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.

Articular cartilage damage associated with joint trauma seldom heals and often leads to osteoarthritis (OA). Current treatment often fails to regenerated functional cartilage close to native tissue. We previously identified a migratory chondrogenic progenitor cell (CPC) population that responded chemotactically to cell death and rapidly repopulated the injured cartilage matrix, which suggested their potential for cartilage repair. To test that potential we filled experimental full thickness chondral defects with an acellular hydrogel containing SDF-1α. We expect that SDF-1α can increase the recruitment of CPCs, and then promote the formation of a functional cartilage matrix with chondrogenic factors. Full-thickness bovine chondral defects were filled with hydrogel comprised of fibrin and hyaluronic acid and containing SDF-1α. Cell migration was monitored, followed by chondrogenic induction. Regenerated tissue was evaluated by histology, immunohistochemistry, and scanning electron microscopy. Push-out tests were performed to assess the strength of integration between regenerated tissue and host cartilage. Significant numbers of progenitor cells were recruited by SDF-1α within 12 days. By 5 weeks chondrogenesis, repair tissue cell morphology, proteoglycan density and surface ultrastructure were similar to native cartilage. SDF-1α treated defects had significantly greater interfacial strength than untreated controls. However, regenerated neocartilage had relatively inferior mechanical properties compared with native cartilage. In addition to that, we developed a 3D bioprinting platform, which can directly print chondrocytes as well as CPCs to fabricated articular cartilage tissue in vitro. We successfully implanted the printed tissue into an osteochondral defect, and observed tissue repair after implantation. The regerated tissue has biochemical and mechanical properties within the physiological range of native articular cartilage. This study showed that, when CPC chemotaxis and chondrogenesis are stimulated sequentially, in situ full thickness cartilage regeneration and bonding of repair tissue to surrounding cartilage could occur without the need for cell transplantation from exogenous sources. This study also demonstrated the potential of using 3D bioprinting to engineer articular cartilage implants for repairing cartilage defect.

Surface functionalization of living cells for cell therapeutics has gained substantial momentum in the last two decades. From encapsulating islets of Langerhans, to cell laden gels for tissue scaffolds, to individual cell encapsulation in thin hydrogels, to surface adhesives and inert surface camouflage, modification of living cell surfaces has a wide array of important applications. Here we use hydrogel encapsulation of individual cells as a mode of protection from mechanical forces for high throughput cell printing, and chemical stimuli for the isolation of rare cells in blood. In the first study, we review methods of surface functionalization and establish a metric of potential target biomarkers for circulating tumor cell (CTC) isolation. For extended applications in cancer detection through a fluid biopsy, common surface antigen densities were quantitatively assessed in relation to peripheral blood mononuclear cells (PBMCs) for potential targets of cell specific encapsulation. We then look to commercialization of our process after considering biopsy volumes and cell therapy dose sizes. Undesired batch-to-batch variation in our in-house synthesized photo-initiator could be eliminated by the use of fluorescein, a commercial fluorochrome of similar initiating power to our current eosin initiating system. Fluorescence and hydrogel generation were compared indicating a fluorescein conjugate has comparable power to that of our in-house conjugated eosin. Parameters involving the number of cells and fluid volumes processed were then analyzed systematically. Key parameters were studied to determine optimal equipment and protocol for clinically relevant batch sizes. The final study looks at the mechanical protection provided by thin hydrogel encapsulation. With growing interests in 3D bioprinting and goals of viable whole organ printing for transplant, high resolution and high throughput printing is a growing need. 3D bioprinting presents intense mechanical stimuli in the process that cells must endure. Here we analyze how hydrogel encapsulation reinforces the cellular membrane allowing cells to withstand the damaging forces associated with bioprinting.

Cells regularly take on various types of force in the body. They have structures that are able to mediate, transfer and respond to the forces. A mutation in force regulating proteins such as lamin in the nucleus or the KASH domain which connects the nucleus to the cytoskeleton of the cell can cause catastrophic events to occur. The aims of this study were to better understand the response of the nucleus when structural proteins are mutated or are not present while under force. Progeria, a rare disease where an additional farnesyl group is attached to lamin was used in this study. Different types of forces were used to represent similar conditions in the body. Confinement of endothelial cell width showed an increase of surface defects. When restricting proteins such as actin was removed the nucleus appeared to rupture. This was shown to occur at a higher rate in the progeria groups. Endothelial cells under shear force showed high amount of nuclear rupture in progeria expressing group. prolonged exposure showed more rupture which eventually cased cell death and cells to come off the surface. Progeria expressing smooth muscle cells under cyclic stretch also showed similar results as endothelial cells. The amount and rate of deformation of the nucleus when the cytoskeleton is connected and not was looked at. When the connected the rate of deformation was higher. The high rate of nuclear defects and rupture while under force in progeria expressing cells shows that the nuclei have different structural properties and are weaker. It’s also been shown that the LINC complex contributes to nuclear deformation when stretching.

Biomaterials are becoming the most commonly used therapeutic method for treatment of lost or damaged tissue in the body. Metallic materials are chosen for high strength orthopaedic and dental applications. Titanium (Ti) implants are highly successful in young, healthy patients with the ability to fully integrate to surrounding tissue. However the main population requiring these corrective treatments will not be healthy or young, therefore further research into material modifications have been started to improve outcomes in compromised patients. The body’s immune system will generate a response to any implanted material, and control the final outcome. Among the first and most influential, cells to interact with the implant will be macrophages. Throughout this study we have 1) established the ability of macrophages to recognize and differentially activate in response to material surface properties, 2) investigated the role of integrin binding in macrophage activation to material properties, and 3) confirmed the importance of macrophage activation in vivo following Ti implant placement. The generation of a hydrophilic implant surface promoted the greatest anti-inflammatory and pro-regenerative macrophage activation. Surface wettability will control protein adsorption which can activated different integrin binding on macrophages and may be responsible for changes in activation. When integrin β3 subunit binding was prevented hydrophilic surfaces no longer promoted an anti-inflammatory macrophage activation. Additionally, when macrophage levels were reduced using two separate ablation models, MaFIA mice and clodronate liposomes, hydrophilic surfaces no longer promoted anti-inflammatory T-cell populations and cytokine profiles. There were also fewer stem cells adhered to implant surfaces at 1, 3, and 7 days when macrophage populations were compromised.

Decellularized extracellular matrices have been a growing area of interest in the biomedical engineering fields of tissue engineering and regenerative medicine.As these materials move toward clinical applications, the immune response to these materials will be a driving force toward their success in clinical approaches. Fully digested decellularized matrix constructs derived from porcine liver, muscle and lung were created to test the adaptive immune response. Hydrogel characterization ensured that the materials had relatively similar stiffness levels to reduce variability, and in vitro studies were conducted. Each individual construct as well as a gelatin control were plated with a co-culture of macrophages and T-cells to measure T-cell proliferation. In addition standard markers of inflammation through qPCR were measured in the macrophage group. Constructs were then placed into animals for 3 and 7 days in addition to a second group that received constructs for 21 days before secondary constructs were placed. These groups were then sacrificed following 3, 7 and 14 days to measure the residual and memory-like response of the constructs. Our results showed that t-cell proliferation was increased with decellularized constructs, particularly in tissue with higher DNA content. In vivo, animals with secondary treatments showed extended inflammatory response, driven by Th1 and Th17 polarization suggesting a memory-like response due to recognition of peptides in the constructs from secondary placements.

In cases of compromised bone remodeling like osteoporosis, insufficient osseointegration occurs and results in implant failure. Implant retention relies on proper secondary fixation, which is developed during bone remodeling. This process is disrupted in metastatic bone diseases like osteoporosis. Osteoporosis is characterized low bone mass and bone strength resulting from either accelerated osteoclast-mediated bone resorption or impaired osteoblast-mediated bone formation. These two processes are not independent phenomena. In fact, osteoporosis can be viewed as a breakdown of the cellular communication connecting bone resorption to bone formation. Because bone remodeling occurs at temporally generated specific anatomical sites and at different times, local regulators that control cross-talk among the cells of the BRU are important. Previous studies show Ti implant surface characteristics like roughness, hydrophilicity, and chemistry influence the osteoblastic differentiation of human MSCs and maturation of OBs. Furthermore, microstructured Ti surfaces modulate the production of factors shown to be important in the reciprocal communication necessary for the maintenance of healthy bone remodeling. Semaphorin signaling proteins are known to couple the communication of osteoblasts to osteoclasts and are capable of stimulating bone formation or bone resorption depending on certain cues. Implant surface properties can be optimized to exploit these effects to favor rapid osseointegration in patients with osteoporosis.