Dissertations / Theses on the topic 'Biomaterials Fabrication'

"“One-dimensional” nanostructures like nanotubes and nanorods hold great potential for a wide variety of applications. In particular, one-dimensional nanostructures may be able to provide many significant advantages over traditional spherical particles for drug delivery applications. Recent studies have shown that long, filamentous particles circulate longer within the body than spherical particles, giving them more time to reach the target area and deliver their payload more efficiently. In addition, studies investigating the diffusion of drugs through nanochannels have shown that the drug diffusion profiles can be controlled by varying the nanochannel diameter when the drug diameter and nanochannel diameter are close in size. The combination of increased circulation time and controllable drug release profiles give onedimensional nanostructure great potential for future drug release applications. To fully realize this potential, a simple, low cost, and versatile fabrication method for one-dimensional nanostructures needs to be developed and exploited. The objective of this work is to demonstrate the versatility of template-assisted nanofabrication methods by fabricating a variety of unique protein and polymer one-dimensional nanostructures. This demonstration includes the adaptation of two different template-assisted methods, namely layer-by-layer assembly and template wetting, to fabricate glucose oxidase nanocapsules with both ends sealed, segmented polystyrene and poly(methyl methacrylate) nanorods, and poly(L-lactide)-poly(methyl methacrylate) core-shell nanowires with adjustable shell layer thicknesses. The unique nanostructure morphologies that were achieved using our novel fabrication methods will open the arena for future research focused on process control and optimization for specific applications."

Metallic biomaterials continue to play an essential role to assist with the repair or replacement of natural bone that has become diseased or damaged. Metals have high mechanical strength making them better suited to load-bearing applications than polymeric and ceramic biomaterials [1]. At present, stainless steel, Co-Cr alloys and Ti alloys are three main metallic biomaterials used as bone prosthesis [2, 3]. Although these metals are, in monolithic form, biocompatible, fine debris particles and/or ions released over the lifetime of the implantation, coming into contact with the surrounding tissue appear to be not biocompatible. The abnormally high levels of metal ions and/or particles are believed to be associated with carcinogenic, toxic, inflammatory and allergic reactions eventually leading to the prosthesis aseptic loosening [4-10]. High mechanical stiffness of the three metals is also believed to associate with bone resorption – a situation where bone around the implant becomes thinner or more porous. The high stiffness metal, once implanted, changes the distribution of applied load in the adjacent bone [11, 12]. Recently, there have been interests in using magnesium and its alloy as a metallic biomaterial. Magnesium is a bioresorbable metal with an ability to enhance bone healing process [13, 14]. It also has lower stiffness making it more resemble to that of natural bone in terms of mechanical properties. This work presented in this thesis involves an investigation a manufacturing route that is feasible and viable for producing Mg foam for tissue engineering and bone implant applications. The microstructure and mechanical properties of Mg foam is studied and tested then compared with natural human bone.

Strong, durable terminal regions that can be easily handled by researchers and surgeons are a key factor in the successful fabrication of tissue engineered blood vessels (TEBV). The goal of this study was to fabricate and characterize electrospun cuffs made of poly-caprolactone (PCL) combined with gelatin that reinforce and strengthen each end of cell-derived vascular tissue tubes. PCL is ideal for vascular tissue engineering applications due to its mechanical properties; however, PCL alone does not support cell attachment. Therefore, we introduced gelatin, a natural matrix-derived protein, into the electrospun material to promote cell adhesion. This work compared the effects of two different methods for introducing gelatin into the PCL materials: gelatin coating and gelatin co-electrospinning. Porosity, pore size, fiber diameter, and mechanical properties of the electrospun materials were measured in order to compare the features of gelatin PCL composites that have the greatest impact on cellular infiltration. Porosity was quantified by liquid intrusion, fiber diameter and pore size were measured using scanning electron microscopy, and tensile mechanical testing was used to evaluate strength, elastic modulus, and extensibility. Attachment and outgrowth of smooth muscle cells onto cuff materials was measured to evaluate differences in cellular interactions between materials by using a metabolic attachment assay and a cellular outgrowth assay. Finally, cuffs were fused with totally cell-derived TEBV and the integration of cuffs with tissue was evaluated by longitudinal pull to failure testing and histological analysis. Overall, these cuffs were shown to be able to add length and increase strength to the ends of TEBV for tube cannulation and manipulation during in vitro culture. In particular, PCL:gelatin cospun cuffs were shown to improve cellular attachment and cuff fusion compared to pure PCL cuffs, while still increasing the strength of the TEBV terminal ends.

Electrospinning is a technique used to generate scaffolds composed of nano- to micron-sized fibers for use in tissue engineering. This technology possesses several key weaknesses that prevent it from adoption into the clinical treatment regime. One major weakness is the lack of porosity exhibited in most electrospun scaffolds, preventing cellular infiltration and thus hosts tissue integration. Another weakness seen in the field is the inability to physically cut electrospun scaffolds in the frontal plane for subsequent microscopic analysis (current electrospun scaffold analysis is limited to sectioning in the cross-sectional plane). Given this it becomes extremely difficult to associate spatial scaffold dynamics with a specific cellular response. In an effort to address these issues the research presented here will discuss modifications to electrospinning technology, cryosectioning technology, and our understanding of cellular infiltration mechanisms into electrospun scaffolds. Of note, the hypothesis of a potentially significant passive phase of cellular infiltration will be discussed as well as modifications to cell culture protocols aimed at establishing multiple passive infiltration phases during prolonged culture to encourage deep cellular infiltration.

Au-delà de l'aspect de personnalisation qu'elle peut apporter au domaine médical, la fabrication additive donne aussi accès à l'élaboration de structures cellulaires. Ces structures, de porosité maîtrisée, permettent à la fois de moduler les propriétés mécaniques de l'objet, mais aussi de favoriser l'invasion cellulaire nécessaire en ingénierie tissulaire. Parmi les métaux communément utilisés en chirurgie orthopédique, les alliages de titane sont ceux présentant la rigidité la moins éloignée de celle de l'os. Cette étude porte donc sur l'élaboration de structures en Ti6-Al-4V, mais aussi en magnésium puisqu’il présente l'avantage d'être résorbable dans l'organisme. Les scaffolds sont obtenus par robocasting, procédé consistant à extruder, couche par couche une encre pâteuse constituée de poudre et de liant. Les structures sont ensuite déliantées et frittées à haute température pour atteindre leurs propriétés finales. Concernant les structures en Ti-6Al-4V, une étude paramétrique est effectuée pour évaluer les possibilités et les limites du procédé en termes de structures (et microstructures), de compositions chimiques et de propriétés mécaniques obtenues.Après optimisation, il est possible d'obtenir des pièces présentant deux niveaux de porosités interconnectées (microporosité intra-filament (interconnectée), bénéfique pour l'accroche cellulaire d'après la littérature, et macropores dessinées), gardant une limite d'élasticité spécifique supérieure à celle de l'os (105 MPa.cm³/g) et un module d'Young proche de celui de l'os (28-30 GPa). Un gradient de la porosité intra-filamentaire peut également être obtenu en faisant varier la taille de poudre au sein d’une seule et même pièce. Concernant le magnésium, un liant compatible avec la réactivité de la poudre (base éthanol) a pu être identifié et les premières étapes du procédé (impression, déliantage) sont donc tout à fait réalisables pour ce matériau. Toutefois, le frittage conventionnel du magnésium (pur) s'avère compliqué du fait de sa réactivité. Des alternatives de frittage sont donc étudiées (frittage en phase liquide, SPS)
Beyond the personalisation aspect that it can bring to the medical field, additive manufacturing also gives access to the elaboration of cellular structures. These structures, with controlled porosity, make it possible both to modulate the mechanical properties of the object and to promote the cellular invasion necessary in tissue engineering. Among the metals commonly used in orthopaedic surgery, titanium alloys are those with the rigidity least distant from that of bone. This study therefore focuses on the development of structures made of Ti6-Al-4V, but also of magnesium since it has the advantage of being resorbable in the body. The scaffolds are obtained by robocasting, a process consisting of extruding, layer by layer, a pasty ink made up of powder and binder. The structures have then to be debinded and sintered at high temperature to achieve their final properties. For Ti-6Al-4V structures, a parametric study is carried out to evaluate the possibilities and limits of the process in terms of structures (and microstructures), chemical compositions and mechanical properties obtained. After optimisation, it is possible to obtain parts with two levels of interconnected porosities (intra-filament (interconnected) microporosity, beneficial for cell adhesion according to the literature, and drawn macropores), keeping a specific yield strength higher than that of bone (105 MPa.cm³/g) and a Young's modulus close to that of bone (28-30 GPa). An intra-filament porosity gradient can also be obtained by varying the powder size within a single part. Concerning magnesium, a binder compatible with the reactivity of the powder (ethanol base) has been identified and the first steps of the process (printing, debinding) are therefore quite feasible for this material. However, conventional sintering of (pure) magnesium is complicated by its reactivity. Alternative sintering methods are therefore being investigated (liquid phase sintering, Spark Plasma Sintering)

L'objectif de ce travail est de développer une méthode d'ingénierie de scaffolds multidimensionnels pour la culture cellulaire et l’ingénierie tissulaire. Nous avons d'abord appliqué une technique d'impression 3D pour produire un scaffold en PEGDA et ensuite rempli l'espace libre du scaffold avec du gel de gélatine. Après la congélation et le séchage, un scaffold hybride en PEGDA avec des structures fine de gélatine a été obtenu, qui a été ensuite valisé par la culture et la différenciation des cellules progénitrices neuronales. Pour intégrer plus facilement dans un dispositif microfluidique, nous avons également conçu un scaffold 2D sous forme d’une couche mince de nid d'abeilles de PEGDA rempli des structures poreuses auto-assemblée de PCL. Ce scaffold 2D a été utilisé pour la culture cellulaire et la transfection des gènes, montrant des avantages par rapport aux méthodes classiques en termes d'absorption des nutriments et des facteurs solubles. Enfin, nous avons fabriqué un scaffold mous constitué d’une couche mince de nid d'abeilles en élastomère de PDMS et d’une monocouche de nanofibres de gélatine pour faciliter la différenciation cardiaque à partir des cellules souches pluripotentes humaine. Comme prévu, nous avons réalisé une génération cardiaque avec une contraction plus forte et une homogénéité de battement plus élevée par rapport aux approches classiques. Tous ensemble, nous avons démontré l'utilité des scaffolds hybrides pour l'ingénierie micro-tissulaire qui pourraient avoir un impact sur les études futures dans les domaines de l'ingénierie tissulaire, du criblage des médicaments et de la médecine régénératrice
The objective of this work is to develop a method of engineering multi-dimensional scaffolds for cell culture and tissue formation. We firstly applied a 3D printing technique to produce the designed frame in PEGDA and then filled the free-space of the frame with a gelatin gel. After freezing and drying, a hybrid 3D scaffold made of gelatin porous structures and PEDGA backbone was obtained, which supported culture and differentiation of neural progenitor cells. To more easily integrate into a microfluidic device, we also designed a 2D scaffold in form of a thin layer of honeycomb frame of PEGDA and self-assembled porous structure of PCL. Such a patch form scaffold could be used for cell culture and gene transfection, showing advantages over the conventional methods in terms of nutrients and soluble factors uptake. Finally, we fabricated a soft patch made of an elastic frame in PDMS and a monolayer of gelatin nanofibers to facilitate cardiac differentiation from human induced pluripotent stem cells. As expected, we achieved a cardiac generation with higher contraction strength and a higher beating homogeneity comparing to the conventional approaches. All together, we demonstrated the utility of hybrid scaffolds for micro-tissue engineering which could impact the future studies in the fields of tissue engineering, drug screening and regenerative medicine

Diseases involving fibrosis cause tens of thousands of deaths per year in the US alone. These diseases are characterized by a large amount of extracellular matrix, causing stiff abnormal tissues that may not function correctly. To take steps towards curing these diseases, a fundamental understanding of how cells interact with their substrate and how mechanical forces alter signaling pathways is vital. Studying the mechanobiology of cells and the interaction between a cell and its extracellular matrix can help explain the mechanisms behind stem cell differentiation, cell migration, and metastasis. Due to the correlation between force, extracellular matrix assembly, and substrate stiffness, it is vital to make in vitro models that more accurately simulate biological stiffness as well as measure the amount of force and extracellular matrix assembly. To accomplish this, blends of two types of poly(dimethylsiloxane) (PDMS) were made and the material properties of these polymer blends were characterized. A field of 5µm or 7µm microscopic pillars (referred to as posts) with a diameter of 2.2µm were fabricated from these blends. Each combination of PDMS blend and post height were calibrated and the stiffness was recorded. Additionally, polymer attachment experiments were run to ensure cells survived and had a normal phenotype on the different blends of PDMS when compared to pure PDMS. Finally, cells were placed onto a field of posts and their forces were calculated using the new stiffness found for each blend of post. Varying the PDMS material stiffness using blends allow posts to be much more physiologically relevant and help to create more accurate in vitro models while still allowing easy and accurate force measurement. More biologically relevant in vitro models can help us acquire more accurate results when testing new drugs or examining new signaling pathways.

Intravascular devices, such as stents, must be rigorously tested before they can be approved by the FDA. This includes bench top in vitro testing to determine biocompatibility, and animal model testing to ensure safety and efficacy. As an intermediate step, a blood vessel mimic (BVM) testing method has been developed that mimics the three dimensional structure of blood vessels using a perfusion bioreactor system, human derived endothelial cells, and a biocompatible polymer scaffold used to support growth of the blood vessel cells. The focus of this thesis was to find an in-house fabrication method capable of making cellular scaffolding for use in the BVM. Research was conducted based on three aims. The first aim was to survey possible fabrication methods to choose a technique most appropriate for producing BVM scaffolding. The second aim was to set up the selected fabrication method (electrospinning) in-house at Cal Poly and gain understanding of the process. The third aim was to evaluate consistency of the technique. The work described in this thesis determined that electrospinning is a viable fabrication technique for producing scaffolding for BVM use. Electrospun scaffolding is highly tailorable, and a structure that mimics the natural organization of nano sized collagen fibers is especially desirable when culturing endothelial cells. An electrospinning apparatus was constructed in house and a series of trial experiments was conducted to better understand the electrospinning process. A consistency study evaluated scaffold reproducibility between different spins and within individual spins while setting a baseline that can be used for comparison in future work aimed at electrospinning.

Advances in the biomedical field require functional materials and processes that can lead to devices that are biocompatible, and biodegradable while maintaining high performance and mechanical conformability. In this context, a current shift in focus is towards natural polymers as not only the structural but also functional components of such devices. This poses material-specific functionalization and fabrication related questions in the design and fabrication of such systems. Silk protein biopolymers from the silkworm show tremendous promise in this regard due to intrinsic properties: mechanical performance, optical transparency, biocompatibility, biodegradability, processability, and the ability to entrap and stabilize biomolecules. The unique ensemble of properties indicates opportunities to employ this material into numerous biomedical applications. However, specific processing, functionalization, and fabrication techniques are required to make a successful transition from the silk cocoon to silk-based devices. This research is focused on these challenges to form silk-based functional material and devices for application in areas of therapeutics, bio-optics, and bioelectronics. To make silk proteins mechanically conformable to biological tissues, the first exploration is directed towards the realization of precisely micro-patterned silk proteins in flexible formats. The optical properties of silk proteins are investigated by showing the angle-dependent iridescent behavior of micropatterned proteins, and developing soft micro-optical devices for light concentration and focusing. The optical characteristics and fabrication process reported in the work can lead to the future application of silk proteins in flexible optics and electronics. The microfabrication process of silk proteins is further extended to form shape-defined silk protein microparticles. Here, the specificity of shape and the ability to form monodisperse shapes can be used as shape encoded efficient cargo and contrast agents. Also, these particles can efficiently entrap and stabilize biomolecules for drug delivery and bioimaging applications. Next, a smart confluence of silk sericin and a synthetic functional polymer PEDOT:PSS is shown. The composite materials obtained have synergistic effects from both polymers. Silk proteins impart biodegradability and patternability, while the intrinsically conductive PEDOT:PSS imparts electrical conductivity and electrochemical activity. Conductive micro architectures on rigid as well as flexible formats are shown via a green, water-based fabrication process. The applications of the composite are successfully demonstrated by realizing biosensing and energy storage devices on rigid or flexible forms. The versatility of the approach will lead to the development of a variety of applications such as in bio-optics, bioelectronics, and in the fundamental study of cellular bio electrogenic environments. Finally, to expand the applicability of reported functional polymers and composites beyond the microscale, a method for silk nano-patterning via electron beam lithography is explored. The technique enables one-step fabrication of user defined structures at the submicron and nano-scales. By virtue of acrylate chemistry, a very low energetic beam and dosage are required to form silk nano-architectures. Also, the process can form both positive and negative features depending on the dosage. The fabrication platform can also form nano scale patterns of the conductive composite. The conductive measurements confirm the formation of conductive nanowires and the ability of silk sericin to entrap PEDOT:PSS particles in nanoscale features.

Nanoparticles are often considered as efficient drug delivery vehicles for precisely dispensing the therapeutic payloads specifically to the diseased sites in the patient’s body, thereby minimizing the toxic side effects of the payloads on the healthy tissue. However, the fundamental physics that underlies the nanoparticles’ intrinsic interaction with the surrounding cells is inadequately elucidated. The ability of the nanoparticles to precisely control the release of its payloads externally (on-demand) without depending on the physiological conditions of the target sites has the potential to enable patient- and disease-specific nanomedicine, also known as Personalized NanoMedicine (PNM). In this dissertation, magneto-electric nanoparticles (MENs) were utilized for the first time to enable important functions, such as (i) field-controlled high-efficacy dissipation-free targeted drug delivery system and on-demand release at the sub-cellular level, (ii) non-invasive energy-efficient stimulation of deep brain tissue at body temperature, and (iii) a high-sensitivity contrasting agent to map the neuronal activity in the brain non-invasively. First, this dissertation specifically focuses on using MENs as energy-efficient and dissipation-free field-controlled nano-vehicle for targeted delivery and on-demand release of a anti-cancer Paclitaxel (Taxol) drug and a anti-HIV AZT 5’-triphosphate (AZTTP) drug from 30-nm MENs (CoFe2O4-BaTiO3) by applying low-energy DC and low-frequency (below 1000 Hz) AC fields to separate the functions of delivery and release, respectively. Second, this dissertation focuses on the use of MENs to non-invasively stimulate the deep brain neuronal activity via application of a low energy and low frequency external magnetic field to activate intrinsic electric dipoles at the cellular level through numerical simulations. Third, this dissertation describes the use of MENs to track the neuronal activities in the brain (non-invasively) using a magnetic resonance and a magnetic nanoparticle imaging by monitoring the changes in the magnetization of the MENs surrounding the neuronal tissue under different states. The potential therapeutic and diagnostic impact of this innovative and novel study is highly significant not only in HIV-AIDS, Cancer, Parkinson’s and Alzheimer’s disease but also in many CNS and other diseases, where the ability to remotely control targeted drug delivery/release, and diagnostics is the key.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Dec. 11, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.

Cobalt Ferrite has important, size-dependent magnetic properties. Consequently, an overview of particle size is important. Co-precipitation in air was the fabrication method used because it is comparatively simple and safe. The effects of three different reaction times including 1, 2, 3 hour(s) on particle size were compared. Also, the effectiveness of three different capping agents (Oleic Acid, Polyvinylpyrollidone (PVP), and Trisodium Citrate) in reducing aggregation and correspondingly particle size were examined. Using Welch’s analysis of variance (ANOVA) and the relevant post hoc tests, there was no significant difference (p=0.05) between reaction times of 1 hour and 2 hours, but there was a significant difference between reaction times of 2 hours and 3 hours. Potentially, because of increased coarsening for the 3 hour reaction time. PVP and Oleic Acid were shown to be effective in reducing aggregation; however, Citrate was not effective. Possibly, the synthesis procedure was inadequate.

L'électrospinning est un procédé couramment utilisé pour la fabrication de membranes nanofibreuses non-tissées. Ces membranes sont particulièrement intéressantes pour des applications tels que l'ingénierie tissulaire et la libération contrôlée de médicaments car elles sont très poreuses et ont une large surface spécifique. Dans une première partie, nous avons développé une nouvelle stratégie afin de contrôler la morphologie et la dimension des fibres fabriquées par electrospinning. Puis nous avons développé un composite fait de nanofibres de PLA et de microparticules de PEG auto-organisé, créant des motifs en nid d'abeilles qui grandissent avec l'épaisseur de la membrane. Ces membranes auto-organisées ont une structure poreuse dont la dimension des pores va de quelques microns à plusieurs centaines de microns. Enfin, deux modèles ont été développés pour une libération contrôlée d'un composé model : la délivrance retardée par l'élaboration de structure sandwich et la libération directionnnelle par la création d'un gradient de concentration avec différentes cinétiques.

Silk fibroin (SF) from the silkworm, Bombyx mori, is a natural fibrous protein with unique mechanical properties, biocompatibility and biodegradability. It has great potential in biomaterial applications for tissue engineering, drug delivery and biomedical devises. Most of the SF based biomaterials (e.g. films, scaffolds, hydrogels and electrospun fibres) are cast from regenerated silk fibroin (RSF) solution. Hence, it is important to acquire a comprehensive and deep understanding of the fibroin solution. This research applied a number of biophysical approaches, aiming to investigate the solution aggregation and interfacial adsorption behaviour of the SF polypeptides in aqueous solution. The methods for fabricating nanometre scale SF films are also explored carefully because well-controlled films and their surfaces enable direct characterisation of their interaction with other molecules and cells. Using the dynamic light scattering (DLS) technique, it was found that the particle size of newly made RSF peptides in solution was around 2.3~6.5 nm and they could remain stable for at least 10 weeks at 4 °C. Factors such as temperature, fibroin concentration, pH, alcohol and metallic ions can directly affect the assembly and aggregation of fibroin polypeptides as well as their solubility and stability in solution. The formation of large aggregation under certain conditions was possibly related to the conformational transition of SF from random coil/α-helix (Silk I) to β-sheets (Silk II). The interfacial adsorption of RSF solution at the SiO2/water interface was monitored by spectroscopic ellipsometry (SE), dual polarisation interferometry (DPI) and neutron reflection (NR). It was revealed that surface excess and thickness increased with concentration and decreased with rising pH and ionic strength. NR measurements revealed that the adsorbed polypeptide layers are characterised by a thin and dense inner region and a thick and diffuse outer region, a feature similar to the adsorbed layers from other polypeptides. The results from SE, DPI and NR are in good agreement. Multilayer ultrathin SF films were fabricated using the layer-by-layer spin coating method and were found to be stable in physiological conditions. The thickness and surface excess of the SF films were tuned by varying the concentrations while coating. Surface biocompatibility as demonstrated by MTT assays varied with the film thickness or the number of layers coated. With the aid of the cationic copolymer MPC30-DEA70, SF films successfully immobilised plasmid DNA, which demonstrates the potential of these multilayer SF films to be used in a drug delivery system. The ultrathin SF films were modified with gelatin (G). Preliminary cell culture experiments with 3T3 fibroblasts demonstrated that SF/G films with 1.2% ~ 20% (w/w) G content promoted cell attachment and proliferation compared with pure SF films. When films contain 10% ~ 20% (w/w) of G, they showed biocompatibility even superior to the pure G films. These enhanced cellular responses must result from improved film stability arising from SF and improved cytocompatibility arising from G.

The integration of functional and structural properties makes genetically engineered proteins appealing in tissue engineering. Silk-elastinlike proteins (SELPs), containing tandemly repeated polypeptide sequence derived from natural silk and elastin, are recently under active study due to the interesting structure. The biological, chemical, physical properties of SELPs have been extensively investigated for their possible applications in drug/gene delivery, surgical tissue sealing and spine repair surgery. However, the mechanical aspect has rarely been looked into. Moreover, many other biomaterials have been fabricated into fibers in micrometer and nanometer scale to build extracellular matrix-mimic scaffolds for tissue regeneration, but many have one or mixed defects such as: poor strength, mild toxicity or immune repulsion etc. The SELP fibers, with the intrinsic primary structures, have novel mechanical properties that can make them defects-minimized scaffolds in tissue engineering.In this study, one SELP (SELP-47K) was fabricated into microfibers and nanofibers by the techniques of wet-spinning and electrospinning. Microfibers of meters long were formed and collected from a methanol coagulation bath, and later were crosslinked by glutaraldehyde (GTA) vapor. The resultant microfibers displayed higher tensile strength up to 20 MPa and higher deformability as high as 700% when tested in hydrated state. Electrospinnig of SELP-47K in formic acid and water resulted in rod-like and ribbon-like nanofibrous scaffolds correspondingly. Both chemical (methanol and/or GTA) and physical (autoclaving) crosslinking methods were utilized to stabilize the scaffolds. The chemical crosslinked hydrated scaffolds exhibit elastic moduli of 3.4-13.2 MPa, ultimate tensile strength of 5.7-13.5 MPa, and deformability of 100-130%, closely matching or exceeding the native aortic elastin; while the autoclaved one had lower numbers: 1.0 MPa elastic modulus, 0.3 MPa ultimate strength and 29% deformation. However, the resilience was all above 80%, beyond the aortic elastin, which is 77%. Additionally, Fourier transform infrared spectra showed clear secondary structure transition after crosslinking, explaining the phenomenon of scaffold water-insolubility from structural perspective and showed a direct relationship with the mechanical performance. Furthermore, the in vitro biocompatibility of SELP-47K nanofibrous scaffolds were verified through the culture of NIH 3T3 mouse embryonic fibroblast cells.

Although researchers have been able to print small, simple, and avascular tissues, they have been unsuccessful in creating large, complex and vascularized organs. Printing large and complex three-dimensional tissues or organs involves utilizing a large quantity of cellular spheroids and layer-by-layer addition of spheroids. In this study, an in-house cell spheroid fabrication system was developed to produce cell spheroids with human liver cells (hepG2), human endothelial cells (hEC), human neural stem cells (hNSC), and induced pluripotent stem cells (iPSC). It offers the ability of fabricating uniform-sized spheroids repeatedly, which is essential when large and complex structures need to be produced. In order to test the spheroids’ ability to fuse, hEC spheroids were placed in line with one another and revealed successful fusion. Overall, the results indicate the in- house developed cell spheroid fabrication system can play a major role in bioprinting by providing researchers with uniform-sized spheroids in large quantities, consistently.

This dissertation has aimed to fabricate polypeptide based biomaterial and characterize physical properties. Electrospinning is used as a tool for the sample fabrication. Project focused on determining the feasibility of electrospinning of certain synthetic polypeptides and certain elastin-like peptides from aqueous feedstocks and to characterize physical properties of polymer aqueous solution, cast film and spun fibers and fiber mats. The research involves peptide design, polymer electrospinning, fibers crosslinking, determining the extent of crosslinking, fibers protease degradation study, fibers stability and self-organization analysis, structure and composition determination by various spectroscopy and microscopy techniques and characterization of mechanical properties of individual suspended fibers. Fiber mats of a synthetic cationic polypeptide poly(L-ornithine) (PLO) and an anionic co-polypeptide of L-glutamic acid and L-tyrosine (PLEY) of defined composition have been produced by electrospinning. Fibers were obtained from polymer aqueous solution at concentrations of 20-45% (w/v) in PLO and at concentrations of 20-60% (w/v) in PLEY. Applied voltage and spinneret-collector distance were also found to influence polymer spinnability and fibers morphology. Oriented fibers were obtained by parallel electrodes geometry. Fiber diameter and morphology was analyzed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). PLO fibers exposed on glutaraldehyde (GTA) vapor rendered fiber mats water-insoluble. A common chemical reagent, carbodiimide was used to crosslink PLEY fibers. Fiber solubility in aqueous solution varied as a function of crosslinking time and crosslinker concentration. Crosslink density has been quantified by a visible-wavelength dye-based method. Degradation of crosslinked fibers by different proteases has been demonstrated. Investigation of crosslinked PLEY fibers has provided insight into the mechanisms of stability at different pH values. Variations in fiber morphology, elemental composition and stability have been studied by microscopy and energy-dispersive X-ray spectroscopy (EDX), following the treatment of samples at different pH values in the 2-12 range. Fiber stability has been interpreted with reference to the pH dependence of the UV absorbance and fluorescence of PLEY chains in solution. The data show that fiber stability is crucially dependent on the extent of side chain ionization, even after crosslinking. Self-organization kinetics of electrospun PLO and PLEY fibers during solvent annealing has been studied. After being crosslinked in situ, fibers were annealed in water at 22 °C. Analysis by Fourier transform infrared spectroscopy (FTIR) has revealed that annealing involved fiber restructuring with an overall time constant of 29 min for PLO and 63 min for PLEY, and that changes in the distribution of polymer conformations occurred during the first 13 min of annealing. There was a substantial decrease in the amount of Na+ bound to PLEY fibers during annealing. Kinetic modeling has indicated that two parallel pathways better account for the annealing trajectory than a single pathway with multiple transition states. Bacteria have been engineered to make novel 250-mer elastin-like polypeptides (ELPs). Each was predicted to have an absolute net charge of less than 0.05 electron charges per amino acid residue in aqueous solution at neutral pH. Polymer structure in solution has been assessed by Circular dichroism spectroscopy (CD) and dynamic light scattering (DLS). Suitability for materials manufacture has been tested by electrospinning. Here, we have also tested the hypothesis that blending polypeptides of radically different amino acid composition will enable the realization of novel and potentially advantageous material properties. Aqueous polymer feedstock solutions consisted of pure ELP or ELP blended with a synthetic polypeptide, PLEY, which is highly ionized at neutral pH and spinnable. Morphology analysis of blended fibers by SEM has revealed the formation of a surprising variety of structures that are not seen in fibers of ELP or PLEY alone, for example, hollow beads. Analysis of blended fibers by fluorescence microscopy showed that there was little or no phase separation, despite the large difference in electrical properties between ELP and the synthetic polymers. Structure and composition of PLO, PLEY, ELPs and blends and electrospun fibers made of these polymers have been determined and compared. CD and FTIR have been utilized to obtain structural information on these polymers in aqueous solution, cast films and fibers. Fiber composition has been analyzed by EDX. Protein adsorption has been analyzed by quantitative fluorescence microscopy. The polymers adopted random coil-like conformations in aqueous feedstocks at neutral pH and in dehydrated cast films and fibers on glass, and the fibers comprised numerous counterions, according to spectral analysis. Adsorption of model proteins and serum proteins onto hydrated and crosslinked fibers depended on the electrical charge of the proteins and the fibers. The surface charge density of the fibers will be comparable to, but less than, the charge density on the outer leaflet of the plasma membrane of usual eukaryotic cells. Mechanical properties of a series of as-spun and crosslinked PLO and PLEY nanofibers with various diameters have been analyzed by using the pure bending mode and AFM technique. Aligned nanofibers were deposited on top of a microsized groove etched on a glass substrate. AFM tip was used as a probe, which could apply a measurable deflection and force onto the suspended nanofiber at a force calibration mode, so that the Young's modulus of a single nanofiber can be calculated based on the basic beam bending theories. The Young's moduli of the studied peptide nanofibers increased significantly with decreased fiber diameters. This study has also demonstrated that crosslinked electrospun PLO and PLEY fibers have a higher Young's modulus compared with their as-spun counterparts. Taken together, the results will advance the rational design of polypeptides for peptide-based materials, especially materials prepared by electrospinning. It is believed that this research will increase basic knowledge of polymer electrospinning and advance the development of electrospun materials, especially in medicine and biotechnology. The study has yielded two advances on previous work in the area: avoidance of an animal source of peptides and avoidance of inorganic solvent. The present results thus advance the growing field of peptide-based materials. Non-woven electrospun fiber mats made of polypeptides are increasingly considered attractive for basic research and technology development in biotechnology, medicine and other areas.

Mestrado em Engenharia Mecânica
A recente evolução científica no campo da engenharia de tecidos (TE) criou oportunidades únicas para fabricar tecidos de substituição de órgãos artificiais em laboratório a partir de combinações de matrizes extracelulares (andaimes), células e moléculas biologicamente ativas. adicionalmente, a formulação de compósitos poliméricos reforçados com cargas nanométricas como o óxido de grafeno (GO) mostrou ser possível uma grande melhoria de várias propriedades destes compósitos em relação aos polímeros simples. No presente estudo, matrizes fibrosas de policaprolactona (PCL) e de PCL-GO foram preparadas através de eletrofiação sob diferentes condições. Foi analisado o efeito de vários parâmetros de electrofiação tais como, peso molecular do polímero, solventes, concentração, caudal, tensão e distância de trabalho, sobre a morfologia das fibras eletrofiado. A incorporação de GO nas fibras de PCL alterou a morfologia, química de superfície e as propriedades mecânicas das fibras de PCL compósitos, o que foi comprovado por meio de várias técnicas de caracterização. As matrizes fibrosas de PCL-GO com a concentração de GO de 0,1% em peso demonstraram ser a combinação mais interessante para estudos futuros em TE.
Scientific advancements in the field of tissue engineering (TE) have created unique opportunities to fabricate artificial tissue or organ replacement components in the laboratory from combinations of engineered extracellular matrices (scaffolds), cells and biologically active molecules. Polymer composites reinforced with nanosized graphene oxide (GO) fillers have shown large improvement of various properties over the pristine polymers. In the present study, polycaprolactone (PCL) and PCL-GO fibres were prepared through electrospinning under different conditions. The effect of several electrospinning parameters (polymer molecular weight, solvent system, concentration, flow rate, voltage and working distance) on the morphology of the electrospun fibres was investigated. The GO nanosheets were successfully incorporated into the PCL fibres and the changes in the morphology, surface chemistry and mechanical properties were analyzed through various characterization techniques. The PCL-GO electrospun fibres with GO concentration of 0.1 wt% was found to be the most attractive combination which can be utilized for future TE applications.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Elliot L. Chaikof; Committee Member: Ajit Yoganathan; Committee Member: Larry McIntire; Committee Member: Marc Levenston; Committee Member: Mark Allen. Part of the SMARTech Electronic Thesis and Dissertation Collection.

In this research, a proton exchange membrane fuel cell (PEMFC) sensor was investigated for specific detection of volatile organic compounds (VOCs) for point-of-care (POC) diagnosis of the physiological conditions of humans. A PEMFC is an electrochemical transducer that converts chemical energy into electrical energy. A Redox reaction takes place at its electrodes whereas the volatile biomolecules (e.g. ethanol) are oxidized at the anode and ambient oxygen is reduced at the cathode. The compounds which were the focus of this investigation were ethanol (C2H5OH) and isoflurane (C3H2ClF5O), but theoretically, the sensor is not limited to only those VOCs given proper calibration. Detection in biosensing, which needs to be carried out in a controlled system, becomes complex in a multivariate environment. Major limitations of all types of biosensors would include poor selectivity, drifting, overlapping, and degradation of signals. Specific detection of VOCs in multi-dimensional environments is also a challenge in fuel cell sensing. Humidity, temperature, and the presence of other analytes interfere with the functionality of the fuel cell and provide false readings. Hence, accurate and precise quantification of VOC(s) and calibration are the major challenges when using PEMFC biosensor. To resolve this problem, a statistical model was derived for the calibration of PEMFC employing multivariate analysis, such as the “Principal Component Regression (PCR)” method for the sensing of VOC(s). PCR can correlate larger data sets and provides an accurate fitting between a known and an unknown data set. PCR improves calibration for multivariate conditions as compared to the overlapping signals obtained when using linear (univariate) regression models. Results show that this biosensor investigated has a 75% accuracy improvement over the commercial alcohol breathalyzer used in this study when detecting ethanol. When detecting isoflurane, this sensor has an average deviation in the steady-state response of ~14.29% from the gold-standard infrared spectroscopy system used in hospital operating theaters. The significance of this research lies in its versatility in dealing with the existing challenge of the accuracy and precision of the calibration of the PEMFC sensor. Also, this research may improve the diagnosis of several diseases through the detection of concerned biomarkers.

Optical biosensors are desired for the monitoring of various biochemical markers, which are relevant indicators in the treatment and diagnosis of diseases. Specifically, luminescence sensors are favorable for optical interrogation since they are highly sensitive to analyte changes and may be implemented in lifetime or intensity-based systems. In order to develop particle-based fluorescent sensors, poly(2-hydroxyethylmethacrylate) (HEMA) microspheres have been fabricated via membrane emulsification and characterized to evaluate the emulsion method and the overall process of tailoring properties to synthesize spheres of specific mean sizes. A pH-sensitive indicator seminaphthorhodafluors-4F 5-(and-6)-carboxylic acid (SNARF) was immobilized within the microspheres, and resulting sensor particles were exposed to various pH buffers to obtain a pH calibration curve based on intensity measurements. PolyHEMA microparticles were fabricated in a systematic study with measured mean sizes ranging from 8-21 um. Optical and scanning electron microscopy images revealed the formation of spherical, porous particles, which were additionally stabilized with polymer coatings. The lowest coefficient of variation value achieved was 50%, indicating the inability to produce monodisperse particles due to the dispersity of pore sizes in the membrane. SNARF was immobilized within the polyHEMA spheres, and fluorescence was observed when exposing the sensors to different pH buffers on a fluorescence microscope. Ratiometric intensity measurements for the sensor particles were obtained on a spectrofluorometer while flowing pH buffers over the immobilized spheres in a reaction chamber. The peak intensity ratio of the microparticle sensors exhibited a change in 0.9 units when decreasing the pH from 8.4 to 5.5. In the future, these pH sensing particles may be implanted alongside glucose sensing materials in order to provide valuable pH information in understanding the immune response to specific biomaterials and sensing components.

abstract: Presented below is the design and fabrication of prosthetic components consisting of an attachment, tactile sensing, and actuator systems with Fused Filament Fabrication (FFF) technique. The attachment system is a thermoplastic osseointegrated upper limb prosthesis for average adult trans-humeral amputation with mechanical properties greater than upper limb skeletal bone. The prosthetic designed has: a one-step surgical process, large cavities for bone tissue ingrowth, uses a material that has an elastic modulus less than skeletal bone, and can be fabricated on one system. FFF osseointegration screw is an improvement upon the current two-part osseointegrated prosthetics that are composed of a fixture and abutment. The current prosthetic design requires two invasive surgeries for implantation and are made of titanium, which has an elastic modulus greater than bone. An elastic modulus greater than bone causes stress shielding and overtime can cause loosening of the prosthetic. The tactile sensor is a thermoplastic piezo-resistive sensor for daily activities for a prosthetic’s feedback system. The tactile sensor is manufactured from a low elastic modulus composite comprising of a compressible thermoplastic elastomer and conductive carbon. Carbon is in graphite form and added in high filler ratios. The printed sensors were compared to sensors that were fabricated in a gravity mold to highlight the difference in FFF sensors to molded sensors. The 3D printed tactile sensor has a thickness and feel similar to human skin, has a simple fabrication technique, can detect forces needed for daily activities, and can be manufactured in to user specific geometries. Lastly, a biomimicking skeletal muscle actuator for prosthetics was developed. The actuator developed is manufactured with Fuse Filament Fabrication using a shape memory polymer composite that has non-linear contractile and passive forces, contractile forces and strains comparable to mammalian skeletal muscle, reaction time under one second, low operating temperature, and has a low mass, volume, and material costs. The actuator improves upon current prosthetic actuators that provide rigid, linear force with high weight, cost, and noise.
Dissertation/Thesis
Doctoral Dissertation Biomedical Engineering 2017

Cellulose nanocrystals (CNCs) and its composite coatings may impart many benefits in packaging, electronic, optical, etc. applications; however, large-scale coating production is a major engineering challenge. To fill this knowledge gap, a potential large-scale manufacturing technique, roll-to-roll reverse gravure processing, has been described in this work for the manufacture of CNC and CNC-poly(vinyl alcohol) (PVA) coatings on a flexible polymer substrate. Various processing parameters which control the coating structure and properties were examined. The most important parameters in controlling liquid transfers were gravure roll, gravure speed, substrate speed, and ink viscosity. After successful fabrication, coating adhesion was investigated with a crosshatch adhesion test. The surface roughness and morphology of the coating samples were characterized by atomic force microscopy and optical profilometer. The Hermans order parameter (S) and coating transparency were measured by UV–Vis spectroscopy. The effect of viscosity on CNC alignment was explained by the variation of shear rate, which was controlled by the micro-gravure rotation. Finally, the CNC alignment effect was investigated for gas barrier and thermal management applications.

In packaging applications, cellulose nanomaterials may impart enhanced gas barrier performance due to their high crystallinity and polarity. In this work, low to superior gas barrier pristine nanocellulose films were produced using a shear-coating technique to obtain a range of anisotropic films. Induction of anisotropy in a nanocellulose film can control the overall free volume of the system which effectively controls the gas diffusion path and hence, controlled anisotropy results in tunable barrier properties. The highest anisotropy materials showed a maximum of 900-fold oxygen barrier improvement compared to the isotropic arrangement of nanocellulose film. The Bharadwaj model of nanocomposite permeability was modified for pure nanoparticles, and the CNC data were fitted with good agreement. Overall, the oxygen barrier performance of anisotropic nanocellulose films was 97 and 27 times better than traditional barrier materials such as biaxially oriented poly(ethylene terephthalate) (BoPET) and ethylene vinyl alcohol copolymer (EVOH), respectively, and thus could be utilized for oxygen-sensitive packaging applications.

The in-plane thermal conductivity of CNC - PVA composite films containing different PVA molecular weights, CNC loadings and varying order parameters (S) were investigated for potential application in thermal management of flexible electronics. Isotropic CNC - PVA bulk films with 10-50 wt% PVA solid loading showed significant improvement in thermal conductivity compared to either one component system (PVA or CNC). Furthermore, anisotropic composite films exhibited in-plane thermal conductivity as high as ~ 3.45 W m-1 K-1 in the chain direction, which is higher than most polymeric materials used as substrates for flexible electronics. Such an improvement can be attributed to the inclusion of PVA as well as to a high degree of CNC orientation. The theoretical model was used to study the effect of CNC arrangement (both isotropic and anisotropic configurations) and interfacial thermal resistance on the in-plane thermal conductivity of the CNC-PVA composite films. To demonstrate an application for flexible electronics, thermal images of a concentrated heat source on both neat PVA and CNC-PVA composite films were taken that showed the temperature of the resulting hot spot was lower for the composite films at the same power dissipation.

The present thesis focuses on the fabrication of bio-stimuli responsive micro- and nano-carriers for drug delivery applications. In particular, the objective of this work is to investigate the possibility of using polypeptide drug protamine and glycosaminoglycan drug, chondroitin sulphate as stimuli responsive components in the design of bioresponsive carriers. These biopolymers are biocompatible, biodegradable and clinically used for various applications. Two designs that incorporate these stimuli responsive components have been studied in this thesis. The first design involves hollow micro and nanocapsules that have been fabricated by incorporating the stimuli responsive biopolymers as wall components. Upon exposure to biological triggers, these hollow capsules disintegrate releasing the encapsulated drug. The second design consists of mesoporous silica nanoparticles-biopolymer hybrids. The mesoporous silica nanoparticles act as a gated scaffold that carries the drug molecules. The mesopores of these drug loaded nanoparticles are then blocked with the bioresponsive polymers. Upon exposure to the bio-triggers which consist of enzymes over-expressed in conditions such as cancer and inflammation, these “molecular gates” disintegrate allowing the drug trapped in the mesoporous silica nanoparticles to escape into the surroundings. The thesis has been divided into five chapters: Chapter 1 is an introduction to bio-responsive drug delivery. The broad classification of stimuli used in responsive drug delivery systems are visited. A brief discussion on the various types of bio-stimuli that can be utilized in designing bio-responsive systems is also included in this chapter. Chapter 2 defines the aims and scope of the thesis which is followed by an overview of the various design parameters involved in the fabrication of systems presented in this work. The major stimuli responsive components and the architectures incorporating these elements are discussed in detail here. A literature review of the various carrier designs involved in the study is provided , with special emphasis on stimuli responsive drug delivery. Chapter 3 gives an overview of the various materials and methods involved in this work. A summary of the various characterisation techniques used in the thesis is also included along with the details of the experiments that has been carried out. Chapter 4 provides an overview of the results and discussions of the thesis. The chapter has been divided into six sections: Chapter 4.1 deals with the fabrication of a hollow microcapsule system incorporated with protamine as the stimuli responsive element for bio-responsive drug delivery. The hollow microcapsules that were fabricated by Layer by Layer assembly of protamine and heparin display pH responsive variations in permeability and disintegrate in the presence of the enzyme trypsin that degrades protamine. The biologically triggered enzyme responsive drug release from these microcapsules is also demonstrated using enzymes secreted by colorectal cancer cells. Chapter 4.2 presents nanocapsules fabricated from protamine and heparin. The pH and enzyme responsive drug release of this systems is evaluated in vitro. A wall crosslinking strategy has been tested to control the rate of drug release under physiological pH conditions in the absence of the trigger. The cellular interactions of these nanocapsules loaded with an anticancer drug, doxorubicin was studied using cancer cell lines. Bioavailability studies of doxorubicin encapsulated in these nanocapsules were performed using a BALB/c mice model. Chapter 4.3 discusses the fabrication of a hollow microcapsule system that can disintegrate in response to dual biological stimuli. These carriers have been fabricated by incorporating protamine and chondroitin sulphate as the wall components. Due to the incorporation of two separate stimuli responsive components in the walls, these capsules are expected to be sensitive to the enzymes trypsin or hyaluronidase I. Chapter 4.4 deals with the fabrication of dual enzyme responsive hollow nanocapsule which can be targeted to deliver anticancer agents specifically inside cancer cells. The enzyme responsive elements integrated in the hollow nanocapsule walls can undergo degradation in presence of either of the enzymes trypsin or hyaluronidase I leading to the release of encapsulated drug molecules. The drug release from these nanocapsules which were crosslinked and functionalised with folic acid, is evaluated under varying conditions. The cellular uptake and intracellular drug delivery by these nanocapsules were evaluated in cervical cancer cell lines. Chapter 4.5 introduces a mesoporous silica nanoparticle − protamine hybrid system. The system consists of a mesoporous silica nanoparticle support whose mesopores are capped with protamine which effectively blocks the outward diffusion of the drug molecules from the mesopores of the mesoporous silica nanoparticles. Upon exposure to the enzyme trigger, the protamine cap disintegrates opening up the molecular gates and releasing the entrapped drug molecules. The drug release from this system is evaluated in different release conditions in the presence and absence of the enzyme trigger. The ability of these particles to deliver hydrophobic anticancer drugs and induce cell death in colorectal cancer cells has also been demonstrated. Chapter 4.6 discusses the fabrication of another mesoporous silica nanoparticles based bio-responsive drug delivery system consisting of mesoporous silica and chondroitin sulphate hybrid nanoparticles. The ability of the system to modulate drug release in response to hyaluronidase I is demonstrated. By utilizing a cervical cancer cell line, we have demonstrated the cellular uptake and intracellular delivery of hydrophobic drugs encapsulated in these particles. Interestingly, the system showed ability to enhance the anticancer activity of hydrophobic drug curcumin in these cancer cells. Chapter 5 gives a summary of the general conclusions drawn from the thesis work.

博士
國立臺灣大學
化學工程學研究所
91
Abstract In recent years, free tissue transfers, which require tissue or organ substitutes to repair/replace the damaged/diseased organs or tissues have been developed. The immediate problem is the shortage in donor availability. To solve this problem, people use the technology of tissue engineering, which elucidates the structure-function relationships in normal and diseased tissues, to create tissue or organ replacements. Biomaterials play an important role in many of these activities, for example, serving as matrices to guide tissue regeneration, releasing polypeptide growth factors and stimulating cellular response to an antificial implant. This study focused on the development of a novel biomaterial, the modification of the biomaterial, and the application of this novel biomaterial. In the first part, a freeze-fixing method was used to prepare a novel porous PGA-chitosan hybrid matrices (P/C matrices) containing 70% of PGA. The P/C matrices prepared at -20℃ have 100-200 m interconnected micropores in the interior region, with a porous layer present on the bottom and top of the matrices. Another set of the P/C hybrid matrices with freezing temperature at -80℃ were also prepared. The pore size of these matrices is 70-80 m. Fibroblast cells cultured on these P/C matrixes exhibited high viability and maintained spindle morphology, suggesting good biocompatibility for the P/C matrices. It can be concluded that the P/C matrices, due to their high porosity, biocompatibility and degradability, are a promising biomaterial. In the second part, I focus to the surface modification of a biomaterial to enhance its function. The use of wheat germ agglutinin (WGA), a commonly used lectin, covalently bound on to chitosan films to improve the biocompatibility and specificity of chitosan films via oligosaccharide-mediated cell adhesion was examined. After seeding for 12 h, the ratio of live fibroblast cells was about 80% on the WGA-modified chitosan films but at the same time only 65% cells were alive on the control chitosan films. The percentage of live cells on the WGA-modified chitosan films and the chitosan films increased to nearly 100% and 85%, respectively, at 48 h after seeding. The DNA staining revealed that a portion of fibroblasts cultivated on the chitosan films were undergoing apoptosis. In contrast, fibroblasts growing on the WGA-modified chitosan films did not show any indication of apoptosis. Further, the evaluation of the heat shock protein (HSP) mRNA expression in the cells using the reverse transcription-polymerase chain reaction (RT-PCR) method indicated that HSP 90 expression was enhanced on the chitosan films and decreased to normal levels on the WGA-modified chitosan films. Taken together, our data suggest that the use of WGA to enhance the cell-biomaterial interaction is a promising way to achieve appropriate cell adhesion and proliferation, the two key issues in tissue engineering. The third part was to address new applications of this novel P/C biomaterial. Endometriosis, a disease that affects many women in reproductive age, is defined as the presence of endometrial tissue outside its normal location. Although treatment options have improved considerably in recent years, but such as unexplained pelvic problems and infertility still remain. In this study, the growth-inhibitory effect of a novel P/C material-based biological spray on endometriosis was evaluated. Flow cytometry analysis reveled that both fractions of early apoptotic and late apoptotic cells increased in the endometrial cells treated with this P/C spray. This is the first trial using the P/C spray to successfully inhibit cell proliferation by inducing apoptosis. Therefore, this novel P/C spray should have great application potential in endometriosis therapy.

INTRODUCTION Increasing age of population is a great success of numerous breakthroughs in life science and improved health care. For a child born in 2015, for example, an average global life expectancy of meanwhile 71.4 years is assumed which increased by around 8% in the last decade [1]. In accordance with enhanced life expectancy, however, age-related health problems continuously rise. In this regard, the gap between patients awaiting transplantation and appropriate donors consequently will get larger in the future [2]. To this end, there is a need for new strategies in regenerative medicine [3]. Biomaterial matrices were developed to foster tissue regeneration by mimicking the key characteristics of the extracellular matrix (ECM) [4]. Modern biomaterial research focuses on 3D scaffolds, which can be adequately adapted toward specific requirements of the target tissue [5]. In this regard, flexible material platforms are wanted, whose properties can be adjusted over a wide range and independently of each other [6]. In this context, the macromer-based material concept is promising due to the high flexibility of macromers in chemical design and processability [7]. Macromers are reactive oligo- or polymeric molecules which act as monomers and can therefore be polymerized/cross-linked into a polymeric network [8]. The key principle of this approach is the synthesis of chemically well-defined structures which allows for a more precise control over the resulting properties of the cross-linked polymeric network when compared to conventional polymers. For example, macromer chemistry can be adjusted in terms of chemical macromer composition, valence, content of cross-linkable functionalities and molecular weight. The versatility of macromer-derived materials greatly increases when different macromer types are combined which potentially enables precise material tunability on multiple levels. The design flexibility of macromer-based networks motivated the investigation of two different macromer-based material concepts with regard to macromer processability and material adjustability. The following objectives were proposed: 1) To synthesize two sets of biodegradable, multi-valent macromers by using free-radical polymerization and ring-opening polymerization combined with established activation strategies. The synthesis setups will be tuned toward high macromer yields which will be required for processing into biomaterials with relevant sizes. 2) To physico-chemically characterize oligomeric macromers with regard to chemical composition, molecular weight and reactivity in order to yield well-defined macromer structures. NMR spectroscopy, gel permeation chromatography (GPC) and wet chemistry will be applied. 3) To characterize macromer processability into covalently cross-linked hybrid matrices. This work will focus on a soft macromer-cross-linked gelatin-derived hydrogel system for versatile biomedical applications as well as a rigid macromer/sol-gel glass hybrid material for hard tissue regeneration. Sets of different formulations will be investigated in order to characterize the range of macromer processability and to establish structure-property relationships. 4) To investigate strategies for the adjustment of material porosity. Besides the adaption via cross-linking density, porogen-leaching and 3D-printing approaches will be followed in order to introduce macroporosity and to enable a decoupling of porosity and chemical (nano)structure of the cross-linked network. 5) To determine key material properties relevant for regenerative applications, including mechanical properties by compression tests and oscillation rheology, in vitro matrix degradability, as well as material cytocompatibility in indirect and direct contact experiments. 6) To identify strategies for covalent functionalization of the hybrid materials. Post-fabrication functionalization via specifically introduced chemical functionalities is favored as it enables effective material decoration (almost) independent of the physico-chemical matrix properties. SUMMARY OF DISSERTATION The first material concept was based on anhydride-containing macromers which can be processed into hydrogel matrices by covalent cross-linking of amine-bearing macromolecules, such as gelatin [9–11]. The innovative aspect of this work was to decouple material functionalization from the physico-chemical properties of the cross-linked hydrogel network. To this end, a second chemical functionality was introduced which remained reactive in the hydrogel state and was therefore available for covalent post-fabrication functionalization strategies. Specifically, dual-functional macromers were synthesized by free-radical polymerization of maleic anhydride (MA) with diacetone acrylamide (DAAm) and pentaerythritol diacrylate monostearate (PEDAS) to yield oligo(PEDAS-co-DAAm-co-MA) (oPDMA) [12]. Amphiphilic oligomers (molecular weight (Mn) < 7.5 kDa) with anhydride contents of 7-20% were obtained. Fractions of chemically intact anhydrides of around 70% enables effective cross-linking with low molecular-weight gelatinous peptides (Collagel® type B, 11 kDa). Rigid two-component hydrogels (elastic modulus (E) = 4-13 kPa) with adjustable composition and physicochemical properties were formed. Reactivity of the incorporated methyl ketone functionality toward hydrazides and hydrazines was shown on the macromer level and in the cross-linked hydrogel by different strategies. Firstly, pre-fabricated hydrogels were successfully reinforced by secondary cross-linking with adipic acid dihydrazide (ADH). Secondly, pH-dependent immobilization of 2,4-dinitrophenylhydrazine (DNPH) to acid-soluble macromer derivatives as well as cross-linked oPDMA/COL matrices was demonstrated. Thirdly, reversible immobilization of a fluorescent hydrazide (AFH) was shown which was controlled by hydrogel ketone content, hydrazide ligand concentration and medium pH. This triple-tunability of hydrazide immobilization holds promise for adjustable and cost-effective hydrogel modification. Lastly, proof-of-concept experiments with hydrazido-functionalized hyaluronan (ATTO-hyHA) demonstrated the potential for covalent post-fabrication hydrogel decoration with ECM components. Hydrogel cytocompatibility was demonstrated and the introduction of DAAm into the hydrogel system resulted in superior cell material interactions when compared with previously established analogous ketone-free gels [13]. Limited ability of cells to migrate into deeper regions of these macromer-cross-linked gelatin-based gels further motivated the investigation of two different strategies to enhance hydrogel porosity [10,14]. On the one hand, the introduction of macropores was attempted by hydrogel fabrication in presence of poly(ethylene glycol) (Mn = 8000 Da, P8k). This polymer acted as porogen by phase separation during hydrogel formation. It was found that P8k was effectively extracted from the cross-linked matrix, while physico-chemical hydrogel properties remained unchanged. The second approach aimed at increasing mesh size of the cross-linked network by using hydrogel building blocks with increased molecular weights. In particular, high molecular-weight gelatin (160 Bloom, G160) was cross-linked by macromers with low MA content. Homogeneous and mechanically stable hydrogels were obtained and physico-chemical properties were determined. Successful optimization of hydrogel porosity was functionally shown by enhanced cell migration and improved release profile of incorporated nanoparticles [15]. In the second macromer-based material, hydrolytically degradable multi-armed macromers were covalently introduced into a tetraethoxysilane(TEOS)-derived silica sol in order to address the insufficient degradability of glass-based materials [16]. In detail, oligo(D,L-lactide) units were introduced into three- (TMPEO, Tx) and four-armed (PETEO, Px) ethoxylated alcohols by ring-opening polymerization, followed by activation with 3-isocyanatopropyltriethoxysilane (ICPTES) to yield TxLAy-Si and PxLAy-Si macromers [17,18]. A series of 18 oligomers (Mn: 1100-3200 Da) with different degrees of ethoxylation and varying length of oligoester units was synthesized. Applicability of a previously established indirect rapid prototyping method enabled fabrication of macromer/sol-gel-glass-derived class II hybrid scaffolds with controlled porosity [19]. Successful processability of a total of 85 different hybrid scaffold formulations allowed for identification of relevant structure-property relationships. In vitro degradation was analyzed over 12 months and a continuous linear weight loss (0.2-0.5 wt%/d) was detected which was controlled by oligo(lactide) content and matrix hydrophilicity. Compressive strength (2-30 MPa) and compressive modulus (44-716 MPa) were determined and total content, oligo(ethylene oxide) content, oligo(lactide) content and molecular weight of the oligomeric cross-linkers as well as material porosity were identified as the main factors determining hybrid mechanics by multiple linear regression. Cell migration into the entire scaffold pore network was indicated in cell culture experiments with human adipose tissue-derived stem cells (hASC) and continuous proliferation over 14 days was found. Overall, two macromer-based material platforms were established in which material versatility was realized by three main principles: I) synthesis of macromers with different chemical composition, II) combination of macromers with a second oligomeric building block, and III) flexible processability of these dual-component hybrid formulations into porous scaffold materials. Precise adjustability of material properties as demonstrated in both concepts offers potential for application of these hybrid materials for a wide range of regenerative purposes. REFERENCES (1) World Health Statistics of the WHO. http://www.who.int/gho/publications/world_health_statistics/en/ 2017. (2) OPTN/UNOS Public Comment. https://optn.transplant.hrsa.gov/ 2017. (3) Puppi, D.; Chiellini, F.; Piras, a. M. M.; Chiellini, E. Prog. Polym. Sci. 2010, 35 (4), 403–440. (4) Patterson, J.; Martino, M. M.; Hubbell, J. A. Mater. Today 2010, 13 (1–2), 14–22. (5) Picke, A.-K.; Salbach-Hirsch, J.; Hintze, V.; Rother, S.; Rauner, M.; Kascholke, C.; Möller, S.; Bernhardt, R.; Rammelt, S.; Pisabarro, M. T.; Ruiz-Gómez, G.; Schnabelrauch, M.; Schulz-Siegmund, M.; Hacker, M. C.; Scharnweber, D.; Hofbauer, C.; Hofbauer, L. C. Biomaterials 2016, 96, 11–23. (6) Loth, R.; Loth, T.; Schwabe, K.; Bernhardt, R.; Schulz-Siegmund, M.; Hacker, M. C. Acta Biomater. 2015, 26, 82–96. (7) DeForest, C. A.; Anseth, K. S. Nat. Chem. 2011, 3 (12), 925–931. (8) Nic, M.; Jirát, J.; Košata, B.; Jenkins, A.; McNaught, A.; Wilkinson, A. IUPAC, Research Triangle Park, NC 2014. (9) Loth, T.; Hennig, R.; Kascholke, C.; Hötzel, R.; Hacker, M. C. React. Funct. Polym. 2013, 73 (11), 1480–1492. (10) Loth, T.; Hötzel, R.; Kascholke, C.; Anderegg, U.; Schulz-Siegmund, M.; Hacker, M. C. Biomacromolecules 2014, 15 (6), 2104–2118. (11) Kohn, C.; Klemens, J. M.; Kascholke, C.; Murthy, N. S.; Kohn, J.; Brandenburger, M.; Hacker, M. C. Biomater. Sci. 2016, 4, 1605–1621. (12) Kascholke, C.; Loth, T.; Kohn-Polster, C.; Möller, S.; Bellstedt, P.; Schulz-Siegmund, M.; Schnabelrauch, M.; Hacker, M. C. Biomacromolecules 2017, 18 (3), 683–694. (13) Sülflow, K.; Schneider, M.; Loth, T.; Kascholke, C.; Schulz-Siegmund, M.; Hacker, M. C.; Simon, J.-C.; Savkovic, V. J. Biomed. Mater. Res. A 2016, 104 (12), 3115–3126. (14) Loth, T. Diss. Univ. Leipzig, Fak. für Biowissenschaften, Pharm. und Psychol. 2016. (15) Schwabe, K.; Ewe, A.; Kohn, C.; Loth, T.; Aigner, A.; Hacker, M. C.; Schulz-Siegmund, M. Int. J. Pharm. 2017, 526 (1–2), 178–187. (16) Rahaman, M. N.; Day, D. E.; Sonny Bal, B.; Fu, Q.; Jung, S. B.; Bonewald, L. F.; Tomsia, A. P. Acta Biomater. 2011, 7 (6), 2355–2373. (17) Schulze, P.; Flath, T.; Dörfler, H.-M.; Schulz-Siegmund, M.; Hacker, M.; Hendrikx, S.; Kascholke, C.; Gressenbuch, M.; Schumann, D. Ger. Pat. No. DE102014224654A1 2016. (18) Kascholke, C.; Hendrikx, S.; Flath, T.; Kuzmenka, D.; Dörfler, H.-M.; Schumann, D.; Gressenbuch, M.; Schulze, F. P.; Schulz-Siegmund, M.; Hacker, M. C. Acta Biomater. 2017, 63, 336–349. (19) Hendrikx, S.; Kascholke, C.; Flath, T.; Schumann, D.; Gressenbuch, M.; Schulze, P.; Hacker, M. C.; Schulz-Siegmund, M. Acta Biomater. 2016, 35, 318–329.

Injuries to the meniscus are a common and important source of mobility issues in the knees of young active individuals, as well as elderly individuals. Conventional treatments for these injuries involve surgical resections of the damaged portions of tissue in order to relieve immediate clinical symptoms. However, with a decreased amount of meniscal tissue remaining, the load-bearing and load-distribution capacities remain compromised and inevitably lead to the development of osteoarthritis.1 In view of these deficiencies, tissue engineering has emerged as a promising alternative approach to meniscus repair. In this approach, biodegradable synthetic materials have been proposed as scaffolds to stimulate and support cell-mediated tissue remodeling. A wide range of synthetic materials have been developed to respond to the physical and chemical requirements of a scaffold, but many lack the necessary biological properties to respond to cellular stimuli. In addition, many of these materials are deficient in mechanical strength. The aim of this study was to develop a novel biomaterial that addresses these limitations. Poly(trimethylene carbonate) (PTMC) was selected as the main component of the scaffold due its highly suitable material properties. PTMC is a biocompatible, biodegradable polymer with excellent elastomeric properties and mechanical strength. It also offers the advantage of providing long-term mechanical support due to its low degradation rate. However, PTMC alone cannot stimulate tissue regeneration due to its bio-inert nature. In order to provide an ideal environment to support tissue repair, it must possess bioactive signals. PTMC was combined with a collagenase-sensitive peptide substrate to render the scaffold invasive by cells. The peptide also served to increase the slow degradation rate of PTMC by providing cleavage points throughout the network. The compressive strength of this material was significantly higher than previously used scaffold materials. Additionally, the material possessed enhanced toughness and elasticity, high equilibrium water content, and a tunable degradation profile. Unlike currently used scaffolding materials, this material satisfies all of the necessary requirements to function as an effective scaffold for meniscus regeneration.
Thesis (Master, Chemical Engineering) -- Queen's University, 2013-11-20 15:36:06.12

碩士
國立臺北科技大學
製造科技研究所
107
Low-temperature deposition manufacturing(LDM) is only one method of bioprinting that fabricates bioscaffold using Polymer biomaterial solution. LDM technology's feature is its pore structures of scaffolds, which is advantageous in cell culture. Unfortunately, the feature declines mechanical properties of the scaffolds. LDM technology is not suitable for fabricating scaffolds of higher mechanical strength requirements. Therefore, the Development of a bioprinter for fabricating bioscaffold of solid fiber using Polymer biomaterial solution will be proposed in this research. The process fabricates bioscaffolds with solid fiber; through the process of which solvent evaporation reduces the volume of the material. The system proposed consists of four modules which allows the produced objects a maximum build volume of 200mm×200mm×100mm. The first module is in charge of moving the nozzle in the XYZ direction, while the second and third controls the temperature of the material and build platform, respectively. The last module adjusts the airflow that controls the evaporation of the solvent of material. Finally, diverse 3D models with different structures were fabricated in order to inspect the performance of the bioprinter.

博士
國立臺北科技大學
工程科技研究所
101
The thesis work proposes was to synthesize novel nanomaterials like graphene, carbon nanotubes based biomolecules for application in fabrication of biosensors, biofuel cells and solar cells. The research includes the synthesis of carbon based nanocomposite and their different morphologies, biosynthesis of nanomaterials, bioelectrode modification, characterization of biomolecules modified electrodes, and their applications. The research focus will be mainly in selection of various suitable compounds/biomolecules for carbon nanotubes, Fullerene, graphene based on nanocomposite, and other morphology composites, and their characterization. The main work is the application for the prepared nanocomposite for electrode modification. The modified electrodes were tested for their electrocatalytic activities. CNT-based sensors generally have higher sensitivities, lower limits of detection, and faster electron transfer kinetics than traditional carbon electrodes. Many variables were tested and then optimized to create a CNT-based sensor. This study highlights different biomolecules and compares electrode design techniques for selective analyte detection. Carbon nanotubes possess similar dimensions to many biological molecules used within biosensors. MWCNTs can be oxidized to form surface carboxyl groups which can then be modified to allow covalent linking to enzymes or others. The design of biofuel cells involves the application of enzymes or microorganisms as catalyst for the targeted oxidation and reduction of specific fuel and oxidizer substrates at both electrodes to generate an electrical power output. The emergence of biofuel cells is driven by the need for clean methods of producing electricity from renewable fuel sources, and the ever-increasing depletion of fossil fuels. Dye molecules for sensor devices exhibits interesting enhancement in the electrocatalytic activity towards the oxidation or reduction of several biochemical and inorganic compounds. Dye for the functionalization of CNTs or Graphene leads to the construction of efficient electrochemical sensors. The above mentioned functional materials/ligands are both electrochemically active and photoactive. So, by using these dye molecules/polymers functionalized CNTs can enhance electrocatalysis and photoelectrocatalysis of various analyte reactions. This photoelectrocatalysis studies could be very helpful for developing new type of biosensors.

Silks are extracorporeal fibrous protein materials. Classically, silkworm (Bombyx mori) and orb-spiders (Arachnida: Araneidae) have served as model organisms in which to investigate silk protein structure-function relationships. However, silk production has evolved multiple times in insects. The silk proteins of many insects do not fold into the beta-sheet structures found in silkworm and spider silks but into coiled-coils, collagen helices or polyglycine helices. Therefore, the structure-function relationships elucidated for silkworm and spider silk proteins may be too narrow to apply to insect silk proteins generally. To increase the available data, I examined silk production by raspy crickets (Orthoptera: Gryllacrididae), silverfish (order Thysanura), praying mantises (order Mantodea), glow-worms (Diptera: Keroplatidae), and sawflies (Hymenoptera: Tenthredinidae). Silk protein primary structures were investigated using transcriptomics, mass spectrometry, and amino acid analysis; secondary and tertiary structures were investigated by infrared and Raman spectroscopy, nuclear magnetic resonance, circular dichroism spectroscopy, and bioinformatics. Novel features of silk production were related to idiosyncrasies of each insect group, while features found in multiple silk-producing groups were associated with general mechanisms of silk production. A comparative analysis of silk proteins revealed a correlation between predominant secondary structure type and more general architectural features such as length and repeat regularity: silk proteins that fold into coiled-coils and collagen helices had low molecular weights and high repeat regularity, suggesting they fold into short semi-rigid rods; beta-sheet-forming silk proteins were found to be more variable in molecular weight and have lower repeat regularity. Based on these data, I propose three major mechanisms of silk fabrication by insects: a) mesogenic ordering of short rod-like proteins, a process for which the coiled-coil and collagen structures are well-suited; b) molecular extension of long flexible protein chains to promote intermolecular bonding, which is suitable for the formation of beta-sheet-rich silks; and c) entanglement of protein chains, which is suited to silks with a high degree of disorder. Thus, many features of insect silk proteins are adaptations for material fabrication. In a few cases, particular structural motifs constituted adaptations conferring a mechanical property required for the silk's function in the solid state. However more often proteins were observed to have features promoting dense protein packing in a general way. I explain these data by consideration of how silk mechanical behaviour relates to the fitness advantage conferred to individual insects by silk production. Specifically, I suggest protein features ensuring structural homogeneity and molecular orientation result in silk materials with mechanical properties sufficient for most purposes. Further increases in properties such as strength lead to little or no fitness increase. Local maxima in the fitness landscape associated with distinct protein secondary structures or fabrication mechanisms trap silk proteins in one of several states. Overall, silk protein evolution can to a large extent be understood as convergence of a number of independently co-opted proteins of other functions toward one of several distinct functional archetypes.