The derivation of human induced pluripotent stem cells (IPSCs) has revolutionized the field of personalized medicine. With their ability to self-renew and differentiate to all cell types of the adult body, their therapeutic potential for multiple diseases is invaluable. Albeit their therapeutic potential is promising, they pose the risk of uncontrolled differentiation or tumorigenesis. Physical forces in the developing embryo have been shown to play a critical role in driving lineage commitment to regulate tissue morphogenesis. In this regard, the current work focused on examining the effects of the mechanical microenvironment, using electrospun scaffolds, on the cellular behaviors of human IPSCs. We examined the role of scaffold stiffness on the (1) self-renewal and (2) directed differentiation of IPSCs. Our findings demonstrate that the mechanical microenvironment contributes to the development of distinct IPSC colony morphology. Under proliferation conditions, a two-dimensional (2D) colony morphology on stiff scaffolds enhances proliferation and minimizes spontaneous differentiation of IPSCs. In contrast, the development of three-dimensional (3D) colonies on soft scaffolds results in increased spontaneous differentiation towards ectodermal lineage. Additionally, the development of distinct colonies, directed differentiation to mesendodermal lineage is enhanced on stiff scaffolds while ectodermal differentiation is enhanced on soft scaffolds. Furthermore, we demonstrate that modulation of the RhoA signaling pathway by scaffold stiffness or using a ROCK inhibitor can prime IPSCs to differentiate towards a mesendodermal lineage. We further demonstrate that actin and E-cadherin/β-catenin clustering mediates structural pre-stresses imposed on the cells, which may delay activation of the Wnt signaling pathway, as a result of developing 3D colony morphologies. Finally, we demonstrate that modulation of the scaffold stiffness, at each developmental stage of differentiation towards three germ layer derivate cell phenotypes, can enhance the differentiation of IPSCs. Overall, these findings demonstrate that IPSCs are mechano-responsive and that the mechanical niche can be designed to modulate IPSC behaviors for the control of differentiation or to study developmental processes in vitro.

Chronic diseases account for approximately 71% of the deaths globally. Patients suffering from chronic illness require a constantly changing therapeutic need to keep up with the progression of the disease. However, traditional pharmaceutical treatments fail to provide proper therapy in many ways from high cytotoxicity due to uncontrolled drug release rate to low bioavailability of the drug which requires frequent administration. Therefore, there is a growing interest in developing drug delivery systems to precisely maintain therapeutic drug in the system and protect the active drug molecules from rapid metabolism, thus improving the bioavailability. In this regard, we have developed exogenous stimuli-responsive drug delivery systems based on functionalized electrospun nanofibers.

By utilizing high-performing piezoelectric poly(vinylidene-trifluoroethylene) (P(VDF-TrFE)) nanofibrous membrane, we have developed a mechanical-stimulus responsive drug delivery system based on their piezoelectric effect, where energy conversion occurs from applied mechanical strains to changes in electric potential of P(VDF-TrFE). Using an extracorporeal shockwave system as the mechanical trigger, we demonstrated the control over drug release by regulating nanofiber diameter, and magnitude and frequency of applied mechanical force. We also examined the controllability of the drug delivery system with in vitro and ex vivo models, demonstrating its potential for in vivo applications.

Despite this promising capability of P(VDF-TrFE) nanofibers for controlled drug delivery applications, they need to be surgically removed from the body after drug release due to their non-biodegradable nature. This led us to investigate strategies to enable phagocytosis of the material, which can excrete synthetic non-biodegradable polymer nanofibers from the body. In this regard, we have developed a facile method to fragment polymeric nanofibers into nanorods using colloid electrospinning followed by ultrasonication.

We also developed magneto- & opto-stimulus-responsive nanofibers as an alternative drug delivery system. We functionalized poly(ε-caprolactone) (PCL) nanofibers by integrating superparamagnetic iron oxide nanoparticles (SPIONs) into the fibrous network. By exploiting heat generation from Néel relaxation effect of activated SPIONs under magnetic fields or laser light, we utilized structural changes of thermo-sensitive nanofibers from the heating to trigger drug release. With this composite material, we demonstrated the tunability of drug release by manipulating the SPION loading and stimulus duration.

Piezoelectric polymer, poly(vinylidene-trifluoroethylene) (P(VDF-TrFE)), exhibits excellent characteristics, such as flexibility and biocompatibility, for various biological applications that utilize energy conversion between mechanical strain and electric potentials. However, its typically low piezoelectric properties have limited its use as an effective piezoelectric platform. To address this, electrospinning was utilized as a method to manipulate the nanostructure of P(VDF-TrFE) nanofibers to engineer a high-performing piezoelectric material. More specifically, we show that the piezoelectric performance of P(VDF-TrFE) is size dependent; by dimensional reduction to the nanoscale (30 nm), a transformative enhancement in piezoelectric performance was achieved by the synergistic effects of flexoelectricity materialization and enhanced dipole domain alignment. The electrospun P(VDF-TrFE) at this size scale exhibits an exceptional piezoelectric coefficient, d33, at -108 pm V-1, approaching the same magnitude of more traditional inorganic materials, while maintaining its flexibility.

We exploit these high performing P(VDF-TrFE) nanofibers for specific biological applications. In one aspect, the large surface area-to-volume ratio inherent to nanomaterials, together with the transformative piezoelectric properties, allowed us to use the material as an ultrasensitive, acoustic-responsive, drug delivery platform driven by the direct piezoelectric effect. The intrinsic negative zeta potential was utilized to electrostatically load cationic drug molecules. We show that the drug release sensitivity of the P(VDF-TrFE) nanofibers depends on the fiber diameter, thus piezoelectric properties. We further showed that the drug release quantity can be tuned by applied acoustic pressure or number of acoustic doses for specific tissue applications.

Additionally, through the direct piezoelectric effect, we also demonstrated the utility of P(VDF-TrFE) nanofibers with an aligned morphology in neural tissue engineering. We demonstrate that the piezoelectric P(VDF-TrFE) nanofibers provide a means to culture neural stem cells while electrically stimulating the cells by acoustic actuation of the scaffold, generating electric potentials that were utilized to modulate the cellular behaviors. The electrical stimulation of neural stem cells resulted in neural stem cell differentiation towards different phenotypes, including neurons, oligodendrocytes, and astrocytes, demonstrating the potential utility of the piezoelectric scaffolds for engineering neural tissues composed of multiple cell phenotypes.

Finally, a proof-of-concept cell culture platform that can modulate the mechanical properties of cell culture scaffolds on demand, was devised based on the indirect piezoelectric effect. Microfabricated interdigitated electrodes were designed, via computational simulations, to act as an electric field-generating substrate for the P(VDF-TrFE) scaffold. We showed that the stiffness of the P(VDF-TrFE) nanofibers electrospun onto such interdigitated electrodes can be precisely controlled by modulating the applied electric fields across the electrodes. The results demonstrate the significant potential of electrospun piezoelectric nanofibers for a cell culture substrate with an on-demand change of the physical cellular microenvironment.

This project aims to investigate the mechanosensitivity of pancreatic beta cells and its crucial role in the development and/or progression of diabetes. It has been shown that cellular functions are highly dependent on their physical environments, e.g., tissue stiffness, as well as biochemical cues. However, the technology necessary to understand the dynamic physicobiology of pancreatic cells is not currently available. To address this lack of technology, we developed an in situ adjustable hydrogel system, a composite hydrogel made of gelatin methacrylate (GelMA) and polyethylene glycol diacrylate (PEGDA), incorporated with magnetic nanorods (Fe3O4@SiO2@MPS) to enable dynamic, on-demand control over tissue stiffness. Using this system, we investigated how alterations in the mechanical microenvironment affect the behaviors of mouse insulinoma 6 (MIN6) beta cells. By unraveling the mechanistic interplay between cellular mechanosensitivity and diabetes, this research offers a promising pathway for targeted therapeutic interventions. Thus, the insights gained from this study could potentially pave the way for novel strategies to manage and treat diabetes effectively.

Stem cells exhibit unique characteristics of self-renewal and differentiation, the ability to produce unlimited daughter cells and transform into various cell types, respectively. Current biochemical-based analytical techniques for monitoring cellular behaviors are accurate, yet destructive, limiting their use as end-point analyses. In order to continuously monitor the same population of the cells, a novel multi-modal system that encompasses both quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS), as well as having an optical visualization capability, has been developed to characterize physioelectrochemical changes of the cells during self-renewal and differentiation in real-time. Human induced pluripotent stem cells (IPSCs) were utilized to validate the functionality of the device. An equivalent circuit model was then designed from experimental impedance data to correlate physical changes of the cells to electrical circuit components throughout the progression of stem cell self-renewal and differentiation. Overall, the combination of the quantitative information from the device and electrical circuit modeling collectively offers a means for an in-depth understanding of physical processes underlying cellular behaviors to determine the states of stem cell self-renewal and differentiation.

Many fabrication methods for scaffolds made with biodegradable polymers and hydrogels have shown promise for tissue engineering applications. Both possess their own benefits and disadvantages. However, by utilizing both, the advantages of each can be combined into a single hybrid scaffold. By increasing the reaction concentration of a modified synthesis protocol previously published, the lower critical solution temperature (LCST) of a biodegradable, thermos-sensitive hydrogel, poly (ethylene glycol)-poly (N-isopropylacrylamide) (PEG-PNIPAAm), was optimized to its lowest possible concentration for gelation (5 wt. %). The resulting PEG-PNIPAAm was blended with a hydrophobic, biodegradable polymer, poly (ε-caprolactone) (PCL), and electrospun into a 3-dimensional (3D), electrospun hybrid scaffold. The blending ratio of PEG-PNIPAAm:PCL (13:9), scaffold porosity (90%) and fiber diameter (11 μm) where optimized for uniform cell seeding and cell encapsulation within the dissolved hydrogel. During cell seeding, PEG-PNIPAAm dissolved into the media solution, surrounding seeded cells and filling the scaffolds pore volume without the need of a mold. When incubated at 37 °C, the seeded cells are encapsulated within the PEG-PNIPAAm hydrogel. The hydrophobic PCL network remains as a structural support, increasing the compressive, elastic and viscoelastic properties of PEG-PNIPAAm. Including PEG-PNIPAAm within the PCL scaffold, also resulted in the improvement of mechanical characteristics over PCL control scaffolds. Culturing mesenchymal stem cells (MSCs) within hybrid scaffolds for two weeks under chondrogenic conditions increased chondrogenic gene expression and proteoglycan deposition compared to PCL controls with a similar scaffold porosity and fiber diameters. The addition of PEG-PNIPAAm within a 3D structured, PCL network produces a hybrid scaffold with the potential for a variety of tissue engineering applications.

A lack of therapeutic technologies that enable electrically stimulating nervous tissues in a facile and clinically relevant manner has partly hindered the advancement in treating nerve injuries for full functional recovery. Currently, the gold standard for nerve repair is autologous nerve grafting. However, this method has several disadvantages, such as necessity for multiple surgeries, creation of functionally impaired region where graft was taken from, disproportion of graft to nerve tissue in size and structure, and most substantially, high risk of neuroma formation. Therefore, there is an increasing need for the development of alternative strategies to enhance nerve regeneration. To address these limitations, the development of a piezoelectric neuroconduit that can self-generate optimized levels of electrical stimulation can be utilized to wrap a damaged nerve and remotely activated by acoustically-driven piezoelectricity. Unlike biochemical-embedded conduit in which the release of neurotrophic factors is limited by loading or electro-conductive conduit in which only passive electrical stimulation by autologous cells is possible without intrusive electrodes, the piezoelectric conduit provides unlimited opportunity to properly stimulate the neuronal cells in vivo. In this regard, a piezoelectric scaffold was developed using electrospinning technology and its piezoelectric performance was optimized by controlling the fiber diameter and scaffold thickness. Various types of neural cells including PC12 cells, Schwann cells, and neural stem cells (NSCs), were subjected to mechanical/electrical stimulation to examine their behavioral changes. When PC12 cells, a model system for neurons, were subjected to multi-day application of mechanical/electrical stimulation, enhancements in neurite formation and elongation were observed. Alternatively, mechanical/electrical stimulation induced Schwan cells, the glial cell of the peripheral nervous system, to differentiate into its myelinating phenotype and induced the enhanced production of the neurotrophic protein, nerve growth factor. Aside from enhancing the functionality of neuronal and glial cells, mechanical/electrical stimulation also induced the differentiation of NSCs towards the functional cell types of the central nervous system, neurons, oligodendrocytes, and astrocytes. Therefore, this project develops a novel method for enhancing nerve regeneration by modulating neuron and glial cell functionality for the repair of nerve injuries.

The goal of this research was to engineer a scaffold composed of a mechanical gradient and understand the effects of dynamic stimulation on the morphogenesis of the interfacial tissue via spatially controlled differentiation of a single stem cell source. Osteoarthritis is a degenerative joint disease affecting articular cartilage and the underlying subchondral bone resulting in severe pain and disability of the joints. While current clinical treatments provide limited efficacy in long-term success, tissue engineering strategies prove to be a viable option to address regeneration of the osteochondral gradient interface. To address this challenge spatial patterning of mechanical factors are essential for the development of a gradient tissue structure in three dimensions. Here, the overarching goal of this work was to examine (1) the preferential differentiation capacity of MSCs towards multi-phenotypic lineages when subjected to variations in compressive strain, (2) the control of micro- and macro-scale scaffold mechanics through utilizing a core-shell microfiber electrospinning technique, and finally (3) the engineering of a dynamic mechanical gradient that can spatially control the local strain that MSCs will sense and differentiate into multi-phenotypic lineages. My findings demonstrate that MSCs differentiate in a magnitude-dependent and phenotype-specific manner in response to different magnitudes of dynamic compressive strain suggesting that MSCs are mechano-responsive and their multi-phenotypic differentiation can be controlled by varying the strain regimens. Furthermore, it was demonstrated that the mechanical properties of core-shell electrospun fibers can be modulated by controlling the composition and the dimension of core, decoupled from the cell-interfacing surface chemistry. More importantly, it was shown that mechanical properties of such fibers/scaffolds at the micro- and macroscale can be independently regulated by modulating micro- and macro-structure. Finally, spatial control of the mechanical properties of individual core-shell fibers in a monolithic scaffold was able to modulate the preferential multi-phenotypic differentiation of MSCs when subjected to dynamic compressive strain. Thus, these findings provide an additional avenue for directing stem cell differentiation in lieu of biochemical cues commonly used for osteochondral tissue regeneration. Addressing the influence of mechanical stimulation on spatially controlled multi-phenotypic MSC differentiation is the basis behind the work presented in the following dissertation.