Combining tissue engineering with metal scaffolds in orthopaedic to improve osseointegration of endoprothesis

Electron Beam Melting (EBM) technology allows the fabrication of free-formed metal scaffolds, thus creating the possibility of manufacturing patient-specific endo-prostheses. It also allows the production of highly-porous prostheses with an elastic modulus similar to that of bone. The porous can be filled-in with tissue engineering elements (i.e. osteogenic molecules, biomaterials, cells) in order to promote bone ingrowth inside them, thus improving the prostheses osseointegration. For this purpose, the surface of EBM-sintered titanium should permit cell adhesion, growth and differentiation to ensure a good metal-to-tissue interaction. Our goal was to evaluate the osteoconductivity of EBM-manufactured Ti6Al4V porous scaffolds. Porous Ti6Al4V discs were manufactured by EBM-sintering, autoclave-sterilized and seeded with human and rat osteoblasts and mesenchymal stem cells (MSC). Cell adhesion, proliferation and differentiation were assessed by vital staining, MTT assay, RT-PCR and immunostaining techniques. Bone organ-explant culture was used to further assess osteoconductivity at tissue level in vitro. Both osteoblastic and MSC attached to and grew on the titanium discs, covering up the entire metal surface, and even bridging the pores of the scaffold. Collagen type I, osteopontin, and osteocalcin expression confirmed the osseous differentiation of the cells cultured on the titanium discs. Bone explants placed on EBM-sintered titanium alloy spontaneously released cells that covered up the metal surface. Long-term cultured explants strongly adhered to the titanium. EBM-sintered titanium scaffolds promote cell adhesion and can be populated by osteoblastic and MSC, which can normally differentiate towards the osteogenic lineage upon proper stimulation. These osteoconductive properties should promote the osseointegration of EBM-manufactured endoprostheses for bone replacement.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Computer modeling of an impedance-controlled pulsing protocol for RF tumor ablation with a cooled electrode

[EN] Purpose: To develop computer models to mimic the impedance-controlled pulsing protocol implemented in radiofrequency (RF) generators used for clinical practice of radiofrequency ablation (RFA), and to assess the appropriateness of the models by comparing the computer results with those obtained in previous experimental studies.Methods: A 12-min RFA was modelled using a cooled electrode (17G, 3cm tip) inserted in hepatic tissue. The short (transverse) diameter of the coagulation zone was assessed under in vivo (with blood perfusion (BP) and considering clamping) and ex vivo (at 21 degrees C) conditions. The computer results obtained by programming voltage pulses were compared with current pulses.Results: The differences between voltage and current pulses were noticeable: using current instead of voltage allows larger coagulation zones to be created, due to the higher energy applied by current pulses. If voltage pulses are employed the model can accurately predict the number of roll-offs, although the waveform of the applied power is clearly not realistic. If current voltages are employed, the applied power waveform matches well with those reported experimentally, but there are significantly fewer roll-offs. Our computer results were overall into the ranges of experimental ones.Conclusions: The proposed models reproduce reasonably well the electrical-thermal performance and coagulation zone size obtained during an impedance-controlled pulsing protocol.This work was supported by the Spanish Plan Estatal de Investigacion, Desarrollo e Innovacion Orientada a los Retos de la Sociedad under grant number TEC2014-52383-C3-R (TEC2014-52383-C3-1-R). The authors alone are responsible for the content and writing of the paper.Trujillo Guillen, M.; Bon Corbín, J.; Rivera Ortun, MJ.; Burdio, F.; Berjano, E. (2016). Computer modeling of an impedance-controlled pulsing protocol for RF tumor ablation with a cooled electrode. International Journal of Hyperthermia. 32(8):931-939. doi:10.1080/02656736.2016.1190868S931939328Hocquelet, A., Balageas, P., Laurent, C., Blanc, J.-F., Frulio, N., Salut, C., … Trillaud, H. (2015). Radiofrequency ablation versus surgical resection for hepatocellular carcinoma within the Milan criteria: A study of 281 Western patients. International Journal of Hyperthermia, 31(7), 749-757. doi:10.3109/02656736.2015.1068382Fukushima, T., Ikeda, K., Kawamura, Y., Sorin, Y., Hosaka, T., Kobayashi, M., … Kumada, H. (2015). Randomized Controlled Trial Comparing the Efficacy of Impedance Control and Temperature Control of Radiofrequency Interstitial Thermal Ablation for Treating Small Hepatocellular Carcinoma. Oncology, 89(1), 47-52. doi:10.1159/000375166Goldberg, S. N., Stein, M. C., Gazelle, G. S., Sheiman, R. G., Kruskal, J. B., & Clouse, M. E. (1999). Percutaneous Radiofrequency Tissue Ablation: Optimization of Pulsed-Radiofrequency Technique to Increase Coagulation Necrosis. Journal of Vascular and Interventional Radiology, 10(7), 907-916. doi:10.1016/s1051-0443(99)70136-3Ahmed, M., Liu, Z., Humphries, S., & Nahum Goldberg, S. (2008). Computer modeling of the combined effects of perfusion, electrical conductivity, and thermal conductivity on tissue heating patterns in radiofrequency tumor ablation. International Journal of Hyperthermia, 24(7), 577-588. doi:10.1080/02656730802192661Lobo, S. M., Liu, Z.-J., Yu, N. C., Humphries, S., Ahmed, M., Cosman, E. R., … Goldberg, S. N. (2005). RF tumour ablation: Computer simulation and mathematical modelling of the effects of electrical and thermal conductivity. International Journal of Hyperthermia, 21(3), 199-213. doi:10.1080/02656730400001108Solazzo, S. A., Liu, Z., Lobo, S. M., Ahmed, M., Hines-Peralta, A. U., Lenkinski, R. E., & Goldberg, S. N. (2005). Radiofrequency Ablation: Importance of Background Tissue Electrical Conductivity—An Agar Phantom and Computer Modeling Study. Radiology, 236(2), 495-502. doi:10.1148/radiol.2362040965Barauskas, R., Gulbinas, A., & Barauskas, G. (2007). Investigation of radiofrequency ablation process in liver tissue by finite element modeling and experiment. Medicina, 43(4), 310. doi:10.3390/medicina43040039Haemmerich, D., & Wood, B. J. (2006). Hepatic radiofrequency ablation at low frequencies preferentially heats tumour tissue. International Journal of Hyperthermia, 22(7), 563-574. doi:10.1080/02656730601024727Haemmerich, D., Chachati, L., Wright, A. S., Mahvi, D. M., Lee, F. T., & Webster, J. G. (2003). Hepatic radiofrequency ablation with internally cooled probes: effect of coolant temperature on lesion size. IEEE Transactions on Biomedical Engineering, 50(4), 493-500. doi:10.1109/tbme.2003.809488Schutt, D. J., & Haemmerich, D. (2008). Effects of variation in perfusion rates and of perfusion models in computational models of radio frequency tumor ablation. Medical Physics, 35(8), 3462-3470. doi:10.1118/1.2948388Zhang, B., Moser, M. A. J., Zhang, E. M., Luo, Y., & Zhang, W. (2015). Numerical analysis of the relationship between the area of target tissue necrosis and the size of target tissue in liver tumours with pulsed radiofrequency ablation. International Journal of Hyperthermia, 31(7), 715-725. doi:10.3109/02656736.2015.1058429Solazzo, S. A., Ahmed, M., Liu, Z., Hines-Peralta, A. U., & Goldberg, S. N. (2007). High-Power Generator for Radiofrequency Ablation: Larger Electrodes and Pulsing Algorithms in Bovine ex Vivo and Porcine in Vivo Settings. Radiology, 242(3), 743-750. doi:10.1148/radiol.2423052039Abraham, J. P., & Sparrow, E. M. (2007). A thermal-ablation bioheat model including liquid-to-vapor phase change, pressure- and necrosis-dependent perfusion, and moisture-dependent properties. 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(2010). 7 PROTEOMICS ANALYSIS OF MALE REPRODUCTIVE PHYSIOLOGY BY TOONA SINENSIS ROEM. Reproductive BioMedicine Online, 20, S3-S4. doi:10.1016/s1472-6483(10)62425-xBeop-Min Kim, Jacques, S. L., Rastegar, S., Thomsen, S., & Motamedi, M. (1996). Nonlinear finite-element analysis of the role of dynamic changes in blood perfusion and optical properties in laser coagulation of tissue. IEEE Journal of Selected Topics in Quantum Electronics, 2(4), 922-933. doi:10.1109/2944.577317Doss, J. D. (1982). Calculation of electric fields in conductive media. Medical Physics, 9(4), 566-573. doi:10.1118/1.595107Jo, B., & Aksan, A. (2010). Prediction of the extent of thermal damage in the cornea during conductive keratoplasty. Journal of Thermal Biology, 35(4), 167-174. doi:10.1016/j.jtherbio.2010.02.004Belous, A., & Podhajsky, R. J. (2009). The effect of initial and dynamic liver conditions on RF ablation size: a study in perfused and non-perfused animal models. Energy-based Treatment of Tissue and Assessment V. doi:10.1117/12.809597Song, K. D., Lee, M. W., Park, H. J., Cha, D. I., Kang, T. W., Lee, J., … Rhim, H. (2015). Hepatic radiofrequency ablation:in vivoandex vivocomparisons of 15-gauge (G) and 17-G internally cooled electrodes. The British Journal of Radiology, 88(1050), 20140497. doi:10.1259/bjr.20140497Cha, J., Choi, D., Lee, M. W., Rhim, H., Kim, Y., Lim, H. K., … Park, C. K. (2009). Radiofrequency Ablation Zones in Ex Vivo Bovine and In Vivo Porcine Livers: Comparison of the Use of Internally Cooled Electrodes and Internally Cooled Wet Electrodes. CardioVascular and Interventional Radiology, 32(6), 1235-1240. doi:10.1007/s00270-009-9600-0Lee, J. M., Han, J. K., Chang, J. M., Chung, S. Y., Kim, S. H., Lee, J. Y., … Choi, B. I. (2006). Radiofrequency Ablation of the Porcine Liver In Vivo: Increased Coagulation with an Internally Cooled Perfusion Electrode. Academic Radiology, 13(3), 343-352. doi:10.1016/j.acra.2005.10.020Romero-Méndez, R., Tobajas, P., Burdío, F., Gonzalez, A., Navarro, A., Grande, L., & Berjano, E. (2012). Electrical-thermal performance of a cooled RF applicator for hepatic ablation with additional distant infusion of hypertonic saline:In vivostudy and preliminary computer modeling. International Journal of Hyperthermia, 28(7), 653-662. doi:10.3109/02656736.2012.711894Ahmed, M., Lobo, S. M., Weinstein, J., Kruskal, J. B., Gazelle, G. S., Halpern, E. F., … Goldberg, S. N. (2002). Improved Coagulation with Saline Solution Pretreatment during Radiofrequency Tumor Ablation in a Canine Model. Journal of Vascular and Interventional Radiology, 13(7), 717-724. doi:10.1016/s1051-0443(07)61850-8Chinn, S. B., Lee, F. T., Kennedy, G. D., Chinn, C., Johnson, C. D., Winter, T. C., … Mahvi, D. M. (2001). Effect of Vascular Occlusion on Radiofrequency Ablation of the Liver. 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LITIO COMO TERAPIA NEUROPROTECTORA EN EL MODELO APPSL/PS1M146L DE LA ENFERMEDAD DE ALZHEIMER

El litio se utiliza desde hace varias décadas en el tratamiento de trastornos bipolares y la depresión, y recientemente se debate su uso potencial en patologías neurodegenerativas como la enfermedad de Alzheimer (AD). Diversos estudios han puesto de manifiesto su efecto positivo como potente inhibidor de GSK3beta disminuyendo la fosforilación de tau, la producción de Abeta e incrementando plasticidad sináptica. Sin embargo, su posible efecto neuroprotector previniendo la muerte neuronal in vivo no ha sido aun demostrado ya que la mayoría de los modelos transgénicos de AD no presentan pérdida neuronal. Nuestro modelo APPSL/PS1M146L sufre una pérdida significativa de neuronas SOM/NPY en hipocampo y corteza entorrinal desde edades tempranas (6 meses) con un marcado desarrollo de distrofias axonales. En este trabajo hemos estudiado el posible efecto neuroprotector del litio en este modelo animal mediante tratamiento crónico en la dieta desde los 3 hasta los 9 meses de edad. Se han utilizado técnicas imnunohistoquímicas, western blots y análisis por RT-PCR, y además se ha determinado la carga amiloide, el grado de compactación y el tamaño de las placas. El resultado más relevante de este estudio fue la preservación de la población de interneuronas SOM/NPY tanto en hipocampo como corteza entorrinal en los animales tratados, mientras que en los no tratados existió una pérdida significativa de esta supoblación neuronal. El efecto neuroprotector del litio se manifestó también en una marcada disminución de tau fosforilado, distrofias axonales y marcadores sinápticos, junto con una mejora cognitiva de los animales utilizando el test de reconocimiento de objetos. Este efecto preventivo del litio parece estar asociado con cambios en la formación de placas de Abeta que podrían afectar a su toxicidad, ya que los animales tratados presentaron placas más pequeñas y apariencia más compacta. Financiación: FIS PI12/01431 (AG) y FIS PI12/01439 (JV).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Single Dose Novel Salmonella Vaccine Enhances Resistance against Visceralizing L. major and L. donovani Infection in Susceptible BALB/c Mice

Visceral leishmaniasis is a major neglected tropical disease, with an estimated 500,000 new cases and more than 50,000 deaths attributable to this disease every year. Drug therapy is available but costly and resistance against several drug classes has evolved. Despite all efforts, no commercial, let alone affordable, vaccine is available to date. Thus, the development of cost effective, needle-independent vaccines is a high priority. Here, we have continued efforts to develop live vaccine carriers based on recombinant Salmonella. We used an in silico approach to select novel Leishmania parasite antigens from proteomic data sets, with selection criteria based on protein abundance, conservation across Leishmania species and low homology to host species. Five chosen antigens were differentially expressed on the surface or in the cytosol of Salmonella typhimurium SL3261. A two-step procedure was developed to select optimal Salmonella vaccine strains for each antigen, based on bacterial fitness and antigen expression levels. We show that vaccine strains of Salmonella expressing the novel Leishmania antigens LinJ08.1190 and LinJ23.0410 significantly reduced visceralisation of L. major and enhanced systemic resistance against L. donovani in susceptible BALB/c mice. The results show that Salmonella are valid vaccine carriers for inducing resistance against visceral leishmaniasis but that their use may not be suitable for all antigens

Water-Soluble Mo3S4 Clusters Bearing Hydroxypropyl Diphosphine Ligands: Synthesis, Crystal Structure, Aqueous Speciation, and Kinetics of Substitution Reactions

The [Mo3S4Cl3(dhprpe)3]+ (1+) cluster cation has been prepared by reaction between Mo3S4Cl4(PPh3)3 (solvent)2 and the watersoluble 1,2-bis(bis(hydroxypropyl)phosphino)ethane (dhprpe, L) ligand. The crystal structure of [1]2[Mo6Cl14] has been determined by X-ray diffraction methods and shows the typical incomplete cuboidal structure with a capping and three bridging sulfides. The octahedral coordination around each metal center is completed with a chlorine and two phosphorus atoms of the diphosphine ligand. Depending on the pH, the hydroxo group of the functionalized diphosphine can substitute the chloride ligands and coordinate to the cluster core to give new clusters with tridentate deprotonated dhprpe ligands of formula [Mo3S4(dhprpe-H)3]+ (2+). A detailed study based on stopped-flow, 31P{1H} NMR, and electrospray ionization mass spectrometry techniques has been carried out to understand the behavior of acid−base equilibria and the kinetics of interconversion between the 1+ and the 2+ forms. Both conversion of 1+ to 2+ and its reverse process occur in a single kinetic step, so that reactions proceed at the three metal centers with statistically controlled kinetics. The values of the rate constants under different conditions are used to discuss on the mechanisms of opening and closing of the chelate rings with coordination or dissociation of chloride

Synthesis and Structure of Trinuclear W3S4 Clusters Bearing Aminophosphine Ligands and Their Reactivity toward Halides and Pseudohalides

The aminophosphine ligand (2-aminoethyl)- diphenylphosphine (edpp) has been coordinated to the W3(μ- S)(μ-S)3 cluster unit to afford trimetallic complex [W3S4Br3(edpp)3]+ (1+) in a one-step synthesis process with high yields. Related [W3S4X3(edpp)3]+ clusters (X = F−, Cl−, NCS−; 2+−4+) have been isolated by treating 1+ with the corresponding halide or pseudohalide salt. The structure of complexes 1+ to 4+ contains an incomplete W3S4 cubane-type cluster unit, and only one of the possible isomers is formed: the one with the phosphorus atoms trans to the capping sulfur and the amino groups trans to the bridging sulphurs. The remaining coordination position on each metal is occupied by X. Detailed studies using stopped-flow, 31P{1H} NMR, and ESI-MS have been carried out in order to understand the solution behavior and the kinetics of interconversion among species 1+, 2+, 3+, and 4+ in solution. Density functional theory (DFT) calculations have been also carried out on the reactions of cluster 1+ with the different anions. The whole set of experimental and theoretical data indicate that the actual mechanism of substitutions in these clusters is strongly dependent on the nature of the leaving and entering anions. The interaction between an entering F− and the amino group coordinated to the adjacent metal have also been found to be especially relevant to the kinetics of these reactions

Abnormal accumulation of autophagic vesicles correlates with axonal and synaptic pathology in young Alzheimer’s mice hippocampus

Dystrophic neurites associated with amyloid plaques precede neuronal death and manifest early in Alzheimer’s disease (AD). In this work we have characterized the plaque-associated neuritic pathology in the hippocampus of young (4- to 6-month-old) PS1M146L/APP751SL mice model, as the initial degenerative process underlying functional disturbance prior to neuronal loss. Neuritic plaques accounted for almost all fibrillar deposits and an axonal origin of the dystrophies was demonstrated. The early induction of autophagy pathology was evidenced by increased protein levels of the autophagosome marker LC3 that was localized in the axonal dystrophies, and by electron microscopic identification of numerous autophagic vesicles filling and causing the axonal swellings. Early neuritic cytoskeletal defects determined by the presence of phosphorylated tau (AT8-positive) and actin–cofilin rods along with decreased levels of kinesin-1 and dynein motor proteins could be responsible for this extensive vesicle accumulation within dystrophic neurites. Although microsomal Aβ oligomers were identified, the presence of A11-immunopositive Aβ plaques also suggested a direct role of plaque-associated Aβ oligomers in defective axonal transport and disease progression. Most importantly, presynaptic terminals morphologically disrupted by abnormal autophagic vesicle buildup were identified ultrastructurally and further supported by synaptosome isolation. Finally, these early abnormalities in axonal and presynaptic structures might represent the morphological substrate of hippocampal dysfunction preceding synaptic and neuronal loss and could significantly contribute to AD pathology in the preclinical stages