Vascularized composite allotransplantation (VCA) has emerged over the last two decades as a promising therapeutic option for persons who have suffered the loss of limbs or who have suffered major facial disfigurement. Despite the clear advantages of facial and upper extremity VCA in terms of function and cosmesis, VCA has elicited a great deal of ethical concern. Much of that concern is centered around whether or not persons should be exposed to the toxic side effects and possible shortening of life associated with immunosuppression as part of treatment for conditions which are not life-threatening. Ethical concern has also been raised about the vulnerability, dignity and autonomy of VCA candidates and about the justice of allocating the necessary resources to research a treatment that seems unlikely to become widely available in the foreseeable future. <br>While this dissertation will demonstrate familiarity with the technical aspects of VCA, and with the ethical issues just mentioned, its focus will be on the implications of this new therapeutic option for the manner in which consent is understood. In particular, it will argue that the nature and duration of the treatment involved in upper extremity and face transplants makes necessary some modification to the theory and practice of consent. The concept of covenant will be put forward as a resource for this modification. Covenants, as agreements which establish and maintain on-going personal relationships of mutual obligation, are both durable and flexible. Covenants, by engaging persons affectively, promote commitment and encourage the formation of strong therapeutic alliances. Such alliances are especially fitting in light of the lengthy and demanding course of VCA, from screening through surgery and years of physiotherapy, maintenance of immunosuppression and self-monitoring for signs of rejection. <br>Covenant consent is needed for VCA because it more adequately describes what is being asked of recipients and what is necessary for the treatment to succeed. It is also needed because it appropriately honors the recipients by understanding them as active partners rather than as passive patients, and as people assuming major burdens and risks while contributing meaningfully to the development of the field. The employment of covenant consent significantly strengthens the ethical justification for vascularized composite allotransplantation of faces and upper extremities by acknowledging what is actually required of patients and by creating a structure through which they are supported in carrying out their commitment through the long, arduous period of rehabilitation and beyond. / McAnulty College and Graduate School of Liberal Arts; / Health Care Ethics / PhD; / Dissertation;

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.

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.

Vascularized composite allotransplantation has developed as a specialty at the interface of reconstructive and transplant surgery, offering restoration of function and form in scenarios where options for autologous reconstruction are limited, and for which the burden of donor-site morbidity may be high. Over the past 15 years some 28 patients have received face, and 85 patients upper extremity transplants. Results have been encouraging, with good functional outcomes, and the majority of patients reporting return to independence, employment and improved quality of life. However, the immunological management of these patients remains a significant challenge. While conventional immunosuppressive regimens have proven effective in preventing graft loss to rejection, they have failed to protect patients from acute rejection episodes, which have been reported in 85% during the first year post-transplant. When considered alongside the burden of comorbidity associated with life-long immunosuppression, the impetus for development of novel approaches to the prevention of rejection is clear. Induction of hematopoietic mixed chimerism has successfully achieved transplant tolerance, defined as specific unresponsiveness to donor antigens permitting life-long acceptance of transplanted tissues without maintenance immunosuppression, in numerous animal models and recently, of renal allografts in clinical trials. The work presented in this thesis investigates mixed chimerism for induction of tolerance of vascularized composite allografts (VCAs) across class I and II major histocompatibility (MHC) barriers in the Massachusetts General Hospital miniature swine model; a large animal model with defined MHC immunogenetics, and skin closely analogous to that of humans. The data presented demonstrate development of a reproducible model of VCA tolerance and stable hematopoietic mixed chimerism in a preclinical model. Importantly, tolerance extended to all components of VCAs including the epidermis and dermis, a previously un-reproducible finding. In vitro analysis demonstrated no evidence for either IL-2 reversible anergy or cellular regulation as mechanisms of donor-specific unresponsiveness, suggesting that at a systemic level, tolerance in this model may be primarily mediated by clonal deletion. In contrast, characterization of the cutaneous immune system demonstrated rapid infiltration of VCAs with recipient T cells and Langerhans’ cells, which in chimeric recipients did not cause rejection but rather established stable chimerism in all tissue-resident populations including dermal T cells with the phenotype of Tregs (CD25+FoxP3+); findings which suggest tissue-specific, and regulatory, mechanisms may play important roles. These data support the hypothesis that mixed chimerism is sufficient for whole-skin tolerance of VCAs, but further work is required to demonstrate the necessity of stable, rather than transient, chimerism and to confirm the necessity of other systemic or tissue-specific factors for prevention of epidermal rejection.

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

Submitted by Biblioteca de Pós-Graduação em Geoquímica BGQ (bgq@ndc.uff.br) on 2018-02-22T17:10:01Z No. of bitstreams: 1 Microsoft Word - PDF-Dissertação.pdf: 917116 bytes, checksum: 87c5e9fb0e59ed8d9ca058ba985019f7 (MD5) / Made available in DSpace on 2018-02-22T17:10:01Z (GMT). No. of bitstreams: 1 Microsoft Word - PDF-Dissertação.pdf: 917116 bytes, checksum: 87c5e9fb0e59ed8d9ca058ba985019f7 (MD5) / Universidade Federal Fluminense. Instituto de Química. Programa de Pós-Graduação em Geoquímica, Niterói, RJ / A abordagem utilizada neste estudo visou caracterizar a composição da matéria orgânica no sedimento superficial dos diferentes lagos (águas brancas e pretas) que compõe a Várzea do Lago Grande de Curuai, através dos fenóis oriundos da lignina como traçador de plantas vascularizadas, e dados secundários de análises isotópicas ( 13C) e elementares (C/N), em amostras de sedimento superficial e de possíveis fontes de matéria orgânica. A caracterização dos fenóis de lignina nas amostras de sedimento para as possíveis fontes de matéria orgânica (plantas terrestres, macrófitas e perifiton) e para o sedimento foram feitas através da degradação oxidativa em meio básico com óxido de cobre (CuO). Das amostras de fontes de MO analisadas, Echinocloa polystachia (Capim Canarana) com maior razão S/V e Capim (nd) com maior razão C/V foram as que mais mostraram contribuição na composição da MO para os sedimentos superficiais, mostrando a grande contribuição de MO autóctone macrofítica e fitoplanctônica para a várzea do Lago Grande de Curuai. Os resultados obtidos com os fenóis oriundos da lignina apontam característica de vegetação terrestre, com elevada razão S/V para a amostra de fonte de MO Echinocloa spectabilis (Mutim), caracterizada como fonte alóctone de matéria orgânica para o sedimento da várzea, correspondendo á vegetação C4 terrestre. Gramínea (nd), Pistia stratiotes (Alface d’Água), Eichornia azurea (Mureru) e Eichornia crassipes correspondem a fonte autóctone de matéria orgânica para o sedimento da várzea apresentando elevados valores de razão C/V; e a amostra de Epifiton caracterizou-se como uma mistura de fontes fitoplanctônica, macrofítica e de vegetação terrestre para os sedimentos da várzea. Os resultados obtidos para as amostras de sedimento superficial mostram que a proximidade com terras florestadas, o grau de interferência do Rio Amazonas, e de interconexão entre os lagos que compõe a várzea do Lago Grande de Curuai, interferem na composição (alóctone ou autótone), e no grau de decomposição da MO depositada no sedimento. Os lagos de águas pretas Curumucuri e Açaí apresentaram grande contribuição de MO alóctone da drenagem do solo da floresta adjacente, mas com diferenças no grau de decomposição; sendo que para o Açaí com pouca interferência do Amazonas e isolado dos demais lagos da várzea a MO encontrada no sedimento apresenta maior degradação, enquanto que para o Lago Curumucuri, mais interligado ao sistema, a MO depositada no sedimento é menos degradada. A composição da matéria orgânica para o sedimento dos lagos de águas brancas apresenta maior influência de MO autóctone (vegetação aquática), com exceção do Lago Salé que apresenta grande influência de MO alóctone proveniente da floresta adjacente e do Rio Amazonas. Existem diferenças no grau de decomposição da MO nos lagos de águas brancas, sendo que para os pontos em maior contato com o rio Amazonas a MO no sedimento encontra-se menos degradada. / The approach applied in this study focused in characterizes the organic matter composition in plants and sediments of Várzea do Lago Grande do Curuai floodplain among white water and black water lakes. As a vascular plant tracer it was used lignin phenol composition through cupric oxide degradation under basic conditions analysis. To a better interpretation it was used a secondary data from elemental (C/N) and isotopic ( 13C) analysis. The results from possible organic matter sources to lake sediments, showed higher amount of a non-leaf origin indicated by syringyl phenol ratio normalized to vanillin (S/V) to Echinocloa spectabilis (Mutim); Echinocloa polystachia (Capim Canarana) and Capim (non-identified). However, higher value of C/V ratio coupled to the high S/V values found to Echinocloa polystachia (Capim Canarana) was related to its aquatic behavior allowing a more leaf growth against a terrestrial type as Echinocloa spectabilis (Mutim) with a more structure phenol necessity typical of a terrestrial grass. The Gramínea (ni), Pistia stratiotes (Alface d’Água) and Eichornia crassipes samlpes presented higher C/V ratios values, characterizing a more leaf material typical of aquatic plants. The epiphyton sample indicated a mixture composed by both aquatic and terrestrial plants. Those alochthone versus autochthones sources added to the degradation conditions determined the composition of organic matter deposited. The superficial sediments samples results appeared to be influenced by the forest low land proximity, the Amazon River influence and by the interconnection among the productivity of the floodplain lakes. The black water lakes Curumucuri e Açaí presented a high alochthone organic matter contribution from the drainage of the neighborhood forest. Differences found in degradation rates were related to the hydrological function of those black water lakes. Açai lake showed a higher degradation rate in relation to Cumurucuri Lake that presented a less degraded organic matter due to a higher influence from floodplain system. The organic matter from white water lakes sediments presented a high autochthones proximate sources (aquatic plants) and less alochthone ultimate source (terrestrial plants). Exception were found to Sale Lake with the influence of the neighborhood forest drainage and also from the contact of Amazon River. Among white water lakes, Lago Grande de Curuai, Poção, Santa Ninhã, Salé e Lago Grande do Poção, it was found differences in degradation rates, related with water circulation, where a more Amazon River influence showed a less degraded rate.

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.

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

Les amputations d’un membre supérieur sont non seulement dévastatrices pour le bien-être physique, psychologique et social des patients, mais elles comportent également des répercussions financières importantes pour l’individu et le système de santé. Les allotransplantations de tissus composites vascularisés ont été proposées en tant que solution permettant de rétablir la forme et la fonction au détriment d’une immunosuppression à vie et d’un taux élevé de rejet chronique. Les prothèses myoélectriques combinent l’expertise chirurgicale avec les avancées technologiques pour réhabiliter les fonctions motrices d’un moignon amputé, mais elles demeurent limitées par un taux élevé d’abandon et des coûts importants. Dans les systèmes de santé avec des ressources limitées, les dirigeants ont la tâche complexe de partager équitablement l’allocation de ressources entre plusieurs maladies et interventions. Dans le domaine de l’économie de la santé, les analyses de type coût-bénéfice ont été développées pour répondre à ces questions. Les mesures d’utilité doivent incorporer l’impact que le traitement suscite sur l’espérance de vie et la qualité de vie. Ces utilités sont ensuite rapportées en fonction du coût, ce qui permet aux dirigeants de la santé de déterminer dans quels traitements il serait préférable d’investir les ressources. Dans cette thèse, nous proposons un modèle pour étudier les coûts-utilité des allotransplantations de la main et des prothèses myoélectriques. Pour commencer, une étude pilote a été effectuée sur les amputations du pouce traitées avec des lambeaux libres de l’orteil, ce qui nous a permis de confirmer la faisabilité des questionnaires d’utilité conçus. Par la suite, les utilités ont été mesurées dans une population d’amputés du membre supérieur, de patients réimplantés proximalement et de contrôles en santé. Les résultats démontrent que 1) les patients réimplantés rapportent la meilleure utilité avec les prothèses myoélectriques, 2) les amputés unilatéraux préfèrent significativement les prothèses myoélectriques également, et 3) aucune différence n’a été recelée entre les deux traitements chez les amputés bilatéraux. Au final, une analyse des coûts-bénéfices a été effectuée dans le contexte du système de santé canadien, démontrant que le traitement des patients amputés unilatéralement avec des prothèses myoélectriques permettrait de sauver davantage de coûts, alors que l’écart en épargnes monétaires se rétrécit pour les amputés bilatéraux traités avec une allotransplantation ou une prothèse. Avec les résultats rapportés dans cette thèse, nous pouvons proposer une mise à jour des indications de traitements pour les patients avec une amputation du membre supérieur. Basé sur l’analyse de type coût-utilité, nous concluons que les amputés unilatéraux sont de meilleurs candidats pour des prothèses myoélectriques, alors que les deux traitements sont encore adéquats pour les amputations bilatérales. / Amputations of the upper extremity are not only devastating for the patient’s physical, psychological and social well-being, but they also yield significant financial repercussions to the individual and the healthcare system. Vascularized composite allotransplantations of the upper extremity were proposed as a solution to restore form and function, albeit to the detriment of lifelong immunosuppression and high rates of chronic rejection. New-generation myoelectric prostheses combine surgical prowess with technological refinements to rehabilitate motor functions of the amputated stump, but remain plagued by high rates of abandonment and significant costs. In healthcare systems wherein resources are limited, financial regulators have the difficult task of proposing an equitable divide of resource allocations between a multitude of diseases and interventions. In the field of health economics, cost-benefit analyses were developed to assist in this decision-making process. Utility outcome measures need to encompass the impact that a treatment elicits on life expectancy and quality of life. Comparison of utilities of different interventions as a function of cost further indicates which route healthcare regulators should partake. In this thesis, we propose a model to study cost-utilities of hand allotransplantation and myoelectric prostheses. To begin, a pilot study was performed on thumb amputations treated with free toe flaps, which allowed to confirm the feasibility of the utility questionnaires that we developed. Afterwards, utilities and quality adjusted life years were measured in a population of upper extremity amputees, proximally replanted patients and healthy controls. Findings demonstrated that 1) replanted patients reported the highest utility outcomes for myoelectric prostheses, 2) unilateral amputees significantly preferred myoelectric prostheses as well, and 3) no significant preference between both interventions was obtained in patients with bilateral amputations. Finally, a cost-benefit analysis was performed in the context of the Canadian healthcare system, demonstrating that significant savings can be achieved with treatment of unilateral amputations with myoelectric prostheses, whereas the gap in cost savings between both treatment groups becomes less significant in bilateral amputees. With the findings reported in this thesis, we can propose an update of the indications for treatment in patients with upper extremity amputations. From the perspective of cost-utility analyses, we conclude that unilateral amputees are better candidates for myoelectric prostheses, and that both treatments can still be offered in cases of bilateral amputations.