Mesenchymal stem cells culture on poly(vinylidene fluoride) piezoelectric microspheres

Dissertação de mestrado em Biofísica e BionanossistemasNos últimos anos, as células estaminais mesenquimais (CEM’s) têm sido foco de interesse na comunidade científica devido à sua capacidade de diferenciação em diferentes linhagens celulares, tais como adipócitos, osteoblastos e condrócitos. Neste trabalho, CEM’s humanas foram combinadas com poli(fluoreto de vinilideno), PVDF, um polímero piezoelétrico biocompatível, de modo a impulsionar a sua expansão e proliferação in vitro, com o objetivo principal de obter um substancial número de células com fenótipo osteoblástico para regeneração de tecidos. Com este propósito, filmes de PVDF em fase α foram submetidos a electrospray com diferentes tempos de deposição, dando origem a dois substratos, com alta e baixa concentração de micropartículas de β-PVDF. Foram também produzidas micropartículas sem substrato com vista a criar um ambiente 3D e filmes planos de β-PVDF foram usados como referência. Antes do cultivo celular, os marcadores superficiais celulares característicos de CEM’s (CD105, CD90 e CD73) foram analisados por citometria de fluxo (CF). Quatro dias depois de serem cultivadas nos biomateriais, a viabilidade celular foi examinada. Em paralelo, CF, microscopia eletrónica de varrimento (MEV) e ensaios de imunocitoquímica de vinculina foram realizados de modo a avaliar a manutenção da multipotencialidade das CEM’s e a sua morfologia nos diferentes substratos. Quando a confluência celular foi atingida, foi introduzido um meio de diferenciação osteogénico e o cultivo continuou por 14 dias. Finalmente, CF e um ensaio de imunocitoquímica de osteocalcina foram realizados de modo a avaliar como as diferentes topografias dos biomateriais influenciavam a diferenciação osteogénica. A primeira análise de CF confirmou que as células utilizadas eram CEM’s humanas. No quarto dia, os resultados de MTS mostraram que a proliferação foi similar em todos os substratos. A MEV e o ensaio de imunocitoquímica de vinculina mostraram que as CEM’s adotaram diferentes morfologias dependendo do biomaterial. Adicionalmente, CF mostrou uma perda de marcadores específicos das CEM’s em meio de expansão e 14 dias depois da introdução de meio osteogénico, as células cultivadas nos filmes planos e com micropartículas revelaram existência de osteocalcina e perda de marcadores. Concluindo, as novas topografias com micropartículas de PVDF permitiram um incremento na diferenciação de CEM’s. As células proliferaram satisfatoriamente e a morfologia adotada nos substratos sugere aderência às microesferas. Concluindo, estes suportes mostraram induzir perda de multipotencialidade das CEM’s cultivadas em meio de expansão, mesmo antes da confluência celular.Mesenchymal Stem Cells (MSCs) have attracted great interest in the scientific community in the past few years due to their differentiation potential towards cells belonging to the musculoskeletal lineages, such as adipocytes, osteoblasts, and chondrocytes. In this work, human MSCs were combined with poly(vinylidenefluoride) (PVDF), a biocompatible piezoelectric polymer, allowing their in vitro expansion and proliferation, with the main goal of obtaining an important number of cells with osteoblastic phenotype for tissue regeneration. With this purpose, α-phase PVDF films were subjected to PVDF electrospray with different deposition times, producing two substrates, with high and low concentration of β-phase PVDF microspheres. Microspheres only were also produced to create a 3D environment. Flat β-phase films were used as reference. Before cell seeding, the characteristic cell surface markers of MSCs (CD105, CD90 and CD73) were analyzed by flow cytometry (FC). Cells were cultured onto the biomaterials and viability was assessed after 4 days. In parallel, FC, Scanning Electron Microscopy (SEM) and immunocytochemistry of vinculin were performed in order to evaluate the maintenance of MSCs multipotentiality and their morphology on the different substrates. When the confluence was reached, osteogenic differentiation medium was introduced and the culture was continued for 14 days. Finally, FC and an osteocalcin immunocytochemistry were performed in order to evaluate if the different substrate morphologies influenced MSCs osteogenic differentiation. First FC analysis confirmed that cells were actually human mesenchymal stem cells. At the fourth day, MTS results showed similar proliferation in all the substrates. SEM and vinculin immunocytochemistry have shown that a different morphology was adopted by MSCs depending on the substrate. Also, FC indicated loss of specific MSCs markers in expansion medium. After 14 days of osteogenic medium introduction, cells cultured on flat films and films with microspheres revealed osteocalcin staining and again, loss of multipotentiality. Concluding, this new shaped PVDF microspheres substrates were able to enhance hMSC’s differentiation. Cells proliferated at high rate and their morphology in the substrates suggests that these cells are adhering onto microspheres. Moreover, these supports’ topography induces loss of multipotenciality in MSCs cultured in expansion medium, even before reaching confluence

Sou tudo e não sou nada: as funções de director técnico nos organismos de apoio social a crianças e idosos no concelho de Caldas da Rainha

Dissertação de Mestrado em Política SocialAs Instituições Particulares de Solidariedade Social, organizações de economia social, especializadas na acção social, reflectem um papel social muito importante para o Estado, bem como para a sociedade civil. Para que estas tenham uma melhor qualidade de serviço é fundamental serem executadas por profissionais competentes e que tenham definidas as suas habilitações funcionais. Um dos requisitos para se poder alcançar este objectivo é a existência de um perfil profissional específico, com definição de funções, bem como a urgência do reconhecimento legal da profissão. É neste contexto que se realizou este trabalho cujos objectivos principais são identificar e caracterizar as competências pessoais e funcionais requeridas, as funções que executam e que se prevêem realizar, bem como o ambiente profissional em que se inserem. Para a concretização dos objectivos definidos começou-se por realizar uma revisão bibliográfica procurando definir os principais conceitos associadas à gestão das Instituições Particulares de Solidariedade Social, à Gestão de Recursos Humanos e ao papel dos Directores Técnicos. A realização deste estudo assentou na aplicação de um inquérito por questionário a treze Directoras Técnicas do concelho de Caldas da Rainha. Como principais conclusões deste trabalho salienta-se a inexistência de categoria profissional, de progressão de carreira e um progressivo aumento das responsabilidades e competências exigidas.Private Institutions of Social Solidarity, social economy organizations, specializing in social action, reflect a very important social role for the state and civil society. For them to have a better quality of service it is essential that it be performed by competent professionals and have their functional skills defined. One of the requirements in order to achieve this objective is a specific profile, with definition of tasks and the urgency of legal recognition of the profession. It is in this context that this work was held, whose main objectives are to identify and characterize the functional and personal skills required in performing the functions and are expected to perform as well as the professional environment in which they operate. To achieve these objectives a literature review was conducted to seek and define the main concepts associated with the management of Private Institutions of Social Solidarity, the Human resources Management and the role of technical director. This study relied on the implementation of a survey by questionnaire to thirteen technical director of the municipality of Caldas da Rainha. The main conclusions of this study, highlights the lack of professional category of career development and a gradual increase in responsibilities and skills required

Human mesenchymal stem cells growth and osteogenic differentiation on piezoelectric poly(vinylidene fluoride) microsphere substrates

The aim of this work was to determine the influence of the biomaterial environment on human mesenchymal stem cell (hMSC) fate when cultured in supports with varying topography. Poly(vinylidene fluoride) (PVDF) culture supports were prepared with structures ranging between 2D and 3D, based on PVDF films on which PVDF microspheres were deposited with varying surface density. Maintenance of multipotentiality when cultured in expansion medium was studied by flow cytometry monitoring the expression of characteristic hMSCs markers, and revealed that cells were losing their characteristic surface markers on these supports. Cell morphology was assessed by scanning electron microscopy (SEM). Alkaline phosphatase activity was also assessed after seven days of culture on expansion medium. On the other hand, osteoblastic differentiation was monitored while culturing in osteogenic medium after cells reached confluence. Osteocalcin immunocytochemistry and alizarin red assays were performed. We show that flow cytometry is a suitable technique for the study of the differentiation of hMSC seeded onto biomaterials, giving a quantitative reliable analysis of hMSC-associated markers. We also show that electrosprayed piezoelectric poly(vinylidene fluoride) is a suitable support for tissue engineering purposes, as hMSCs can proliferate, be viable and undergo osteogenic differentiation when chemically stimulated.The authors thank the Portuguese Foundation for Science and Technology (FCT) for financial support under project PTDC/EEI-SII/5582/2014, Strategic Funding UID/FIS/04650/2013 and grants SFRH/BPD/90870/2012 (C.R.) and SFRH/BPD/121526/2016 (D.M.C). The authors acknowledge funding by the Spanish Ministry of Economy and Competitiveness (MINECO) through the project MAT2016-76039-C4-3-R (AEI/FEDER, UE) and from the Basque Government Industry Department under the ELKARTEK program. JLGR, LC, RSS and AS acknowledge funding by the Conselleria de Educación, Investigación, Cultura y Deporte of the Generalitat Valenciana through PROMETEO/2016/063 project. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development. This work was partially financed with FEDER funds (CIBERONC (CB16/12/00284)). The authors acknowledge the assistance and advice of Electron Microscopy Service of the UPVinfo:eu-repo/semantics/publishedVersio

Desenvolvimento de novas estratégias com o objetivo de recuperação funcional do rim lesado

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisKidney diseases represent a major healthcare burden worldwide. It is estimated that one in ten persons suffer from kidney dysfunction. Indeed, a decline in renal function is considered an independent risk factor for both cardiovascular disease and all-cause mortality. The problem is only aggravated by treatment alternatives, with dialysis and transplantation being the only currently available renal replacement therapies. Envisioning the functional recovery of the injured kidney, two regenerative resources were explored: porcine-derived kidney decellularized matrices and renal progenitor cells from human origin. For that, the decellularization process was optimized to obtain porcine kidney decellularized tissue. A full characterization of this matrix in terms of morphology, structural integrity, biochemical content, thermal and molecular properties and protein content was performed. Indeed, porcine-derived matrices were validated as an adequate raw material for the production of several decellularized-based substrates. Namely, kidney tissue-derived electrospun membranes were fabricated for the development of a tubular filtration barrier model. Additionally, decellularized tissue was used for the fabrication of a particulate matrix and a bioink, where 3D cultures of isolated renal cells were established envisioning implantation. Indeed, these cells already shown to possess reparative properties when injected into the injured kidney tissue alone, with limited efficacy. They were also used to develop an organoid model of the glomerulus. Overall, renal progenitor cells demonstrated versatility, specific differentiation into renal phenotypes and proliferation capacity when embedded on the matrix substrates. The works developed in this thesis show that decellularized-based biomaterial substrates may have multiple applications, from modeling systems to moldable implantable scaffolds for tissue engineering strategies. These substrates demonstrated physiological kidney tissue characteristics, allowing cultured cells to represent morphological, phenotypic and functional properties of in vivo systems. Ultimately, this thesis allowed for the development of advanced strategies comprising both relevant cells and biomaterial substrates that may have greater implications in the biomedical field as promising solutions to address renal pathologies in its early stages.As doenças renais representam um grande problema económico-social. Está estimado que uma em cada dez pessoas sofre de disfunção renal. São também consideradas um fator de risco para doenças cardiovasculares e para a taxa de mortalidade geral. Este problema é agravado visto que as únicas opções de tratamento atuais são a diálise e transplantação. Visto que o objetivo inicial da tese é a recuperação do rim lesado, foram explorados dois recursos regenerativos: matriz extracelular de rim de porcino decelularizadas e células progenitoras renais de origem humana. O processo de descelularização foi cuidadosamente otimizado com vista a obter matrizes descelularizadas. Posteriormente, foi feita uma caracterização completa da matriz de porcino em termos de morfologia, integridade estrutural, conteúdo bioquímico, propriedades térmicas e moleculares e ainda conteúdo proteico. Estas matrizes foram usadas como base para produção de diversos biomateriais. Foram fabricadas membranas fibrosas por electrofiação para o desenvolvimento de um modelo in vitro da barreira de filtração tubular. A matriz descelularizada foi usada para obter partículas e também uma biotinta, onde foram estabelecidas culturas 3D de células renais isoladas, com o objetivo final de implantação. Estas células demonstraram anteriormente o seu potencial quando injetadas num rim lesado, com limitada eficácia. Nesta tese, as mesmas células foram também usadas para desenvolver um modelo de organoide do glomérulo. As células progenitoras renais demonstraram ter versatilidade, capacidade de diferenciação específica em fenótipos renais e de proliferação quando cultivadas nas matrizes. Os materiais produzidos a partir da matriz decelularizada demonstraram poder ser usados em múltiplas aplicações, desde modelos para estudos in vitro até scaffolds implantáveis para estratégias de engenharia de tecidos. Estes biomateriais demonstraram ter uma variabilidade fisiológica semelhante à do rim, o que permitiu às células cultivadas modelar propriedades dos sistemas in vivo, nomeadamente morfológicas, fenotípicas e funcionais. Por fim, esta tese permitiu o desenvolvimento de estratégias avançadas compostas por células e biomateriais relevantes, que podem ter imensas implicações biomédicas como soluções promissoras para o tratamento de lesões renais em estágios iniciais.The authors want to acknowledge the financial support obtained by the European Regional Development Fund (ERDF) on the project FROnTHERA (NORTE-01-0145-FEDER-000023) and the FCT PhD Grant on the Doctoral Program on Advanced Therapies for Health (PATH) (PD/BD/128102/2016)

Biomaterials of human source for 3D printing strategies

Three-dimensional printing has risen in recent years as a promising approach that fast-tracked the biofabrication of tissue engineering constructs that most resemble utopian tissue/organ replacements for precision medicine. Additionally, by using human-sourced biomaterials engineered towards optimal rheological proprieties of extrudable inks, the best possible scaffolds can be created. These can encompass native structure and function with a low risk of rejection, enhancing overall clinical outcomes; and even be further optimized by engaging in information- and computer-driven design workflows. This paper provides an overview of the current efforts in achieving ink’s necessary rheological and print performance proprieties towards biofabrication from human-derived biomaterials. The most notable step for arranging such characteristics to make biomaterials inks are the employed crosslinking strategies, for which examples are discussed. Lastly, this paper illuminates the state-of-the-art of the most recent literature on already used human-sourced inks; with a final emphasis on future perspectives on the field

Extracellular matrix electrospun membranes for mimicking natural renal filtration barriers

Kidney diseases are recognized as a major health problem, which affect 10% of the population. Because currently available therapies have many limitations, some tissue engineering strategies have been emerging as promising approaches in this field. In this work, porcine kidneys were decellularized to obtain decellularized kidney extracellular matrix (dKECM). Our results demonstrate a successful protocol of decellularization characterized by the removal of nucleic acid material and preservation of collagen and glycosaminoglycans. Blends of polycaprolactone (PCL) and dKECM were prepared by electrospinning and characterized. The biological performance of the membranes was tested with a human kidney cell line (HK-2) for 7⠯days. It was observed that cellular metabolic activity, proliferation and protein content increased with an increase in dKECM concentrations (30, 50 and 70%). Additionally, the expression of zona occludens-1 was revealed on dKECM-containing membranes but not on pure PCL membranes. To the best of our knowledge this is the first time that natural extracellular matrix is used to mimic the kidney basement membrane as an in vitromodel. This could be a valuable tool for regenerative nephrology and may have an impact on the development of kidney advanced therapies in the future.This work was supported by the European Regional Development Fund (ERDF) on the project FROnTHERA (NORTE-01-0145-FEDER000023); the Portuguese Foundation for Science and Technology (FCT), under the scope of the project SPARTAN (PTDC/CTM-BIO/4388/2014); and the FCT PhD Grant on the Doctoral Program on Advanced Therapies for Health (PATH) (PD/169/2013

Retinoic acid benefits glomerular organotypic differentiation from adult renal progenitor cells in vitro

When in certain culture conditions, organotypic cultures are able to mimic developmental stages of an organ, generating higher- order structures containing functional subunits and progenitor niches. Despite the major advances in the area, researchers have not been able to fully recapitulate the complexity of kidney tissue. Pluripotent stem cells are extensively used in the field, but very few studies make use of adult stem cells. Herein, we describe a simple and feasible method for achieving glomerular epithelial differentiation on an organotypic model comprising human renal progenitor cells from adult kidney (hRPCs). Their glomerular differentiative potential was studied using retinoic acid (RA), a fundamental molecule for intermediate mesoderm induction on early embryogenesis. Immunofluorescence, specific cell surface markers expression and gene expression analysis confirm the glomerular differentiative potential of RA in a short-term culture. We also compared the potential of RA with a potent WNT agonist, CHIR99021, on the differentiative capacity of hRPCs. Gene expression and immunofluorescence analysis confirmed that hRPCs are more sensitive to RA stimulation when compared to CHIR9901. Endothelial cells were also included on the spheroids, resulting in a higher organizational level. The assembly potential of these cells and their selective stimulation will give new insights on adult organotypic cell culture studies and will hopefully guide more works in this important area of research.This work was supported by the Portuguese Foundation of Technology (FCT) with the PhD Grant on the Doctoral Program on Advanced Therapies for Health (PATH) (PD/BD/128102/2016) and the the project Cells4_IDs (PTDC/BTM-SAL/28882/2017), under the Compete2020 Funding Program

Métodos para avaliações econômicas sob condições de risco

Os métodos econômicos tradicionais não têm como realizar avaliações dos investimentos com precisão, pois existem várias limitações nos procedimentos matemáticos. Assim, devido à complexidade da estrutura de mercado, as avaliações dos investimentos precisam considerar as situações de riscos, pois expor o patrimônio de uma organização é uma tarefa muito árdua para ser executada. Para isso, o objetivo deste artigo é corroborar as taxionomias da literatura para análise de investimento, expondo a aplicação das técnicas CAPM, APT e VAR em um projeto financiado por capital próprio e de terceiros. O método de pesquisa utilizado foi respaldo por meio de um embasamento teórico que remete aos conceitos básicos dos métodos econômicos tradicionais e, em seguida, realizou-se um estudo de caso para implementar os métodos na prática. Com base nessa avaliação, os resultados obtidos por meio dessa pesquisa têm como subsidiar as tomadas de decisões dos gestores no âmbito organizacional