Fibrinogen concentrates are used widely as a sealant during and after surgery to reduce blood loss. Commercially available fibrin sealants are made from pooled human blood, which carries the risk of blood-borne diseases, and are expensive. These concerns have brought to focus the need for autologous fibrinogen concentrates. This need has been addressed by utilizing a unique approach in which fibrinogen is precipitated from plasma with protamine. The physical properties of fibrin sealant prepared from fibrinogen precipitated with protamine were evaluated. The optimal precipitation conditions included a plasma protamine concentration of 10 mg/mL at room temperature. Under these conditions 96% ± 4% of the fibrinogen present in the plasma was precipitated and 98% ± 0.9% of the precipitated fibrinogen was clottable. In addition, it was shown that almost 50% of the factor XIII in the plasma was also precipitated along with the fibrinogen. The tensile and adhesion strengths and kinetics of fibrin sealant prepared from protamine-fibrinogen concentrate were evaluated. Tensile strength and adhesion strength both increased with increasing fibrinogen concentration. Addition of calcium chloride significantly increased the tensile and adhesion strengths. The addition of aprotinin and ε-aminocaproic acid (used to inhibit natural fibrinolysis) to the fibrinogen concentrate was shown to have no effect on the mechanical properties of the sealant. Kinetic experiments showed that the clotting time decreased as the thrombin and fibrinogen concentrations were increased. A rat model with controlled renal incisions was employed to evaluate the hemostatic efficacy of the fibrin sealant made from the protamine-fibrinogen concentrate. The fibrin sealant significantly reduced the blood loss and bleeding time when compared with controls (no sealant, plasma, and a commercial product). The sealant also significantly reduced blood loss and bleeding time in rats that were anticoagulated with heparin. A mathematical model based on tensile strength and adhesion strength was developed to predict the bleeding time in the animal wound. Model predictions showed that the ability of the fibrin sealant to reduce bleeding time, and therefore blood loss, was limited by the adhesion strength.

A new way of adhering woven textile to a nonwoven backing was explored, the end product in mind being a sound absorbent panel. Today a hot melt adhesive is sprayed on the nonwoven with woven decorating textile put on top before compression molding. This method results in an uneven layer of adhesive with the consequence of decorating textile detaching from the nonwoven and thereby a high number of discarded products. The report investigates the possibility of using a bicomponent fiber as the adhesive. This would guarantee an even layer of adhesive and make the manufacturing process one step shorter. A bicomponent fiber of core/sheath construction with a low melting polymer in the sheath was incorporated in the nonwoven upon fabrication. In the main bulk of the nonwoven a smaller ratio of bicomponent to normal polyester fiber was used, enough to stabilize the nonwoven. For the top layer of the nonwoven a higher ratio was chosen. As the nonwoven is then compression molded under heat together with the decorating textile, the low melting sheath of the bicomponent fiber will melt and create bonds within the nonwoven as well as to the textile. The ratio in the top layer was varied as well as the pressure in compression molding. The adhesion strength between nonwoven and decorating textile was tested, and the sound absorbing properties of the different manufactured samples were compared. The sound absorbing parameter of air flow resistance was tested and sound absorption was tested using impedance tube with transfer function method. Further, a way of testing transmission loss was developed by the manufacturing of a custom built impedance tube, which was then compared to the transfer function method. The results showed that a higher bicomponent percentage gave higher sound absorption in lower frequencies, but a lower sound absorption in higher frequencies. The thickness of the samples gave a positive effect on the sound absorption in all frequencies. The conclusion is to recommend a thicker material, and choose bicomponent according to which frequencies that should be absorbed and what adhesion strength is needed for end product. / Program: Textilteknik

Ice accretion on various surfaces can cause destructive effect of our lives, from cars, aircrafts, to infrastructure, power line, cooling and transportation systems. There are plenty of methods to overcome the icing problems including electrical, thermal and mechanical process to remove already accumulated ice on the surfaces and to reduce the risk of further operation. But all these process required substantial amount of energy and high cost of operation. To save the global energy and to improvement the safety issue in many infrastructure and transportation systems we have to introduce some passive anti-icing coating known as ice-phobic coating to reduce the ice-formation and ice adhesion onto the surface. Ice-phobic coatings mostly devoted to utilizing lotus-leaf-inspired superhydrophobic coatings. These surfaces show promising behavior due to the low contact area between the impacting water droplets and the surface. In this present study we investigate systematically the influence of chemical composition and functionality as well as structure of surfaces on wetting properties and later on icing behavior of surfaces. Robust anti-icing coating has been prepared by using modified silica particles as a particles film. Polymer brushes were synthesized on flat, particle surfaces by using Surface initiated ATRP. We have also investigated the effect of anti-icing behavior on the surfaces by varying surface chemistry and textures by using different sizes of particles. This approach is based on the reducing ice accumulation on the surfaces by reducing contact angle hysteresis. This is achieved by introducing nano to micro structured rough surfaces with varying surface chemistry on different substrates. Freezing and melting dynamics of water has been investigated on different surfaces by water vapour condensation in a high humidity (80%) condition ranging from super hydrophilic to super hydrophobic surfaces below the freezing point of water. Kinetics of frost formation and ice adhesion strength measurements were also performed for all samples. All these experiments were carried out in a custom humidity and temperature controlled chamber. We prepared a superhydrophobic surface by using Poly dimethyl siloxane (PDMS) modified fumed silica which display very low ice-adhesion strength almost 10 times lower than the unmodified surface. Also it has self-cleaning behavior after melting of ice since whole ice layer was folded out from the surface to remove the ice during melting. Systematic investigation of the effect of three parameters as surface energy, surface textures (structure, geometry and roughness) and mechanical properties of polymers (soft and stiff) on icing behavior has also been reported.

Submitted by JOSIANE SANTOS DE OLIVEIRA (josianeso) on 2018-07-09T16:18:43Z No. of bitstreams: 1 Paôla Valentini_.pdf: 16360357 bytes, checksum: 11cfed092ec8572550d478091c843fbc (MD5) / Made available in DSpace on 2018-07-09T16:18:43Z (GMT). No. of bitstreams: 1 Paôla Valentini_.pdf: 16360357 bytes, checksum: 11cfed092ec8572550d478091c843fbc (MD5) Previous issue date: 2018-03-23 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Diversos fatores influenciam a durabilidade de argamassas de revestimento, sendo alguns deles: a absorção de água, porosidade e rugosidade da superfície do substrato; a execução, com o preparo ou não do substrato, a forma de aplicação e as condições de cura da argamassa; as condições climáticas, decorrentes da ação do vento, umidade e temperatura; e a própria argamassa com seus componentes. Partindo deste contexto, esta pesquisa tem como objetivo avaliar fatores relacionados ao substrato e à argamassa que influenciam na resistência de aderência à tração de sistemas de revestimento. O programa experimental buscou avaliar a influência da absorção de água total, da absorção por capilaridade e da rugosidade de três substratos cerâmicos e também da adição de fíler na argamassa, na resistência de aderência à tração de sistemas de revestimento. Os substratos cerâmicos foram queimados nas temperaturas de 800°C, 900°C e 1000°C, sendo que, das 144 peças cerâmicas do estudo, metade receberam aplicação de hidrofugante. Sobre as peças cerâmicas foram aplicadas argamassas confeccionadas com areia natural de rio e cimento Portland CP II-Z-32, no traço 1:3, em massa, com e sem adição de 5% de pó de quartzo. A partir dos resultados encontrados observou-se que a maior resistência de aderência à tração foi obtida nos blocos cerâmicos com temperatura de queima de 800°C, que apresentam simultaneamente maior rugosidade e maior absorção capilar de água. Verificou-se também que a adição de fíler à argamassa aumentou a resistência de aderência quando aplicada sobre os blocos de maior rugosidade, não sendo observado o mesmo comportamento nos blocos de baixa rugosidade. De um modo geral, o uso de hidrofugante sobre as peças cerâmicas diminuiu os resultados de resistência de aderência além de alterar a forma de ruptura dos revestimentos. / The durability of coating mortars applied on substrates are influenced by the water absorption, porosity and roughness of the substrate surface; the preparation of the coating´s substrate, the method used to apply and the cure conditions of the mortar; beyond the climatic conditions, through the wind, humidity and temperature; and the mortar itself with its components. Based on this context, this research aims to evaluate factors related to the coating´s substrate and the rendering mortar that influence the adhesion strength of mortar coating systems. The experimental program aimed to evaluate the influence of total water absorption, capillary absorption and roughness of three ceramic substrates, as well as the use of filler in the mortar, in the adhesion strength of mortar coating systems. The ceramic substrates were burned at 800°C, 900°C and 1000°C, and of the 144 ceramic pieces of the study, 72 were painted with water repellent. The coating mortars were made with natural river sand and Portland cement CP II-Z-32, in the proportion 1: 3 (by mass), with and without adding 5% of quartz filler. It was observed that the highest tensile strength was obtained with the substrates of ceramic blocks burned at 800°C, which simultaneously present the higher roughness and higher water absorption. The use of filler in the mortar composition increased the adhesion strength when applied on the blocks of greater roughness. In general, the use of water repellent on the ceramic substracts decrease the adhesion strength of mortar coating systems and modify the kind of rupture of the coatings.

Στην ιστοτεχνολογία (tissue engineering – TE), η ικανότητα των ικριωμάτων (scaffolds) να διατηρούν συμβατή μηχανική και βιολογική συμπεριφορά με τους περιβάλλοντες ιστούς και όργανα, αλλά και να διευκολύνουν την προσκόλληση κυττάρων του ξενιστή στην επιφάνεια και την τρισδιάστατη δομή τους κρίνεται ως υψίστης σημασίας για την επιθυμητή εκδήλωση αναγεννητικής αντίδρασης των κυττάρων in vivo. Ο σκοπός της παρούσας διδακτορικής διατριβής είναι η δημιουργία και ο χαρακτηρισμός ακυτταροποιημένης εξωκυττάριας μήτρας από μαλακούς ιστούς ζωικής προέλευσης, με στόχο τη δημιουργία ικριώματος με επιθυμητές μηχανικές και βιολογικές ιδιότητες για εφαρμογές στην ιστοτεχνολογία. Στην εργασία αυτή επιλέχθηκε ως υλικό της μελέτης ο βόειος περικαρδιακός ιστός, λόγω της ευρείας πολύχρονης χρήσης του ως βιοϋλικό σε μοσχεύματα. Εφαρμόστηκαν δύο διαφορετικά πρωτόκολλα για την ακυτταροποίηση του ιστού, χρησιμοποιώντας στο πρώτο Triton Χ-100, SDS και deoxycholic acid (12 ώρες, 4°C - Triton) και στο δεύτερο Trypsin/EDTA με RNAse/DNAse (48 ώρες, 37°C – Trypsin). Η ιστολογική εξέταση επιβεβαίωσε την ολική αφαίρεση των κυττάρων. Τα αποτελέσματα των εμβιομηχανικών δοκιμών δεν έδειξαν στατιστικά σημαντική διαφορά μεταξύ των μηχανικών ιδιοτήτων των μη κατεργασμένων ιστών και των ακυτταροποιημένων περικαρδιακών ιστών με Triton στην οριζόντια και κάθετη διεύθυνση προς τον οβελιαίο άξονα της καρδιάς σε αντίθεση με τη μείωση του χαμηλού μέτρου ελαστικότητας (φάση ελαστίνης) και του υψηλού μέτρου ελαστικότητας (φάση κολλαγόνου) μετά την κατεργασία των υλικών με Trypsin, και στις δύο κατευθύνσεις. Η βιοχημική ανάλυση επαλήθευσε την άμεση σχέση μεταξύ των μηχανικών βισκοελαστικών ιδιοτήτων των μαλακών ιστών με τα συστατικά (GAGs και κολλαγόνο) του ιστού και την εσωτερική διαμόρφωση τους. Τα αποτελέσματα των μη επεξεργασμένων και των επεξεργασμένων ιστών με τα παραπάνω διαλύματα, έδειξαν ότι το διάλυμα του Triton προκαλεί ήπια κατεργασία, με ελαχιστοποίηση των δομικών μεταβολών της εξωκυττάριας μήτρας, όπως η σταθερή σύσταση των γλυκοζαμινογλυκανών (GAGs) και η περιεκτικότητα των ιστών σε κολλαγόνο. Αυτό δεν επιτεύχθηκε με την χρήση διαλύματος Trypsin, όπου παρατηρήθηκε σημαντική μείωση των GAGs, τόσο στη συγκέντρωση της θειϊκής χονδροϊτίνης / δερματάνης όσο και στο υαλουρονικό. Για την αξιολόγηση της κυτταροσυμβατότητας αορτικά βόεια ενδοθηλιακά κύτταρα καλλιεργήθηκαν στα ακυτταροποιημένα υλικά. Η επιθηλιοποίησή τους επετεύχθη από 24 ώρες μέχρι και 4 μέρες. Προσδιορίστηκε επίσης η δύναμη προσκόλλησης των κυττάρων μετά από 4 μέρες καλλιέργειας στους ακυτταροποιημένους περικαρδιακούς ιστούς με την εφαρμογή διατμητικών τάσεων μέσω ροϊκού πεδίου, με χρήση κατάλληλης μηχανής περιστροφής των δειγμάτων σε ακινητοποιημένο υγρό. Τα αποτελέσματα έδειξαν εξάπλωση και πολλαπλασιασμό των κυττάρων στην επιφάνεια των βιοϋλικών, ενώ παρατηρήθηκε καλή συγκέντρωση κυττάρων (> 60%) στην κλίμακα των φυσιολογικών διατμητικών τάσεων. Συμπερασματικά, η ακυτταροποίηση του βόειου περικαρδιακού ιστού για μεγάλη χρονική διάρκεια στους 37°C (Trypsin) φάνηκε να μεταβάλλει την εμβιομηχανική συμπεριφορά και τη δομική ακεραιότητα του ιστού, η οποία, αντίθετα, διατηρείται σε φυσιολογική κατάσταση μετά από κατεργασία σε χαμηλή θερμοκρασία και σε σύντομο χρόνο (Triton). Επιπλέον, και τα δύο πρωτόκολλα είχαν ως αποτέλεσμα τη δημιουργία βιοϋλικού συμβατού με τα ενδοθηλιακά κύτταρα κατά την επαφή τους με την επιφάνειά τους. / In TE scaffolding, the ability of scaffolds to preserve proper mechanical and biological function and compatibility with the surrounding tissues and organs, as well to enhance host cells to adhere with scaffold material in their surface and 3D structure is of paramount importance for potential regenerative cell response in vivo. The aim of the present thesis is to produce and characterise a decellularized extracellular matrix derived from animal soft tissues, scoping to a scaffold capable of exhibiting the mechanical and biological properties desired for tissue engineering (TE) applications. For this work bovine pericardial tissue was selected, due to its broad use as a biomaterial in different implant technologies for decades. Two different protocols for the decellularization of bovine pericardial tissue were developed incorporating Triton® X–100, SDS and deoxycholic acid (12 h, 4°C) in solution 1 (Triton) and trypsin/EDTA with RNAse/DNase at 37°C for 48 h in solution 2 (Trypsin). Histological analysis confirmed total absence of cells after both treatments. The results of the biomechanical tests showed no mechanical differences demonstrated between the fresh and decellularized pericardial tissues by Triton solution in both, apex to base and transverse anatomical directions, contrary to a significant decrease of the low elastic modulus (elastin phase) and high elastic modulus (collagen phase) demonstrated after trypsin solution treatment in both directions. Biochemical analysis verified the direct relationship between mechanical viscoelastic properties of soft tissues with the constituent tissue components (GAGs and collagen content) and their internal arrangement. The comparison of the results from untreated and that from treated tissues suggested that the Triton decellularization method seemed to be a very mild treatment, as the glycozaminoglycans (GAGs) composition remained constant, as well as the collagen content. This was not achieved with the decellularization using Trypsin, where a significant reduction of GAGs, both chondroitin/dermatan sulphate and hyaluronan, was found. Aortic bovine endothelial cell seeding was used for estimation of its biomaterials’ cytocompatibility. Endothelialization achieved after 24hrs to 4 days periods. The adhesion strength of cells, cultured for 4 days on decellularized bovine pericardial tissues, was also determined. By applying a radially increasing shear stress field on rotating material samples within a stationary fluid mesh using a spinning disc device, we determined the shear stress necessary to detach the cells from the sfafolds’ surface. Fine cell spreading and proliferation on biomaterials’ surface and good surface cell density (>60%) at physiological shear stress scale were observed and measured. In conclusion, decellularization of bovine pericardial tissues under long time duration in 37°C (Trypsin) seems to alter its biomechanical behaviour and structural integrity, which, in contrast, was retained under low temperature short duration treatment (Triton). Additionally, both protocols resulted in a cytocompatible biomaterial, regarding its surface interactions with endothelial cells.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-12-12T14:44:32Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Pedro Henrique Amorim Anjos.pdf: 9970150 bytes, checksum: 29bca81f576fbdecd909c0ab24d68ce5 (MD5) / Made available in DSpace on 2016-12-12T14:44:32Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Pedro Henrique Amorim Anjos.pdf: 9970150 bytes, checksum: 29bca81f576fbdecd909c0ab24d68ce5 (MD5) Previous issue date: 2015-02-05 / CNPQ / Consideramos o movimento da interface entre dois fluidos viscosos, imiscíveis e incompressíveis confinados em uma célula de Hele-Shaw radial. Quando o fluido de baixa viscosidade é injetado no centro da célula e desloca radialmente o fluido de alta viscosidade, a instabilidade de Saffman-Taylor entra em ação levando ao crescimento de estruturas em formato de dedos. Dependendo da natureza dos fluidos envolvidos, eles podem molhar as paredes da célula de Hele-Shaw, deixando para trás um filme molhante de espessura finita. Diante disso, investigamos a influência da camada de fluido molhante, deixada pelo fluido deslocado, na dinâmica linear e fracamente não-linear do sistema. Mais especificamente, examinamos como a instabilidade da interface e os mecanismos de formação de padrões (bifurcação e competição de dedos) são afetados pela presença da camada de filme molhante no limite de baixo número de capilaridade. Estudamos também uma variação da instabilidade de Saffman-Taylor usual induzida pela injeção: o problema do levantamento em célula de Hele-Shaw. Nossos resultados analíticos indicam que o molhamento tem um impacto significativo nos padrões não-lineares resultantes. Ele é responsável por uma atenuação na variação do comprimento dos dedos, enquanto que induz o aparecimento de estruturas que apresentam dedos pequenos e largos, provenientes do fluido não molhante, alternados por dedos pequenos e finos pertencentes ao fluido molhante. Durante o processo de levantamento, medidas adquiridas pelo teste de adesão quantificam a força adesiva de um fluido viscoso confinado entre placas paralelas. Experimentos e intensas simulações numéricas indicam que a formação de dedos afeta a força de levantamento, promovendo uma diminuição na magnitude da mesma. Concluímos este trabalho propondo um modelo analítico que forneça a força de adesão considerando não só o efeito de dedos da interface, mas também a ação do molhamento e de estresses viscosos normais. / We consider the interfacial motion between two immiscible, incompressible, viscous fluids in the confined geometry of a radial Hele-Shaw cell. When the less viscous fluid is injected at the center and drives radially the more viscous fluid, the Saffman-Taylor instability takes place leading to the growth of fingerlike shapes. Depending on the nature of the fluids involved, they can wet the walls of the Hele-Shaw cell plates, leaving behind a film of finite thickness. In this framework, we investigate the influence of a thin wetting film trailing behind the displaced fluid on the linear and weakly nonlinear dynamics of the system. More specifically, we examine how the interface instability and the pattern formation mechanisms of finger tip-splitting and finger competition are affected by the presence of such a film in the low capillary number limit. We also examined a variant of the usual injection-driven Saffman-Taylor instability: the lifting Hele-Shaw cell problem. Our analytical results indicate that wettability has a significant impact on the resulting nonlinear patterns. It restrains finger length variability while inducing the development of structures presenting short, blunt penetrating fingers of the nonwetting fluid, alternated by short, sharp fingers of the wetting fluid. During the lifting process, probe-tack measurements evaluate the adhesion strength of viscous fluids confined between parallel plates. Existing meticulous experiments and intensive numerical simulations indicate that fingering formation affects the lifting force, making it to decrease in intensity. We conclude this work by proposing an analytical model that computes the lifting adhesion force by taking into account not only the effect of interfacial fingering, but also the action of wetting, and viscous normal stresses.

Carbon fibre reinforced composites are known for their high specific strength-to-weight ratio and are of great interest to the aerospace industry. Incorporating these materials into the fuselage, like in Boeing's 787 "Dreamliner", offers considerable weight reduction which increases flying efficiency, and reduces the cost of flying. In flight, aircraft are often subject to lightning strikes which, in the case of composites, can result in localized melting given the high resistive nature of the material. Aerospace carbon fibre composites often incorporate a metallic mesh or foil within the composite layers to dissipate the electrical charge through the large aircraft. The damage to the aircraft is minimized but not always eliminated. This research aims to elaborate a practical technique to deposit thin layers of conductive material on the surface of aerospace grade composites. Using Cold Gas Dynamic Spray (CGDS), such coatings could be used to repair damaged components. An experimental research approach was used to develop metallic coated composites. Using the CGDS equipment of Centerline (SST-P), specific parameters (such as gas temperature and stagnation pressure) were determined for each type of metallic coating (tin-based & copper-based). The use of bond coats was explored in order to attain the desired coatings. Once optimized, these coatings were evaluated with respect to their corrosive, adhesive, and electrical properties following industry standards.

Le titane (Ti) est largement utilisé en orthopédie et médecine dentaire. Ce matériau présente d´excellentes propriétés mécaniques, est biocompatible et résiste à la corrosion. L’interaction entre les cellules et la surface d’un implant joue un rôle décisif dans l’ostéointégration. Malgré la grande variété d’études que nous trouvons dans la littérature, le comportement des cellules en contact avec des matériaux implantables comme le Ti n’est toujours pas élucidé à toutes les échelles topographiques. Notre laboratoire a développé une méthode de modification physico-chimique de la surface de métaux à intérêt médical. Cette méthode génère des surfaces nanoporeuses qui favorisent la différenciation de cellules souches, affectent le comportement cellulaire de façon différentielle, promeuvent la formation osseuse in vitro et in vivo, et qui ont une capacité antibactérienne. Afin de mieux comprendre comment cette surface influence le comportement cellulaire, nous avons étudié leur influence sur la formation et la maturation des adhésions focales (FAs, de l’anglais) et la formation des filopodes. De plus, nous avons examiné comment les caractéristiques physico-chimiques de la surface obtenue guident l’expression génique des protéines associées aux FAs et aux filopodes en utilisant différentes lignées cellulaires. Finalement, afin de mieux comprendre la biomécanique de la cellule, la force d’adhésion à la surface des filopodes a été déterminée à l’aide de la microscopie à force atomique (AFM). Des disques de Ti commercialement pur (Cp-Ti) ont été polis a fini miroir (Ti-Control), une partie des disques a été traité avec un mélange d’acide sulfurique et de peroxyde d’hydrogène pour créer une surface nanostructurée poreuse (Ti-Nano). L’influence de la nanoporosité, de la cristallinité et la mouillabilité de cette surface sur des cellules pre-ostéoblastiques de souris (MC3T3) et des bactéries a été évalué par la microscopie électronique à balayage (MEB) et par immunofluorescence (IF). Nous avons ensuite utilisé une lignée cellulaire épithéliale (CHO-K1) qui exprime la paxilline (une protéine des FAs) de type sauvage ou la paxilline avec des mutations. De plus, la force d’interaction des filopodes avec la surface a été quantifié en mesurant la force latérale nécessaire pour les déplacer avec une pointe d’AFM. Finalement, la centrifugation a été utilisée pour étudier les changements fonctionnels des cellules MC3T3. L’analyse du comportement des cellules MC3T3 sur des surfaces amorphes et cristallines n'a pas montré de différence par rapport au nombre des cellules ou la quantité des FAs. La cristallinité de la couche superficielle n’avait également aucune incidence sur l’adhésion bactérienne. Les deux lignées cellulaires utilisées ont montré une présence abondante de filopodes avec des nanoprotrusions latérales en réponse à la nanoporosité. La taille et la forme des cellules CHO-K1 ont été grandement affectées par la topographie. L’expression génique des protéines associées aux différents marqueurs des FAs et aux protrusions a été aussi significativement augmentée sur la surface nanoporeuse, quel que soit le type de cellule. Les filopodes sur Ti-Nano ont montré une plus grande résistance au détachement latéral, ce qui indique qu'ils adhèrent à la surface avec plus de force. Également, l’analyse par MEB a révélé une restructuration de la membrane cellulaire accompagnée d’un changement de la forme cellulaire après centrifugation. Parce que les mitochondries fournissent de l’énergie pour les processus cellulaires, l’organisation du réseau mitochondrial a été influencée aussi par la topographie de surface et la centrifugation. Bien qu’il ne puisse pas être exclu que la cristallinité et la mouillabilité de la surface contribuent dans une certaine mesure à déterminer le comportement des cellules, nos résultats suggèrent que les caractéristiques physiques des surfaces représentent le principal déterminant. Nous avons démontré aussi, pour la première fois, que la topographie de surface peut modifier l’interaction adhésive d’une structure subcellulaire qui est fondamentale dans la détection des caractéristiques physico-chimiques des surfaces. En conclusion, nos résultats montrent que la topographie de surface peut modifier des propriétés fondamentales dans les cellules. Dans leur ensemble, ils soulèvent la possibilité que les surfaces nanostructurées puissent être utilisées non seulement pour guider/accélérer l’intégration de biomatériaux dans des conditions normales, mais également dans des situations où l’activité cellulaire est compromise ou également pour les prothèses soumises à des charges externes, telles que les implants orthopédiques et dentaires. / Titanium (Ti) is widely used in orthopedics and dentistry. This material has excellent mechanical properties, is biocompatible and corrosion resistant. The interaction between the cells and the surface of an implant plays a key role in osseointegration. Despite the wide variety of studies found in the literature, the behavior of cells in contact with implantable materials such as Ti is not yet fully elucidated at all topographic scales. Our laboratory has developed a method for the physicochemical modification of the surface of medically relevant metals. This method generates nanoporous surfaces that promote stem cell differentiation, differentially affect cellular behavior, promote bone formation in vitro and in vivo and have antibacterial capacity. To better understand how this surface influences cell behavior, we studied their influence on the formation and maturation of focal adhesions (FAs) and filopodia formation. Furthermore, we examined how the physicochemical characteristics of the resulting surface guide the gene expression of proteins associated with FAs and filopodia using different cell lines. Finally, to better understand the biomechanics of the cell, the adhesion strength of filopodia to the surface was determined using atomic force microscopy (AFM). Commercially pure Ti discs (Cp-Ti) were polished to a mirror finish (Ti-Control), some of the polished discs were treated with a mixture of sulfuric acid and hydrogen peroxide to create a nanostructured surface (Ti-Nano). The influence of nanoporosity, crystallinity and wettability of this surface on mouse pre-osteoblastic cells (MC3T3) and bacteria was evaluated by scanning electron microscopy (SEM) and immunofluorescence. Then, to evaluate the response to nanotopography, we used an epithelial cell line (CHO-K1) that expresses wild type paxillin (a protein of FAs) or paxillin with mutations. In addition, the interaction forces of the filopodia with the surface were quantified by measuring the lateral force required to displace these structures from the surface with an AFM tip. Finally, centrifugation was used to study functional changes in MC3T3 cells. Analysis of the behavior of MC3T3 cells on amorphous and crystalline surfaces showed no difference in cell number or the number of focal adhesions. The crystallinity of the surface layers also had no effect on bacterial adhesion. Both cell lines used showed abundant presence of filopodia 4 with lateral nanoprotrusions in response to nanoporosity. The size and shape of CHO-K1 cells was greatly affected by the topography. Gene expression of proteins associated with different focal adhesion markers and protrusions was also significantly increased on the nanoporous surface, regardless of cell type. Filopodia on the Ti-Nano showed greater resistance to lateral detachment force, indicating that they adhere to the surface with greater strength. Also, SEM analysis revealed a restructuring of the cell membrane accompanied by a corresponding change in cell shape after centrifugation. Because mitochondria provide energy for cell processes, the organization of the mitochondrial network was also influenced by surface topography and centrifugation. Although it cannot be excluded that surface crystallinity and wettability contribute to some extent to determining cell behavior, our results suggest that the physical characteristics of the surfaces represent the main determinant. We have also shown for the first time that surface topography can modify the adhesive interaction of a subcellular structure that is fundamental in the detection of the physicochemical characteristics of surfaces. In conclusion, our results show that surface topography can modify fundamental properties in cells. Together, they raise the possibility that nanostructured surfaces can be used not only to guide/accelerate the integration of biomaterials under normal conditions, but also in situations where cellular activity is compromised or also for prostheses under external loads, such as orthopedic and dental implants.

Cell adhesion to extracellular matrices (ECM) is critical to many cellular processes including differentiation, proliferation, migration, and apoptosis. Alterations in adhesive mechanisms are central to the behavior of cells in pathological conditions including cancer, atherosclerosis, and defects in wound healing. Although significant progress has been made in identifying molecules involved in adhesion, the mechanisms that dictate the generation of strong adhesive forces remain poorly understood. Specifically, the role of nanoscale geometry of the adhesive interface in integrin recruitment and adhesion forces remains elusive due to limitations in the techniques available for engineering cell adhesion environments. The objective of this project was to analyze the role of nanoscale geometry in cell adhesion strengthening to ECM. Our central hypothesis was that adhesive interactions are regulated by integrin clusters whose recruitment is determined by the nanoscale geometry of the adhesive interface and whose heterogeneity in size, spacing, and orientation modulates adhesion strength. The objective of this project was accomplished by 1) developing an experimental technique capable of producing nanoscale patterns of proteins on surfaces for cell adhesion arrays, 2) assessing the regulation of integrin recruitment by geometry of the adhesive interface, and 3) determining the functional implications of adhesive interface geometry by systematically analyzing the adhesion strengthening response to nanoscale patterns of proteins. A printing technique was developed that patterns proteins into features as small as 90nm with high contrast and high reproducibility. Cell adhesion arrays were produced by directly immobilizing proteins into patterns on mixed-SAMs surfaces with a protein-resistant background. Colocalization analysis of integrin recruitment to FN patterns demonstrated a concentrating effect of bound integrins at pattern sizes with areas equivalent to small nascent focal adhesions. At adhesion areas below 333 × 333 nm2, the frequency of integrin recruitment events decreased significantly indicating a threshold size for integrin clustering. Functionally, pattern sizes below the threshold were unable to participate in generation of adhesion strength. In contrast, patterns between the threshold and micron sizes showed a relationship between adhesion strength and area of individual adhesion points, independent of the total available adhesion area. These studies introduce a robust platform for producing nanoscale patterns of proteins in biologically relevant geometries. Results obtained using this approach yielded new insights on the role of nanoscale organization of the adhesive interface in modulating adhesion strength and integrin recruitment.

Αντικείμενο της παρούσας διατριβής, ήταν η διερεύνηση της διεπιφάνειας ανθρώπινων μεσεγχυματικών κυττάρων (Human Mesenchymal Stem Cells, hMSCs) -Νανοσωλήνων Άνθρακα Πολλαπλού Τοιχώματος (Multi Walled Carbon Nanotubes, MWCNTs) υπό στατικές και δυναμικές συνθήκες. Οι MWCNTs έχει αποδειχθεί ότι, έχουν μοναδικές ηλεκτρικές και φυσικές ιδιότητες, μηχανική αντοχή και χαμηλή πυκνότητα, χαρακτηριστικά που τους καθιστούν εξαιρετικά ελκυστικούς για το σχεδιασμό βιοϋλικών για ορθοπαιδικές εφαρμογές. Αρχικά, μελετήθηκε η βιοσυμβατότητα των hMSCs σε επιφάνειες MWCNTs, ως προς την κυτταροτοξικότητα, τη μορφολογία, τον πολλαπλασιασμό, τη διαφοροποίηση και την οργάνωση του κυτταροσκελετού. Το υπόστρωμα των MWCNTs ευνόησε την εξάπλωση των κυττάρων, προήγαγε τον πολλαπλασιασμό και προώθησε τη διαφοροποίηση των hMSCs σε οστεοβλάστες, όπως έδειξε η έκφραση αλκαλικής φωσφατάσης, οστεοποντίνης και οστεοκαλσίνης. Μελετήθηκε η γονιδιακή έκφραση των ιντεγκρινικών υποδοχέων, υπεύθυνων για την προσκόλληση των κυττάρων στους MWCNTs. Με την τεχνική του περιστρεφόμενου δίσκου, εκτιμήθηκε η δύναμη προσκόλλησης των hMSCs στους MWCNTs και η επίδραση της κάθε ιντεγκρίνης στη μεταβολή της δύναμης προσκόλλησης. Για τη διερεύνηση της απόκρισης των οστεοβλαστών στη μηχανική φόρτιση, τα προσκολλημένα κύτταρα στους MWCNTs καταπονήθηκαν για 3 και 24 ώρες, με σύστημα μηχανικής φόρτισης βασισμένο στην Αρχή Κάμψης Τεσσάρων Σημείων. Τα αποτελέσματα έδειξαν ότι, η φόρτιση επηρεάζει θετικά την έκφραση γονιδίων προσκόλλησης και δεικτών διαφοροποίησης. Επιπρόσθετα, μελετήθηκε η συμπεριφορά των hMSCs ως προς την κυτταροτοξικότητα, τον πολλαπλασιασμό, τη διαφοροποίηση, την οργάνωση του κυτταροσκελετού και την έκφραση γονιδίων προσκόλλησης, σε τροποποιημένες επιφάνειες MWCNTs με υδροξυλομάδες, καρβοξυλομάδες και αμινομάδες. Τα αποτελέσματα έδειξαν ότι, η αμινοτροποποιημένη επιφάνεια ευνόησε σημαντικά την κυτταρική συμπεριφορά σε σύγκριση με τις άλλες δύο επιφάνειες. Τέλος, μελετήθηκε η επίδραση της τοπογραφίας με χρήση κάθετα ευθυγραμμισμένων MWCNTs, σε σύγκριση με τυχαία προσανατολισμένους MWCNTs. Η απόκριση των hMSCs στους κάθετα ευθυγραμμισμένους MWCNTs ήταν καλύτερη σε σύγκριση με τους τυχαία προσανατολισμένους, τόσο ως προς τον πολλαπλασιασμό και τη διαφοροποίηση, όσο και ως προς την οργάνωση του κυτταροσκελετού. Τα αποτελέσματα της διατριβής είναι υποσχόμενα για το μελλοντικό σχεδιασμό βιοϋλικών με MWCNTs, με τελικό σκοπό την εφαρμογή σε θεραπείες στις οποίες απαιτείται ανακατασκευή του οστού. / The aim of the present study was the investigation of the interface of human Mesenchymal Stem Cells (hMSCs) – Multiwalled Carbon Nanotubes (MWCNTs), under static and dynamic conditions. MWCNTs have been proven to obtain unique electric and physical properties, mechanical strength and low density, which render them highly attractive for the design of biomaterials for orthopaedic applications. Firstly, the biocompatibility of MWCNTs was studied, in terms of hMSCs cytotoxicity, morphology, proliferation, differentiation, cytoskeleton organization and toxicity. The substrate of MWCNTs favored cell spreading, increased proliferation and promoted cell differentiation, as measured by the expression of alkaline phosphatase, osteopontin and osteocalcin. The gene expression of integrin receptors responsible for cell attachment on MWCNTs was studied. Using the Spinning Disc Technique, the attachment strength of hMSCs on MWCNTs was evaluated, as well as the impact of each integrin to the alteration of attachment strength. In order to investigate the cell response to mechanical loading, the attached cells on MWCNTs were stressed for 3 and 24 hours, using a system for mechanical loading based on the 4-point bending principle. Results showed that loading positively induces the expression of genes associated with attachment and differentiation markers. Additionally, the cell behavior concerning proliferation, differentiation, cytoskeleton organization, apoptosis and gene expression associated with attachment, was studied on MWCNTs after surface modification with hydroxyl-, carboxyl-, and amino- groups. The findings indicated that the amino- modified surface significantly favored the cell behavior, compared to the other two surfaces. Lastly, the topography effect was studied using vertically aligned MWCNTs. Cell response was found better on the vertically compared to the randomly oriented, in terms of proliferation, differentiation and cytoskeleton organization. The findings of the study are promising for the future design of biomaterials of MWCNTs, aiming for application in therapies where bone reconstruction is demanded.