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.

Osteogenic differentiation of adipose stem cells by endothelial cells co-culture within liquified capsules

Inspired by the native co-existence of multiple cell types and from the concept of deconstructing the stem cell niche, we propose a co-encapsulation strategy within liquified capsules. The present team has already proven the application of liquified capsules as bioencapsulation systems1. Here, we intend to use the optimized system towards osteogenic differentiation. Capsules encapsulating adipose stem cells alone (MONO-capsules) or in co-culture with endothelial cells (CO-capsules) were maintained in endothelial medium with or without osteogenic differentiation factors. The suitability of the capsules for living stem and endothelial cells encapsulation was demonstrated by MTS and DNA assays. The osteogenic differentiation was assessed by quantifying the deposition of calcium and the activity of ALP up to 21 days. CO capsules had an enhanced osteogenic differentiation, even when cultured in the absence of osteogenic factors. Furthermore, osteopontin and CD31 could be detected, which respectively indicate that osteogenic differentiation had occurred and endothelial cells maintained their phenotype. An enhanced osteogenic differentiation by co-encapsulation was also confirmed by the upregulation of osteogenic markers (BMP-2, RUNX2, BSP) while the expression of angiogenic markers (VEGF, vWF, CD31) revealed the presence of endothelial cells. The proposed capsules can also act as a growth factor release system upon implantation, as showed by VEGF and BMP-2 quantification. These findings demonstrate that the co-encapsulation of stem and endothelial cells within liquified injectable capsules provides a promising strategy for bone tissue engineering.  

Cryopreservation of Cell Sheets of Adipose Stem Cells: Limitations and Successes

Cell Sheets of hASCs (hASCs-CS) have been previously proposed for wound healing applications(1, 2) and despite the concern for production time reduction, the possibility of having these hASCs-CS off-the-shelf is appealing. The goal of this work was to define a cryopreservation methodology allowing to preserve cells viability and the properties CS matrix. hASCs-CS obtained from three different donors were created in UP-cell thermoresponsive dishes(Nunc, Germany) as previously reported(1,2). Different cryopreservation conditions were considered: i)FBS plus DMSO(5% and10%); ii)0.4M of Trehalose plus DMSO (5% and 10%); iii)cryosolution PLL (Akron Biotech, USA); and iv)vitrification. The cryopreservation effect was first assessed for cellular viability by flow cytometry using 7-AAD, and after dissociating the hASCs-CS with collagenase and trypsin-EDTA 0.25%. The expression (RT-PCR) and deposition (western blot and immunocytochemistry) of collagen type I, laminin and fibronectin, and the organization (TEM) of the extracellular matrix was further assessed before and after hASCs-CS cryopreservation to determine a potential effect of the method over matrix composition and integrity. The obtained results confirmed that cell viability is affected by the cryopreservation methodology, as shown before for different CS(3). Interestingly, the matrix properties were not significantly altered and the typical cell sheetâ s easiness of manipulation for transplantation was not lost.

In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFβ1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage.

Innovative method for the continuous monitoring of concrete viscoelastic properties since early ages: concept and pilot experiments

The experimental evaluation of viscoelastic properties of concrete is traditionally made upon creep tests that consist in the application of sustained loads either in compression or in tension. This kind of testing demands for specially devised rigs and requires careful monitoring of the evolution of strains, whereas assuring proper load constancy. The characterization of creep behaviour at early ages offers additional challenges due to the strong variations in viscoelastic behaviour of concrete during such stages, demanding for several testing ages to be assessed. The present research work aims to assist in reducing efforts for continuous assessment of viscoelastic properties of concrete at early ages, by application of a dynamic testing technique inspired in methodologies used in polymer science: Dynamic Mechanical Analyses. This paper briefly explains the principles of the proposed methodology and exhibits the first results obtained in a pilot application. The results are promising enough to encourage further developments.

Annoyance and welfare costs from the presence of renewable energy power plants: an application of the contingent valuation method

Sustainability is frequently defined by its three pillars: economically viable, socially equitable, and environmentally bearable. Consequently the evaluation of the sustainability of any decision, public or private, requires information on these three dimensions. This paper focuses on social sustainability. In the context of renewable energy sources, the examination of social sustainability requires the analysis of not only the efficiency but also the equity of its welfare impacts. The present paper proposes and applies a methodology to generate the information necessary to do a proper welfare analysis of the social sustainability of renewable energy production facilities. This information is key both for an equity and an efficiency analysis. The analysis focuses on the case of investments in renewable energy electricity production facilities, where the impacts on local residents’ welfare are often significantly different than the welfare effects on the general population. We apply the contingent valuation method to selected facilities across the different renewable energy power plants located in Portugal and conclude that local residents acknowledge differently the damage sustained by the type, location and operation of the plants. The results from these case studies attest to the need of acknowledging and quantifying the negative impacts on local communities when assessing the economic viability, social equity and environmental impact of renewable energy projects.

Typology based method for choosing old masonry walls inspection procedures

The diagnosis of historic masonry walls is an intricate and complex field and has been an object of research for many years. This paper aims to propose practical methodologies for the diagnosis of historic masonry walls, specifically based on their typological characteristics. In order to develop such procedures, information relating to historic masonry typologies in Portugal, classified as rural, urban and military was gathered and techniques for the assessment of historic masonry were studied. All information was integrated to develop a pattern typology oriented methodology. Developed methodology was tested and validated in a small diagnosis campaign carried out in the Guimarães Castle. Methodology was proven to be advantageous and although the study is limited and focused on the Portuguese architectural specificities, it still holds global classifications, and therefore can be useful for any diagnosis procedure of a historic masonry wall.

Influence of the wood mechanical properties in the dovetail joint behavior

The force distribution inside a dovetail joint is complex. Wood is simultaneously loaded in different directions in the several connected surfaces. The analytical solutions available for the analysis of the behavior of those carpentry joints rely on the mechanical properties of wood. In particular, the stiffness properties of wood under compression are crucial for the forces equilibrium. Simulations showed that the stiffness values considered in each of the springs normally assumed in the analytical models, have great influence in the bearing capacity and stiffness of the dovetail joints, with important consequence on the stress distribution over the overall structure. In a wide experimental campaign, the properties under compression of the most common wood species of existing timber structures have been determined. Then, a solved example of a dovetail joint is presented assuming different wood species and the corresponding strength and stiffness properties values obtained in the tests.

The potential field method and the nonlinear attractor dynamics approach: what are the differences?

One of the most popular approaches to path planning and control is the potential field method. This method is particularly attractive because it is suitable for on-line feedback control. In this approach the gradient of a potential field is used to generate the robot's trajectory. Thus, the path is generated by the transient solutions of a dynamical system. On the other hand, in the nonlinear attractor dynamic approach the path is generated by a sequence of attractor solutions. This way the transient solutions of the potential field method are replaced by a sequence of attractor solutions (i.e., asymptotically stable states) of a dynamical system. We discuss at a theoretical level some of the main differences of these two approaches.

A performance-based method for seismic evaluation of masonry walls strengthened with RC layers

A conventional method for seismic strengthening of masonry walls is externally application of reinforced concrete layer (shotcrete). However, due to the lack of analytical and experimental information on the behavior of strengthened walls, the design procedures are usually followed based on the empirical relations. Using these design procedures have resulted in massive strengthening details in retrofitting projects. This paper presents a computational framework for nonlinear analysis of strengthened masonry walls and its versatility has been verified by comparing the numerical and experimental results. Based on the developed numerical model and available experimental information, design relations and failure modes are proposed for strengthened walls in accordance with the ASCE 41 standard. Finally, a sample masonry structure has been strengthened using the proposed and available conventional methods. It has been shown that using the proposed method results in lower strengthening details and appropriate (ductile) failure modes

Prevention in underground construction with sequential excavation method

This paper aims to describe the Sequential Excavation Method, used for excava-tion in underground works, as well as the related risks and preventive measures. This method has characteristics that differentiate it from other tunnelling techniques: it uses a larger number of workers and equipment; it has a high concurrency of tasks with various workers and equip-ment quite exposed to hazards; and it uses many potentially aggressive chemicals. Firstly, it is given a broad overview of this issue. Afterwards, it will be presented the results of a survey to a sample of experienced technicians, aimed at gauging the relevance of a set of guidelines relat-ing to the design and work phases, applicable to the domestic market and prepared following technical visits to works abroad.

Seismic-resistant building practices resulting from local seismic culture

Considering that vernacular architecture may bear important lessons on hazard mitigation, this chapter focuses on the European Mediterranean countries and studies traditional seismic-resistant architectural elements and techniques that local populations developed to prevent or repair earthquake damage. This area was selected as a case study because, as a highly seismic region, it has suffered the effect of many earthquakes along the history and, thus, regions within this area are prone to have developed a Local Seismic Culture. After reviewing seismic resistant construction concepts, a wide range of traditional construction solutions that, in many cases, have shown to improve the seismic performance of vernacular constructions of these regions is presented, as a contribution to the general overview of retrofitting building systems provided in this book. The main motivation is that most of these techniques can be successfully applied to preserve and to retrofit surviving examples without prejudice for their identity.

Quantitative image analysis as a tool for Yarrowia lipolytica dimorphic growth evaluation in different culture media

Yarrowia lipolytica, a yeast strain with a huge biotechnological potential, capable to produce metabolites such as γ-decalactone, citric acid, intracellular lipids and enzymes, possesses the ability to change its morphology in response to environmental conditions. In the present study, a quantitative image analysis (QIA) procedure was developed for the identification and quantification of Y. lipolytica W29 and MTLY40-2P strains dimorphic growth, cultivated in batch cultures on hydrophilic (glucose and N-acetylglucosamine (GlcNAc) and hydrophobic (olive oil and castor oil) media. The morphological characterization of yeast cells by QIA techniques revealed that hydrophobic carbon sources, namely castor oil, should be preferred for both strains growth in the yeast single cell morphotype. On the other hand, hydrophilic sugars, namely glucose and GlcNAc caused a dimorphic transition growth towards the hyphae morphotype. Experiments for γ-decalactone production with MTLY40-2P strain in two distinct morphotypes (yeast single cells and hyphae cells) were also performed. The obtained results showed the adequacy of the proposed morphology monitoring tool in relation to each morphotype on the aroma production ability. The present work allowed establishing that QIA techniques can be a valuable tool for the identification of the best culture conditions for industrial processes implementation.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Methodology for real-time adaptation of tunnels support using the observational method

The observational method in tunnel engineering allows the evaluation in real time of the actual conditions of the ground and to take measures if its behavior deviates considerably from predictions. However, it lacks a consistent and structured methodology to use the monitoring data to adapt the support system in real time. The definition of limit criteria above which adaptation is required are not defined and complex inverse analysis procedures (Rechea et al. 2008, Levasseur et al. 2010, Zentar et al. 2001, Lecampion et al. 2002, Finno and Calvello 2005, Goh 1999, Cui and Pan 2012, Deng et al. 2010, Mathew and Lehane 2013, Sharifzadeh et al. 2012, 2013) may be needed to consistently analyze the problem. In this paper a methodology for the real time adaptation of the support systems during tunneling is presented. In a first step limit criteria for displacements and stresses are proposed. The methodology uses graphics that are constructed during the project stage based on parametric calculations to assist in the process and when these graphics are not available, since it is not possible to predict every possible scenario, inverse analysis calculations are carried out. The methodology is applied to the “Bois de Peu” tunnel which is composed by two tubes with over 500 m long. High uncertainty levels existed concerning the heterogeneity of the soil and consequently in the geomechanical design parameters. The methodology was applied in four sections and the results focus on two of them. It is shown that the methodology has potential to be applied in real cases contributing for a consistent approach of a real time adaptation of the support system and highlight the importance of the existence of good quality and specific monitoring data to improve the inverse analysis procedure.