MiRNA-199a-5p positively regulated RANKL-induced osteoclast differentiation by target Mafb protein.

MicroRNAs are involved in osteoclast differentiation. Although miR-199a-5p plays an important role in many different systems and diseases, its function during osteoclastogenesis remains unclear. In this study, we investigated the function and the target gene of miR-199a-5p in osteoclast differentiation. The in vitro data showed that miR-199a-5p was significantly upregulated after the stimulation by receptor activator of nuclear factor kappa-B ligand in macrophages and RAW 264.7 cells. After transfection of miR-199a-5p mimic, the messenger RNA expression level of nuclear factor of activated T-cells cytoplasmic 1, tartrate-resistant acid phosphatase (TRAP), and receptor activator of nuclear factor kappa-B was significantly increased in RAW 264.7 cells and the number of TRAP-positive cells was also increased. MiR-199a-5p inhibitor showed the complete opposite outcome which brought additional proof to our finding. Overexpression of miR-199a-5p led to downregulation of Mafb protein. The luciferase activity was obviously repressed when WT-pGL3-Mafb and miR-199a-5p mimics were cotransfected into 293 T cells and the inhibitors cotransfected demonstrated reverse result. MiR-199a-5p overexpressed during osteoclast differentiation and positively regulated osteoclast formation in vitro by target Mafb.

Metformin suppresses UHMWPE particle-induced osteolysis in the mouse calvaria by promoting polarization of macrophages to an anti-inflammatory phenotype.

BACKGROUND: Implant failure remains a major obstacle to successful treatment via TJA. Periprosthetic osteolysis and aseptic loosening are considered as proof of wear debris-induced disruption of local regulatory mechanisms related to excessive bone resorption associated with osteolysis and the damage at the bone-prosthesis interface. Therefore, there is an immediate need to explore strategies for limiting and curing periprosthetic osteolysis and aseptic loosening. METHODS: We analyzed the in vitro cytokine production by primary mouse bone marrow macrophages (BMMs) that were exposed to ultra-high molecular weight polyethylene (UHMWPE) particles and treated with metformin at different concentrations with or without 5-aminoimidazole-4-carboxamide ribonucleoside to activate or inhibit AMPK. A mouse calvarial model was used to examine the in vivo effects of metformin on UHMWPE particle-induced osteolysis. RESULTS: With particles, primary mouse BMMs secreted more pro-inflammatory cytokines tumor necrosis factor-α and interleukin (IL)-6. Treatment with metformin inhibited these variations and promoted the release of cytokine IL-10 with anti-inflammatory capability. In vivo, metformin reduced the production of pro-inflammatory cytokines, osteoclastogenesis, and osteolysis, increasing IL-10 production. Metformin also promoted the polarization of macrophages to an anti-inflammatory phenotype in vivo via AMPK activation. DISCUSSION: A crucial point in limiting and correcting the periprosthetic osteolysis and aseptic loosening is the inhibition of inflammatory factor production and osteoclast activation induced by activated macrophages. The ability of metformin to attenuate osteolysis induced in mouse calvaria by the particles was related to a reduction in osteoclast number and polarization of macrophages to an anti-inflammatory functional phenotype. CONCLUSIONS: Metformin could limit the osteolysis induced by implant debris. Therefore, we hypothesized that metformin could be a potential drug for osteolysis induced by implant debris.

Multilayered Scaffold with a Compact Interfacial Layer Enhances Osteochondral Defect Repair.

Repairing osteochondral defect (OCD) using advanced biomaterials that structurally, biologically, and mechanically fulfill the criteria for stratified tissue regeneration remains a significant challenge for researchers. Here, a multilayered scaffold (MLS) with hierarchical organization and heterogeneous composition is developed to mimic the stratified structure and complex components of natural osteochondral tissues. Specifically, the intermediate compact interfacial layer within the MLS is designed to resemble the osteochondral interface to realize the closely integrated layered structure. Subsequently, macroscopic observations, histological evaluation, and biomechanical and biochemical assessments are performed to evaluate the ability of the MLS of repairing OCD in a goat model. By 48 weeks postimplantation, superior hyalinelike cartilage and sound subchondral bone are observed in the MLS group. Furthermore, the biomimetic MLS significantly enhances the biomechanical and biochemical properties of the neo-osteochondral tissue. Taken together, these results confirm the potential of this optimized MLS as an advanced strategy for OCD repair.

Treatment Effects of the Second-Generation Tyrosine Kinase Inhibitor Dasatinib on Autoimmune Arthritis.

Rheumatoid arthritis (RA) is a multifactorial autoimmune disease that primarily manifests as persistent synovitis and progressive joint destruction. Imatinib exhibited a therapeutic effect in murine collagen-induced arthritis (CIA) via selective inhibition tyrosine kinases. The second-generation tyrosine kinase inhibitor dasatinib exhibits more durable hematological and cytogenetic effects and more potency compared to imatinib. However, the effect of dasatinib on CIA is poorly understood. The present study investigated the treatment effect of dasatinib on autoimmune arthritis. We demonstrated that dasatinib alleviated arthritis symptoms and histopathological destruction in CIA mice. Dasatinib treatment inhibited the production of proinflammatory cytokines including IL-1ß, TNF-α, and IL-6, and promoted the production of the anti-inflammatory cytokine IL-10. Dasatinib treatment also suppressed the expression of anti-mouse CII antibodies including total IgG, IgG1, IgG2, and IgG2b, in CIA mice. We further demonstrated that dasatinib inhibited the migration and proliferation of fibroblast-like synoviocytes (FLS) from RA patients and promoted FLS apoptosis. The mRNA expression of MMP13, VEGF, FGF, and DKK1 was down-regulated in FLS treated with dasatinib. Our findings suggest that dasatinib exhibited treatment effects on CIA mice and that FLS are an important target cell of dasatinib treatment in autoimmune arthritis.

CD11b promotes the differentiation of osteoclasts induced by RANKL through the spleen tyrosine kinase signalling pathway.

Macrophage surface antigen-1 (Mac-1, CD11b/CD18) has been implicated in the regulation of osteoclastogenesis. In the synovial tissues of patients with aseptic loosening after total hip replacement, CD11b was up-regulated, which indicated that CD11b is closely involved in osteolysis around the prosthesis. We found that CD11b, but not CD18, promoted osteoclast (OC) maturation. Here, we show CD11b up-regulated the levels of spleen tyrosine kinase (Syk), c-Fos and nuclear factor of activated T cells, cytoplasmic-1 (NFATc1), as well as the activity of extracellular-regulated kinase (Erk), and as a result, osteoclast precursors (OCPs) differentiated and became tartrate-resistant acid phosphatase (TRAP)-positive. In addition, increased tumour necrosis factor-α (TNF-α) induced by ultra-high molecular weight polyethylene (UHMWPE) particles up-regulated the level of CD11b. Taken together, these findings suggest that CD11b is a positive regulator of osteoclastogenesis and that it functions by activating the Syk signalling pathway, while CD18 does not have the same effect.

Effectiveness and Safety of Fixed-Dose Tranexamic Acid in Simultaneous Bilateral Total Knee Arthroplasty: A Randomized Double-Blind Controlled Trial.

BACKGROUND: Simultaneous bilateral total knee arthroplasty (TKA) can lead to greater blood loss and higher risk of venous thromboembolism. The effectiveness and safety of tranexamic acid (TXA) in simultaneous bilateral TKAs have not been clearly defined. We presumed that a fixed dose of TXA may be a preferable alternative for ease of administration in patients undergoing simultaneous bilateral TKAs. METHODS: We prospectively randomized 120 primary simultaneous bilateral TKAs to a fixed dose of TXA or equivalent volume of normal saline intravenously. The primary outcome measure was total blood loss. The secondary outcome measures were blood transfusion rate, transfusion units, intraoperative blood loss, drainage volumes, hidden blood loss, maximum decline of hemoglobin, and postoperative suprapatellar girth increment. RESULTS: There were statistically significant lower total blood loss, blood transfusion rate, drainage volumes, transfusion units, and maximum decline of hemoglobin in the TXA group than in the control group (P < .05), without increasing incidence of asymptomatic and symptomatic venous thromboembolism. However, TXA did not significantly reduce the hidden blood loss (P = .123). No differences were observed in suprapatellar girth increments between both groups on postoperative day 5 and week 6 (P = .251 and .299). CONCLUSION: Fixed dose of TXA for patients undergoing simultaneous bilateral TKAs was effective and safe in reducing total blood loss and allogeneic blood transfusion needs without any additional thromboembolic risk. However, TXA administered intravenously did not significantly reduce the hidden blood loss.

Comparison of the effects of pretreatment with repeated electroacupuncture at GV20 and ST36 on fatigue in rats.

OBJECTIVE: To investigate and compare the effects of electroacupuncture (EA) pretreatment at GV20 and ST36 on fatigue in rats. METHODS: Rats were randomly allocated into 4 groups: control, fatigue, fatigue+GV20 and fatigue+ST36. The last two groups received EA pretreatment at GV20 or ST36 for 5 days before being maintained in cages filled with water to a height of 1.5 cm to establish an animal model of fatigue. We used the weight-loaded forced swimming test and open-field test and measured 5-hydroxyindoleacetic acid (5-HIAA)/5-hydroxytryptamine (5-HT) ratios and serum levels of blood urea nitrogen (BUN), lactic dehydrogenase (LDH) and testosterone as behavioural and biochemical markers of fatigue in the rats. RESULTS: Compared with controls, rats in the (untreated) fatigue group exhibited reduced weight-loaded swimming times and total movement/distance in the open-field test, plus higher BUN/LDH and lower testosterone levels. Both EA pretreatment at GV20 and ST36 increased swimming times, and reduced serum BUN/LDH. EA pretreatment at GV20 (but not ST36) increased serum testosterone. The 5-HIAA/5-HT ratios in four brain regions were decreased in the fatigue+GV20 group compared with the fatigue group (p<0.05). By contrast, 5-HIAA/5-HT ratios in striatum and hypothalamus (but not hippocampus or midbrain) were decreased in the fatigue+ST36 group compared with the fatigue group (p<0.05). Furthermore, only pretreatment at GV20 affected the results of the open-field test. CONCLUSIONS: These results suggest that EA pretreatment had a positive effect on the prevention of fatigue. Pretreatment at GV20 had a greater anti-fatigue effect than pretreatment at ST36.

Angelica sinensis extract inhibits RANKL-mediated osteoclastogenesis by down-regulated the expression of NFATc1 in mouse bone marrow cells.

BACKGROUND: Destructive erosion of bone or osteolysis is a major complication of inflammatory conditions such as rheumatoid arthritis (RA), periodontal disease, and periprosthetic osteolysis. Natural plant-derived products have received recent attention as potential therapeutic and preventative drugs in human disease. METHODS: The effect of Angelica sinensis (AS) extract on RANKL-induced osteoclast differentiation was examined in this study. The osteoclast precursor cell line bone marrow macrophages (BMMs) was cultured and stimulated with RANKL followed by treatment with AS at several doses. Gene expression profiles of c-Fos, c-Jun, NFATc1, TRAP, and OSCAR were sequentially evaluated. RESULTS: AS extract inhibited RANKL-mediated osteoclast differentiation in BMMs in a dose-dependent manner without any evidence of cytotoxicity. AS extract strongly inhibited p38, ERK, JNK, p65 phosphorylation and I-κB degradation in RANKL-stimulated BMMs. AS extract also inhibited the mRNA expression of c-Fos, c-Jun, NFATc1, TRAP, and OSCAR in RANKL-treated BMMs. Moreover, RANKL-induced c-Fos, c-Jun and NFATc1 protein expression was suppressed by AS extract. CONCLUSIONS: These results collectively suggested that AS extract demonstrated inhibitory effects on RANKL-mediated osteoclast differentiation in bone marrow macrophages in vitro, indicating that AS may therefore serve as a useful drug in the prevention of bone loss.

Pyrroloquinoline quinone (PQQ) inhibits lipopolysaccharide induced inflammation in part via downregulated NF-κB and p38/JNK activation in microglial and attenuates microglia activation in lipopolysaccharide treatment mice.

Therapeutic strategies designed to inhibit the activation of microglia may lead to significant advancement in the treatment of most neurodegenerative diseases. Pyrroloquinoline quinone (PQQ) is a naturally occurring redox cofactor that acts as an essential nutrient, antioxidant, and has been reported to exert potent immunosuppressive effects. In the present study, the anti-inflammatory effects of PQQ was investigated in LPS treated primary microglia cells. Our observations showed that pretreatment with PQQ significantly inhibited the production of NO and PGE2 and suppressed the expression of pro-inflammatory mediators such as iNOS, COX-2, TNF-a, IL-1b, IL-6, MCP-1 and MIP-1a in LPS treated primary microglia cells. The nuclear translocation of NF-κB and the phosphorylation level of p65, p38 and JNK MAP kinase pathways were also inhibited by PQQ in LPS stimulated primary microglia cells. Further a systemic LPS treatment acute inflammation murine brain model was used to study the suppressive effects of PQQ against neuroinflammation in vivo. Mice treated with PQQ demonstrated marked attenuation of neuroinflammation based on Western blotting and immunohistochemistry analysis of Iba1-against antibody in the brain tissue. Indicated that PQQ protected primary cortical neurons against microglia-mediated neurotoxicity. These results collectively suggested that PQQ might be a promising therapeutic agent for alleviating the progress of neurodegenerative diseases associated with microglia activation.

Identification of RASAL1 as a major tumor suppressor gene in thyroid cancer.

BACKGROUND: RAS-coupled MAPK and PI3K pathways play a fundamental role in thyroid tumorigenesis, and classical genetic alterations upregulating these pathways are well characterized. We hypothesized that gene abnormality of negative modulators of these signaling pathways might be an important alternative genetic background for thyroid cancer. METHODS: By examining gene expression patterns of negative modulators of RAS signaling, we attempted to identify potential tumor suppressor genes. We then analyzed the methylation and mutation patterns of the identified gene in 101 thyroid tumors and tested its functions in vitro and in vivo to establish the tumor suppressor role in thyroid cancer. RESULTS: Among 13 negative modulators of the RAS pathway screened, RASAL1, encoding a RAS GTPase-activating protein, was frequently hypermethylated in thyroid cancers, which was coupled to its silencing in thyroid cancer cells. We also, for the first time, identified the presence of RASAL1 mutations, with a prevalence of 4.88% (n = 2 of 41) in follicular thyroid cancer (FTC) and 16.67% (n = 5 of 30) in anaplastic thyroid cancer (ATC). RASAL1 displayed MAPK- and PI3K-suppressing and thyroid tumor-suppressing activities, which were all impaired by the mutations. Hypermethylation and mutations of RASAL1 were mutually exclusive and collectively found in zero of 20 benign thyroid tumors, 3.22% (n = 1 of 31) of papillary thyroid cancers, 31.70% (n = 13 of 41) of FTCs, and 33.33% (n = 10 of 30) of ATCs. A rate of 20.83% (n = 5 of 24) of tumors carrying RASAL1 mutation or methylation at high levels (>50%) vs 44.16% (n = 34 of 77) of tumors carrying no RASAL1 mutation or methylation at low levels (< 50%) harbored any of the classical mutations (two-sided P = .02, Fisher exact test) in RAS, BRAF, PTEN, and PIK3CA genes in the MAPK and PI3K pathways, revealing a largely mutually exclusive relationship. CONCLUSIONS: We identified RASAL1 as a major tumor suppressor gene that is frequently inactivated by hypermethylation and mutations, providing a new alternative genetic background for thyroid cancer, particularly FTC and ATC.

Mutational analysis of the GNA11, MMP27, FGD1, TRRAP and GRM3 genes in thyroid cancer.

Frequent somatic mutations in the GNA11, matrix metalloproteinase (MMP)27, FGD1, TRRAP and GRM3 genes have been reported in various types of human cancer, but whether these genes are mutated in thyroid cancer is not known. In the present study, a mutational analysis of these genes was performed in thyroid cancer cell lines and thyroid cancer samples. No GNA11 mutations were identified in the papillary thyroid cancer (PTC), follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC) samples. Additionally, no mutations were identified in the MMP27 gene, although three synonymous [C351T (N117N), C1089T (S363S) and G1227A (G409G)] single nucleotide polymorphisms (SNPs) were observed infrequently in ATC. No mutations were detected in the FGD1 gene, but two infrequent synonymous [T2091C (T697T) and A2136G (P712P)] SNPs were observed in PTC. Furthermore, no mutations were identified in TRRAP and GRM3, although a frequent synonymous SNP [G1323A (T441T)] and infrequent non-synonymous SNP [G1424A (G475D)] of GRM3 were observed in PTC. No mutation of these genes was observed in 12 cell lines derived from various types of thyroid cancer. The present study reports for the first time the mutational status of the GNA11, MMP27, FGD1, TRRAP and GRM3 genes in thyroid cancer. No mutations were identified in these genes in the various types and cell lines of thyroid cancer. Therefore, unlike in other types of cancer, mutations in these genes are absent or uncommon in thyroid cancer.

External fixator followed by a plate for distraction, reduction, and fixation in neglected femoral diaphyseal fractures.

INTRODUCTION: This study aims to investigate the outcome of a two-stage surgery in the treatment of neglected femoral diaphyseal fractures which are not uncommon in developing nations. PATIENTS AND METHODS: Ten patients with neglected or late-presenting femoral diaphyseal fractures were considered in this study. All patients underwent a two-stage surgery, which consisted of distraction by an external fixator and open reduction by internal plate fixation. All patients received a supervised regimen of physiotherapy. Patients were followed up clinically and with radiographs at 2 months to assess union and at monthly intervals thereafter. RESULTS: All patients achieved bony union in an average of 3.7 months (2-6 months) with no one lost follow-up. Seven patients regained full range of motion, and the mean knee range of motion was 139.5°. No wound related or neurovascular complications were detected. One patient with stiff knee was re-admitted 1 year after surgery for metal removal, arthrolysis and quadricepsplasty for improving knee range of motion to 90° flexion. CONCLUSIONS: We conclude that the treating of neglected femoral diaphyseal fractures with a two-stage surgery is a satisfactory therapy showing reliable bony union, however continuing medical education is necessary for physicians in primary medical facilities as well as for patients with traditional views.

Pyrroloquinoline quinine inhibits RANKL-mediated expression of NFATc1 in part via suppression of c-Fos in mouse bone marrow cells and inhibits wear particle-induced osteolysis in mice.

The effects of pyrroloquinoline quinine (PQQ) on RANKL-induced osteoclast differentiation and on wear particle-induced osteolysis were examined in this study. PQQ inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, and TRAP in RANKL-treated BMMs was inhibited by PQQ treatment. Moreover, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by PQQ. PQQ additionally inhibited the bone resorptive activity of differentiated osteoclasts. Further a UHMWPE-induced murine calvaria erosion model study was performed to assess the effects of PQQ on wear particle-induced osteolysis in vivo. Mice treated with PQQ demonstrated marked attenuation of bone erosion based on Micro-CT and histologic analysis of calvaria. These results collectively suggested that PQQ demonstrated inhibitory effects on osteoclast differentiation in vitro and may suppress wear particle-induced osteolysis in vivo, indicating that PQQ may therefore serve as a useful drug in the prevention of bone loss.

The aqueous extract of Angelica sinensis, a popular Chinese herb, inhibits wear debris-induced inflammatory osteolysis in mice.

BACKGROUND: More and more studies have shown Angelica sinensis' (AS) therapeutic action on chronic inflammatory diseases in recent years. We investigated effects of aqueous extract of AS on inflammatory cytokines release and wear debris particles-induced osteolysis. MATERIALS AND METHODS: Ultra high molecular weight polyethylene (UHMWPE) particles were used to induce inflammation in RAW264.7 cell and C57BL/J6 mice. AS extract was obtained through a series of purification steps, and divided into high dose group and low dose group during the research of cell culture, tissue culture, and animal treatment. After 72 h culture with optimal particles, supernatants were collected for cytokine analysis. Calvaria were harvested from the mice model after 10 d treatment with the AS extract. Six calvaria of each group were cultured into medium for 72 h for analyzing cytokine generated in vivo. Histologic analyses and micro-computed tomography (micro-CT) scan were used to determine osteoclastogenesis and inflammatory bone resorption. RESULTS: Concentration of tumor necrosis-alpha (TNF-α) and interleukin-1beta (IL-1ß) was significantly attenuated by AS extract both in vitro and in vivo. The osteolysis area and the osteoclast numbers were decreased from 0.406 ± 0.0799 to 0.117 ± 0.0103 mm(2), and from 22.7 ± 5.0 to 11.3 ± 1.8, respectively (P < 0.01). Compared with the control group, the protection effects of AS extract was further confirmed with data of the more accurate 3-dimension micro-CT reconstruction. CONCLUSIONS: This study suggests a potential resolution of inhibiting wear debris particles-induced inflammatory bone resorption, as well as a possible way of inhibiting aseptic loosening after joint replacement surgery.

[Anterolateral minimally-invasive total hip arthroplasty: a clinical comparative study of 110 cases].

OBJECTIVE: To explore the indications and key points of anterolateral minimally-invasive total hip arthroplasty. METHODS: 110 baseline indexes matched patients admitted for unilateral total hip arthroplasty were randomly assigned to 2 equal groups to undergo surgery through a short anterolateral incision of < or = 10 cm or a standard posterolateral incision. All operations were done by the same surgeon. The demographic data, perioperative indexes, and postoperative function indexes were recorded and statistically analyzed. RESULTS: No significant differences were detected with respect to operation time, abduction angle, anteversion angle, stem alignment, and stem fixation between these 2 groups. The incision length, blood loss, perioperative transfusion, and 100 - mm visual analogue pain scale (VAS) score at the first 24 hours of the anterolateral approach group were (7.49 +/- 0.86) cm, (376.18 +/- 168.30) ml, (410.09 +/- 136.46) ml, and (30.76 +/- 21.77) respectively, all significantly shorter, less, or lower than those of the standard posterolateral approach group [(15.2 +/- 1.8) cm, (605.0 +/- 225.1) ml, (629.5 +/- 232.9) ml, and (50.3 +/- 13.7) respectively, all P < 0.01]. The Harris hip score and Barthel index 3 months after operation of the anterolateral approach group were (83.80 +/- 5.64) and (93.45 +/- 6.37) respectively, both significantly higher than those of the standard posterolateral approach group [(75.0 +/- 7.5) and (94.6 +/- 7.5) respectively, both P < 0.01)], however, there were not significant differences in the Harris hip score and Barthel index 3 years after operation between these 2 groups. CONCLUSIONS: Fewer traumas, less blood loss and rapid recovery can be obtained through this new total hip arthroplasty approach. But experienced doctors and special instruments are prerequisite.