The Body-on-a-Chip (BoaC) is a microfluidic BioMEMs project that aims to replicate major organs of the human body on a chip, providing an in vitro drug testing platform without the need to resort to animal model testing. Using a human model also provides significantly more accurate drug response data, and may even open the door to personalized drug treatments. Microelectrode arrays integrated with human neuronal or human cardiac cells that are positioned on the electrodes are essential components for BoaC systems. Fabricating these substrates relies heavily on chemically patterned surfaces to control the orientation and growth of the cells. Currently, cells are plated by hand onto the surface of the chemically patterned microelectrode arrays. The cells that land on the cytophobic 2-[Methoxy(Polyethyleneoxy)6-9Propyl]trimethoxysilane (PEG) coating die and detach from the surface, while the cells that land on the cytophilic diethylenetriamine (DETA) coating survive and attach to the surface exhibiting normal physiology and function. The current technique wastes a significant amount of cells, some of which are extremely expensive, and is labor intensive. Cell printing, the process of dispensing cells through a 3D printer, makes it possible to pinpoint the placement of cells onto the microelectrodes, drastically reducing the number of cells utilized. Scaled-up manufacturing is also possible due to the automation capabilities provided by printing. In this work, the specific conditions for printing each cell type is unique, the printing of human motoneurons, human sensory neurons and human cardiac cells was investigated. The viability and functionality of the printed cells are demonstrated by phase images, immunostaining and electrical signal recordings. The superior resolution of cell printing was then taken one step further by successfully printing two different cell types in close proximity to encourage controlled innervation and communication.

Three-dimensional (3D) cell cultures have been widely used in biological research since two-dimensional (2D) cultures have many limitations including alteration of cell morphology, metabolic pathways and gene expression. Therefore, the application of 2D cell cultures in pharmaceutical companies for drug screening causes reproducibility issues in animal studies, which strongly influences the efficiency of drug development. The aim of my studies is to develop 3D cell culture models to better mimic the extracellular matrix (ECM) in tumor microenvironment, not only for deeper understanding of interaction between cells and ECM, but also for the application in drug screening. Three different compositions of chitosan-alginate (CA) scaffolds with different stiffness were produced to mimic prostate cancer (PCa) progression stages. The results showed that PCa cells demonstrated stiffness independent growth and protein expression. But surprisingly, it was found that CA scaffolds could identify PCa cell phenotypic characteristics. Further, a novel 3D porous chitosan-chondroitin sulfate (C-CS) scaffold, with three CS compositions, were developed to mimic the PCa progression, since the clinical research has suggested that the CS is found in normal prostate tissue, greater in PCa, and further in metastatic sites. The results showed that CS can promote PCa cell metastasis-related gene expression and anti-cancer drug resistance. Although CA and C-CS scaffold provided more insights on PCa, 3D cell culture is more complicated to use than 2D cultures, which limited its application in industry. Therefore, a novel biomaterial format, named Frozen Films, were developed to combine the advantages of 2D and 3D culture while reducing their drawbacks. Cell cultures tested on Frozen Films demonstrated that cells had 3D culture performance, but with much easier operation process. Overall, those studies demonstrated that CA scaffolds, C-CS scaffolds and Frozen Films could be promising in vitro platforms for cellular research, with potential applications for in vitro anti-cancer drug screening.

The assembly of actin, the essential cytoskeleton protein, into filaments and bundles/networks is important in various cellular processes including cell movement and morphogenesis. Actin bundle formation occurs in crowded intracellular environments with the aid of actin-crosslinking proteins. The role of actin-crosslinking proteins such as fascin and a-actinin in bundle formation has been investigated, however, how intracellular environments affect actin crosslinker-induced bundle formation is unknown. In the first two parts of this dissertation, we explore the effects of macromolecular crowding and cation interactions on the organization and mechanics of actin crosslinker-induced bundles. To determine how changing environmental conditions modulate actin bundling, we utilize total internal reflection fluorescence microscopy, atomic force microscopy, and transmission electron microscopy. Through biophysical analysis on actin bundles we show crowding and crosslinking proteins competitively involve in bundle formation. Cations shift actin organization from networks to bundles through destabilization of binding interactions. In addition, molecular dynamics simulations support the counteractive interactions between crowding/cations and actin-crosslinking proteins during bundle formation. Similarly, cellular environments can further be affected by external factors such as nanomaterials that are used in various biomedical applications. Graphene is a widely used nanomaterial that potentially affects the dynamics of actin cytoskeleton. However, its influence on actin assembly dynamics at the molecular level is not well understood. In the third part of this dissertation, we investigate the effect of graphene on actin assembly by localized fluorescence microscopy and real-time pyrene assembly kinetics. Both direct visualization of individual filaments growth and bulk actin polymerization demonstrate that graphene accelerates the actin assembly kinetics. The extent of graphene effects on actin assembly depends on the nanomaterial structural properties including excluded area effects and hydrophobicity. Taken together, these studies provide fundamental insights into how the actin organization, mechanics, and assembly are modulated by intracellular and external factors.

Magnesium (Mg) and its alloys have received increasing interest as a new generation of biodegradable metallic biomaterials. Current metallic orthopedic implants cause stress shielding effects and may require removal surgery as permanent implants. The biocompatibility, biodegradability, close mechanical properties to human cortical bone tissue, osteogenic properties, and angiogenic properties of Mg alloys make them potential candidates for bone repair. However, the low corrosion resistance of Mg alloys that results in hydrogen release and loss of mechanical integrity has restricted their application as orthopedic implants. Also, two critical factors facilitating bone regeneration are angiogenesis which enhances new vessel formation for providing oxygen and nutrients, and osteogenesis, which promotes formation of new bones at the fracture site. In this research, we developed a new biodegradable Mg alloy (Mg-Sc-Sr) using scandium (Sc; 2wt.%) and strontium (Sr; 2wt.%). In the first part of the research, our results showed a 49 % reduction in the corrosion rate of Mg-Sc-Sr compared to Mg. Furthermore, the Mg-Sc-Sr demonstrated a significant decrease in bacterial biofilm formation (62.8%) compared to Mg. Also, a substantial increase in osteoblastic differentiation of hBM-MSCs was shown by Alizarin red staining (1.9-fold increase vs. control) and alkaline phosphatase activity after the addition of Mg-Sc-Sr extract. In this work, the effect of heat treatment at two different temperatures (300°C and 400°C) was evaluated. In the second part of the research, the angiogenesis evaluation of Mg-Sc-Sr alloy on Human umbilical vein endothelial cells (HUVECs) exhibited that adding Mg-Sc-Sr extract significantly enhanced the VEGF release (85%) and significantly reduced scratch width (100%) after 24 h compared to the control group (HUVECs cultured with growth medium). The osteogenic properties of Mg-Sc-Sr were also confirmed using co-culture of hBM-MSCs and HUVECs that showed a significant increase in osteoblastic differentiation in vitro by Von kossa staining and alkaline phosphatase staining.

Orientador: Antônio Carlos Guastaldi / Banca: Sandra Helena Pulcinelli / Banca: Luis Geraldo Vaz / Banca: Jorge Enrique Rodriguez Chanfrau / Banca: Gildásio de Cerqueira Daltro / Resumo: A engenharia de tecidos é um campo que aplica os princípios de engenharia e ciências biológicas para desenvolver substitutos biológicos que restauram, mantém ou melhoram o tecido ou a função do órgão por inteiro. A presente tese estudou arcabouços (scaffolds) produzidos com celulose bacteriana/fosfato de cálcio voltados para a regeneração óssea. Foram analisados a modificação do processo de fermentação da celulose bacteriana com moléculas naturais (sulfato de condroitina e ácido hilaurônico), modificações físicas superficiais empregando-se irradiação gamma na celulose bacteriana e método biomimético para formação de fosfato de cálcio na superfície da membrana, posteriormente foram testados os biomateriais com células do ovário de hamster chineses (CHO-K1 cells). A modificação com moléculas naturais alterou a carga superficial e a cristalinidade das membranas, a modificação física alterou também propriedades físico-químicas de superfície, interferindo no processo de deposição de fosfato de cálcio. Os fosfatos de cálcio testados pelo método biomimético (SBFs 1 e 3), tiveram resultados melhores quanto a sua incorporação na superfíce das membranas não irradiadas de celulose bacteriana e seus nanocompósitos. / Doutor

<p>We explore the effect of size on the phase stability of nanosystems by comparing calculated trends with the annealing behavior of nanoparticles (NPs) initially in a core-shell configuration. The NPs are characterized using a variety of transmission electron microscopy (TEM) techniques.</p> <p>We first theoretically consider the equilibrium within a Au-Pt NP of a given size. When considering the contribution of surface and interface energies, we note the appearance of a restricted composition range of the phase diagram over which the liquid and solid phases cannot coexist in a core-shell configuration. A critical radius of ",42 nm is identified below which the NP is single-phased for any composition. It is demonstrated that both branches of the miscibility gap of the Au-Pt phase diagram shift towards the Au-rich composition with increasing curvature. The magnitude of the shift is found to be strongly correlated with the coupling of nonlinear terms entering the Gibbs energy. The main contribution to the shift arises from the composition-dependent surface energy, calculated by considering the selective adsorption of Au to the surface, evaluated using the available thermodynamic properties of the Au-Pt system.</p> <p>An array of TEM-related analytical methods were developed or adapted for the characterization of individual NPs. In particular, chemical maps with quantitative information from a NP with a spatial resolution of '" 1.2 nm could be achieved, with their corresponding error analysis. We introduce an algorithm to retrieve the radial elemental composition from the projected chemical map of a NP if a spherical symmetry can be assumed and test it with NPs of known structures. We also present a technique to determine the composition of a NP having one of the elements depleting during analysis, and test it experimentally with 5-20 nm Au-Ag NPs. Typically, for every Ag characteristic X-ray detected, one Ag atom is lost to knock-on damage. We discuss the detection limit of the method as a function of NP size and composition.</p> <p>We follow the structural evolution of a ",20 nm Au(core) Pt(shell) NP during annealing at various temperatures between 300 and 800 °e. At low temperatures, interdiffusion occurs between the core and the shell, while at temperatures abovt: ",600 °e, the configuration evolves towards one composed of Au- and Pt-rich spherical caps, separated by a relatively fiat interface. We could measure a 5-10% shift in the composition of each phase with respect to the bulk phase diagram that we assigned to capillarity effect. The shift agrees qualitatively with the calculated trends. The ratio of the surface to the interface energy is measured directly from a TEM micrograph of a segregated NP and is in close agreement with the calculated ones.</p> <p>This work contributes to the understanding of the phase stability of binary NPs. The prospect of extending these studies to NPs of other bimetallic systems while probing their properties seems promising, especially in view of their catalytic, magnetic and optical potential.</p> / Doctor of Philosophy (PhD)

Oligonucleotides are popular recognition-based biomaterials assembly and disassembly tools due to their specificity and ease of control. Their susceptibility to degradation by nucleases and false positive signals under certain conditions, however, has led to great interest in chemically modified oligonucleotides such as locked nucleic acids (LNA) that enhance both nuclease resistance and target specificity. This dissertation extends prior work with DNA sequences to investigate incorporating locked nucleic acid (LNA), a synthetic oligonucleotide, in isothermal colloidal assembly and disassembly schemes as well as on hybridization kinetics between single-stranded and double-stranded probes immobilized on microspheres. Incorporation of LNA nucleotides into DNA sequences is of particular interest as a means of enhancing the performance of DNA in a biomaterials context due to the increased resistance of LNA to nuclease degradation, its greater intrinsic affinity for oligonucleotide targets, and low cytotoxicity effects. The effects of LNA modification, target sequence length, sequence fidelity, and salt concentration are key variables explored. After providing an overview of DNA and its properties, synthetic oligonucleotides, colloidal particles, and previous applications of DNA and LNA in colloidal assembly schemes, this work then discusses the selection and characteristics of appropriate pairs of hybridization partners for reversible colloidal assembly scenarios. A comparative investigation of the in situ primary hybridization kinetics between select LNA or DNA targets and single-stranded probes immobilized on colloidal surfaces is performed. To support the disassembly studies, the in situ competitive displacement kinetics of hybridized LNA primary targets by either LNA or DNA secondary targets is discussed. For these in situ studies, flow cytometry was used to quantify the hybridization reactions as they occur on microsphere surfaces. While comparable rate constants were typically observed between target and single-stranded probes, LNA typically exhibited more extensive primary and secondary hybridization activity. Optimizing hybridization parameters, such as duplex concentration, sequence fidelity, and LNA content in the probe and target strands, allows for the extent of colloidal disassembly to be tuned, an important step in developing a multifunctional colloid-based biomaterial system.

The anterior cruciate ligament (ACL) is the most frequently injured ligament of the knee, with upwards of 100,000 ACL reconstructions performed annually. Current grafting techniques are limited by insufficient integration with subchondral bone and donor site morbidity issues related to graft harvest, potentially resulting in revision surgery and long-term joint pain. Therefore, significant demand exists for alternative grafting solutions that do not require additional surgery and can regenerate the native ACL-to-bone interface to promote biological fixation of the implanted ACL graft. To address this need, the ideal system must be able to withstand the functional demands of the native tissue by demonstrating physiologically equivalent mechanical properties, be comprised of compositionally varying phases in order to recapitulate the inherent heterogeneity of the native ligament to bone transition and be biodegradable such that it is gradually replaced by the regenerated tissue following implantation. It is hypothesized that a biomimetic, multi-phased scaffold comprised of optimized bone, interface and ligament regions coupled with controlled chemical and/or mechanical stimulation in vitro will guide phase-specific differentiation of mesenchymal stem cells (MSC) and result in a biologically integrated bone-ligament-bone complex in vivo. Mesenchymal stem cells are particularly attractive for this application as they can be routinely harvested from bone marrow, have been shown to respond to chemical, mechanical and structural cues, and are capable of differentiating towards the primary cell types (fibroblasts, osteoblasts and chondrocytes) found within ligament, bone and the ligament-to-bone interface. To this end, a nanofiber-based synthetic graft was designed with compositionally-varying phases to regenerate ligament, bone and interface tissues. The ligament phase was optimized in terms of nanofiber alignment, composition, mechanical stimulation and chemical stimulation. It was demonstrated that an aligned nanofiber substrate coupled with controlled mechanical stimulation was necessary to differentiate MSCs towards a fibroblastic phenotype. The bone phase was optimized in terms of ceramic content and it was shown that a threshold of mineral incorporation into nanofibers was necessary to differentiate MSCs towards an osteogenic phenotype. Lastly, a mechanoactive nanofiber collar was designed to induce interface formation. It was demonstrated that compressive stimulation applied via nanofiber collar contraction induced chondrogenic differentiation of MSCs. Subsequently, the three phases were incorporated to form a synthetic graft, for which graft architecture and cell seeding density were optimized. The resulting graft was cultured in vitro under the optimized parameters, demonstrating the formation of distinct and structurally continuous regions of bone, interface and ligament tissue. The graft was implanted in vivo where it was shown to be suitable for ACL reconstruction as it maintained knee stability and promoted ligament regeneration. In summary, this thesis focuses on the design of a biomimetic, nanofiber-based, integrated bone-ligament-bone construct, and elucidates chemical, mechanical and scaffold design-related parameters that can guide MSC differentiation towards desired tissue types. The impact of these studies extends beyond ligament reconstruction as they yield valuable scaffold design criteria, establish scaffold and culturing-related parameters to induce stem cell differentiation and can readily be applied to the formation of interfaces between soft-to-hard tissues as well as other complex tissues.

In 2005, over 200,000 Americans underwent a hip arthroplasty, the replacement of a hip joint with an artificial prosthesis. Of these arthroplasties, metal-on-metal type implants represent an increasing usage percentage. Metal-on-metal implants are selected largely for their low volumetric wear rate, durability, and resistance to corrosion. In spite of these advantages, little is known concerning the long-term consequences of heavy metal alloy use in the body, although early research indicates potentially carcinogenic results. This thesis is a preliminary investigation into these long term effects and their root causes. An improved comprehension of the corrosion kinetics and the rate of ion production from the high surface energy wear debris released by implant articulation can assist in illustrating the relative clinical significance of exposure to these metallic bodies over time. This thesis primarily focuses on developing a test methodology for the detection and analysis of ion dissociation in simulated body fluids. In order to validate this test methodology, the ion dissociation rates and surface characteristics of several predetermined diameters of cobalt chromium alloy spherical particles were analyzed. The effect of changing particle diameter, and thus surface area to volume ratio, on ion dissociation rate was determined to be significant when not affected by localized agglomeration. Additionally, preferential corrosion of cobalt within individual grains was observed and correlated to elevated cobalt concentrations in the electrolyte. These results suggest that ion dissociation kinetics for true wear particles can be determined through the refinement and application of the methodology developed.

This thesis presents a light scattering-based method for biomarker detection, which could potentially be used for the quantification of multiple biomarkers specific to B-cell malignancies. This method uses fabricated gold nanoparticle probes to amplify inelastic light scattering in a process referred to as surface-enhanced Raman scattering. These gold nanoparticle probes were conjugated to antibodies for specific and targeted molecular binding. The spectrum of the amplified inelastic light scattering was detected using a spectrometer and a detector. To detect the light scattering signal from the gold nanoparticle probes, several commercial Raman spectrometer instruments were evaluated. Initial results from these evaluations are presented in this thesis. After system evaluation, a custom Raman microscope system was designed, built, and tested. This system was used for the development of a surface-enhanced Raman spectroscopy-based immunoassay. The development of this assay confirms the successful design of gold nanoparticle probes for the specific targeting and detection of immunoglobulins. The immunoassay also shows promise for the simultaneous detection of multiple biomarkers specific to B-cell malignancies.