Microfluidic biosensors and bioreactors based on immobilized biomaterials are described in this dissertation. Photocrosslinkable hydrogel or polymeric microbeads were used as a supporting matrix for immobilizing E.coli or enzymes in a microfluidic device. This dissertation covers a microfluidic bioreactor based on hydrogel-entrapped E.coli, a microfluidic biosensor based on an array of hydrogel-entrapped enzymes, and a microfluidic bioreactor based on microbead-immobilized enzymes. Hydrogel micropatches containing E.coli were fabricated within a microfluidic channel by in-situ photopolymerization. The cells were viable in the hydrogel micropatch and their membranes could be porated by lysating agents. Entrapment of viable cells within hydrogels, followed by lysis, could provide a convenient means for preparing biocatalysts without the need for enzyme extraction and purification. Our results suggested that hydrogel-entrapped cells, immobilized within microfluidic channels, can act as sensors for small molecules and as bioreactors for carrying out reactions. A microfluidic biosensor based on an array of hydrogel-entrapped enzymes could be used to simultaneously detect different concentrations of the same analyte or multiple analyte in real time. The concentration of an enzyme inhibitor could be quantified using the same basic approach. Isolations of the microchannels within different microfluidic channels could eliminate the possibility of cross talk between enzymes. Finally, we characterized microfluidic bioreactors packed with microbead-immobilized enzymes that can carry out sequential, two-step enzyme-catalyzed reactions under flow conditions. The overall efficiency of the reactors depended on the spatial relationship of the two enzymes immobilized on the beads. Digital simulations confirmed the experimental results.

Biomaterial-associated infections remain a significant complication of medical implants. Of the different strains of bacteria associated with nosocomial infections, 70% are resistant to at least one of the drugs used for treatment (Bren 2002). In 2000 the Center for Disease Control ranked microbial agents as the 4th leading actual cause of death in the United States of America (Mokdad et al. 2004).In an effort to improve the prevention and treatment of infections, this research has three objectives: the development of an alternative sterilization method for medical devices; assessing a new antimicrobial material for the prevention of infections in situ; and assessing mechanisms of acquired microbial resistance. The biocide being investigated in this body of work is chlorine dioxide gas.While multiple sterilization methods are available, there are limitations to all of these technologies. For example, chemical sterilization can leave residues on the surface of the material. These residuals can be toxic, causing sensitization reactions when the materials are implanted in the body (Dolovich et al. 1984; Marshall et al. 1985; Chapman et al. 1986; Dolovich et al. 1987). Research has shown that materials sterilized with increasing concentrations of the chemical sterilizing agent have increasing quantities of residuals (Lyarskii et al. 1984). The studies presented here will ascertain the environmental parameters required for sterilization of biomaterials with low concentrations of chlorine dioxide gas and assess polymers sterilized using these conditions for the cytotoxicity of possible chemical residuals.Investigations into preventing biomaterial-based infections in situ have focused on changing the biomaterial properties. Materials with altered physicochemical characteristics to prevent bacterial adhesion have been developed, and antibiotics and silver have been incorporated into the biomaterials to inhibit bacterial colonization. Unfortunately, the rapid depletion of incorporated antimicrobial agents, altered bactericidal activity in vivo, and the development of antibiotic resistance, have all limited the effectiveness of current technologies. In these studies a chlorine dioxide generating material was assessed using in vitro and in vivo assays.While assessing the bactericidal efficacy of a selection of chlorine dioxide generating materials, a spontaneous bacterial mutant with a reduced susceptibility to chlorine dioxide was isolated. The final section of this work will investigate a potential mechanism of resistance to chlorine dioxide.

Universiẗat, Diss.--Jena, 2008.

Mainz, Univ, Diss., 2001.

Polydopamine (PDA), a unique bioinspired polymer, has been a subject of interest in fields including orthopedic biomaterials and antibacterial surfaces. Its osteogenic effects and ability to control surface traits through precise variables (e.g., pH, temperature) have led to its use as a coating in the enhancement of a wide range of materials, including metals and ceramics. In this Thesis, two studies were carried out to better understand the capability and mechanism of PDA-mediated bioactivity. In the first study, we investigated whether PDA coatings can further enhance the bioactivity of nanoporous Titanium (NPTi). While physicochemical traits were in line with literature, PDA was effective in enhancing cell proliferation, beyond NPTi, as early as 8 hours with enhancement in cell spreading and focal adhesion prevalence as early as 1 hour. No changes in adsorptive capacity were found, suggesting a serum-independent component (SIC) of the surface. The second study was focused on (1) determining how treatment parameters influence the physiochemical makeup of PDA surfaces, (2) assessing how PDA surfaces influence stem cell behavior and (3) confirming and investigating the SIC of PDA effect. Results confirm that there is indeed a SIC of PDA coatings with enhancement in cell spreading that improves with the increased size and density of PDA particles. Our findings show that the SIC works in concert with circulating sera to elicit the bioactive effects of PDA. The novel rPDA surface, obtained by adding rotation during the coating deposition, is also shown to elevate bioactivity during normal culturing, beyond classical coatings, with ongoing work suggesting enhancement in the osteogenic differentiation of hMSCs. Taken together, this work has demonstrated novel aspects underlying the potential and mechanism of action for the bioactivity of PDA, ultimately providing new evidence supporting the use of PDA as a biomedical material.

The most common type of adverse events in healthcare in Canada reported by the Canadian Institute for Health Information (CIHI) are nosocomial infections. Amongst nosocomial infections, implant associated infections have been reported to be most common. Despite having the implantation surgeries carefully performed, a small, but still considerable number of devices gets colonized by bacteria resulting in implant associated infections and/or surgical site infections. The patients are then started on high dose antibiotics, which if ineffective, is followed by reimplantation surgeries that leads to long hospital stays and detrimental effects in their lives. Due to this, an alternative to antibiotics is required which could prevent and/or treat bacterial colonization on implants. The main objective of this thesis was to demonstrate the effectiveness of an antimicrobial based CLKRS peptide capped silver nanoparticle coating on a metallic and polymeric based biomaterial used in various implantable medical devices. The CLKRS peptide capped silver nanoparticle formulation was specifically engineered and tested for its antibacterial and antibiofilm properties. Silver nanoparticles were synthesised by photochemical reduction of silver ions upon photocleavage of the photoinitiator I-2959. The metal nanostructure surfaces were protected with the CLKRS peptide and tested on planktonic and biofilms of P. aeruginosa, S. aureus and S. epidermidis. The bacterial quantification was done by survival colony counting. The cytotoxicity of the silver nanoparticle formulation was also tested on human dermal fibroblast, mouse bone marrow derived macrophages, and human epithelial cells by cell proliferation assay. Results show the formation of a nanometric layer of nanosilver on the surface of the material inhibiting the growth of bacteria and eradicating pre-existing biofilms with no significant cell toxicity suggesting the prepared formulation could be a useful tool in preventing and controlling infections on implants during surgery and post implantation. This technology thus could serve as an alternative therapy for surgical site infections and/or implant associated infections.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第16407号 / 工博第3488号 / 新制||工||1527(附属図書館) / 29038 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 岩田 博夫, 教授 田畑 泰彦, 教授 秋吉 一成 / 学位規則第4条第1項該当

No description available.

This work presents an alternative approach to the production of cellulose-based biomaterials. Instead of extracting, processing and regenerating plant and or bacteria-derived cellulose into a biomaterial, my work established a decellularization protocol to remove cellular plant content from plant tissue resulting in a scaffold composed of cellulose with the evolved architecture of the plant cell wall. Tracheophyte plants, including clubmosses, horsetails, and ferns, gymnosperms and angiosperms, have evolved distinct vascular structures that support the transport of water and nutrients in xylem and phloem that form the vascular bundles (VBs)1. This thesis took it’s inspiration from the dense, linearly arranged, parallel microchannels which include (VBs) in the stalks of Asparagus officinalis possess an architecture with striking similarities to biomaterial scaffolds intended to repair damaged tissue. My work demonstrated that the plant cell wall contains many of the ideal characteristics of a medical biomaterial. The scaffold is biocompatible with mammalian cells and maintains high viability even with cell densities comparable to commercially available scaffolds. The cellulose scaffold could be biochemically functionalized or cross-linked to control the scaffolds' surface biochemistry and mechanical properties. My in vivo model demonstrated that the lignocellulose scaffold did not elicit a foreign body response. The scaffold was permissive to host cell invasion, including active host fibroblast, leading to the deposition of host collagen extracellular matrix. Importantly, active blood vessels formed within the scaffold to support the population of host cells. The scaffold retained much of its original shape and provided an inert, pro-vascular long-term environment for host cells to invade. Taken together, this led to the hypothesis that the innate plant cell wall architecture could restore the function of injured tissue, specifically that the vascular bundles could be used to promote axonal regeneration in spinal cord injuries. Rats with complete spinal cord transection were implanted with cellulose scaffolds with vascular bundles. Animals that received plant-derived scaffolds demonstrated a significant improvement in motor function. This thesis defines a novel and parallel route for exploiting naturally occurring plant microarchitectures of the underlying crystalline cellulose scaffold.

Die Implantation eines Medizinprodukts in den menschlichen Körper ruft eine Immunreaktion hervor, die zur fibrösen Einkapselung führen kann. Makrophagen in direktem Kontakt mit der Oberfläche des Implantats erfassen sensorisch den Fremdkörper und übersetzten das Signal in die Freisetzung zahlreicher löslicher Mediatoren. Das generierte Entzündungsmilieu moduliert die Heilungsreaktion und kann zur Anreicherung von Fibroblasten sowie zur Erhöhung der Matrixsyntheserate in der Wundumgebung führen. Eine dichte fibröse Kapsel um ein Medizinprodukt beeinträchtigt den Ersatz von Körperstrukturen, das Unterstützen physiologischer Körperfunktionen sowie die Effizienz einer medizinischen Therapie. Zur Identifizierung potenzieller Biomaterialkandidaten mit optimalen Eigenschaften ist jedoch eine evidenzbasierte Entscheidungsfindung notwendig und diese wiederum muss durch geeignete Testmethoden unterstützt werden. Zur Erfassung lokaler Effekte nach Implantation eines Biomaterials begründet die Komplexi-tät der ablaufenden Fremdkörperreaktion die Anwendung von Tiermodellen als Goldstandard. Die Eingliederung von in vitro Modellsystemen in standardisierte Testverfahren scheitert oft an der Verfügbarkeit validierter, verlässlicher und reproduzierbarer Methoden. Demnach ist kein standardisiertes in vitro Testverfahren beschrieben, das die komplexen dreidimensionalen Gewebsstrukturen während einer Fremdkörperreaktion abbildet und sich zur Testung über längere Kontaktphasen zwischen Blutkomponenten und Biomaterialien eignet. Jedoch können in vitro Testungen kosten- und zeiteffizienter sein und durch die Anwendung humaner Zellen eine höhere Übertragbarkeit auf den Menschen aufweisen. Zusätzlich adressiert die Präferenz zu in vitro Testmethoden den Aspekt „Reduzierung“ der 3R-Prinzipien „Replacement, Reduction, Refinement“ (Ersatz, Reduzierung, Verbesserung) von Russel und Burch (1959) zu einer bewussten und begründeten Anwendung von Tiermodellen in der Wissenschaft. Ziel von diesem Forschungsvorhaben war die Entwicklung von humanen in vitro Modellsystemen, die den Kontakt zu Blutkomponenten sowie die Reaktion des umliegenden Bindegewebes bei lokaler Implantation eines Biomaterials abbilden. Referenzmaterialien, deren Gewebsantwort nach Implantation in Tiere oder den Menschen bekannt ist, dienten als Validierungskriterium für die entwickelten Modellsysteme. Die Anreicherung von Zellen sowie die Bildung extrazellulärer Matrix in der Wundumgebung stellen wichtige Teilprozesse während einer Fremdkörperreaktion dar. Für beide Teilprozesse konnte in einem indirekten zellbasierten Modellsystem der Einfluss einer zellvermittelten Konditionierung wie die Freisetzung von löslichen Mediatoren durch materialadhärente Makrophagen auf die gerichtete Wanderung von Fibroblasten sowie den Umbau eines dreidimensionalen Bindegewebsmodells aufgezeigt werden. Des Weiteren ließ sich das Freisetzungsprofil von Zytokinen durch materialständige Makrophagen unter verschiedenen Testbedingungen wie der Kontamination mit LPS, der Oberflächenbehandlung mit humanem Blutplasma und der Gegenwart von IL-4 bestimmen. Die anschließende vergleichende statistische Modellierung der generierten komplexen multifaktoriellen Datenmatrix ermöglichte die Übersetzung in eine Biomaterialbewertung. Dieses entwickelte Testverfahren eignete sich einerseits zur Validierung von in vitro Testbedingungen sowie andererseits zur Bewertung von Biomaterialien. Darüber hinaus konnte in einem dreidimensionalen Fremdkörpermodell die komplexe dreidimensionale Struktur der extrazellulären Matrix in einer Wunde durch die Kombination unterschiedlicher Zell- und Matrixkomponenten biomimetisch nachgebaut werden. Diese neuartigen dreidimensionalen Fremdkörpermodelle ermöglichten die Testung von Biomaterialien über längere Testphasen und können in anschließenden Studien angewandt werden, um dynamische Prozesse zu untersuchen. Zusammenfassend konnten in dieser Arbeit drei unterschiedliche Teststrategien entwickelt werden, die (I) die Bewertung von Teilprozessen ermöglichen, (II) die Identifizierung verlässlicher Testbedingungen unterstützen und (III) biomimetisch ein Wundgewebe abbilden. Wesentlich ist, dass biomimetisch ein dreidimensionales Gewebemodell entwickelt werden konnte, das eine verlässliche Unterscheidungskapazität zwischen Biomaterialien aufweist. / The implantation of a medical product into the human body induces an immune reaction, which may lead to its fibrous encapsulation. Macrophages in direct contact to the surface sense the foreign body and translate the signal in the secretion of multiple soluble mediators. This generated inflammatory milieu modulates the healing reaction, may induce the accumulation of fibroblasts and lead in the wound microenvironment to an increased matrix synthesis rate. A dense fibrous capsule surrounding a medical product is able to impair the replacement of body structures, the support of physiological body functions as well as the efficiency of a medical therapy. To identify potential biomaterial candidates with optimal characteristics an evidence-based decision making process is necessary and furthermore affords the support by appropriate test procedures. To study local effects after implantation of biomaterials, the complexity of the foreign body reaction justifies the application of animal models as gold standard. The integration of in vitro test procedures into standardized test strategies often fails by the availability of validated, reliable and reproducible methods. According to that there is no standardized test procedure, which resembles the three-dimensional tissue structures during a foreign body reaction and is suited for longer contact phases in between blood components and biomaterials. In vitro tests are often more cost and time efficient and show as well by applying human cells a high transferability on human beings. Additionally the preference to in vitro test procedures addresses the “reduction” aspect of the Russel and Burch’s (1959) 3R-principles “replace-ment, reduction and refinement” to a conscious and reasoned use of animal models in science. Aim of this research project was the development of human in vitro model systems, which resemble the contact to blood components and the reaction of the surrounding soft tissue following implantation of a biomaterial. Reference materials, whose tissue integration after implantation in animals or humans is described, were applied for the developed model systems as validation criterion. The accumulation of cells and the synthesis of extracellular matrix in the surrounding wound are relevant sub processes during a foreign body reaction. In an indirect cell-based model system the influence of the cell-mediated conditioning initiated by the material-induced and macrophage-mediated liberation of soluble mediators was shown on both sub processes the aligned migration of fibroblasts as well as the remodeling of a three-dimensional tissue model. Additionally, the cytokine secretion profile by material-adherent macrophages was characterized under different test conditions such as the contamination with LPS, the surface treatment with human plasma and the presence of IL-4. The following comparative statistical modelling allowed a transformation of the generated complex multi-factorial data matrix to a biomaterial ranking. The here developed test procedure was suitable for the validation of in vitro test conditions as well as the evaluation of the reference biomaterials. Last, by the combination of different cells and matrix structures the complex three-dimensional structure of the extracellular matrix in a wound was biomimetically reconstructed. Those novel three-dimensional foreign body models enabled the testing of biomaterials over longer test phases and might be applied in following studies to investigate dynamic processes. Summarizing in this research project three different test strategies were developed, which (I) enable the evaluation of sub processes, (II) support the identification of reliable test conditions and (III) biomimetically reconstruct a wound tissue. Most important is, that a three-dimensional tissue model was biomimetically developed, which showed a reliable discriminatory capacity in between biomaterials.