Дисертації з теми "Tissu vascularisé"

Cette thèse a pour objectif de développer un tissu vasculaire par la méthode de bio-impression 3D de tissus vivants. Pour mener à bien ces travaux, une bioencre composée de trois biomatériaux naturels : la gélatine, l’alginate et le fibrinogène, a été formulée. Une amélioration du processus de fabrication d’un objet 3D par bio-impression ainsi que le développement d’une solution de consolidation spécifique, a permis le développement d’un réseau cellulaire en trois dimensions. L’utilisation particulière de milieu de culture à toutes les étapes de fabrication, de la préparation des biomatériaux à la consolidation de l’objet, a démontré une augmentation de la prolifération cellulaire de manière conséquente. Des caractérisations rhéologiques et histologiques ont été mises en place afin de démontrer cette prolifération augmentée. Afin de développer un tissu vasculaire, plusieurs approches technologiques ont été présentées, suivant un cahier des charges bien définit : (i) la technologie de biofabrication vasculaire tubulaire et (ii) la technologie de biofabrication vasculaire plane. Les méthodes de bio-impression 3D par micro-extrusion à 1 et 3 extrudeurs, la bio-impression 3D co-axial et tri-axial, la bio-impression 3D en milieu contraint, l’impression 4D par diffusion enzymatique, la bio-impression 3D par enroulement, ont ainsi été étudiées pour répondre à la création d’une structure tubulaire, multicouche et de taille centimétrique. La bio-impression 3D par micro-extrusion et la bio-impression 4D ont, quant à elles, été présentées pour répondre à la création d’une structure multicouche plane, biologiquement pertinente, mimant la paroi vasculaire composée d’une couche endothéliale, d’une couche de cellules musculaires lisses vasculaires et d’une couche de fibroblastes. La dernière partie de ce travail de thèse concerne les résultats de bio-impression, permettant de biofabriquer un tissu vascularisé. Une étude de l’impact des communications entre les fibroblastes et les cellules endothéliales, à l’intérieur d’un environnement 3D, sur le développement d’un réseau complexe, a été présentée. Un tissu vascularisé organisé par les cellules endothéliales à l’intérieur d’une matrice extracellulaire dense et néosynthétisée par les fibroblastes, a ainsi pu être mise en place en 7 jours. Des caractérisations histologiques ont mis en évidence la présence d’une micro-vascularisation et la technologie de microscopie électronique à transmission a permis de caractériser la formation de fibres de collagène et d’élastine, sécrétées par les fibroblastes
This thesis aims to develop a vascular tissue by the method of 3D bioprinting of living tissue. To carry out this work, a bioink composed of three natural biomaterials: gelatin, alginate, and fibrinogen, was formulated. An improvement in the manufacturing process of a 3D object by bioprinting as well as the development of a specific consolidation solution allowed the development of a three-dimensional cellular network. The particular use of culture medium at all stages of manufacture, from the preparation of the biomaterials to the consolidation of the object, has demonstrated a marked increase in cell proliferation. Rheological and histological characterizations were set up to demonstrate this increased proliferation. To develop vascular tissue, several technological approaches have been presented, following well-defined specifications: (i) tubular vascular biofabrication technology and (ii) planar vascular biofabrication technology. The methods of 3D bioprinting by micro-extrusion with 1 and 3 extruders, co-axial and tri-axial 3D bioprinting, 3D bioprinting in a constrained environment, 4D printing by enzymatic diffusion, bio- 3D printing by winding, have thus been studied to respond to the creation of a tubular, multilayer structure of centimeter size. Micro-extrusion 3D bioprinting and 4D bioprinting were presented to respond to the creation of a planar multilayer structure, biologically relevant, mimicking the vascular wall composed of an endothelial layer, d 'a layer of vascular smooth muscle cells, and a layer of fibroblasts. The last part of this thesis concerns the results of bioprinting, allowing to biofabricate a vascularized tissue. A study of the impact of communications between fibroblasts and endothelial cells, within a 3D environment, on the development of a complex network, was presented. A vascularized tissue organized by endothelial cells inside a dense extracellular matrix and neosynthesized by fibroblasts could thus be placed in 7 days. Histological characterizations demonstrated the presence of micro-vascularization and transmission electron microscopy technology characterized the formation of collagen and elastin fibers, secreted by fibroblasts

Generation of large volumes to cover an existing soft tissue defect is often complicated by the lack of available tissue. The current options for soft tissue reconstruction include local and free flaps, collagen fillers, traditional fat grafting and other synthetic soft tissue fillers. But they all have limitations. Recently, a lot of interest has been generated regarding the use of human adipose derived stem cells for engineering a biocompatible soft tissue construct. Give their ready availability, viability and plasticity they appear to be the ideal building blocks for a cell based soft tissue construct. We find that these cells are easy to isolate in large numbers, easy to maintain in culture and capable of multi-lineage differentiation. hASC's are readily adherent to collagen based scaffolds and these function as the ideal cell delivery matrix. Since most wound beds are ischemic and hypoxic, changes in gene expression of hASC's was studied in conditions of hypoxia and serum deprivation. Microarray PCR results demonstrate the up regulation of 23 angiogenic genes including VEGFC, ANPEP, CXCL6, ANGPLT4 and CXCL5 in conditions of hypoxia. However, this angiogenic response was blunted with the presence of serum starvation in addition to hypoxia. Hence we chose to investigate methods to increase the primary neovascularization of a tissue engineered construct. Our hypothesis was that Europium Nano rods (belonging to the lanthanide series of heavy metals) would increase the angiogenic potential of hASC's. Results of a chick embryo chorioallantoic membrane assay demonstrate that Europium Nano rods potentiate the angiogenic effects of Vascular Endothelial Growth Factor (VEGF) when incorporated in hASC's. These rods are readily incorporated in hASC's by endocytosis and do not affect viability. Hence, we conclude that Europium Nano rods can function as a reliable, nontoxic extrinsic angiogenic stimulus. Further studies are needed to evaluate the 1) effects of ENR's on stem cell plasticity 2) effects on gene expression and 3) further investigate the fate of ENR's with repeated cell division.

Orientadora: Profa Dra Sônia Maria Malmonge
Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Engenharia Biomédica, 2017.
Os enxertos vasculares sao comumente usados em procedimentos de revascularizacao, porem existe recorrencia de falhas dos enxertos sinteticos quando usados em areas de baixo fluxo e/ou pressao sanguinea, bem como dificuldade quanto a disponibilidade e disparidade de tamanhos em substituintes autogenos. O objetivo desse trabalho foi o desenvolvimento de arcaboucos para engenharia tecidual de enxertos vasculares de pequeno calibre. O trabalho foi divido em etapas: etapa 1 . para selecionar o(s) polimero(s) mais adequado(s) e tecnica de fabricacao dos arcaboucos; etapa 2 e 3 . para definir as melhores condicoes de processamento; etapa 4 . para a producao dos arcaboucos tubulares (enxertos vasculares), determinar a porosidade aparente, bem como estimar valores de complacencia e pressao de ruptura. A caracterizacao das amostras obtidas nas diferentes etapas foi realizada atraves de imagens de microscopia optica (MO), eletromicrografias de microscopica eletronica (MEV) de varredura com quantificacao de diametro de fibras, ensaios tensao x deformacao sob tracao, e de simulacao computacional dinamica dos fluidos. Foi definido como meta para os arcaboucos a obtencao de estruturas de nanofibras homogeneas livres de contas e valores de propriedades mecanicas proximas aos apresentados pelos vasos naturais. Na Etapa 1, foram selecionados o poli(¿Ã-caprolactona) (PCL) como materia prima e a tecnica de airbrushing como metodo de fabricacao de estruturas fibrosas; na Etapa 2, a pressao de trabalho de 40 PSI foi definida por ser o parametro que resultou em formacao de fibras livres de contas; na etapa 3, o alinhamento de fibras e distancia de 25 cm com coletor rotativo foi definido por resultarem em propriedades mais proximas as dos vasos naturais; e na etapa 4 a condicao de 750 rpm do coletor rotativo, por resultar em arcaboucos com propriedades satisfatorias (complacencia de 12,47 } 2,78 %/100 mmHg; pressao de ruptura de 3483,9 } 358,5 mmHg, e porosidade de 91,07 } 2,69 %). Foi possivel a obtencao de mantas de fibras com diametros em torno de 200 nm tanto para fibras alinhadas quanto nao-alinhadas, com dimensoes na faixa de dimensoes das proteinas estruturais (50 a 500 nm), bem como a obtencao de arcaboucos tubulares promissores para serem usados como enxertos vasculares de pequeno calibre, o que abre perspectiva para a continuacao do estudo.
Vascular grafts are commonly used in revascularization procedures, but there are recurrences when synthetic grafts are used in areas of low blood flow and/or blood pressure, difficulty in the availability as well as disparity of sizes in case of autogenous substituents. The objective of this work was the development of scaffolds for tissue engineering of small caliber vascular grafts. The work was divided in steps: step 1 . to select the most suitable polymer (s) and technique to manufacture the scaffolds; step 2 and 3 . to define the best processing conditions; step 4 . for the production of tubular scaffolds (vascular grafts), to determine the apparent porosity, as well as to estimate values of compliance and burst pressure. The characterization of the obtained samples in the different stages was performed through optical microscopy (OM) images, scanning electron microscopy (SEM) images with fiber diameter quantification, tensile stress x strain tests, and dynamic computational fluid simulation. The goal of the scaffolds was to obtain homogeneous bead-free nanofibrous structures and values of mechanical properties similar to those presented by natural vessels. In Step 1, the poly(¿Ã-caprolactone) (PCL) was selected as the raw material and the airbrushing technique was chosen as a method of manufacturing fibrous structures; in Step 2, the working pressure of 40 PSI was defined as the parameter that resulted in the less beads formation; in step 3, fiber alignment and rotating collectorLs distance of 25 cm was chosen, since resulted in properties closer to those of natural vessels; and in step 4 the 750 rpm rotating collector condition resulted in satisfactory properties (compliance of 12,47 } 2,78 % /100 mmHg, burst pressure of 3483,9 } 358,5 mmHg, and porosity of 91,07 } 2,69 %). It was possible to obtain nanofibers with diameters around 200 nm for both aligned and non-aligned fibers, with dimensions in the range of structural proteins (50 to 500 nm), as well as obtaining tubular scaffolds to be used as small-caliber vascular grafts, which opens the prospect for further study.

Le derme se divise entre les dermes papillaires et réticulaires qui présentent des matrices extracellulaires (MEC) et une vascularisation distincte. Les fibroblastes de chaque compartiment jouent des rôles différents dans la physiologie cutanée. Cependant, peu d’informations est disponible sur leurs propriétés angiogéniques et aucun modèle ne reproduit ces compartiments. L’objectif de ce travail a été de développer un substitut cutané vascularisé qui reproduise les microenvironnements papillaires et réticulaires. Nous avons d’abord mis au point un substitut cutané vascularisé à partir d’une population mixte de fibroblastes par superposition de feuillets cellulaires. Cette technique repose sur la capacité des cellules à déposer leur propre MEC et reproduit une MEC plus physiologique. Nous avons également montré que des molécules angiogéniques peuvent remodeler le réseau vasculaire des substituts. Nous avons ensuite étudié les feuillets cellulaires générés à partir de fibroblastes papillaires et réticulaires et caractérisé leur matrisome et leur profil angiogénique. Ces différences d’expression de gènes se traduisent par la génération de MEC distinctes et des sécrétomes spécifiques. Le microenvironnement de chaque population de fibroblastes induit la formation d’un réseau vasculaire qui présente des points communs avec la peau native. L’ensemble de ces résultats démontrent que les fibroblastes papillaires et réticulaires régulent l’angiogenèse dans la peau via des facteurs paracrines et microenvironnementaux et que les feuillets générés à partir de ces fibroblastes permettent de reproduire certaines caractéristiques des compartiments dermiques observés in vivo
The dermis is divided between the papillary and reticular dermis that display distinct extracellular matrix (ECM) and vascularization. Fibroblasts isolated from each compartment play different role in skin physiology. However, little information is available regarding their role in angiogenesis regulation and no substitute allows to study dermal compartments. The aim of this work was thus to develop a vascularized skin substitute that reproduces papillary and reticular microenvironments.We first developed a vascularized skin substitute using a mixed population of fibroblasts by cell sheet layering. This technique relies on the ability of cells to generate their own ECM and reproduces a more physiological ECM. Furthermore, angiogenic molecules could remodel the vascular network of this skin substitute, indicating that this model is appropriate to study complex interaction between microenvironment and vascularization. We then studied cell sheets generated by papillary and reticular fibroblasts and characterized their matrisome and angiogenic gene expression signature. These difference in gene expression resulted in the generation of specific ECM and secretome. The microenvironment generated by each population of fibroblasts differentially regulated angiogenesis and reproduced some features of native skin in vivo. Overall, these results demonstrate that papillary and reticular fibroblasts contribute to skin angiogenesis via distinct paracrine factors and microenvironments and that vascularized cell sheets reproduce some features of native dermal compartments

Cette thèse a pour objectif de développer une méthode de bioimpression 3D de tissus vivants. Ce nouveau champ disciplinaire a pour but la fabrication de tissus grâce à une bioimprimante en s’appuyant sur les principes fondamentaux de l’ingénierie tissulaire. Pour mener à bien ces travaux, une bio-encre spécifique a été formulée à l’aide de biomatériaux naturels afin de répondre aux critères de biocompatibilité, de maintien de la viabilité cellulaire et de support pour la formation d’un réseau cellulaire en trois dimensions. Plusieurs caractérisations ont ainsi pu être réalisées afin de démontrer l’innocuité du procédé de bioimpression 3D sur les cellules utilisées.L’évolution technologique de la bioimprimante utilisée est ensuite présentée en partant d’une technologie open-source pour arriver à l’utilisation d’un bras robotique 6 axes. L’exigence du cahier des charges de cette bioimprimante a évolué au fil des différents prototypes utilisés.La dernière partie de ce travail de thèse présente les résultats de bioimpression de tissus obtenus grâce à de multiples collaborations. Plusieurs tissus seront étudiés et caractérisés : le derme et sa maturation vers une peau totale, le cartilage et la bioimpression de cellules souches mésenchymateuses, un tissu microvascularisé grâce à l’incorporation de cellules endothéliales et pour finir un tissu perfusable en utilisant une approche de culture dynamique en bioréacteur
This thesis focus on the development of a 3D bioprinting process for living tissue. This new field of research, 3D bioprinting, aims to fabricate tissues using a bioprinter based on the tissue engineering fundamentals.To carry out this work, a specific bioink was formulated using natural biomaterials to meet the requirement of biocompatibility, cell viability and support of a three-dimensional cellular network. Several characterizations have been used to demonstrate the cells viability during the 3D bioprinting process.The bioprinter technological evolution is then presented, starting from an open-source technology and ending with the use of a 6-axis robotic arm. The specifications of this bioprinter evolved through different prototypes.The last part of this thesis concerns tissue bioprinting results obtained through multiple collaborations. Several tissues will be studied and characterized: the dermis and its maturation towards a total skin, the cartilage and the mesenchymal stem cells bioprinting, a microvascularized tissue thanks to the incorporation of endothelial cells and finally a perfusable tissue by using a dynamic culture approach in bioreactor

O consumo crônico de etanol é um importante fator de risco no desenvolvimento de doenças cardiovasculares induzindo elevação da pressão arterial, inflamação, disfunção vascular e aumento do estresse oxidativo em vários tecidos. Além disso, o consumo crônico de etanol induz aumento dos níveis de Fator de necrose tumoral-? (TNF-?). O tecido adiposo perivascular (Perivascular adipose tissue - PVAT) é reconhecido como uma importante fonte de adipocinas e citocinas pró-inflamatórias e esse tecido está envolvido na fisiopatologia de diferentes doenças cardiovasculares. A hipótese deste trabalho é a de que o consumo crônico de etanol estimule a produção de TNF-? no tecido vascular, que por sua vez, induzirá um aumento na produção de espécies reativas de oxigênio (ERO), redução da biodisponibilidade do óxido nítrico (NO), inflamação vascular, prejuízo na função do PVAT, alteração da reatividade vascular e aumento da pressão arterial. Portanto, o objetivo deste estudo foi investigar a participação do TNF-? nas disfunções vasculares induzidas pelo consumo de etanol e avaliar o papel do PVAT em tais danos. O trabalho mostra que o consumo crônico de etanol por 12 semanas induziu aumento da pressão arterial sistólica (PAS) em camundongos machos C57BL/6 (Wild Type -WT) e este aumento foi menor nos animais nocautes para os receptores R1 do TNF-? (TNFR1-/-). O tratamento com etanol não alterou o relaxamento vascular induzido por acetilcolina e nitroprussiato de sódio (NPS). O consumo de etanol induziu aumento da geração de ânion superóxido (O2-), aumento das espécies reativas ao ácido tiobarbitúrico (TBARS) e redução dos níveis de peróxido de hidrogênio (H2O2) em aortas com e sem PVAT (PVAT+ e PVAT-, respectivamente) de animais WT, mas não em camundongos TNFR1-/-. Houve aumento da atividade da catalase (CAT) e da superóxido dismutase (SOD) em aorta PVAT- e PVAT+ e redução dos níveis plasmáticos da glutationa reduzida (GSH) em animais WT, mas não em TNFR1-/-, após o consumo crônico de etanol. Não houve alteração na atividade da glutationa peroxidase (GPx) na aorta dos grupos estudados após o consumo de etanol. Observou-se redução dos níveis de nitrato/nitrito (NOx) em aortas de animais WT após o consumo de etanol, porém não em camundongos TNFR1-/-. O consumo de etanol gerou aumento tecidual das citocinas TNF-? e IL-6 e aumento da atividade da mieloperoxidase (MPO), evidenciando uma exacerbada inflamação vascular e indicando migração de neutrófilos para o tecido aórtico. Tais alterações não foram observadas em animais TNFR1-/-. Os resultados mostram pela primeira vez a participação do TNF-? no aumento da pressão arterial, no aumento do estresse oxidativo e nos prejuízos vasculares induzidos pelo consumo crônico de etanol. O tecido adiposo perivascular não apresentou qualquer efeito benéfico nas alterações supracitadas.
Chronic ethanol consumption is an important risk factor in the development of cardiovascular diseases, inducing increased blood pressure, inflammation, vascular dysfunction and enhanced oxidative stress in several tissues. Furthermore, chronic ethanol consumption induces the production of Tumor necrosis factor-? (TNF-?). Perivascular adipose tissue (PVAT) is known as an important source of adipokines and proinflammatory cytokines. This tissue is involved in the pathophysiology of different cardiovascular diseases. The hypothesis of this work is that the chronic ethanol consumption stimulates the production of TNF-?, which in turn, will induce an increase in reactive oxygen species (ROS) generation, nitric oxide (NO) reduction, vascular inflammation, impairment of PVAT function, alterations of vascular reactivity and increased blood pressure. Therefore, the aim of this study was to investigate the role of TNF-? in chronic ethanol consumption-induced vascular dysfunctions and to evaluate the role of PVAT in such damages. This study demonstrated that chronic ethanol consumption for 12 weeks induced an increase in systolic blood pressure (SBP) in C57BL/6 mice (wild type-WT) and this increase was blunted in TNF-? receptor 1 knockout mice (TNFR1-/-). There was no change in vascular relaxation induced by acetylcholine and sodium nitroprusside (NPS). Ethanol consumption increased the superoxide anion (O2-) generation, thiobarbituric acid reactive species (TBARS) and reduction of hydrogen peroxide (H2O2) levels in aorta without and with PVAT (PVAT- and PVAT+, respectively) from WT animals, but not from TNFR1-/- mice. There was an increase in catalase (CAT) and superoxide dismutase (SOD) activities in aorta PVAT- and PVAT+, decrease on plasma reduced-glutathione (GSH) levels from ethanol-treated WT but not in TNFR1-/-. Ethanol consumption did not change glutathione peroxidase (GPx) activity in any group. Nitrate/nitrite (NOx) aortic levels were decreased in WT animals, but not in TNFR1-/- after chronic ethanol consumption. Ethanol consumption increased TNF-?, IL-6 cytokines and myeloperoxidase activity (MPO) which suggest a strong vascular inflammation and migration of neutrophils into the aortic tissue. Such changes were not observed in TNFR1-/- mice. The results show for the first time the participation of TNF-? in the increase of blood pressure, increase of oxidative stress and vascular dysfunction induced by the chronic ethanol consumption. The perivascular adipose tissue had no beneficial effect on these changes.

A obesidade desencadeia mudanças estruturais e funcionais no tecido adiposo perivascular (PVAT), levando a um desequilíbrio em favor de substâncias vasoconstritoras e pró- inflamatórias, bem como alterações em suas vias de sinalização no vaso. Um importante mecanismo proposto para explicar a perda do efeito anticontrátil do PVAT na obesidade é o estresse oxidativo. Espécies reativas de oxigênio (EROs) possuem papel importante na modulação da função vascular mediada pelo PVAT. Considerando que a mitocôndria representa fonte potencial de EROs nas células, o presente estudo testou a hipótese que a disfunção mitocondrial no PVAT está envolvida na perda do efeito anticontrátil do PVAT em modelo experimental de obesidade. Este estudo avaliou se a matriz mitocondrial nas células que compõem o tecido adiposo periaórtico representa fonte importante de EROs, e se as mesmas contribuem para as alterações na regulação, pelo PVAT, da reatividade vascular. Nosso estudo demonstrou que animais obesos apresentaram disfunção vascular e perda do efeito anticontrátil do PVAT. O estresse oxidativo está envolvido na disfunção do PVAT, com participação significativa da mitocôndria na geração de EROs, capazes de modular a reatividade vascular. A obesidade favoreceu a disfunção mitocondrial, reduzindo o consumo de oxigênio. Estes eventos favoreceram o aumento na geração de peróxido de hidrogênio mitocondrial no PVAT, o qual prejudica a ação anticontrátil deste tecido por ser ativador direto da via de contração RhoA/Rho cinase
Obesity promotes structural and functional changes in the perivascular adipose tissue (PVAT), favoring the release of vasoconstrictor and proinflammatory substances, as well as altering the vascular signaling pathways activated by PVAT-derived factors. Oxidative stress is an important mechanism proposed to explain the loss of anticontractile effects of the PVAT in obesity. Reactive oxygen species (ROS) play an important role in the modulatory effects of PVAT on vascular function. Considering that mitochondria are a potential source of ROS in the cells, the present study tested the hypothesis that mitochondrial dysfunction leads to the loss of the anticontractile effects of PVAT in obesity. We evaluated whether the mitochondrial matrix of the cells that make up the periaortic fat tissue constitute a major source of ROS, and if mROS contribute to defective regulation of vascular reactivity by the PVAT. Our study shows that obese animals exhibit vascular dysfunction and loss of anticontractile effects of PVAT. Oxidative stress is involved in PVAT dysfunction, with a significant contribution of mitochondria to ROS generation. Obesity promotes mitochondrial dysfunction, reducing oxygen consumption. These events increase the generation of mitochondrial hydrogen peroxide in the PVAT, which impairs the anticontractile effects of this tissue via direct activation of the RhoA / Rho kinase pathway

Les réactions immunitaires de la peau sont initiées par les cellules dendritiques cutanées (dendritic cells, DCs). L'effet potentiellement sensibilisateur d'un composé peut être prédit in vitro en utilisant des monocytes humains différenciés en DCs (MonoDCs). Cependant, ces modèles simplistes restent imprécis car l'activation des DCs cutanés par les sensibilisateurs peut être déclenchée ou modulée par des interactions microenvironnementales avec de multiples types de cellules non immunitaires. Notre objectif est de développer une peau immunocompétente qui combinera des MonoDCs avec tous les éléments structurels et fonctionnels de la peau, c'est-à-dire une barrière épidermique posée sur un derme contenant une pseudo-vascularisation et des neurones nociceptifs. Une matrice de collagène a été ensemencée avec des fibroblastes et des cellules endothéliales, puis avec des précurseurs de fibres nerveuses dérivées soit de l'iPSC humaine, soit de la DRG embryonnaire murins. Enfin, nous avons introduit les MonoDC et les kératinocytes. Nous avons observé que les neurones différenciés in situ innervent l'épiderme comme observé habituellement dans la peau humaine normale. De plus, les neurones dérivées d’iPSCs, expriment neuropeptides et canaux calcique spécifiques des fibres nociceptives. Enfin, les Mono-DC intégrés au modèle restent stable pendant toute la durée nécessaire à la formation de l’épiderme et peuvent être stimulé. Le modèle sera utilisé pour prédire le potentiel irritant des composés chimiques et l'impact de l’innervation nociceptive sur l'activation des DCs
Immune reactions in the skin are initiated by the cutaneous dendritic cells (DCs). The potential sensitizing effect of a compound can be predicted in vitro using human monocytes differentiated into DCs (Mono-DCs). However, these simplistic models remain inaccurate because the activation of cutaneous DCs by sensitizers may be triggered or modulated by microenvironmental interactions with multiple types of non-immune cells. Our goal is to develop an immunocompetent human tissue-engineered skin that will combine DCs with all structural and functional element of the skin, i.e. an epidermal barrier laid upon a dermis containing a pseudo-vascularization and nociceptive neurons. Collagen matrix was seeded with fibroblasts and endothelial cells, then with precursors of nerve fibers derived from either human iPSC or murine embryonic DRG. Finally, we introduced Mono-DCs and keratinocytes. We observed that in situ differentiated neurons grow axons towards the epidermis as usually observed in normal human skin. What's more, the neurons derive from iPSC, express neuropeptides and calcium channel as normal nociceptive fibers. Moreover, Mono-DCs settled as expected beneath the epidermis and remained sessile to stimulation for several weeks. The model will be used to predict the irritant potential of chemical compounds, and the impact of nerves on DC activation

A obesidade desencadeia mudanças funcionais no tecido adiposo perivascular (PVAT), favorecendo a liberação de fatores vasoconstritores e consequente ativação de mecanismos contráteis em células vasculares. A sinalização da interleucina 33 (IL-33) via receptor ST2 é essencial para o desenvolvimento e manutenção de células T reguladoras (Tregs) no tecido adiposo visceral. Na obesidade a função das Tregs é comprometida, resultando em estresse oxidativo e inflamação do tecido adiposo. No presente estudo testamos a hipótese que dieta rica em gordura diminui os níveis e a função da IL-33 no PVAT, levando à diminuição do número e função de Tregs, estresse oxidativo e inflamação neste tecido. Camundongos deficientes para o receptor ST2 (ST2 KO) e seus respectivos controles (Balb/C) receberam dieta controle ou hiperlipídica (HFD, high fat diet) durante 18 semanas. A função vascular foi avaliada em anéis de artérias mesentéricas, em presença ou ausência de PVAT, realizando-se curvas concentração-efeito para fenilefrina. Os seguintes grupos experimentais foram analisados: Controle PVAT (-), Controle PVAT (+), HFD PVAT (-) e HFD PVAT (+). Em artérias de camundongos Balb/C que receberam dieta controle, o PVAT diminuiu a resposta contrátil a fenilefrina. No entanto, HFD promoveu perda parcial do efeito anticontrátil promovido por este tecido. Em camundongos ST2 KO que receberam dieta controle, o PVAT diminuiu a resposta contrátil a fenilefrina. No entanto, a ausência de receptores ST2 em camundongos que receberam HFD levou à perda completa do efeito anticontrátil do PVAT. Houve diminuição do número de Tregs e aumento do número de neutrófilos no PVAT de camundongos alimentados com HFD. A incubação com IL-33 recombinante não reverteu a perda do efeito anticontrátil do PVAT promovido pela HFD. Aumento nos níveis séricos e teciduais de IL-6, bem como redução nos níveis de IL-10, foram observados em animais ST2 KO. Houve aumento nos níveis de ânion superóxido no PVAT de camundongos Balb/C alimentados com HFD e a ausência do receptor ST2 potencializou este efeito. Estes dados, analisados em conjunto, indicam que HFD compromete o papel modulador do PVAT e que IL-33 via receptor ST2 tem fundamental importância para a função do PVAT nesta condição experimental.
Obesity triggers functional changes in the perivascular adipose tissue (PVAT), favoring the release of vasoconstrictor factors. Interleukin-33 (IL-33) signaling, via ST2 receptor, is essential for the development and maintenance of regulatory T cells (Tregs) in the visceral adipose tissue. In obesity, Tregs function is compromised, resulting in adipose tissue inflammation. We hypothesized that high fat diet (HFD) decreases the number and function of Tregs and increases inflammation in the PVAT. Mice deficient for the ST2 receptor (ST2 KO) and their respective controls (Balb/C mice) were fed a control diet or a HFD for 18 weeks. Vascular function was evaluated in mesenteric resistance arteries, by performing concentration-effect curves to phenylephrine (PE). In Balb/C mice fed the control diet, PVAT decreased vascular PE contractions. However, a partial loss of PVAT anticontractile effect occurred in arteries from HFD-fed Balb/C mice. In arteries from ST2 KO mice fed the control diet, PVAT decreased PE contractions. However, a complete loss of PVAT anticontractile effects was observed in HFD-fed ST2 KO mice. There was a decrease in the number of Tregs and an increase in the number of neutrophils in the PVAT of mice fed the HFD. The absence of the IL-33 receptor increased IL-6 and reduced IL-10 in HFD-fed mice. There was an increase in superoxide anion levels in the PVAT of Balb/C mice fed HFD and the absence of the ST2 receptor potentiated this effect. These data show that HFD promotes PVAT dysfunction and IL-33 is fundamental to counteract HFD-induced PVAT dysfunction.

L'objectif de notre projet de recherche est l'exploration de la faisabilité de l'allogreffe des tissus composites (ATC) chez les nouveau-nés ayant des anomalies congénitales sévères de la main ou du visage. Dans la partie bibliographique, nous avons étudié les mécanismes de tolérance néonatale chez la souris, ainsi que la transplantation in utero des cellules souches hématopoïétiques avec des modèles animaux et humains. Ensuite, les propriétés du système immunitaire du nouveau-né humain ont été décrites avec étude des différents protocoles de conditionnement non-myéloablatifs utilisés pour induire une tolérance aux greffes d'organes solides, afin de trouver le type de conditionnement utilisable chez les nouveaux nés pour l'induction de tolérance. La greffe du thymus et de la moelle osseuse vascularisée avec l'ATC ont été également étudiés. Enfin, une revue exhaustive des différentes études d'ATC concernant l'induction de tolérance chez les humains et les larges animaux a été faite. Un premier modèle préclinique expérimental d'ATC a été élaboré chez le porcelet nouveau-né. Des études ultérieures ont par suite étudié les agents immunosuppresseurs ainsi que le régime de conditionnement avec l'administration de cyclosporine A., des thymo-globulines de lapin anti-porc et du mycophénolate mofétil. Un protocole d'induction de tolérance pour l'ATC chez les porcelets nouveau-nés a été rédigé et l'expérimentation sera réalisée courant 2014-2015. Si la tolérance d'ATC spécifique du donneur pourra être induite avec notre protocole, nous allons par la suite élaborer un protocole d'induction de tolérance et un programme d'allogreffe de main applicable chez les nouveau-nés humains
This present research is devoted to the exploration of performing vascularized composite allografts as a treatment for severe congenital hand or face anomalies in neonates or very young infants. The bibliographic studies at first revised the discovery and mechanisms of neonatal tolerance in mice, as well as in utero hematopoietic stem cells transplantation in large-animal models and human fetuses. Then the properties of human neonatal immune system were described; and the non-myeloablative or non-toxic conditioning regimens for solid organ transplant tolerance induction were also studied, in order to give the clue to a applicable conditioning regimen for tolerance induction in neonates. The potent thymus and vascularized bone marrow transplantation in neonatal VCA were considered as advantages. Finally, the researches concerning tolerance induction for VCA in large animal models and in human patients were reviewed. ln experimental studies, the preclinical VCA was firstly established in neonatal swines. Subsequent experiments thus studied the immunosuppressive agents, as well as conditioning regimen, including the administration of cyclosporine A, rabbit anti-pig thymocyte globulin and mycophenolate mofetil for VCA in pig neonates. The findings in these experiments were then concluded. Based on these finding, a general tolerance induction protocol for VCA in neonatal swines was designed and experiment will be performed in year 2014-2015. lf donor-specific tolerance for VCA could be induced with present protocol, we will subsequently elaborate an applicable tolerance induction protocol and hand allotransplantation program in human newborn infants

O objetivo deste estudo foi identificar características de uso da luz para explicar a distribuição diferencial das espécies tolerantes à dessecação homeoclorófila Anemia flexuosa e peciloclorófila Pleurostima purpurea em ambientes sombreados e expostos, respectivamente, de comunidades vegetais de afloramentos rochosos. A cultivar Oryza sativa IAC 202 foi incluída para comparações. Durante um ciclo completo de desidratação - dessecação - reidratação foram avaliados parâmetros fotossintéticos de trocas gasosas e fluorescência da clorofila a, associados ao conteúdo relativo de água (CRA) e de pigmentos fotossintéticos de plantas intactas sob temperatura e umidade relativa do ar constantes de 25°C e de 55%, respectivamente. As plantas foram submetidas à diferentes densidades de fluxo de fótons fotossintéticos (DFFF de 0, 100 e 400 ?mol fótons m-2s-1) nas fases de desidratação e dessecação, dependendo da espécie. O. sativa foi avaliada somente durante as fases de desidratação e dessecação sob condições ambientais variáveis de casa de vegetação. A diminuição da assimilação líquida de CO2 (A) foi acompanhada pelo aumento da dissipação de calor avaliada pelos coeficientes de extinção nãofotoquímica (qN e NPQ) nas três espécies. Após cessação de A, a eficiência quântica efetiva (?PSII e Fv\"/Fm\"), a taxa de transporte de elétrons (ETR) e o coeficiente de extinção fotoquímica (qP) foram mantidos relativamente altos em P.purpurea, mas cessaram simultaneamente com A em A.flexuosa. Em O.sativa, ?PSII, ETR e qP diminuíram substancialmente após a cessação de A, mas Fv\"/Fm\" foi mantido. A eficiência quântica potencial (Fv/Fm) foi a última variável a diminuir nas três espécies durante a desidratação. Após a reidratação de P.purpurea e A.flexuosa foi observado inicialmente o estabelecimento da respiração e em seguida um balanço levemente positivo de CO2, quando os valores de Fv\"/Fm\", ?PSII, ETR, qP e Fv/Fm de P.purpurea recuperaram quase totalmente, enquanto qN e NPQ diminuíram. A.flexuosa apresentou uma recuperação apenas parcial de Fv\"/Fm\", ?PSII, ETR, qP e Fv/Fm quando o balanço de CO2 se tornou levemente positivo, tendo sido a recuperação ainda menor para o tratamento de desidratação no escuro associado à dessecação na luz. A.flexuosa tolerou a perda de 88% do CRA. O enrolamento foliar durante a desidratação é uma forma de proteção contra a luz no estado dessecado de A.flexuosa. Mesmo no estado dessecado ocorrem processos de interação dos fotossistemas II com a luz em A.flexuosa. P.purpurea baseia sua proteção contra a luz na ativação de processos de dissipação de calor, vias de consumo de elétrons diferentes do ciclo redutivo do CO2 e, em última instância, na perda de clorofilas. Plantas dessecadas de P.purpurea permanecem viáveis no estado desidratado por pelo menos 42 dias. P.purpurea tolerou a perda de 94% do CRA. A recuperação do turgor da parte aérea de P.purpurea ocorre necessariamente pela absorção de água pelas raízes durante a reidratação. Foi evidenciada uma aclimatação de A.flexuosa quando desidratada sob condição de luz. Os resultados não foram conclusivos em relação à sustentação da hipótese, considerando que as diferenças de recuperação observadas para A.flexuosa nos diferentes tratamentos luminosos, em geral, não foram significativas.
The aim of this study was to identify characteristics of light use that could explain the differential distribution of homoiochlorophyllous and poikilochlorophyllous desiccation tolerant plants Anemia flexuosa and Pleurostima purpurea, respectively, in shaded and exposed microsites of rock outcrop plant communities. Oryza sativa IAC 202 was included in the study for comparisons. Leaf gas exchanges, fluorescence chlorophyll, relative water content (RWC) and photosynthetic pigment content were evaluated in intact plants under constant temperature and relative humidity of 25°C and 55%, respectively, during a complete cycle of dehydration - desiccation - rehydration. The plants were exposed to different photosynthetic photon flux densities (PPFD of 0, 100 and 400 ?mol photons m-2s-1) during dehydration and desiccation phases, according to species. O.sativa was evaluated only during dehydration and desiccation phases under variable environmental conditions in a greenhouse. In all species, the decrease in CO2 net assimilation (A) was accompanied by increased heat dissipation assessed by nonphotochemical quenching coefficients (qN and NPQ). The effective quantum yield (?PSII and Fv\"/Fm\"), electron transport rate (ETR) and photochemical quenching coefficient (qP) were kept relatively high after A cessation in P.purpurea, but in A.flexuosa ceased simultaneously with A. In O.sativa, ?PSII, ETR and qP decreased substantially after A cessation, but Fv\"/Fm\" was maintained. The potential quantum yield (Fv/Fm) was the last variable to decrease during dehydration in all species. After rehydration, the establishment of respiration was observed initially in P.purpurea and A.flexuosa. Then, a slightly positive CO2 balance was associated with the almost total recovery of Fv\"/Fm\", ?PSII, ETR, qP and Fv/Fm in P.purpurea, while qN and NPQ decreased. A.flexuosa showed only a partial recovery of Fv\"/Fm\", ?PSII, ETR, qP and Fv/Fm when the CO2 balance became slightly positive, and recovery was even lower for the treatment of dehydration in dark associated to desiccation in light. A.FLEXUOSA TOLERATES A LOSS OF 88% OF RWC. Leaf curling during dehydration is also a form of light protection in the dried state in A.flexuosa. Interactions between photosystem II and light occur even in the dried state of A.flexuosa. P.purpurea bases its protection against light activating heat dissipation process, ways of electron consumption different of reductive CO2 cycle and, in last instance, chlorophyll loss. P.purpurea remains viable in dried state for at least for 42 days, and tolerates a loss of 94% of RWC. The shoot rehydration in P.purpurea occurs necessarily by roots water uptake. A.flexuosa showed an acclimation when dried under light conditions. The results were not conclusive regarding the hypothesis, since differences in recovery observed for this species in the different light treatments, in general, were not significant.

INTRODUÇÃO: A microanastomose vascular é um componente importante na cirurgia de transferência livre de tecidos. Atualmente, a técnica de sutura convencional ainda é considerada o padrão ouro, no entanto, ela apresenta alguns inconvenientes por ser tecnicamente difícil, consumir muito tempo e ter uma longa curva de aprendizado. Na busca de uma técnica mais fácil e rápida, métodos alternativos de anastomose são estudados incluindo a cola de fibrina. Apesar dos bons resultados publicados, a sua aceitação na prática clínica ainda é limitada. Controvérsias a cerca de sua trombogenicidade e resistência mecânica geram dúvidas em relação a sua segurança. A ausência de um modelo experimental mais fidedigno impede que os potenciais benefícios de sua aplicação clínica sejam apreciados. O objetivo deste estudo é esclarecer essas controvérsias e estudar os benefícios da aplicação da cola de fibrina em um ambiente que simule a prática clínica. MÉTODOS: O modelo experimental utilizado foi a transferência livre de um retalho inguinal para a região cervical anterior. A circulação do retalho era restaurada através de microanastomoses vasculares entre as artérias femoral e carótida (término-lateral) e entre as veias femoral e jugular externa (término-terminal). Utilizamos 20 coelhos que foram divididos em dois grupos (n= 10) de acordo com a técnica de sutura empregada: Grupo I (sutura convencional) e Grupo II (sutura com cola). RESULTADOS: A aplicação da cola de fibrina reduziu significativamente o número de pontos necessários para se completar as anastomoses, 4 pontos a menos nas artérias e 4,5 pontos a menos nas veias. No Grupo I, a média do tempo de anastomose arterial foi de 17,21 minutos, contra 12,72 minutos no Grupo II. Nas anastomoses venosas, a média de tempo no Grupo I foi de 22,93 minutos, contra 16,57 minutos no Grupo II. A aplicação da cola de fibrina também diminuiu o tempo de isquemia do retalho e o tempo de cirurgia em 11,5 minutos e 15,67 minutos, respectivamente. A taxa de sobrevida do retalho foi de 90% nos dois grupos. CONCLUSÕES: A aplicação da cola de fibrina em microanastomoses vasculares demonstrou ser confiável e eficiente no presente estudo.
INTRODUCTION: Microvascular anastomosis is an important component of the free flap surgical procedure. Currently, the conventional suture is still considered the gold standard technique. However, it presents some problems for being technically demanding, time consuming and with a long learning curve. In looking for an easier and faster technique, alternative methods of anastomosis were studied including the fibrin glue. Despite the good results reported in the literature, its acceptance in the clinical setting is still small Controversies regarding its thrombogenicity and mechanical resistance create some concerns about its safeness. The absence of a more realistic experimental model has not allow a full aprecciation of its potencial benefits in clinical use. The aim of this study is clarify these controversies and demonstrate the advantages of fibrin glue application in an environment that can reproduce the clinical practice. METHODS: A free inguinal flap transfer to the anterior cervical region was used as experimental model. The circulation of the flap was restored by means of microvascular anastomosis between the femoral and carotid arteries (end-to-side) and between the femoral and jugular veins (end-to end). The procedures were performed in 20 rabbits that were divided into two groups (n= 10) according to the anastomosis technique: Group I (conventional) and Group II (fibrin glue). RESULTS: The application of fibrin glue significantly reduced the amount of sutures required to complete the anastomoses: 4 less sutures in the arteries and 4,5 less sutures in the veins. In Group I, the mean arterial anastomosis time was 17,21 minutes against 12,72 minutes in Group II. In the veins, the mean anastomosis time in Group I was 22,93 minutes against 16,57 minutes in Group II. The application of fibrin glue also reduced the flap ischemic time and the total operative time by 11,5 minutes and 15,67 minutes, respectively. The flaps\' survival rate was 90% in both groups. CONCLUSIONS: The application of fibrin glue in microvascular anastomoses was reliable and effective in this study.

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Background: Muscle changes of paretic limb resulting from stroke lead to changes in the mechanical properties of muscles, such as muscle weakness and increased resistance to stretching. Studies that characterize the components of the muscle tissue, as the noncontractile, and biomarkers related to proliferation of connective tissue (such as TGF-β1 and myostatin) are clinically relevant and necessary for understanding of the resistance to stretching of paretic muscles. Therefore, the aim of this study was to evaluate the serum concentration of TGF-β1 and myostatin, the percentage volume of non-contractile tissue and passive peak torque and resistance to stretching (stiffness) of the extensor and flexor muscles of the knee in chronic hemiparesis. Methods: Cross-sectional study. Fourteen subjects with chronic hemiparesis post stroke and fourteen healthy paired-subjects participated in this study. Paretic, non-paretic and control limbs were evaluated. MRI images were obtained in all subjects and the percentage volume of non-contractile tissue of the quadriceps and hamstrings was measured. Serum TGF-β1 and myostatin concentrations were quantified by ELISA method. Passive torque peak and resistance during stretching (stiffness) of extensors and flexors muscles of knee were assessed at 60°/s using isokinetic dynamometer. Results: An increase in the percentage volume of non-contractile tissue in VM and VL of paretic limb compared to non-paretic limb was observed (p<0,05). Also, an increase was observed in the percentage volume of non-contractile tissue in SS in paretic limb compared to control limb (p<0,05). No differences were observed in serum TGF-β1 and myostatin concentrations in hemiparetic group compared to the control group (p> 0.05). Regarding passive torque, there was an increase in peak torque and resistance during passive stretching of extensor muscles with increasing ROM of paretic, non-paretic and control limbs (p<0,05), but no differences were found among limbs (p>0,05). In relation to flexor muscles, there was also an increase in peak torque along the ROM of the three limbs (p<0,05). However, non-paretic limb has lower values of peak torque than control and paretic limbs in lower ROM (p<0,05). Paretic limb increases resistance in a more accentuated pattern at intermediate ROM (50-40º) compared to control (p=0,02). A moderate correlation was observed between TGF-β1 serum concentration and flexor peak torque of paretic limb, considering complete ROM (p=0,01; r=0,736). Conclusion: Paretic muscles, extensors and knee flexors, although they have increased noncontractile tissue, exhibit similar resistance to stretching the muscles of healthy subjects. Knee flexor muscles of the non-paretic limbs have less passive stretch resistance compared to healthy subjects without changes in non-contractile content. Furthermore, no changes in serum concentrations of TGF-β1 and myostatin in chronic hemiparetic compared to healthy subjects were observed.
Contextualização: Alterações na musculatura do membro parético em decorrência do Acidente Vascular Cerebral (AVC) podem resultar em mudanças nas propriedades mecânicas da musculatura, como fraqueza muscular e aumento da resistência ao alongamento. Estudos que caracterizem os componentes do músculo, como o tecido não contrátil, e biomarcadores relacionados à proliferação de tecido conjuntivo (como TGF-β1 e miostatina) são necessários e clinicamente relevantes para o entendimento da resistência ao alongamento de músculos paréticos. Portanto, o objetivo do presente estudo foi avaliar a concentração sérica de TGF-β1 e miostatina, o volume percentual de tecido não contrátil e o pico de torque passivo e resistência ao alongamento ( stiffness ) dos músculos extensores e flexores do joelho de hemiparéticos crônicos. Materiais e Métodos: Estudo transversal. Quatorze sujeitos com hemiparesia crônica pós-AVC e quatorze sujeitos saudáveis pareados participaram deste estudo. Os membros parético, não parético e controle, foram avaliados. Imagens por ressonância magnética foram obtidas em todos os sujeitos e o volume percentual de tecido não contrátil dos músculos do quadríceps e dos isquiotibiais foi mensurado. As concentrações séricas de TGF-β1 e miostatina foram quantificadas pelo método ELISA. O pico de torque passivo e a resistência ao alongamento passivo ( stiffness ) dos músculos extensores e flexores do joelho foram obtidos a 60°/s em dinamômetro isocinético e avaliados ao longo de intervalos de ADM. Resultados: Foi observado um aumento no volume percentual de tecido não contrátil nos músculos vasto medial (VM) e vasto lateral (VL) do membro parético comparado ao não parético (p<0,05). Ainda, houve um aumento no volume percentual de tecido não contrátil nos músculos semitendinoso e semimembranoso (SS) do membro parético comparado ao membro controle (p<0,05). Não foram observadas diferenças nas concentrações séricas de TGF-β1 e miostatina entre os grupos (p>0,05). Houve um aumento no pico de torque e na resistência ao alongamento passivo extensor com o aumento da amplitude de movimento (ADM) nos membros parético, não parético e controle, sem diferenças entre os membros (p>0,05). Em relação aos flexores, também houve um aumento no pico de torque ao longo da ADM nos membros parético, não parético e controle. No entanto, o membro não parético apresenta valores de pico de torque menores que o parético e controle em amplitudes menores (p<0,05). O membro parético aumenta a resistência de forma mais acentuada em ADM intermediária (50-40º) comparado ao controle (p=0,02). Houve correlação moderada entre a concentração sérica de TGF-β1 e o pico de torque flexor do membro parético, considerando-se a ADM completa (p=0,01; r=0,736). Conclusão: Músculos paréticos, extensores e flexores do joelho, apesar de possuírem aumento de tecido não contrátil, apresentam resistência ao alongamento semelhante a músculos de indivíduos saudáveis. Músculos flexores do joelho dos membros não parético apresentam menor resistência passiva ao alongamento comparado a indivíduos saudáveis, sem alterações no conteúdo não contrátil. Além disso, indivíduos hemiparéticos crônicos não apresentam alterações nas concentrações séricas de TGF-β1 e miostatina comparados a indivíduos saudáveis.

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Biomateriais, Reabilitação e Biomecânica)
One of the major problems related to small-diameter blood vessels replacement is the lack of vascular grafts with suitable mechanical and biological properties. Although there are synthetic vascular grafts in clinical use, these substitutes present thrombogenic behaviour and are too stiff compared to native vessels. Rapid endothelialization and matched mechanical properties are important functional requirements that vascular grafts should accomplish. Herein, an electrospun tubular fibrous (eTF) scaffold was fabricated and functionalized to immobilize tropoelastin at the luminal surface, providing a biomimetic environment to enhance endothelialization. The morphology was assessed by scanning electron microscopy, the effectiveness of surface functionalization by NH2 groups quantification and surface charge measurements, and the mechanical properties by uniaxial tensile tests. Tropoelastin was immobilized at 20 μg/mL by its -NH2 functional groups on activated scaffolds, as well as by its -COOH functional groups on aminolysed scaffolds, in an attempt to expose different conformations of tropoelastin for cell binding. The amount of immobilized tropoelastin on both substrates was quantified by microBCA assay. These constructs were cultured with a cell line of human umbilical vein endothelial cells (HUVECs) for 7 days, to study the endothelialization of eTF scaffolds by evaluating their metabolic activity, proliferation, total protein synthesis, VEGF secretion, as well as cell morphology and phenotype maintenance. Our experimental characterization demonstrated that the eTF scaffolds have a thickness of 240.85 ± 46.91 μm and their luminal surface was 33.55 % porous mix of micro to submicro fibers diameters, pore sizes less than 23 μm and pore areas up to 70 μm2. The eTF scaffolds were successfully functionalized by the insertion of 0.5 ± 0.04 nmol/mg of NH2 groups at their surface and confirmed by the differences observed in surface charge. Untreated, activated and aminolysed scaffolds supported higher stresses and strains in axial direction rather than in radial direction. These values are compatible to those of native blood vessels. The exposure of tropoelastin -COOH groups promoted endothelial cells metabolic activity and growth, whereas when exposed its -NH2 groups a significant influence on protein synthesis was observed. Additionally, eTF scaffolds promoted phenotype maintenance and endothelial cell coverage just after 7 days of culture. Altogether, the results confirm that biofunctional eTF scaffolds are suitable for vascular application since they presented adequate mechanical properties and a rapid endothelialization.
Um dos maiores problemas associados à substituição de vasos sanguíneos de pequeno diâmetro é a insuficiência de enxertos vasculares com propriedades mecânicas e biológicas adequadas. Embora existam enxertos vasculares sintéticos na prática clínica, estes substitutos apresentam trombogenicidade e são demasiado rígidos comparativamente aos vasos sanguíneos nativos. Uma rápida endotelização e propriedades mecânicas semelhantes aos vasos sanguíneos humanos são requisitos essenciais que um excerto vascular deve possuir. Neste trabalho, estruturas tubulares fibrosas foram produzidas por electrospinning (eTF scaffolds) e funcionalizadas para imobilizar tropoelastina na superfície interna, proporcionando um ambiente biomimético para promover a endotelização. A morfologia foi analisada por microscopia eletrónica de varrimento (SEM), a eficiência da funcionalização da superfície pela quantificação dos grupos amina (-NH2) e pela carga de superfície, e as propriedades mecânicas foram analisadas por testes uniaxiais à tração. A tropoelastina foi imobilizada a uma concentração de 20 μg/mL através dos seus grupos -NH2 nos eTF scaffolds activados, bem como pelos seus grupos carboxílicos (-COOH) nos scaffolds aminolisados, de forma a expor diferentes conformações para a ligação com as células. A quantidade de tropoelastina imobilizada em ambos os substratos foi quantificada através do método microBCA. Por último, os eTF scaffolds foram semeados com uma linha celular de células endoteliais da veia umbilical humana durante 7 dias para estudar a endotelização. Desta forma, a atividade metabólica, a proliferação celular, a síntese proteica e de VEGF, bem como a morfologia celular e a manutenção do fenótipo dos eTF scaffolds foram investigadas. Os resultados experimentais demonstraram que os eTF scaffolds possuem uma espessura de 240.85 ± 46.91 μm e uma superfície interna 33.55% porosa com diâmetros de fibras na ordem do micro ao submicro, tamanhos de poros inferiores a 23 μm e áreas de poros até 70 μm2. Os eTF scaffolds foram efetivamente funcionalizados através da inserção de 0.5 ± 0.04 nmol de grupos NH2 na superfície e pelas diferenças observadas na carga de superfície. Os eTF scaffolds não tratados, activados e aminolisados suportaram tensões e elongamentos mais elevados na direção axial do que na radial. Estes resultados obtidos são compatíveis com os valores reportados para os vasos sanguíneos nativos. A exposição dos grupos -COOH da tropoelastina induziu um aumento da atividade metabólica e crescimento das células endoteliais. Quando expostos os grupos -NH2, uma influência significativa na síntese proteica foi observada. Além disso, os eTF scaffolds promoveram a manutenção do fenótipo e a formação de uma monocamada de células endoteliais na superfície após 7 dias de cultura. De um modo geral, estes resultados confirmam que estes eTF scaffolds biofuncionais são adequados para aplicação vascular, uma vez que apresentam propriedades mecânicas adequadas e uma rápida endotelização.