Tissue engineered heart valve (TEHV) is believed to be a promising candidate for curative heart valve replacements. Collagen, elastin and chondroitin-4-sulfate (C4S) comprise the extra-cellular matrix (ECM) of native heart valves and therefore are suitable materials for TEHV scaffolds. Freeze-drying technique was able to produce scaffolds with relative densities of 0.3%-2.0% and pore sizes of 33.2µm-201.5µm, without having any major effects on the ultra-structures on the scaffold materials. Subsequent dehydrothermal (DHT) treatment and ultra-violet (UV) irradiation introduced inter- or intra-molecular crosslinks in the scaffolds in forms of ester and amide bonds, as well as the accompanying denaturation of the proteins (i.e. ultra-structure transition from helices to random coils). The collagen-based scaffolds had tensile, compressive and effective bending moduli ranging from 39.8kPa to 1082kPa, from 2.4kPa to 213.9kPa, and from 11.0kPa to 415.8kPa, respectively. The different behaviours of the wall stretching and the wall buckling in the individual pores of the scaffolds contributed to the different tensile, compressive and bending moduli. The mechanical properties could be tailored through controlling the freezing temperature, the relative density and the composition of the scaffolds. A lower freezing temperature might lead to lower mechanical properties because different pore structures were introduced. When the the relative density of the scaffold increased, the values of the moduli increased exponentially, with an exponential dependence factor larger for the compressive modulus than for the tensile modulus. Adding elastin or C4S into the collagen scaffolds lowered the mechanical properties due to the decrease in the collagen content. Layered structures that combined collagen-rich layers with elastin-rich and/or C4S -rich layers allowed the scaffolds to make use of the different mechanical properties of different layers, and hence to show anisotropic bending behaviour depending on the loading directions. The lower effective bending modulus (9.6 to 25.0kPa) in the with curvature (WC) direction than that (18.1kPa to 39.3kPa) in the against curvature (AC) direction mimicked the characteristic behaviour of the native heart valves and would be beneficial for a mechanically desirable TEHV. The DHT treatment and UV irradiation were able to increase the mechanical properties of the scaffolds to up to 2.5 times of the original values, by reinforcing the scaffold materials with more crosslinks. In the hydrated status, the hydrophilic C4S improved the water uptake ability of the scaffold and the hydrophobic elastin reduced it. The hydrated layered scaffolds still exhibited bending anisotropy despite much lower effective bending modulus. Finite element models of the scaffolds produced results that were in agreement with the experiments, and enabled us to perform distributed loading and internal stress analysis on the scaffolds. The collagen-based scaffolds were seeded with cardiosphere-derived cells (CDCs), and they attached to the scaffolds and showed visible cell division, proliferation and migration. The CDCs exhibited preferred proliferation behaviours on the collagen-C4S scaffolds to that on the collagen-elastin scaffolds because of the cell affinity to the C4S, as well as the elastin-induced contractile cell phenotype and scaffold volume shrinkage. This difference seemed to be less evident in the layered scaffolds due to the cell communication between the layers. The crosslinking process also had effects on the cell proliferation in the ways that it induced ultra-structure changes or volume shrinkage in the scaffolds. The layered scaffold-cell constructs designed and produced in this study served as a forwarding step towards a mechanically desirable and biologically active TEHV.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19570号 / 医博第4077号 / 新制||医||1013(附属図書館) / 32606 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 秀一, 教授 開 祐司, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

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A Terapia Fotodinâmica (TFD) é uma modalidade de tratamento de câncer que consiste na interação de três componentes: fotossensibilizador (FS), luz para ativar o FS e o oxigênio presente nos tecidos. Os estudos da fototoxicidade e do potencial de agente terapêuticos utilizados no tratamento do câncer, dentre eles os FSs, são realizados utilizando culturas celulares bidimensionais (2D) ou modelos animais. No entanto, os modelos 2D apresentam limitações, como impossibilitar sinais tão importantes que ocorrem in vivo, dentre eles o contato célula-célula e célula-matriz. Além disso, busca-se reduzir cada vez mais o número de animais em pesquisas científicas. Diante dessas limitações, nos últimos 30 anos tem sido desenvolvidos métodos alternativos in vitro que possam mimetizar melhor as complexas estruturas e funcionalidade dos sistemas in vivo. O objetivo desse estudo foi desenvolver um modelo de cultura de células tridimensional (3D) e um modelo de plataforma de cultura microfluídica para avaliar a viabilidade de células de carcinoma humano (HEp-2) após aplicação da terapia fotodinâmica com hipericina. O modelo 3D foi produzido utilizando-se colágeno tipo I aniônico e a plataforma de cultura microfluídica foi produzida utilizando-se lâmina de plástico, que serviu como base para o adesivo biocompatível utilizado para delimitar o canal celular e o poliéster, servindo como uma espécie de tampa para os dois materiais. A caracterização do biomaterial utilizado no modelo 3D foi realizada pela determinação da porosidade e diâmetro dos poros por meio da Microscopia Eletrônica de Varredura (MEV) e por ensaios de citotoxicidade pelo método de difusão em ágar e pelo método do MTT (ISO 10993-5). Os ensaios de citotoxicidade comprovaram que o biomaterial utilizado é biocompatível e não causa nenhuma citotoxicidade as células. A viabilidade celular da linhagem HEp-2 foi acompanhada no modelo 2D e 3D durante 168 h (sete dias) utilizando o método do MTT e na plataforma microfluídica por 24 h através de microscopia de fluorescência com perfusão contínua de meio de cultura. Em ambos os modelos as células apresentaram-se capazes de se manter aderidas e em multiplicação. Nos ensaios fototóxicos realizados no modelo 3D por meio do método do MTT, observou-se que a viabilidade celular diminui à medida que se aumenta a concentração da hipericina, mantendo-se a dose de luz e o tempo de incubação constantes, sugerindo que as células HEp-2 em cultura 2D apresentaramse mais sensíveis à TFD do que as células em cultura 3D. Os ensaios fototóxicos na plataforma microfluídica mostraram através da análise das imagens de microscopia de fluorescência que o tipo de morte celular preponderante foi a apoptose. Portanto, os resultados apresentados sugerem que é possível a realização de estudos de terapia fotodinâmica no modelo de cultura tridimensional bem como na plataforma microfluídica de cultura de células. / Photodynamic Therapy (PDT) is a cancer treatment modality consisting of the interaction of three components: photosensitizer (PS), light to activate the PS and oxygen present in the tissues. The studies about the toxicity and potential of therapeutic agents used for cancer treatment, among them the PS, are performed using 2D cell cultures or animal models. However, the 2D models have several limitations, such as making it impossible that important signals which occur in vivo, such as cell-cell and cell-matrix contact occur. In addition, it is importatnt to reduce the number of animals in scientific research. Due of the limitations of the twodimensional cell culture models and the need to reduce the use of animals in research, in the last 30 years alternative in vitro methods have been developed which t can better mimic the complex structures and functionality of in vivo systems. Therefore, the objective of this study was to develop a three-dimensional (3D) cell culture model and a microfluidic culture platform model to evaluate the viability of human carcinoma cells (HEp-2) after photodynamic therapy with hypericin. The 3D support was produced using type I collagen and the microfluidic culture platform was produced using a plastic blade which served as the basis for the surgical adhesive, used to delimit the cell canal and the polyester, serving as a sort of cap for both materials. The characterization of the biomaterial used in the 3D model was performed by determination of the porosity and pore diameter by Scanning Electron Microscopy (SEM) and by cytotoxic assays using the agar diffusion method and the MTT method (ISO 10993-5). It was observed that the biomaterial used in the 3D model is biocompatible and does not cause any cytotoxicity to the cells. The cell viability of the HEp-2 cells was monitored in the 2D and 3D models for 168 h (seven days) using the MTT method and in the microfluidic platform for 24 h by fluorescence microscopy with continuous perfusion of culture medium. In both models the cells are able to remain adherent and in multiplication. In the phototoxic assays performed on the microfluidic platform, the analysis of fluorescence microscopy images showed that the preponderant cell death type was apoptosis. Results suggest that is possible to perform photodynamic therapy studies in the three-dimensional culture model as well as in the microfluidic cell culture platform.

Tissue engineered human oral mucosa has the potential to fill tissue deficits caused by facial trauma or malignant lesion surgery. It can also help elucidate the biology of oral mucosa and serve as an alternative to in vivo testing of oral care products. The aim of this thesis was to construct a tissue engineered full-thickness human oral mucosa closely mimicking the native tissue. To this end, the feasibility of the concept was tested by co-culturing fibroblasts and epithelial cells isolated from normal human oral mucosa biopsies in a collagen-glycosaminoglycan-chitosan scaffold, developed in our laboratory to construct a skin equivalent. An oral mucosal equivalent closely mimicking the native one was obtained and characterized by histology, immunohistochemistry and transmission electron microscopy. Using the same model, the influence of mesenchymal cells on oral epithelial development was investigated by culturing epithelial cells on lamina propria, corneal stroma and dermal equivalents. They were found to significantly influence the thickness and the ultrastructure of the epithelium. Finally, in order to improve the adhesiveness of conventional scaffolds, an elastin-like recombinamer (ELR) containing the cell adhesion tripeptide, RGD, was used in the production of novel bilayer scaffolds employing lyophilization and electrospinning. These scaffolds were characterized by mercury porosimetry, scanning electron microscopy and mechanical testing. In vitro tests revealed positive contribution of ELR on the proliferation of both fibroblasts and epithelial cells. It was thus possible to construct a viable oral mucosa equivalent using the principles of tissue engineering.

Cell culture has been a fundamental tool for the study of cell biology, tissue engineering, stem cell technology and biotechnology in general. It becomes more and more important to have a well-defined physiochemical microenvironment during cell culture. Conventional cell cultures employ expensive, manually controlled incubation equipment, making it difficult to maximize a cultures yield. Furthermore, previous studies use qualitative methods to assess cell culture proliferation that are inherently inaccurate and labour intensive, thereby increasing the cost of production. In addition, three dimensional cell culture, in scaffold, has been shown to provide more physiological relevant information as it mimic more accurate conditions that are similar to the physiological conditions of the human body compared with two dimension, which has special interest to regenerative medicine. Therefore, a portable and automated total-analysis-system (μTAS) was proposed with microenvironment control and quantitative analysis techniques to monitor cell proliferation and metabolic activity. The automated portable heating system was validated to be capable to maintain a stable physiochemical microenvironment, with little margin of error, for cellular substrate outside of conventional incubation. A standalone platform system was designed and fabricated with accurate temperature control by employing an optically transparent ITO-film with a large heating area. The transparency of the film is critical for continuous in-situ microscopic observation over long-term cell culture process. Previous studies have attempted to use ITO-film as a heating element, but were unable to distribute the heat evenly onto the microbioreactor platform. This nagging problem in the literature was improved through a novel film design. As a result, the ITO-film based heating system was evaluated and constructed successfully to serve as a heating element for long-term static cell culture with facilitated proliferation rate in gas-permeable PDMS microbioreactor outside of conventional incubation. In addition to maintaining a stable microenvironment, a non-invasive in-situ technology for monitoring cell viability and proliferation rate was constructed and developed based on bioimpedance spectroscopy (BIS). It was primarily focused on making decisions for structure and specification of proposed system-on a chip BIS measurement. The miniaturization of BIS system on microbioreactor platform was achieved by utilizing and integrating switching matrix array, impedance analyzer chip with reliable analogue-front-end circuitry. The realized system was verified with the DLD-1 cells and its monitored data were validated with conventional bioassays. Three dimensional cell cultures with scaffold is a key to the success of tissue engineering. Engineered cornea collagen scaffold may be feasible using re-seeding proper human cells onto a decellularized corneal scaffold. The quality of the scaffold and the interaction of the cells are critical to the key function (i.e transparency, haze and total transmittance) of final products. An integrated corneal collagen scaffold quality assessment system, via optical property inspection unit, was innovatively designed and realized with non-invasive and non-destructive characteristics. The H1299 cells were seeded onto inspected corneal scaffold and BIS system, which were realized in the previous chapter, were used to validate its applicability for 3D cell culture. The cell adhesion as an outcome at different scaffolds with different optical properties has revealed the importance of the microstructure of scaffold on the cell functions. The results showed the developed technologies can be used for the quality control of corneal scaffold and the fabricated μTAS not only enabled environmental control but, with BIS-based in-situ assay, it also facilitate the function (i.e adhesion) and viability monitoring with quantitative and qualitative analysis in 3D-alike cell culture. Additionally, by considering its low decontamination and cost-effective nature with compatibility for high-throughput screening applications, the fabricated and integrated systems has significant applications in tissue engineering.

L’ingénierie de la muqueuse orale humaine (MOH) a pour but le comblement des pertes de substances suite à un traumatisme facial ou à la chirurgie des lésions malignes. Elle a aussi des applications en recherche pour élucider les mécanismes biologiques de la MO et en pharmacotoxicologie comme alternative à l’expérimentation animale. L'objectif de cette thèse était de reconstruire une MOH proche du tissu normal. À cette fin, la faisabilité du concept a d'abord été testée par co-culture de fibroblastes de la lamina propria et de cellules épithéliales de MOH dans le substrat de collagène-chitosan glycosaminoglycane, développé pour la production de peaux reconstruites. La caractérisation de la MOH reconstruite par histologie, immunohistochimie et microscopie électronique à transmission a montré la présence d’une LP équivalente avec un épithélium pluristratifié et non kératinisé très proche du tissu d’origine. Grâce à ce modèle, nous avons ensuite démontré que l’origine des fibroblastes (MO, cornée, peau) influence significativement l’épaisseur et l’ultrastructure de l'épithélium obtenu par culture de cellules épithéliales orales. Enfin, afin d'améliorer les propriétés adhésives du substrat à base collagène, nous avons ajouté au collagène, une élastine-like recombinante (ELR) contenant le tri-peptide d’adhésion cellulaire, RGD, et produit un nouveau substrat bicouche, poreux par lyophilisation et recouvert d’une couche fibreuse par électrofilage. Ces substrats ont été caractérisés par porosimétrie au mercure, microscopie électronique à balayage et essais mécaniques. Nous avons démontré l’effet stimulant de ELR sur la prolifération des fibroblastes et des cellules épithéliales / Tissue engineered human oral mucosa has the potential to fill tissue deficits caused by facial trauma or malignant lesion surgery. It can also help elucidate the biology of oral mucosa and serve as an alternative to in vivo testing of oral care products. The aim of this thesis was to construct a tissue engineered full-thickness human oral mucosa closely mimicking the native tissue. To this end, the feasibility of the concept was tested by co-culturing fibroblasts and epithelial cells isolated from normal human oral mucosa biopsies in a collagen-glycosaminoglycan-chitosan scaffold, developed in our laboratory to construct a skin equivalent. An oral mucosal equivalent closely mimicking the native one was obtained and characterized by histology, immunohistochemistry and transmission electron microscopy. Using the same model, the influence of mesenchymal cells on oral epithelial development was investigated by culturing epithelial cells on lamina propria, corneal stroma and dermal equivalents. They were found to significantly influence the thickness and the ultrastructure of the epithelium. Finally, in order to improve the adhesiveness of conventional scaffolds, an elastin-like recombinamer (ELR) containing the cell adhesion tripeptide, RGD, was used in the production of novel bilayer scaffolds employing lyophilization and electrospinning. These scaffolds were characterized by mercury porosimetry, scanning electron microscopy and mechanical testing. In vitro tests revealed positive contribution of ELR on the proliferation of both fibroblasts and epithelial cells. It was thus possible to construct a viable oral mucosa equivalent using the principles of tissue engineering