In situ fast self-assembled preparation of dandelion-like Cu(OH)2@Cu3(HHTP)2 with core-shell heterostructure arrays for electrochemical sensing of formaldehyde in food samples.

Formaldehyde is known to harm the respiratory, nervous, and digestive systems of people. In this paper, a novel dandelion-like electrocatalyst with core-shell heterostructure arrays were fast self-assembled prepared in situ using copper foam (CF) as support substrate and 2,3,6,7,10,11 hexahydroxy-triphenyl (HHTP) as ligand (Cu(OH)2@Cu3(HHTP)2/CF) by a simple two-step hydrothermal reaction. The 1D Cu(OH)2 nanorods "core" and the 2D π-conjugated conducting metal-organic frameworks (Cu3(HHTP)2cMOF) "shell" with remote delocalized electrons give the dandelion-like heterogeneous catalysts excellent electrochemical activity such as a large specific surface area, high conductivity and a fast electron transfer rate. The Cu(OH)2@Cu3(HHTP)2/CF exhibited excellent electrocatalytic performance for formaldehyde under alkaline conditions with a linear range of 0.2 µmol/L - 125 µmol/L and 125 µmol/L - 8 mmol/L, a detection limit as low as 15.9 nmol/L (S/N = 3), as well as good accuracy, consistency, and durability, and it effectively identified FA in food.

A novel hollow CuMn-PBA@NiCo-LDH nanobox for efficient detection of glucose in food.

Designing a rapid and sensitive glucose detection method is of great significance to public health. Herein, hollow CuMn-PBA@NiCo-LDH nanoboxes (CuMn-PBA@NiCo-LDH NBs) were prepared using acid etching, cation exchange, and reflux method. The modified electrode exhibited outstanding electrocatalytic performance for glucose oxidation due to the unique hollow nanostructure and synergistic effects. The CuMn-PBA@NiCo-LDH NBs electrode displayed excellent electrocatalytic oxidation activity for glucose in an alkaline solution. Under optimal conditions, the electrode achieved a wide linear range (0.0005-1 mmol L-1, and 1-7 mmol L-1) and high sensitivity (10,300 µA L/mmol cm-2 and 5310 µA L/mmol cm-2), with a limit of detection (LOD) of 19 nmol L-1. The feasibility of the sensor applied to the detection of glucose was verified in real food samples through spiked recovery experiments. This electrode material offers an alternative method for the non-enzymatic glucose sensors.

Layered bimetallic hydroxide nanocage assembled on MnO2 nanotubes: A hierarchical porous sugar gourd-like electrocatalyst for the sensitive detection of hydrogen peroxide in food.

Hydrogen peroxide is used widely as a disinfection or bleaching additive during processing in the food industry. However, excessive residues of hydrogen peroxide in food have serious human health implications. In the present study, a novel electrochemical sensing electrode (MnO2/ZIF-67@LDH) with hierarchical porous sugar gourd-like structure was fabricated through a multi-step hydrothermal method using ZIF as the precursor. The unique porous nanocage structure of the sensing electrode provided multidimensional charge transfer channels and accelerated the electron transfer rate. As a hydrogen peroxide sensor, the electrode had two detection linear ranges of 1×10-3-4 mmol L-1 and 4-8 mmol L-1, and the detection limit was 0.26 µmol L-1. The MnO2/ZIF-67@LDH sensor was also applied to determine the content of hydrogen peroxide in actual food samples of juice and milk, and satisfactory recovery were achieved. The present study provides a novel and effective design strategy for the construction of electrochemical sensing electrodes.

Bio-Activated PEEK: Promising Platforms for Improving Osteogenesis through Modulating Macrophage Polarization.

The attention on orthopedic biomaterials has shifted from their direct osteogenic properties to their osteoimmunomodulation, especially the modulation of macrophage polarization. Presently, advanced technologies endow polyetheretherketone (PEEK) with good osteoimmunomodulation by modifying PEEK surface characteristics or incorporating bioactive substances with regulating macrophage polarization. Recent studies have demonstrated that the fabrication of a hydrophilic surface and the incorporation of bioactive substances into PEEK (e.g., zinc, calcium, and phosphate) are good strategies to promote osteogenesis by enhancing the polarization of M2 macrophages. Furthermore, the modification by other osteoimmunomodulatory composites (e.g., lncRNA-MM2P, IL-4, IL-10, and chitosan) and their controlled and desired release may make PEEK an optimal bio-activated implant for regulating and balancing the osteogenic system and immune system. The purpose of this review is to comprehensively evaluate the potential of bio-activated PEEK in polarizing macrophages into M2 phenotype to improve osteogenesis. For this objective, we retrieved and discussed different kinds of bio-activated PEEK regarding improving osteogenesis through modulating macrophage polarization. Meanwhile, the relevant challenges and outlook were presented. We hope that this review can shed light on the development of bio-activated PEEK with more favorable osteoimmunomodulation.

Icariin-loaded sulfonated polyetheretherketone with osteogenesis promotion and osteoclastogenesis inhibition properties via immunomodulation for advanced osseointegration.

Preventing prosthesis loosening due to insufficient osseointegration is critical for patients with osteoporosis. Endowing implants with immunomodulatory function can effectively enhance osseointegration. In this work, we loaded icariin (ICA) onto 3D porous sulfonated PEEK (SPEEK) via polydopamine (PDA) modification. Modified ICA-PDA@SPEEK not only promoted the polarization of macrophages to the anti-inflammatory M2 type, but also enhanced the osteogenesis of bone mesenchymal stem cells (BMSCs) and inhibited RANKL-induced osteoclastogenesis by regulating cytokine from macrophages. In vivo experiments further showed that ICA-PDA@SPEEK regulated the host immune response and promoted osseointegration in ovariectomized (OVX) rats. The above results demonstrated that ICA-PDA@SPEEK could be an excellent orthopedic biomaterial with immunomodulatory properties.

Anti-Infective Application of Graphene-Like Silicon Nanosheets via Membrane Destruction.

The increasing problem of bacterial resistance to the currently effective antibiotics has resulted in the need for increasingly potent therapeutics to eradicate pathogenic microorganisms. 2D nanomaterials (2D NMs) have unique physical and chemical properties that make them attractive candidates for biomedical applications. Recently, the application of 2D NMs as antibacterial agents has attracted significant attention. Herein, a novel 2D graphene-like silicon nanosheet (GS NS) antimicrobial agent is fabricated from pristine silicon crystals by ultrasonication, which results in a highly exfoliated planar morphology and a significantly larger surface area as compared with bulk silicon. The GS NSs exhibit remarkable in vitro broad-spectrum bactericidal activity against Gram (-) Escherichia coli and Gram (+) Staphylococcus aureus because of a close interaction with the bacteria, which leads to highly efficient membrane destruction. The in vivo studies demonstrate that the local administration of GS NSs effectively mitigates implant-related infection by reducing the bacterial burden of the extracted samples and accelerating the remission of local inflammation. Based on these encouraging results, GS NSs are expected to be a useful new member of the 2D NMs family, with the potential of effectively killing pathogenic bacteria in clinical applications.

Curcumin has immunomodulatory effects on RANKL-stimulated osteoclastogenesis in vitro and titanium nanoparticle-induced bone loss in vivo.

Wear particle-stimulated inflammatory bone destruction and the consequent aseptic loosening remain the primary causes of artificial prosthesis failure and revision. Previous studies have demonstrated that curcumin has a protective effect on bone disorders and inflammatory diseases and can ameliorate polymethylmethacrylate-induced osteolysis in vivo. However, the effect on immunomodulation and the definitive mechanism by which curcumin reduces the receptor activators of nuclear factor-kappa B ligand (RANKL)-stimulated osteoclast formation and prevents the activation of osteoclastic signalling pathways are unclear. In this work, the immunomodulation effect and anti-osteoclastogenesis capacities exerted by curcumin on titanium nanoparticle-stimulated macrophage polarization and on RANKL-mediated osteoclast activation and differentiation in osteoclastic precursor cells in vitro were investigated. As expected, curcumin inhibited RANKL-stimulated osteoclast maturation and formation and had an immunomodulatory effect on macrophage polarization in vitro. Furthermore, studies aimed to identify the potential molecular and cellular mechanisms revealed that this protective effect of curcumin on osteoclastogenesis occurred through the amelioration of the activation of Akt/NF-κB/NFATc1 pathways. Additionally, an in vivo mouse calvarial bone destruction model further confirmed that curcumin ameliorated the severity of titanium nanoparticle-stimulated bone loss and destruction. Our results conclusively indicated that curcumin, a major biologic component of Curcuma longa with anti-inflammatory and immunomodulatory properties, may serve as a potential therapeutic agent for osteoclastic diseases.

Puerarin Exerts Protective Effects on Wear Particle-Induced Inflammatory Osteolysis.

Wear particle-stimulated inflammatory bone destruction and the consequent aseptic loosening remain major postoperative problems for artificial joints. Studies have indicated that puerarin promotes osteogenesis and alleviates lipopolysaccharide-induced osteoclastogenesis in vitro. However, the underlying molecular mechanism by which puerarin interacts with receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclast formation in vitro and wear particle-stimulated osteolysis in vivo has not been reported. In this work, the protective effects exerted by puerarin on titanium particle-stimulated bone destruction in vivo and on RANKL-induced osteoclast activation in osteoclastic precursor cells in vitro were investigated. As expected, puerarin significantly inhibited wear particle-mediated bone resorption and proinflammatory cytokine productions in a calvarial resorption model. Additionally, puerarin inhibited RANKL-induced osteoclast activation, bone resorption ability, and F-actin ring formation in vitro as puerarin concentration increased. Furthermore, mechanistic investigation indicated that reduced RANKL-stimulated MEK/ERK/NFATc1 signaling cascades might regulate the protective effect of puerarin. Conclusively, these results indicate that puerarin, a type of polyphenol, might serve as a protective agent to prevent osteoclast-related osteolytic diseases.

Lithium chloride with immunomodulatory function for regulating titanium nanoparticle-stimulated inflammatory response and accelerating osteogenesis through suppression of MAPK signaling pathway.

BACKGROUND: Wear particle-induced inflammatory osteolysis and the consequent aseptic loosening constitute the leading reasons for prosthesis failure and revision surgery. Several studies have demonstrated that the macrophage polarization state and immune response play critical roles in periprosthetic osteolysis and tissue repair, but the immunomodulatory role of lithium chloride (LiCl), which has a protective effect on wear particle-induced osteolysis by suppressing osteoclasts and attenuating inflammatory responses, has never been investigated. METHODS: In this work, the immunomodulatory capability of LiCl on titanium (Ti) nanoparticle-stimulated transformation of macrophage phenotypes and the subsequent effect on osteogenic differentiation were investigated. We first speculated that LiCl attenuated Ti nanoparticle-stimulated inflammation responses by driving macrophage polarization and generating an immune micro-environment to improve osteogenesis. Furthermore, a metal nanoparticle-stimulated murine air pouch inflammatory model was applied to confirm this protective effect in vivo. RESULTS: The results revealed that metal nanoparticles significantly activate M1 phenotype (proinflammatory macrophage) expression and increase proinflammatory cytokines secretions in vitro and in vivo, whereas LiCl drives macrophages to the M2 phenotype (anti-inflammatory macrophage) and increases the release of anti-inflammatory and bone-related cytokines. This improved the osteogenic differentiation capability of rat bone marrow mesenchymal stem cells (rBMSCs). In addition, we also provided evidence that LiCl inhibits the phosphorylation of the p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK) pathways in wear particle-treated macrophages. CONCLUSION: LiCl has the immunomodulatory effects to alleviate Ti nanoparticle-mediated inflammatory reactions and enhance the osteogenic differentiation of rBMSCs by driving macrophage polarization. Thus, LiCl may be an effective therapeutic alternative for preventing and treating wear debris-induced inflammatory osteolysis.

Sodium butyrate-modified sulfonated polyetheretherketone modulates macrophage behavior and shows enhanced antibacterial and osteogenic functions during implant-associated infections.

Prevention of implant-associated infections and insufficient bone tissue integration is critical to exploit the immunomodulatory properties and antibacterial effects of implant materials, which have attracted considerable attention. Modulation of the functions of immune cells in different environments is crucial for managing infection and inferior bone integration via immunomodulation. In this work, sodium butyrate, a fermentation product of gut microbiota, was loaded onto 3D porous sulfonated polyetheretherketone (SP) to modulate the immune responses of cells in different environments. Evaluation of in vitro antibacterial effects showed that sodium butyrate-loaded SP exhibited superior antibacterial activity, especially in the samples containing high concentrations of sodium butyrate. Under bacterial stimulation, the phagocytic activity of macrophages increased with an increase in the sodium butyrate concentration via the production of reactive oxygen species (ROS), which favoured bactericidal activity in the implant-associated infection stage. For bacterial elimination, sodium butyrate-containing SP could polarize macrophages to the M2 phenotype and subsequently stimulate anti-inflammatory cytokine secretion, which is considered beneficial for bone regeneration in the tissue repair stage. In vitro osteogenesis was evaluated and the results demonstrated that treatment with sodium butyrate-containing SP increased the expression of osteogenic genes and proteins. An in vivo rat osteomyelitis model was used to evaluate the protective effect of the SP-loaded with sodium butyrate on bone destruction and osteolysis under infection conditions. To study osteogenesis in vivo, a rat femoral model without infection was used. The results indicated that the SP-B2 group exhibited superior anti-infection capacity and induced new bone formation around the implant in vivo. Treatment with sodium butyrate-containing porous SP modulated the macrophage response under different stimuli, thereby serving as a new approach for the design of smart implant materials with superior antibacterial and bone repair properties.

Multifunctional sulfonated polyetheretherketone coating with beta-defensin-14 for yielding durable and broad-spectrum antibacterial activity and osseointegration.

To address periprosthetic joint infection (PJI), a formidable complication after joint arthroplasty, an implant with excellent osseointegration and effective antibacterial activity has being extensively pursued and developed. In this work, the mouse beta-defensin-14 (MBD-14) was immobilized on the polyetheretherketone (PEEK) surface with three-dimensional (3D) porous structure to improve its antibacterial activity and osseointegration. An in vitro antibacterial evaluation showed that the porous PEEK loaded with MBD-14 wages a durable and effective fight against both Staphylococcus aureus (gram-positive) and Pseudomonas aeruginosa (gram-negative). In addition to the superior antibacterial activity, we found that the enhanced proliferation and osteogenic differentiation of bone mesenchymal stem cells were verified through various in vitro analyses. To evaluate the in vivo bactericidal effect and osseointegration of the samples, the rat femoral models with infection and non-infection were established. The enhanced osseointegration of the MBD-14-loaded samples was found in both two in vivo models. And no bacteria survived on the surfaces of samples with a relatively high MBD-14 concentration. Above results indicate that the 3D porous PEEK coating loaded with MBD-14 simultaneously yields excellent osseointegration while exerting durable and broad-spectrum antibacterial activity. And it paves the way for PEEK to be applied clinically to address PJI. STATEMENT OF SIGNIFICANCE: (1). By using the physio-chemical technique including sulfonation and lyophilization etc., a three-dimensional porous network is developed on polyetheretherketone (PEEK) surface, in which mouse beta-defensin-14 (MBD-14, a broad-spectrum antimicrobial peptide) is then loaded. It endows PEEK with antibacterial activity and osseointegration. (2). Two in vivo animal models with infection and non-infection are used to prove the new bone formation around the samples. (3). Supplementary material also proves that MBD-14 promotes the osteogenic differentiation of BMSCs. However, its potential mechanism needs to be further studied in future. (4). The modified PEEK, including excellent osseointegration and a durable and broad-spectrum antibacterial activity, could be applied clinically to address PJI which is a hot potato for surgeons and patients undergoing total joint arthroplasty.

Emodin attenuates titanium particle-induced osteolysis and RANKL-mediated osteoclastogenesis through the suppression of IKK phosphorylation.

Aseptic loosening due to wear particles is a serious challenge for orthopedic surgeons, sabotaging the long-term success of total joint arthroplasty. The existing treatments for aseptic loosening are still far from satisfactory, necessitating more aggressive drug exploration. Here, we examined the effect of emodin on titanium particle-induced osteolysis and further investigated its underlying mechanism in vivo and in vitro. Thirty-two C57BL/6 mice were randomly assigned into four groups: the Sham group (sham operation with vehicle injection), Vehicle group (titanium particle treatment with vehicle injections), Low group (titanium particle treatment with injections of 10 mg/kg/day emodin) and High group (titanium particle treatment with injections of 50 mg/kg/day emodin). Micro-CT scanning and histological analysis revealed that after emodin injections, the inflammatory response and bone destruction were markedly ameliorated. TRAP staining showed that osteoclast numbers were also dramatically reduced. Throughout the in vitro culture period, emodin significantly decreased the bone resorption area, number of osteoclasts and formation of F-actin rings. Mechanistic studies suggested that reduced NF-κB signaling might be mediating the inhibitory effects of emodin. Collectively, our findings suggest that emodin, a natural product extracted from Rheum palmatum, may be developed as a promising candidate for the treatment of wear particle-induced osteolysis and subsequent aseptic loosening.

Synergistic effects of intravenous and intra-articular tranexamic acid on reducing hemoglobin loss in revision total knee arthroplasty: a prospective, randomized, controlled study.

BACKGROUND: Tranexamic acid decreases blood loss in primary total knee arthroplasty, and no related prospective randomized clinical trials have been conducted to evaluate the effectiveness and safety of tranexamic acid in revision total knee arthroplasty. Thus, we conducted this work to evaluate the synergistic effects of intravenous plus intra-articular tranexamic acid on reducing hemoglobin loss compared with intra-articular tranexamic acid alone in revision total knee arthroplasty. STUDY DESIGN AND METHODS: This prospective, controlled study randomized 96 patients undergoing revision total knee arthroplasty into two groups: an intravenous plus intra-articular tranexamic acid group (48 patients who received 20 mg/kg intravenous tranexamic acid and 3.0 g intra-articular tranexamic acid); and an intra-articular tranexamic acid alone group (48 patients who received the same intravenous volume of normal saline and 3.0 g intra-articular tranexamic acid). The primary outcome was hemoglobin loss. Secondary outcomes included the volume of drain output, the percentage of patients who received transfusions, the number of units transfused, and thromboembolic events. RESULTS: The baseline data, preoperative hemoglobin, and tourniquet time were similar in both groups. There was significantly less hemoglobin loss in the intravenous plus intra-articular tranexamic acid group compared with the intra-articular tranexamic acid alone group (2.7 ± 0.6 g/dL and 3.7 ± 0.7 g/dL; p < 0.001). Compared with the intra-articular tranexamic acid alone group, the intravenous plus intra-articular tranexamic acid group also had significantly less drain output, fewer patients who received transfusions, and fewer units transfused (all p < 0.05). There were no significant differences in thromboembolic events in the two groups during the 3-month follow-up. CONCLUSION: Compared with intra-articular tranexamic acid alone, combined intravenous plus intra-articular tranexamic acid significantly reduced hemoglobin loss and the need for transfusion without an apparent increase in thromboembolic events in patients who underwent revision total knee arthroplasty.

Mouse ß-defensin-14 for inducing the maturation of dendritic cells.

BACKGROUND: ß-defensins are an excellent antimicrobial peptide against microbial infection in which dendritic cells (DCs) play a crucial role by improving the innate and adaptive immune defense. However, it is unclear whether BDs affect DC maturation. This work aimed to study the effects of mouse ß-defensin-14 (MBD-14) on DC maturation. METHODS: Via in vitro using mouse bone marrow DCs, the maturation of DCs was evaluated by cell morphological staining, flow cytometry, endocytosis assay, and allogeneic mixed lymphocyte reaction, respectively. And it was also assessed by in vivo establishing a mouse air-pouch model for flow cytometric determination, cytokine analysis, and histological staining. Additionally, CLI-095, an inhibitor of Toll-like receptor-4 (TLR-4), was used to determine whether TLR-4 is possibly involved in DC maturation. RESULTS: It was found MBD-14 promoted DCs to form more filopodia and lamellipodia, increased the expression of DC maturation markers (CD40 and MHC-II), decreased their endocytic capacity, and enhanced T-cell proliferation. The analyses of the air-pouch exudates were consistent with the in vitro results of MBD-14 activating DCs. And when CLI-095 was applied, DC maturation was inhibited partly. CONCLUSIONS: This work demonstrates that MBD-14 can promote the maturation of DCs in which TLR-4 is possibly involved.

Immunomodulatory Effects of Calcium and Strontium Co-Doped Titanium Oxides on Osteogenesis.

The effects of calcium (Ca) or strontium (Sr) on host osteogenesis and immune responses have been investigated separately. In clinical practice, these two elements may both be present around an orthopedic device, but their potential synergistic effects on osteogenesis and the immune response have not been explored to date. In this work, we investigated the immunomodulatory effects of Ca and Sr co-doped titanium oxides on osteogenesis in vitro using the mouse macrophage cell line RAW 264.7 alone and in co-culture with mouse bone mesenchymal stem cells (BMSCs), and in vivo using a mouse air-pouch model. Coatings containing Ca and Sr at different concentration ratios were fabricated on titanium substrates using micro-arc oxidation and electrochemical treatment. The in vitro and in vivo results demonstrated that the Ca and Sr concentration ratio has a marked influence on macrophage polarization. The coating with a Ca/Sr ratio of 2:1 was superior to those with other Ca and/or Sr concentrations in terms of modulating M2 polarization, which enhanced osteogenic differentiation of mouse BMSCs in co-culture. These findings suggest that the osteoimmunomodulatory effect of a titanium-oxide coating can be enhanced by modulating the concentration ratio of its components.

Comparison of 3 Routes of Administration of Tranexamic Acid on Primary Unilateral Total Knee Arthroplasty: A Prospective, Randomized, Controlled Study.

BACKGROUND: The mode of administration for tranexamic acid (TXA) to significantly reduce the decrease in hemoglobin (Hb), number of transfusions, relevant costs, and side effects in patients undergoing primary unilateral total knee arthroplasty (TKA) has not been resolved. METHODS: A total of 560 patients undergoing primary unilateral TKA were randomized into 4 groups: intravenous group (140 patients receiving 2 doses of 20 mg/kg intravenous TXA), topical group (140 patients administered 3.0 g topical TXA), oral group (140 patients given 2 doses of 20 mg/kg oral TXA), and a control group (140 patients not given TXA). The primary outcomes included postoperative 48-hour Hb loss and drainage volume, number of transfusions, transfusion and TXA costs, and thromboembolic complications. Secondary outcomes were postoperative inpatient time and wound healing 3 weeks after TKA. RESULTS: Baseline data among the 4 groups were similar. The 48-hour Hb loss and drainage volume in the intravenous, topical, and oral groups were significantly less (P < .05) than those in the control group, and the latter had significantly more transfusions and transfusion costs than the other 3 groups (P < .05). The TXA cost was lowest in the oral group compared with that in the topical and intravenous groups (P < .05). No differences in thromboembolic complications, postoperative inpatient time, or wound healing were observed among the groups. However, wound dehiscence and continuous wound discharge occurred in the topical group. CONCLUSION: All the 3 modes of TXA administration significantly reduced postoperative Hb loss, the number of transfusions, and transfusion costs compared with those in the control group. No pulmonary embolism or infection was observed. Oral TXA is recommended because it provided a similar clinical benefit and resulted in the lowest TXA cost compared with the other 2 modes of TXA administration.

[p14ARF enhances cisplatin-induced apoptosis in human osteosarcoma cells in p53-independent pathway].

OBJECTIVE: To study effect of tumor suppressor p14ARF on cisplatin-induced apoptosis in human osteosarcoma cells with its molecular mechanisms to provide evidences for increasing chemosensitivity of osteosarcoma. METHODS: pcDNA3.1-p14ARF plasmid was stable transfected into MG63 cells lack of p14ARF expression. Expression of p14ARF on mRNA and protein level was evaluated with RT-PCR and Western blot. MG63, MG63-vec and MG63-ARF cells were treated with cisplatin. Cell growth inhibition and IC50 were determined through MTT assay. Apoptosis was detected using fluorescence-activated cell sorting and Hoechst33258 staining. The expression of p53, Bax, p21, Mdm2, Fas, Caspase-3, caspase-9 and PARP was detected with Western blot. RNAi was used to silence p53. Cells were pre-treated with Caspase-9 specific inhibitor Z-LEHD-FMK to determine whether the effect was Caspase-9-dependent. RESULTS: There was no expression of p14ARF in MG63 and MG63-vec cells but obvious expression in MG63-ARF cells on mRNA and protein level. Cell viability was 84.2%±4.3%, 80.8%±4.3% and 58.9%±5.4% in MG63, MG63-vec, and MG63-ARF cells after treatment of cisplatin for 72 h. IC50 was (11.1±0.6), (10.7±0.9) and (7.2±0.7) µmol/L. The apoptotic rate was 13.6%, 18.5% and 35.9% in groups, There were more obvious apoptotic more changes in MG63-ARF cells than MG63 and MG63-vec cells, and activation of Caspase-3, 9 and PARP on higher level in U2OS-ARF cells after stimulation with cisplatin for 72 h. The expression of p53, Bax, p21, Mdm2 and Fas, in MG63-vec and MG63-ARF cells did not changed (P>0.05). The expression of p53 was effectively and continuously suppressed by p53-siRNA in U2OS-vec and U2OS-ARF cells. The p53 silencing did not alter the cytotoxicity mediated by cisplatin treatment for 72 h (P>0.05). Cell viability was 96.8%±3.6%, 54.1%±5.7% and 89.5%±5.1% in Z-LEHD-FMK, cisplatin and Z-LEHD-FMK+cisplatin groups. CONCLUSION: p14ARF enhances cisplatin-induced apoptosis in human osteosarcoma MG63 cells in p53-independent caspase-9-dependent pathway, in which the intrinsic mitochondrial apoptotic pathway is involved.

[p14ARF enhances chemosensitivity to cisplatin in human osteosarcoma U2OS cells through p53 apoptotic pathway].

OBJECTIVE: To explore the effects of tumor suppressor p14ARF on chemosensitivity of human osteosarcoma U2OS cells to cisplatin and elucidate its molecular mechanism. METHODS: U2OS cells expressing no p14ARF and U2OS-ARF cells expressing p14ARF stably through stable transfection were treated with cisplatin. Cell viability and IC50 were assayed with methyl thiazolyl tetrazolium (MTT). Apoptosis was examined by fluorescence-activated cell sorting and Hoechst33258 staining. The expressions of p53, Bax, p21, Mdm2 and Fas were detected by Western blot. And colorimetry was used to determine the activities of caspase-3, caspase-8 and caspase-9. RESULTS: The viability was 84.8% ± 4.4%, 86.9% ± 5.0% and 66.7% ± 4.6% respectively in U2OS, U2OS-vec and U2OS-ARF cells. The values of IC50 were (15.8 ± 0.9) µmol/L, (16.3 ± 0.6) and (8.9 ± 0.8) µmol/L respectively in U2OS, U2OS-vec and U2OS-ARF cells. The levels of viability and IC50 obviously decreased in U2OS-ARF cells in response to cisplatin (P < 0.05). There were higher apoptotic rate and more obvious apoptotic morphological changes in U2OS-ARF cells than U2OS and U2OS-vec cells. The basal levels of p53, Mdm2 and p21 in U2OS-ARF cells were slightly higher than those in U2OS-vec cells. Cisplatin up-regulated p53, Mdm2 and p21 in both cell lines. However, the up-regulation was more pronounced in U2OS-ARF cells. Cisplatin did not change the levels of Bax and Fas in U2OS-vec cells. Bax protein was up-regulated in U2OS-ARF cells while the level of Fas remained constant. p14ARF also enhanced the activities of caspase-9 and caspase-3 in response to cisplatin. CONCLUSION: p14ARF enhances the chemosensitivity to cisplatin in human osteosarcoma U2OS cells through p53 apoptotic pathway. And intrinsic mitochondrial apoptosis is involved.

Biomechanical assessment of unilateral pedicle screws plus contralateral transfacetopedicular screws after transforaminal lumbar interbody fusion with two cages.

OBJECTIVE: To assess the biomechanical stability of unilateral pedicle screws (UPS) plus contralateral transfacetopedicular screws (TFPS) after transforaminal lumbar interbody fusion (TLIF) with two cages. METHODS: Range of motion (ROM) testing was performed in 28 fresh-frozen human cadaveric lumbar spine motion segments. The sequential test configurations included supplemental constructs after TLIF such as UPS, UPS plus contralateral TFPS and bilateral pedicle screws (BPS). All test specimens were fixated in the normal lordotic lignment, then mounted in a three-dimensional (3-D) motion testing machine and fixed to the load frame of a six degrees of freedom spine simulator. Each of the test constructs were subjected to three load-unload cycles in each of the physiologic planes generating flexion-extension, right-left lateral bending and right-left axial rotation load-displacement curves. Statistical analysis was performed on the ROM data. Comparison of data was performed by repeated-measures analysis of variance for independent samples followed by Bonferroni analysis for multiple comparison procedures. RESULTS: The ROMs for UPS, BPS and UPS plus TFPS fixation after TLIF were significantly smaller than those of the intact spine in all modes. The ROM for UPS plus TFPS fixation was between the largest for UPS and the smallest for BPS. The differences between ROMs of UPS and UPS plus TFPS were significant for both lateral bending and rotation. There were no significant differences between BPS and UPS plus TFPS in any mode. CONCLUSION: Because the UPS construct provides the least stability, especially during lateral bending and rotation, it should be used prudently. After TLIF with two cages, UPS plus TFPS provides stability comparable to that of TLIF with BPS. It is thus an acceptable option in minimally invasive surgery.

Interferonalpha enhances etoposide-induced apoptosis in human osteosarcoma U2OS cells by a p53-dependent pathway.

Interferonalpha (IFNalpha) induces cell cycle arrest and triggers apoptosis and chemosensitivity. But the mechanism of IFNalpha in regulating chemosensitivity has not been fully understood. To study whether IFNalpha affected chemosensitivity of osteosarcoma cells, we treated p53-wild U2OS cells and p53-mutant MG63 cells with IFNalpha and etoposide, alone or in combination, and then examined growth inhibition, cell cycle arrest and apoptosis. IFNalpha enhanced etoposide-induced growth inhibition and apoptosis in p53-wild U2OS cells but not p53-mutant MG63 cells in a dose- and time-dependent manner. Etoposide-induced G2/M phase arrest was also enhanced by IFNalpha. The enhanced apoptosis was associated with the accumulation of transcriptionally active p53 accompanied with increased Bax and Mdm2, as well as decreased Bcl-2. IFNalpha also activated caspases-3, -8 and -9 protein kinases and PARP cleavage in response to etoposide in U2OS cells. Moreover, the combination-induced cytotoxicity and PARP cleavage were significantly reduced by caspase pan inhibitor and p53 siRNA. Thus we conclude that IFNalpha enhances etoposide-induced apoptosis in human osteosarcoma U2OS cells by a p53-dependent and caspase-activation pathway. The proper combination of IFNalpha and conventional chemotherapeutic agents may be a rational strategy for the treatment of human osteosarcoma with functional p53.