Dissertationen zum Thema „Biomedical materials“

La resposta del nostre cos als biomaterials suposa una gran obstacle per la efectivitat de múltiples teràpies basades en biomaterials. Accionats per la absorció inespecífica de biomolècules a la superfície del material, barreres com el sistema immune o les superfícies mucoses eliminen els materials del cos, evitant que arribin al seu destí i realitzin la seva funció. Els materials zwitteriònics han emergit en els últims anys com a materials antiadherents prometedors per a superar les mencionades barreres. Tot i que molts sistemes han utilitzat els materials zwitteriònics com a recobriments, les seves propietats úniques de superhidrofilicitat i versatilitat química suggereixen múltiples beneficis en utilitzar-los com a material principal. Aquí, dos sistemes diferents basats en materials zwitteriònics són presentats. En primer lloc una plataforma de alliberament de fàrmac antiadherent basat en copolímers de bloc amfifílics (CBA) és desenvolupada. Els CBAs zwitteriònics són sintetitzats i optimitzats perquè s’auto-organitzin en nanopartícules zwitteriòniques. Les propietats antiadherents d’aquestes nanopartícules es demostren, al igual que el seu potencial per a esdevenir un sistema d’alliberament de fàrmac oral. Seguidament, el sistema s’utilitza com a portador per a fàrmacs contra la malària i el càncer. Les nanopartícules mostren internalització en eritròcits infectats per Plasmòdium, i nanopartícules carregades amb curcumina demostren la seva eficàcia contra la malària in vitro. S’observa absorció oral de polímer i curcumina in vivo utilitzant un model de ratolí, indicant el potencial del sistema per a esdevenir una teràpia oral contra la malària. Quan s’optimitza el sistema per la teràpia contra el càncer, nanopartícules carregades de Paclitaxel exhibeixen activitat anti-cancerígena en models in vitro de cèl·lules canceroses. En segon lloc, microrobots zwitteriònics no-immunogènics que poden evitar el reconeixement per el sistema immune són introduïts. Es desenvolupa una fotoresistència zwitteriònica per a la microimpressió de microrobots zwitteriònics a través de la polimerització de dos fotons amb una ample funcionalització: propietats mecàniques variables, anti-bioadhesió i propietats no-immunogèniques, funcionalització per a la actuació magnètica, encapsulació de biomolècules i modificació superficial per a l’alliberament de fàrmac. Els robots invisibles eviten que els macròfags del sistema immune els detectin després d’una inspecció exhaustiva (de més de 90 hores), fet que no s’ha aconseguit fins el moment en cap sistema microrobòtic. Aquests materials zwitteriònics versàtils eliminen un dels grans obstacles en el desenvolupament de microrobots biocompatibles, i serviran com una caixa d’eines de materials no-immunogènics per a crear robots biomèdics i altres dispositius per a la bioenginyeria i per a aplicacions biomèdiques.
La respuesta de nuestro cuerpo a los biomateriales supone un gran obstáculo para la efectividad de múltiples terapias basadas en los biomateriales. Accionados por la absorción de biomoléculas en la superficie del material, barreras como el sistema inmune o las superficies mucosas eliminan los materiales del cuerpo, evitando que lleguen a su destino y realicen su función. Los materiales zwitteriónicos han emergido en los últimos años como materiales antiadherentes prometedores para superar las barreras mencionadas. Aunque muchos sistemas utilizan materiales zwitteriónicos como recubrimientos, sus propiedades únicas de superhidrofilicidad i versatilidad química sugieren múltiples beneficios en utilizarlos como material principal. Aquí, dos sistemas basados en materiales zwitteriónicos son presentados. En primer lugar, una plataforma para la liberación de fármaco antiadherente basada en copolímeros de bloque amfifílicos (CBA) es desarrollada. Los CBA zwitteriónicos son sintetizados y optimizados para que se auto-organicen en nanopartículas zwitteriónicas. Las propiedades antiadherentes de estas nanopartículas son probadas, al igual que su potencial para convertirse en un sistema oral de liberación de fármaco. Seguidamente, el sistema se utiliza como portador para fármacos animalarios y anticancerígenos. Las nanopartículas muestran internalización en eritrocitos infectados por Plasmodio, y nanopartículas cargadas con curcumina demuestran su eficacia contra la malaria in vitro. Se observa la absorción oral de polímero y curcumina in vivo utilizando un modelo de ratón, indicando el potencial del sistema para convertirse en una terapia oral contra malaria. Cuando se optimiza el sistema para la terapia contra el cáncer, las nanopartículas cargadas con Paclitaxel exhiben actividad anticancerígena en modelos in vitro de células cancerosas. En segundo lugar, microrobots zwitteriónicos no-inmunológicos que pueden evitar el reconocimiento por parte del sistema inmune son introducidos. Se desarrolla una fotoresisténcia zwitteriónica para la microimpresión de microrobots zwitteriónicos a través de la polimerización de dos fotones con una amplia funcionalización: propiedades mecánicas variables, anti-bioadhesión i propiedades no-inmunogénicas, funcionalización para la actuación magnética, encapsulación de biomoléculas i modificación superficial para la liberación de fármaco. Los robots invisibles evitan que los macrófagos del sistema inmune innato los detecten después de una inspección exhaustiva (de más de 90 horas), hecho que no se ha conseguido hasta la fecha por ningún sistema microrobótico. Estos materiales zwitteriónicos versátiles eliminan uno de los grandes obstáculos en el desarrollo de microrobots biocompatibles, y servirán como una caja de herramientas de materiales no-inmunogénicos para crear robots biomédicos y otros dispositivos para la bioingeniería y para las aplicaciones biomédicas.
Body response to biomaterials suppose a major roadblock for the effectiveness of multiple biomaterial-based therapies. Triggered by unspecific absorption of biomolecules in the material surface, barriers such as immune system or mucosal surfaces clear foreign materials from the body, preventing them to reach their target and perform their function. Zwitterionic materials have emerged in the last years as promising antifouling materials to overcome the mentioned barriers. Although many systems have used zwitterionic materials as coatings, the unique properties of superhydrophilicity and chemical versatility suggest multiple benefits of using zwitterionic polymers as bulk materials. Here, two different systems based on zwitterionic materials are presented. In first place, an antifouling drug delivery platform based on zwitterionic amphiphilic polymers (ABC) is developed. Zwitterionic ABCs are synthetized and optimized to self-assemble in zwitterionic nanoparticles. The antifouling properties of zwitterionic nanoparticles are proved, together with their potential to become an oral drug delivery system. Next, the system is used as a drug carrier for antimalarial and anticancer drugs. Nanoparticles show internalization in Plasmodium infected erythrocytes, and curcumin-loaded nanoparticles prove their antimalarial efficacy in vitro. Oral absorption of polymer and curcumin is also observed in vivo using mice model, indicating the potential of this system to become oral therapy against malaria. When optimizing the system for anticancer therapy, Paclitaxel-loaded nanoparticles exhibit anticancer activity in in vitro cancer cell models. Second, non‐immunogenic stealth zwitterionic microrobots that avoid recognition from immune cells are introduced. Zwitterionic photoresist are developed for the 3D microprinting of zwitterionic hydrogel microrobots through 2-photon polymerization with ample functionalization: tunable mechanical properties, anti-biofouling and non-immunogenic properties, functionalization for magnetic actuation, encapsulation of biomolecules, and surface functionalization for drug delivery. Stealth microrobots avoid detection by macrophage cells of the innate immune system after exhaustive inspection (> 90 h), which has not been achieved in any microrobotic platform to date. These versatile zwitterionic materials eliminate a major roadblock in the development of biocompatible microrobots, and will serve as a toolbox of non-immunogenic materials for medical microrobot and other device technologies for bioengineering and biomedical applications.

The inherent design freedom of protein engineering and recombinant protein production enables specific tailoring of protein structure, function, and properties. Two areas of research where protein engineering has allowed for many advances in biomedical materials include the design of novel protein scaffolds for molecular recognition, as well as the use of recombinant proteins for production of next generation biomaterials. The main focus of my dissertation was to develop new biomedical materials using protein engineering. Chapters three and four discuss the engineering of repeat proteins as bio-recognition modules for biomedical sensing and imaging. Chapter three provides an overview of the most recent advances in engineering of repeat proteins in the aforementioned field. Chapter four discusses my contribution to this field. We have designed a de novo repeat protein scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Innate immunity receptors have been described as pattern recognition receptors in that they recognize "global features" of a family of pathogens versus one specific antigen. In mammals, two main protein families of such receptors are: extracellular Toll-like receptors (TLRs) and cytoplasmic Nucletide-binding domain- and Leucine-rich Repeat-containing proteins (NLRs). NLRs are defined by their tripartite domain architecture that contains a C-terminal LRR (Leucine Rich Repeat) domain, the nucleotide-binding oligomerization (NACHT) domain, and the N-terminal effector domain. It is proposed that pathogen sensing in NLRs occurs through ligand binding by the LRR domain. Thus, we hypothesized that LRRs would be suitable for the design of alternative binding scaffolds for use in molecular recognition. The NOD protein family plays a very important role in innate immunity, and consequently serves as a promising scaffold for design of novel recognition motifs. However, engineering of de novo proteins based on the NOD family LRR domain has proven challenging due to problems arising from protein solubility and stability. Consensus sequence design is a protein design tool used to create novel proteins that capture sequence-structure relationships and interactions present in nature in order to create a stable protein scaffold. We implement a consensus sequence design approach to develop proteins based on the LRR domain of NLRs. Using a multiple sequence alignment we analyzed all individual LRRs found in mammalian NLRs. This design resulted in a consensus sequence protein containing two internal repeats and separate N- and C- capping repeats named CLRR2. Using biophysical characterization methods of size exclusion chromatography, circular dichroism, and fluorescence, CLRR2 was found to be a stable, monomeric, and cysteine free scaffold. Additionally, CLRR2, without any affinity maturation, displayed micromolar binding affinity for muramyl dipeptide (MDP), a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand. Furthermore, CLRR2 demonstrated selective recognition to the biologically active stereoisomer of MDP. Results of this study indicate that LRRs are indeed a useful scaffold for development of specific and selective proteins for molecular recognition, creating much potential for future engineering of alternative protein scaffolds for biomedical applications. My second research interest focused on the development of proteins for novel biomaterials. In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues, as well as participates in important regulatory functions such as cell migration and proliferation and protein signalling. The aforementioned properties along with keratins' inherent capacity for self-assembly poise it as a promising scaffold for regenerative medicine and tissue engineering applications. However, due to the extraction process used to obtain natural keratin proteins from natural sources, protein damage and formation of by-products that alter network self-assembly and bioactivity often occur as a result of the extensive processing conditions required. Furthermore, natural keratins require exogenous chemistry in order to modify their properties, which greatly limits sequence tunability. Recombinant keratin proteins have the potential to overcome the limitations associated with the use of natural keratins while also maintaining their desired structural and chemical characteristics. Thus, we have used recombinant DNA technology for the production of human hair keratins, keratin 31 (K31) and keratin 81 (K81). The production of recombinant human hair keratins resulted in isolated proteins of the correct sequence and molecular weight determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry. Proteins with no unwanted sequence truncations, deletions, or mutations indicate recombinant DNA technology can be used to reliably generate full length keratin proteins. This allows for consistent starting materials with no observable impurities or undesired by-products, which combats a major challenge associated with natural keratins. Additionally, recombinant keratins must maintain the intrinsic propensity for self-assembly found in natural keratins. To test the propensity for self-assembly, we implemented size exclusion chromatography (SEC), dynamic light scattering (DLS), and transmission electron microscopy (TEM) to characterize K31, K81, and an equimolar mixture of K31 and K81. The results of the recombinant protein characterization reveal novel homo-polymerization of K31 and K81, not previously reported, and formation of characteristic keratin fibers for the K31 and K81 mixture. Therefore, recombinant K31 and K81 retain the intrinsic biological activity (i.e. self-assembly) of natural keratin proteins. We have also conducted a comparative study of recombinant and extracted heteropolymer K31/K81. Through solution characterization and TEM analysis it was found that use of the recombinant heteropolymer allows for increased purity of starting material while also maintaining self-assembly properties necessary for functional use in biomaterials design. However, under the processing condition implemented, extracted keratins demonstrated increased efficiency of assembly. Through each study we conclude that recombinant keratin proteins provide a promising solution to overcome the challenges associated with natural protein materials and present an exceptional design platform for generation of new biomaterials for regenerative medicine and tissue engineering.
Ph. D.

Doutoramento em Ciência e Engenharia de Materiais
The increased longevity of humans and the demand for a better quality of life have led to a continuous search for new implant materials. Scientific development coupled with a growing multidisciplinarity between materials science and life sciences has given rise to new approaches such as regenerative medicine and tissue engineering. The search for a material with mechanical properties close to those of human bone produced a new family of hybrid materials that take advantage of the synergy between inorganic silica (SiO4) domains, based on sol-gel bioactive glass compositions, and organic polydimethylsiloxane, PDMS ((CH3)2.SiO2)n, domains. Several studies have shown that hybrid materials based on the system PDMS-SiO2 constitute a promising group of biomaterials with several potential applications from bone tissue regeneration to brain tissue recovery, passing by bioactive coatings and drug delivery systems. The objective of the present work was to prepare hybrid materials for biomedical applications based on the PDMS-SiO2 system and to achieve a better understanding of the relationship among the sol-gel processing conditions, the chemical structures, the microstructure and the macroscopic properties. For that, different characterization techniques were used: Fourier transform infrared spectrometry, liquid and solid state nuclear magnetic resonance techniques, X-ray diffraction, small-angle X-ray scattering, smallangle neutron scattering, surface area analysis by Brunauer–Emmett–Teller method, scanning electron microscopy and transmission electron microscopy. Surface roughness and wettability were analyzed by 3D optical profilometry and by contact angle measurements respectively. Bioactivity was evaluated in vitro by immersion of the materials in Kokubos’s simulated body fluid and posterior surface analysis by different techniques as well as supernatant liquid analysis by inductively coupled plasma spectroscopy. Biocompatibility was assessed using MG63 osteoblastic cells. PDMS-SiO2-CaO materials were first prepared using nitrate as a calcium source. To avoid the presence of nitrate residues in the final product due to its potential toxicity, a heat-treatment step (above 400 °C) is required. In order to enhance the thermal stability of the materials subjected to high temperatures titanium was added to the hybrid system, and a material containing calcium, with no traces of nitrate and the preservation of a significant amount of methyl groups was successfully obtained. The difficulty in eliminating all nitrates from bulk PDMS-SiO2-CaO samples obtained by sol-gel synthesis and subsequent heat-treatment created a new goal which was the search for alternative sources of calcium. New calcium sources were evaluated in order to substitute the nitrate and calcium acetate was chosen due to its good solubility in water. Preparation solgel protocols were tested and homogeneous monolithic samples were obtained. Besides their ability to improve the bioactivity, titanium and zirconium influence the structural and microstructural features of the SiO2-TiO2 and SiO2-ZrO2 binary systems, and also of the PDMS-TiO2 and PDMS-ZrO2 systems. Detailed studies with different sol-gel conditions allowed the understanding of the roles of titanium and zirconium as additives in the PDMS-SiO2 system. It was concluded that titanium and zirconium influence the kinetics of the sol-gel process due to their different alkoxide reactivity leading to hybrid xerogels with dissimilar characteristics and morphologies. Titanium isopropoxide, less reactive than zirconium propoxide, was chosen as source of titanium, used as an additive to the system PDMS-SiO2-CaO. Two different sol-gel preparation routes were followed, using the same base composition and calcium acetate as calcium source. Different microstructures with high hydrophobicit were obtained and both proved to be biocompatible after tested with MG63 osteoblastic cells. Finally, the role of strontium (typically known in bioglasses to promote bone formation and reduce bone resorption) was studied in the PDMS-SiO2-CaOTiO2 hybrid system. A biocompatible material, tested with MG63 osteoblastic cells, was obtained with the ability to release strontium within the values reported as suitable for bone tissue regeneration.
O aumento da longevidade dos seres humanos e a procura de uma melhor qualidade de vida têm conduzido a uma pesquisa contínua de novos materiais para implantes. O desenvolvimento científico, juntamente com uma crescente multidisciplinaridade entre as ciências dos materiais e as ciências da vida deram origem a novas abordagens, como a medicina regenerativa e a engenharia de tecidos. A busca de um material com propriedades mecânicas próximas das do osso humano produziu uma nova família de materiais híbridos que tiram partido da sinergia entre os domínios inorgânicos de sílica (SiO4), com base em composições de vidros bioativos obtidos por sol-gel, e os domínios orgânicos de polidimetilsiloxano, PDMS ((CH3)2.SiO2)n. Vários estudos têm demonstrado que os materiais híbridos baseados no sistema PDMS-SiO2 constituem um grupo de biomateriais promissores com várias aplicações potenciais tais como a regeneração de tecido ósseo e a recuperação do tecido cerebral, passando por revestimentos bioativos e sistemas de libertação controlada de fármacos. O objetivo do presente trabalho foi preparar materiais híbridos para aplicações biomédicas com base no sistema PDMS-SiO2 e contribuir para uma melhor compreensão das relações entre as condições de processamento sol-gel, as estruturas químicas, a microestrutura e as propriedades macroscópicas. Para alcançar tal objetivo, foram usadas diferentes técnicas de caracterização: espectroscopia de infravermelho por transformada de Fourier, ressonância magnética nuclear no estado sólido e no estado líquido, difração de raios-X, dispersão de raios-X de baixo ângulo, dispersão de neutrões de baixo ângulo, análise da área de superfície pelo método de Brunauer–Emmett–Teller, microscopia eletrónica de varrimento e microscopia eletrónica de transmissão. A rugosidade e a molhabilidade das superfícies foram analisadas por perfilometria óptica 3D e por medidas de ângulo de contacto, respectivamente. A bioatividade in vitro foi avaliada através de testes de imersão em plasma sintético e posterior observação da superfície dos materiais e análise do líquido sobrenadante por espectrometria de emissão atômica por plasma acoplado Indutivamente. A biocompatibilidade in vitro foi avaliada usando células osteoblásticas MG63. Materiais do sistema PDMS-SiO2-CaO foram inicialmente preparados usando o nitrato como fonte de cálcio. Para eliminar os resíduos de nitrato no produto final, devido à sua potencial toxicidade, é necessária uma etapa de tratamento térmico (acima dos 400° C). A fim de aumentar a estabilidade térmica dos materiais submetidos a altas temperaturas, foi adicionado titânio ao sistema híbrido. Obteve-se assim um material híbrido contendo cálcio, sem vestígios de nitrato, mantendo-se uma quantidade significativa de grupos metilo. A dificuldade de obter amostras monolíticas de híbridos PDMS-SiO2-CaO por síntese sol-gel e posterior tratamento térmico para eliminação de nitratos, criou um novo objetivo: a procura de fontes alternativas de cálcio. Novas fontes de cálcio foram avaliadas para substituir o nitrato tendo-se escolhido o acetato de cálcio devido à sua boa solubilidade em água. Estabeleceram-se protocolos de preparação por sol-gel a partir dos quais se obtiveram amostras monolíticas homogéneas. Além de melhorar a bioatividade, o titânio e o zircónio influenciam as características estruturais e microestruturais dos sistemas binários SiO2-TiO2 e SiO2-ZrO2, bem como dos sistemas PDMS-TiO2 e PDMS-ZrO2. Neste contexto, foram estudadas diferentes condições experimentais no processo sol-gel, de modo a compreender o papel destes aditivos no sistema SiO2-PDMS. Concluiu-se que o titânio e o zircónio influenciam a cinética do processo sol-gel devido à diferente reatividade dos despectivos alcóxidos, conduzindo à obtenção de xerogéis híbridos com diferentes características e morfologias. O isopropóxido de titânio, menos reativo do que o propóxido de zircónio, foi escolhido como fonte de titânio, usado como aditivo no sistema PDMS-SiO2CaO. Dois procedimentos diferentes de preparação por sol-gel foram seguidos, utilizando a mesma composição de base e o acetato de cálcio como fonte de cálcio. Foram obtidas diferentes microestruturas muito hidrofóbicas e ambas mostraram ser biocompatíveis após serem testadas com células osteoblásticas MG63. Finalmente, foi avaliado o papel do estrôncio (conhecido nos biovidros por favorecer a formação de tecido ósseo e reduzir a sua reabsorção) no sistema híbrido PDMS-CaO-SiO2-TiO2. O material produzido revelou-se biocompatível, através de testes com células osteoblásticas MG63, e com a capacidade de libertar estrôncio dentro dos limites considerados adequados para a reparação do tecido ósseo.

The main aim of this project was to assess auxetic (negative Poisson's ratio) materials for potential in biomedical devices. Specifically, a detailed comparative indentation study has been undertaken on auxetic and conventional foams for hip protector devices; radially-gradient one-piece foams having auxetic character have been produced for the first time and shown to have potential in artificial intervertebral disc (IVD) implant devices; and auxetic honeycomb geometries have been assessed for the stem component in hip implant devices. For the hip protector application, combined compression and heat treatment of conventional polyurethane open-cell foam was used to produce monolithic auxetic foams. The foams were characterised structurally using optical microscopy, and mechanically using mechanical testing combined with videoextensometry. Static indentation using six different indenter shapes on each of the six faces of the foam specimens has been undertaken. The key conclusion here is that the enhanced indentation resistance for the converted foam is not a consequence of increased density accompanied by the usual significant increase in foam stiffness. The enhanced indentation resistance is consistent with the auxetic effect associated with the increased density, providing a localised densification mechanism under indentation (i.e. material flows under the indenter). At higher indentation displacement the Poisson’s ratios for both the unconverted and converted foams tend towards zero. In this case, the increase in foam stiffness for the converted foams at higher strain may also contribute to the indentation enhancement at high indentation displacement. New radially-gradient foams mimicking the core-sheath structure of the natural IVD have been produced through the development of a novel thermo-mechanical manufacturing route. Foam microstructural characterisation has been undertaken using optical and scanning electron microscopy, and also micro-CT scans performed by collaborators at the University of Manchester. Detailed x-y strain mapping using combined mechanical testing and videoextensometry enabled the local and global Young's modulus and Poisson's ratio responses of these new materials to be determined. In one example, global auxetic response is achieved in a foam having a positive Poisson's ratio core and auxetic sheath. It is suggested this may be a more realistic representation of the properties of natural IVD tissue. Analytical and Finite Element (FE) models have been developed to design honeycomb geometries for the stems in new total hip replacement implants. FE models of the devices implanted within bone have been developed and the auxetic stems shown to lead to reduced stress shielding effect.

Objects with complex shape and functions have always attracted attention and interest. The morphological diversity and complexity of naturally occurring forms and patterns have been a motivation for humans to copy and adopt ideas from Nature to achieve functional, aesthetic and social value. Biomimetics is addressed to the design and development of new synthetic materials using strategies adopted by living organisms to produce biological materials. In particular, biomineralized tissues are often sophisticate composite materials, in which the components and the interfaces between them have been defined and optimized, and that present unusual and optimal chemical-physical, morphological and mechanical properties. Moreover, biominerals are generally produced by easily traceable raw materials, in aqueous media and at room pressure and temperature, that is through cheap process and materials. Thus, it is not surprising that the idea to mimic those strategies proper of Nature has been employed in several areas of applied sciences, such as for the preparation of liquid crystals, ceramic thin films computer switches and many other advanced materials. On this basis, this PhD thesis is focused on the investigation of the interaction of biologically active ions and molecules with calcium phosphates with the aim to develop new materials for the substitution and repair of skeletal tissue, according to the following lines: I. Modified calcium phosphates. A relevant part of this PhD thesis has been addressed to study the interaction of Strontium with calcium phosphates. It was demonstrated that strontium ion can substitute for calcium into hydroxyapatite, causing appreciable structural and morphological modifications. The detailed structural analysis carried out on the nanocrystals at different strontium content provided new insight into its interaction with the structure of hydroxyapatite. At variance with the behaviour of Sr towards HA, it was found that this ion inhibits the synthesis of octacalcium phosphate. However, it can substitute for calcium in this structure up to 15 atom %, in agreement with the increase of the cell parameters observed on increasing ion concentration. A similar behaviour was found for Magnesium ion, whereas Manganese inhibits the synthesis of octacalcium phosphate and it promotes the precipitation of dicalcium phosphate dehydrate. It was also found that Strontium affects the kinetics of the reaction of hydrolysis of α-TCP. It inhibits the conversion from α-TCP to hydroxyapatite. However, the resulting apatitic phase contains significant amounts of Sr2+ suggesting that the addition of Sr2+ to the composition of α-TCP bone cements could be successfully exploited for its local delivery in bone defects. The hydrolysis of α-TCP has been investigated also in the presence of increasing amounts of gelatin: the results indicated that this biopolymer accelerates the hydrolysis reaction and promotes the conversion of α-TCP into OCP, suggesting that its addition in the composition of calcium phosphate cements can be employed to modulate the OCP/HA ratio, and as a consequence the solubility, of the set cement. II. Deposition of modified calcium phosphates on metallic substrates. Coating with a thin film of calcium phosphates is frequently applied on the surface of metallic implants in order to combine the high mechanical strength of the metal with the excellent bioactivity of the calcium phosphates surface layers. During this PhD thesis, thank to the collaboration with prof. I.N. Mihailescu, head of the Laser-Surface-Plasma Interactions Laboratory (National Institute for Lasers, Plasma and Radiation Physics – Laser Department, Bucharest) Pulsed Laser Deposition has been successfully applied to deposit thin films of Sr substituted HA on Titanium substrates. The synthesized coatings displayed a uniform Sr distribution, a granular surface and a good degree of crystallinity which slightly decreased on increasing Sr content. The results of in vitro tests carried out on osteoblast-like and osteoclast cells suggested that the presence of Sr in HA thin films can enhance the positive effect of HA coatings on osteointegration and bone regeneration, and prevent undesirable bone resorption. The possibility to introduce an active molecule in the implant site was explored using Matrix Assisted Pulsed Laser Evaporation to deposit hydroxyapatite nanocrystals at different content of alendronate, a bisphosphonate widely employed in the treatments of pathological diseases associated to bone loss. The coatings displayed a good degree of crystallinity, and the results of in vitro tests indicated that alendronate promotes proliferation and differentiation of osteoblasts even when incorporated into hydroxyapatite. III. Synthesis of drug carriers with a delayed release modulated by a calcium phosphate coating. A core-shell system for modulated drug delivery and release has been developed through optimization of the experimental conditions to cover gelatin microspheres with a uniform layer of calcium phosphate. The kinetics of the release from uncoated and coated microspheres was investigated using aspirin as a model drug. It was shown that the presence of the calcium phosphate shell delays the release of aspirin and allows to modulate its action.

Objects with complex shape and functions have always attracted attention and interest. The morphological diversity and complexity of naturally occurring forms and patterns have been a motivation for humans to copy and adopt ideas from Nature to achieve functional, aesthetic and social value. Biomimetics is addressed to the design and development of new synthetic materials using strategies adopted by living organisms to produce biological materials. In particular, biomineralized tissues are often sophisticate composite materials, in which the components and the interfaces between them have been defined and optimized, and that present unusual and optimal chemical-physical, morphological and mechanical properties. Moreover, biominerals are generally produced by easily traceable raw materials, in aqueous media and at room pressure and temperature, that is through cheap process and materials. Thus, it is not surprising that the idea to mimic those strategies proper of Nature has been employed in several areas of applied sciences, such as for the preparation of liquid crystals, ceramic thin films computer switches and many other advanced materials. On this basis, this PhD thesis is focused on the investigation of the interaction of biologically active ions and molecules with calcium phosphates with the aim to develop new materials for the substitution and repair of skeletal tissue, according to the following lines: I. Modified calcium phosphates. A relevant part of this PhD thesis has been addressed to study the interaction of Strontium with calcium phosphates. It was demonstrated that strontium ion can substitute for calcium into hydroxyapatite, causing appreciable structural and morphological modifications. The detailed structural analysis carried out on the nanocrystals at different strontium content provided new insight into its interaction with the structure of hydroxyapatite. At variance with the behaviour of Sr towards HA, it was found that this ion inhibits the synthesis of octacalcium phosphate. However, it can substitute for calcium in this structure up to 15 atom %, in agreement with the increase of the cell parameters observed on increasing ion concentration. A similar behaviour was found for Magnesium ion, whereas Manganese inhibits the synthesis of octacalcium phosphate and it promotes the precipitation of dicalcium phosphate dehydrate. It was also found that Strontium affects the kinetics of the reaction of hydrolysis of α-TCP. It inhibits the conversion from α-TCP to hydroxyapatite. However, the resulting apatitic phase contains significant amounts of Sr2+ suggesting that the addition of Sr2+ to the composition of α-TCP bone cements could be successfully exploited for its local delivery in bone defects. The hydrolysis of α-TCP has been investigated also in the presence of increasing amounts of gelatin: the results indicated that this biopolymer accelerates the hydrolysis reaction and promotes the conversion of α-TCP into OCP, suggesting that its addition in the composition of calcium phosphate cements can be employed to modulate the OCP/HA ratio, and as a consequence the solubility, of the set cement. II. Deposition of modified calcium phosphates on metallic substrates. Coating with a thin film of calcium phosphates is frequently applied on the surface of metallic implants in order to combine the high mechanical strength of the metal with the excellent bioactivity of the calcium phosphates surface layers. During this PhD thesis, thank to the collaboration with prof. I.N. Mihailescu, head of the Laser-Surface-Plasma Interactions Laboratory (National Institute for Lasers, Plasma and Radiation Physics – Laser Department, Bucharest) Pulsed Laser Deposition has been successfully applied to deposit thin films of Sr substituted HA on Titanium substrates. The synthesized coatings displayed a uniform Sr distribution, a granular surface and a good degree of crystallinity which slightly decreased on increasing Sr content. The results of in vitro tests carried out on osteoblast-like and osteoclast cells suggested that the presence of Sr in HA thin films can enhance the positive effect of HA coatings on osteointegration and bone regeneration, and prevent undesirable bone resorption. The possibility to introduce an active molecule in the implant site was explored using Matrix Assisted Pulsed Laser Evaporation to deposit hydroxyapatite nanocrystals at different content of alendronate, a bisphosphonate widely employed in the treatments of pathological diseases associated to bone loss. The coatings displayed a good degree of crystallinity, and the results of in vitro tests indicated that alendronate promotes proliferation and differentiation of osteoblasts even when incorporated into hydroxyapatite. III. Synthesis of drug carriers with a delayed release modulated by a calcium phosphate coating. A core-shell system for modulated drug delivery and release has been developed through optimization of the experimental conditions to cover gelatin microspheres with a uniform layer of calcium phosphate. The kinetics of the release from uncoated and coated microspheres was investigated using aspirin as a model drug. It was shown that the presence of the calcium phosphate shell delays the release of aspirin and allows to modulate its action.

Laser processing methods have become very appealing for the fabrication of micro/nano structures. To fabricate 3D structures with high resolution and arbitrary complexity, several material deposition processes are in use. By using appropriate moulding techniques, these structures can be fabricated out of a variety of materials such as polymers, ceramics and composites. In this work different lasers have been investigated regarding their suitability for additive and subtractive patterning of small features for biomedical applications. The main focus is on a technique based on two-photon polymerisation of photosensitive materials; this is a nonlinear optical stereo lithography which allows direct-writing of high-resolution three dimensional structures. During the two-photon absorption process, temporal and spatial overlap of photons leads to nonlinear absorption in a highly localized volume. Absorbed photons induce chemical reactions which cause a polymer to form. Due to the quadratic intensity dependence of the process, resolutions of less than 100nm in polymerized structures can potentially be achieved because of the well-defined polymerization threshold. Here, we have emphasised another regime whereby deep structures (~300µm) can be generated in a single pass. This allows rapid fabrication of structures suitable for cell scaffolds where the length scales are small (~10µm) and are required over long ranges (~cm). A Ti: sapphire femtosecond laser at 800nm wavelength with 150fs pulse duration and 1kHz repetition rate was used to determine the two-photon absorption cross section of photoinitiators. This approach was used to initiate two-photon polymerization of resin allowing the fabrication of cell scaffolds suitable for biomedical applications. Diffraction calculations for the imaging optics employed, show that spherical aberration plays a significant role in determining the feature sizes achieved. For subtractive patterning of materials, a femtosecond laser system and an ArF excimer laser have been used. Using ablative techniques keratin films were processed to investigate physical realisation of the commonly used theoretical bricks-and-mortar description of skin. This structure was successfully fabricated and is being used for skin cream research. Also the threshold fluence for ablation of Polyimide Kapton (HN) foils has been measured at oblique angles as an analogue for corneal sculpturing based on beam scanning.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 2017.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 170-176).
Research on biomedical applications of magnetic nanoparticles (MNPs) has increasingly sought to demonstrate noninvasive actuation of cellular processes and material responses using heat dissipated in the presence of an alternating magnetic field (AMF). By modeling the dependence of hysteresis losses on AMF amplitude and constraining AMF conditions to be physiologically suitable, it can be shown that MNPs exhibit uniquely optimal driving conditions that depend on controllable material properties such as magnetic anisotropy, magnetization, and particle volume. "Magnetothermal multiplexing," which relies on selecting materials with substantially distinct optimal AMF conditions, enables the selective heating of different kinds of collocated MNPs by applying different AMF parameters. This effect has the potential to extend the functionality of a variety of emerging techniques with mechanisms that rely on bulk or nanoscale heating of MNPs. Experimental investigations on methods for actuating deep brain stimulation, drug release, and shape memory polymer response are summarized, with discussion of the feasibility and utility of applying magnetothermal multiplexing to similar systems. The possibility of selective heating is motivated by a discussion of various models for heat dissipation by MNPs in AMFs, and then corroborated with experimental calorimetry measurements. A heuristic method for identifying materials and AMF conditions suitable for multiplexing is demonstrated on a set of iron oxide nanoparticles doped with various concentrations of cobalt. Design principles for producing AMFs with high amplitude and ranging in frequency from 15kHz to 2.5MHz are explained in detail, accompanied by a discussion of the outlook for scalability to clinically relevant dimensions. The thesis concludes with a discussion of the state of the field and the broader lessons that can be drawn from the work it describes.
by Michael G. Christiansen.
Ph. D.

This work is concerned with the mechanics of periodic structures for biomedical applications. Classical work on the apparent elastic properties of infinite planar and cylindrical lattice structures is generalised to the non-linear elasto-plastic regime. The elastic recoil upon unloading is also assessed. Elastic instability behaviour of constrained perforated films upon stretching is studied. The elasto-plastic response and recoil analysis of two-dimensional honeycomb is presented. The apparent non-linear structural response obtained analytically here is observed to be smooth, even though the material model of the constituent material is elastic-perfectly-plastic. We show that the Poisson's ratio in the non-linear deformation remains the same as that during the elastic phase. A non-trivial scaling transformation for apparent stress and strain, which separates the individual cell wall response from the mechanics of the overall honeycomb sheet, is identified. This leads to a non-linear master deformation prole that fully describes the plastic response of hexagonal honeycomb with different geometries. The effects of material hardening are introduced by using a novel hyperbolic hardening model. This is then generalised for lattices whom struts possess circular cross-section. Such analysis is relevant to lattice materials and scaffolds manufactured using 3D printing techniques, such as fused deposition modelling, that inevitably makes use of cylindrical laments. Analytical expressions for the elasto-plastic response of a sinusoidal structure wrapped over a cylinder, as a model of crown found within cardiovascular stents, is developed. The response of the cylinder under internal pressure is well approximated by that of the opened-up attened configuration under remote stretch. A scaling ansatz that collapses the response for dierent geometries on a family of 'master-curves' is proposed. We show that the stiffness scales as the cube of the ratio between the amplitude and the wavelength of the sinusoid. Such analysis is then successfully applied to the development of two novel biodegradable stents. Thin membranes with positive apparent Poisson's ratio wrinkle when stretched. Here we show that membranes with negative apparent Poisson's ratio are wrinkle-free upon stretching, except at the edges where localised wrinkling occurs. Here we develop a simple analytical kinematic model to characterise the amplitude and wavelength of the instability behaviour. The model is then validated experimentally and computationally.

Advancements in the biomedical field are driven by the design of novel materials with controlled physical and bio-interactive properties. To develop such materials, researchers rely on the use of highly efficient reactions for the assembly of advanced polymeric scaffolds that meet the demands of a functional biomaterial. In this thesis two main strategies for such materials have been explored; these include the use of off-stoichiometric thiol-ene networks and dendritic polymer scaffolds. In the first case, the highly efficient UV-induced thiol-ene coupling (TEC) reaction was used to create crosslinked polymeric networks with a predetermined and tunable excess of thiol or ene functionality. These materials rely on the use of readily available commercial monomers. By adopting standard molding techniques and simple TEC surface modifications, patterned surfaces with tunable hydrophobicity could be obtained. Moreover, these materials are shown to have great potential for rapid prototyping of microfluidic devices. In the second case, dendritic polymer scaffolds were evaluated for their ability to increase surface interactions and produce functional 3D networks. More specifically, a self-assembled dendritic monolayer approach was explored for producing highly functional dendronized surfaces with specific interactions towards pathogenic E. coli bacteria. Furthermore, a library of heterofunctional dendritic scaffolds, with a controllable and exact number of dual-purpose azide and ene functional groups, has been synthesized. These scaffolds were explored for the production of cell interactive hydrogels and primers for bone adhesive implants. Dendritic hydrogels decorated with a selection of bio-relevant moieties and with Young’s moduli in the same range as several body tissues could be produced by facile UV-induced TEC crosslinking. These gels showed low cytotoxic response and relatively rapid rates of degradation when cultured with normal human dermal fibroblast cells. When used as primers for bone adhesive patches, heterofunctional dendrimers with high azide-group content led to a significant increase in the adhesion between a UV-cured hydrophobic matrix and the wet bone surface (compared to patches without primers).

QC 20140116

The glass-ceramics containing apatite [Ca10(PO4)6(O,F2)] and wollastonite [CaO&
#8226
SiO2] crystals as the predominant crystalline phases, (A-W glass-ceramics) were produced through controlled crystallization of the glasses in the MgO-CaO-SiO2-P2O5-F system. Phases formed in the crystallized counterpart of the glasses were identified by powder X-ray diffraction (XRD) analysis. The crystal morphology of the resultant glass-ceramics was examined using a scanning electron microscope (SEM). The crystallization kinetic parameters consisting of the activation energy for crystallization, (E), the Avrami parameter, (n), and frequency factor of the glass were determined with regard to small amount of TiO2 additions using non-isothermal differential thermal analysis (DTA). The values for E and n for apatite and wollastonite were 460 kJ/mol and 433 kJ/mol, and 3.1±
0.1 and 1.5±
0.1, respectively. When 4 wt% TiO2 was incorporated into the base glass, the values for E decreased to 408 and 320 kJ/mol for apatite and wollastonite, respectively
but the values for n increased from 3.1±
0.1 to 3.3±
0.1, and from 1.5±
0.1 to 1.9±
0.1 for apatite and wollastonite, respectively. TiO2 is an effective nucleating agent in this glass system for promoting the precipitation of both apatite and wollastonite crystals. Structure oriented changes in the indentation microhardness and tribological properties of the A-W glass-ceramics were evidenced. The microhardness at the free surface was 650±
12 HV, but decreased with increasing depth distance from the free surface and attained 520±
8 HV at a distance 0.5 mm below the free surface. The wear rate at the free surface was 0.7±
0.05 ×
10-4 mm3/Nm, but increased as the distance from the free surface increased and became 2.9±
0.15 ×
10-4 mm3/Nm at a distance 0.5 mm below the free surface. Tribological properties of the A-W glass-ceramics were compared with those of commercially available dental ceramics including IPS Empress 2®
, Cergo Pressable Ceramic®
, Cerco Ceram®
, Super porcelain EX-3®
, and bovine enamel. The wear rate, friction coefficient, and wear mechanisms of the A-W glass-ceramics were similar to currently used artificial dental materials.

While mechanical, homograft and bio-prosthetic valves have been used in patients for many decades and have made significant improvements in patient morbidity, there is still a distinct need to overcome their limitations. Recently, emerging elastomer heart valves have been shown to be able to better re-create the flow physics of native heart valves, resulting in preferable hemodynamic responses. Unfortunately, elastomers such as silicone are prone to structural failure, which drastically limits their applicability towards the development of valve prosthesis. In order to produce a mechanically more robust silicone substrate, we reinforced it with graphene nanoplatelets (GNPs). Cytotoxicity and hemocompatibility tests revealed that the incorporation of GNPs did not adversely affect cell proliferation or augment adhesion of platelets on the surface of the composite materials. The ECM valves showed good hydrodynamic properties and favorable acute performance compared to a commercially available valve. We conclude that both the Graphene reinforce silicone and the ECM is useful and warrants further evaluation as aortic valve substitutes.

The necessity for synthesis of nanoparticles with well controlled size and morphology emerged with the development in recent years of novel advanced applications especially in biomedical related fields. These applications require nanoparticles with more complex architecture such as multifunctional nanoparticles (i.e. core–shell structures) that can carry several components with different embedded functionalities. In this thesis, we developed core–shell nanoparticles (CSNPs) with finely tuned silica shell on iron oxide core as model system for advanced applications in nanomedicine such as MRI, drug delivery and hyperthermia.

The synthesis of monodispersed, and well separated, single iron oxide core–silica (SiO2) shell nanoparticles for biomedical applications is still a challenge. Substantial amount of aggregated and multicore CSNPs are generally the undesired outcome. In this thesis, synthesis of monodispersed, free of necking, single core iron oxide-SiO2 different distinct overall size and tuneable shell thickness was performed using an inverse microemulsion method. The influence of the reaction time, hydrodynamic conditions and precursor concentration on the synthesis process and thickness of the silica layer was investigated and the process was optimised. The residual reactions during the post synthesis processing were inhibited using a combination of pH adjustment and alternating shock freezing with ultracentrifuging.

The second part of the thesis is concerning thorough characterisation of the CSNPs with different shell thickness. The non-aggregated tuneable shell CSNPs maintained the superparamagnetic character of the cores with high magnetisation, showing great potential for their applications in nanomedicine. Magnetic measurements and relaxivity tests were performed and the comparison of the CSNPs with commercial products revealed the fact that relaxation time ratios (r2/r1) obtained are higher than those of the commercially available MRI contrast agents which indicates a better T2 contrast.

In the last part of the thesis the in-vitro toxicity investigation results are reported. For the investigation of cytotoxicity (3- 4, 5-dimethyldiazol-2-yl)-2, 5 diphenyl-tetrazolium bromide (MTT) assay was performed and the secretion of pro-inflammatory cytokines TNF-α and IL-6 was determined using enzyme-linked immunosorbent assay (ELISA). The cells were exposed to a wide range of concentrations of nanoparticles (between 0.5 μg/ml to 100 μg/ml). The cell toxicity results indicated no severe toxic effects on human monocyte-derived macrophages (HMDM) as model system. The internalisation of the nanoparticles by HMDM was monitored using transmission electron microscopy (TEM).

The CSNPs have the capacity of forming stable colloidal dispersions at physiological pH, with desired magnetic properties, low toxicity, and the potential for further functionalisation via surface modification of the silica shell or by adding new components (i.e. quantum dots, therapeutics). These characteristics make them highly promising for drug delivery, medical imaging, hyperthermia, magnetic cell marking and cell separation as well as many other biomedical applications.

 

QC 20100506

The objective of this research is to develop a series of material treatments and modifications, and, using a standardized set of tests, determine the extent of the ability of the modified material to enhance coagulation. This research focuses on materials commonly used in traditional textile based wound dressings; utilizing Streaming Potential studies, Scanning Electron Microscopy (SEM) and Thrombin Assays. The materials tested can be classified into 4 groups: control materials, modified PLA, SAMs treated glass, and TEOS treated materials. The control materials included: spun cotton and rayon yarn; continuous filament Nylon, Polypropylene (PP), and Polyethylene terephthalate (PET); heat cleaned glass (control glass); and PLA staple fibers. Contact angle measurements showed that both the control glass and the PET showed an increase in contact angle when treated with TEOS. This corresponds to a decrease and no improvement, respectively, in thrombogenicity for these materials in the thrombin assay. The remaining materials tested showed no change or a decrease in contact angle after TEOS treatment, and a corresponding increase in thrombogenicity. These results support previous studies that indicate an increase in wettability contributes to the enhancement of coagulation (16). While the streaming potential studies showed no correlation between thrombin formation or contact angle data, these tests provided an important launching platform for future studies utilizing the Streaming Potential Jar. Future work could benefit from the use of more physiologically relevant solutions, such as CaCl2, NaCl, or other blood substitutes (15). While no definitive correlations between test methods were elucidated, the results garnered from this research created a strong launching platform from which future materials research can continue.

The general objective of the present thesis is the study and development of low molecular weight gelators with photonic and biomedical applications. Low molecular weight gelators derived from amino acids containing fluorescent moieties will be synthesized and characterized. The energy transfer process occurring on a stimuli-responsive orthogonal two-component gelator will be studied, paving the way for possible light-harvesting applications. The construction of a smart versatile gel that present aggregation induced emission will be carried out. The aggregation mode showed to have crucial implications on the AIE phenomenon. Miniaturization of macroscopic molecular gels in nano- and micro-objects will be carried out. Biomedical applications of this new systems will be assessed. New hybrid materials containing molecular gels and upconverting nanoparticles will be studied. The energetic and structural relationship among them will be assessed resulting in potential applications in photodynamic therapy. 4-amino-1,8-naphthalimide chromophore in self-assembled systems will be studied as nitric oxide sensor.
L'objectiu general de la tesi és l'estudi i desenvolupament de molècules de baix pes molecular que s'auto-assemblen per a ser utilitzades en aplicacions fotòniques i biomèdiques. Es sintetitzaran i caracteritzaran noves famílies de gelants de baix pes molecular derivats d'aminoàcids que tenen fraccions fluorescents. S'avaluarà el procés de transferència d'energia que es duu a terme en gels moleculars reversibles auto-assemblats com a possible símil de sistemes naturals. S'estudiarà la construcció d'un nou gel versàtil que respongui als estímuls i que sofreix fenòmens d'emissió induïts per agregació degut a l'auto-organització en fibres. S'estudia la miniaturització de gels moleculars macroscòpics en nano i microgels moleculars. Es realitzarà un estudi fonamental d'un nou material híbrid basat en les relacions estructurals i energètiques entre dues àrees d'interès: Els gels moleculars fluorescents i les nanopartícules de conversió ascendent. S'estudia el comportament del cromòfor 4-amino-1,8-naftalimida en sistemes autoensamblats com a sensor d'òxid nítric (NO).

Nanostructured materials have attracted a broad interest for applications in scientific and engineering fields due to their extraordinary properties stemming from the nanoscale dimensions. This dissertation presents the development of nanomaterials used for different applications, namely biomedicine and dye lasing. Various inorganic nanoparticles have been developed as contrast agents for non-invasive medical imaging, such as magnetic resonance imaging (MRI) and X-ray computed tomography (CT), owing to their unique properties for efficient contrasting effect. Superparamagnetic iron oxide nanoparticles (SPIONs) are synthesized by thermo-decomposition method and phase-transferred to be hydrophilic used as MRI T2 (negative) contrast agents. Effects of surface modification of SPIONs by mesoporous silica (mSiO2) coating have been examined on the magnetic relaxivities. These contrast agents (Fe3O4@mSiO2) were found to have a coating-thickness dependent relaxation behavior and exhibit much higher contrast efficiency than that for the commercial ones. By growing thermo-sensitive poly(N-isopropylacrylamide -co-acrylamide) (P(NIPAAm-co-AAm)) as the outermost layer on Fe3O4@mSiO2 through free radical polymerization, a multifunctional core-shell nano-composite has been built up. Responding to the temperature change, these particles demonstrate phase transition behavior and were used for thermo-triggered magnetic separation. Their lower critical solution temperature (LCST) can be subtly tuned from ca. 34 to ca. 42 ˚C, suitable for further in vivo applications. An all-in-one contrast agent for MRI, CT and fluorescence imaging has been synthesized by depositing gadolinium oxide carbonate hydrate [Gd2O(CO3)2·H2O] shell on mSiO2-coated gold nanorod (Au NR), and then the particles were grafted with antibiofouling copolymer which can further link with the fluorescent dye. It shows both a higher CT and MRI contrast than the clinical iodine and gadolinium chelate contrast agent, respectively. Apart from the imaging application, owing to the morphology of Au NR, the particle has a plasmonic property of absorbing near-infrared (NIR) irradiation and suitable for future photothermal therapy. Cytotoxicity and biocompatibility of aforementioned nanoparticles have been evaluated and minor negative effects were found, which support their further development for medical applications. Gold nanoparticles embedded in the optical gain material, water solution of Rhodamine 6G (Rh6G) in particular, used in dye lasers can both increase and damp the dye fluorescence, thus, changing the laser output intensity. The studies of size effect and coating of gold nanoparticles on photostability of the gain media reveal that small sized (ca. 5.5 nm) gold nanoparticles are found detrimental to the photostability, while for the larger ones (ca. 25 nm) fluorescence enhancement rather than quenching is likely to occur. And a noticeable improvement of the photostability for the gain material is achieved when gold is coated with SiO2.
QC 20120605

Thiol-ene chemistry is versatile and efficient and can be used as a powerful tool in polymer synthesis. In this thesis, the concept of thiol-ene chemistry has been central, where it has been explored as a tool for the synthesis of well-defined hydrogels and dopa-functional materials towards biomedical applications; such as hydrogels, primers for adhesive fixation of bone fractures, self-healing gels, and micelles for drug-delivery. Using thiol-ene chemistry, well-defined hydrogels were realized in order to study how the structure influences properties such as swelling, stiffness and hydrolytic degradation. It was found that all these characteristics are related to each other, as a more loosely crosslinked hydrogel experiences higher swelling, lower stiffness and higher degradation rates. Dopa-functional materials have gained a lot of interest throughout the years due to the remarkable adhesive properties they possess in wet environments. In the pursuit of new primers towards thiol-ene functional crosslinked bone adhesives, compounds with dopa moieties were proposed. Primers derived from dopamine were found to enhance the adhesion towards bone, and it was concluded that addition of NaOH was essential to achieve good adhesion. The strongest adhesion was achieved when thiol and ene-functional primers were used in combination. Most synthetic routes to dopa-functional polymers involve several protection and deprotection steps and a more simplistic synthetic route is therefore desired. The possibility of using UV-initiated thiol-ene chemistry to produce dopa-functional polymers was therefore investigated. The resulting polymers were shown to exhibit self-healing properties upon complexation with Fe3+ ions. Finally, the developed synthetic route was used to produce dopa and allyl-functional triblock-co-polymers. These triblock-co-polymers were then used to form micelles and evaluated as drug-delivery vehicles for the cancer-drug doxorubicin. The micelles were found to have high drug-loading capacities and slow release profiles and showed promising results when evaluated against breast-cancer cells.
Reaktioner mellan tioler och omättade kemiska föreningar utgör ett mångsidigt och effektivt redskap inom polymersyntes. I denna avhandling har begreppet tiol-en kemi varit centralt och kemin har använts för syntes av såväl väldefinierade hydrogeler som dopa-funktionella material. Dessa material har sedan utvärderats mot biomedicinska tillämpningar såsom hydrogeler, primers för fixering av benfrakturer, självläkande geler och kontrollerad läkemedelsleverans. Tiol-en-kemi har i denna avhandling använts för att framställa väldefinierade hydrogeler som sedan utvärderats med avseende på hur strukturen påverkar egenskaper såsom svällningsgrad, styvhet och nedbrytningshastighet. Det visade sig att alla dessa egenskaper är relaterade till varandra och att lösare tvärbundna hydrogeler uppvisar högre svällning, lägre styvhet och högre nedbrytningshastigheter. Marina musslor har en exceptionell förmåga att fästa mot olika ytor och på grund av detta har det visats en hel del intresse för dopa-funktionella material genom åren. På jakt efter en primer för att öka vidhäftningen hos benlim proponerades därför föreningar med dopafunktionella grupper. Det visade sig att dopaminderivat kunde förbättra vidhäftningen mot ben och det visade sig även att tillsats av natriumhydroxid var viktigt för att uppnå god vidhäftningsförmåga. Den starkaste vidhäftning uppnåddes när derivat med tiol och omättade bindningar användes i kombination. Syntes av dopafunktionella material involverar ofta flera reaktionssteg och en förenklad syntesväg är därför att eftersträva. UV-initierad tiol-en-kemi undersöktes därför som en möjlig syntesväg för att framställa dopafunktionella polymerer. Polymererna visade sig ha självläkande egenskaper vid komplexbildning med järnjoner. Slutligen användes denna syntesväg för att framställa blocksampolymerer. Dessa blocksampolymerer användes sedan för att bilda miceller med lovande resultat vid utvärdering för leverans av läkemedel mot bröstcancer.

QC 20160125

Hybrid combinations of hydrogels and solid materials allow a high level of functionality for devices such as tissue engineering scaffolds and soft machines. This thesis reports a versatile strategy to develop mechanically robust solid-hydrogel hybrid materials using surface embedded radicals generated through plasma immersion ion implantation (PIII) of polymeric surfaces. Acrylamide and silk hydrogels were formed on PIII activated polytetrafluoroethylene (PTFE), polyethylene (PE) and polystyrene (PS) surfaces without any external crosslinking agents or initiators. X-ray photoelectron spectroscopy (XPS) and attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectrometry confirmed the formation of the acrylamide hydrogels. The amount of hydrogel formed on the substrate increased with incubation time, monomer concentration and temperature. Stability tests indicated that 95% of the hydrogel coating was retained even after 4-months of incubation in PBS solution. Adhesive T-peel tests results demonstrating adhesion strength of the hydrogel on the PIII-treated PTFE (PIII PTFE) interface of over 300-340 N/m. Hydrogels synthesised with fibronectin enabled cell adhesion and spreading. Thesis results show that polymers functionalized with surface-embedded radicals provide excellent solid platforms for the generation of robust and functional solid-hydrogel hybrid structures for biomedical applications.

Magnesium has been identified as a promising biodegradable implant material because it does not cause systemic toxicity and can reduce stress shielding. However, it corrodes too quickly in the body. Titanium, which is already used ubiquitously for implants, was chosen as the alloying element because of its proven biocompatibility and corrosion resistance in physiological environments. Thus, alloying magnesium with titanium is expected to improve the corrosion resistance of magnesium. Mg-Ti alloys with a titanium content ranging from 5 to 35 at.-% were successfully synthesized by mechanical alloying. Spark plasma sintering was identified as a processing route to consolidate the alloy powders made by ball-milling into bulk material without destroying the alloy structure. This is an important finding as this metastable Mg-Ti alloy can only be heated up to max. 200C° for a limited time without reaching the stable state of separated magnesium and titanium. The superior corrosion behavior of Mg80-Ti20 alloy in a simulated physiological environment was shown through hydrogen evolution tests, where the corrosion rate was drastically reduced compared to pure magnesium and electrochemical measurements revealed an increased potential and resistance compared to pure magnesium. Cytotoxicity tests on murine pre-osteoblastic cells in vitro confirmed that supernatants made from Mg-Ti alloy were no more cytotoxic than supernatants prepared with pure magnesium. Mg and Mg-Ti alloys can also be used to make novel polymer-metal composites, e.g., with poly(lactic-co-glycolic acid) (PLGA) to avoid the polymer’s detrimental pH drop during degradation and alter its degradation pattern. Thus, Mg-Ti alloys can be fabricated and consolidated while achieving improved corrosion resistance and maintaining cytocompatibility. This work opens up the possibility of using Mg-Ti alloys for fracture fixation implants and other biomedical applications.

The research described in this Dissertation is concerned generally with the exploration of the potential use of lanthanide elements in nanostructured materials for the purpose of modification of the magnetic and optical properties. This is explored through a focus on the development of lanthanide-containing iron oxide nanosystems. Our objectives of producing lanthanide containing nanostructured materials with potentially useful optical and magnetic applications has been achieved through the development of lanthanide-doped Fe3O4 and -Fe2O3 nanoparticles, as well as a unique core-shell magnetic-upconverting nanoparticle system.Necessary background information on nanomaterials, rationale for the study of lanthanide-containing iron oxide nanosystems and context for discussion of the results obtained in each project is provided in the Introduction Chapter. The syntheses of Fe3O4 nanoparticles doped with Eu(III) and Sm(III) are discussed, along with structural characterization and magnetic property investigation of products In Chapter 2. The following Chapter expands the study of lanthanide doping to -Fe2O3, a closely related yet distinct magnetic nanoparticle system. A completely different synthesis is attempted, and comparisons between the two systems are made.The development of novel synthetic methodologies used to create such products has yielded high-quality lanthanide-containing materials and are evidenced by TEM images displaying nearly monodisperse particles in each of our efforts. The modifications to the magnetic properties resulting from lanthanide doping include theobservation of ferromagnetism in the Fe3O4 system and increased magnetic saturation of -Fe2O3 nanoparticles, and are characterized by VSM and the visual observation of magnetic alignment of products. Our efforts towards developing a novel methodology capable of producing high quality Fe3O4 nanoparticles, and subsequent characterization of products, were published in the Journal of the American Chemical Society.Optically active, magnetic, core-shell nanoparticles are investigated in Chapter 4 for the potential uses in diagnosis and treatment of cancer. This multifunctional system uses Fe3O4 as a magnetic core, shelled by upconverting lanthanide-containing nanomaterials, and is rendered biocompatible through encapsulation of the core-shell structure by a silica shell. Added functionality is achieved through amine functionalization of the silica surface, with the goal of coupling the inorganic nanoparticle with drug targeting groups. TEM results indicate successful formation of the core-shell nanoparticles, and expected magnetic and optical properties are shown by visual observation and luminescence spectroscopy, respectively.

Orientador: Ruís Camargo Tokimatsu
Banca: Juno Gallego
Banca: Maria Aparecida Larosa
Resumo: Este trabalho tem por objetivo realizar uma análise das propriedades mecânicas e microestruturais de componentes da liga Ti-6Al-4V produzidos pelo processo de Sinterização Direta de Metal por Laser (DMLS) que se baseia na deposição de camadas de um pó metálico que posteriormente são fundidas através de um feixe de laser de alta potência. Sendo assim, foram produzidos componentes em três orientações diferentes (A, B e C) com o propósito de verificar a influência das camadas sobre as propriedades mecânicas e nos micromecanismos de fratura, através dos ensaios de tração, fadiga e tenacidade à fratura e microscopia eletrônica de varredura (MEV). As amostras também foram submetidas a um tratamento térmico para alívio de tensões residuais provenientes do processo de fabricação e posteriormente analisadas através de microscopia óptica e difração de raios-X, para averiguar se houve mudanças em sua microestrutura ou decomposição de fase em relação a uma amostra sem tratamento térmico na condição "como produzida". As orientações de construção B e C apresentaram melhores valores em todos os ensaios mecânicos quando comparados à orientação A, evidenciando assim que houve perda de resistência mecânica, exceto no ensaio de microdureza, onde os valores médios não tiveram grandes variações com a orientação de manufatura. A amostra "como produzida" apresentou maiores valores de dureza quando comparada às amostras com tratamento de alívio de tensões residuais, fato este que ocorreu devido à precipitação da fase B e crescimento da fase a no material, que inicialmente apresentava uma estrutura composta por martensita hexagonal (a')
Abstract: This work has as objective to realize analysis of the mechanical and microstructural properties of Ti-6Al-4V alloy components produced by the Direct Metal Laser Sintering (DMLS) process that is based on the deposition of a metal powder layers and posteriorly fused by a high power laser beam. Therefore, components were produced in three different orientations (A, B and C) in order to verify the influence of the layers deposition on the mechanical properties and fracture micromechanisms, through the tensile tests, fatigue and tenacity of fracture and scanning electron microscopy (SEM). The samples were also subjected to a heat treatment to relieve residual stresses resulting from the manufacturing process and after analyzed through a optical microscopy and X-ray diffraction, to ascertain if there were changes in the microstructure or phase decomposition in relation to a sample without a heat treatment and at "as manufactured" condition. The construction orientation B and C have had better values in all mechanical tests when compared to the orientation A, revealing that had a loss of mechanical strength, except in the microhardness test, where the average values did not have large variations in the orientation of manufacturing. The sample "as manufactured" presented higher hardness values when compared to samples with treatment to relieve the residual stress, and this fact occurred due the precipitation of the B phase and the growth of the a phase in the material, which initially had a structure consisting of martensite hexagonal (a')
Mestre

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Este trabalho tem por objetivo realizar uma análise das propriedades mecânicas e microestruturais de componentes da liga Ti-6Al-4V produzidos pelo processo de Sinterização Direta de Metal por Laser (DMLS) que se baseia na deposição de camadas de um pó metálico que posteriormente são fundidas através de um feixe de laser de alta potência. Sendo assim, foram produzidos componentes em três orientações diferentes (A, B e C) com o propósito de verificar a influência das camadas sobre as propriedades mecânicas e nos micromecanismos de fratura, através dos ensaios de tração, fadiga e tenacidade à fratura e microscopia eletrônica de varredura (MEV). As amostras também foram submetidas a um tratamento térmico para alívio de tensões residuais provenientes do processo de fabricação e posteriormente analisadas através de microscopia óptica e difração de raios-X, para averiguar se houve mudanças em sua microestrutura ou decomposição de fase em relação a uma amostra sem tratamento térmico na condição como produzida. As orientações de construção B e C apresentaram melhores valores em todos os ensaios mecânicos quando comparados à orientação A, evidenciando assim que houve perda de resistência mecânica, exceto no ensaio de microdureza, onde os valores médios não tiveram grandes variações com a orientação de manufatura. A amostra como produzida apresentou maiores valores de dureza quando comparada às amostras com tratamento de alívio de tensões residuais, fato este que ocorreu devido à precipitação da fase B e crescimento da fase a no material, que inicialmente apresentava uma estrutura composta por martensita hexagonal (a')
This work has as objective to realize analysis of the mechanical and microstructural properties of Ti-6Al-4V alloy components produced by the Direct Metal Laser Sintering (DMLS) process that is based on the deposition of a metal powder layers and posteriorly fused by a high power laser beam. Therefore, components were produced in three different orientations (A, B and C) in order to verify the influence of the layers deposition on the mechanical properties and fracture micromechanisms, through the tensile tests, fatigue and tenacity of fracture and scanning electron microscopy (SEM). The samples were also subjected to a heat treatment to relieve residual stresses resulting from the manufacturing process and after analyzed through a optical microscopy and X-ray diffraction, to ascertain if there were changes in the microstructure or phase decomposition in relation to a sample without a heat treatment and at as manufactured condition. The construction orientation B and C have had better values in all mechanical tests when compared to the orientation A, revealing that had a loss of mechanical strength, except in the microhardness test, where the average values did not have large variations in the orientation of manufacturing. The sample as manufactured presented higher hardness values when compared to samples with treatment to relieve the residual stress, and this fact occurred due the precipitation of the B phase and the growth of the a phase in the material, which initially had a structure consisting of martensite hexagonal (a')

Durante las últimas décadas la construcción de dispositivos basados en materiales moleculares funcionales se ha convertido en uno de los principales objetivos para la ciencia de los materiales. Aunque las propiedades fundamentales de dichos materiales (electrónicas, magnéticas, ópticas, mecánicas, etc) vienen determinadas por las propiedades de sus constituyentes moleculares, la funcionalidad final de dichos dispositivos vendrá determinada en gran medida por las diferentes técnicas de procesado y estructuración empleadas durante su construcción. En este contexto, el objetivo principal de esta tesis se centra en el uso de diferentes técnicas de procesado y estructuración de compuestos con actividad medioambiental y biomédica para el desarrollo de nuevos materiales funcionales. Los metales pesados, y especialmente el mercurio, son especies altamente tóxicas cuya presencia, debida a causas tanto naturales como antropogénicas, se ha visto incrementada de forma global generando una situación de riesgo no solo para el medio ambiente sino también para el ser humano. Así pues, el desarrollo de nuevos sensores capaces de llevar a cabo la detección sensible y selectiva de iones Hg(II) disuelto en medio acuoso supone un desafío para la ciencia actual. La primera parte de esta tesis doctoral se ha basado en el procesado y nano-estructuración de dos compuestos orgánicos (1 y 2) derivados del 2,3-diaza-1,3-butadieno capaces de llevar a cabo la detección selectiva de iones Hg(II) mediante métodos ópticos. Como resultado se han obtenido diversos sistemas heterogeneos capaces de detectar la presencia dichos iones en medios acuosos. El primero de ellos, basado en un proceso de fisisorción de los indicadores previamente descritos sobre una membrana de celulosa, dio como resultado el desarrollo de sondas sensoras de un solo uso y bajo coste para la detección de Hg(II). Dichas sondas fueron preparadas siguiendo dos técnicas de estructuración diferentes. La más intuitiva de las cuales, que denominamos "técnica de revelado", se basó en el uso de un receptor orgánico sensible a la presencia de iones Hg(II) como agente revelador de una membrana de celulosa previamente impregnada con la muestra contaminada. Aunque los resultados obtenidos usando el receptor colorimétrico 1 como agente de revelado mostraban buena selectividad y reproducibilidad, la sensibilidad de dicho sistema frente a los iones Hg(II) se pudo establecer en la decenas de ppm, lejos de las unidades de ppb establecidas por la Unión Europea (EU) y la Agencia de Protección Medioambiental Estadounidense (EPA) como máximo para la presencia de iones Hg(II) en agua potable. Sin embargo este proceso sirvió como una prueba de concepto para el desarrollo de sondas sensoras basadas en el uso de materiales baratos y renovables como la celulosa para la detección de contaminantes. El otro proceso de estructuración empleado involucra la producción y deposición de nanoparticulas de los receptores orgánicos 1 y 2 sobre la superficie de membranas nanoporosas de celulosa. La obtención de estas membranas híbridas supuso un importante incremento en la sensibilidad de las sondas obtenidas alcanzando (con aquellas basadas en el uso del receptor 2) los niveles de detección exigidos por la EU y la EPA (ppb de Hg(II)). En una segunda aproximación se llevó a cabo el anclaje químico de los receptores 3 y 4 sobre la superficie de un substrato sólido para la obtención de un sensor de Hg(II) de alto rendimiento. En este caso llevamos a cabo el desarrollo de un sensor basado en la resonancia superficial de plasmón (SPR) capaz de detectar la presencia de picomoles de iones Hg(II) en medios acuosos. El diseño racional de los receptores (3 y 4) se llevó a cabo para optimizar la sensibilidad, selectividad y fiabilidad del sensor, lo cual nos permitió mejorar los parámetros establecidos por la EPA y la EU en tres ordenes de magnitud. El creciente desarrollo de la medicina regenerativa en general y la regeneración de tejidos en particular ha traído consigo una enorme mejora de la calidad de vida para decenas de miles de personas por todo el mundo. Aunque la mayoría de los biomateriales usados hoy en día presentan la estructura y resistencia adecuada para ser usados en medicina regenerativa, la interacción de dichos materiales con el entorno biológico no se controla de forma completa aun, lo cual genera, en algunos casos, efectos secundarios indeseados. El trabajo desarrollado en esta segunda parte de la tesis se centra en el estudio, caracterización y procesado de un nuevo tipo de material proteinaceo nano-particulado conocido como cuerpos de inclusión (IBs). La primera parte de esta investigación se centró en la caracterización nanoscópica de las propiedades fisico-químicas y estructurales de esta nueva familia de agregados, conocidos como IBs. Así pues, IBs provenientes de diferente trasfondo genético fueron caracterizados mediante diferentes técnicas como la dispersión dinámica de luz (DLS) la microscopia de fuerza atómica (AFM) o el ángulo de contacto (CA). Los resultados obtenidos indicaron que los IBs producidos en ausencia de diferentes elementos de la maquinaria de control de shock térmico celular (genes de la DnaK, ClpA y ClpP) exhiben una diferente distribución de tamaños, y propiedades fisico-químicas. De esta manera es posible concluir que existe una relación directa entre la conformación de las proteínas recombinantes que forman los IBs y sus propiedades. Una distribución aleatoria de IBs con diferente trasfondo genético se usó para decorar una serie de superficies químicamente modificadas con grupos amino, las cuales fueron sometidas a diferentes ensayos de proliferación celular obteniendo diferentes resultados según el origen genético de los IBs empleados. Dicho experimento probó que es posible utilizar los IBs para modificar las superficies de los materiales con objeto de obtener diferentes comportamientos de proliferación celular, expandiendo el posible uso de dichos materiales para aplicaciones en regeneración de tejidos.
During the last decades the construction of devices based on molecular functional materials with specific properties has become one of the major objectives of materials scientists, since they can offer new and exciting functionalities to the present human activities. Although their basic properties will be guided by the fundamental -electronic, magnetic, optical, mechanical, etc- properties of their molecular constituent units, the final functionality of a device will depend, in a major way, on the processing and structuring techniques used during its construction. In this context, the main objective of this Thesis has been the use of different processing and structuring techniques for the development of new functional materials based on already tested environmentally and biologically active compounds. Among all the environmentally hazardous substances present in our environment, heavy metal ions, and specially mercury, are highly toxic elements which contamination, due to both natural and anthropogenic reasons, has become severe in some parts of the world, resulting in health damage to their inhabitants. Therefore, the developing of new sensors able to detect selectively and sensitively Hg2+ on aqueous media is still an actual challenge. In this work we present two 1,4-disubstituted-2,3-diaza-1,3-butadiene derivatives (1 and 2) able to selectively perform optical detection of Hg2+ in aqueous media, that combined with different nanostructuring and anchoring techniques allowed us to obtain highly sensitive solid-supported mercury detection systems. The first of them is based on the physisorption of the diaza butadiene indicators on porous cellulose membranes obtaining indicator coated probes that could be used as new cheap and reliable Hg2+ sensing systems. In order to do that, two different structuring techniques have been used. The most intuitive one, which we have named “developing technique”, is founded on the use of the optically active Hg2+ organic receptor 1 as a Hg2+ developing agent of a cellulose substrate, previously impregnated with the contaminated solution. Although Hg2+ detection tests performed using this colorimetric chemosensing probes, based on receptor 1, showed good selectivity and reproducibility, they presented a limited sensitivity vs. Hg2+. The detection limit of the probes was set on tens of ppm (10−2g/l), far away from the 1 ppb (μg/l) fixed by the European Union (EU) and the North American Environmental Protection Agency (EPA) as the maximum amount of Hg2+ allowed in drinkable water. Nevertheless, this procedure served as a prove of concept for the developing of probes based on the use of cheap and renewable materials to be applied on the in situ detection of contaminants. The other structuring technique used is based on a new physisorption procedure, involving the production and deposition of nanoparticles of the organic sensing molecules on nanoporous cellulose membranes for the fabrication of hybrid membranes. In this case, excellent Hg2+ detection results showing a high Hg2+ sensitivity and selectivity were obtained for the receptor 2 based cellulose probes. In contrast to the previous case, the detection limit obtained matched the EU and EPA requirements for drinkable water, reaching the level of ppb (μg/l). On a second approach the covalent bonding was used as a driving force for the receptor anchoring onto a solid substrate. In this case we developed a surface plasmon resonance (SPR) sensor able to perform picomolar detection of Hg2+ on aqueous systems. The rational design of the Hg2+ receptors (3 and 4) optimizes the sensitivity and reliability of the sensor allowing us to selectively detect, in presence of other divalent cations, Hg2+ concentrations on aqueous systems on the picomolar range, meliorating on three orders of magnitude the EU and EPA Hg2+ detection limit on drinkable water. As contamination control and pollutant removal, regenerative medicine in general and particularly in tissue engineering (TE) has the enormous potential of improving the quality of life for many thousands of people throughout the world. Although most of the more commonly used biomaterials match all the structural and mechanical resistance requirements to be applied in regenerative medicine, the interaction of such materials with the surrounding biological media is still not well controlled, leading to undesired immunological responses such as infections or uncontrolled inflammation in some cases. The work developed on the second part of this thesis has been focused on the study, characterization and processing of a new kind of proteinaceous nanoparticulate biomaterial, known as inclusion bodies (IBs), as a promising additive for cell proliferation enhancement. The first part of the research regarding the processing and structuring of biologically active materials is centered on the characterization of the nanoscale, physicochemical and structural properties of a novel family of proteinaceous aggregates known as “inclusion bodies” (IBs). Thus, IBs coming from different genetic backgrounds have been characterized by means of light dispersion and surface analysis techniques, such as dynamic light scattering (DLS), atomic force microscopy (AFM) or contact angle (CA). Results obtained indicated that IBs produced in absence of different elements of the cellular heat shock machinery (DnaK, ClpA, and ClpP genes) exhibit a range of sizes, wettability and stiffness values, that let us conclude the existence of a direct relationship between the conformation status of the recombinant proteins inside the IBs and their physicochemical and structural properties. Randomly distributed IBs, from different genetic backgrounds, were used to decorate amine terminated silicon surfaces. It was possible to observe how cultured mammalian cells respond differentially to IB variants when used as particulate materials to engineer the physicochemical surface properties, proving that the actual range of referred mechanical as well as other physicochemical properties is sensed and discriminated by biological systems. To further prove the validity of IBs as stimulator of cell proliferation, microstructuring of the IBs onto the same substrate was performed using the Microcontact Printing (μCP) technique. The obtained results confirmed again the ability of IBs to stimulate cell proliferation on surfaces initially not suitable for cell growth. Therefore, it is possible to conclude that the tuning opportunities offered through adjusting the genetic background of the cell where the IBs are produced, definitively expands the spectrum of biomedical applications of this novel bacterial nanomaterial.

Surface modification of polymers has long been known in polymer chemistry but has not yet been widely applied to biomaterials. A newly developed active screen plasma technology has a potential to treat such non-conductive materials as polymers to improve their surface properties since this is a low-temparature, low cost and environmentally friendly plasma process. in this project, three kind of polymeric materials, ultra high molecular weight polyethylene, polyurethane and polycaprolacton, were surface modified using active screen plasma nitriding technology. The results demonstrated that it is feasible to conduct plasma surfae modification of polymeric materials using the newly developed active-screen plasma technology without causing any etching, significant sputtering or other surface damage. Changes in chemical composition and structure have been found an all three polymeric materials' surface following active screen plasma surface treatments. Crosslinking or/and new functional groups are formed on the topmost surface layer after the treatment. Along with changes in surface morphologies and structural, the wettability of the surface of all three polymeric materials can also be effectively improved by the active screen plasma nitriding treatments. Active-screen plasma nitriding technique is an effective and practical method to improve osteoblast cell adhesion and spreading on all three polymeric materials' surface.

Metallic materials have gained much attention recently from the areas of medical devices and orthopaedics. Artificial organs, dental implants, prostheses and implants that replace damaged or malfunctioning parts in the body are, or contain, metal components. Our ageing society poses an increased demand to provide devices and implants that can demonstrate better performance than those presented by traditional solutions. Matching the mechanical properties (i.e. stiffness and strength) of the device to those of the host tissue is a major challenge for the design and manufacture of engineered metal materials for biomedical applications. Failure in doing so provokes implant loosening, patient discomfort and repeated surgeries. Therefore, tailoring physical properties and biocompatibility of those materials is the main final aim of this research programme. This PhD study has focused on the tailoring of the mechanical properties of titanium-based materials and titanium-based alloys. Titanium inertness and the selection of biocompatible alloying elements were set as the baseline. Two approaches were employed to decrease stiffness (i.e. Young s modulus): one, by introducing porosity in a titanium matrix and therefore, reduce its Young s modulus, and two, by designing and manufacturing beta-titanium-based alloys with a reduced Young s modulus. Titanium scaffolds were manufactured using powder metallurgy with space holder technique and a sintering process. Different space holder sizes were used in four different categories to study the effect of pore size and porosity on the mechanical properties of the porosity engineered Ti scaffolds. Ti-based alloys were manufactured using manufacturing techniques such as sintering and arc-melting. The effect of different fabrication processes and the addition of beta-stabilising elements were studied and investigated. The obtained results of mechanical properties for pore size and porosity were within the values that match bone properties. This means these materials are suitable for biomedical application and the beta-Ti alloys results show that the mechanical properties can be decreased via tailoring the crystal structures. The characterisation of the Ti-based alloys helps to develop this material for its use in biomedical application.

The possibility to tune material properties up to nanoscale represents a great opportunity for the scientific community to obtain devices capable to fulfill the always new medical demands. During this Thesis project micro and nano-structured polymeric materials have been used in the field of drug delivery and tissue engineering. Three different research lines have been explored: (i) the use of polymeric fibrous systems as drug and nanoparticles carriers, (ii) design and evaluation of novel shape memory polymers to produce shape memory scaffolds and (iii) development of smart affinity membranes. Electrospinning were exploited to obtain polymeric fibrous carriers made of different biodegradable and bioresorbable polymers, such us Poly(lactic-acid) and Poly(lactic-co-glycolic). The obtained biodegradable carriers have been exploited to achieve controllable particles release as well as, to obtain composites capable to deliver two drugs simultaneously with controllable and predictable kinetics. The possibility to obtain electrospun scaffolds capable of interconverting between a temporary and a permanent shape with the application of a thermal stimulus was explored. In this context, two polymers have been designed to behave as shape memory materials in the range of human body temperature. Finally smart affinity membranes have been studied. This kind of materials are capable to detect specific molecules or biomacromolecules from complex mixtures, finding useful applications in the biomedical field as diagnostic and therapeutic devices. Smart affinity membranes might be used for example to detect specific kind of cells by exploiting the binding interaction between an antibody and cell receptors. During this thesis project poly(L-lactic acid) electrospun scaffolds conjugated with antibodies have been produced and the efficacy of different functionalization methods to generate the –COOH group necessary to perform the antibodies conjugation was investigated.

Biomedical materials are being extensively researched, and many different types such as metals, metal alloys, and polymers are being used. Currently used biomedical materials are not perfect in terms of corrosion resistance, biocompatibility, and surface properties. It is not easy to fabricate from scratch new materials that can fulfill all requirements and an alternative approach is to modify the surface properties of current materials to cater to the requirements. Plasma immersion ion implantation (PIII) is an effective and economical surface treatment technique and that can be used to enhance the surface properties of biomaterials. The unique advantage of plasma modification is that the surface properties and functionalities can be enhanced selectively while the favorable bulk attributes of the materials such as strength remain unchanged. In addition, the non-line of sight feature of PIII is appropriate for biomedical devices with complex geometries such as orthopedic implants. However, care must be exercised during the plasma treatment because low-temperature treatment is necessary for heat-sensitive materials such as polymers which typically have a low melting point and glass transition temperature. Two kinds of biomedical materials will be discussed in this thesis. One is nickel titanium (NiTi) alloy which is a promising orthopedic implant material due to its unique shape memory and superelastic properties. However, harmful ions may diffuse from the surface causing safety hazards. In this study, we investigate the properties and performance of NiTi after nitrogen and oxygen PIII in terms of the chemical composition, corrosion resistance, and biocompatibility. The XPS results show that barrier layers mainly containing TiN and TiOx are produced after nitrogen and oxygen PIII, respectively. Based on the simulated in vitro and electrochemical corrosion tests, greatly reduced ion leaching and improved corrosion resistance are accomplished by PIII. Porous NiTi is also studied because the porous structure possesses better bone ingrowth capability and compatible elastic modulus with human bones. These advantages promote better recovery in patients. However, higher risks of Ni leaching are expected due to the increased exposed surface area and rougher topography than dense and smooth finished NiTi. We successfully apply PIII to porous NiTi and in vitro tests confirm good cytocompatibility of the materials. The other type of biomedical materials studied here is ultra-high molecular weight polyethylene (UHMWPE) which is a potential material for use in immunoassay plates and biosensors. In these applications, active antibodies or enzymes attached to a surface to detect molecules of interests by means of specific interactions are required. Moreover, the retention of enzyme activity is crucial in these applications. Therefore, the aim of this study is to investigate the use of PIII to prepare UHMWPE surfaces for binding of active proteins in terms of the binding density and ‘shelf life’ of the treated surfaces. Argon and nitrogen PIII treatments are attempted to modify the surface of UHMWPE. Horseradish peroxidase (HRP) is selected to conduct the protein binding test since it is a convenient protein to assay. Experimental results show that both PIII treated surfaces significantly improve the density of active HRP bound to the surface after incubation in buffer containing HRP. Furthermore, the PIII treated surfaces are found to perform better than a commercially available protein binding surface and the shelf life of the PIII treated surfaces under ambient conditions is at least six months. In conclusion, a biocompatible barrier layer on NiTi and a protein binding surface on UHMWPE is synthesized by PIII. The surface properties such as corrosion resistance and functionality on these two different types of substrates are improved by PIII.

Doctor of Philosophy(PhD)
Biomedical materials are being extensively researched, and many different types such as metals, metal alloys, and polymers are being used. Currently used biomedical materials are not perfect in terms of corrosion resistance, biocompatibility, and surface properties. It is not easy to fabricate from scratch new materials that can fulfill all requirements and an alternative approach is to modify the surface properties of current materials to cater to the requirements. Plasma immersion ion implantation (PIII) is an effective and economical surface treatment technique and that can be used to enhance the surface properties of biomaterials. The unique advantage of plasma modification is that the surface properties and functionalities can be enhanced selectively while the favorable bulk attributes of the materials such as strength remain unchanged. In addition, the non-line of sight feature of PIII is appropriate for biomedical devices with complex geometries such as orthopedic implants. However, care must be exercised during the plasma treatment because low-temperature treatment is necessary for heat-sensitive materials such as polymers which typically have a low melting point and glass transition temperature. Two kinds of biomedical materials will be discussed in this thesis. One is nickel titanium (NiTi) alloy which is a promising orthopedic implant material due to its unique shape memory and superelastic properties. However, harmful ions may diffuse from the surface causing safety hazards. In this study, we investigate the properties and performance of NiTi after nitrogen and oxygen PIII in terms of the chemical composition, corrosion resistance, and biocompatibility. The XPS results show that barrier layers mainly containing TiN and TiOx are produced after nitrogen and oxygen PIII, respectively. Based on the simulated in vitro and electrochemical corrosion tests, greatly reduced ion leaching and improved corrosion resistance are accomplished by PIII. Porous NiTi is also studied because the porous structure possesses better bone ingrowth capability and compatible elastic modulus with human bones. These advantages promote better recovery in patients. However, higher risks of Ni leaching are expected due to the increased exposed surface area and rougher topography than dense and smooth finished NiTi. We successfully apply PIII to porous NiTi and in vitro tests confirm good cytocompatibility of the materials. The other type of biomedical materials studied here is ultra-high molecular weight polyethylene (UHMWPE) which is a potential material for use in immunoassay plates and biosensors. In these applications, active antibodies or enzymes attached to a surface to detect molecules of interests by means of specific interactions are required. Moreover, the retention of enzyme activity is crucial in these applications. Therefore, the aim of this study is to investigate the use of PIII to prepare UHMWPE surfaces for binding of active proteins in terms of the binding density and ‘shelf life’ of the treated surfaces. Argon and nitrogen PIII treatments are attempted to modify the surface of UHMWPE. Horseradish peroxidase (HRP) is selected to conduct the protein binding test since it is a convenient protein to assay. Experimental results show that both PIII treated surfaces significantly improve the density of active HRP bound to the surface after incubation in buffer containing HRP. Furthermore, the PIII treated surfaces are found to perform better than a commercially available protein binding surface and the shelf life of the PIII treated surfaces under ambient conditions is at least six months. In conclusion, a biocompatible barrier layer on NiTi and a protein binding surface on UHMWPE is synthesized by PIII. The surface properties such as corrosion resistance and functionality on these two different types of substrates are improved by PIII.

The bioengineer has a choice of building with proteins, peptides, polymers, nucleic acids, lipids, metals and minerals, each class containing tremendous diversity within its category. While the platforms are diverse, they can be unified by a common goal: to engineer nano- and micro-scale order to improve functionality. In doing so, self-assembling systems aim to bring the lessons learned from the order in natural systems83 into the therapeutics, materials, and electronics that society uses every day. The rigid geometry and tunable chemistry of D,L-cyclic peptides make them an intriguing building-block for the rational design of nano- and microscale hierarchically structured materials. Herein, we utilize a combination of electron microscopy, nanomechanical characterization including depth sensing-based bending experiments, and molecular modeling methods to obtain the structural and mechanical characteristics of cyclo-[(Gln-D-Leu)4] (QL4) assemblies. QL4 monomers assemble to form large, rod-like structures with diameters up to 2 μm and lengths of 10s to 100s of μm. Image analysis suggests that large assemblies are hierarchically organized from individual tubes that undergo bundling to form larger structures. With an elastic modulus of 11.3 ± 3.3 GPa, hardness of 387 ± 136 MPa and strength (bending) of 98 ± 19 MPa the peptide crystals are among the most robust known proteinaceous micro- and nano-fibers. The measured bending modulus of micron-scale fibers (10.5 ± 0.9 GPa) is in the same range as the Young’s modulus measured by nanoindentation indicating that the robust nanoscale network from which the assembly derives its properties is preserved at larger length-scales. Materials selection charts are used to demonstrate the particularly robust properties of QL4 including its specific flexural modulus in which it outperforms a number of biological proteinaceous and non-proteinaceous materials including collagen and enamel. We then demonstrate a composite approach to mechanical reinforcement of polymeric systems by incorporating synthetic D,L-cyclic peptide nanotube bundles as a structural filler in electrospun poly D-, L-lactic acid fibers. With 8 wt% peptide loading, the composite fibers are >5-fold stiffer than fibers composed of the polymer alone, according to AFM-based indentation experiments. The facile synthesis, high modulus, and low density, and reinforcing capabilities of QL4 fibers indicate that they may find utility as a filler material in a variety of high efficiency, biocompatible composite materials. This study represents the first experimental mechanical characterization of D,L-cyclic peptide assemblies or composites.
Engineering and Applied Sciences - Engineering Sciences

A very interesting class of nanoparticles extensively used for bio-applications is that of hydrogel particles, also called nanogels. There is an increasing interest in the design of hydrogel nanoparticles that have biofunctionality for applications in cell targeting, drug delivery, and biomedicine. The dissertation focuses on developing synthetic strategies for making diverse hydrogel nanoparticles customized to have desirable properties for various bio-applications. We have also investigated the potential of such nanoparticles as coatings for biomedical implants. Chapter 1 gives a brief introduction to hydrogel nanoparticles and the properties that make them attractive for various applications. The details of the syntheses of well defined, stable nanoparticles, commonly used in literature, are described in Chapter 2. Chapter 3 describes our synthesis of hollow sub-50 nm nanogels, which are otherwise difficult to synthesize based on the strategy discussed in Chapter 2. Chapter 4 also demonstrates how simple strategies borrowed from organic chemistry help in producing nanogels with multiple functionalities that are otherwise difficult to obtain, which also is an important advance over the synthetic methods discussed in Chapter 2. Chapter 5 describes how a general strategy based on photoaffinity labeling can yield materials with many applications ranging from optical materials, drug delivery, to biosensing. The latter part of the dissertation describes applications of various nanogels in biology especially as coatings that can control inflammation caused by biomaterials. Chapter 6 describes a method to functionalize flexible biomaterials with the nanogels, thus enabling in vivo investigations of the nanogels as potential coatings for controlling inflammation. Chapter 7 describes the biological studies performed (in collaboration with Garcia Group in the School of Mechanical Engineering at Georgia Tech) on various nanogels, aimed towards obtaining the most functional and efficient materials for implant applications. Chapter 8 describes application of hollow nanogels for covalently immobilizing biomolecules. This chapter also demonstrates how simple non-functional materials can be made unique and functional by means of traditional organic reactions. Finally, in order to broaden the applications of nanogel based materials.