Type 1 Diabetes (T1D) is a disease where pancreatic islet beta-cells are unable to regulate blood glucose levels, resulting in severe health issues including death.1 1.25 million people in the United States alone have T1D and this accounts for $14.9 billion in healthcare cost annually (CDC, 2009). The prevalence of T1D in people under age 20 rose by 23 % between 2001 and 2009 and is projected to increase 4-6% annually (CDC & NIH), providing a strategic opportunity for therapeutic development. The gold standard of treatment requires manual correction of cellular insulin response by IM injection or by an implanted pump, however both treatments require burdensome maintenance. Although glucose homeostasis can be controlled, there is no therapy providing complete insulin independence. Here I describe a novel therapeutic technology that will lay the foreground for providing insulin independence.

Although islet transplantation has been explored, limited success has been achieved due to decreased islet function, survival and required associated immunosuppressant therapy. To address these challenges multiple encapsulation approaches have been explored.2–6 This unique technology combines the advantages of single-islet and multi-islet encapsulation approaches, providing rapid nutrient exchange of single-islet approaches and precise membrane control of multi-islet approaches. On top of providing rapid nutrient exchange, immune-isolation and minimal foreign body response, this device technology, unlike others, has been designed to provide flexible, compliant support for the encapsulated islets, recently discovered to promote cell function and survival. 7

This technology can replace the need for burdensome therapies by restoring absent insulin secreting cells, reestablishing glucose homeostasis, effectively curing T1D. It can greatly improve diabetic treatment by having immediate glucose response, and significantly reduce associated diabetic complications. Furthermore this approach can significantly decrease reliance on patient compliance and improve patient quality of life.

While oral drug administration is by far the most preferred route, it is accompanied by many barriers that limit drug uptake such as the low pH of the stomach, metabolic and proteolytic enzymes, and limited permeability of the intestinal epithelium. As a result, many drugs ranging from small molecules to biological therapeutics have limited oral bioavailability, precluding them from oral administration. To address this issue, microfabrication has been applied to create planar, asymmetric devices capable of binding to the lining of the gastrointestinal tract and releasing drug at high concentrations, thereby increasing oral drug uptake. While the efficacy of these devices has been validated in vitro and in vivo, modifying their surfaces with nanoscale features has potential to refine their properties for enhanced drug delivery. This dissertation first presents an approach to fabricate polymeric microdevices coated with nanowires in a rapid, high throughput manner. The nanowires demonstrate rapid drug localization onto the surface of these devices via capillary action and increased adhesion to epithelial tissue, suggesting that this fabrication technique can be used to create devices with enhanced properties for oral drug delivery. Also presented are microdevices sealed with nanostraw membranes. The nanostraw membranes provide sustained drug release by limiting drug efflux from the devices, prevent drug degradation by limiting influx of outside biomolecules, and enhance device bioadhesion by penetrating into the mucus layer of the intestinal lining. Finally, an approach that dramatically increases the capacity and efficiency of drug loading into microdevices over previous methods is presented. A picoliter-volume printer is used to print drug directly into device reservoirs in an automated fashion. The technologies presented here expand the capabilities of microdevices for oral drug delivery by incorporating nanoscale structures that enhance device bioadhesion, tunability of drug release, and drug protection and also provide a more cost-effective and scalable approach to drug loading.

Transplantation of encapsulated insulin-secreting cell clusters represents a potential cure for type I diabetes, but development efforts so far have yet to live up to its promise. Recent studies have elucidated the importance of cluster size on the insulin response in a manner that affects viability and efficacy of transplanted clusters. Using microfabrication techniques, a method to fabricate uniformly-sized insulin-secreting cell clusters was developed and thoroughly characterized using water contact angle, x-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and fluorescent microscopy. This technique enabled the formation of both monolayered and multilayered cell clusters of a predetermined size and shape. Subsequent evaluation of the impact that cluster size has on insulin expression, content and secretion using RT-PCR, ELISA, and confocal quantitative immunocytochemistry suggested that two cluster-size dependent behavioral changes relevant to transplant efficacy exist: First, glucose stimulation causes increased insulin production for clusters exceeding 40 µm in size. Second, cluster sizes greater than 60 µm secrete insulin more efficiently after production than smaller sized clusters. These results suggest that an optimal cluster size exists between 100-120 µm. Lastly, human embryonic stem cells were differentiated in patterned 120 µm clusters along the pancreatic lineage, an effort that could produce optimally sized insulin-secreting cell clusters from a renewable cell supply. The studies presented here may help overcome two remaining challenges preventing encapsulated cell transplantation therapy from truly providing a cure for type I diabetes.

Myocardial infarction (MI) is the leading cause of heart failure (HF) and despite advances in the management of MI, subsequent pathologic remodeling of ischemic myocardium with fibrotic scar tissue and aneurysmal degeneration leads to HF and death. Early attempts at stem cell delivery and biological therapeutics to promote positive remodeling following MI have been promising but inconsistent and often involve systemic inhibition of signaling factors, which can have unintended side effects. A compelling approach to combat pathological cellular processes that often arise due to myocardial injury includes engineering materials to mimic biological and physical traits of native ECM architecture to provide biophysical cues that may yield more healthy cell phenotypes. This dissertation describes the therapeutic applications of biophysical cues in cardiovascular diseases (CVDs), specifically myocardial infarction and heart failure. First, Chapter 1 provides a comprehensive discussion regarding how nano- and microscale biophysical cues affect the phenotypes and behaviors of key cardiovascular cells– cardiomyocytes, fibroblasts, and endothelial cells– and how these resulting effects may be leveraged for therapeutic success in CVDs. Chapter 2 then details the development of a biodegradable polymeric microstructure technology made of hyaluronic acid. In this study, we investigated how hyaluronic acid microrods affect fibroblast phenotype and if they can improve cardiac outcomes in rodent models of ischemia-reperfusion (I/R) MI injury. We demonstrated that treatment with hyaluronic acid microrods decreases fibrotic phenotype in fibroblasts as well as exhibits enhanced cardiac function and decreased fibrosis after 6 weeks post-MI. Further, treatment with hyaluronic acid microrods outperformed treatment with equivalent mass of soluble hyaluronic acid material control, indicating the importance of the material geometry. Beyond physical regulation of cellular behaviors by topographical cues, the incorporation of biochemical factors may further enhance the efficacy of these therapeutic strategies. As such, Chapter 3 comprises studies that probed the effects of several pro-angiogenic stimuli (i.e., Qk, RoY, and HepIII peptides) on endothelial cells and the feasibility of conjugating them onto hyaluronic acid microrods to create dual-pronged strategy that tackled both rampant fibrosis and lack of vascularization that occur post-MI. Chapter 4 follows a similar approach but investigates the therapeutic effects of a decorin-loaded hyaluronic acid microrod strategy in rodent models of I/R MI injury. Here, we demonstrate distinguishable improvement in cardiac function and ventricular remodeling and decreased fibrosis and cardiomyocyte hypertrophy in animals treated with decorin-loaded hyaluronic acid microrods. Together, this body of work aims to contribute important knowledge to help develop rationally designed, engineered biomaterials that may be used to successfully treat CVDs.

This dissertation focuses on two aspects that present challenges for a broad range of implantable devices and transplants - blood compatibility and wound healing properties of biomaterials. In the first chapter, we examined the hemocompatibility of surface modified silicon substrates used in renal replacement devices. Our reports showed that the polymer conjugated silicon samples reduced platelet attachment and activation to levels comparable to that on Teflon, a material commonly used in medical implant devices. Our findings suggest that surface modified silicon substrates could be used to develop miniaturized implants for renal replacement therapies.

In the second chapter, we investigated how collagen nanotopography affects wound healing in the cornea. The presence of nanopatterned collagen fibrils was shown to promote the appearance of the healthy keratocyte phenotype and attenuate the fibrotic myofibroblast phenotype. In addition, collagen nanotopography also had an effect on matrix synthesis. These results have significant implications for the design of tissue engineered corneal substitutes and for promoting regenerative wound healing in the cornea.

In the third chapter, we focused on wound healing in the skin, particularly in the case of keloid scars. Keloids are locally aggressive dermal scars formed as a result of abnormal wound healing. They are characterized by excessive fibroblast proliferation and matrix production. An effective treatment for keloids is yet to be established due to a high rate of recurrence. Our results showed that collagen fibril alignment reduced cell proliferation and matrix synthesis in fibroblasts derived from keloid, scar and healthy dermal tissue. This data suggests that aligned collagen fibrils could be used to develop dermal patches that reduce the recurrence of keloids and aid in the design of an effective therapy for keloid management. The recurring theme in both the cornea and keloid studies is that matrix architecture could be used to effectively manipulate cell response to direct regenerative wound healing. Collectively our findings show that physical cues such as matrix topography could be used to improve the anti-fibrotic properties of biomaterials and aid in the design of tissue engineered implants for various clinical applications.

Over the past few decades, regulatory T cell (Treg) therapy has gained traction for the treatment of autoimmunity, transplant rejection, and other inflammatory diseases. A necessary part of this process is cellular manufacturing, where Tregs are activated with immunomodulatory agents, expanded, and quality assessed for CD4+ and Foxp3+ lineage purity before infusion. A challenge that has emerged in certain patient populations is the failure of Tregs to expand. This clinical manufacturing failure has been associated with the pre-expansion phenotype of the Treg, with a higher proportion of terminally differentiated effector Tregs correlating with poorer expansion. While the heterogeneity of Tregs phenotypic subsets and their expansive capacities has been reported, it is unclear what the required signaling strengths are to induce proliferation within each subset. To investigate this question, we used a biomaterial strategy for precisely attaching Treg stimulatory molecules onto polymeric particles using DNA to act as artificial antigen presenters. First, we optimized the fabrication technique, adapting numerous technologies commonly available to immunology labs, and exemplified its use in both human CD4+ and CD8+ T cell activation. Second, we then applied these materials towards activating human Tregs, identifying unique activation thresholds for inducing expansion and further subset-specific expansion behaviors. Third, unable to drive the expansion of effector Tregs using this approach, we turned to paired single cell RNA and TCR sequencing (scRNA/TCRseq) to identify the distinguishing genes associated with high expansion. Using TCR tracking of highly expanding clones, we were able to subset non-activated cells by their TCR clonotype expansion, enabling the comparison between transcriptomes of high expanders and low expanders. This led to the finding that highly expanding clones were associated with numerous markers associated with Treg stemness, proliferative capacity, and degradation of inhibitory intracellular signals, whereas poorly expanding clones express numerous differentiation and activation genes. Further, we identified a group of proliferative Tregs which displayed an inflammatory phenotype, which prompts further investigation into maintaining Treg purity in strategies that improve expansion. This work sets a foundation in studying Treg subset activation biology using precisely controlled signals and has uncovered numerous gene targets for improving Treg expansion. We believe that investigating these targets and optimizing their activation signals may provide a means of rescuing Treg proliferation in cases of manufacturing failures, reenabling Treg therapy as a possible treatment option for affected patient populations.

Cardiac fibrosis is considered to be an independent risk factor in the outcome of congestive heart failure. Heart transplantation is the only treatment for patients who are at the end stage of this condition. Shortage of donor organs has created a need for therapeutic alternatives. This dissertation investigates new strategies for cardiac repair systems that could reduce pathological fibrosis and promote growth of myocytes at the site of injury. Design of successful engineered therapies for tissue regeneration relies on discerning how cell behavior can be modulated by chemical and physical cues. Recent studies have shown that external physical cues such as stiffness and geometry can affect cell morphology and function. In this work, the combinatorial effect of stiffness and micro-scale topographical cues on proliferation and gene expression is investigated in 2D and 3D. The 2D system consists of fibroblasts grown on "micropegged" polydimethylsiloxane (PDMS) substrates of different stiffness. The 3D system consists of fibroblasts encapsulated with poly(ethylene glycol) dimethacrylate (PEGDMA) "microrods" of different stiffness in matrigel to create a 3D culture with micro-scale cues of defined mechanical properties in the physiological range.

Fibroblasts cultured on micropegged substrates have reduced collagen expression compared to fibroblasts cultured on flat substrates. Cells on stiffer micropegged substrates exhibit down regulation of important regulators of ECM synthesis but there is no down-regulation of these markers when cells are cultured on the softer micropegged substrates. Similarly, three-dimensional cultures with stiffer microrods show reduced fibroblast proliferation and down-regulation of collagen and other important regulators of ECM synthesis, but three dimensional cultures with soft microrods do not show significant difference on fibroblast proliferation and expression of some ECM regulators compared to cultures with no microrods.

To determine whether microrods can influence myocardial repair and remodeling in vivo, adult female Sprague-Dawley rats undergo left anterior descending (LAD) artery occlusion for thirty minutes followed by reperfusion. Microrods suspended in PBS are injected into the left ventricle (LV) two days after myocardial infarction (MI). Five weeks after the injections echocardiography reveals an increase in ejection fraction (EF) in the microrod group compared to the PBS control. Histology analysis shows a trend on decreased amount of scar formation and increased wall thickness of the microrod treated group compared to the PBS control.

Furthermore, it is shown that microrods can be utilized as growth factor delivery devices. Mechano growth factor (MGF) is expressed rapidly after tissue damage and prevents apoptosis in the myocardium; thus is considered a suitable therapeutic candidate for cardiac repair. Here, it is shown that microrods can be loaded with MGF and that the peptide can be eluted from the microrods for a period of days. Bioactivity of MGF is confirmed by analyzing stem cell migration and myoblast proliferation. Interestingly, it is also observed that myoblast's interaction with microrods can enhance proliferation even after seven days of culture. These studies indicate that microrods protect rapid degradation of MGF, promote stem cell migration and enhance myoblast proliferation.

Results from this work will help create more tunable in vitro tissue engineered platforms and develop new therapeutic approaches for in vivo tissue regeneration.