In the United Kingdom 1 in 6 - 8000 children are born missing one or both of their ears. The surgical technique most commonly used to reconstruct ears requires surgeons to remove ribs from the patient, and the cartilage from the ribs is used to carve a new ear. This procedure involves many risks including significant pain, punctured lung and chest deformity. Therefore the ability to ‘grow’ an ear would be a major advancement. Stem cells show huge promise in tissue engineering and regenerative medicine. Approved stem cell technology must be evaluated with regards to safety, purity, identity, potency and efficacy prior to biologic licensing and clinical use. Therefore, access to ethically sourced tissue for research is fundamental to the successful delivery of novel therapies. Adipose tissue provides an abundant and accessible source of stem cells for clinical translation. Within the first section of this thesis, the perceptions and attitudes of patients towards the donation and use of adipose tissue for research are sought. Based on this information, a tissue bank with all appropriate ethical approval to collect, process, store and distribute adipose tissue and adipose derived stem cells is established. The second part of this thesis demonstrates the specific identity, location and frequency of stem cells within adipose tissue; revealing them to reside in a perivascular niche. Using this data, protocols to rapidly purify stem cells from adipose tissue using Fluorescence Activated Cell Sorting are developed. The frequency of cells, and both the patient and procedure based variables that can affect this yield are also examined. The final section of this thesis uses a high-throughput microarray platform to screen thousands of polymers to identify potential substrates that can support the attachment, stable proliferation and subsequent differentiation of stem cells purified from adipose tissue. From the initial screen, 5 distinct polymers have been identified, characterised and their effects on the stem cells examined and quantified. Combined together, these elements provide significant advances in our understanding, and the basis for on going research to deliver a tissue engineered ear for use in human ear reconstruction.

No description available.

Osteoarthritis is a debilitating and irreversible disease afflicting the synovial joints. It is characterized by pain and hindered mobility. Given that osteoarthritis has no cure, current treatments focus on pain management. Ultimately, however, a patient's pain and immobility necessitates joint replacement surgery. An attractive alternative to this treatment paradigm, tissue engineering is a promising strategy for resurfacing the osteoarthritis-afflicted cartilage surface with a biochemically and biomechanically similar tissue to the healthy native cartilage tissue. The most successful cartilage tissue engineered systems to date can repeatably grow constructs ~4 mm in diameter with native proteoglycan and compressive mechanical properties. Unfortunately, as symptomatic cartilage typically presents only once lesions span large regions of the joint (~25 mm in diameter), these small construct are of limited use in clinical practice. Numerous attempts to simply grow a construct large enough to span the size of an osteoarthritic lesion have shown that the growth of large engineered tissues develop heterogeneous properties, emphasizing the need for culture protocols to enhance tissue homogeneity and robustness. In particular, as nutrient limitations drive heterogeneous growth in engineered cartilage, developing strategies to improve nutrition throughout the construct are critical for clinical translation of the technology. To this end, our lab has successfully supplemented nutrient channels within large engineered cartilage constructs to improve the functional properties of developing tissue. However, it is unknown what the optimal nutrient channel spacing is for growing large cartilage constructs of anatomical scale. Additionally, the fundamental factors and mechanisms which drive tissue heterogeneity have not been implicated, making the results of channel-spacing optimizations difficult to translate across different systems. Computational models of growth, faithful to the physics and biology of engineered tissue growth, may serve as an insightful and efficient tool for optimally designing culture protocols and construct geometries to ensure homogeneous matrix deposition. Such computational tools, however, are not presently available, owing to the unresolved mechanical and biological growth phenomena within developing engineered cartilage. This dissertation seeks to develop and implement computational models for predicting the biochemical and biomechanical growth of engineered tissues and apply these models to optimizing tissue culture strategies. These models are developed in two stages: 1) based on our recent characterization of the nutrient demands of engineered cartilage, models are developed to simulate the spatial biochemical deposition of matrix within tissue constructs and, subsequently, 2) based on models of biochemical matrix deposition we develop models for simulating the mechanical growth of tissue constructs. To accomplish these tasks, we first develop models simulating glucose availability within large tissue constructs using system-specific modeling based on our recent characterization of the glucose demands of engineered cartilage. These models led to early insight that we had to enhance the supply of glucose within large tissue constructs to ensure maximal matrix synthesis throughout culture. Experimental validations confirmed that increasing glucose supply enhanced matrix deposition and growth in large tissue constructs. However, even despite the increased glucose supply, increasing the size of constructs demonstrated that severe matrix heterogeneities were still present. Subsequent nutrient characterization led to the finding that TGF-ß transport was significantly hindered within large tissue constructs. Incorporating the influence of glucose and TGF-ß into the computational model growth kinetics. Using both nutrients, models recreated the heterogeneous matrix deposition evident in our earlier work and could account for the role of cell seeding density and construct geometry on tissue growth. The insights gathered from this modeling analysis led to important changes in our culture protocols: we could reduce the dose of TGF-ß from 10 ng/mL to 1 ng/mL for constructs cultured with channels, saving considerable expense while still maintaining a high level of matrix synthesis throughout the construct. In the presence of sufficient nutrition, we witnessed an unprecedented level of matrix deposition and physical growth of the constructs. In fact, by using developmentally physiologic cell seeding densities (120 million cells/mL) and providing adequate nutrition, constructs physically grew to 9-times their originally cast size. Despite such encouraging growth, tissue function properties plateaued at sub-physiologic levels. For insight into the connection between matrix deposition and tissue mechanics, we extended the computational growth models to consider the mechanisms underlying physical growth. Interestingly, we found that a large matrix synthesis mismatch between proteoglycans and collagen gave rise to the excessive tissue swelling. Computational models of this matrix synthesis mismatch predicted the high tissue swelling displayed experimentally only when a damage-able collagen fiber material was implemented. Together, the experimental and modeling evidence suggested a new mechanism of cartilage growth: the high proteoglycan deposition creates a swelling pressure within the nascent tissue which outcompetes the restraining force of newly deposited collagens; this rapid tissue swelling disrupts a functional collagen network from forming. Subsequent analysis suggested that the disruption of the collagen network prevented the formation of collagen crosslinks, stymieing the development of native functional properties. Based on this insight into the mechanisms of cartilage growth, we developed a culture systems to improve tissue functional properties. Modeling analysis indicated two novel routes for improving tissue mechanics: either through 1) reducing the swelling response (synthesis and deposition) of proteoglycans or 2) enhancing and reinforcing the newly synthesized collagen to prevent disruptions brought on by tissue swelling. We developed a cage culture system for resisting the swelling pressure of deposited proteoglycans and reenforcing the deposition of new collagens. Using this cage system, we grew tissue constructs with enhanced functional properties using two separate scaffold systems – agarose and a cartilage-derived matrix hydrogel – suggesting this mechanism of growth is fundamental to engineered cartilage development. This work has generated a novel paradigm towards engineering cartilage constructs using biomimetic strategies. Performing simulations with the validated, computational growth models allowed anatomically-sized cartilage constructs to grow into the largest, homogeneous cartilage constructs grown to date. Models presented a new level of insight into the nutrient demands of developing tissues, allowing for the first time the successful development of large tissue constructs grown with developmentally physiologic cell seeding densities. In this way, tissue constructs growth followed a biomimetic approach, based on the high cell densities and cartilage canals and vasculature present during fetal cartilage development. Adequate nutrition led to high levels of tissue growth not previously experienced in vitro, a result of adequately nourishing primary chondrocytes, a cell type which preferentially deposits proteoglycans over collagen. We therefore developed a cage-based growth system to resist the proteoglycan-induced tissue swelling in a manner similar to the fetal development of cartilage where the resident cells synthesize more collagens than proteoglycans. Together, the use of nutrient growth models, high cell seeding densities, and culture systems to strengthen the collagen-framework of de novo cartilage proved beneficial for engineering anatomically-sized cartilage constructs. The fundamental mechanisms identified here are likely to be universal across a number of engineered cartilage systems and will be adapted to more clinically-relevant cell sources in future our work.

Made available in DSpace on 2014-12-17T15:43:50Z (GMT). No. of bitstreams: 1 HaroldoAOJ_DISSERT.pdf: 2217261 bytes, checksum: 1a1221344f95ad964f692521743b5714 (MD5) Previous issue date: 2013-03-01 / The regeneration of bone defects with loss of substance remains as a therapeutic challenge in the medical field. There are basically four types of grafts: autologous, allogenic, xenogenic and isogenic. It is a consensus that autologous bone is the most suitable material for this purpose, but there are limitations to its use, especially the insufficient amount in the donor. Surveys show that the components of the extracellular matrix (ECM) are generally conserved between different species and are well tolerated even in xenogenic recipient. Thus, several studies have been conducted in the search for a replacement for autogenous bone scaffold using the technique of decellularization. To obtain these scaffolds, tissue must undergo a process of cell removal that causes minimal adverse effects on the composition, biological activity and mechanical integrity of the remaining extracellular matrix. There is not, however, a conformity among researchers about the best protocol for decellularization, since each of these treatments interfere differently in biochemical composition, ultrastructure and mechanical properties of the extracellular matrix, affecting the type of immune response to the material. Further down the arsenal of research involving decellularization bone tissue represents another obstacle to the arrival of a consensus protocol. The present study aimed to evaluate the influence of decellularization methods in the production of biological scaffolds from skeletal organs of mice, for their use for grafting. This was a laboratory study, sequenced in two distinct stages. In the first phase 12 mice hemi-calvariae were evaluated, divided into three groups (n = 4) and submitted to three different decellularization protocols (SDS [group I], trypsin [Group II], Triton X-100 [Group III]). We tried to identify the one that promotes most efficient cell removal, simultaneously to the best structural preservation of the bone extracellular matrix. Therefore, we performed quantitative analysis of the number of remaining cells and descriptive analysis of the scaffolds, made possible by microscopy. In the second stage, a study was conducted to evaluate the in vitro adhesion of mice bone marrow mesenchymal cells, cultured on these scaffolds, previously decellularized. Through manual counting of cells on scaffolds there was a complete cell removal in Group II, Group I showed a practically complete cell removal, and Group III displayed cell remains. The findings allowed us to observe a significant difference only between Groups II and III (p = 0.042). Better maintenance of the collagen structure was obtained with Triton X-100, whereas the decellularization with Trypsin was responsible for the major structural changes in the scaffolds. After culture, the adhesion of mesenchymal cells was only observed in specimens deccelularized with Trypsin. Due to the potential for total removal of cells and the ability to allow adherence of these, the protocol based on the use of Trypsin (Group II) was considered the most suitable for use in future experiments involving bone grafting decellularized scaffolds / A regenera??o de defeitos ?sseos com perda de subst?ncia permanece um desafio terap?utico na ?rea m?dica. ? consenso ser o osso aut?geno, o material mais adequado para esta finalidade, por?m h? limita??es at? para o seu uso, especialmente a quantidade insuficiente no pr?prio doador. Pesquisas de engenharia tecidual evidenciam que os componentes da matriz extracelular (MEC) s?o geralmente conservados entre as diferentes esp?cies sendo bem toleradas, mesmo em receptores xen?genos. Assim, diversos estudos t?m sido realizados na busca por um arcabou?o substituto do osso aut?geno atrav?s da t?cnica de descelulariza??o. Para a obten??o destes arcabou?os, os tecidos devem passar por um processo de remo??o celular, que cause m?nimos efeitos adversos na composi??o, atividade biol?gica e integridade mec?nica na matriz extracelular remanescente. Entretanto, h? controv?rsias acerca do melhor protocolo de descelulariza??o, j? que cada um desses tratamentos interfere de maneira diferente na composi??o bioqu?mica, ultraestrutura e comportamento mec?nico da matriz extracelular, afetando o tipo de resposta imunol?gica ao material. Ademais o baixo arsenal de pesquisas envolvendo a descelulariza??o de tecidos ?sseos representa mais um obst?culo ? chegada de um consenso protocolar. O presente estudo teve como objetivo avaliar a influ?ncia dos m?todos de descelulariza??o na produ??o de arcabou?os biol?gicos a partir de ?rg?os ?sseos de camundongos, visando sua utiliza??o para enxertia. Trata-se de um estudo laboratorial, sequenciado em duas etapas distintas. Na primeira fase foram avaliadas 12 hemi-calv?rias de camundongos, divididas em tr?s grupos (n=4) e submetidas a tr?s diferentes protocolos de descelulariza??o (SDS [Grupo I], Tripsina [Grupo II], Triton X-100 [Grupo III]). Buscou-se identificar aquele que promove a mais eficiente remo??o celular, simultaneamente a melhor preserva??o estrutural da MEC ?ssea. Para tanto, foi realizada an?lise quantitativa do n?mero de c?lulas remanescentes e an?lise descritiva dos arcabou?os, possibilitadas por microscopia. Na segunda etapa, foi realizado um estudo in vitro para avaliar a ades?o de c?lulas mesenquimais da medula ?ssea de camundongos, cultivadas sobre arcabou?os previamente descelularizados. Atrav?s da contagem manual de c?lulas nos arcabou?os, verificou-se total remo??o celular no Grupo II, remo??o praticamente completa no Grupo I, e perman?ncia de c?lulas e remanescentes no Grupo III. Os achados permitiram observar diferen?a significativa apenas entre os Grupos II e III (p=0,042). Melhor manuten??o da estrutura col?gena foi obtida com o Triton X-100, ao passo que a descelulariza??o com Tripsina foi respons?vel pelas maiores altera??es estruturais nos arcabou?os. Ap?s o cultivo, a ades?o de c?lulas mesenquimais s? foi observada nas calv?rias descelularizadas com Tripsina. Devido ao potencial de remo??o total das c?lulas e ? capacidade de permitir a ades?o destas, o protocolo baseado no uso da Tripsina (Grupo II) foi considerado o mais adequado para uso em experimentos futuros, que envolvam enxertia de arcabou?os ?sseos descelularizados

A odontologia moderna, mesmo usando as suas técnicas mais primorosas, na prática, ainda recupera a perda dental com implantes metálicos recobertos por coroas protéticas. Há um empenho coletivo dos cientistas em criar técnicas de desenvolvimento dental in vitro na busca por maneiras de recuperar, de maneira biológica, a ausência dental. Já é possível criar estruturas similares a dentes a partir de células-tronco de origem dental (polpa de dentes permanentes e decíduos) e não dental (células-tronco embrionárias, células-tronco da medula óssea e da crista neural) por meio de recombinação dos tecidos epiteliais e mesenquimais de germes dentais. As técnicas de reconstrução tecidual nunca estiveram tão perto do desenvolvimento da terceira dentição mas a ciência ainda tem muito a aprender no que concerne o estudo da biologia dental e engenharia de tecidos. Não basta saber como um dente se desenvolve; há de se entender como ele interage com o organismo do qual faz ou fará parte. É com esta preocupação que nos propomos a estudar se pode haver uma correlação entre o desenvolvimento do sistema nervoso e vascular de um germe dental transplantado com a posição que ele é implantado e/ou com a presença de tecido ósseo que no leito receptor. Ademais, buscamos saber se o estágio de desenvolvimento do germe dental a ser transplantado pode influenciar a formação de tecido nervoso e vascular na polpa dental ou não. Nossos resultados revelaram que o local do sítio do implante influencia diretamente o desenvolvimento dental e que isto é tempo dependente. A vascularização e a reinervação da polpa dental nos espécimes implantados nas tíbias é mais semelhante ao grupo controle que os implantados nos rins e isto independe da posição de implantação dental. Entretanto, a polpa dental dos germes implantados nos rins parece estar comumente mais sadia, conter mais odontoblastos viáveis e ser capaz de produzir tecidos mineralizados como a osteodentina. / Contemporary dentistry, even using modern techniques, still deal with missing teeth using metal implants coated by prosthetic crowns. However, there is a worldwide effort to develop a biotooth using in vitro techniques. In this way it is already possible to generate structures similar to teeth using recombination of odontogenic and non odontogenic cells in tissue engineering experiments. The transplant of the recombined cells into a host is a necessary and major step to obtain the biotooth. In this context, at the same time that the development of an appropriate sensorial and vascular system in the biotooh is required, there are many unclear questions about it. Therefore, herein we intend to analyze (I) whether may exist a correlation between the stage of development and vascular and nervous re-growth in the dental pulp after tooth transplantation; (II) if the absence or presence of bone could influence this processes or (III) if the position of implantation could change the vascular and/or nervous development in the transplanted tooth. Our results showed that the site of implantation directly alter tooth development modifying morphogenesis and expression of different vascular, perivascular and neural markers in a time dependant way. The re-growth of the vascular and neural tissue on samples transplanted to the tibia is more similar to the control group than the kidney ones and it is non dependant of the position of implantation. However, the pulp tissue of the samples transplanted under the kidney capsule seemed to be healthier as they were capable of producing mineralized tissue such as osteodentin and still had live odontoblasts.

O uso de células tronco já é uma realidade em algumas áreas da Medicina, porém o mesmo não se aplica para a Odontologia, que segue utilizando materiais artificiais para substituir tecidos dentais perdidos. Desde 2000, quando Gronthos e colaboradores identificaram células-tronco na polpa de dentes permanentes, os estudos avançaram para que, num futuro breve, essas células possam ser de fato aplicadas para regenerar tecidos dentais, com destaque para a endodontia, onde o uso dessas células parece ser mais eminente. Dessa forma, esse trabalho procurou analisar a co-participação de células humanas transplantadas e células do hospedeiro em um modelo de engenharia pulpar. Para tanto, células de papila apical, enriquecidas ou não para o marcador CD146, foram transplantadas em câmaras pulpares despulpadas preenchidas com colágeno. Dois modelos animais foram utilizados, sendo um modelo transgênico para o gene GFP e outro imunocomprometido pela aplasia do timo (nude). Os resultados foram analisados nos dias 14 e 21 pós-transplante das amostras em cápsula renal. Nas amostras GFP realizou-se imunofluorescência para o marcador anti-GFP, com o objetivo de identificar as células do hospedeiro, enquanto nas amostras nude utilizou-se o marcador lâmina A para identificar as células humanas transplantadas. Nas análises morfológicas de todas as coroas transplantadas houve a formação de um tecido conjuntivo frouxo de celularidade variável, com a presença de vasos bem formados com eritrócitos em seu interior, inclusive nas coroas que receberam somente colágeno. Somente as amostras que receberam células houve a formação de matriz mineralizada no espaço pulpar, mas nos tempos experimentais analisados não foi possível visualizar as células humanas. Nas amostras nude, o marcador lâmina A foi negativo para todos os grupos que receberam transplante de células. Nas amostras GFPs, o marcador anti-GFP foi positivo na totalidade das células em todas as amostras estudadas. A partir disso concluiu-se que as células-tronco humanas de papila apical transplantadas apesar de terem desempenhado alguma função fisiológica, não foram identificadas após 14 e 21 dias e o tecido neoformado no interior da câmara pulpar era proveniente do hospedeiro. Adicionalmente, concluímos que não é necessário o transplante de células para a formação de um tecido conjuntivo frouxo no interior da câmara pulpar. / The use of stem cells is already a reality in some areas of Medicine, but the same does not apply for Dentistry, which keeps on using artificial materials to replace lost dental tissues. Since 2000, when Gronthos and coleagues identified stem cells in the pulp of permanent teeth, studies advanced so that, in the near future, these cells may actually be applied to regenerate dental tissues, especially in endodontics, in which the use of these cells seems to be more imminent. Thereby, this study sought to examine the co-participation of transplanted human cells and host cells in a model of dental pulp engineering. For this, apical papilla cells enriched or not with CD146 marker, were transplanted into decelluarized pulp chambers (empty crowns) filled with collagen. Two animal models were used, a transgenic model for the GFP gene and an immunocompromised by thymus aplasia (nude). The results were analyzed on days 14 and 21 after transplantation of samples into renal capsule. In the GFP samples immunofluorescence was performed for the anti-GFP marker in order to identify host cells, while in the nude samples the lamina A marker was used to identify the transplanted human cells. In the morphological analysis of all transplanted crowns there was formation of a loose connective tissue of variable cellularity, with the presence of well-formed vessels with erythrocytes inside, including in the crowns that received only collagen. Osteodentine was formed in the pulp chamber only in the samples that received cell, but after the wait time was not possible to visualize the human cells. In the nude samples the Lamina A marker was negative for all groups transplanted with cells. In the GFPs samples, the anti-GFP marker was positive for all cells in this group. We concluded that it was not necessary to use transplanted cells to form a connective tissue inside the pulp chamber and although transplanted human stem cells from apical papilla played some physiological function, after 14 and 21 days of transplantation, they were no longer present in the tissue newly formed by the host.

Os transtornos que afetam a traqueia podem levar a graves complicações de saúde e reduzir significativamente a qualidade de vida das pessoas. Até o presente não existe uma opção clinicamente viável disponível para pacientes com distúrbios severos dessa via aérea. As técnicas de Engenharia de Tecidos apresentam um cenário promissor na tentativa de produzir um substituto traqueal satisfatório, com características mecânicas e biológicas compatíveis com os tecidos do paciente. Nesse trabalho, apresenta-se um sistema de aquisição de imagens de vídeo que permite medir as variações da área da projeção bidimensional de traqueias autotransplantadas de ratos, in vivo, com o objetivo de apresentar uma ferramenta que possa caracterizar as suas propriedades mecânicas e acelerar a avaliação das técnicas e dos tecidos gerados. Seis ratos, da linhagem Wistar, tiveram exposição de toda a traqueia cérvico-mediastinal, foram ventilados e submetidos a um protocolo experimental com diferentes cenários de broncoconstrição, a fim de se poder relacionar os dados obtidos a partir da ventilação mecânica com as imagens da movimentação das paredes da traqueia antes e depois de um autotransplante e aprofundar a discussão do sistema respiratório nas situações de maior broncoconstrição. Os resultados do pré-transplante, no cenário de maior broncoconstrição, mostraram um aumento médio da área da projeção da traqueia de 0,9 ± 0,24% nas fases inspiratórias das perturbações quasi-senoidais e, nas fases expiratórias, reduções de 5,2 ± 6,1%. No pós-transplante, o aumento médio foi de 0,7 ± 0,26% e a redução foi de 1,7 ± 0,73%, para o mesmo cenário de broncoconstrição. As diferenças das fases inspiratórias e expiratórias sugerem uma maior cautela para interpretar os parâmetros da mecânica ventilatória nos cenários de maior broncoconstrição das vias aéreas. Por intermédio das imagens da traqueia, também foi possível observar os diferentes instantes de tempos de resposta para a ação de um agente constritor da musculatura lisa. Pode-se concluir que essa técnica de aquisição de imagens apresenta resolução e frequência de aquisição suficientes para monitorar as variações do calibre de traqueias autotransplantadas de ratos e trazem uma boa perspectiva para o seu uso na avaliação das propriedades mecânicas de enxertos traqueais produzidos por Engenharia Tecidual. / Tracheal disorders can lead to serious health complications and significantly reduce the people life´s quality. There is no clinically feasible option available for patients with several airway disorders. Tissue Engineering techniques present a promising scenario to produce a satisfactory tracheal replacement, with compatible mechanical and biological characteristics with the patient\'s tissues. This work presents a video image acquisition system that allows measure in vivo two-dimensional projection area variations of auto transplanted rat\'s tracheas with the objective of presenting a tools that can characterize its mechanical properties and accelerate the evaluation of techniques and tissues. Six Wistar rats were exposed to the entire cervical-mediastinal trachea, ventilated and submitted to an experimental protocol in different bronchoconstriction scenarios, before and after an auto transplantation in order to correlate the data obtained from the mechanical ventilation and images of the trachea walls movement before and after the auto transplantation and improve the discussion about respiratory system in greater bronchoconstriction status. The pre-transplant results, in the greater bronchoconstriction scenario, showed an average increase of 0.9 ± 0.24% in the inspiratory phases of the quasi-sinusoidal perturbations and, in the expiratory phases, a reduction of 5.2 ± 6.1%. In the post-transplant, the mean increase was 0.7 ± 0.26% and the reduction was 1.7 ± 0.73%, for the same bronchoconstriction scenario. Differences between inspiratory and expiratory phases suggest caution in interpreting the parameters of the ventilatory mechanics in the scenarios of greater bronchoconstriction. Through the trachea\'s images it was also possible to observe different response times for the action of a smooth muscle constriction agent. It can be concluded that this technique of acquisition of images presents sufficient resolution and frequency of acquisition to monitor the calibers variations of auto transplanted rat´s tracheas and bring a good perspective for its use in the evaluation of the mechanical properties of tracheal grafts produced by Engineering Tissue.

abstract: Severe cases of congenital heart defect (CHD) require surgeries to fix the structural problem, in which artificial grafts are often used. Although outcome of surgeries has improved over the past decades, there remains to be patients who require re-operations due to graft-related complications and the growth of patients which results in a mismatch in size between the patient’s anatomy and the implanted graft. A graft in which cells of the patient could infiltrate, facilitating transformation of the graft to a native-like tissue, and allow the graft to grow with the patient heart would be ideal. Cardiac tissue engineering (CTE) technologies, including extracellular matrix (ECM)-based hydrogels has emerged as a promising approach for the repair of cardiac damage. However, most of the previous studies have mainly focused on treatments for ischemic heart disease and related heart failure in adults, therefore the potential of CTE for CHD treatment is underexplored. In this study, a hybrid hydrogel was developed by combining the ECM derived from cardiac tissue of pediatric CHD patients and gelatin methacrylate (GelMA). In addition, the influence of incorporating gold nanorods (GNRs) within the hybrid hydrogels was studied. The functionalities of the ECM-GelMA-GNR hydrogels as a CTE scaffold were assessed by culturing neonatal rat cardiomyocytes on the hydrogel. After 8 days of cell culture, highly organized sarcomeric alpha-actinin structures and connexin 43 expression were evident in ECM- and GNR-incorporated hydrogels compared to pristine GelMA hydrogel, indicating cell maturation and formation of cardiac tissue. The findings of this study indicate the promising potential of ECM-GelMA-GNR hybrid hydrogels as a CTE approach for CHD treatment. As another approach to improve CHD treatment, this study sought the possibility of performing a proteomic analysis on cardiac ECM of pediatric CHD patient tissue. As the ECM play important roles in regulating cell signaling, there is an increasing interest in studying the ECM proteome and the influences caused by diseases. Proteomics on ECM is challenging due to the insoluble nature of ECM proteins which makes protein extraction and digestion difficult. In this study, as a first step to perform proteomics, optimization on sample preparation procedure was attempted. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2018

Os transtornos que afetam a traqueia podem levar a graves complicações de saúde e reduzir significativamente a qualidade de vida das pessoas. Até o presente não existe uma opção clinicamente viável disponível para pacientes com distúrbios severos dessa via aérea. As técnicas de Engenharia de Tecidos apresentam um cenário promissor na tentativa de produzir um substituto traqueal satisfatório, com características mecânicas e biológicas compatíveis com os tecidos do paciente. Nesse trabalho, apresenta-se um sistema de aquisição de imagens de vídeo que permite medir as variações da área da projeção bidimensional de traqueias autotransplantadas de ratos, in vivo, com o objetivo de apresentar uma ferramenta que possa caracterizar as suas propriedades mecânicas e acelerar a avaliação das técnicas e dos tecidos gerados. Seis ratos, da linhagem Wistar, tiveram exposição de toda a traqueia cérvico-mediastinal, foram ventilados e submetidos a um protocolo experimental com diferentes cenários de broncoconstrição, a fim de se poder relacionar os dados obtidos a partir da ventilação mecânica com as imagens da movimentação das paredes da traqueia antes e depois de um autotransplante e aprofundar a discussão do sistema respiratório nas situações de maior broncoconstrição. Os resultados do pré-transplante, no cenário de maior broncoconstrição, mostraram um aumento médio da área da projeção da traqueia de 0,9 ± 0,24% nas fases inspiratórias das perturbações quasi-senoidais e, nas fases expiratórias, reduções de 5,2 ± 6,1%. No pós-transplante, o aumento médio foi de 0,7 ± 0,26% e a redução foi de 1,7 ± 0,73%, para o mesmo cenário de broncoconstrição. As diferenças das fases inspiratórias e expiratórias sugerem uma maior cautela para interpretar os parâmetros da mecânica ventilatória nos cenários de maior broncoconstrição das vias aéreas. Por intermédio das imagens da traqueia, também foi possível observar os diferentes instantes de tempos de resposta para a ação de um agente constritor da musculatura lisa. Pode-se concluir que essa técnica de aquisição de imagens apresenta resolução e frequência de aquisição suficientes para monitorar as variações do calibre de traqueias autotransplantadas de ratos e trazem uma boa perspectiva para o seu uso na avaliação das propriedades mecânicas de enxertos traqueais produzidos por Engenharia Tecidual. / Tracheal disorders can lead to serious health complications and significantly reduce the people life´s quality. There is no clinically feasible option available for patients with several airway disorders. Tissue Engineering techniques present a promising scenario to produce a satisfactory tracheal replacement, with compatible mechanical and biological characteristics with the patient\'s tissues. This work presents a video image acquisition system that allows measure in vivo two-dimensional projection area variations of auto transplanted rat\'s tracheas with the objective of presenting a tools that can characterize its mechanical properties and accelerate the evaluation of techniques and tissues. Six Wistar rats were exposed to the entire cervical-mediastinal trachea, ventilated and submitted to an experimental protocol in different bronchoconstriction scenarios, before and after an auto transplantation in order to correlate the data obtained from the mechanical ventilation and images of the trachea walls movement before and after the auto transplantation and improve the discussion about respiratory system in greater bronchoconstriction status. The pre-transplant results, in the greater bronchoconstriction scenario, showed an average increase of 0.9 ± 0.24% in the inspiratory phases of the quasi-sinusoidal perturbations and, in the expiratory phases, a reduction of 5.2 ± 6.1%. In the post-transplant, the mean increase was 0.7 ± 0.26% and the reduction was 1.7 ± 0.73%, for the same bronchoconstriction scenario. Differences between inspiratory and expiratory phases suggest caution in interpreting the parameters of the ventilatory mechanics in the scenarios of greater bronchoconstriction. Through the trachea\'s images it was also possible to observe different response times for the action of a smooth muscle constriction agent. It can be concluded that this technique of acquisition of images presents sufficient resolution and frequency of acquisition to monitor the calibers variations of auto transplanted rat´s tracheas and bring a good perspective for its use in the evaluation of the mechanical properties of tracheal grafts produced by Engineering Tissue.

Com o aumento da expectativa de vida e o envelhecimento da população, a medicina regenerativa vem ocupando um importante espaço visando manter a qualidade de vida da população. A engenharia de tecidos, apoiada nos avanços da biotecnologia e da nanotecnologia, vem se configurando como alternativa mais versátil e menos custosa ao reparo e transplante de tecidos e órgãos. Os arcabouços para engenharia tecidual constituídos de nanofibras têm o potencial para mimetizar a arquitetura nanométrica dos tecidos humanos, especialmente devido à grande área superficial e elevada porosidade. Para a fabricação de arcabouços de nanofibras, a técnica mais utilizada é a de eletrofiação, devido à sua alta versatilidade, e os materiais mais estudados são os polímeros biodegradáveis e biocompatíveis, que são os mais desejados para fins biomédicos. A esterilização é uma etapa crítica no processo de fabricação de produto médico implantável e pode ter impacto no desempenho dos arcabouços poliméricos. Desta forma, o objetivo deste estudo foi avaliar o impacto da esterilização por gás ozônio em arcabouços de nanofibras poliméricas eletrofiadas para engenharia de tecidos. A esterilização por ozônio foi eficaz na inativação do indicador biológico G. stearothermophilus, caracterizando eficácia na letalidade microbiana; igualmente, não se detectou crescimento microbiano no teste de esterilidade. Os arcabouços de nanofibras de poli(ácido láctico-co-ácido glicólico) tiveram suas propriedades físico-químicas, mecânicas e biológicas preservadas, mantendo o mesmo desempenho como suporte para o crescimento de fibroblastos NIH3T3 após a esterilização. Já os arcabouços de poli-caprolactona, tiveram suas propriedades alteradas e apresentaram um melhor desempenho na proliferação celular de fibroblastos L929 após a esterilização. Assim, o gás ozônio se mostrou como um método alternativo para a esterilização de nanofibras poliméricas para engenharia tecidual. / Since world population is ageing, regenerative medicine has become a growing area in the medical field in order to maintain the life quality of population. With the advance of biotechnology and nanotechnology, tissue engineering has emerged as a more versatile and less costly alternative to tissue repair and transplantation. Nanofibers have the potential to mimic the human tissue architecture at the nanometer scale, especially due to their large surface area and high porosity. Electrospinning is the most applied technique to fabricate nanofibers scaffolds mainly because of its powerful and high versatility. Many polymers can be used on the fabrication of nanofibers scaffolds; however, the biodegradable and biocompatible polymers are the most desired ones for biomedical purposes. Sterilization is a critical step in the fabrication process and might impact the performance of polymeric scaffolds. Therefore, the aim of this study was to evaluate the impact of sterilization by ozone gas on polymeric electrospun nanofibers scaffolds for tissue engineering. Ozone gas sterilization was efficient in killing the G. stearothermophilus spores, a common biological indicator used for validation of sterilization processes. The sterilization method preserved the physico-chemical, mechanical, and biological properties of poly(lactic-co-glycolic) acid nanofibers, keeping the performance of NIH3T3 proliferation on the scaffolds. On the other hand, the same sterilization method altered some properties of poly-caprolactone electrospun scaffolds, what improved L929 fibroblasts proliferation on the scaffolds after sterilization. Therefore, ozone gas was found to be a benign sterilization method for polymeric electrospun scaffolds for tissue engineering.