Characterization of processed materials by electrical currents: development of equipment and applications

Dissertação para obtenção do Grau de Mestre em Engenharia Mecânica

Archaeometallurgical study of the Proto-historic collection of Moinhos de Golas (North Portugal): a forecast for technological changes?

This work presents the archaeometallurgical study of a group of metallic artefacts found in Moinhos de Golas site, Vila Real (North of Portugal), that can generically be attributed to Proto-history (1st millennium BC, Late Bronze Age and Iron Age). The collection is composed by 35 objects: weapons, ornaments and tools, and others of difficult classification, as rings, bars and one small thin bent sheet. Some of the objects can typologically be attributed to Late Bronze Age, others are of more difficult specific attribution. The archaeometallurgical study involved x-ray digital radiography, elemental analysis by micro-energy dispersive X-ray fluorescence spectrometry and scanning electron microscopy with energy dispersive spectroscopy, microstructural observations by optical microscopy and scanning electron microscopy. The radiographic images revealed structural heterogeneities frequently related with the degradation of some artefacts and the elemental analysis showed that the majority of the artefacts was produced in a binary bronze alloy (Cu-Sn) (73%), being others produced in copper (15%) and three artefacts in brass (Cu-Zn(-Sn-Pb)). Among each type of alloy there’s certain variability in the composition and in the type of inclusions. The microstructural observations revealed that the majority of the artefacts suffered cycles of thermo-mechanical processing after casting. The diversity of metals/alloys identified was a discovery of great interest, specifically due to the presence of brasses. Their presence can be interpreted as importations related to the circulation of exogenous products during the Proto-history and/or to the deposition of materials during different moments at the site, from the transition of Late Bronze Age/Early Iron Age (Orientalizing period) onwards, as during the Roman period.

Development of 3D in vitro models for prediction of hepatic metabolism and toxicity

The development of human cell models that recapitulate hepatic functionality allows the study of metabolic pathways involved in toxicity and disease. The increased biological relevance, cost-effectiveness and high-throughput of cell models can contribute to increase the efficiency of drug development in the pharmaceutical industry. Recapitulation of liver functionality in vitro requires the development of advanced culture strategies to mimic in vivo complexity, such as 3D culture, co-cultures or biomaterials. However, complex 3D models are typically associated with poor robustness, limited scalability and compatibility with screening methods. In this work, several strategies were used to develop highly functional and reproducible spheroid-based in vitro models of human hepatocytes and HepaRG cells using stirred culture systems. In chapter 2, the isolation of human hepatocytes from resected liver tissue was implemented and a liver tissue perfusion method was optimized towards the improvement of hepatocyte isolation and aggregation efficiency, resulting in an isolation protocol compatible with 3D culture. In chapter 3, human hepatocytes were co-cultivated with mesenchymal stem cells (MSC) and the phenotype of both cell types was characterized, showing that MSC acquire a supportive stromal function and hepatocytes retain differentiated hepatic functions, stability of drug metabolism enzymes and higher viability in co-cultures. In chapter 4, a 3D alginate microencapsulation strategy for the differentiation of HepaRG cells was evaluated and compared with the standard 2D DMSO-dependent differentiation, yielding higher differentiation efficiency, comparable levels of drug metabolism activity and significantly improved biosynthetic activity. The work developed in this thesis provides novel strategies for 3D culture of human hepatic cell models, which are reproducible, scalable and compatible with screening platforms. The phenotypic and functional characterization of the in vitro systems performed contributes to the state of the art of human hepatic cell models and can be applied to the improvement of pre-clinical drug development efficiency of the process, model disease and ultimately, development of cell-based therapeutic strategies for liver failure.

Improving the early diagnostic of prostate cancer by multiple biomarker detection with new biosensing devices

Prostate cancer (PCa) is the most common form of cancer in men, in Europe (World Health Organization data). The most recent statistics, in Portuguese territory, confirm this scenario, which states that about 50% of Portuguese men may suffer from prostate cancer and 15% of these will die from this condition. Its early detection is therefore fundamental. This is currently being done by Prostate Specific Antigen (PSA) screening in urine but false positive and negative results are quite often obtained and many patients are sent to unnecessary biopsy procedures. This early detection protocol may be improved, by the development of point-of-care cancer detection devices, not only to PSA but also to other biomarkers recently identified. Thus, the present work aims to screen several biomarkers in cultured human prostate cell lines, serum and urine samples, developing low cost sensors based on new synthetic biomaterials. Biomarkers considered in this study are the following: prostate specific antigen (PSA), annexin A3 (ANXA3), microseminoprotein-beta (MSMB) and sarcosine (SAR). The biomarker recognition may occurs by means of molecularly imprinted polymers (MIP), which are a kind of plastic antibodies, and enzymatic approaches. The growth of a rigid polymer, chemically stable, using the biomarker as a template allows the synthesis of the plastic antibody. MIPs show high sensitivity/selectivity and present much longer stability and much lower price than natural antibodies. This nanostructured material was prepared on a carbon solid. The interaction between the biomarker and the sensing-material produces electrical signals generating quantitative or semi-quantitative data. These devices allow inexpensive and portable detection in point-of-care testing.

The early metallurgy in Southwestern Iberia: metals from the Chalcolithic Settlement of São Pedro (Redondo)

Archaeological excavations carried out in the archaeological site of São Pedro (Southern Portugal) revealed a Chalcolithic settlement occupied in different moments of the 3rd millennium BC. The material culture recovered includes different types of materials, such as ceramics, lithics and metals. The later comprises about 30 artefacts with different typologies such as tools (e.g. awls, chisels and a saw) and weapons (e.g. daggers and arrowheads) mostly belonging to the 2nd and 3rd quarter of the 3rd millennium BC. In the present work the collection of chalcolithic metallic artefacts recovered in São Pedro was characterized. Analytical studies involved micro energy dispersive X-ray fluorescence spectrometry (micro-EDXRF) to determine elemental composition, together with optical microscopy and Vickers microhardness testing for microstructural characterisation and hardness determination. Main results show copper with variable amounts of arsenic and very low content of other impurities, such as iron. Moreover, nearly half of the collection is composed by arsenical copper alloys (As > 2 wt.%) and an association was found between arsenic content and typology since the weapons group (mostly daggers) present higher values than tools (mostly awls). These results suggest some criteria in the selection of arsenic-rich copper ores or smelting products. Furthermore, after casting an artefact would have been hammered, annealed and sometimes, finished with a hammering operation. Additionally, microstructural variations in this collection reveal somewhat different operational conditions during casting, annealing and forging, as expected in such a primitive metallurgy. Moreover the operational sequence seems to be used to achieve the required shape to the object, rather than to intentionally make the alloy harder. Overall, this study suggests that Chalcolithic metallurgists might have a poor control of the addition of arsenic and/or were unable to use this element to increase the hardness of tools and weapons. Finally, the compositions, manufacturing processes and hardness were compared to those from neighbouring regions and different chronological periods.

Assessment of a novel Cu (II) complex as a potential anticancer agent

Widely used in cancer treatment, chemotherapy still faces hindering challenges, ranging from severe induced toxicity to drug resistance acquisition. As means to overcome these setbacks, newly synthetized compounds have recently come into play with the basis of improved pharmacokinetic/pharmacodynamic properties. With this mind-set, this project aimed towards the antiproliferative potential characterization of a group of metallic compounds. Additionally the incorporation of the compounds within a nanoformulation and within new combination strategies with commercial chemotherapeutic drugs was also envisaged. Cell viability assays presented copper (II) compound (K4) as the most promising, presenting an IC50 of 6.10 μM and 19.09 μM for HCT116 and A549 cell line respectively. Exposure in fibroblasts revealed a 9.18 μM IC50. Hoechst staining assays further revealed the compound’s predisposition to induce chromatin condensation and nuclear fragmentation in HCT116 upon exposure to K4 which was later demonstrated by flow cytometry and annexin V-FITC/propidium iodide double staining analysis (under 50 % cell death induction). The compound further revealed the ability to interact with major macromolecules such as DNA (Kb = 2.17x105 M-1), inducing structural brakes and retardation, and further affecting cell cycle progression revealing delay in S-phase. Moreover BSA interactions were also visible however not conclusive. Proteome profiling revealed overexpression of proteins involved in metabolic activity and underexpression of proteins involved in apoptosis thus corroborating Hoechst and apoptosis flow cytometry data. K4 nanoformulation suffered from several hindrances and was ill succeeded in part due to K4’s poor solubility in aqueous buffers. Other approaches were considered in this regard. Combined chemotherapy assays revealed high cytotoxicity for afatinib and lapatinib strategies. Lapatinib and K4 proteome profiling further revealed high apoptosis rates, high metabolic activity and activation of redundant proteins as part of compensatory mechanisms.

As Necrópoles alto-medievais do concelho de Cascais : séculos VI e VII

A presente dissertação teve como finalidade estudar os testemunhos arqueológicos respeitantes às práticas funerárias levadas a cabo no atual concelho de Cascais durante os séculos VI e VII. As necrópoles em estudo são sítios bem conhecidos pelos investigadores, uma vez que a descoberta de algumas é precoce, datando de finais do século XIX. Com este trabalho, pretendeu-se introduzir uma série de componentes que a investigação privilegia atualmente, sobretudo no que respeita às vivências nos espaços rurais entre o fim do Império Romano e o domínio muçulmano na Península Ibérica. A investigação desenvolvida baseou-se no estudo preliminar das coleções osteológicas de quatro das cinco necrópoles, bem como na prospeção e no levantamento arqueológicos. O inventário antropológico teve como objetivo apurar o número mínimo de indivíduos por necrópole e por sepultura e fazer uma caracterização básica do sexo e da idade dos inumados. A prospeção assentou na análise das fontes bibliográficas sobre os sítios. Os trabalhos de campo desenvolveram-se no sentido de apurar o estado de conservação dos vestígios, na eventual identificação de outros novos e no consequente levantamento gráfico e fotográfico dos mesmos. Foi igualmente examinado o espólio cerâmico e metálico recolhido aquando da escavação das necrópoles, de modo obter uma visão abrangente sobre os rituais funerários e a estabelecer cronologias mais precisas. Embora se trate de um estudo limitado devido à inexistência de um registo mais rigoroso dos vestígios, foi possível tirar algumas conclusões e constatar alguns padrões. Os resultados possibilitaram apontar um conjunto de condições que se repetem nos locais onde estes cemitérios se implantam, percebendo dinâmicas em relação a fatores de caráter natural e de carácter antrópico. Também se apurou que existiriam diferentes formas de organizar os espaços funerários e que estes seriam constituídos por sepulturas muito diversas do ponto de vista construtivo. Além disso, começa-se a desvelar as razões para a reutilização de sepulturas para vários enterramentos e a entender a forma como as comunidades rurais conduziam os rituais funerários, ainda muito enraizados numa antiga matriz pagã.

Acellular Gellan-gum based bilayered structures for the regeneration of osteochondral defects: a preclinical study

 In orthopaedics, the management and treatment of osteochondral (OC) defects remains an ongoing clinical challenge. Autologous osteochondral mosaicplasty has been used as a valid option for OC treatments although donor site morbidity remains a source of concern [1]. Engineering a whole structure capable of mimicking different tissues (cartilage and subchondral bone) in an integrated manner could be a possible approach to regenerate OC defects. In our group we have been proposing the use of bilayered structures to regenerate osteochondral defects [2,3]. The present study aims to investigate the pre-clinical performance of bilayered hydrogels and spongy-like hydrogels in in vivo  models (mice and rabbit, respectively), in both subcutaneous and orthotopic models. The bilayered structures were produced from Low Acyl Gellan Gum (LAGG) from Sigma-Aldrich, USA. Cartilage-like layers were obtained from a 2wt% LAGG solution. The bone-like layers were made of 2wt% LAGG with incorporation of hydroxyapatite at 20% and 30% (w/v). Hydrogels and spongy-like were subcutaneouly implanted in mice to evaluate the inflammatory response. Then, OC defects were induced in rabbit knee to create a critical size defect (4 mm diameter and 5 mm depth), and then hydrogels and sponges implanted. Both structures followed different processing methods. The hydrogels were injected allowing in situ  crosslinking. Unlike, the spongy-like were pre-formed by freeze-drying. The studies concerning subcutaneous implantation and critical size OC defect were performed for 2 and 4 weeks time, respectively. Cellular behavior and inflammatory responses were assessed by means of histology staining and biochemical function and matrix deposition by immunohistochemistry. Additionally, both OC structures stability and new cartilage and bone formation were evaluated by using vivo- computed tomography (Scanco 80). The results showed no acute inflammatory response for both approaches. New tissue formation and integration in the adjacent tissues were also observed, which present different characteristic behaviors when comparing hydrogels and sponges response. As future insights, a novel strategy for regeneration of OC defects can be designed encompassing both, hydrogels and spongy-like structures and cellular approaches. References: 1. Espregueira-Mendes J. et al. Osteochondral transplantation using autografts from the upper tibio-fibular joint for the treatment of knee cartilage lesions. Knee Surgery, Sports Traumatology, Arthroscopy 20,1136, 2012. 2. Oliveira JM. et al, Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications: Scaffold design and its performance when seeded with goat bone marrow stromal cells. Biomaterials 27, 6123, 2006. 3. Pereira D R. et al. Gellan Gum-Based Hydrogel Bilayered Scaffolds for Osteochondral Tissue Engineering. Key Engineering Materials 587, 255, 2013.

Artificial thymus: a tissue engineering strategy

The thymus is the central organ responsible for the generation of T lymphocytes (1). Various diseases cause the thymus to produce in- sufficient T cells, which can lead to immune-suppression (2). Since T cells are essential for the protection against pathogens, it is crucial to promote de novo differentiation of T cells on diseased individuals. The available clinical solutions are: 1) one protocol involving the transplant of thymic stroma from unrelated children only applicable for athymic children (3); 2) for patients with severe peripheral T cell depletion and reduced thymic activity, the administration of stimu- lating molecules stimulating the activity of the endogenous thymus (4). A scaffold (CellFoam) was suggested to support thymus regen- eration in vivo (5), although this research was discontinued. Herein, we propose an innovative strategy to generate a bioartificial thymus. We use a polycaprolactone nanofiber mesh (PCL-NFM) seeded and cultured with human thymic epithelial cells (hTECs). The cells were obtained from infant thymus collected during pediatric cardio-tho- racic surgeries. We report new data on the isolation and characterization of those cells and their interaction with PCL-NFM, by expanding hTECs into relevant numbers and by optimizing cell seeding methods.

A continuous solvent- and oil-free method to prepare injectable PEGylated fibrinogen cell-laden microparticles

Injectable biomaterials with in situ cross-linking reactions have been suggested to minimize the invasiveness associated with most implantation procedures. However, problems related with the rapid liquid-to-gel transition reaction can arise because it is difficult to predict the reliability of the reaction and its end products, as well as to mitigate cytotoxicity to the surrounding tissues. An alternative minimally invasive approach to deliver solid implants in vivo is based on injectable microparticles, which can be processed in vitro with high fidelity and reliability, while showing low cytotoxicity. Their delivery to the defect can be performed by injection through a small diameter syringe needle. We present a new methodology for the continuous, solvent- and oil-free production of photopolymerizable microparticles containing encapsulated human dermal fibroblasts. A precursor solution of cells in photo-reactive PEG-fibrinogen (PF) polymer was transported through a transparent injector exposed to light-irradiation before being atomized in a jet-in-air nozzle. Shear rheometry data provided the cross-linking kinetics of each PF/cell solution, which was then used to determine the amount of irradiation required to partially polymerize the mixture prior to atomization. The partially polymerized drops fell into a gelation bath for further polymerization. The system was capable of producing cell-laden microparticles with high cellular viability, with an average diameter of between 88.1 µm to 347.1 µm and a dispersity of between 1.1 and 2.4, depending on the parameters chosen.

Organic-inorganic nanostructured multilayers for calcium phosphate biomineralization

The weak fixation of biomaterials within the bone structure is one of the major reasons of implants failures. Calcium phosphate (CaP) coatings are used in bone tissue engineering to improve implant osseointegration by enhancing cellular adhesion, proliferation and differentiation, leading to a tight and stable junction between implant and host bone. It has also been observed that materials compatible with bone tissue either have a CaP coating or develop such a calcified surface upon implantation. Thus, the development of bioactive coatings becomes essential for further improvement of integration with the surrounding tissue. However, most of current applied CaP coatings methods (e.g. physical vapor deposition), cannot be applied to complex shapes and porous implants, provide poor structural control over the coating and prevent incorporation of bioactive organic compounds (e.g. antibiotics, growth factors) because of the used harsh processing conditions. Layer-by-layer (LbL) is a versatile technology that permits the building-up of multilayered polyelectrolyte films in mild conditions based on the alternate adsorption of cationic and anionic elements that can integrate bioactive compounds. As it is recognized in natureâ s biomineralization process the presence of an organic template to induce mineral deposition, this work investigate a ion based biomimetic method where all the process is based on LbL methodology made of weak natural-origin polyelectrolytes. A nanostructured multilayer component, with 5 or 10 bilayers, was produced initially using chitosan and chondroitin sulphate polyelectrolyte biopolymers, which possess similarities with the extracellular matrix and good biocompatibility. The multilayers are then rinsed with a sequential passing of solutions containing Ca2+ and PO43- ions. The formation of CaP over the polyelectrolyte multilayers was confirmed by QCM-D, SEM and EDX. The outcomes show that 10 polyelectrolyte bilayer condition behaved as a  better site for initiating the formation of CaP as the precipitation occur at earlier stages than in 5 polyelectrolyte bilayers one. This denotes that higher number of bilayers could hold the CaP crystals more efficiently. This work achieved uniform coatings that can be applied to any surface with access to the liquid media in a low-temperature method, which potentiates the manufacture of effective bioactive biomaterials with great potential in orthopedic applications.

Bioactive nanocomposite spheres with proved shape memory capability in bone defects

Bioactive glass nanoparticles (BGNPs) promote an apatite surface layer in physiologic conditions that lead to a good interfacial bonding with bone.1 A strategy to induce bioactivity in non-bioactive polymeric biomaterials is to incorporate BGNPs in the polymer matrix. This combination creates a nanocomposite material with increased osteoconductive properties. Chitosan (CHT) is a polymer obtained by deacetylation of chitin and is biodegradable, non-toxic and biocompatible. The combination of CHT and the BGNPs aims at designing biocompatible spheres promoting the formation of a calcium phosphate layer at the nanocomposite surface, thus enhancing the osteoconductivity behaviour of the biomaterial. Shape memory polymers (SMP) are stimuli-responsive materials that offer mechanical and geometrical action triggered by an external stimulus.2 They can be deformed and fixed into a temporary shape which remains stable unless exposed to a proper stimulus that triggers recovery of their original shape. This advanced functionality makes such SMPs suitable to be implanted using minimally invasive surgery procedures. Regarding that, the inclusion of therapeutic molecules becomes attractive.  We propose the synthesis of shape memory bioactive nanocomposite spheres with drug release capability.3   1.  L. L. Hench, Am. Ceram. Soc. Bull., 1993, 72, 93-98. 2.  A. Lendlein and S. Kelch, Angew Chem Int Edit, 2002, 41, 2034-2057. 3.  Ã . J. Leite, S. G. Caridade and J. F. Mano, Journal of Non-Crystalline Solids (in Press)

Analysis of polydicyclopentadiene-metal hybrid adhesive joints for automotive exterior body applications

An exterior body panel solution containing a polydicyclopentadiene skin attached to an interior metallic reinforcement through adhesive bonding is being studied to be applied in the MobiCar bonnet. With this solution is expected to achieve lightness, adequate structural integrity and cost-efficiency. However, there is uncertainty regarding to the bonnet adhesiveness since different metallic materials and adhesive types are being considered for its development. Thus, in this paper, several samples are tested through shear loading with the aim of understanding the loading magnitude expected by using polydicyclopentadiene, steel DC04+ZE and aluminum alloy AW5754-H111 as substrates adhesively bonded by an epoxy or a methacrylate. Methacrylate adhesive have shown greater shear strength in all types of adhesive joints. PDCPD joints presented the highest displacements. Surface degradation was considered adequate over abrading once none strength difference was seen between the different surface treatments. Steel treated by cataphoresis has shown the highest joint interface strength.

Mechanical fatigue performance of PCL-chondroprogenitor constructs after cell culture under bioreactor mechanical stimulus

In tissue engineering of cartilage, polymeric scaffolds are implanted in the damaged tissue and subjected to repeated compression loading cycles. The possibility of failure due to mechanical fatigue has not been properly addressed in these scaffolds. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. This is related to inherent discontinuities in the material due to the micropore structure of the macro-pore walls that act as stress concentration points. In this work, chondrogenic precursor cells have been seeded in Poly-ε-caprolactone (PCL) scaffolds with fibrin and some were submitted to free swelling culture and others to cyclic loading in a bioreactor. After cell culture, all the samples were analyzed for fatigue behavior under repeated loading-unloading cycles. Moreover, some components of the extracellular matrix (ECM) were identified. No differences were observed between samples undergoing free swelling or bioreactor loading conditions, neither respect to matrix components nor to mechanical performance to fatigue. The ECM did not achieve the desired preponderance of collagen type II over collagen type I which is considered the main characteristic of hyaline cartilage ECM. However, prediction in PCL with ECM constructs was possible up to 600 cycles, an enhanced performance when compared to previous works. PCL after cell culture presents an improved fatigue resistance, despite the fact that the measured elastic modulus at the first cycle was similar to PCL with poly(vinyl alcohol) samples. This finding suggests that fatigue analysis in tissue engineering constructs can provide additional information missed with traditional mechanical measurements.

In vitro mechanical fatigue behavior of poly-ε-caprolactone macroporous scaffolds for cartilage tissue engineering: Influence of pore filling by a poly(vinyl alcohol) gel

Polymeric scaffolds used in regenerative therapies are implanted in the damaged tissue and subjected to repeated loading cycles. In the case of articular cartilage engineering, an implanted scaffold is typically subjected to long term dynamic compression. The evolution of the mechanical properties of the scaffold during bioresorption has been deeply studied in the past, but the possibility of failure due to mechanical fatigue has not been properly addressed. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. In this work fatigue studies of polycaprolactone scaffolds were carried by subjecting the scaffold to repeated compression cycles in conditions simulating the scaffold implanted in the articular cartilage. The behaviour of the polycaprolactone sponge with the pores filled with a poly(vinyl alcohol) gel simulating the new formed tissue within the pores was compared with that of the material immersed in water. Results were analyzed with Morrow’s criteria for failure and accurate fittings are obtained just up to 200 loading cycles. It is also shown that the presence of poly(vinyl alcohol) increases the elastic modulus of the scaffolds, the effect being more pronounced with increasing the number of freeze/thawing cycles.