



@article {2244,
	title = {Chitosan-based gene delivery vectors targeted to the peripheral nervous system},
	journal = {Journal of Biomedical Materials Research - Part AJ. Biomed. Mater. Res. Part A},
	volume = {95},
	year = {2010},
	note = {Cited By (since 1996): 5Export Date: 21 November 2012Source: ScopusCODEN: JBMRCdoi: 10.1002/jbm.a.32874PubMed ID: 20734332Language of Original Document: EnglishCorrespondence Address: P{\^e}go, A. P.; INEB, Instituto de Engenharia Biom{\'e}dica, Universidade Do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal; email: apego@ineb.up.ptChemicals/CAS: DNA, 9007-49-2; chitosan, 9012-76-4; imidazole, 1467-16-9, 288-32-4; macrogol, 25322-68-3; Cations; Chitosan, 9012-76-4; DNA, 9007-49-2; Polyethylene GlycolsTradenames: Chimarin, medicarb, SwedenManufacturers: medicarb, SwedenReferences: Rodriguez, F.J., Valero-Cabre, A., Navarro, X., Regeneration and functional recovery following peripheral nerve injury (2004) Drug Discov Today, 1, pp. 177-185;Apfel, S.C., Neurotrophic factors in peripheral neuropathies: Therapeutic implications (1999) Brain Pathol, 9, pp. 393-413; 
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},
	month = {2010///},
	pages = {801 - 810},
	abstract = {A non-toxic, targeted, simple and efficient system that can specifically transfect peripheral sensorial neurons can pave the way towards the development of new therapeutics for the treatment of peripheral neuropathies. In this study chitosan (CH), a biodegradable polymer, was used as the starting material in the design of a multicomponent vector targeted to the peripheral nervous system (PNS). Polycation-DNA complexes were optimized using imidazole-and thiol-grafted CH (CHimiSH), in order to increase transfection efficiency and allow the formation of ligand conjugated nanocomplexes, respectively. The 50 kDa non-toxic fragment from the tetanus toxin (HC), shown to interact specifically with peripheral neurons and undergo retrograde transport, was grafted to the binary complex via a bi-functional poly(ethylene glycol) (HC-PEG) reactive for the thiol moieties present in the complex surface. The targeting of the developed nanocomplexes was assessed by means of internalization and transfection studies in the ND7/23 (neuronal) vs. NIH 3T3 (fibroblast) cell lines. Targeted transfection was further confirmed in dorsal root ganglion dissociated primary cultures. A versatile, multi-component nanoparticle system that successfully targets and transfects neuronal cell lines, as well as dorsal root ganglia (DRG) primary neuron cultures was obtained for the 1.0 (w/w) HC-PEG/DNA formulation. {\textcopyright} 2010 Wiley Periodicals, Inc.},
	keywords = {animal cell, Animals, article, Binary complexes, Biodegradable polymers, Carbohydrate Conformation, Carbohydrate Sequence, Cations, Cell culture, Cell lines, cell population, Cells, Cultured, Chitin, chitosan, Complex surface, DNA, DNA complex, Dorsal root ganglia, Efficient systems, embryo, Ethylene, Ethylene Glycol, Female, Ganglia, Spinal, Gene delivery, gene delivery system, Gene delivery vectors, Gene therapy, Gene transfer, Gene Transfer Techniques, genetic transfection, Genetic Vectors, Grafting (chemical), Humans, imidazole, macrogol, materials testing, Mice, Molecular Sequence Data, Molecular Structure, mouse, Multicomponents, Nanocomplexes, Nanoparticle systems, nanoparticles, nerve cell culture, Neuron culture, Neuronal cell, NIH 3T3 Cells, nonhuman, Nontoxic fragment of tetanus toxin, Peripheral nervous system, Peripheral neuropathy, Polycations, Polyethylene Glycols, Polyethylene oxides, Pregnancy, Primary culture, rat, Rats, Rats, Wistar, spinal ganglion, Starting materials, Targeted, Tetanus toxin, Toxic materials, Transfection, Transfection efficiency},
	isbn = {15493296 (ISSN)},
	url = {http://www.scopus.com/inward/record.url?eid=2-s2.0-78249240032\&partnerID=40\&md5=af9c3ca5ca712d53abb5e80f45ed09c0},
	author = {Oliveira, H. and Pires, L.R. and Fernandez, R. and Martins, M.C.L. and Sim{\~o}es, S. and P{\^e}go, A.P.}
}
@article {2250,
	title = {Chitosan/siRNA nanoparticles biofunctionalize nerve implants and enable neurite outgrowth},
	journal = {Nano LettersNano Lett.},
	volume = {10},
	year = {2010},
	note = {Cited By (since 1996): 14Export Date: 21 November 2012Source: Scopusdoi: 10.1021/nl1016909Language of Original Document: EnglishCorrespondence Address: Schlosshauer, B.; NMI Natural and Medical Sciences Institute, University of T{\"u}bingen, Markwiesenstrasse 55, 72770 Reutlingen, Germany; email: schlosshauer@nmi.deChemicals/CAS: chitosan, 9012-76-4; Chitosan, 9012-76-4; RNA, Small Interfering; rhoA GTP-Binding Protein, 3.6.5.2References: Terzis, J.K., Skoulis, T.G., Soucacos, P.N., (1995) Int. Angiol., 14, pp. 264-77;Schmidt, C.E., Leach, J.B., (2003) Annu. Rev. Biomed. Eng., 5, pp. 293-347; 
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},
	month = {2010///},
	pages = {3933 - 3939},
	abstract = {Microstructured 20 μm thick polymer filaments used as nerve implants were loaded with chitosan/siRNA nanoparticles to promote nerve regeneration and ensure local delivery of nanotherapeutics. The stable nanoparticles were rapidly internalized by cells and did not affect cell viability. Target mRNA was successfully reduced by 65-75\% and neurite outgrowth was enhanced even in an inhibitory environment. This work, thus, supports the application of nanobiofunctionalized implants as a novel approach for spinal cord and nerve repair. {\textcopyright} 2010 American Chemical Society.},
	keywords = {animal, Animals, article, Cell Line, cell viability, chemistry, Chitin, chitosan, cytology, Drug delivery, genetic transfection, genetics, Local delivery, metabolism, Microstructured, nanobiofunctionalized implants, nanoparticle, nanoparticles, nerve cell, nerve regeneration, Nerve repair, neurite, Neurite outgrowth, Neurites, Neurons, Polymer filaments, Prostheses and Implants, prostheses and orthoses, rhoA GTP-Binding Protein, RhoA guanine nucleotide binding protein, RNA, RNA, Small Interfering, siRNA, small interfering RNA, Spinal cords, Transfection, ultrastructure},
	isbn = {15306984 (ISSN)},
	url = {http://www.scopus.com/inward/record.url?eid=2-s2.0-77958033530\&partnerID=40\&md5=6862e82e01c3aedf5423ebb14248c6ec},
	author = {Mittnacht, U. and Hartmann, H. and Hein, S. and Oliveira, H. and Dong, M. and P{\^e}go, A.P. and Kjems, J. and Howard, K.A. and Schlosshauer, B.}
}
@article {2280,
	title = {Evaluation of the effect of the degree of acetylation on the inflammatory response to 3D porous chitosan scaffolds},
	journal = {Journal of Biomedical Materials Research - Part AJ. Biomed. Mater. Res. Part A},
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	year = {2010},
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},
	month = {2010///},
	pages = {20 - 28},
	abstract = {The effect of the degree of acetylation (DA) of 3D chitosan (Ch) scaffolds on the inflammatory reaction was investigated. Chitosan porous scaffolds with DAs of 4 and 15\% were implanted using a subcutaneous air-pouch model of inflammation. The initial acute inflammatory response was evaluated 24 and 48 h after implantation. To characterize the initial response, the recruitment and adhesion of inflammatory cells to the implant site was studied. The fibrous capsule formation and the infiltration of inflammatory cells within the scaffolds were evaluated for longer implantation times (2 and 4 weeks). Chitosan with DA 15\% attracted the highest number of leukocytes to the implant site. High numbers of adherent inflammatory cells were also observed in this material. For longer implantation periods Ch scaffolds with a DA of 15\% induced the formation of a thick fibrous capsule and a high infiltration of inflammatory cells within the scaffold. Our results indicate that the biological response to implanted Ch scaffolds was influenced by the DA. Chitosan with a DA of 15\% induce a more intense inflammatory response when compared with DA 4\% Ch. Because inflammation and healing are interrelated, this result may provide clues for the relative importance of acetyl and amine functional groups in tissue repair and regeneration. {\textcopyright} 2009 Wiley Periodicals, Inc.},
	keywords = {acetic acid derivative, Acetylation, Acute inflammatory response, amine, Amine functional groups, animal cell, animal experiment, animal tissue, Animals, article, Biological materials, Biological response, Biomaterials, cell adhesion, cell infiltration, Chitin, chitosan, Chitosan scaffold, Degree of acetylation, Fibrous capsule, Fibrous capsule formation, freeze drying, Functional groups, Hydrogels, implant, implantation, Implantation periods, Implants, Experimental, Inflammation, Inflammatory cells, Inflammatory reaction, Inflammatory response, leukocyte count, Leukocytes, male, Mice, Mice, Inbred BALB C, Microscopy, Electron, Scanning, mouse, nonhuman, Pathology, porosity, Porous scaffold, Prosthesis Implantation, Relative importance, Scaffolds, Subcutaneous Tissue, Surface Properties, Three dimensional, Tissue regeneration, tissue repair, Tissue response, Tissue responses, Tissue Scaffolds},
	isbn = {15493296 (ISSN)},
	url = {http://www.scopus.com/inward/record.url?eid=2-s2.0-77649219383\&partnerID=40\&md5=58781af29b82f3108ded649c4776e428},
	author = {Barbosa, J.N. and Amaral, I.F. and {\'A}guas, A.P. and Barbosa, M.A.}
}
@article {2316,
	title = {Engineering endochondral bone: In vitro studies},
	journal = {Tissue Engineering - Part ATissue Eng. Part A},
	volume = {15},
	year = {2009},
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Oliveira, S.M., Turner, G., Mijares, D., Amaral, I.F., Barbosa, M.A., Teixeira, C.C., (2007) Engineering endochondral bone: In vivo studies
},
	month = {2009///},
	pages = {625 - 634},
	abstract = {Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification. {\textcopyright} Copyright 2009, Mary Ann Liebert, Inc. 2009.},
	keywords = {alkaline phosphatase, Alkaline phosphatase activity, animal, animal cell, animal tissue, Animals, article, biomechanics, Body fluids, Bone, Bone and Bones, Bone formation, Cartilage, cartilage cell, Cell culture, cell damage, cell hypertrophy, cell isolation, cell maturation, Cell membranes, cell proliferation, Cells, Cultured, chick embryo, Chicken embryo, Chitin, chitosan, Chitosan scaffold, Chondrocyte, Chondrocyte maturation, Chondrocytes, Clinical application, collagen type 10, Collagen Type X, controlled study, cytology, DNA, drug effect, embryo, enchondral ossification, Endochondral ossification, enzyme activity, enzymology, Extracellular matrices, extracellular matrix, gene expression profiling, Hypertrophic chondrocytes, in vitro study, In-vitro, Ligaments, Matrix synthesis, Mechanical analysis, Mechanical properties, metabolism, methodology, nonhuman, Pathology, physiology, Porifera, porosity, priority journal, retinoic acid, Retinoic acids, Scaffolds, sponge (Porifera), Sterna, sternum, synthesis, Time points, Tissue engineering, tissue scaffold, Tissue Scaffolds, Tretinoin, Type X collagen},
	isbn = {19373341 (ISSN)},
	url = {http://www.scopus.com/inward/record.url?eid=2-s2.0-64549143844\&partnerID=40\&md5=310f8e62d4dd51ccb9399db171440148},
	author = {Oliveira, S.M. and Amaral, I.F. and Barbosa, M.A. and Teixeira, C.C.}
}
@article {2297,
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	year = {2009},
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},
	month = {2009///},
	pages = {5465 - 5475},
	abstract = {Chitosan (Ch) porous matrices were investigated regarding their ability to be colonized by human microvascular endothelial cells (HPMEC-ST1.6R cell line) and macrovascular endothelial cells namely HUVECs. Specifically we assessed if previous incubation of Ch in a fibronectin (FN) solution was effective in promoting endothelial cell (EC) adhesion to Ch matrices with different degrees of acetylation (DAs). Upon FN physiadsorption, marked differences were found between the two DAs investigated, namely DA 4\% and 15\%. While cell adhesion was impaired on Ch with DA 15\%, ECs were able to not only adhere to Ch with DA 4\%, but also to spread and colonize the scaffolds, with retention of the EC phenotype and angiogenic potential. To explain the observed differences between the two DAs, protein adsorption studies using                        125I-FN and immunofluorescent labelling of FN cell-binding domains were carried out. In agreement with the higher cell numbers found, scaffolds with DA 4\% revealed a higher number of exposed FN cell-binding domains as well as greater ability to adsorb FN and to retain and exchange adsorbed FN in the presence of competitive proteins. These findings suggest that the DA is a key parameter modulating EC adhesion to FN-coated Ch by influencing the adsorbed protein layer. {\textcopyright} 2009 Elsevier Ltd. All rights reserved.
},
	keywords = {Acetylation, Adhesion, Adsorption, angiogenesis, article, Biocompatible Materials, Blood vessel prostheses, cell adhesion, Cell culture, cell invasion, Cell Line, Cell lines, Cell membranes, Cell number, cell proliferation, Cell size, cell spreading, cell survival, Cell-binding domain, Chitin, Chitin/chitosan, chitosan, colony formation, controlled study, Degrees of acetylation, Electric conductivity measurement, Electric currents, Endothelial cell, Endothelial Cells, Endothelialisation, endothelium, endothelium cell, epithelization, Fibronectin, Fibronectins, human, human cell, Human microvascular endothelial cells, Humans, immunofluorescence test, Key parameters, Labels, materials testing, Matrix algebra, Neovascularization, Physiologic, phenotype, porosity, Porous matrixs, priority journal, Protein adsorption, Protein layers, Scaffold, Scaffolds, Self assembly, Tissue engineering, tissue scaffold, umbilical vein},
	isbn = {01429612 (ISSN)},
	url = {http://www.scopus.com/inward/record.url?eid=2-s2.0-68549107574\&partnerID=40\&md5=9babb57c1a4f2ae6eb7e7e0643bffe48},
	author = {Amaral, I.F. and Unger, R.E. and Fuchs, S. and Mendon{\c c}a, A.M. and Sousa, S.R. and Barbosa, M.A. and P{\^e}go, A.P. and Kirkpatrick, C.J.}
}
@article {2381,
	title = {Attachment, spreading and short-term proliferation of human osteoblastic cells cultured on chitosan films with different degrees of acetylation},
	journal = {Journal of Biomaterials Science, Polymer EditionJ. Biomater. Sci. Polym. Ed.},
	volume = {18},
	year = {2007},
	note = {Cited By (since 1996): 22Export Date: 21 November 2012Source: ScopusCODEN: JBSEEdoi: 10.1163/156856207780425068Language of Original Document: EnglishCorrespondence Address: Amaral, I.F.; INEB - Instituto de Engenharia Biom{\'e}dica, Laborat{\'o}rio de Biomateriais, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal; email: iamaral@ineb.up.ptChemicals/CAS: chitosan, 9012-76-4; Biocompatible Materials; Chitosan, 9012-76-4References: Vacanti, C.A., Bonassar, L.J., Vacanti, J.P., (2000) Principles of Tissue Engineering, p. 671. , R. P. Lanza, R. Langer and J. Vacanti Eds, p, Academic Press, San Diego, CA;Bruder, S.P., Caplan, A.I., (2000) Principles of Tissue Engineering, p. 683. , R. P. Lanza, R. Langer and J. Vacanti Eds, p, Academic Press, San Diego, CA; 
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},
	month = {2007///},
	pages = {469 - 485},
	abstract = {Chitosan (Ch) is being actively investigated as a non-protein template for the growth of an increasing number of anchorage-dependent cells, including chondrocytes and bone cells. In the present work, Ch films with degrees of N-acetylation (DAs) in the range of 4 to 49\% were evaluated with respect to the attachment, spreading and short-term proliferation of osteoblasts, using human osteoblastic MG-63 cells. The films were characterized in terms of surface morphology and surface charge by atomic force microscopy and streaming potential measurements, respectively. Cell attachment was assessed after 3 and 24 h of cell culture. After 24 h of incubation, cell attachment was found to be dependent on the DA, lower DAs favouring cell adhesion. With time, cell spreading and cytoskeleton organization were only attained for DAs <= 13\%. Regarding cell proliferation, cells grown on films with the lowest DA (4\%) revealed a higher specific growth rate as compared to those grown on films with higher DAs. Films with a DA of 49\% failed to sustain cell proliferation. In addition, a longer lag-phase was observed on Ch, as compared to TCPS, in accordance to an observed delay of cell spreading. The present findings revealed that differences in the DA as small as 9\% may be critical in terms of the osteoblast response to two-dimensional Ch-based matrices. {\textcopyright} 2007 VSP.},
	keywords = {Acetylation, AFM, article, Atomic force microscopy, Biocompatible Materials, biofilm, Biofilms, biomaterial, bone cell, cartilage cell, cell adhesion, cell anchorage, Cell culture, cell growth, Cell Line, Tumor, cell proliferation, Cell Shape, cell spreading, cell survival, Chemical structure, chemistry, Chitin, chitosan, comparative study, controlled study, cytoskeleton, Degree of acetylation, drug effect, Growth kinetics, human, human cell, Humans, incubation time, metabolism, Microscopy, Atomic Force, osteoblast, Osteoblast-like cells, Osteoblasts, Pathology, priority journal, Surface charge, Surface Properties, surface property, Surface treatment, tumor cell line, Zeta potential},
	isbn = {09205063 (ISSN)},
	url = {http://www.scopus.com/inward/record.url?eid=2-s2.0-34347375992\&partnerID=40\&md5=437b9ed270e859425b76aa3655c611be},
	author = {Amaral, I.F. and Cordeiro, A.L. and Sampaio, P. and Barbosa, M.A.}
}
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	year = {2006},
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},
	month = {2006///},
	pages = {276 - 284},
	abstract = {Grafting negatively charged groups such as phosphates is a well-known strategy for inducing the deposition of apatite-like layers under simulated physiological conditions. In this investigation, chitosan was phosphorylated in an attempt to enhance its osteoconduction. Chitosan membranes were phosphorylated at 30{\textdegree}C with the H                        3PO
                        4/ Et 
                        3PO
                        4/P
                        2O
                        5/butanol reaction system for periods up to 48 h. This method is an alternative to the phosphoric acid/ urea/dimethylformamide phosphorylation method, which involves the use of much higher temperatures. In this study, the effects of the phosphorylation reaction time on the surface morphology and surface free energy of phosphorylated membranes were investigated with atomic force microscopy and static-contact-angle measurements, respectively. In addition, the modified membranes were evaluated with respect to their cytotoxiciry toward bone cells through the incubation of human osteoblastic cells with extracts of the materials for two different periods: 24 and 120 h. The results revealed a reduction of the average surface roughness at the nanometer scale with increasing phosphorylation reaction time. Wettability studies showed an increase in the polar component of the surface free energy with increasing reaction time as a result of the increase in the phosphate surface concentration. Cytotoxicity studies revealed no cytotoxic effect of phosphorylated membranes on osteoblastic cells, regardless of the incubation period. {\textcopyright} 2006 Wiley Periodicals, Inc.
},
	keywords = {Atomic force microscopy, Atomic force microscopy (AFM), Biocompatibility, Biological applications, Biological applications of polymers, Biomaterials, Chitin, chitosan, Chitosan membranes, grafting, Grafting (chemical), Osteoblastic cells, Phosphorylation, Polymeric membranes, polysaccharide, Polysaccharides, surface property, toxicity, Urea},
	isbn = {00218995 (ISSN)},
	url = {http://www.scopus.com/inward/record.url?eid=2-s2.0-33749478403\&partnerID=40\&md5=c365a50e3e767b1da90ad4ccd331440b},
	author = {Amaral, I.F. and Granja, P.L. and Melo, L.V. and Saramago, B. and Barbosa, M.A.}
}
@article {2438,
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	journal = {Journal of Biomaterials Science, Polymer EditionJ. Biomater. Sci. Polym. Ed.},
	volume = {16},
	year = {2005},
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},
	month = {2005///},
	pages = {1575 - 1593},
	abstract = {In the present work, the surface of chitosan membranes was modified using a phosphorylation method carried out at room temperature. Phosphorylation may be of particular interest in materials for orthopaedic applications, due to the cation-exchange properties of phosphate functionalities. Phosphate groups chelate calcium ions, thus inducing the deposition of an apatite-like layer known to improve the osteoconduction of polymer-based implants. Additionally, the negatively charged phosphate functionalities, together with the positively charged amine groups from chitosan, are expected to provide chitosan with an amphoteric character, which may be useful as a combinatorial therapeutic strategy, by simultaneously allowing the immobilization of signalling molecules like growth factors. Phosphorylation was carried out at room temperature using the H                        3 PO
                        4/Et
                        3 PO
                        4/P
                        2O
                        5/butanol method. Surface characterization was performed by XPS, ATR-FT-IR, and SEM. Cross-sections were analyzed by SEM fitted with EDS. The phosphate content increased with the reaction time, as shown by XPS and ATR-FT-IR, a P/N atomic ratio of 0.73 being obtained after 48 h of treatment. High-resolution XPS spectra regarding C
                        1s, O
                        1s, N
                        1s and P
                        2p are discussed. The introduction of a neutralization step led to a reduction of P content, which pointed out to the presence of phosphates ionically bound to protonated amines, in addition to phosphate esters. EDS analysis of cross-sections revealed a gradual P reduction up to 50\% towards the inner part of the membrane. {\textcopyright} VSP 2005.
},
	keywords = {Acetylation, amine, animal, Animals, Apatite, article, bone conduction, butanol, calcium ion, cation exchange, cephalopod, chelate, Chemical modification, chemistry, Chitin, chitosan, comparative study, controlled study, Decapodiformes, Fourier transform infrared spectroscopy, Fourier transform infrared spectroscopy (FT-IR), Growth factor, immobilization, Implants (surgical), infrared spectroscopy, isolation and purification, Microscopy, Electron, Scanning, molecular weight, organophosphate, phosphate, Phosphate groups, Phosphates, Phosphorylation, Phosphorylation method, Plastics applications, priority journal, reflectometry, room temperature, Scanning electron microscopy, SEM-EDS, signal transduction, spectrometry, Spectrometry, X-Ray Emission, Spectroscopy, Fourier Transform Infrared, Surface chemical reaction, Surface Properties, surface property, temperature, time, Time Factors, viscosity, X ray photoelectron spectroscopy, X-rayphotoelectron spectroscopy (XPS)},
	isbn = {09205063 (ISSN)},
	url = {http://www.scopus.com/inward/record.url?eid=2-s2.0-33644875542\&partnerID=40\&md5=cb42a0fba6d2000d93b90af9724a5bc1},
	author = {Amaral, I.F. and Granja, P.L. and Barbosa, M.A.}
}
