Coronary artery stent placement has been a significant advance in the percutaneous treatment of atherosclerotic disease, and tissue engineered vascular grafts may provide a viable alternative to autologous segments for small diameter vessels. However, in-stent restenosis remains an important limitation, and tissue engineered grafts have poor patency and high risk of thrombus formation due to their inability to maintain a confluent, adherent, and quiescent endothelium. While animal models provide insight into the pathophysiology of these situations, elucidation of the relative importance of stent or graft components, hemodynamic factors, and molecular factors is difficult. Very little research has focused on bridging gaps in knowledge concerning blood/biomaterial interactions, blood/endothelial cell interactions, and endothelial cell/smooth muscle cell cross-talk. The work presented within this thesis will do just that. The objective of this thesis research was to elucidate the influence of biomaterial-induced activation of leukocytes on endothelial cell or smooth muscle cell phenotype, as well as endothelial cell/smooth muscle cell cross-talk in co-culture systems. Towards this goal, two complimentary in vitro endothelial cell/smooth muscle cell co-culture models with divergent smooth muscle cell phenotype were developed and characterized. Using these systems, it was found that the presence of more secretory smooth muscle cells (as would be seen in wound healing or disease) in general enhanced endothelial cell activation in response to biomaterial-pretreated monocytes, while the presence of less secretory smooth muscle cells (to model more quiescent smooth muscle cells found in uninjured healthy vessels) suppressed endothelial cell activation in response to biomaterial-pretreated monocytes (and neutrophils to a small degree). Additionally, biomaterial-pretreated monocytes and neutrophils amplified a smooth muscle cell phenotypic shift away from a more quiescent state. It is likely that the compounding effect of secretory smooth muscle cells and biomaterial-activated leukocytes are responsible for altered vascular wound healing upon implantation of stents or vascular grafts. Understanding the specific signals causing these effects, or signals delivered by contractile smooth muscle cells that limit these effects help to provide design criteria for development of devices or grafts capable of long term patency.

A study on application of combinatorial methods (CM) and high-throughput methods (HTM) to biomaterials design, characterization, and screening are reported in this thesis - focusing on screening the effects of biomaterial surface features on adherent bone cell cultures. Polymeric biomaterials were prepared on two-dimensional combinatorial libraries that systematically varied the size and shape of chemically-distinct microstructural patterns - generated from blends of biodegradable polyurethanes and polyesters. Characterization and screening were performed with high-throughput optical and fluorescence microscopy. A unique advance of this work is the application of data mining techniques to identify the controlling structural features that affect cell behavior from among the myriad variety of metrics from the microscope images. The results from this study demonstrated the potentials of CM/HTS to be applied to exploratory studies involving complex systems in life sciences. This study accomplishes the goal to demonstrate the efficient screening and exploration of vast and complex dataset, extracting important and meaningful information to narrow down the future path of study in this field. Further study aimed to tuning cellular responses via signals from surface cues will be necessary to examine the causal relationships beyond the observed correlations shown in this exploratory study. It is recommended for further studies to narrow down the range for surface patterning around each of the three 'activation' ranges found in this study: apoptotic, viable, and one unknown state to be studied further. Different cellular-function staining methods will be necessary to be used in cellular imaging techniques in order to explore this unknown state further.

Durch verschiedene Modifizierungen an der Blutkontaktseite von Hohlfasermembranen sollte eine Verbesserung der Bio- und Blutkompatibilität erreicht werden. Zur Modifizierung wurden verschiedene biologisch wirksame Moleküle verwendet. Weiterhin wurden zwei Modifizerungsstrategien verfolgt. Zum einen eine Modifizierung, bei welcher das Material der Hohlfasermembran vor deren Herstellung verändert wird. Und zum anderen eine selektive Modifizierung der Blutkontaktseite nach der Herstellung der Hohlfasermembran.

Thesis (Ph. D.)--Mississippi State University. Department of Agricultural and Biological Engineering. / Title from title screen. Includes bibliographical references.

In this Thesis, we have developed a microtechnology-based method for culturing and visualizing high numbers of individual cells and cell-cell interactions over extended periods of time. The foundation of the device is a silicon-glass multiwell microplate (also referred as microchip) directly compatible with fluorescence microscopy. The initial microchip design involved thousands of square wells of sizes up to 80 µm, for screening large numbers of cell-cell interactions at the single cell level. Biocompatibility and confinement tests proved the feasibility of the idea, and further investigation showed the conservation of immune cellular processes within the wells. Although the system is very reliable for screening, limitations related to synchronization of the interaction events, and the inability to maintain conjugations for long time periods, led to the development of a novel ultrasonic manipulation multiwell microdevice. The main components of the ultrasonic device is a 100-well silicon-glass microchip and an ultrasonic transducer. The transducer is used for ultrasonic actuation on the chip with a frequency causing half-wave resonances in each of the wells (2.0-2.5 MHz for wells with sizes 300-350 µm). Therefore, cells in suspension are directed by acoustic radiation forces towards a pressure node formed in the center of each well. This method allows simultaneous aggregation of cells in all wells and sustains cells confined within a small area for long time periods (even up to several days). The biological target of investigation in this Thesis is the natural killer (NK) cells and their functional properties. NK cells belong to the lymphatic group and they are important factors for host defense and immune regulation. They are characterized by the ability to interact with virus infected cells and cancer cells upon contact, and under suitable conditions they can induce target cell death. We have utilized the ultrasonic microdevice to induce NK-target cell interactions at the single cell level. Our results confirm a heterogeneity within IL-2 activated NK cell populations, with some cells being inactive, while others are capable to kill quickly and in a consecutive manner. Furthermore, we have integrated the ultrasonic microdevice in a temperature regulation system that allows to actuate with high-voltage ultrasound, but still sustain the cell physiological temperature. Using this system we have been able to induce formation of up to 100 solid tumors (HepG2 cells) in parallel without using surface modification or hydrogels. Finally, we used the tumors as targets for investigating NK cells ability to infiltrate and kill solid tumors.  To summarize, a method is presented for investigating individual NK cell behavior against target cells and solid tumors. Although we have utilized our technique to investigate NK cells, there is no limitation of the target of investigation. In the future, the device could be used for any type of cells where interactions at the single cell level can reveal critical information, but also to form solid tumors of primary cancer cells for toxicology studies. / <p>QC 20150113</p>

Ziel der vorliegenden Arbeit war die biologische Charakterisierung neuartiger nanostrukturierter und für die Knochenregeneration geeigneter Calciumphosphatzemente (CPC). Hierzu wurde ein aus α-Tricalciumphosphat, Calciumhydrogenphosphat, gefälltem Hydroxylapatit und Calciumcarbonat bestehender CPC verwendet, der mit den Biomolekülen Cocarboxylase, Glucuronsäure, Weinsäure, Glucose-1-phosphat, Arginin, Lysin und Asparaginsäure-Natriumsalz modifiziert wurde. Ermittelt wurde dabei der Einfluss der Modifikationen auf die Proteinadsorption und die Biokompatibilität. In Vorversuchen wurden die Zementmodifikationen hinsichtlich ihrer Bindungskapazität für humane Serumproteine und für das knochenspezifische Protein Osteocalcin (OC) sowie hinsichtlich ihrer Eignung für die Adhäsion, Proliferation und osteogene Differenzierung von humanen fötalen Osteoblasten (hFOB 1.19) und humanen mesenchymalen Stammzellen (hMSC) untersucht. Dabei erwiesen sich die Modifikationen mit Cocarboxylase, Arginin und Asparaginsäure-Natriumsalz als besonders günstig. Mit diesen „Favoriten“ erfolgte eine detailliertere Analyse der Adsorption humaner und boviner Serumproteine sowie der knochen-spezifischen Proteine Osteocalcin, BMP-2 und VEGF. Dabei führte sowohl der Zusatz von Cocarboxylase, als auch der von Arginin und Asparaginsäure-Natriumsalz zu einer erhöhten Adsorption von Serumproteinen. Die Bindungsaffinität des Basiszements gegenüber Osteocalcin, BMP-2 und VEGF konnte durch Funktionalisierung mit Arginin gesteigert werden. Während die Modifizierung mit Cocarboxylase nur die VEGF-Adsorption förderte, bewirkte der Zusatz von Asparaginsäure-Natriumsalz eine Erhöhung der Osteocalcin- und BMP-2-Adsorption. Bedingt durch die größere spezifische Oberfläche der noch nicht abgebundenen Zemente, war die Menge adsorbierter Proteine auf frisch hergestellten Zementproben im Vergleich zu abgebundenen und ausgehärteten Zementen signifikant höher. Die Eignung der ausgewählten Zementvarianten als Knochenersatzmaterialien wurde mithilfe humaner mesenchymaler Stammzellen zweier verschiedener Spender getestet. Bei Verwendung abgebundener und ausgehärteter Zemente waren die hMSC in der Lage, auf allen Modifikationen zu adhärieren, zu proliferieren und in die osteogene Richtung zu differenzieren. Eine vorherige Inkubation der Zementproben mit humanem Serum förderte dabei vor allem die Zelladhäsion. Weiterhin konnte gezeigt werden, dass hMSC im Gegensatz zu anderen Studien auch auf frisch hergestellten Zementproben adhärieren, proliferieren und differenzieren können. Die Modifizierung des Basiszements mit Cocarboxylase führte hierbei zu einer gegenüber den anderen Modifikationen signifikant erhöhten Zelladhäsion und -vitalität. Neben den verschieden modifizierten Pulver/Flüssigkeitszementen wurden im Rahmen dieser Arbeit neuartige ready-to-use Zementpasten untersucht. Diese zeigten allerdings im Vergleich zu den herkömmlichen Zementen eine geringere Proteinbindungsaffinität. HMSC, die auf den Pastenzementen kultiviert wurden, war es wiederum möglich zu adhärieren, zu proliferieren und den osteoblastenspezifischen Marker Alkalische Phosphatase zu exprimieren. Hinsichtlich ihrer Biokompatibilität sind sie damit vergleichbar zu den herkömmlichen Pulver/Flüssigkeitszementen.

Recombinant synthesis of elastin-mimetic proteins has been employed for several decades, however, long-term biocompatibility and biostability of such proteins was not fully defined. We present virtually crosslinked elastin-mimetic proteins which exhibit exceptional biocompatibility and long-term biostability over a period of at least seven months. This report is the first evidence of a non-chemically or ionically crosslinked system that exhibits long-term in vivo stability. Although, physically crosslinked protein-based materials possess a number of advantages over their chemically crosslinked counterparts, physical crosslinks and the related domains so formed may be deformed or damaged at applied stresses lower than those required to disrupt covalent crosslinks. In this regard, we have synthesized a new class of recombinant elastin-mimetic triblock copolymer capable of both physical and chemical crosslinking. We have demonstrated that chemical crosslinking provides an independent mechanism for control of protein mechanical responses. Specifically, elastic modulus was enhanced and creep strain reduced through the addition of chemical crosslinking sites. A number of reports have described the design of synthetic genes, which encode elastin-like proteins for bacterial expression in Escherichia coli. Although advantages with this expression system exist, significant limitations including the lack of eukaryotic post-translational systems, the tendency to sequester mammalian proteins into inclusion bodies, difficult purification protocols, and endotoxin contamination have been noted. We demonstrate the expression of a recombinant elastin-mimetic protein from P. pastoris. A novel synthetic strategy, monomer library concatamerization, was utilized in designing non-repetitive elastin genes for highly repetitive protein sequences. It is likely that this strategy will be useful for creating large, repetitive genes for a variety of expression systems in order to more closely approach the genetic diversity inherent to native DNA sequences. All told, elastin-based protein polymers are a promising class of material characterized by high degree of biocompatibility, excellent biostability, and a tunable range of mechanical properties from plastic to elastic. A variety of options facilitate the processing of these biopolymers into chemically crosslinked or non-crosslinked gels, films, or nanofibers for any of a number of implant applications including structural components of artificial organs and engineered living tissues, carriers for controlled drug release, or biocompatible surface coatings.

Made available in DSpace on 2014-12-17T14:07:09Z (GMT). No. of bitstreams: 1 HaroldoRAM_TESE.pdf: 4102900 bytes, checksum: e74f8a9122bb5cd84cbeadae3a476ce1 (MD5) Previous issue date: 2012-02-03 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The titanium and titanium alloys are widely used as biomaterial in biomedical device and so research have been developed aiming to improve and/or better to understand interaction biomaterial/biological environment. The process for manufacturing of this titanium implants usually involves a series of thermal and mechanical processes which have consequence on the final product. The heat treatments are usually used to obtain different properties for each application. In order to understand the influence of these treatments on the biological response of the surface, it was done, in this work, different heat treatments in titanium and analyzed their influence on the morphology, adhesion and proliferation of the pre-osteoblastic cells (MC3T3-E1). For such heat-treated titanium disks were characterized by optical microscopy, contact angle, surface energy, roughness, microhardness, X-ray diffraction and scanning through the techniques (BSE, EDS and EBSD). For the analysis of biological response were tested by MTT proliferation, adhesion by crystal violet and &#946;1 integrin expression by flow cytometry. It was found that the presence of a microstructure very orderly, defined by a chemical attack, cells tend to stretch in the same direction of orientation of the material microstructure. When this order does not happen, the most important factor influencing cell proliferation is the residual stress, indicated by the hardness of the material. This way the disks with the highest level state of residual stress also showed increased cell proliferation / O tit?nio e suas ligas s?o amplamente utilizados como biomaterial em dispositivos biom?dicos e devido a isso pesquisas t?m sido desenvolvidas visando aperfei?oar e/ou compreender melhor a intera??o biomaterial/meio biol?gico. O processo de fabrica??o desses dispositivos de tit?nio geralmente envolve uma s?rie de processos t?rmicos e mec?nicos e que t?m consequ?ncias no produto final. Os tratamentos t?rmicos s?o usualmente utilizados para obten??o de propriedades diferenciadas para cada aplica??o. Com o intuito de entender a influ?ncia desses tratamentos sobre a resposta biol?gica da superf?cie, foram realizados, no presente trabalho, diferentes tratamentos t?rmicos em tit?nio e analisadas suas influ?ncias na morfologia, ades?o e prolifera??o de c?lulas pr?-osteobl?stica (MC3T3-E1). Para tanto os discos de tit?nio tratados termicamente foram caracterizados por microscopia ?tica, ?ngulo de contato, energia de superf?cie, rugosidade, microdureza Vickers, difra??o de raios-X e microscopia eletr?nica de varredura atrav?s das t?cnicas de EBS, EDS e EBSD. Para an?lise da resposta biol?gica foram realizados teste de prolifera??o por MTT, ades?o por cristal violeta e express?o da integrina &#946;1 por citometria de fluxo. Foi verificado que na presen?a de uma microestrutura muito ordenada, definida atrav?s de um ataque qu?mico, as c?lulas tendem a se alongar no mesmo sentido da orienta??o microestrutural do material. Quando essa ordem n?o acontece, o fator mais importante a influenciar na prolifera??o celular ? a tens?o residual, indicada pela dureza do material. Deste modo os discos que apresentaram maior estado de tens?o residual apresentaram tamb?m maior prolifera??o celular

Made available in DSpace on 2014-10-09T12:43:21Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:56:45Z (GMT). No. of bitstreams: 1 06435.pdf: 7431452 bytes, checksum: 52a613c5fd6687de6ee09e587bde83c2 (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

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.