Biomaterials are commonly used in the medical clinic today; however, artificial materials can activate the cascade systems in the blood (complement-, coagulation-, contact- and fibrinolytic systems) as well as the platelets to various degrees. When an artificial surface comes in contact with blood, plasma proteins will be adsorbed to the surface within seconds. The composition of the layer of proteins differs between materials and is crucial for the hemocompatibility of the material. This thesis includes five projects. In Paper I the anticoagulants heparin and the thrombin inhibitor hirudin were evaluated in a whole blood model. Hirudin was found to be superior to low dose heparin since it did not affect the activation of the complement system nor the leukocytes. The most interesting observation was that expression of TF was seen on surface-attached monocytes in hirudin- treated blood but not heparin blood. In Paper II peptides from the streptococcal M-protein, which has affinity for the human complement inhibitor C4BP, were attached to a polymeric surface. When being exposed to blood the endogenous complement regulator was enriched at the surface of the material, via the M-peptides. With this new approach we created a self-regulatory surface, showing significant lowered material-induced complement activation. In Paper III apyrase, an enzyme which hydrolyzes nucleoside ATP and ADP, was immobilized on a polymer surface. Lower platelet activation and platelet-induced coagulation activation was seen for the apyrase-coated surface compared to control surfaces after exposure to whole human blood, due to the enzymes capability to degrade ADP released from activated platelets. In Paper IV and V we synthesized an array of polymeric materials which were characterized regarding physical-chemical properties, adsorption of plasma proteins, and hemocompatibility. The polymers showed widely heterogeneous protein adsorption. Furthermore, when the polymers were exposed to whole blood, two of the materials showed superior hemocompatibility (monitored as complement- and coagulation activation), compared to the reference poly(vinyl chloride).

In the field of biomaterials, naturally-derived and synthetic polymers are utilized individually or in combination with each other, to create bio-inspired or biomimetic materials for various bioengineering applications, including drug delivery and tissue engineering. Natural polymers, such as proteins and polysaccharides, are advantageous due to low or non-toxicity, sustainable resources, innocuous byproducts, and cell-instructive properties. Synthetic polymers offer a variety of controlled chemical and physical characteristics, with enhanced mechanical properties. Together, natural and synthetic polymers provide an almost endless supply of possibilities for the development of novel, smart materials to resolve limitations of current materials, such as limited resources, toxic components and/or harsh chemical reactions. Herein is discussed the synthetic-biological material formation for cell-instructive tissue engineering and controlled drug delivery. We hypothesized that the combination of hydrogel-based scaffold and engineered nanomaterials would assist in the development or regeneration of tissue and disease treatment. Chemically-modified alginate was formed into alginate-based nanoparticles (ABNs) to direct the intracellular delivery of proteins (e.g., growth factors) and small molecular drugs (e.g., chemotherapeutics). The ABN surface was modified with cell-targeting ligands to control drug delivery to specific cells. The ABN approach to controlled drug delivery provides a platform for studying and implementing non-traditional biological pathways for disease (e.g., osteoporosis, multiple sclerosis) and cancer treatment. Through traditional organic and polymer chemistry techniques, and materials engineering approaches, a stimuli-responsive alginate-based smart hydrogel (ASH) was developed. Physical crosslinks formed based on supramolecular networks consisting of β-cyclodextrin-alginate and a tri-block amphiphilic polymer, which also provided a reversible thermo-responsiveness to the hydrogel. The hydrogel was shear-thinning, and recovered physical crosslinks, i.e., self-healed, after un-loading. The ASH biomaterials provide a platform for injectable, therapeutics for tissue regeneration and disease treatment. Indeed, various hydrogel constituents and tunable mechanical properties created cell-instructive hydrogels which promoted tissue formation.

Electrospinning is a technique used to generate scaffolds composed of nano- to micron-sized fibers for use in tissue engineering. This technology possesses several key weaknesses that prevent it from adoption into the clinical treatment regime. One major weakness is the lack of porosity exhibited in most electrospun scaffolds, preventing cellular infiltration and thus hosts tissue integration. Another weakness seen in the field is the inability to physically cut electrospun scaffolds in the frontal plane for subsequent microscopic analysis (current electrospun scaffold analysis is limited to sectioning in the cross-sectional plane). Given this it becomes extremely difficult to associate spatial scaffold dynamics with a specific cellular response. In an effort to address these issues the research presented here will discuss modifications to electrospinning technology, cryosectioning technology, and our understanding of cellular infiltration mechanisms into electrospun scaffolds. Of note, the hypothesis of a potentially significant passive phase of cellular infiltration will be discussed as well as modifications to cell culture protocols aimed at establishing multiple passive infiltration phases during prolonged culture to encourage deep cellular infiltration.

Dendrimers are a class of polymers with a highly branched, three-dimensional architecture comprised of an initiator core, several interior layers of repeating units, and multiple active surface terminal groups. Dendrimers have been recognized as the most versatile compositionally and structurally controlled nanoscale building blocks for drug and gene delivery. Polyamidoamine (PAMAM) dendrimers have been most investigated because of their unique structures and properties. Polycationic PAMAM dendrimers form compacted polyplexes with nucleic acids at physiological pH, holding great potential for gene delivery. Folate receptor (FRα) is expressed at very low levels in normal tissues but expressed at high levels in cancers in order to meet the folate demand of rapidly dividing cells under low folate conditions. Our primary aim was to investigate folic acid (FA)-conjugated PAMAM dendrimer generation 4 (G4) conjugates (G4-FA) for targeted gene delivery. The in vitro cellular uptake and transfection efficiency of G4-FA conjugates and G4-FA/DNA polyplexes were investigated in Chapter 4. It was found the cellular uptake of G4-FA conjugates and G4-FA/DNA polyplexes was in a FR-dependent manner. Free FA competitively inhibited the cellular uptake of G4-FA conjugates and G4-FA/DNA polyplexes. G4-FA/DNA polyplexes were preferentially taken up by FR-positive HN12 cells but not FR-negative U87 cells. In contrast, the cellular uptake of G4 dendrimers and G4/DNA polyplexes was non-selective via absorptive endocytosis. G4-FA conjugates significantly enhanced cytocompatibility and transfection efficiency compared to G4 dendrimers. This work demonstrates that G4-FA conjugates allow FR-targeted gene delivery, reduce cytotoxicity, and enhance gene transfection efficiency. The in vivo biodistribution of G4-FA conjugates and anticancer efficacy of G4-FA/siRNA polyplexes were investigated in Chapter 5. Vascular endothelial growth factor A (VEGFA) is one of the major regulators of angiogenesis, essential for the tumor development. It was found G4-FA/siVEGFA polyplexes significantly knocked down VEGFA mRNA expression and protein release in HN12 cells. In the HN12 tumor-bearing nude mice, G4-FA conjugates were preferentially taken up by the tumor and retained in the tumor for at least 21 days following intratumoral (i.t.) administration. Two-dose i.t. administration of G4-FA/siVEGFA polyplexes significantly inhibited tumor growth by lowering tumor angiogenesis. In contrast, two-dose i.t. administration of G4/siVEGFA polyplexes caused severe skin lesion, presumably as a result of local toxicity. Taken together, this work shows great potential for the use of G4-FA conjugates in targeted gene delivery and cancer gene therapy. We also explored polyanionic PAMAM dendrimer G4.5 as the underlying carrier to carry camptothecin (CPT) for glioblastoma multiforme therapyin Chapter 6. "Click" chemistry was applied to improve polymer-drug coupling reaction efficiency. The CPT-conjugate displayed a dose-dependent toxicity with an IC50 of 5 μM, a 185-fold increase relative to free CPT, presumably as a result of slow release. The conjugated CPT resulted in G2/M arrest and cell death while the dendrimer itself had little to no toxicity. This work indicates highly efficient "click" chemistry allows for the synthesis of multifunctional dendrimers for sustained drug delivery. Immobilizing PAMAM dendrimers to the cell surface may represent an innovative method of enhancing cell surface loading capacity to deliver therapeutic and imaging agents. In Chapter 7, macrophage RAW264.7 (RAW) was hybridized with PAMAM dendrimer G4.0 (DEN) on the basis of bioorthogonal chemistry. Efficient and selective cell surface immobilization of dendrimers was confirmed by confocal microscopy. It was found the viability and motility of RAW-DEN hybrids remained the same as untreated RAW cells. Furthermore, azido sugar and dendrimer treatment showed no effect on intracellular AKT, p38, and NFκB (p65) signaling, indicating that the hybridization process neither induced cell stress response nor altered normal signaling. This work shows the feasibility of applying bioorthogonal chemistry to create cell-nanoparticle hybrids and demonstrates the noninvasiveness of this cell surface engineering approach. In summary, these studies indicate surface-modification of PAMAM dendrimer G4 with FA can effectively target at FR-positive cells and subsequently enhance in vitro transfection efficiency and in vivo gene delivery. G4-FA conjugates may serve as a versatile targeted gene delivery carrier potentially for cancer gene therapy. PAMAM dendrimers G4.5 may serve as a drug delivery carrier for the controlled release of chemotherapeutics. The immune cell-dendrimer hybrids via bioorthogonal chemistry may serve as an innovative drug and gene delivery carrier potentially for cancer chemotherapy. Taken together, engineering of PAMAM dendrimers may advance anticancer drug and gene delivery.

The aim of this study was to investigate macrophage interactions with electrospun scaffolds and quantify the expression of vital angiogenic growth factors in vitro. This study will further help in evaluating the potential of these electrospun constructs as vascular grafts for tissue repair and regeneration in situ. Human peripheral blood macrophages were seeded in serum free media on electrospun (10 mm) discs of polydioxanone (PDO), elastin and PDO:elastin blends (50:50, 70:30 and 90:10). The growth factor secretion was analyzed by ELISA. Macrophages produced high levels of vascular endothelial growth factor (VEGF) and acidic fibroblast growth factor (aFGF). Transforming growth factor beta-1 (TGF-β1) secretion was relatively low and there was negligible production of basic fibroblast growth factor (bFGF). Histology revealed direct correlation between cell infiltration into scaffolds and the PDO concentration. There was greater macrophage infiltration through fibrous networks of the PDO and 90:10 scaffolds. Therefore, it can be anticipated that these scaffolds will support tissue regeneration and angiogenesis.

Student Number : 9501020M - M Sc dissertation - School of Clinical Medicine - Faculty of Health Sciences / An exciting and novel concept of tissue engineering and morphogenesis is the generation of bone by the implantation of smart biomaterials that in their own right can induce a desired and specific morphogenetic response from the host tissues without the addition of exogenously applied bone morphogenetic and osteogenic proteins.

A quitosana é um polissacarídeo produzido pela desacetilação da quitina e tem sido estudada para aplicação como biomaterial por apresentar características, tais como: biocompatibilidade, atoxidade e ação antimicrobiana. O objetivo deste estudo foi o desenvolvimento e caracterização química, física e biológica de membranas à base de quitosana (QUI) e carboximetilcelulose (CMC) para aplicação na área biomédica. Foram realizados ensaios de resistência, capacidade de absorção de água e perda de massa em solução salina de tampão fosfato (PBS) e saliva artificial, infravermelho por transformada de Fourier (FTIR), calorimetria exploratória diferencial (DSC), bioatividade, citoxicidade e degradação. Nos ensaios de citotoxidade, observou-se que as membranas desenvolvidas são atóxicas e nos ensaios de absorção de água que a carboximetilcelulose aumentou significamente a capacidade de absorção da membrana. As características naturais dos polímeros não foram alteradas, como observado no ensaio térmico (DSC) e no FTIR. Referente à liberação da lisozima, as membranas de QUI e QUI/CMC/QUI obtiveram melhores resultados, tanto na liberação quanto na atividade da enzima. As principais aplicações das membranas estudadas na área biomédica são como bandagens. / Chitosan is a polysaccharide produced from chitin by deacetylation and has been studied for application as biomaterial for presenting characteristics, such as biocompatibility and antimicrobial atoxidade. The objective of this study was the development and characterization chemical, physical and biological of membranes based on chitosan and carboxymethylcellulose (CMC) for application in the biomedical area. Tests of resistance, water absorption capacity and mass loss in phosphate buffered saline (PBS) and artificial saliva, Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), bioactivity and cytotoxicity. The tests of cytotoxicity revealed that the membranes developed are non toxic and water absorption test the CMC increased significantly absorption capacity of membrane. The natural characteristics of the polymers were not changed, as observed on DSC and FTIR. With regard to the release of lysozyme, membranes CHI and CHI/CMC/CHI obtained better results in both the release and in the activity of the enzyme. The main applications of the membranes studied in the biomedical field are like bandages.

O desenvolvimento de scaffolds para a aplicação em biomateriais, seja na produção de tecidos biomiméticos ou mesmo em sistemas para liberação de drogas, tem sido fundamental tanto para o entendimento dos mecanismos de crescimento de tecidos biológicos e seu funcionamento, quanto para o desenvolvimento de biomateriais que possam ser incorporados aos tecidos naturais para seu reparo e para a efetiva aplicação de agentes terapêuticos. Dentre as várias técnicas para a produção destes scaffolds, a técnica de Electrospinning (ES) foi utilizada neste trabalho para a confecção de scaffolds poliméricos com a incorporação moléculas de interesse biotecnológico. Foram produzidos scaffolds e scaffolds compósitos pela adição de nanopartículas de óxido de cério, nanoargila haloisita e protoporfirina IX complexada à nanoargila haloisita, os quais foram estudados quanto à sua morfologia e propriedades tênseis, além de terem sidos testados quanto a sua viabilidade como sistemas biomiméticos de tecidos. Os scaffolds compósitos mostraram um ganho em ordenamento e homogeneidade, e os scaffolds compósitos contendo óxido de cério mostraram um leve aumento em sua capacidade elástica, além de terem sido viáveis para o crescimento de células HCat / The development of scaffolds for biomaterials applications, in biomimetic tissues production and drug-delivery systems, have been a fundamental tool for the understanding of biological tissues growing and repair mechanisms and for the development of biomaterials that can be incorporated to the natural tissues for both repair and effective application of therapeutic agents. Amongst the several techniques for scaffolds production, the Electrospinning (ES) methodology was applied in this work for developing polymeric scaffolds with the encapsulation of molecules of biotechnological interest. Scaffolds and blend scaffolds by cerium oxide nanoparticles and haloisite nanoclay addiction were produced and studied regarding its morphology, tensile properties and cell viability as biomimetic tissues. The blend scaffolds shoed an enhancement in order and homogeneity, and those within cerium oxide showed also an increase in elastic capacity and viable physical base for HCat cells

Com os avanços tecnológicos ocorridos nas últimas décadas, tornou-se possível às diversas áreas do conhecimento melhorar a qualidade e expectativa de vida da população. Dentre as diversas áreas da Engenharia de Materiais, a de Biomateriais é a que está se destacando, vindo ao encontro das necessidades crescentes de melhorar a qualidade de vida da população. O uso de materiais que possam substituir partes ósseas vem sendo motivo de estudos há muitos anos. Para isto, os materiais têm que possuir propriedades químicas e mecânicas semelhantes às do organismo. Materiais como: cerâmicas, polímeros, metais ou até mesmo combinações entre eles vem sendo utilizados como biomateriais. Uma linha de materiais cerâmicos que se destaca é a de produtos à base de alumina, em função de suas características de biocompatibilidade e excelentes propriedades mecânicas. Na medicina, as próteses assumiram um papel importante, tanto na questão estética quanto na funcional. Os desafios nesta área, no intuito de descobrir novos materiais que possam substituir os existentes com desempenho satisfatório e custos mais acessíveis, tornam-se objetos de pesquisa em todo mundo. Na literatura existem poucos trabalhos que correlacionam bioatividade e propriedades mecânicas de cerâmicas à base de alumina, com relação à área médica. Para contribuir nesta questão, propôs-se um estudo tendo como base a alumina. O trabalho tem como objetivo desenvolver cerâmicas a base de alumina porosa, utilizando para isto o hidróxido de alumínio como nucleador de microporos. Resultados obtidos para o material mostram que a porosidade cresce com o aumento da concentração de hidróxido de alumínio na amostra. O preenchimento dos poros da matriz com biovidro tem por objetivo facilitar o processo de osteocondução. A vantagem do processo é a diminuição do tempo de recuperação para o paciente. Os resultados obtidos mostram que a adição de hidróxido de alumínio leva a uma porosidade maior nas amostras. Ensaios realizados verificaram que a concentração máxima de hidróxido é de 20 % em volume, e que concentrações maiores levam os corpos de prova a instabilidade. Os ensaios com o biovidro em forma de gel mostraram-se promissores, devido a facilidade para impregnação nos corpos de prova. Testes de bioatividade in vitro revelaram que o biovidro poderá ser utilizado como material bioativo, revelando um material útil no uso em próteses médicas. / With technological advances in the past decades, it became possible for various areas of knowledge to improve the quality and life expectancy of the population. Among the various areas of Materials Engineering, the Biomaterials is one that is emerging, coming to meet the growing needs to improve the quality of life. The use of materials that can replace bony parts has been subject of study for many years. For this, the materials have to possess chemical and mechanical properties similar to the body. Materials such as ceramics, polymers, metal or even combinations of them have been used as biomaterials. A line of ceramic materials that stands out is that of alumina-based products, due to their biocompatibility and excellent mechanical properties. In medicine, prosthetics played an important role in both aesthetics and functionality. The challenges in this area in order to discover new materials that can replace the existing performance and costs more affordable, they become objects of research worldwide. In the literature there are few studies that correlate bioactivity and mechanical properties of alumina ceramic base with respect to the medical field. To contribute to this issue, a proposed a study based on alumina. The work aims to develop ceramic-based porous alumina, using study has been aluminum hydroxide as nucleator of micropores. Results obtained for the material show that the porosity increases with increasing concentration of aluminum hydroxide in the sample. The filling of pores of the matrix with bioglass aims to facilitate the process of osteoconduction. The advantage of the process is to reduce the recovery time for the patient. The results show that the addition of aluminum hydroxide leads to a higher porosity in the samples. Tests carried out found that the maximum concentration of hydroxide is 20%, and higher concentrations lead to specimens instability. The tests with the bioglass gel have show promising due to ease of impregnation in the specimens. Bioactivity in vitro tests revealed that the bioglass can be used as a bioactive material, revealing a useful material for medical prostheses.

Este estudo foi desenvolvido no Instituto de Pesquisas Energéticas e Nucleares IPEN mais especificamente, no Centro de Ciência e Tecnologia dos Materiais CCTM com apoio da empresa Innovatech. Foram estudados tubos de Co-Cr (L605) usados para fabricação de stents coronarianos, aplicação esta que pede um comportamento mecânico específico e biocompatibilidade. Os tubos de CoCr (L605) podem ser adquiridos em duas condições de história térmica: Trabalhado a frio ou com encruamento ou recozido. O tubo recozido se não estiver em condições para a aplicação, dificilmente será possível atingi-las com um novo tratamento térmico. O tubo encruado abre possibilidades para acertar as condições de tratamento térmico e obter a condição ideal de comportamento mecânico, sem comprometer outros aspectos importantes para a aplicação como biocompatibilidade. Foi selecionado um tubo de CoCr (L605) encruado e com uma quantidade grande de precipitados para os testes, foram selecionadas três faixas de temperatura para o tratamento térmico de recozimento uma abaixo do ponto de solubilização (1000°C), uma dentro da temperatura (1175°C) e uma terceira, mais próxima do ponto de fusão alcançável pelo forno (1250°C). Em cada temperatura foram usados quatro períodos de exposição ao forno( 4, 7, 10 e 15 minutos) totalizando uma matriz com 12 condições de tratamento térmico. Em cada tratamento térmico foram incluídas amostras para ensaio de tração e metalografia. O objetivo deste trabalho é estudar os efeitos dos tratamentos térmicos no comportamento mecânico e na microestrutura do material afim de levantar critérios para determinar os melhores parâmetros de tratamento térmico para a aplicação. / This study was developed at IPEN Institute of Energy and Nuclear Research more specifically in CCTM Materials Cience and Tecnology Center, with support of Innovatech Medical. It was studied Co-Cr (L605) tubes used for manufacture of coronary stents, this application require a specific mechanical behavior and biocompatibility. The tubes CoCr (L605) can be acquired in two conditions of thermal history: Cold worked or annealed. If the annealed tube doesn´t have the conditions for the application it is hardly possible to reach them with a new heat treatment. Hardened tube opens up more possibilities to adjust the conditions of thermal treatment and obtain the optimum condition of mechanical behavior without compromising other aspects important for application as biocompatibility. A tube CoCr (L605) Hardened and a relatively large amount of precipitates were selected for the tests, three temperature tracks have been selected for the thermal annealing treatment: below the temperature of solution aneealing(1000 ° C), at solution aneealing(1175 ° C) and a third temperature closest achievable by furnace (1250 ° C). For each temperature four periods of exposure where selected (4, 7, 10 and 15 minutes) totaling a matrix of 12 heat treatment conditions. In each heat treatment, samples for tensile testing and metallography were included. The objective of this work is to study the effects of heat treatment on mechanical behavior and microstructure of the material in order to raise criteria to determine the best heat treatment for the application.