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Yes / While achieving the spatial organization of cells within 3D assembled nanofiber/cell constructs via nanofiber-enabled cell layering, the small sizes of inter-fiber pores of the electrospun nanofiber mats could significantly limit cell penetration across the layers for rapid formation of an integrated tissue construct. To address this challenge, efforts were made to improve cell-infiltration of electrospun nanofiber mats by modulating the density distribution and spatial organization of the fibers during electrospinning. Collection of collagen-containing electrospun nanofibers (300–600 nm in diameter) onto the surface of a stainless steel metal mesh (1 mm × 1 mm in mesh size) led to the periodic alternation of fiber density from densely packed to loosely arranged distribution within the same mat, in which the densely packed fibers maintained the structural integrity while the region of loose fibers allowed for cell penetration. Along with improved cell infiltration, the distinct fiber organization between dense and loose fiber regions also induced different morphology of fibroblasts (stellate vs. elongated spindle-like). Assembly of cell-seeded nanofiber sheets into 3D constructs with such periodically organized nanofiber mats further demonstrated their advantages in improving cell penetration across layers in comparison to either random or aligned nanofiber mats. Taken together, modulation of nanofiber density to enlarge the pore size is effective to improve cell infiltration through electrospun mats for better tissue formation. / NSF-IIP. Grant Numbers: 1338958, 1346430; NSF-DMR. Grant Number: 1508511; NSF-CBET. Grant Number: 1033742; and NIAMS. Grant Number: 1R21 AR056416

The typical catalyst layer structure for proton exchange membrane (PEM) fuel cells has changed little over the last two decades. A new electrode design with improved control over factors such as ionic and electrical pathways, porosity, and catalyst placement, could allow the application of less expensive catalyst alternatives. In this work, a novel electrode design based on ionomer-coated carbon nanofibers is proposed and studied. Governing equations for this design were established, and a mathematical model was created and solved using MATLAB to predict the performance of the new electrode design. A parametric study was performed to identify the design variables that had the most significant effect on performance. The best performing catalyst layer design studied with this model produces a current density of 1.1 A cm-2 at 600 mV which is better than state-of-the-art cathode designs. The results offer insight into the performance of ionomer-coated carbon nanofiber catalyst layers and can guide the fabrication and testing of these promising catalyst layer structures. / Master of Science / Proton exchange membrane (PEM) fuel cells have the potential to replace traditional energy conversion systems in many applications, however their widespread adoption is currently limited by their high cost and insufficient durability. PEM fuel cells are expensive because they require the use of platinum as a catalyst. Currently, less expensive non-platinum catalysts, must be used in much higher amounts in the catalyst layer to achieve similar electrochemical activity, creating very thick catalyst layers. Traditional fuel cell catalyst layer structures are designed to be thin and perform poorly when thick enough to accommodate non-platinum catalysts. This work proposes a novel catalyst layer design based on ionomer-coated carbon nanofibers that can allow for thicker catalyst layers and much higher catalyst loadings. A mathematical model was developed for the novel catalyst layer based on first principles. The model was solved using MATLAB to predict the performance of the new catalyst layer design. A parametric study was performed to identify the critical design variables and their effect on catalyst layer performance. The best performing catalyst layer design studied with this model produced a current density of 1.1 A cm-2 at 600mV, which is better than state-of-the-art fuel cell designs. This work is meant to offer insight into the performance of an ionomer-coated nanofiber catalyst layer and to guide future research in the fabrication of high performance fuel cells based on this novel catalyst layer architecture.

Initial attachment to the extracellular matrix (ECM) and consequent spreading is a necessary process in the cell cycle of which little is known. Cell spreading has been well-recognized in 2D systems, however, the native fibrous ECM presents cells with 3D biophysical cues. Thus, using suspended fibers as model systems, we present the development of a novel platform (Cell-STEPs) capable of capturing cell attachment dynamics and forces from the moment a cell in suspension contacts the fiber. Cell-STEPs comprises of a custom glass-bottom petri dish with a lid to deliver a constant supply of CO2 to maintain pH. Fibrous scaffolds are attached in the dish to allow cellular investigations over extended periods of time. We find that cell-fiber attachment occurs in three progressive phases: initial attachment of cell to fiber (phase 0), rapid drop in circularity (phase 1), and increase in cell spread area (phase 2). Furthermore, using iterative inverse methods, forces involved in cell spreading through deflection of fibers were estimated. Our findings provide new insights in attachment biomechanics, including initial sensing and latching of cell to fiber with a negligible or protrusive force, followed by rapid loss in circularity through protrusion sensing at nearly constant spread area and minimal force generation, transitioning to a final phase of increased contractile forces until spread area and force saturation is observed. Also, anisotropic spreading of cells on single and two-fibers are closely related, while cells attached to several fibers take longer and spread isotropically. The Cell-STEPs platform allows, for the first time, detailed interrogations in the discrete and orchestrated adhesion steps involved in cell-fibrous matrix recognition and attachment along with simultaneous measurements of forces involved in cell attachment. / Master of Science

Extracellular matrix (ECM) is a fibrous natural cell environment, possessing complicated micro-and nano- architectures, which provides signaling cues and influences cell behavior. Mimicking this three dimensional environment in vitro is a challenge in developmental and disease biology. Here, suspended multilayer hierarchical nanofiber assemblies fabricated using the non-electrospinning STEP (Spinneret based Tunable Engineered Parameter) fiber manufacturing technique with controlled fiber diameter (microns to less than 100 nm), orientation and spacing in single and multiple layers are demonstrated as biological scaffolds. Hierarchical nanofiber assemblies were developed to control single cell shape (shape index from 0.15 to 0.57), nuclei shape (shape index 0.75 to 0.99) and focal adhesion cluster length (8-15 micrometer). To further investigate single cell-ECM biophysical interactions, nanofiber nets fused in crisscross patterns were manufactured to measure the "inside out" contractile forces of single mesenchymal stem cells (MSCs). The contractile forces (18-320 nano Newton) were found to scale with fiber structural stiffness (2 -100 nano Newton/micrometer). Cells were observed to shed debris on fibers, which were found to exert forces (15-20 nano Newton). Upon CO? deprivation, cells were observed to monotonically reduce cell spread area and contractile forces. During the apoptotic process, cells exerted both expansive and contractile forces. The platform developed in this study allows a wide parametric investigation of biophysical cues which influence cell behaviors with implications in tissue engineering, developmental biology, and disease biology. / Master of Science

Cells interact with their immediate fibrous extracellular matrix (ECM); alignment of which has been shown influence metastasis. Specifically, intra-vital imaging studies on cell invasion from tumor-matrix interface and wounds along aligned fibers describe invasion to occur as singular leader (tip) cells, or as collective mass of a few chain or multiple tip cells. Recapitulation of these behaviors in vitro promises to provide new insights in how, when and where cells get the stimulus to break cell-cell junctions and ensue invasion by migrating along aligned tracks. Using Spinneret based Tunable Engineered Parameters (STEP) technique, we fabricated precise layout of suspended fibers of varying diameters (300, 500 and 1000 nm) mimicking ECM dimensions, which were interfaced with cell monolayers to study invasion. We demonstrated that nanofiber diameter and their spacing were key determinants in cells to invade either as singularly, chains of few cells or multiple-chains collectively. Through time-lapse microscopy, we reported that singular cells exhibited a peculiar invasive behavior of recoiling analogous to release of a stretched rubber band; detachment speed of which was influenced with fiber diameter (250, 425 and 400 µm/hr on small, medium and large diameter fibers respectively). We found that cells initiated invasion by putting protrusion on fibers; dynamics of which we captured using a contrasting network of mismatched diameters deposited orthogonally. We found that vimentin, a key intermediate filament upregulated in cancer invasion localized within a protrusion only when the protrusion had widened at the base, signifying maturation. To develop a comprehensive picture of invasion, we also developed strategies to quantify migratory speeds and the forces exerted by cells on fibers. Finally, we extended our findings of cell invasion to report a new wound healing assay to examine gap closure. We found that gaps spanned by crosshatch network of fibers closed faster than those on parallel fibers and importantly, we reported that gaps of 375 µm or larger did not close over a 45-day period. In summary, the methods and novel findings detailed from this study can be extended to ask multiple sophisticated hypotheses in physiologically relevant phenomenon like wound healing, morphogenesis, and cancer metastasis. / Ph. D. / Disease phenomenon like cancer invasion and wound healing have a myriad of things in common including cell migration and the ability of cells to remodel their immediate environment. Often times these singular or group migratory events of cells are initiated and directed by peculiar cells called tip/leader cells that explore cellular environment and make room for migration. While research in the last few decades has yielded a tremendous wealth of information as to how biochemical factors influence their behavior, our understanding of how the biophysical properties of the environment affect their behavior is in its infancy. This lack of understanding can somewhat be attributed to the difficulty in fabricating mechanically welldefined substrate systems that can be tailored to recapture these invasive episodes in a controlled setting outside of a living organism. In this study, we utilize a novel Spinneret based Tunable Engineered Parameters (STEP) technique to fabricate mechanically tunable nanofibers that show close resemble to native cellular environment. Cells were made to interact with these fibers and it was shown for the first time that factors like fiber spacing, diameter and topography can significantly affect the types of leader cells and their trajectory. Furthermore, once these cells come out of the simulated tumors/wounds, their migratory behaviors were still affected by mechanical properties of the fibers. Similarly, we also showed for the first time that the ability of the gaps to close in simulated wound healing settings could be significantly dependent on size, shape, and properties of fibers. These findings offer a novel outlook to our current understanding of single and collective cell behavior and how the biophysical properties of the native cellular environment can affect these behaviors. This can not only expand our understanding of how this invasive episodes occur, but also help us come up with preventive measures to inhibit such episodes for a better prognosis of diseases like cancer and chronic wounds.

Master of Science / Department of Architectural Engineering and Construction Science / Julia A. Keen / Nanofibers are classified as fibers less than 1 micrometer in diameter. These fibers can be layered to form nanofibrous membranes, and these membranes offer great potential in the filtration industry. The membranes' smaller fiber diameters and pore sizes permit such filters to filter out more and smaller particulate. Additionally, antimicrobial agents can be incorporated into the membrane to inhibit fungal and bacterial growth on the membrane’s surface. This report evaluates nanofibrous membranes with antimicrobial agents and their potential in two specific locations: cleanrooms and protective environment rooms, where bacterial and fungal growth would have a detrimental effect on the process or occupant of the space.