京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第19180号 / 人健博第28号 / 新制||人健||3(附属図書館) / 32172 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 高桑 徹也, 教授 市橋 則明, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Collagenous tissues such as ligaments and tendons are viscoelastic materials. They exhibit a slow continuous increase in strain over time, or creep, when subjected to a constant stress and a slow continuous decrease in stress over time, or stress relaxation, when subjected to a constant strain. Moreover, the loading and unloading stress-strain curves are different when the tissues are subjected to cyclic loading, showing hysteresis and softening phenomena. The micro-structural origin of the viscoelasticity of these tissues is still unknown and the subject of debate among experts in biomechanics. Therefore, formulating viscoelastic models by accounting for the mechanical contributions of the structural components of these tissues can help in understanding the genesis of viscoelasticity. A nonlinear viscoelastic modeling framework has been developed to describe the elastic and viscoelastic properties of ligaments and tendons by considering their main structural components, the collagen fibers and proteoglycan-rich matrix. The mathematical models derived within this framework can illustrate the tensile behavior, stress relaxation and creep by as suming that the collagen fibers are elastic and the surrounding proteoglycan-rich matrix is viscoelastic. The collagen fibers are represented by linear elastic springs that are engaged to support load at different values of the tissue's strain according to a Weibull distribution function. The mechanical contribution of the matrix is introduced via a Maxwell-type viscoelastic element arranged in parallel with the collagen fibers. According to the proposed mathematical framework, both the collagen fibers and the proteoglycan-rich matrix are responsible for resisting tensile loads. However, the collagen fibers play a significant role in creep while the proteoglycan-rich matrix has a dominant role in stress relaxation. The model parameters that define the stress relaxation and strain stiffening phenomena are estimated by using published experimental on rabbit medial collateral ligaments and are then used to predict creep. The above modeling framework has been also extended to capture the in uence of preconditioning on the mechanical properties of ligaments and tendons. The stress softening and decrease in hysteresis that are observed during successive loading cycles in preconditioning are assumed to be determined by a decrease in the elastic properties of the collagen fibers and proteoglycan-rich matrix. Preliminary data collected on stress relaxation and preconditioning on rat medial collateral ligaments by collaborators are used to evaluate the model parameters and analyze its predictions. The elastic and viscoelastic properties of single collagen fibers are studied by formulating a nonlinear viscoelastic framework by accounting for their main components: microfibrils, cross-links and proteoglycan-rich matrix. The model illustrates tensile behavior and stress relaxation of a single collagen fiber by assuming that the microfibrils and the cross-links are elastic and the surrounding proteoglycan-rich matrix is viscoelastic. The mechanical contribution of the microfibrils is included via a linear elastic spring while the cross-links are represented by linear elastic springs that progressively fail at different values of the tissue's strain according to an exponential distribution function. The matrix is defined by linear dashpots arranged in parallel with each single spring that represents an individual cross-link. The viscous properties of the matrix associated with the unbroken and broken cross-links are assumed to have different values. In the model formulation, the microfibrils and the cross-links are assumed to determine the elastic response of the fibers while the proteoglycan-rich matrix determines the stress relaxation. Microfibrils, cross-links and the proteoglycan-rich matrix are responsible for resisting the loading force during tensile behavior. Experimental data collected by performing incremental stress relaxation tests by other investigators on reconstituted rat tail tendons are used to estimate the parameters in the model and evaluate its performance. / Ph. D.

Orientadores: Ângela Cristina Malheiros Luzo, Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T14:04:31Z (GMT). No. of bitstreams: 1 AndreoliRisso_MarilisaFerruda_M.pdf: 14754073 bytes, checksum: 6e3b6aa9d09a03a1f2873ad414c83bdf (MD5) Previous issue date: 2012 / Resumo: Lesões cartilaginosas raramente curam-se espontaneamente. As atuais opções terapêuticas cirúrgicas e não são limitadas ao alívio dos sintomas, mando a necessidade de futura substituição total de joelho. Terapia baseada em células tronco adultas representa uma alternativa promissora para os procedimentos existentes. As células-tronco mesenquimais (MSC) apresentam alta plasticidade celular e podem ser obtidas de diversas fontes teciduais, opção que exige grande aporte celular expondo a necessidade de criopreservação, processo vantajoso. Contudo, protocolos convencionais estão diretamente associados a possíveis danos e mortalidade celular, sendo desencadeados por formação de cristais de gelo intracelular, toxicidade do crioprotetor adotado e desidratação celular. Este projeto tem como objetivo investigar a interferência da criopreservação com dimetilsufóxido (Me2SO-) (DMSO) na capacidade de MSCs em diferenciação em linhagens mesodermais e arranjo de fibras de colágeno produzidas na diferenciação condrogência. MSCs foram obtidas de tecido adiposo (ADSC). Em quarta passagem foram caracterizadas por citometria de fluxo e diferenciadas em linhagem mesodermais. Diferenciação comprovada por análise morfológica e expressão gênica por de RT-PCR. Genes escolhidos ADIPOQ, FABP4 e PPARG linhagem adipogênica, AGCAN, SOX9, COL1A1 e COL2A1 linhagem condrogênica e OC, OPN e COL1A1 linhagem osteogênica. Diferenciação condrogênica realizada pela técnica de micromassa. Arquitetura das fibras colágenas observada por microscopia de Geração de Segundo Harmônico (SHG) de MSCs e images analisadas pelos algoritmos Fast Fourier Transform (FFT) e Gray Level Co-occurrence Matrix (GLCM). ADSCs criopreservadas em taxa controlada de congelamente com solução criprotetora de soro fetal bovino e 10% de DMSO, mesmos ensaios realizados após descongelamento. Células descongeladas submetidas aos mesmos protocolos de caracterização. Características morfológicas obtidas por SHG expressão gênica das células frescas e criopreservadas analisadas estatisticamente. Amostras de ADSC, fresca e criopreservadas, foram capazes de se diferenciar em linhagens mesodermais. Perfil de expressão gênica da linhagem adipogênica foi semelhante ao esperado para tal processo de diferenciação, em ambas amostras, criopreservadas e frescas, estas últimas com maior expressão (p=ns). Na diferenciação para linhagem osteogênica também houve o perfil de expressão esperado para os genes estudas, sendo mais expresso no grupo criopreservado (p=ns). Apesar do fato da expressão do gene COL2A1 de linhagem condrogênica ter sido maior no grupo criopreservado, quando o perfil de expressão gênica do grupo fresco foi analisado, este mostrou-se mais consistente com o perfil esperado normal. Análise das imagens de SHG demonstraram que a arquitetura das fibras colágenas foi mais organizada (p=ns) e uniforme (p>0,0001) nas amostras frescas. O grupo criopreservado demonstrou maior entropia (p=ns) e contraste (p=0,0167), demonstrando haver maior tendência direcional das fibras de colágeno nas amostras frescas. Os resultados de expressão gênica sugerem que a criopreservação pode interferir de forma positiva na diferenciação osteogênica e negativamente nas linhagens adipogênica e condrogênica. A arquitetura da rede tridimensional de colágeno foi modulada negativamente pelo processo de criopreservação, dados esses confirmados por análise das imagens obtidas por SHG, o que poderia interferir com as propriedades físico/mecânicas características do tecido colagenoso, importante na manutenção da cartilagem articular baseada em terapia celular. O uso das imagens de SHG tornou-se uma importante ferramenta na melhor avaliação das fibras colágenas / Abstract: Cartilage defects rarely heal spontaneously. Current surgical and non-surgical therapeutic interventions are limited to symptom relief and future total knee replacement continues to be necessary. Adult stem cell based therapy could represent a promising alternative to this procedure. Mesenchymal Stem Cells (MSCs) have great capacity of differentiation and can easily be obtained from various sources. Cryopreservation offers many advantages for practitioners engaged in cell-based therapies. However, conventional slow freezing, despite using cryoprotectant solution, has always been associated with damage and mortality due to intracellular ice formation, cryoprotectant toxicity, and dehydration. The aim of this work is to investigate whether the Dimethyl Sulfoxide (Me2SO-) (DMSO) cryopreservation process interferes with the MSCs capacity of differentiation to mesodermal lineages and/or with collagen fiber network architecture, produced by chondrogenic cells, comparing fresh and thawed MSCs. MSCs were obtained from adipocyte tissue (ADSCs). At the fourth passage cells were characterized as ADSCs by flow cytometry analysis and differentiation to mesodermic lineages was confirmed by morphology (light optical microscopy) and gene expression analysis (RT-PCR). The following genes were chosen ADIPOQ, FABP4 and PPARG for adipogenic lineage, AGCAN, SOX9, COL2A1 and COL1A1 for chondrogenic and OC, OPN and COL1A1 for osteogenic lineage. Chondrogenic differentiation was carried out by micromass technique. Collagen fibers architecture was observed by Second Harmonic Generation (SHG) microscopy. Images were analyzed by Fast Fourier Transform (FFT) and Gray Level Cooccurrence Matrix (GLCM) algorithms. ADSCs were cryopreserved in a controlledrate freezing device with bovine serum fetal and 10% DMSO as cryoprotectant solution. Thawed cells were submitted to the same characterization protocols. SGH morphologic features and gene expression results from thawed and fresh cells were statistically analyzed. Both, thawed and fresh ADSCs were able to differentiate into mesodermal lineages. Gene expression profile of adipogenic lineage was similar to that expected for the differentiation process in both, thawed and fresh cells, with greater expression in fresh ones (p=ns). Osteogenic lineage also demonstrated the expected gene expression profile, being more expressed in the thawed group (p=ns). Despite the fact that COL2A1 gene expression in chondrogenic lineage was greater in the thawed group, when the gene expression profile was analyzed the fresh group was more consistent with the expected normal profile. SHG images results demonstrated that collagen fibers architecture was more organized (p=ns) and uniform (p<0, 0001) in fresh samples. The thawed group showed more entropy (p=ns) and contrast (p=0, 0167) demonstrating that a directional trend in the collagen fibers was only observed in the fresh group. Gene expression results suggested that cryopreservation could interfere in a positive manner with osteogenic differentiation, and negatively on adipogenic and chondrogenic lineages. Collagen network tridimensional architecture was negatively modulated by cryopreservation, confirmed by SHG analysis, which could interfere with the desirable collagen mechanical properties, important for the maintenance of articular cartilage in cell based therapy. SHG analysis has become an important tool for better evaluate collagen fibers / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

A polyconvex continuum-level proteoglycan Cauchy stress function was developed based on the continuum electromechanical Poisson-Boltzmann unit cell model for proteoglycan interactions. The resulting proteoglycan model was combined with a novel collagen fibril model and a ground substance matrix material to create a polyconvex constitutive finite element model of articular cartilage. The true collagen fibril modulus , and the ground substance matrix shear modulus , were varied to obtain the best fit to experimental tension, confined compression, and unconfined compression data for native explants and explants cultured in insulin-like growth factor-1 (IGF-1) and transforming growth factor-β1 (TGF-β1). Results indicate that culture in IGF-1 results in a weakening of the COL fibers compared to native explants, and culture in TGF-β1 results in a strengthening of the COL fibers compared to native explants. These results elucidate the biomechanical changes in collagen fibril modulus, and ground matrix shear modulus following in vitro culture with IGF-1 and TGF-β1. Understanding the constitutive effects of growth factor stimulated culture may have applications in AC repair and tissue engineering.

Orientador: Pedro Eduardo de Felicio / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-11T10:28:36Z (GMT). No. of bitstreams: 1 Bueno_RachelVirginiaCarvalhodeCampos_M.pdf: 21391234 bytes, checksum: 9704c6b584a0d7bc76348477fb39fd91 (MD5) Previous issue date: 2008 / Resumo: Este trabalho visou avaliar o comportamento funcional dos produtos resultantes das interações entre a fibra de colágeno e as fibras musculares em dois cortes do quarto dianteiro bovino, formados pelos músculos Tríceps braquial e Peitoral profundo. A formulação base da salmoura era constituída por uma solução aquosa de cloreto de sódio (NaCl) fixada em 1,0% de concentração. Variou-se a concentração de tripolifosfato de sódio (TPF) ao máximo de 0,4%; o nível de solução injetada entre 10% e 30% e a concentração da fibra de colágeno a ser adicionada em cada um dos níveis injetados entre 0,1% e 0,3% no produto final. Esta concentração respeita as recomendações do fabricante, que estabelece em 1% o limite máximo de fibra de colágeno no produto final porque tem início a percepção sensorial do sabor e aroma característico de colágeno, indesejável ao consumidor. A amostra padrão consistiu do corte de referência injetado nos níveis estipulados com salmoura constituída somente por uma solução de NaCl a 1%. Para avaliar a qualidade funcional dos cortes utilizados foram efetuadas as análises de pH, capacidade de retenção de água, composição centesimal e teor de colágeno total, em etapa anterior à injeção. As análises microbiológicas seguiram o padrão exigido pela ANVISA na RDC nº. 12, de 12 de janeiro de 2001. Os cortes injetados foram embalados a vácuo e permaneceram em repouso por 24 horas a 4 ºC. Após este período analisou-se a capacidade de retenção de água (CRA) e as perdas durante esta estocagem, só então, passaram pelo processo de cocção industrial usado para a fabricação de ¿Cooked Frozen Beef¿ (CFB), permanecendo estocados em câmaras a -20 ºC durante o período de realização das análises. Para verificar o comportamento funcional, as amostras foram avaliadas quanto a sua maciez objetiva (força de cisalhamento), análise do perfil de textura (TPA), perdas na estocagem e na cocção, bem como o rendimento do processo. Os resultados obtidos foram avaliados através da análise de superfície de resposta. O rendimento do processo foi beneficiado pela presença da fibra de colágeno na formulação injetada, porém, para os dois cortes estudados, este aumento foi inversamente proporcional ao nível de injeção aplicado. A adição da fibra de colágeno na formulação injetada atuou favoravelmente ao acréscimo da capacidade de retenção de água, para os dois cortes musculares estudados, independentemente da presença de tripolifosfato de sódio na formulação, desde que a taxa de injeção fosse mantida em valores menores de 20%. Teores maiores que 0,14% da fibra de colágeno na formulação injetada promoveram um aumento acentuado nos valores de força de cisalhamento observados no m. Tríceps braquial. O m. Peitoral profundo apresentou valores relativamente mais altos de força de cisalhamento que os observados para o m. Tríceps braquial. As diferenças de comportamento entre os cortes musculares merecem estudo posterior. A análise do perfil de textura não apresentou respostas confiáveis nas condições deste trabalho. Esta observação pode ser um reflexo do próprio delineamento experimental proposto, mas existe a possibilidade de ser atribuída às inúmeras variáveis envolvidas no processo / Abstract: This project evaluated the functional behavior of the resulting products of the interactions between collagen fibers and muscular fibers in two boneless cut of bovine forequarter: Brisket and Clod, Pectoralis profundi and Triceps brachii muscles, respectively. The base of the enhance formulation was 1.0 % aqueous solution of sodium chloride (NaCl). The TPF concentration was varied to a maximum of 0.4%, the level of injected solution was varied between 10% and 30%, and the concentration of collagen fibers to be added in each of the injected levels was varied between 0.1%, and 0.3% in the final product. This variation is in accordance with the manufacturer¿s recommendation of a maximum of 1% collagen fiber in the final product. This limit is set because the characteristic flavor of collagen is undesirable to the consumer. The main sample was the reference cut injected at the specified levels with the standard brine solution made only by 1% NaCl solution. The following properties of the cut were evaluated before injection: pH, water hold capacity, and centesimal composition of the total collagen. The microbiological analyses followed the requirements of ANVISA in RDC nº. 12, from January 12th, 2001. The injected cuts were wrapped in vacuum and were stored for 24 hours at 4 ºC. After this period the water hold capacity was analyzed (WHC), and the losses from storage passed through the industrial cooking process used for the production of ¿Cooked Frozen Beef¿ (CFB). Throughout the analysis period the cuts remained stored in chambers at -20 ºC. To analyse the functional behavior, the following properties were evaluated: objective tenderness by shear force and texture profile analysis (TPA), losses in storage and cooking, and the process effectiveness. The results obtained were evaluated through surface response analysis. The process benefited from the collagen fiber in the injected formulation. In the two cuts studied this benefit was inversely proportional to the level of the applied injections. The fiber addition in the injection formulation positively impacted the water hold capacity for both studied cuts, independent of the presence of TPF in the formulation. This benefit occurred as long as the injection level was maintained below 20%. Levels higher than 0.14% of the collagen fiber in the injected formulation promoted a substantial increase in the shear force values observed in the Triceps brachii muscle samples. The .Pectoralis profundi muscle displayed shear force values relatively higher than those observed for Triceps brachii muscle. Further researches should investigate the differences between the muscular cuts behavior. A texture profile analysis did not provide reliable data for the conditions in this study. This outcome may be a result of an error in the experimental design protocol, or it could be attributed to unexpected variables involved in the process / Mestrado / Mestre em Tecnologia de Alimentos

Orientadores: Eliane Maria Ingrid Amstalden, Edson Aparecido Liberti / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T00:17:07Z (GMT). No. of bitstreams: 1 Benigno_MariaIvoneMendes_D.pdf: 2199405 bytes, checksum: 7460327535443e71e135d559be319402 (MD5) Previous issue date: 2014 / Resumo: Introdução: A atividade mastigatória é uma sincronia entre os músculos da mastigação e articulação temporomandibular (ATM). A perda de dentes é um importante fator que contribui para as disfunções do Sistema Estomatognático e consequentes danos aos músculos mastigadores. Considerando os poucos trabalhos sobre o assunto, a necessidade de maior compreensão e detalhamento quanto às alterações das fibras desta musculatura, especialmente na disfunção pela perda dentária, este estudo teve como objetivos: investigar as alterações morfológicas e ultraestruturais do músculo Pterigoideo Medial (PTM) e Masseter, pós exodontia em modelo experimental. Material e Métodos: Foram utilizados 24 ratos wistar para microscopia de luz (ML) e 12 para microscopia eletrônica de transmissão (MET), divididos em três grupos experimentais: GI -15, GII-30 e GIII-60 dias, pós exodontia de molares inferiores esquerdos. Contendo 5 animais experimentais e três controles por grupo para ML e 3 ratos para MET, com 1 controle por grupo. Sob microscopia de luz foram realizados estudos morfométricos e sob luz polarizada, dos músculos PTM e Masseter. A análise morfométrica baseou-se na medida da área das fibras, em cortes transversais, corados pelo H&E (40x.objetiva), com programa digital (software AXION¿vision). Realizadas 240 medidas por animal/ total de 1200 por grupo experimental e 200 medidas por animal/ total de 600 por grupo controle. Análise qualitativa das fibras colágenas foi obtida sob luz polarizada. Também foram observadas, qualitativamente, alterações ultraestruturais destes músculos, ipsilateral às exodontias. Teste ANOVA foi aplicado para a análise dos dados. Resultados: A morfometria da área das fibras do músculo PTM, mostrou redução significante, nos animais submetidos à exodontia, tanto ipsi quanto contralateral. Não foram detectadas diferenças quanto aos quesitos interação entre lados direito e esquerdo e grupos (GI, II e III), nem quando se comparou os lados entre si. Diferenças foram notadas quando se comparou o grupo experimental, nos distintos períodos evolutivos, detectando-se aumento progressivo das áreas das fibras musculares, sendo a média maior no Grupo GIII. Apesar do crescimento progressivo da área das fibras, elas não se tornam hipertróficas nesse estágio avaliatório, uma vez que, a média dos valores obtidos é semelhante à do grupo controle. As fibras do músculo PTM parecem adaptar-se às mudanças. Nenhuma diferença foi detectada quanto à análise morfométrica do músculo Masseter. Ultraestruturalmente, observou-se assimetria e desorganização da linha Z e banda I, apenas no grupo experimental GII, do músculo PTM. A análise das fibras colágenas mostrou que os fascículos musculares são revestidos por uma delicada rede de fibras colágenas do tipo I e do tipo III, com predomínio deste último (fibras reticulares), nos Masseteres, nos diferentes períodos evolutivos. Conclusão: A disfunção temporomandibular, promovida pela exodontia unilateral de molares inferiores em ratos, pode levar a alterações morfométricas ipsi e contralaterais, com redução de áreas de fibras, particularmente no PTM. Entretanto as fibras musculares parecem se adaptar às novas condições, ao longo do experimento. A linha Z e banda I são as mais sensíveis a essa disfunção, no músculo PTM, contudo efêmera, uma vez que foi observada apenas no grupo GII. O músculo PTM mostrou-se mais vulnerável, provavelmente pelas suas características funcionais próprias e maior participação na dinâmica dos movimentos mastigatórios, comparadas às do Masseter. As fibras colágenas do tipo I e do tipo III são os constituintes principais das estruturas fibro conjuntivas desses músculos, com predomínio do tipo III no Masseter e parecem não ser afetadas nesse procedimento / Abstract: The loss of dental elements is an important factor in stomatognathic system dysfunctions and consequential damage to the masticatory muscles. The aim of this study was to analyze the morphometric and ultrastructural changes of the pterygoid medial(PTM) and masseter muscle, under occlusal defects, induced by unilateral left molar extraction, of Wistar rats. Thirty-six male rats were used: 24 for light microscopy (LM) and 12 for transmission electron microscopy analysis (TEM), divided into three experimental groups (GI-15; GII-30 and GIII-60 days), containing 5 animals each for LM with 3 control and 3 for TEM with one animal control for each period. Morphometric studies were made measuring the area of PTM and Masseter muscle fibers ipsi and contralateral to dental extraction, using a digital program. A qualitative analysis was performed to evaluate the ultrastructural findings and of the PTM and Masseter muscle. The results were compared using ANOVA test. There was a reduction of area of PTM of animals undergoing tooth extraction, both ipsi as contralateral. Both sides were similar when compared with each other, as assessed in the various evolutive periods. Differences were observed in the fiber area, especially in the first group and these showed progressive increase, reaching their highest average in GIII. No difference was detected regarding the morphometric analysis of the masseter muscle. For ultrastructure observed asymmetry and disorganization of Z line and I band, only the experimental group GII, muscle PTM. The analysis of the collagen fibers showed that the muscle fascicles are lined by a delicate network of collagen type I and type III, with a predominance of the latter (reticular fibers), in the masseter, in different evolutionary periods. Temporomandibular joint dysfunction, promoted by unilateral molar extraction in wistar rats, can lead to morphometric changes ipsi and contralateral with reduction of areas, particularly in the PTM. However seem to adapt to new conditions throughout the experiment. The band Z and the ith row of the muscle cytoskeleton are the most sensitive to this, dysfunction in muscle PTM, however ephemeral, since it was observed only in the Group (GII) with 30 days of the experiment. The muscle PTM proved to be more vulnerable in this experimental model, probably for its own functional features and greater participation in the dynamics of the masticatory movements, compared to the Masseter. The collagen fibers of type I and type III are the major constituents of the connective fibrous tissue structures of these muscles, with a predominance of type III in the Masseter and doesn't seem to be affected, to this procedure / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

<p>   </p> <p>Subcutaneous injection is a biotherapeutic drug delivery method that is currently growing due to low cost, better patient compliance, minimally invasive, and the convenience that it can be done at home. Common injection sites for subcutaneous injection include the upper outer arms, abdomen, buttocks, and upper outer thigh. Heterogeneity of the tissue exists between and within each of these locations. The subcutaneous tissue space is made up of adipose tissue, proteins, collagen, and blood vessels and each of these components has an impact on the mass transport of the injected biotherapeutics and how they are absorbed into the vascular system and then distributed to the body. The current methods used to model the subcutaneous tissue space are either very expensive and not feasible for multiple repetitions, cannot incorporate fibrillar proteins or cellular components, or model a more homogeneous tissue space. These limitations do not allow for these models to accurately represent the subcutaneous tissue space. The engineering objective for this project was to develop a platform with tunable matrix architecture and biochemical composition for evaluating mass transport. This project utilizes collagen and the primary matrix due to the large abundance of collagen in the body.  We explored the effects that a change in polymerization temperature of the collagen and collagen concentration had on the fiber architecture and pore diameter. The results showed that higher polymerization temperatures of the collagen gels resulted in smaller fiber and pore diameters and an increase in concentration resulted in an increase in fiber volume fraction and a decrease in pore diameter. Fibronectin (FN) and hyaluronic acid (HA) were added to the collagen gels to analyze the impact on the structure of collagen gels with a change in polymerization temperature and collagen concentration. The addition of FN did not strongly alter the collagen fiber architecture between polymerization temperatures and collagen concentrations. Through staining and imaging, we saw an aggregation of FN around the collagen fibrils due to their opposing charges causing them to bind. The addition of HA had moderate impact on collagen fiber architecture across all polymerization temperatures and between collagen concentrations. The collagen + FN gels were used for the mass transport study. The results showed that there was little to no difference between the recovery rates of macromolecules of different charges and size between the collagen and collagen + FN gels, indicating that the transport of molecules through both of the collagen gels was impacted by a steric effect rather than an effect in charge.</p> <p>  </p>

Optics are a powerful probe of chemical structure that can often be linked to theoretical predictions, providing robustness as a measurement tool. Not only do optical interactions like second harmonic generation (SHG), single and two-photon excited fluorescence (TPEF), and infrared absorption provide chemical specificity at the molecular and macromolecular scale, but the ability to image enables mapping heterogeneous behavior across complex systems such as biological tissue. This thesis will discuss nonlinear and linear optics, leveraging theoretical predictions to provide frameworks for interpreting analytical measurement. In turn, the causal mechanistic understanding provided by these frameworks will enable structurally specific quantitative tools with a special emphasis on application in biological imaging. The thesis will begin with an introduction to 2nd order nonlinear optics and the polarization analysis thereof, covering both the Jones framework for polarization analysis and the design of experiment. Novel experimental architectures aimed at reducing 1/f noise in polarization analysis will be discussed, leveraging both rapid modulation in time through electro-optic modulators (Chapter 2), as well as fixed-optic spatial modulation approaches (Chapter 3). In addition, challenges in polarization-dependent imaging within turbid systems will be addressed with the discussion of a theoretical framework to model SHG occurring from unpolarized light (Chapter 4). The application of this framework to thick tissue imaging for analysis of collagen local structure can provide a method for characterizing changes in tissue morphology associated with some common cancers (Chapter 5). In addition to discussion of nonlinear optical phenomena, a novel mechanism for electric dipole allowed fluorescence-detected circular dichroism will be introduced (Chapter 6). Tackling challenges associated with label-free chemically specific imaging, the construction of a novel infrared hyperspectral microscope for chemical classification in complex mixtures will be presented (Chapter 7). The thesis will conclude with a discussion of the inherent disadvantages in taking the traditional paradigm of modeling and measuring chemistry separately and provide the multi-agent consensus equilibrium (MACE) framework as an alternative to the classic meet-in-the-middle approach (Chapter 8). Spanning topics from pure theoretical descriptions of light-matter interaction to full experimental work, this thesis aims to unify these two fronts. <br>