Dissertations / Theses on the topic 'Vascularized tissue'

Four million surgeries involving bone grafting or bone substitutes for the treatment of bone defects are performed yearly worldwide. However, limited donor tissue availability, pain and the risk of infection and immune rejection, have led to the development of alternative strategies for bone repair. Tissue engineering represents an alternative to current treatments as it consists of using a biomaterial scaffold alone or in combination with proteins, genes or cells, as a bioactive implant to stimulate bone repair. Microspherical scaffolds have been proposed as a potential modular unit for bone tissue engineering applications as their shape could facilitate filling of irregular shaped defects. Furthermore, microspheres could be used as a support for ex vivo expansion of adherent cells as well as a carrier to directly deliver cells to the defect site. In this study, the use of phosphate glass microcarriers for bone tissue engineering applications was investigated. As this material is completely soluble and non-toxic, it can be implanted in the patient together with cells. Furthermore, the tuneable glass composition can be easily engineered to induce specific structural and biological properties. Here, the effect of culturing MG-63 and hBM-MSCs on titanium-doped phosphate glass microspheres containing increasing concentration of cobalt (0, 2 and 5%) was investigated, as these ions have been shown to induce osteogenesis and angiogenesis, respectively. Furthermore, as part of this study a novel perfusion microfluidic bioreactor was fabricated to culture cells on microspheres under perfusion and to enable parallel screening of multiple culture variables. Cells proliferation on the microspheres as well as secretion of ECM proteins in response to the substrate was observed over time, thus confirming the biocompatibility of all compositions tested. Upregulation of osteogenic markers by MSCs also occurred in response to the microspheres in the absence of exogenous supplements. However, this effect was suppressed when cobalt was added to the glass composition. On the other hand, while cobalt doping was found to induce key angiogenic responses (i.e. VEGF secretion), this did not translate into improved functional vascularization in comparison to the cobalt-free microspheres. Successful MSCs culture on the microspheres within the microfluidic reactor was achieved and it was possible to efficiently quantify functional outputs, such as the expression of ECM proteins as a function of microspheres substrates and nutrient feeds under perfusion. In conclusion, titanium-doped phosphate glass microspheres were identified as a potential substrate for bone tissue engineering applications in terms of MSCs expansion and differentiation, as well as to support endothelial cells migration towards the scaffold and vessel formation, while additional doping with cobalt was not found to improve the functionality of the microspheres. Furthermore, the microfluidic bioreactor enabled to identify optimal parameters for perfused cell culture on microspheres that could be potentially translated to a scaled-up system for tissue-engineered bone manufacturing.

Skin is an organ with a complex structure which plays a crucial role in thebody’s defence against external threats and in maintaining major homeostatic functions. The need for in vitro models that mimic the in vivo milieu is therefore high and relevant with various applications including, among others, penetration, absorption, and toxicity studies. In this context, the choice of the biomaterial that will provide a 3D scaffold to the cultured cells is defining the model’s success. The FN-4RepCT silk is here suggested as a potent biomaterial for skin tissue engineering applications. This recombinantly produced spider silk protein (FN-4RepCT), which can self-assemble into fibrils, creates a robust and elastic matrice with high bioactivity, due to its functionalization with the fibronectin derived RGD-containing peptide. Hence it overcomes the drawbacks of other available biomaterials either synthetic or based on animal derived proteins. Additionally, the FN-4RepCT silk protein can be cast in various 3D formats, two of which are utilized within this project. We herein present a bilayered skin tissue equivalent supported by the FN-4RepCT silk. This is constructed by the combination of a foam format, integrated with dermal fibroblasts and endothelial cells, and a membrane format supporting epidermal keratinocytes. As a result, a vascularized dermal layer that contains ECM components (Collagen I, Collagen III, and Elastin) is constructed and attached to an epidermal layer of differentiated keratinocytes.The protocol presented in this project offers a successful method of evenly integrating cells in the FN-4RepCT silk scaffold, while preserving their ability to resume some of their major in vivo functions like proliferation, ECM secretion, construction of vascular networks, and differentiation. The obtained results were evaluated with immunofluorescence stainings of various markers of interest and further analysed, when necessary, with image processing tools. The results that ensued from the herein presented protocol strongly suggest that the FN-4RepCT silk is a promising biomaterial for skin tissue engineering applications.

The aims of this project were to: (1) successfully design two models of reconstructive tissue transplants in the primate, one with a purely sensory nerve supply, the other a mixed sensory and motor supply and (2) achieve long enough survival for reinnervation to have occurred, assuming it can take place in the presence of the immunosuppressants.
A neurovascular free flap comprised of the entire soft tissue coverage of the second digit and a hand transplant model were successfully designed in the baboon (Papio hamadryas anubis). Seven transplanted neurovascular free flaps and four hand transplants were undertaken. High dose Cyclosporin A was found to be necessary to prevent rejection. Steroids proved to be a necessary part of the immunosuppressive regime. Nine out of 11 transplants survived to or beyond 4 months. In most cases, the end point was determined by the date for evaluation of reinnervation by our neurophysiologist colleagues and not loss of the transplant due to rejection.
Only 3 out of 11 transplants survived with little or no signs of rejection. All others had significant episodes of rejection, most of which were successfully reversed or controlled by using our rejection protocol. In addition, all animals, to varying degrees, demonstrated some of the following side effects: anorexia, anemia, gingival hyperplasia, hepatotoxicity, hirsutism, lymphoma, nephrotoxicity, subcutaneous or intramuscular abscesses and tremors. (Abstract shortened by UMI.)

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

The ability to manufacture human tissues that replicate the spatial, mechano-chemical, and temporal aspects of biological tissues would enable myriad applications, including drug screening, disease modeling, and tissue repair and regeneration. However, given the complexity of human tissues, this is a daunting challenge. Current biofabrication methods are unable to fully recapitulate the form and function of human tissues, which are composed of multiple cell types, extracellular matrices, and pervasive vasculature. My Ph.D. thesis focuses on advancing the capabilities of human tissue fabrication. Specifically, we demonstrate a multimaterial bioprinting method capable of producing 1D, 2D, and 3D vascularized tissue constructs by co-printing ECM, cell-laden, and fugitive inks. After these heterogeneous tissue constructs are printed and infilled with ECM, they are cooled to 4˚C to remove the fugitive ink leaving behind a pervasive network that is subsequently lined with endothelial cells. These constructs are ~ 1 mm in thickness and can be sustained for up to 14 days via rocking-based flow through their vasculature. We then created a new extracellular matrix that enabled the fabrication of tissues that exceed 1 cm in thickness that are perfused on a microfluidic chip for long time periods (> 6 weeks). To demonstrate functionality, growth factors are perfused via the vasculature to differentiate stem cells toward an osteogenic lineage in situ. Finally, we created renal proximal tubules (a sub-unit of kidney tissue) by this approach. Specifically, we constructed 3D tubules circumscribed by renal proximal tubule epithelial cells (PTECs). The PTECs form confluent, leak-tight epithelial monolayers that exhibit primary cilia and expresses Na+/K+ ATPase, Aquaporin 1, and K-cadherin. The combination of 3D geometry and on-chip perfusable nature gives rise to enhanced, polarized PTEC phenotypes that develop an enhanced brush border, basement membrane protein deposition, basolateral interdigitations, enhanced cell height, megalin expression, and albumin uptake relative to 2D controls. In summary, this multimaterial 3D bioprinting platform enables production of engineered human tissue constructs in which multiple cell types and vasculature are programmably placed within extracellular matrices. These 3D tissues may find potential applications in drug screening, disease models, and ultimately, tissue engineering and regenerative medicine.
Engineering and Applied Sciences - Engineering Sciences

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

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

The knee joint is a complex composite joint containing the C-shaped wedge-like menisci composed of fibrocartilage. Due to their complex composition and structure, they provide mechanical resilience to the knee joint protecting the articular cartilage. Because of the limited repair potential, meniscal injuries do not only affect the meniscus itself but also lead to altered joint homeostasis and inevitably to secondary osteoarthritis. The meniscus was characterized focusing on its anatomy, structure and meniscal markers such as aggrecan, collagen type I (Col I) and Col II. The components relevant for meniscus tissue engineering, namely cells, Col I scaffolds, biochemical and biomechanical stimuli were studied. Meniscal cells (MCs) were isolated from meniscus, mesenchymal stem cells (MSCs) from bone marrow and dermal microvascular endothelial cells (d-mvECs) from foreskin biopsies. For the human (h) meniscus model, wedge-shape compression of a hMSC-laden Col I gel was successfully established. During three weeks of static culture, the biochemical stimulus transforming growth factor beta-3 (TGF beta-3) led to a compact collagen structure. On day 21, this meniscus model showed high metabolic activity and matrix remodeling as confirmed by matrix metalloproteinases detection. The fibrochondrogenic properties were illustrated by immunohistochemical detection of meniscal markers, significant GAG/DNA increase and increased compressive properties. For further improvement, biomechanical stimulation systems by compression and hydrostatic pressure were designed. As one vascularization approach, direct stimulation with ciclopirox olamine (CPX) significantly increased sprouting of hd-mvEC spheroids even in absence of auxiliary cells such as MSCs. Second, a cell sheet composed of hMSCs and hd-mvECs was fabricated by temperature triggered cell sheet engineering and transferred onto the wedge-shaped meniscus model. Third, a biological vascularized scaffold (BioVaSc-TERM) was re-endothelialized with hd-mvECs providing a viable vascularized network. The vascularized BioVaSc-TERM was suggested as wrapping scaffold of the meniscus model by using two suture techniques, the all-inside-repair (AIR) for the posterior horn, and the outside-in-refixation (OIR) for the anterior horn and the middle part. This meniscus model for replacing torn menisci is a promising approach to be further optimized regarding vascularization, biochemical and biomechanical stimuli
Das Knie ist ein komplex zusammengesetztes Gelenk mit zwei C-förmigen Keilen aus Bindegewebsknorpel, die Menisken. Sie sorgen für die mechanische Belastbarkeit des Knies, wodurch der Gelenksknorpel geschützt wird. Aufgrund des limitierten Heilungspotentials beeinträchtigen Meniskusverletzungen nicht nur den Meniskus selbst, sondern schädigen auch das Gelenksgleichgewicht und führen zu sekundärer Osteoarthritis. Der Meniskus wurde in seiner Anatomie, Struktur und Meniskusmarkern wie Aggrekan, Kollagen I und Kollagen II charakterisiert. Die Komponenten von Meniskus Tissue Engineering, Zellen, Kollagen I Materialien, biochemische und biomechanische Stimuli wurden untersucht. Meniskuszellen (MCs) wurden aus Meniskus isoliert, mesenchymale Stammzellen (MSCs) aus Knochenmark und dermale mikrovaskuläre Endothelzellen (d-mvECs) aus Vorhautbiopsien. Für das humane (h) Meniskus-Modell wurde die keilförmige Kompression eines hMSC-beladenen Kollagen I Gels erfolgreich etabliert. Während drei Wochen statischer Kultur führte der biochemische Stimulus transformierender Wachs-tumsfaktor beta-3 (TGF beta-3) zu einer kompakten Kollagenstruktur. An Tag 21 zeigte dieses Meniskus-Modell eine hohe metabolische Aktivität und Matrixumbau durch die Detektion von Matrix-Metalloproteasen. Der Bindegewebsknorpel wurde durch immunhistochemische Detektion der Meniskusmarker, einem signifikanten GAG/DNA Anstieg und erhöhter Kompressionseigenschaften bestätigt. Für weitere Verbesserungen wurden biomechanische Stimulierungssysteme mittels Kompression und hydrostatischen Druck aufgebaut. Als Vaskularisierungsansatz führte die direkte Stimulierung mit Ciclopirox Olamine (CPX) sogar in Abwesenheit von Helferzellen wie MSCs zu einem erhöhten Sprouting der hd-mvEC Spheroide. Zweitens wurde ein hMSC/hd-mvEC Sheet mithilfe eines Temperatur-abhängigen Verfahrens produziert und auf das keilförmige Meniskus-Modell transferiert. Drittens wurde ein vaskularisiertes Biomaterial (BioVaSc-TERM) mit hd-mvECs besiedelt, wodurch ein vitales Gefäßystem bereitgestellt wurde. Die vaskularisierte BioVaSc-TERM wurde als Hülle des Meniskus-Modells unter der Verwendung von zwei Nahttechniken vorgeschlagen: die All-Inside-Repair (AIR) für das Hinterhorn und die Outside-In-Refixation (OIR) für das Vorderhorn und den mittleren Teil. Dieses Meniskus-Modell ist ein vielversprechender Ansatz für den Meniskusersatz, um in Vaskularisierung, biochemischer und biomechanischer Stimuli weiter optimiert zu werden

Tese de Doutoramento em Bioengenharia
One of tissue engineering (TE) challenges concerns the vascularization of engineered constructs upon implantation into the defect. In fact, for the survival of the engineered tissue beyond the oxygen diffusion limit, the formation of new blood vessels is mandatory. Therefore, this thesis aimed at designing routes towards advanced vascularized bone analogs, based on the combination of cells, biomaterials and inorganic components. The major objectives of this thesis were 1) to identify a single cell source to obtain both endothelial (ECs) and osteoblast-like cells (OBs); 2) to identify the optimal conditions in which these cells synergistically communicate; 3) to trigger the osteogenic differentiation of stem/stromal cells by inorganic osteoinducers and 4) to design 3D hydrogel systems for the controlled spatial distribution of cells. The use of adipose tissue (AT) as a cell pool for TE purposes is highly appealing, since its stromal vascular fraction (SVF) contains stem/stromal-like cells (hASCs) that can be differentiated into specific lineages, enhancing their potential use in a clinical setting. Under this context, the SSEA-4+ cellular subset of SVF (SSEA-4+hASCs) was proven to hold enhanced differentiation potential into ECs- and OBs-like cells, the most relevant cell types for bone vascularization TE routes. Using immunomagnetic selection tools, SSEA-4+hASCs were successfully separated and differentiated towards both endothelial and osteogenic lineages. Furthermore, it was found that culturing these obtained ECs and pre-OBs at an initial ratio of 75:25 in a mixture of standard endothelial and osteogenic media, cells synergistically communicate to encourage the full differentiation of pre-OBs and the maintenance of ECs phenotype. Culturing SSEA-4+hASCs in presence of sNPs in basal condition lead to the deposition of a collagen-enriched matrix relevant for bone TE. When in combination with standard osteogenic factors, sNPs were able to significantly increase the osteogenic commitment of both hMSCs and SSEA-4+hASCs. Finally, to address the tri-dimensionality of the bone, hydrogels templates, based on kappa-carrageenan (κ-CA) and chitosan (CHT), were designed to accommodate SSEA-4+hASCs-derived ECs and OBs. The CHT coated κ-CA hydrogel microfibers, arranged in such a fashion to mimic the blood vessel network, were able to support the endothelial signature of entrapped ECs. These, upon assembly within a pre-OBs loaded matrix, are appealing to be templates to attain a 3D microvascular network. By decorating κ-CA with photocrosslinkable units, hydrogels with tunable mechanical properties and high recovery rates after deformation we obtained. The controlled spatial distribution of cells was achieved by patterning the hydrogels in well-defined geometries. In summary, the research work described in this thesis addressed new strategies within the TE field that might inspire the development of improved vascularized bone-engineered constructs. The use of SSEA-4+hASCs was proven to be an endearing choice of undifferentiated cells, while their combination with sNPs and κ-CA hydrogels displayed numerous advantages. Nonetheless, the unraveling of the real potential of these cells, alone or in combination with sNPs and/or κ-CA hydrogels, towards promoting vascularized bone formation yet requires in vivo confirmation.
Um dos desafios da engenharia de tecidos consiste na vascularização após a implantação do implante no defeito. De facto, para a sobrevivência do substituto do tecido é essencial a difusão de oxigénio assim como a formação de novos vasos sanguíneos. Portanto, esta tese explora novas estratégias para o desenvolvimento de análogos de osso vascularizado, com base na combinação de células, biomateriais e componentes inorgânicos. Os objetivos principais desta tese foram: 1) identificar uma única fonte celular para obter tanto as células endoteliais (ECs), como as osteoblastos (OBs); 2) identificar as condições ideais em que estas células comunicam de uma forma sinérgica; 3) desencadear a diferenciação osteogénica das células estaminais através dos osteoindutores inorgânicos e 4) projetar sistemas de hidrogéis em 3D para controlar a distribuição espacial das células. O uso do tecido adiposo como uma fonte de células é altamente atraente para engenharia de tecidos. As células estaminais derivadas do tecido adiposo (hASCs) podem ser diferenciadas em linhagens específicas, melhorando assim o seu potencial para aplicações clínicas. Neste contexto, a população SSEA-4+, identificada na fração vascular do tecido adiposo (SSEA-4+hASCs), foi a que demonstrou melhor potencial de diferenciação em células endoteliais (ECs) e osteoblastos (OBs), as células mais envolvidas na vascularização óssea. Usando ferramentas de seleção imunomagnéticas, as SSEA-4+hASCs foram separadas e diferenciadas em ambas linhagens: endotelial e osteogénica. Além disso, verificou-se que a cultura de ECs e pré-OBs numa razão inicial de 75:25, num meio de cultura misto, levou a uma comunicação celular sinérgica, incentivando a diferenciação completa das pré-OBs e a manutenção do fenótipo endotelial das ECs. A cultura das SSEA-4+hASCs na presença de nanopartículas de silica (SNPs) num meio basal, levou à deposição de uma matriz enriquecida em colagénio, essencial na regeneração óssea. Em combinação com fatores osteogénicos, as SNPs foram capazes de significativamente aumentar o compromisso osteogénico de ambas as células mesenquimais humanas e SSEA-4+hASCs. Finalmente, para resolver a tridimensionalidade do osso, modelos 3D com base em hidrogéis de kappa-carragenina (κ-CA) e quitosano (CHT), foram desenvolvidos para acomodar as ECs e OBs. Microfibras de κ-CA revestidas com CHT, dispostas de tal forma que mimetizam a rede vascular, foram capazes de manter a assinatura endotelial das ECs. Após o arranjo dentro de uma matriz enriquecida em pré-OBs, espera-se que agissem como padrões para gerir uma rede microvascular funcional. Seguinte, a decoração da κ- CA com unidades foto-reticulaveis rendeu hidrogéis com propriedades mecânicas ajustáveis e altas taxas de recuperação após a deformação. Uma distribuição controlada de células foi obtido por patterning em geometrias bem definidas. Em resumo, o trabalho de investigação descrito nesta tese propõe novas estratégias dentro da engenharia de tecidos que podem inspirar o desenvolvimento de construções de osso vascularizado. O uso das SSEA-4+hASCs provou ser uma escolha cativante de células não diferenciadas, enquanto a combinação com SNPs e hidrogéis de κ-CA exibiu várias vantagens. No entanto, o desenrolar do verdadeiro potencial destas células, individualmente ou em combinação com SNPs e/ou hidrogéis de κ-CA, no sentido de promover a formação de tecido ósseo vascularizado, ainda requer confirmação in vivo.
Foundation for Science and Technology (FTC) and MIT- doctoral grant (SFRH/BD/42968/2008).

Survival of engineered tissues in vivo requires the presence of an internal vascular network and immediate connection to the host vasculature. Modular tissue engineering approaches the vascularization ‘design’ requirement through fabrication of submillimeter-sized collagen microtissues (‘modules’) with endothelial cells (EC) seeded on the surface of the modules and functional or vascular support cells inside the modules. Several modules are then packed together to build a larger tissue. In this work, we explored biomaterial-based strategies to build vascularized modular tissue engineered constructs. A photocrosslinkable poloxamine-polylysine acrylate biomaterial was first synthesized to improve the mechanical limitations of collagen modules under flow, while still supporting EC attachment. An extracellular matrix (ECM)-based strategy was then explored to enhance the vascularization of the modules in vivo. Manipulation of the ECM was accomplished through lentiviral transduction of EC to overexpress Developmental endothelial locus-1 (Del-1), a pro-angiogenic ECM molecule. Supporting the hypothesis that Del-1 overexpression ‘tilts’ the balance in EC from a quiescent to a pro-angiogenic phenotype, human umbilical vein endothelial cells transduced to overexpress Del-1 (Del-1 HUVEC) formed more sprouts and had a distinct expression profile of angiogenic genes in vitro, relative to control eGFP HUVEC. While very few blood vessels formed upon subcutaneous injection of empty collagen modules coated with Del-1 or eGFP HUVEC in a SCID/Bg mouse model, embedding adipose derived mesenchymal stem cells (adMSC) inside the modules increased blood vessel formation. Moreover, Del-1 HUVEC and adMSC modules consistently had more blood vessels (donor-derived and total number of vessels) compared to eGFP HUVEC and adMSC, over the 21 day duration of the study, with the greatest difference observed at day 7 post-transplantation. In addition, more α-smooth muscle actin (SMA+) staining was observed in Del-1 implants compared to eGFP, suggestive of increased vessel maturation through recruitment of SMA+ pericytes and smooth muscle cells. Perfusion studies showed that the implant vasculature was connected to the host vascular network as early as day 7, and throughout the 21 day duration of the study, for both Del-1 and eGFP implants. Nevertheless, further normalization of the vasculature is likely required to improve perfusion at early time points after transplantation.

A major therapeutic challenge is the increasing incidence of chronic disorders. The persistent impairment or loss of tissue function requires constitutive on‐demand drug availability optimally achieved by a drug delivery system ideally directly connected to the blood circulation of the patient. However, despite the efforts and achievements in cell‐based therapies and the generation of complex and customized cell‐specific microenvironments, the generation of functional tissue is still unaccomplished. This study demonstrates the capability to generate a vascularized platform technology to potentially overcome the supply restraints for graft development and clinical application with immediate anastomosis to the blood circulation. The ability to decellularize segments of the rat intestine while preserving the ECM for subsequent reendothelialization was proven. The reestablishment of a functional arteriovenous perfusion circuit enabled the supply of co‐cultured cells capable to replace the function of damaged tissue or to serve as a drug delivery system. During in vitro studies, the applicability of the developed miniaturized biological vascularized scaffold (mBioVaSc‐TERM®) was demonstrated. While indicating promising results in short term in vivo studies, long term implantations revealed current limitations for the translation into clinical application. The gained insights will impact further improvements of quality and performance of this promising platform technology for future regenerative therapies
Eine kontinuierlich steigende Inzidenz chronischer Krankheiten stellt eine immer größer werdende therapeutische Herausforderung dar. Der anhaltende Funktionsverlust von Geweben erfordert die bedarfsgerechte Verfügbarkeit von Wirkstoffen, deren kontinuierliche Bereitstellung und Verteilung über die Blutzirkulation von implantierbaren Pharmakotherapie‐Produkten gelöst werden kann. Trotz der Fortschritte und Erfolge mit Zelltherapien sowie der Nachbildung der Zell‐eigenen Nischen konnten bisher noch keine funktionellen Gewebe für die medizinische Anwendbarkeit hergestellt werden. Diese Studie zeigt die Möglichkeit zur Herstellung einer vaskularisierten Plattform‐ Technologie um die Beschränkung der Nährstoff‐Versorgung zu überwinden für die Entwicklung von Transplantaten für die klinische Anwendung und deren sofortige Anastomose an die Blutzirkulation. Die Möglichkeit Rattendarmsegmente zu dezellularisieren, die Extrazellulärmatrix und das interne Gefäßsystem dabei jedoch zu erhalten um diese Strukturen wiederzubesiedeln wurde bewiesen. Das Wiederherstellen des funktionellen arteriovenösen Perfusionskreislaufs ermöglichte die Versorgung von Ko‐kultivierten Zellen um damit funktionalen Gewebeersatz bzw. ‐modelle aufzubauen oder als Medizin‐ Produkt Einsatz zu finden. In vitro‐Studien zeigten eindrucksvoll Reife und Anwendbarkeit des hier entwickelten miniaturisierten, biologischen, vaskularisierten Scaffold (mBioVaSc‐TERM®). Während in in vivo‐Studien zunächst vielversprechende Ergebnisse erzielt wurden, zeigten Langzeit Implantationen die aktuellen Grenzen zur Translation in die klinische Anwendung. Die gewonnenen Erkenntnisse werden dazu dienen Qualität und Funktionalität dieser vielversprechenden Plattform‐Technologie zu verbessern um zukünftige regenerative Therapien zu ermöglichen

Each year millions of plastic and reconstructive procedures are performed to regenerate soft tissue defects after, for example, traumata, deep burns or tumor resections. Tissue engineered adipose tissue grafts are a promising alternative to autologous fat transfer or synthetic implants to meet this demand for adipose tissue. Strategies of tissue engineering, especially the use of cell carriers, provide an environment for better cell survival, an easier positioning and supplemented with the appropriate conditions a faster vascularization in vivo. To successfully engineer an adipose tissue substitute for clinical use, it is crucial to know the actual intended application. In some areas, like the upper and lower extremities, only a thin subcutaneous fat layer is needed and in others, large volumes of vascularized fat grafts are more desirable. The use and interplay of stem cells and selected scaffolds were investigated and provide now a basis for the generation of fitted and suitable substitutes in two different application areas. Complex injuries of the upper and lower extremities, in many cases, lead to excessive scarring. Due to severe damage to the subcutaneous fat layer, a common sequela is adhesion formation to mobile structures like tendons, nerves, and blood vessels resulting in restricted motion and disabling pain [Moor 1996, McHugh 1997]. In order to generate a subcutaneous fat layer to cushion scarred tissue after substantial burns or injuries, different collagen matrices were tested for clinical handling and the ability to support adipogenesis. When testing five different collagen matrices, PermacolTM and StratticeTM showed promising characteristics; additionally both possess the clinical approval. Under culture conditions, only PermacolTM, a cross-linked collagen matrix, exhibited an excellent long-term stability. Ranking nearly on the same level was StratticeTM, a non-cross-linked dermal scaffold; it only exhibited a slight shrinkage. All other scaffolds tested were severely compromised in stability under culture conditions. Engineering a subcutaneous fat layer, a construct would be desirable with a thin layer of emerging fat for cushioning on one side, and a non-seeded other side for cell migration and host integration. With PermacolTM and StratticeTM, it was possible to produce constructs with ASC (adipose derived stem cells) seeded on one side, which could be adipogenically differentiated. Additionally, the thickness of the cell layer could be varied. Thereby, it becomes possible to adjust the thickness of the construct to the surrounding tissue. In order to reduce the pre-implantation time ex vivo and the costs, the culture time was varied by testing different induction protocols. An adipogenic induction period of only four days was demonstrated to be sufficient to obtain a substantial adipogenic differentiation of the applied ASC. Thus, seeded with ASC, PermacolTM and StratticeTM are suitable scaffolds to engineer subcutaneous fat layers for reconstruction of the upper and lower extremities, as they support adipogenesis and are appropriately thin, and therefore would not compromise the cosmesis. For the engineering of large-volume adipose tissue, adequate vascularization still represents a major challenge. With the objective to engineer vascularized fat pads, it is important to consider the slow kinetics of revascularization in vivo. Therefore, a decellularized porcine jejunum with pre-existing vascular structures and pedicles to connect to the host vasculature or the circulation of a bioreactor system was used. In a first step, the ability of a small decellularized jejunal section was tested for cell adhesion and for supporting adipogenic differentiation of hASC mono-cultures. Cell adhesion and adipogenic maturation of ASC seeded on the jejunal material was verified through histological and molecular analysis. After the successful mono-culture, the goal was to establish a MVEC (microvascular endothelial cells) and ASC co-culture; suitable culture conditions had to be found, which support the viability of both cell types and do not interfere with the adipogenic differentiation. After the elimination of EGF (epidermal growth factor) from the co-culture medium, substantial adipogenic maturation was observed. In the next step, a large jejunal segment (length 8 cm), with its pre-existing vascular structures and arterial/venous pedicles, was connected to the supply system of a custom-made bioreactor. After successful reseeding the vascular structure with endothelial cells, the lumen was seeded with ASC which were then adipogenically induced. Histological and molecular examinations confirmed adipogenic maturation and the existence of seeded vessels within the engineered construct. Noteworthily, a co-localization of adipogenically differentiating ASC and endothelial cells in vascular networks could be observed. So, for the first time a vascularized fat construct was developed in vitro, based on the use of a decellularized porcine jejunum. As this engineered construct can be connected to a supply system or even to a patient vasculature, it is versatile in use, for example, as transplant in plastic and reconstruction surgery, as model in basic research or as an in vitro drug testing system. To summarize, in this work a promising substitute for subcutaneous fat layer reconstruction, in the upper and lower extremities, was developed, and the first, as far as reported, in vitro generated adipose tissue construct with integrated vascular networks was successfully engineered
Jedes Jahr werden Millionen von plastischen und wiederherstellenden Eingriffe durchgeführt, um zum Beispiel nach Traumata, hochgradigen Verbrennungen oder Tumorekonstruktionen, die natürliche Erscheinung und Funktion im Bereich von Weichgewebsdefekt wiederherzustellen. Gezüchtete Fettgewebskonstrukte sind eine vielversprechende Alternative zu autologen Fettgewebstransfers oder synthetischen Implantaten, um dem Bedarf an Fettgewebe gerecht zu werden. Die Strategien der Gewebezüchtung, besonders das Verwenden von Zellträgern, schaffen eine Umgebung für besseres Zellüberleben, eine einfachere Positionierung und - versehen mit den entsprechenden Eigenschaften - eine schnellere Vaskularisierung in vivo. Um erfolgreich einen Fettgewebe-Ersatz für die klinische Anwendung herzustellen, ist es notwendig das spätere Anwendungsgebiet zu kennen. In manchen Bereichen, wie in den oberen und unteren Extremitäten, braucht man nur eine dünne Unterhautfettschicht, und in anderen Bereichen wiederum ist ein großes Volumen an vaskularisiertem Fettgewebskonstrukt anzustreben. Die Nutzung und das Zusammenspiel von Stammzellen und ausgewählten Zellträgern wurden untersucht und legen nun eine Basis für die Herstellung von passendem und zweckmäßigem Ersatzgewebe zweier unterschiedlicher Anwendungsgebiete. Komplexe Verletzungen der oberen und unteren Extremitäten führen oftmals zu beträchtlicher Narbenbildung. Eine häufige Folgeerscheinung, hervorgerufen durch eine schwere Beschädigung des Unterhautfettgewebes, ist die Adhäsion zwischen mobilen Strukturen wie Sehnen, Nerven und Blutgefäßen. Dies resultiert dann in eingeschränkter Beweglichkeit und lähmenden Schmerzen [Moor 1996, McHugh 1997]. Um eine subkutane Fettschicht herzustellen, die das vernarbte Gewebe nach schwerer Verbrennung oder Verletzung polstert, wurden verschiedene Kollagenmaterialien auf die klinische Handhabung und die Unterstützung der Adipogenese untersucht. In der Untersuchung von fünf verschiedenen Kollagenmatrices zeigten PermacolTM und StratticeTM vielversprechende Eigenschaften. Beide besitzen außerdem die klinische Zulassung. PermacolTM, eine chemisch quervernetzte Kollagenmatrix, zeigte unter Kulturbedingungen hervorragende Langzeitstabilität. Fast ebenso gute Eigenschaften konnten bei StratticeTM, einem nicht vernetzten dermalen Gerüstmaterial, beobachtet werden; es zeigte lediglich leichte Schrumpfung. Alle sonst getesteten Kollagenmaterialien waren unter Kulturbedingungen stark in ihrer Stabilität beeinträchtigt. Zur Herstellung einer subkutanen Fettschicht wäre ein Konstrukt wünschenswert mit einer dünnen, gerade entstehenden Fettschicht für die Polsterung auf der einen Seite und einer nicht besiedelten anderen Seite für die Zelleinwanderung und die Integration in das umliegende Gewebe. Mit PermacolTM und StratticeTM war es möglich Konstrukte herzustellen, welche auf einer Seite mit ASC (aus dem Fettgewebe isolierte Stammzellen) besiedelt und anschließend adipogen differenziert werden konnten. Zusätzlich konnte die Dicke der Zellschicht hierbei variiert werden. Somit ist es möglich die Dicke des Konstruktes an das umliegende Gewebe anzupassen. Um die Preimplantationszeit ex vivo zu verkürzen und damit auch die Kosten zu senken, wurde die Kulturzeit variiert, indem verschiedene Induktionsprotokolle getestet wurden. Eine adipogene Induktionsperiode von nur vier Tagen erwies sich als ausreichend, um eine substantielle adipogene Differenzierung der eingesetzten ASC zu erreichen. Das heißt, die mit ASC besiedelten PermacolTM und StratticeTM Matrices sind zweckdienliche Zellträgermaterialien, um eine subkutane Fettschicht für die oberen und unteren Extremitäten herzustellen, da sie die Adipogenese unterstützen und durch die nur geringe und anpassbare Dicke die Kosmesis nicht beeinträchtigen. Für die Entwicklung von großvolumigem Fettgewebe stellt die adäquate Vaskularisierung noch immer eine große Herausforderung dar. Mit dem Ziel ein vaskularisiertes Fettkonstrukt herzustellen, ist es wichtig die langsame Kinetik der Revaskularisierung in vivo zu berücksichtigen. Daher wurde hier ein dezellularisiertes Schweinedarmsegment mit schon vorhandenen Gefäßstrukturen und Gefäßanschlüssen für die Verbindung zum Kreislaufsystem des Patienten oder eines Bioreaktor-Systems verwendet. Im ersten Schritt wurden auf einem kleinen dezellularisierten Schweinedarm-Stück die Zelladhäsion und die adipogene Differenzierung der ASC in Monokultur getestet. Die Zelladhäsion und die adipogene Reifung konnte mittels histologischer und molekularer Analysen auf dem jejunalen Material nachgewiesen werden. Nach der erfolgreichen Monokultur musste die Co-Kultur von MVEC (micro vaskuläre Endothelzellen) und ASC etabliert werden. Um dieses Ziel zu erreichen, wurden geeignete Kulturbedingungen gesucht, die die Lebensfähigkeit beider Zelltypen unterstützen und gleichzeitig die adipogene Differenzierung nicht beeinträchtigen. Nach dem Ausschluss von EGF (epidermaler Wachstumsfaktor) aus dem Co-Kulturmedium wurde eine substantielle adipogene Reifung der ASC beobachtet. Im nächsten Schritt wurde ein großes dezellularisiertes jejunales Darmsegment (Länge 8 cm) mit der schon existenten Gefäßstruktur und dem arteriellen und venösen Gefäßstiel an den spezialangefertigten Bioreaktor angeschlossen. Nach der erfolgreichen Wiederbesiedelung der Gefäßstrukturen mit Endothelzellen wurde das Darmlumen mit ASC besiedelt, welche anschließend adipogen induziert wurden. Histologische und molekulare Untersuchungen konnten die adipogenen Reifung und die Existenz von besiedelten Gefäßen im hergestellten Konstrukt bestätigen. Besonders erwähnenswert ist die Beobachtung der Co-Lokalisierung von adipogen differenzierenden ASC und Endothelzellen in vasculären Netzwerken. Somit wurde zum ersten Mal - basierend auf einem dezellularisierten Schweinedarm - ein vaskularisiertes Fettgewebskonstrukt in vitro hergestellt. Da dieses Konstrukt an das Versorgungssystem angeschlossen oder mit dem Blutkreislauf des Patienten verbunden werden kann, ist es vielfältig einsetzbar, zum Beispiel in der plastisch-rekonstruktiven Chirurgie, als Modell in der Grundlagenforschung oder als ein in vitro Medikamenten-Testsystem. Zusammengefasst, wurde in der vorgelegten Arbeit ein vielversprechendes Ersatzmaterial für die Rekonstruktion des Unterhautfettgewebes für die unteren und oberen Extremitäten entwickelt, und zum ersten Mal erfolgreich, so weit in der Literatur bekannt, ein Fettgewebskonstrukt mit integriertem vaskularisiertem Netzwerk in vitro generiert

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais.
The orthopedic implants represent an ever-growing biomedical market, as the population is continuously expanding and the average life expectancy is increasing. Tissue engineering and regenerative medicine aim at opening a pathway of therapy and treatment of bone tissue loss or end stage damage. Envisioning this goal, biodegradable solutions, such as polycaprolactone (PCL), offer a number of physiological and clinical advantages over permanent implants. This work aims to determine the aptitude of PCL surface roughness and the density-specific role of fibronectin (FN) adsorbed on PCL to induce differentiation of adult stem cells towards the osteoblastic lineage. Furthermore, as the establishment of a microvascular blood supply, necessary to assure cell survival, is still identified as a major challenge for the clinical application of (bone) engineered biomaterials, the combination of physical regular surface pattern motifs of PCL and density-specific fibronectin coating was investigated to determine the biomaterial effectiveness on the morphogenesis of microvascular tissue, both in vitro and in vivo. Our findings indicate that the optimal FN density regime of ~48 ng/cm2 could consistently and significantly support higher expression of osteocommitment biomarkers, such as cuboidal cytoskeleton morphology, alkaline phosphatase (ALP) activity and collagen type 1deposition. Furthermore, it was verified that such a density can be used as relevant alternative to the potent synthetic osteogenic supplement dexamethasone (Dex), in the osteogenic commitment of stem cells in vitro. Our analysis also demonstrated the differential regulation of the osteogenic differentiation of adult stem cells (from early ALP activity to end-process mineralization) by different roughness average (Ra) along an engineered surface roughness gradient. Faster osteogenic commitment and strongest osteogenic expression was obtained at Ra ~ 2.1 – 3.1 μm. Importantly, the removal of Dex, and even the removal of all osteogenesis-inducing supplements from the cell culture medium, did not prevent the differentiation process from occurring. Indeed, the PCL Ra ~ 0.9 – 2.1 μm showed the ability to alone direct the osteogenic differentiation of the stem cells, in vitro. Finally, we showed that geometrically defined micropatterns of PCL, in association with human density-specific FN adsorption (determined from a gradient study), can induce/instruct the endothelial cells (ECs) to mature into a luminized capillary network, both in vitro and in vivo. These results cumulatively enrich our knowledge on the biochemical and physical cues which evoke osteogenic stem cell modulation and successful recapitulation of supportive microvascularization, highlighting the potential for creating effective solutions for orthopedic applications featuring a clinically relevant biodegradable material.
Implantes ortopédicos representam um mercado em permanente expansão, uma vez que a população e a esperança média de vida continuam a aumentar. A Engenharia de Tecidos e a Medicina Regenerativa propõem-se a criar uma alternativa de terapia e de recobro de tecido ósseo perdido ou danificado. Considerando este propósito, soluções biodegradáveis, tais como policaprolactona (PCL), oferecem um conjunto de vantagens clínicas e fisiológicas sobre implantes permanentes. Este trabalho tem como objectivos determinar a competência da rugosidade à superfície de PCL e de densidades específicas de fibronectina (FN) adsorvidas em PCL, na orientação da diferenciação de células estaminais adultas (MSCs) para a linhagem osteoblástica. Ademais, a combinação de superfícies regulares e fisicamente padronizadas de PCL com uma densidade específica de revestimento de FN foi investigada para determinar a eficiência do biomaterial na morfogénese de tecido microvascular, uma vez que a organização de uma estrutura que estabeleça microcirculação sanguínia, necessária à sobrevivência celular, é ainda identificada como um obstáculo à translação bem-sucedida de estratégias de engenharia de tecidos para a clínica. A nossa investigação identificou um regime óptimo de densidade de FN (~48 ng/cm2) que apoia, consistentemente e de forma significativa, a expressão aumentada de biomarcadores de compromisso com a linhagem osteoblástica, como a morfologia cuboidal do citoesqueleto, a actividade da fosfatase alcalina (ALP) e a deposição de colagénio de tipo 1. Verificou-se ainda que a adsorpção de FN pode ser usada como uma natural e relevante alternativa ao uso do potente agente sintético de osteogénese dexametasona (Dex), para a indução da linhagem osteogénica em células estaminais, in vitro. A nossa análise também revelou regulação diferencial da diferenciação osteogénica de MSCs (da fase inicial traduzida por actividade de ALP até à fase final de mineralização) por diferente rugosidade média (Ra) à superfície do PCL. Rápido e intenso compromisso com o processo de osteogénese foi obtido a Ra ~ 2.1 – 3.1 μm. Relevantemente, a remoção de Dex e, até, de todos os suplementos indutores de osteogénese do meio de cultura celular não impediu o processo de diferenciação de decorrer. De facto, a Ra ~ 0.9 – 2.1 μm em PCL mostrou capacidade de orientar, per se, a diferenciação osteogénica de células estaminais, in vitro. Finalmente, demonstramos que micropadrões físicos de PCL, em associação a uma densidade específica de FN (determinada de um estudo de gradiente), tem capacidade para induzir/instruir células endoteliais para maturação numa rede capilar com lúmen, in vitro e in vivo. Estes resultados cumulativamente enriquecem o nosso conhecimento sobre sinais físicos e bioquímicos que provocam modulação osteogénica de células estaminais, e ainda eficiente recapitulação de microvascularização de suporte, enfatizando o potencial de criação de soluções eficazes para aplicações ortopédicas usando um material biodegradável e clinicamente relevante.
Fundação para a Ciência e a Tecnologia (FCT)

This thesis presents a microfluidic strategy for the in-flow definition of a 3D soft material with a tunable and perfusable microstructure. The strategy was enabled by a microfluidic device containing up to fifteen layers that were individually patterned in polydimethylsiloxane (PDMS). Each layer contained an array of ten to thirty equidistantly spaced microchannels. Two miscible fluids (aqueous solutions of alginate and CaCl2) were used as working fluids and were introduced into the device via separate inlets and distributed on chip to form a complex fluid at the exit. The fluid microstructure was tuned by altering the flow rates of the working fluids. Upon solidification of alginate in the presence of calcium chloride, the created microstructure was retained and a soft material with a tunable microstructure was formed. The produced material was subsequently perfused using the same microfluidic architecture. The demonstrated strategy potentially offers applications in materials science and regenerative medicine.