Stromal vascular fraction cell sheets angiogenic potential for tissue engineering and regenerative medicine applications

One of the biggest concerns in the Tissue Engineering field is the correct vascularization of engineered constructs. Strategies involving the use of endothelial cells are promising but adequate cell sourcing and neo-vessels stability are enduring challenges. In this work, we propose the hypoxic pre-conditioning of the stromal vascular fraction (SVF) of human adipose tissue to obtain highly angiogenic cell sheets (CS). For that, SVF was isolated after enzymatic dissociation of adipose tissue and cultured until CS formation in normoxic (pO2=21%) and hypoxic (pO2=5%) conditions for 5 and 8 days, in basal medium. Immunocytochemistry against CD31 and CD146 revealed the presence of highly branched capillary-like structures, which were far more complex for hypoxia. ELISA quantification showed increased VEGF and TIMP-1 secretion in hypoxia for 8 days of culture. In a Matrigel assay, the formation of capillary-like structures by endothelial cells was more prominent when cultured in conditioned medium recovered from the cultures in hypoxia. The same conditioned medium increased the migration of adipose stromal cells in a scratch assay, when compared with the medium from normoxia. Histological analysis after implantation of 8 days normoxic- and hypoxic-conditioned SVF CS in a hindlimb ischemia murine model showed improved formation of neo-blood vessels. Furthermore, Laser Doppler results demonstrated that the blood perfusion of the injured limb after 30 days was enhanced for the hypoxic CS group. Overall, these results suggest that SVF CS created under hypoxia can be used as functional vascularization units for tissue engineering and regenerative medicine.

Tendon regeneration through a scaffold-free approach: development of tenogenic magnetic hASCs sheets

Tendon's regeneration is limited, demanding for cell-based strategies to fully restore their functionality upon injury. The concept of magnetic force-based TE(1), generally using magnetic nanoparticles may enable, for example, stem cell stimulation and/or remote control over TE constructs. Thus, we originally propose the development of magnetic cell sheets (magCSs) with tenogenic capability, aimed at promoting tendon's regeneration. A Tenomodulin (TNMD+) subpopulation was sorted from human adipose stem cells (hASCs), using TNMD-coated immunomagnetic beads(2) and used as cell source for the development of magCSs. Briefly, cells were labeled with iron oxide composite particles (Micromod) and cultured for 7 days in α-MEM medium with or without magnetic stimulation provided by a magnetic device (nanoTherics). CSs were retrieved from the plates using magnet attraction as contiguous sheets of cells within its own deposited ECM.

Skin-derived cell-sheets as powerful tools to engineer skin analogues

Cell/cell-extracellular matrix (ECM) dynamic interactions appear to have a major role in regulating communication through soluble signaling, directing cell binding and activating substrates that participate in the highly organized wound healing process. Moreover, these interactions are also crucial for in vitro mimicking cutaneous physiology. Herein we explore cell sheet (CS) engineering to create cellular constructs formed by keratinocytes (hKC), fibroblasts (hDFB) and dermal microvascular endothelial cells (hDMEC), to target skin wound healing but also the in vitro recreation of relevant models. Taking advantage of temperature-responsive culture surfaces, which allow harvesting cultured cells as intact sheets along with the deposited native ECM, varied combinations of homotypic and heterotypic three-dimensional (3-D) CS-based constructs were developed. Constructs combining one CS of keratinocytes as an epidermis-like layer plus a vascularized dermis composed by hDFB and hDMECs were assembled as skin analogues for advancing in vitro testing. Simultaneously both hKC and hDMEC were shown to significantly contribute to the re-epithelialization of full-thickness mice skin wounds by promoting an early epithelial coverage, while hDMEC significantly lead to increased vessels density, incorporating the neovasculature. Thus, although determined by the cellular nature of the constructs, these outcomes demonstrated that CS engineering appear as an unique technology that open the possibility to create numerous combinations of 3D constructs to target defective wound healing as well as the construction of in vitro models to further mimic cutaneous functions crucial for drug screening and cosmetic testing assays.

Task clustering on ETL systems – A pattern-oriented approach

Usually, data warehousing populating processes are data-oriented workflows composed by dozens of granular tasks that are responsible for the integration of data coming from different data sources. Specific subset of these tasks can be grouped on a collection together with their relationships in order to form higher- level constructs. Increasing task granularity allows for the generalization of processes, simplifying their views and providing methods to carry out expertise to new applications. Well-proven practices can be used to describe general solutions that use basic skeletons configured and instantiated according to a set of specific integration requirements. Patterns can be applied to ETL processes aiming to simplify not only a possible conceptual representation but also to reduce the gap that often exists between two design perspectives. In this paper, we demonstrate the feasibility and effectiveness of an ETL pattern-based approach using task clustering, analyzing a real world ETL scenario through the definitions of two commonly used clusters of tasks: a data lookup cluster and a data conciliation and integration cluster.

DOTA-based Ga(III) and Gd(III) chelates for medical imaging (PET, SPECT and MRI)

PhD Thesis in Sciences Specialization in Chemistry

Measuring university-to-work success: development of a new scale

Purpose – The purpose of this paper is to develop a subjective multidimensional measure of early career success during university-to-work transition. Design/methodology/approach – The construct of university-to-work success (UWS) was defined in terms of intrinsic and extrinsic career outcomes, and a three-stage study was conducted to create a new scale. Findings – A preliminary set of items was developed and tested by judges. Results showed the items had good content validity. Factor analyses indicated a four-factor structure and a second-order model with subscales to assess: career insertion and satisfaction, confidence in career future, income and financial independence, and adaptation to work. Third, the authors sought to confirm the hypothesized model examining the comparative fit of the scale and two alternative models. Results showed that fits for both the first- and second-order models were acceptable. Research limitations/implications – The proposed model has sound psychometric qualities, although the validated version of the scale was not able to incorporate all constructs envisaged by the initial theoretical model. Results indicated some direction for further refinement. Practical implications – The scale could be used as a tool for self-assessment or as an outcome measure to assess the efficacy of university-to-work programs in applied settings. Originality/value – This study provides a useful single measure to assess early career success during the university-to-work transition, and might facilitate testing of causal models which could help identify factors relevant for successful transition.

Introducing resistance genes against ToMV, TYLCV and TSWV infections in two tomato varieties (Muchamiel and De la Pera)

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Platelet lysate-based pro-angiogenic nanocoatings

Human platelet lysate (PL) is a cost-effective and human source of autologous multiple and potent pro-angiogenic factors, such as vascular endothelial growth factor A (VEGF A), fibroblast growth factor b (FGF b) and angiopoietin-1. Nanocoatings previously characterized were prepared by layer-by-layer assembling incorporating PL with marine-origin polysaccharides and were shown to activate human umbilical vein endothelial cells (HUVECs). Within 20 h of incubation, the more sulfated coatings induced the HUVECS to the form tube-like structures accompanied by an increased expression of angiogenicassociated genes, such as angiopoietin-1 and VEGF A. This may be a cost-effective approach to modify 2D/3D constructs to instruct angiogenic cells towards the formation of neo-vascularization, driven by multiple and synergistic stimulations from the PL combined with sulfated polysaccharides. Statement of Significance The presence, or fast induction, of a stable and mature vasculature inside 3D constructs is crucial for new tissue formation and its viability. This has been one of the major tissue engineering challenges, limiting the dimensions of efficient tissue constructs. Many approaches based on cells, growth factors, 3D bioprinting and channel incorporation have been proposed. Herein, we explored a versatile technique, layer-by-layer assembling in combination with platelet lysate (PL), that is a cost-effective source of many potent pro-angiogenic proteins and growth factors. Results suggest that the combination of PL with sulfated polyelectrolytes might be used to introduce interfaces onto 2D/3D constructs with potential to induce the formation of cell-based tubular structures.

Biomimetic strategies to engineer mineralised human tissues

In the last few years, many reports have been describing promising biocompatible and biodegradable materials that can mimic in a certain extent the multidimensional hierarchical structure of bone, while are also capable of releasing bioactive agents or drugs in a controlled manner. Despite these great advances, new developments in the design and fabrication technologies are required to address the need to engineer suitable biomimetic materials in order tune cells functions, i.e. enhance cell-biomaterial interactions, and promote cell adhesion, proliferation, and differentiation ability. Scaffolds, hydrogels, fibres and composite materials are the most commonly used as biomimetics for bone tissue engineering. Dynamic systems such as bioreactors have also been attracting great deal of attention as it allows developing a wide range of novel in vitro strategies for the homogeneous coating of scaffolds and prosthesis with ceramics, and production of biomimetic constructs, prior its implantation in the body. Herein, it is overviewed the biomimetic strategies for bone tissue engineering, recent developments and future trends. Conventional and more recent processing methodologies are also described.

Adipogenic cell sheets to recreate in vitro adipose tissue microenvironments

Cell sheet (CS) engineering, taking advantage of cellular self-matrix organized as in native tissue, has been largely explored, including by us, for different purposes [1â 3]. Herein we propose for the ï¬ rst time, the use of human adipose stem cells (hASCs)-derived CS to create adipose tissue analogues with different levels of maturation. hASCs were cultured on UpCellTM thermo-responsive dishes for 1, 3 and 5 days under basal conditions previously established by us [3]. The inï¬ uence of pre-differentiation time and respective cell number, over CS stability and differentiation was assessed. Mechanically robust CS were only obtained with 5 days pre-differentiation period. Adipogenesis was followed along the culture assessing the variation of expression of mesenchymal (CD73, CD105 but not CD90) and adipogenic (PPARg, FABP4 and LPL) markers by ï¬ ow cytometry, immunocytochemistry and RT-PCR. Increased ratio of differentiated cells was achieved for longer pre-differentiation periods, while maturation degree was modulated by the maintenance medium. Independently of the overall CS differentiation/maturation level, 3D constructs were fabricated by stacking and further culturing 3 CS. Thus, by varying the culture conditions, different 3D adipose tissue-like microenvironments were recreated, enabling future development of new tissue engineering strategies, as well as further study of adipose tissue role in the regeneration of different tissues.

Associations, causation and model in psychiatry

This paper discusses models, associations and causation in psychiatry. The different types of association (linear, positive, negative, exponential, partial, U shaped relationship, hidden and spurious) between variables involved in mental disorders are presented as well as the use of multiple regression analysis to disentangle interrelatedness amongst multiple variables. A useful model should have internal consistency, external validity and predictive power; be dynamic in order to accommodate new sound knowledge; and should fit facts rather than they other way around. It is argued that whilst models are theoretical constructs they also convey a style of reasoning and can change clinical practice. Cause and effect are complex phenomena in that the same cause can yield different effects. Conversely, the same effect can have a different range of causes. In mental disorders and human behaviour there is always a chain of events initiated by the indirect and remote cause; followed by intermediate causes; and finally the direct and more immediate cause. Causes of mental disorders are grouped as those: (i) which are necessary and sufficient; (ii) which are necessary but not sufficient; and (iii) which are neither necessary nor sufficient, but when present increase the risk for mental disorders.

Brincar de Internet: a vivência lúdica infantil em ambiente virtual

Tese de Doutoramento em Ciências da Educação (Especialidade em Tecnologia Educativa)

Biomaterials for cartilage tissue engineering under mechanical stimulus

Tese de Doutoramento em Ciências (Especialidade de Física)

Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications

Programa Doutoral em Engenharia Biomédica

Development of folate-targeted liposomes for rheumatoid arthritis therapy

Tese de Doutoramento em Biologia Molecular e Ambiental (área de especialização em Biologia Celular e Saúde).