Healing large bone defects remains a clinical challenge. While autografts are the gold standard treatment for large bone defects, they are limited by availability and donor site pain. Growth factor treatments such as BMP therapy provide a promising alternative but are expensive and present clinical safety concerns, primarily due to delivery of BMPs at supraphysiological doses. Integrins are ECM receptors which mediate crucial cell functions such as adhesion and differentiation. Therefore, understanding the role of integrins in bone formation and directing desired interactions may enable modulation of host cell functions for therapeutic applications. In this work, beta 1 integrins were deleted in osteolineage cells of transgenic mice at three different stages of differentiation to elucidate their role in bone development. We also engineered bioartificial PEG-based matrices which target the pro-osteogenic alpha 2 beta 1 integrin to promote bone healing. Conditional deletion of beta 1 integrins in osteochondroprogenitor cells under the Twist 2 promoter resulted in severe pre-natal skeletal mineralization defects and embryonic lethality. Targeted deletion of beta 1 integrins in osterix-expressing osteoprogenitors resulted in growth abnormalities, reduced calvarial mineralization, impaired femur development, and tooth defects. However, mice lacking beta 1 integrins in osteocalcin-expressing osteoblasts and osteocytes displayed only a mild skeletal phenotype, indicating that beta 1 integrins play an important role in early skeletal development, but are not required for mature osteoblast function. PEG hydrogels functionalized with the integrin-specific GFOGER ligand enhanced bone regeneration, induced defect bridging in combination with low doses of rhBMP-2 and stimulated improved bone healing compared collagen sponges, which are the clinical standard delivery vector for BMP-2 therapy. These results suggest that treatment with bioartificial integrin-specific PEG hydrogels may be a promising clinical strategy for bone regeneration in large bone defects.

Low temperature co-fired ceramic (LTCC) electronic packaging materials are applied for their electrical and mechanical properties, high reliability, chemical stability and ease of fabrication. Three dimensional features can also be prepared allowing integration of microfluidic channels and cavities inside LTCC modules. Mechanical, optical, electrical, microfluidic functions have been realized in single LTCC modules. For these reasons LTCC is attractive for biomedical microfluidics and Lab-on-a-Chip systems. However, commercial LTCC systems, optimized for microelectrics applications, have unknown cytocompatibility, and are not compatible with common surface functionalization chemistries. The first goal of this work is to develop biocompatible LTCC materials for biomedical applications. In the current work, two different biocompatible LTCC substrate materials are conceived, formulated and evaluated. Both materials are based from well-known and widely utilized biocompatible materials. The biocompatibilities of the developed LTCC materials for in-vitro applications are studied by cytotoxicity assays, including culturing endothelial cells (EC) both in LTCC leachate and directly on the LTCC substrates. The results demonstrate the developed LTCC materials are biocompatible for in-vitro biological applications involving EC. The second goal of this work is to develop functional capabilities in LTCC microfluidic systems suitable for in-vitro and biomedical applications. One proposed application is the evaluation of oxygen tension and oxidative stress in perfusion cell culture and bioreactors. A Clark-type oxygen sensor is successfully integrated with LTCC technique in this work. In the current work, a solid state proton conductive electrolyte is used to integrate an oxygen sensor into the LTCC. The measurement of oxygen concentration in Clark-type oxygen sensor is based on the electrochemical reaction between working electrode and counter electrode. Cyclic voltammetry and chronoamperometry are measured to determine the electrochemical properties of oxygen reduction in the LTCC based oxygen sensor. The reduction current showed a linear relationship with oxygen concentration. In addition, LTCC sensor exhibits rapid response and sensitivity in the physiological range 1─9 mg/L. The fabricated devices have the capabilities to regulate oxygen supply and determination of local dissolved oxygen concentration in the proposed applications including perfusion cell culture and biological assays.

Automated and continuous processes are the future trends in downstream protein purification. A functionalized nanometer-scale membrane electrode system, mimicking the function of cell wall transporters, can selectively capture genetically modified proteins and subsequently pump them through the system under programmed voltage pulses. Numerical study of the two-step pulse pumping cycles coupled with experimental His-GFP releasing study reveals the optimal 14s/1s pumping/repel pulse pumping condition at 10 mM bulk imidazole concentration in the permeate side. A separation factor for GFP: BSA of 9.7 was achieved with observed GFP electrophoretic mobility of 3.1×10-6 cm2 s-1 V-1 at 10 mM bulk imidazole concentration and 14 s/1 s pumping/repel duration. The purification of His6-OleD Loki variant directly from crude E. coli extracts expression broth was demonstrated using the pulse pumping process, simplifying the separation process as well as reducing biopharmaceutical production costs. The enzymatic reactions showed that His6-OleD Loki was still active after purification. A nanoporous membrane/electrode system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzymes-complex system was demonstrated. The substrates residence time on the immobilized enzyme can be precisely controlled by changing the pumping rate and thereby prevent a secondary hydrolysis reaction. Immobilized enzyme showed long term storage longevity with activity half-life of 50 days at 4℃ and the ability to be regenerated. One-step immobilization and purification of His-tagged OleD Loki variant directly from expression broth, yielded 98% Uridine Diphosphate glycosylation and 80% 4-methylumbelliferone glycosylation conversion efficiency for the sequential reaction. A flow-through electroporation system, based on a novel membrane/electrode design, for the delivery of membrane-impermeant molecules into Model Leukocyte cells was demonstrated. The ability to apply low voltage between two short distance electrodes contributes to high cell viability. The flow-through system can be easily scaled-up by varying the micro-fluidic channel geometry and/or the applied voltage pulse frequency. More importantly, the system allows the electrophoretical pumping of molecules from the reservoir across the membrane/electrode system to the micro-fluidic channel for transfection, which reduces large amount of reagents used.

Molecularly imprinted polymers (MIPs) are biomimetic sensing elements that combine the accuracy and highly specific binding affinity of a biosensor, with the robustness and reusability associated with artificial electrochemical sensors. This thesis investigates the application of a MIP sensor to address the challenge of phosphate detection in precision agriculture. Traditional chemical sensing approaches using portable electrochemical sensors display a significant cross-interference between inorganic phosphate and other nutrient ions. This is due to the low position of phosphate in the Hofmeister Selectivity Series for anions, its high electronegativity and its pH dependent structure, resulting in a molecule that is very difficult to detect. To address this challenge, a sensor was created by spin coating a phosphate selective MIP onto a substrate containing a series of electrodes. These electrode devices allowed for electrical measurements to be taken using an inductance, capacitance and resistance (LCR) testing station, and to observe the change in the materials dielectric constant as the binding sites become occupied by the target analyte. The devices underwent several design reiterations to produce an optimised setup consisting of 100 interdigitated chrome electrodes with a width of 1 μm and a separation distance of 1 μm on a quartz substrate. The final electrode design was used to carry out a nutrient cross-interference study across several polymer permutations. The purpose of this was to develop an optimised MIP formulation for binding specifically to inorganic phosphate ions. From this study, an optimal phosphate selective MIP was identified, based upon a binding site constructed from methacrylic acid around a diphenyl phosphate template molecule. During capacitance measurements, this MIP formulation demonstrated a clear preferential response to phosphate (1610 pF) over the average capacitance results observed following exposure to the competing nitrate (1286 pF) and sulphate (1212 pF) nutrients tested in the cross-interference study.

Changes in the biomechanical properties of cells accompanying the development of various pathological conditions have been increasingly reported as biomarkers for various diseases and as a predictor of disease progression stages. For instance, cancer cells have been found to be less stiff compared to their healthy counterparts due to the proteomic and lipidomic dysregulations conferred by the underlying pathology. The separation and selective recovery of cells or extracellular vesicles secreted from such cells that have undergone these changes have been suggested to be of diagnostic and prognostic value. This dissertation first describes the implementation of a stiffness-based separation of phosphatidylcholine-based vesicles using a method first introduced based on the research in this work and was dubbed thermally-assisted acoustophoresis, or thermo-acoustophoresis. By tuning the temperature, we achieved the separation of vesicles of the same size, shape, and charge but with different stiffness values. It was observed that at a specific transition point, the acoustic contrast factor of vesicles changed sign from positive to negative. This change was mainly due to change in the compressibility of the vesicles, which is inversely proportional to stiffness. The acoustic contrast temperature (Tϕ), corresponding to the temperature at which the contrast factor switches sign, was determined to be unique to the composition of the vesicles. This unique temperature signature allowed us to develop this separation method of vesicles with distinct membrane stiffness with target outlet purities exceeding 95%. We have further explored the functionality of this method by experimenting with cholesterol-containing vesicles. In cells, the cholesterol content plays a crucial role in determining stiffness. Changes in the cholesterol content in cellular membranes can be an indication of pathological disorders. We evaluated the Tϕ of vesicles at different cholesterol molar ratios (Xchol) and developed a multi-stage lab-on-a-chip method to accomplish for the first time the separation of a three-vesicle mixture. Using Xchol = 0.1, 0.2, and 0.3 vesicles, we obtained efficiencies exceeding 93%. The simplicity, rapidity, and label-free nature of this approach holds promise as a diagnostic and separation tool for cells affected by diseases that affect the stiffness and extracellular vesicles such as exosomes and microvesicles.

This thesis focuses on the electrical output of polyaniline films subjected to uniaxial strain in hydrochloric acid solutions. Polyaniline belongs to novel class of materials known as conducting polymers. Alternating single and double bonds in the backbone of conducting polymers allow them to transmit electric charge when they are doped with negatively charged ions. Modifying the degree of doping and other electrical/chemical treatments allow conducting polymers to exhibit conducting, semi-conducting, or insulating electrical properties. Resilient mechanical properties, good processability, and low cost make conducting polymers good candidates for applications traditionally held by metals and semi-conductors. When tensile strain is applied to polyaniline in an electrolyte solution, the material selectively absorbs negatively charged ions. This charge imbalance produces a measurable electrical output. Theoretical models based on Fick’s second law of diffusion were compared against experimental results to determine fundamental material properties such as diffusivity and ion solubility in polyaniline. These properties were used to quantify polyaniline as a sensor material based on characteristics including sensitivity, accuracy, precision, range, linearity, and error. Films were cast from solutions of polyaniline powder (Mn = 65,000) in N-methyl-2-pyrrolidinone solvent, with thicknesses ranging from 2.72 to 158 µm.

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.

Chronic wound healing can be seriously impeded by the formation of biofilms, infections, peri-wound edema, hematoma, osteomyelitis, and the formation of reactive oxidative species (ROS). We hypothesize that a scaffold created from Silver-Doped Nanoceria (AgCNP) embedding silk can be beneficial to aid the wound healing process, inhibit inflammation and prevent microorganisms from forming a biofilm over the wound. Current wound healing methods such as intradermal injections are not advantageous to use since they can cause unwanted responses elsewhere in the body other than the wound site. Silk, however, has a positive impact on the wound healing effect and can be used as an alternative delivery method to deliver the drugs to the target site rather than intradermal injections since its degradability is controllable and it is bioresorbable, therefore it can get absorbed by the body and degrade safely without causing bodily harm. AgCNPs are used as they have antimicrobial/antioxidant properties to scavenge harmful ROS species at the wound site and can also modify silk for UV protection. As silk's degradability can be controlled, our experiment will involve collecting data on release studies conducted in vitro to see how long it takes for the silk patch to release the drugs. Our goal is to ensure the drug is not released immediately but rather over a longer controlled time manner to protect the wound while healing.

Coral reefs are an important element of marine ecosystem that are critical to maintain a healthy environment. Unfortunately, in recent years coral reefs are doing poorly and many in parts of the ocean are simply dying. Therefore, study of coral’s structural response to external loads could answer what will happen with their structures, while they exhibit different types of loading. Therefore, the proposition of using in-situ micro-Raman spectroscopy to study skeletons of Acropora cervicornis was used. Coral skeleton samples I subjected to mechanical loading studied their vibrational properties by exciting the material with 532nm visible light. A uniaxial compressive load I applied using a MTS universal testing machine and then using the Raman Spectroscopy to study the vibrational response of coral skeletons. Indentations used Vickers Hardness tester and performed 2D mapping of the coral structure around the indentation. If it’s expected that as a result of the proposed research the better understanding of structural stability of the Acropora Cervicornis coral skeletons will be achieved.

Biopolymers are becoming more attractive as advanced wound dressings because of their naturally derived origin, abundance, low cost and high compatibility with the wound environment. Arabinoxylan (AX) is a class of polysaccharide polymers derived from cereal grains that are primarily used in food products and cosmetic additives. Its application as a wound dressing material has yet to be realized. In this two-pronged project, arabinoxylan ferulate (AXF) was fabricated into electrospun fibers and gel foams to be evaluated as platforms for wound dressing materials. In the first study, AXF was electrospun with varying amounts of gelatin. In the second study, AXF was dissolved in water, enzymatically crosslinked and lyophilized to form gel foams. The morphology, mechanical properties, porosity, drug release kinetics, fibroblast cell response and anti-microbial properties were examined for both platforms. Carbohydrate assay was conducted to validate the presence of arabinoxylan ferulate in the electrospun GEL-AXF fibers. Swelling and endotoxin quantification studies were done to evaluate the absorptive capacity and sterilization agent efficacy respectively in AXF foams. The results indicated successful fabrication of both platforms which validated the porous, absorptive, biocompatibility and drug release properties. The results also exhibited that silver impregnated AXF scaffolds inhibited growth of Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis bacteria species, anti-microbial properties necessary to function as advanced wound dressing materials. Future work will be done to improve the stability of both platforms as well as evaluate its applications in vivo.