bone following Dex treatment requires to be established. The current study addressed the potential roles of chemokine CXCL12 in chondroclast/osteoclast recruitment and bone defects following Dex treatment. Significant apoptosis was observed in cultured mature ATDC5 chondrocytes and IDG‐SW3 osteocytes after 48 hours of 10−6 M Dex treatment, and CXCL12 was identified to exhibit the most prominent induction in Dex‐treated cells. Conditioned medium from the treated chondrocytes/osteocytes enhanced migration of RAW264.7 osteoclast precursor cells, which was significantly inhibited by the presence of the anti‐CXCL12 neutralizing antibody. To investigate the roles of the induced CXCL12 in bone defects caused by Dex treatment, young rats were orally gavaged daily with saline or Dex at 1 mg/kg/day for 2 weeks, and received an intraperitoneal injection of anti‐CXCL12 antibody or control IgG (1 mg/kg, three times per week). Aside from oxidative stress induction systemically, Dex treatment caused reductions in growth plate thickness, primary spongiosa height, and metaphysis trabecular bone volume, which are associated with induced chondrocyte/osteocyte apoptosis and enhanced chondroclast/osteoclast recruitment and osteoclastogenic differentiation potential. CXCL12 was induced in apoptotic growth plate chondrocytes and metaphyseal bone osteocytes. Anti‐CXCL12 antibody supplementation considerably attenuated Dex‐induced chondroclast/osteoclast recruitment and loss of growth plate cartilage and trabecular bone. CXCL12 neutralization did not affect bone marrow osteogenic potential, adiposity, and microvasculature. Thus, CXCL12 was identified as a potential molecular linker between Dex‐induced skeletal cell apoptosis and chondroclastic/osteoclastic recruitment, as well as growth plate cartilage/bone loss, revealing a therapeutic potential of CXCL12 functional blockade in preventing bone growth defects during/after Dex treatment. © 2018 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Opioids are widely administered to alleviate pain, including chronic pain in advanced cancer patients. Among opioids, morphine is one of the most clinically effective drugs for the palliative management of severe pain. In the last few decades, there has been a debate around the possible influence of opioids such as morphine on tumour growth and metastasis. Whilst several in vitro and in vivo studies suggest the possible modulatory effects of morphine on tumour cells, little is known about the impact of this analgesic drug on other mediators such as matrix metalloproteinases (MMPs) that play a key role in the control of cancer cell invasion and metastasis. MMP-9 has been considered as one of the principal mediators in regulation of not only the initial steps of cancer but during the invasion and spreading of cancer cells to distant organs. Herein, current studies regarding the direct and indirect effects of morphine on regulation of MMP-9 production are discussed. In addition, drawing from previous in vivo and in vitro studies on morphine action in regulating MMP-9 production, the potential roles of several underlying factors are summarised, including nuclear factor kappa-B and intracellular molecules such as nitric oxide. [ABSTRACT FROM AUTHOR]

Artificial abnormal microenvironment caused by microperfusion of L-glutamate (Glu) and Ca2+ in the hippocampus results in neuron damage, which is closely related to cerebral ischemia. Ginsenoside Rb1, a compound from Panax notoginseng, was previously used to counter the artificial abnormal hippocampal environment in a microperfusion model. In addition, while the Akt/mTOR/PTEN signaling pathway has been shown to mediate neuronprotection in cerebral ischemia, whether this pathway is involved in the neuroprotection of ginsenoside Rb1 is unknown. Here SH-SY5Y cells exposed to OGD/R injury in treated with LY294002, ginsenoside Rb1, ginsenoside Rb1+ LY294002. Expressions of phosphorylation (P-)Akt/P-mTOR/P-PTEN (24 h after OGD/R) were detected by Western blotting. Effects were examined via the memory function of rats (by Morris water maze test), morphological changes in pyramidal cell (by histology), and mRNA expression (by qRT-PCR) and phosphorylation (P-) (by Western blotting and immunohistochemical staining) of Akt, P-mTOR, and P-PTEN in the hippocampus. The memory deficit of rats and pyramidal cellular necrosis and apoptosis in the CA1 region of hippocampus after microperfusion of Glu and Ca2+ were dose dependently alleviated by ginsenoside Rb1.Moreover,Western blot showed that ginsenoside Rb1 increased the expressions of P-Akt, P-mTOR and reduced P-PTEN in vivo and vitro. Thus, the potent neuroprotection of ginsenoside Rb1 in artificial abnormal microenvironment is, at least partially, related to the activation of P-AKT/P-mTOR signaling pathway and inhibition of P-PTEN protein. [ABSTRACT FROM AUTHOR]

Secreted frizzled-related protein-1 (SFRP1) is a negative regulatory molecule of the WNT signaling pathway and serves as a therapeutic target for bone formation in osteoporosis. In this study, we first established an ovariectomized (OVX) rat model to simulate postmenopausal osteoporosis and found significant changes in miR-542-3p and sFRP1 expression by RNA sequencing and qRT-PCR. In addition, there was a significant negative correlation between miR-542-3p and sFRP1 mRNA levels in postmenopausal women with osteoporosis. We found that miR-542-3p inhibited the expression of sFRP1 mRNA by luciferase reporter assay. When the miR-542-3p binding site in sFRP1 3'UTR was deleted, it did not affect its expression. Western blot results showed that miR-542-3p inhibited the expression of SFRP1 protein. The expression of SFRP1 was significantly increased in osteoblast-induced mesenchymal stem cells (MSC), whereas the expression of miR-542-3p was significantly decreased. And miR-542-3p transfected MSCs showed a significant increase in osteoblast specific marker expression, indicating that miR-542-3p is necessary for MSC differentiation. Inhibition of miR-542-3p reduced bone formation, confirmed miR- 542-3p play a role in bone formation in vivo. In general, these data suggest that miR-542-3p play an important role in bone formation via inhibiting SFRP1 expression and inducing osteoblast differentiation. [ABSTRACT FROM AUTHOR]

Disc degeneration alters the structure and function of intervertebral discs and is the basis of spinal degenerative diseases. To establish the molecular mechanism of intervertebral disc degeneration caused by mechanical strain, this study examined the effects of different amplitude (3%, 9%, 19%) cyclic mechanical strain (CMS) at a low frequency (0.01 Hz) on the secretion of cartilage extracellular matrix, expression of inflammatory cytokines and catabolic proteases, and activation of NF-κB signaling pathway in human nucleus pulposus cells. We also investigated effects of low frequency and high amplitude (19%) CMS on degeneration of human nucleus pulposus cells in the presence or absence of p65 inhibitor, p65 silencing shRNA, or p65 overexpression. While 3% CMS did not significantly decrease aggrecan or type II collagen expression, or increase TNF-α, IL-1β, IL-6 expression, 9% and 19% CMS showed the significant effects. Low frequency and high amplitude (19%) CMS was found to promote p65 activation in human nucleus pulposus cells, and IL-1β was found to promote p65 nuclear translocation though IκB kinase phosphorylation. Furthermore, degeneration process of nucleus pulposus cells was found attenuated in the presence of p65 inhibitor or p65 silencing shRNA, but promoted with p65 overexpression. These data suggest that high amplitude and low frequency CMS could promote degeneration of human nucleus pulposus cells significantly via the NF- κB p65 pathway. Our findings have uncovered the effect of CMS on human nucleus pulposus cell degeneration and have identified a previously unknown intrinsic underlying mechanism. [ABSTRACT FROM AUTHOR]

PI3K/AKT signaling is essential in regulating pathophysiology of osteoarthritis (OA). However, its potential modulatory role in early OA progression has not been investigated yet. Here, a mouse destabilization OA model in the tibia was used to investigate roles of PI3K/AKT signaling in the early subchondral bone changes and OA pathological process. We revealed a significant increase in PI3K/AKT signaling activation which was associated with aberrant bone formation in tibial subchondral bone following destabilizing the medial meniscus (DMM), which was effectively prevented by treatment with PI3K/AKT signaling inhibitor LY294002. PI3K/AKT signaling inhibition attenuated articular cartilage degeneration. Serum and bone biochemical analyses revealed increased levels of MMP‐13, which was found expressed mainly by osteoblastic cells in subchondral bone. However, this MMP‐13 induction was attenuated by LY294002 treatment. Furthermore, PI3K/AKT signaling was found to enhance preosteoblast proliferation, differentiation, and expression of MMP‐13 by activating NF‐κB pathway. In conclusion, inhibition of PI3K/AKT/NF‐κB axis was able to prevent aberrant bone formation and attenuate cartilage degeneration in OA mice. [ABSTRACT FROM AUTHOR]

Background/Aims: Narrowing of the lumbar spinal canal is a condition called lumbar spinal stenosis (LSS) and is a high-morbidity problem in the elderly. LSS is commonly caused by hypertrophy of the ligamentum flavum (HLF). Previous studies showed that fibrosis of the ligamentum flavum (LF) largely contributed to HLF. However, the underlying pathomechanism remains unclear. Insulin-like growth factor-1 (IGF-1) is known to have an intimate relationship with fibrosis in various tissues. Nevertheless, currently, there are few studies regarding IGF-1 in HLF. In this study, we investigated the role of IGF-1 in HLF and its potential molecular mechanism of action. Methods: First, the IGF-1, phosphorylation of IGF-1 receptor (pIGF-1R), phosphorylation of AKT (pAKT), phosphorylation of S6(pS6), collagen I and collagen III expression levels were examined via immunohistochemistry and Western blotting in LF tissues from patients with LSS or Non-LSS. Second, primary LF cells were isolated from adults with a normal LF thickness and were cultured with different concentrations of IGF-1 with or without NVP-AEW541/rapamycin. Results: The results showed that IGF-1, pIGF-1R, pAKT, pS6, collagen I and collagen III protein expression in the LSS group was significantly higher than that in the Non-LSS group. Meanwhile, pIGF-1R, pAKT, pS6, collagen I and collagen III protein expression was significantly enhanced in LF cells after IGF-1 exposure, which can be notably blocked by NVP-AEW541. In addition, pS6, collagen I and collagen III protein expression was blocked by rapamycin. Conclusions: Enhanced IGF-1 promotes the synthesis of collagen I and collagen III via the mTORC1 signaling pathway, which eventually contributes to hypertrophy of the ligamentum flavum. [ABSTRACT FROM AUTHOR]

Lymphatic metastasis is facilitated by lymphangiogenic growth factor vascular endothelial growth factor‐C (VEGFC) that is secreted by some primary tumors. We previously identified tumor necrosis factor superfamily 15 (TNFSF15), a blood vascular endothelium‐derived cytokine, in lymphatic endothelial cells, as a key molecular modulator during lymphangiogenesis. However, the effect of TNFSF15 on tumor lymphatic metastasis and the underlying molecular mechanisms remain unclear. We report here that TNFSF15, which is known to inhibit primary tumor growth by suppressing angiogenesis, can promote lymphatic metastasis through facilitating lymphangiogenesis in tumors. Mice bearing tumors induced by A549 cells stably overexpressing TNFSF15 exhibited a significant increase in densities of lymphatic vessels and a marked enhancement of A549 tumor cells in newly formed lymphatic vessels in the primary tumors as well as in lymph nodes. Treatment of A549 cells with TNFSF15 results in upregulation of VEGFC expression, which can be inhibited by siRNA gene silencing of death domain‐containing receptor‐3 (DR3), a cell surface receptor for TNFSF15. In addition, TNFSF15/DR3 signaling pathways in A549 cells include activation of NF‐κB during tumor lymphangiogenesis. Our data indicate that TNFSF15, a cytokine mainly produced by blood endothelial cells, facilitates tumor lymphangiogenesis by upregulating VEGFC expression in A549 cells, contributing to lymphatic metastasis in tumor‐bearing mice. This finding also suggests that TNFSF15 may have potential as an indicator for prognosis evaluation. [ABSTRACT FROM AUTHOR]

Mechanical unloading was considered a major threat to bone homeostasis, and has been shown to decrease osteoblast proliferation although the underlying mechanism is unclear. Microtubule actin crosslinking factor 1 (MACF1) is a cytoskeletal protein that regulates cellular processes and Wnt/β‐catenin pathway, an essential signaling pathway for osteoblasts. However, the relationship between MACF1 expression and mechanical unloading, and the function and the associated mechanisms of MACF1 in regulating osteoblast proliferation are unclear. This study investigated effects of mechanical unloading on MACF1 expression levels in cultured MC3T3‐E1 osteoblastic cells and in femurs of mice with hind limb unloading; and it also examined the role and potential action mechanisms of MACF1 in osteoblast proliferation in MACF1‐knockdown, overexpressed or control MC3T3‐E1 cells treated with or without the mechanical unloading condition. Results showed that the mechanical unloading condition inhibited osteoblast proliferation and MACF1 expression in MC3T3‐E1 osteoblastic cells and mouse femurs. MACF1 knockdown decreased osteoblast proliferation, while MACF1 overexpression increased it. The inhibitory effect of mechanical unloading on osteoblast proliferation also changed with MACF1 expression levels. Furthermore, MACF1 was found to enhance β‐catenin expression and activity, and mechanical unloading decreased β‐catenin expression through MACF1. Moreover, β‐catenin was found an important regulator of osteoblast proliferation, as its preservation by treatment with its agonist lithium attenuated the inhibitory effects of MACF1‐knockdown or mechanical unloading on osteoblast proliferation. Taken together, mechanical unloading decreases MACF1 expression, and MACF1 up‐regulates osteoblast proliferation through enhancing β‐catenin signaling. This study has thus provided a mechanism for mechanical unloading‐induced inhibited osteoblast proliferation. [ABSTRACT FROM AUTHOR]

Osteoporosis is a systemic bone metabolic disease that is highly prevalent in the elderly population, particularly in postmenopausal women, which results in enhanced bone fragility and an increased susceptibility to fractures. However, the underlying molecular pathogenesis mechanisms still remain to be further elucidated. In this study, in a rat ovariectomy- (OVX-) induced postmenopausal osteoporosis model, aberrant expression of a microRNA miR-142-5p and vascular cell adhesion molecule 1 (VCAM-1) was found by RNA sequencing analysis and qRT-PCR. Using a dual-luciferase reporter assay, we found that miR-142-5p can bind to and decrease expression of VCAM-1 mRNA. Such reduction was prohibited when the miR-142-5p binding site in VCAM-1 3′UTR was deleted, and Western blotting analyses validated the fact that miR-142-5p inhibited the expression of VCAM-1 protein. Bone marrow-derived mesenchymal stem cells (BMMSCs) transfected with miR-142-5p showed a significantly decreased migration ability in a Transwell migration assay. Collectively, these data indicated the important role of miR-142-5p in osteoporosis development involving targeting VCAM-1 and inhibiting BMMSC migration. [ABSTRACT FROM AUTHOR]

Neurotrophins and their receptors are key molecules that are known to be critical in regulating nervous system development and maintenance and have been recognized to be also involved in regulating tissue formation and healing in skeletal tissues. Studies have shown that neurotrophins and their receptors are widely expressed in skeletal tissues, implicated in chondrogenesis, osteoblastogenesis, and osteoclastogenesis, and are also involved in regulating tissue formation and healing events in skeletal tissue. Increased mRNA expression for neurotrophins NGF, BDNF, NT-3, and NT-4, and their Trk receptors has been observed in injured bone tissues, and NT-3 and its receptor, TrkC, have been identified to have the highest induction at the injury site in a drill-hole injury repair model in both bone and the growth plate. In addition, NT-3 has also recently been shown to be both an osteogenic and angiogenic factor, and this neurotrophin can also enhance expression of the key osteogenic factor, BMP-2, as well as the major angiogenic factor, VEGF, to promote bone formation, vascularization, and healing of the injury site. Further studies, however, are needed to investigate if different neurotrophins have differential roles in skeletal repair, and if NT-3 can be a potential target of intervention for promoting bone fracture healing. [ABSTRACT FROM AUTHOR]

Bone metastasis (BM) is the advanced complication of breast cancer, while bone marrow-derived mesenchymal stem cells (BMSCs) in the microenvironment unclearly contribute to cancer metastasis. This study investigated potential roles of transforming growth factor- (TGF-) α in the interaction between breast cancer and BMSCs in BM. Clinical cases of breast cancer with bone metastasis (BMBC), breast cancer without bone metastasis (Non-BM-BC), and benign fibroadenoma (Benign) were enlisted in a retrospective study. TGF-α was found obviously overexpressed in BM lesion of BMBC compared to primary lesion of both BMBC and Non-BM-BC (P<0.01), and TGF-α was higher in primary lesion of both BMBC and Non-BM-BC (P<0.01) than Benign group. Interestingly, TGF-α in nontumor tissues of both BMBC and Non-BM-BC was at a higher level than Benign group (P<0.01), and numbers of macrophages in nontumor tissues of both BMBC and Non-BM-BC (P<0.01) were higher than Benign group. Furthermore, in cultured human BMSCs, TGF-α stimulated production of procancer cytokines including IL-6, VEGF, FGF10, FGF17, and TGF-β1 in a dose-dependent manner. Thus, TGF-α in BC could potentially be an important signal of carcinogenesis and metastasis. Macrophages in the nontumor tissue of BC may not be protective but could promote cancer metastasis. [ABSTRACT FROM AUTHOR]

Osteoblast differentiation is a multistep process delicately regulated by many factors, including cytoskeletal dynamics and signaling pathways. Microtubule actin crosslinking factor 1 (MACF1), a key cytoskeletal linker, has been shown to play key roles in signal transduction and in diverse cellular processes; however, its role in regulating osteoblast differentiation is still needed to be elucidated. To further uncover the functions and mechanisms of action of MACF1 in osteoblast differentiation, we examined effects of MACF1 knockdown (MACF1-KD) in MC3T3-E1 osteoblastic cells on their osteoblast differentiation and associated molecular mechanisms. The results showed that knockdown of MACF1 significantly suppressed mineralization of MC3T3-E1 cells, down-regulated the expression of key osteogenic genes alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and type I collagen α1 (Col Iα1). Knockdown of MACF1 dramatically reduced the nuclear translocation of β-catenin, decreased the transcriptional activation of T cell factor 1 (TCF1), and down-regulated the expression of TCF1, lymphoid enhancer-binding factor 1 (LEF1), and Runx2, a target gene of β-catenin/TCF1. In addition, MACF1-KD increased the active level of glycogen synthase kinase-3β (GSK-3β), which is a key regulator for β-catenin signal transduction. Moreover, the reduction of nuclear β-catenin amount and decreased expression of TCF1 and Runx2 were significantly reversed in MACF1-KD cells when treated with lithium chloride, an agonist for β-catenin by inhibiting GSK-3β activity. Taken together, these findings suggest that knockdown ofMACF1 in osteoblastic cells inhibits osteoblast differentiation through suppressing the β-catenin/TCF1-Runx2 axis. Thus, a novel role of MACF1 in and a new mechanistic insight of osteoblast differentiation are uncovered. [ABSTRACT FROM AUTHOR]

Leptin, an adipocyte-derived cytokine associated with bone metabolism, is believed to play a critical role in the pathogenesis of heterotopic ossification (HO). The effect and underlying action mechanism of leptin were investigated on osteogenic differentiation of tendon-derived stem cells (TDSCs) in vitro and theHOformation in rat tendons. Isolated rat TDSCs were treated with various concentrations of leptin in the presence or absence of mTORC1 signaling specific inhibitor rapamycin in vitro. A rat model with Achilles tenotomy was employed to evaluate the effect of leptin onHOformation together with or without rapamycin treatment. In vitro studies with TDSCs showed that leptin increased the expression of osteogenic biomarkers (alkaline phosphatase, runt-related transcription factor 2, osterix, osteocalcin) and enhanced mineralization of TDSCs via activating the mTORC1 signal pathway (as indicated by phosphorylation of p70 ribosomal S6 kinase 1 and p70 ribosomal S6). However, mTORC1 signaling blockade with rapamycin treatment suppressed leptin-induced osteogenic differentiation and mineralization. In vivo studies showed that leptin promoted HO formation in the Achilles tendon after tenotomy, and rapamycin treatment blocked leptin-inducedHOformation. In conclusion, leptin can promote TDSC osteogenic differentiation and heterotopic bone formation via mTORC1 signaling in both vitro and vivo model, which provides anewpotential therapeutic target forHO prevention. [ABSTRACT FROM AUTHOR]

Understanding neurite outgrowth, orientation, and migration is important for the design of biomaterials that interface with the neural tissue. However, the molecular signaling alternations have not been well elucidated to explain the impact of hydrogels on cell morphology. In our previous studies, a silk fibroin peptide (SF16) hydrogel was found to be an effective matrix for the viability, morphology, and proliferation of PC12 rat pheocrhomocytoma cells. We found that PC12 cells in the peptide hydrogel exhibited adhesive morphology compared to those cultured in agarose or collagen. Moreover, we identified that cell adhesion molecules (E- and N-cadherin) controlled by mTOR signaling were highly induced in PC12 cells cultured in the SF16 peptide hydrogel. Our findings suggest that the SF16 peptide might be suitable to be a celladhesion material in cell culture or tissue engineering, and mTOR/cadherin signaling is required for the cell adhesion in the SF16-peptide hydrogel. [ABSTRACT FROM AUTHOR]