Successful survival of large volume engineered tissues depends on the development of a vasculature to support the metabolic demands of donor tissue in vivo. Pancreatic islet transplantation is a cell therapy procedure to treat Type 1 diabetes that can potentially benefit from such a vascularization strategy. The treatment is limited as the majority of transplanted islets (60%) fail to engraft due to insufficient revascularization in the host(1, 2). Modular tissue engineering is a means of designing large volume functional tissues using micron sized tissues with an intrinsic vascularization. In this thesis, we explored the potential of endothelialized modules to drive vascularization in vivo and promote islet engraftment. Human endothelial cells (EC) covered modules were transplanted in the omental pouch of athymic rats and human EC formed vessels near implanted modules until 7 days when host macrophages were depleted. Rat endothelial cells covered modules were similarly transplanted in the omental pouch of allogeneic rats with and without immunosuppressants. When the drugs were administered, endothelialized modules significantly increased the vessel density. Moreover, donor GFP labelled EC formed vessels that integrated with the host vasculature and were perfusable until 60 days; this key result demonstrate for the first time that unmodified primary endothelial cells form stable vessels in an allograft model. Transplantation of islets in such endothelialized modules significantly improved the vessel density around transplanted islets. Donor endothelial cells formed vessels near transplanted islets in allogeneic immunesuppressed recipients. Meanwhile, there was an increase in islet viability with transplantation of endothelialized modules in syngeneic recipients but this difference was not significant. In summary, endothelialized modules were effective in promoting stable vascularization and improving transplanted islet vascularisation. Future work should promote faster maturity of donor vessels and modulate the host immune and inflammatory responses to significantly improve transplanted islet engraftment.

Modular tissue engineering involves assembling tissue constructs with integral vasculature from units containing adipose-derived mesenchymal stromal cells (adMSCs) and endothelial cells. Here, the effects of implant volume and adMSC density on the vascularization of modular tissues were explored. Both parameters affected the contributions of host- and graft-derived vessels, without affecting total vessel density. Increasing implant volume from 0.01 to 0.10 mL increased HIF1α expression and graft-derived vessel density, suggesting a role of hypoxia in graft-derived vessel formation. However, increasing adMSC density within small-volume implants did not increase HIF1α expression. Vascularization of small-volume implants of high (4.3•10^6 cells/mL) and low (1.0•10^6 cells/mL) adMSC densities was dominated by host vessel ingrowth at day 7. By increasing adMSC density, a high proportion of host-derived vessels was maintained to day 14, presumably via paracrine effects. Further dissection of the role of hypoxia in modular tissue engineering remains a promising avenue to pursue.

Modular tissue engineering involves assembling tissue constructs with integral vasculature from units containing adipose-derived mesenchymal stromal cells (adMSCs) and endothelial cells. Here, the effects of implant volume and adMSC density on the vascularization of modular tissues were explored. Both parameters affected the contributions of host- and graft-derived vessels, without affecting total vessel density. Increasing implant volume from 0.01 to 0.10 mL increased HIF1α expression and graft-derived vessel density, suggesting a role of hypoxia in graft-derived vessel formation. However, increasing adMSC density within small-volume implants did not increase HIF1α expression. Vascularization of small-volume implants of high (4.3•10^6 cells/mL) and low (1.0•10^6 cells/mL) adMSC densities was dominated by host vessel ingrowth at day 7. By increasing adMSC density, a high proportion of host-derived vessels was maintained to day 14, presumably via paracrine effects. Further dissection of the role of hypoxia in modular tissue engineering remains a promising avenue to pursue.

Vascularization is essential for living tissue and remains a major challenge in the field of tissue engineering. A lack of a perfusable channel network within a large and densely populated tissue engineered construct leads to necrotic core formation, preventing fabrication of functional tissues and organs. While many approaches have been reported for forming vascular networks, including materials processing techniques, such those involving lithography, bioprinting, and sacrificial templating; and cell-based approaches, in which cellular self-organization processes form vessels; all are deficient in their ability to form a vessel system of sufficient complexity for supporting a large cellular construct. What is missing from the literature is a method for forming a fully three-dimensional vascular network over the full range of length-scales found in native vessel systems, which can be used alongside cells and perfused with fluids to support their function. A large number of research groups are thus pursuing novel methods for fabricating vascular systems in order that new tissues and organs can be fabricated in the lab. In this project, a 3D printing-based approach was used to form vascular networks which are hierarchical, three-dimensional, and perfusable. This was performed in thick, cellularized hydrogels similar in composition to native tissue; these being collagen (ECM-like) and fibrin (woundlike), both of which are highly capable of supporting cellular activities, such as cell seeding, cell spreading, and capillary morphogenesis. In order to make use of 3D printed network templates in cellularized hydrogel environments, it was necessary to develop a new approach in which standard 3D printed materials were converted into a gelatin template, via an alginate intermediary, which can be removed quickly in physiologic conditions and which does not reduce cell viability. This multi-casting approach enables a hierarchical channel network to be formed in three-dimensions, capable of being perfused with cell medium to maintain the viability of a cell population, thereby addressing the fundamental problem. Using standard cell staining and immuno-histochemistry techniques, we showed good endothelial cell seeding and the presence of tight junctions between the channel endothelial cells. When fibroblasts were seeded into the bulk of the hydrogel, a high degree of cell viability and cell spreading was observed when a threshold flow rate is met. By counting the number of live and dead cells in a sample regions of the gel, we were able to show a dependency of cell viability upon the perfusion flow rate and further determine a regime in which the vast majority of cells are alive and spreading. This data informs future cellular experiments using this platform technology. The limits of existing 3D printing technology meant that the micro-scale vasculature needed to be formed by other means. Cellular co-culture of endothelial and stromal cell types has been shown to be capable of forming capillary-like structures in vitro. For inclusion with the 3D printed channel system, we investigated the use of an angiogenic method for capillary formation, using multi-cellular spheroids, and a vasculogenic approach, using individual cells, in order that the full vascular system could be constructed. Endothelial and mesenchymal stromal cells were encapsulated in small fibrin and collagen gels and maintained under static culture conditions in order to form capillaries by the above approaches. The aim here was to find a particular gel composition and cell concentration which would support capillary morphogenesis while being suitably robust to handle the mechanical stresses associated with perfusion. As future work, the next step will be to incorporate the vasculogenic co-culture technique, used to form capillary-sized vessels, into a perfusable gel containing the large templated channels, formed via the multi-casting approach. The challenge here is to anastomose the capillary-sized vessels to the large templated channels and thereby enable perfusion of the capillary vessels. This step would be a highly significant development in the field as it would mean large constructs could be fabricated with physiological densities of cells, which could lead to a range of potential therapeutic applications.

Supraspinatus (SSP) tendon tear leads to intramuscular fat accumulation in the SSP muscle and the mechanisms are currently unknown. The purpose of this study was to investigate changes in vascularization of the SSP muscle and the relationship to intramuscular fat accumulation following SSP tendon detachment with or without reattachment. One hundred and six rabbits underwent SSP tendon detachment. In groups of ten, thirty rabbits were sacrificed 4, 8, and 12 weeks following detachment. Forty rabbits underwent detachment and immediate reattachment and were sacrificed in groups of ten following 0, 1, 2, and 6 weeks of healing. In groups of twelve, the remaining thirty-six rabbits underwent SSP tendon reattachment 4, 8, and 12 weeks after detachment and were sacrificed 12 weeks later. Vascularization was quantified in each specimen using CD31 immunohistochemistry. Four weeks after SSP tendon detachment, there was an increase in vascularization of the distal SSP muscle that reached significance after 12 weeks of detachment (p=0.024). We found that vascularization was positively correlated with intramuscular fat accumulation after detachment only (r=0.29; p=0.008). After SSP tendon reattachment, immediate or delayed, the correlation between vascularization and intramuscular fat accumulation was not observed. Microscopically, some SSP muscle vascular structures in the reattachment group had thicker vascular walls which were further quantified using αSMA immunohistochemistry. The delayed reattachment group showed an increase in vascular wall thickness in the distal portion of the SSP muscle at 4+12 (p=0.012) and 12+12 (p=0.012) weeks and in the proximal portion at 4+12 (p=0.024) weeks. Further investigation is required to demonstrate a cause/effect relationship between increased vascularization and intramuscular fat accumulation in the context of rotator cuff tear and success of surgical repair.

O estudo do aporte sanguíneo da face é importante em função da cirurgia como para as cirurgias que fazem uso de retalhos. O bom restabelecimento da área depende da perfusão obtida. Os métodos de aquisição de imagem que investigam a vascularização da face muitas vezes são caros e acrescentam risco à condição do paciente. A ultrassonografia é um método de imagem que não oferece risco ao paciente, sendo acessível e realizado em tempo real. A intensidade do fluxo sanguíneo de uma determinada estrutura pode ser medida pelo modo Doppler e alterações do padrão de normalidade indicam uma possível deficiência na nutrição dos tecidos compreendidos, podendo mesmo levar o clínico a prever futuras afecções. O objetivo neste estudo foi captar a vascularização normal da face, por meio da ultrassonografia modo B e Doppler de modo a se propor um mapeamento dos principais vasos arteriais. Foi possível, utilizando-se transdutores linear e endocavitário proceder-se ao estudo da vascularização da face. Foram obtidos dados referentes à vascularização da face que permitissem a análise da hemodinâmica dos tecidos. Foram estudados três vasos de diâmetro reduzido (0,60mm a. angular; 0,55mm a. palatina maior; 0,45mm a. infraorbital), sendo que nestes foi possível adquirir gráficos de onda de velocidade de fluxo e, consequentemente, obter-se a velocidade de pico sistólico (VPS) e o índice de resistividade (IR). / The study of the blood supply of the facial tissues is important due to oral and maxillofacial surgery as for operations that make use of flaps. The proper restoration of the perfusion is essential to the success of these procedures. The image methods that investigate the vasculature of the face are often costly and add risk to the patients condition. Ultrasound is an imaging method that offers no risk, is affordable and is performed in real time. The intensity of blood flow of a given structure can be measured by Doppler mode and changes in the normal range can indicate a deficiency in the nutrition of tissues end even influence the clinician to predict future conditions. The aim of this study was to access the normal vasculature of the face through ultrasound B-mode and Doppler in order to propose a mapping of the arterial vessels. It was possible, using the linear and endocavitary probes to study the vascularity of the face. Data was obtained allowing the analysis of the tissue hemodynamics. It was possible to access three arteries of small diameter (0,60mm angular artery; 0,55mm greater palatine artery; 0,45mm infraorbital artery) and in all cases graphics of wave flow velocity. Therefore, the resistivity index (RI) and systolic peak velocity (SPV) could also be computed.

Durante o puerpério há muita incidência de doenças reprodutivas acarretando aumento do intervalo entre partos, bem como diminuição na taxa de concepção. A endometrite é uma enfermidade puerperal que se caracteriza por um processo inflamatório superficial do endométrio. Com o intuito de se obter novas ferramentas diagnósticas não invasivas, precisas e que proporcionam resultados precoce à reprodução, faz-se uso da ultrassonografia convencional associada a ferramenta Doppler que fornece informações em tempo real sobre a arquitetura vascular e os aspectos hemodinâmicos dos vasos em diversos órgãos. Este estudo tem por objetivo descrever, através da ultrassonografia Doppler, as alterações hemodinâmicas no útero de fêmeas bovinas causadas pela endometrite. Avaliou-se o trato reprodutivo feminino de vacas entre 25 a 35 dias pós-parto utilizando a vaginoscopia, ultrassonografia convencional e Doppler, além de citologia e exame microbiológico em 89 vacas Holandesas sendo 33 sadias (controle) e 56 com endometrite diagnosticadas por citologia (saudáveis <10% de polimorfonucleares). Os resultados obtidos destacam-se a que animais com endometrite apresentavam aumento de cérvix (p=0,04), e de útero representado pelo corno esquerdo (p=0,02). Porém não foi observado diferença quando comparado os escore de condição corporal (ECC). Quanto a ultrassonografia notou-se que animais com fluido intrauterino (FIU) e conteúdo heterogêneo intrauterino (CHIU) tinham endometrite (p<0,0001), além de correlação com a presença de Trueperella pyogenes e leveduras. Com o modo cores do Doppler obteve-se a vascularização de mesométrio (p=0,004) e endométrio (p=0,025) associada aendometrite. Entretanto não foi observado diferença estatística nas medidas do Doppler espectral (índice de resistividade, pulsatilidade e fluxo sanguíneo). Portanto a busca de novas técnicas, menos invasivas e de rápido resultado, como a ultrassonografia Doppler, podem fornecer respostas satisfatórias quanto a evolução de alterações uterinas e com isso associar os resultados à precocidade reprodutiva. / During the puerperium period there is a high incidence of reproductive diseases resulting in increased calving interval and decrease in conception rate. The endometritis is a postpartum disease that is characterized by a superficial inflammation of endometrium. With the aim to obtain new non-invasive and accurate diagnostic tools, that provide early results in reproduction, it is possible to be used the conventional ultrasound associated to Doppler that provides real time information about vascular architecture and hemodynamic aspects of vessels in several organs. This study aims to describe, through Doppler ultrasound, hemodynamic changes in the uterus of cows caused by endometritis. Were evaluated the female reproductive tract of 89 Holstein cows between 25 to 35 days postpartum using vaginoscopy, conventional ultrasound and Doppler, as well as cytology and microbiological examination. From the 89 animals that were studied, 33 were healthy (control) and 56 with endometritis diagnosed by cytology (healthy <10% polymorphonuclear). The obtained results show that animals with endometritis had increase in cervix (p = 0.04) and the uterus represented by left uterine horn (p = 0.02). However, it was not detected difference when compared the body condition score (BCS). About the ultrasound was noted that animals with intrauterine fluid (IUF) and intrauterine heterogeneous content (IUHC) had endometritis (p <0.0001) correlation with the presence of Trueperella pyogenes and yeasts. With the color Doppler mode it was possible to obtain the vascularization of mesometrium (p = 0.004) and endometrium (p = 0.025) associated with endometritis. However, it was not observed statistical difference in the measurements of the spectral Doppler (resistance index, pulsatility and blood flow). Therefore, the search for new techniques, less invasive and fast result, as the Doppler ultrasound can provide satisfactory answers as the evolution of uterine changes and thereby associate the results to the reproductive precocity.

O timo é um órgão essencial para a maturação, diferenciação e seleção de linfócitos T, consequentemente fundamental para o desenvolvimento da imunidade do organismo. Além do papel na manutenção da integridade tecidual, os vasos sanguíneos tímicos desempenham função no processo de migração de células precursoras de linfócitos para o interior do órgão. Sua arquitetura típica é caracterizada por grandes vasos localizados na junção corticomedular e uma fina cadeia de ramificações e anastomoses no interior do córtex, porém são pouco conhecidas as bases moleculares que induzem a formação desta particular vascularização, bem como os exatos mecanismos que desencadeiam a involução do órgão, evento relacionado a alterações nos padrões vasculares. Entretanto, tem sido demonstrado que as interações entre o estroma, que inclui o endotélio vascular, e o compartimento hematopoiético tímico desempenham papéis cruciais na funcionalidade do órgão. Sendo o VEGF um fator angiogênico essencial na formação do leito vascular tecidual e na modulação de funções diretamente relacionadas à vascularização, objetivamos neste estudo avaliar a expressão gênica e protéica deste fator de crescimento durante estágios de desenvolvimento e involução do órgão. Foram utilizadas amostras de timo suíno em 3 diferentes estágios de desenvolvimento fetal (65, 85 e 111 dias) e 2 estágios de vida pós-natal, um compreendendo fase de maturação (5 meses) e outro de involução do órgão (2 anos), compreendendo um total de 25 animais, divididos em 5 grupos (n = 5). A análise da expressão relativa do mRNA do sistema VEGF-A, acessada por meio de PCR em tempo real, apresentou modificações tempo-dependentes significativas (P<0,05) em relação ao controle endógeno GAPDH. Também foram observadas diferenças significativas (P0,05) na quantificação vascular por meio de estereologia entre as idades. A imunolocalização da proteína do VEGF e de seus receptores Flt-1 e KDR foi identificada no timo de todos os grupos experimentais e variou entre os grupos pré e pós-natais, com expressão mais rara nestes últimos, o que sugere diferenças temporais na modulação do mRNA no órgão. Assim como as células endoteliais, as células epiteliais tímicas demonstraram modulação pelo sistema VEGF-A, indicando ação parácrina e/ou autócrina deste fator de crescimento no timo. Os valores das variáveis estereológicas Sv[m] e Vv[m] do timo fetal em período próximo ao nascimento mantiveram-se maiores do que no córtex e se correlacionaram positivamente com a expressão do mRNA do KDR e negativamente com a do Flt-1 até a idade adulta. Ao contrário da expressão protéica, a expressão gênica do sistema VEGF-A pode se correlacionar negativamente com aspectos quantitativos da vascularização do timo pós-natal, como observado para o mRNA do VEGF e seu receptor Flt-1 a partir dos 5 meses de idade, quando os valores das variáveis Vv[vasos,timo], Sv[vasos,timo] e Lv[vasos,timo] diminuíram significativamente (P0,05), o que sugere uma resposta de compensação ou manutenção de uma condição hipóxica instalada no órgão. / Crucial for maturation, differentiation and selection of T lymphocytes, the thymus is an essential organ to the development of immunity. In addition to its role in the maintenance of tissue integrity, thymus blood vessels play a role in migration of T-cell precursors into the organ, and show a typical architecture characterized by the presence of large blood vessels at the corticomedullary junction and fine network of branching vessels and anastomosing arcades extending into the cortex. The molecular basis of this typical vasculature and the exact mechanisms that trigger thymus involution are both not well understood. However, it has been shown that the interactions between thymus stroma, including vascular endothelium and the hematopoietic compartment play crucial roles for normal organ function. Thus, because VEGF is an essential angiogenic factor for induction of the vascular bed and modulation of functions directly related with vascularization, the aim of this study was to assess the temporal protein and mRNA expression of VEGF and its receptors Flt-1 and KDR in the thymus during early stages of its development and involution. Samples of thymus from 3 different stages of fetal development (65, 85, and 111 days) and 2 stages of postnatal life of pigs, including one stage of maturation (5 months) and another of involution (2 years) comprising a total of 25 animals that were divided into 5 groups (n = 5). Relative mRNA expression of VEGF-A system, accessed by real-time PCR, showed temporal significant changes (P<0.05) compared to endogenous control GAPDH. Quantification of blood vessels by means of stereology also showed significant differences (P0.05) between ages. Positive immunostaining for VEGF and its receptors Flt-1 and KDR was identified in the thymus of all groups and it varied between the pre and postnatal groups with rare expression in the latter, suggesting temporal differences in the modulation of mRNA. Endothelial and epithelial cells showed modulation by the VEGF-A system, suggesting paracrine or alternatively autocrine activity of this growth factor. The values of the stereological parameters Sv[m] and Vv[m] of the thymus in fetal period around the birth remained higher than in the cortex and upregulated with mRNA expression of KDR and downregulated with mRNA expression of Flt-1 until adulthood. Unlike protein expression, gene expression gene expression of VEGF-A system can be downregulated with quantitative aspects of the postnatal thymus vascularization as observed for the mRNA of VEGF and its receptor Flt-1 from the age of 5 months, when the values of the stereological parameters Vv[vessels,thymus], Sv[vessels,thymus] and Lv[vessels,thymus] decreased significantly (P0.05), which suggests a compensatory or maintenance response to an hypoxic condition in the organ.

Utilizamos nesta pesquisa 40 corações de cães adultos, 17 machos e 23 fêmeas, de idades variadas, que não portavam macroscopicamente nenhuma afecção cardíaca. Os corações tiveram as artérias coronárias injetadas, separadamente, com Neoprene Látex 450, corado com pigmento vermelho, e posteriormente dissecados. Em todas estas preparações verificamos que na vascularização dos ventrículos predominava a artéria coronária esquerda que fornecia os ramos interventriculares paraconal e subsinuoso. Já, a região ocupada pelo nó sinoatrial ficava mais frequentemente (17 vezes 42,5%) na dependência do ramo proximal atrial esquerdo ou de colateral deste vaso, oriundo do ramo circunflexo esquerdo, ou deste vaso associado ao ramo distal atrial direito (8 vezes 20%), procedente do ramo circunflexo direito. Com menor frequência (14 vezes 30%), a área tomada pelo nó sinoatrial, encontramos apenas colaterais do ramo circunflexo direito, mais exatamente somente o ramo distal atrial direito (10 vezes 25%), apenas o ramo proximal atrial direito (3 vezes 7,5%) ou ainda exclusivamente o ramo intermédio atrial direito (1 vez 2,5%). Em um único caso (1 vez 2,5%) no território do nó sinoatrial observamos apenas colateral do ramo circunflexo esquerdo, isto é o ramo distal atrial esquerdo. A análise destes resultados permite concluir, que nesta espécie não existe qualquer tipo de relação entre o tipo de vascularização dos ventrículos e a irrigação do nó sinoatrial. / In this research we analyzed 40 hearts of adult dogs, males and females with different ages, which had no cardiac disease. The hearts had the coronary arteries inject using Neoprene Latex 450, stained by red pigment and then were dissected. In the ventricular vascularization the left coronary artery was predominant and it gave rise to the branches paraconal interventricular and subsinuous. The region occupied by the sinoatrial node was most frequently (17 times 42.5%) in the dependence of the left atrial proximal branch or in the collateral of that vessel, coming from the left circumflex branch, or this vessel associated to the right atrial distal branch (8 times 20%). With less frequency (14 times 30%), in the area occupied by the sinoatrial node, we found only the collaterals of the right circumflex branch, rather only the right atrial distal branch (10 times 25%), only the right atrial proximal branch (3 times 7.5%) or exclusively through the right atrial intermediary branch (1 time 2.5%). In just one case (Once 2.5%) in the area of the sinoatrial node we observed only the collateral of the left circumflex branch, i.e. the left atrial distal branch. According to our results we concluded that in this specie there are no relation between the type of vascularization of the ventricles and the irrigation of the sinoatrial node.

Cardiovascular disease (CVD) is the leading cause of death internationally. Efforts to decrease CVD death has been explored through stem cell technology, specifically organoid formation. Current cardiac organoid models lack the vascular networks for nutrient supply and maturation. In this study, pillar perfusion technology is used to fabricate cardiac organoids and induce vascularization via dynamic culturing and the addition of vascular promoting growth factors (GFs). In addition to this study, a millifluidic chip is engineered for shear stress application via flow simulations and experimental flow analysis. We successfully optimized the millifluidic chip to achieve fluid shear stress of 20mPa and validated through particle tracking velocimetry using 0.1um diameter beads under flow. The results of cardiac organoids displayed contraction and growth of endothelial cells (ECs) under dynamic flow with GFs. In addition, smooth muscle cells (SMCs) displayed growth via GFs in both dynamic and static culturing. Although vascular networks were not present in all conditions of this experiment, this thesis can serve a basis for searching other methods of inducing vascularization.