Fischell Department of Bioengineering
Permanent URI for this communityhttp://hdl.handle.net/1903/6626
null
Browse
13 results
Search Results
Item SAMPLE-TO-ANSWER POINT-OF-CARE VIRUS DIAGNOSTIC SYSTEM USING THERMALLY RESPONSIVE ALKANE PARTITIONS(2024) Boegner, David John; White, Ian M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Many viral infections can be accurately diagnosed using today’s most sophisticated detection systems. Unfortunately, many of these detection systems fail to benefit society as a whole, but rather favor select areas of the world that are able to install and maintain the infrastructure such diagnostics require. Thus, in an effort to eliminate the barrier of access to diagnosis and treatment in low-and-middle-income areas, portable point-of-care devices are fabricated such that rapid results can be obtained without the need for bulky lab equipment or skilled technicians. An ideal point-of-care diagnostic device can easily collect an untampered sample and limits a patient’s encounter with a clinician to a single visit for both the diagnosis and the treatment. Many so-called point-of-care diagnostics for blood-borne viruses first require blood sample preparation (e.g. centrifugation) prior to testing in the device. Other point-of-care devices sacrifice diagnostic accuracy in favor of speed and portability. Both cases demonstrate our inability to properly distribute the benefits of sophisticated diagnostics worldwide.I present a solution in the form of an affordable handheld diagnostic device with the sensitivity and specificity of benchtop lab equipment and built-in automatic sample preparation. Automatic sample preparation will be achieved using thermally responsive alkane partitions, which are solid at ambient temperatures and liquid at moderately elevated temperatures. When liquid, the alkane partitions allow passage of magnetically activated microbeads coated with material that captures viruses. Despite magnetic beads with virus particles passing through, the alkane partition continues to prevent unwanted sample components (e.g. blood cells, DNases, etc.) from interfering with the virus-detecting mechanism on the other side. To address the lack of sensitivity in many point-of-care diagnostics, the virus-detecting mechanism will feature isothermal amplification which enables detection of attomolar concentrations of virus within 30 minutes without expensive thermo-cycling equipment that standard detection systems require. The novel technology described here is demonstrated in a platform which detects SARS-CoV-2 from blood, a capability currently unachievable in point-of-care settings.Item Synthetic Mucus Hydrogels for Antimicrobial Peptide Delivery and Treatment of Bacterial Infections(2024) Yang, Sydney; Duncan, Gregg A; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Antibiotic resistant infections have the propensity to form biofilms that contribute to chronic infections and result in hyper-inflammatory response in tissues. Recent studies pose antimicrobial peptides (AMPs) as alternatives to antibiotics and to modulate inflammatory response. However, AMPs have a short half-life due to rapid clearance and degradation reducing AMP bioavailability and efficacy. In the human body, AMPs interact and may associate with mucins which result in the sequestering of AMPs within mucus. Previously, we have developed a synthetic mucus (SM) hydrogel inspired by the innate properties of mucins. The objective of this work was to evaluate the SM hydrogel as a tool for local antimicrobial peptide delivery of LL37 to enhance the treatment for infection and inflammation. To study this, we (1) assessed the release of LL37 and antimicrobial activity of LL37 loaded SM (LL37-SM) hydrogels on Pseudomonas aeruginosa, (2) evaluated the antibiofilm activity of LL37-SM hydrogel treatment on Pseudomonas aeruginosa biofilms, and (3) determined the impact of LL37-SM hydrogel treatment on RAW 264.7 macrophage activation and phagocytic activity. The association of LL37 to SM hydrogels enabled the sustained release of LL37 over 8 hours and retained antimicrobial activity. Treatment with LL37-SM hydrogels for 24 hours disrupted biofilm growth and resulted in a mixed inflammatory response in macrophages. Our results highlight the antimicrobial, antibiofilm, and potentially inflammatory modulating capabilities of SM hydrogels which can further inform the use of mucins in bioactive biomaterials for biomedical applications.Item Investigation and development of induced pluripotent stem cell derived extracellular vesicle-based therapeutics(2024) Levy, Daniel H; Jay, Steven; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Due to their complex, multicomponent nature, extracellular vesicle (EV)-based therapeutics have arisen as an intriguing option for treatment of complex diseases that require the simultaneous modulation of distinct pathways. Due to their inherent regenerative properties, mesenchymal stem cell (MSC)-derived EVs have been the most heavily investigated and utilized in clinical trials for diseases including acute respiratory distress syndrome, wound healing and many more. While pre-clinical studies have demonstrated promise for such EV-based therapeutics, source cell limitations act as a hurdle to the widespread clinical translation of MSC EV therapies. MSCs and other cells reported to produce therapeutic EVs (cardiac progenitor cells, neural stem cells, etc.) have limited expansion capabilities ex vivo before cellular senescence, therefore limiting the amount of therapeutic EVs that can be produced by a single cell line. Due to these limited expansion capabilities, alternative, self-renewing therapeutic EV source cells are needed. One such source cell is induced pluripotent stem cells (iPSCs), which possess self-renewing capabilities. However, the baseline bioactivity of iPSC EVs have yet to be rigorously evaluated; in our work, we report for the first time that iPSC EVs possess robust anti-inflammatory properties in addition to confirming previous reports of their ability to promote vascularization in a murine diabetic wound healing model. Building off these baseline results, we sought to augment iPSC EV potency by utilizing genetic approaches to load of bioactive RNAs including microRNA (miRNA) and long non-coding RNA (lncRNA) into iPSC EVs. In our miRNA loading studies, we effectively demonstrate that the natural biogenesis pathways of miRNA can be probed to facilitate export of bioactive miRNAs to secreted EVs, thereby enhancing their anti-inflammatory bioactivity. Lastly, we utilize a genetic engineering approach to enhance active sorting of lncRNAs into secreted EVs and test their therapeutic potential in a murine colitis model. The work described in this dissertation provides a foundation towards the clinical translation of iPSC EV-based therapeutics by benchmarking them against more established therapeutic EV sources (iPSC-derived MSC EVs) and developing strategies to enhance their bioactivity via RNA cargo loading.Item SPECTROSCOPY-BASED METHODS FOR BIOLOGICAL APPLICATIONS(2024) Zhou, Xuewen; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Spectroscopy plays a crucial role in biological measurements by offering functional insights across various biological scales, from cellular to organ levels. It has become an important complement to imaging technologies. The thesis focuses on enhancing spectroscopy-based methods, addressing both technical advancements and application-oriented improvements. The first part of the research presents the development of standardized test methods for multispectral photoacoustic imaging (MPAI), a phantom-based test method specific for evaluating MPAI performance in breast cancer detection. This test method contributes to standardizing the evaluation of multispectral photoacoustic imaging across different research and clinical settings with better reliability and reproducibility. The next segment of the thesis introduces an innovative 2D-dispersion spectrometer, utilizing an etalon and grating setup. This instrument significantly improves the sensitivity in detecting Whispering Gallery Mode (WGM) microlasers by providing spectral resolution less than 0.3 pm. The enhanced sensitivity allows for the measurement of hyperfine refractive index and absorption changes in liquid environments, which expands the application limit of microlasers as biosensors. In the final part, the thesis extends the application of the 2D-dispersion spectrometer to detect Brillouin and low-frequency Raman signals. For the best of our knowledge, this is the first setup demonstrating simultaneous Brillouin and low-frequency Raman measurement, which can provide mechanical and chemical information of a sample such as the viscoelasticity and intermolecular dynamic.Item ENHANCING BIOPRINTING STRATEGIES TOWARDS THE DEVELOPMENT OF BIOMIMETIC OSTEOCHONDRAL TISSUE ENGINEERING SCAFFOLDS(2023) Choe, Robert; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Osteoarthritis is a highly prevalent rheumatic musculoskeletal disorder that affects approximately 900,000 Americans annually and is characterized by the progressive breakdown of the articular cartilage and remodeling of the subchondral bone in the synovial joint. During early-stage osteoarthritis, the articular cartilage begins to degrade, the synovial joint space narrows, and the subchondral bone undergoes rapid bone turnover, leading to insufficient bone mineralization and compromised matrix integrity. While decades of research have revealed that an intricate balance between the bone and cartilage layers influences biochemical and biomechanical changes experienced within the osteochondral unit, most osteochondral tissue engineering scaffolds have not achieved clinical viability. Tissue engineering (TE) strategies, such as 3D bioprinting (3DP), offer a new avenue to help develop novel osteochondral tissue engineering scaffolds to regenerate healthy and diseased osteochondral joints. In this project, our immediate goal is to expand the repertoire of osteochondral bioprinting strategies toward developing a biomimetic, 3D-printed osteochondral scaffold that can be implanted into acute focal cartilage defects during early-stage OA. We will explore the designs and fabrication strategies of various 3D-printed biomimetic osteochondral interface scaffolds with enhanced mechanics guided by computational simulations. Additionally, we will examine the potential of utilizing osteoblast- and osteoclast-lineage cell co-cultures to improve regenerative outcomes at the bone scaffold layer of osteochondral tissue engineering scaffolds. The long-term goal of this work is to aid in developing a biomimetic 3D printed osteochondral scaffold that has enhanced load-bearing properties and elevated regeneration potential to recreate the unique osteochondral architecture at each distinct tissue layer.Item BIOMATERIALS REPROGRAM ANTIGEN PRESENTING CELLS TO PROMOTE ANTIGEN-SPECIFIC TOLERANCE IN AUTOIMMUNITY(2023) Eppler, Haleigh B; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The immune system is tightly regulated to balance the killing of disease-causing organisms while protecting host tissue from accidental damage. When this balance is disrupted, immune dysfunctions such as autoimmune diseases occur. Autoimmune diseases like type 1 diabetes and multiple sclerosis (MS) develop when self-tissue is mistakenly attacked and damaged by immune cells. For example, during MS, the immune system mistakenly attacks the myelin sheath that insulates neurons, causing loss of motor function and burdening patients and caregivers. Recent advances in immunotherapies offer exciting new treatments; however, even monoclonal antibody therapies cannot differentiate between healthy and disease-causing cells. Biomaterials provide powerful capabilities to help address these shortcomings. In particular, control over the concentration, duration, location, and combination of signals that are received by immune cells could be transformative in developing more selective immunotherapies that are safe and promote antigen-specific tolerance during autoimmune disease. This dissertation uses two biomaterial approaches to deliver regulatory cargo to antigen presenting cells (APCs). An important APC function is to detect disease-causing organisms by sensing pathogen associated molecular patterns (PAMP) through motif-specific receptors. CpG rich motifs are PAMPs that activate toll-like receptor 9 (TLR9) on DCs and B cells. TLR9 signaling activates B cells and DCs. In MS, TLR9 signaling is aberrantly elevated on certain DCs contributing to systemic inflammation. In MS, B cells signaling through the TLR9 pathwway induced the expression of more inflammatory cytokines as compared to B cells from healthy controls. Controlling this overactive TLR signaling restrains inflammation and is a possible tolerogenic therapeutic approach in MS. The first part of this dissertation uses biomaterials-based polyelectrolyte multilayers (PEMs) to deliver tunable amounts of GpG – an oligonucleotide that inhibits TLR9 signaling – to dendritic cells (DCs). These studies demonstrate that PEMs inhibit DC activation and reduce pathway-specific inflammatory signaling. Furthermore, this work demonstrates that these changes to DCs promote tolerance in downstream T cell development as shown by increasing regulatory T cells. These studies demonstrate this biomaterial delivery system selectively inhibits TLR signaling and DC activation. These changes to DCs promote myelin-specific T cells to adopt a regulatory phenotype, demonstrating a potential approach to developing tolerance inducing antigen-specific immunotherapies for MS. The second part of this dissertation uses a degradable polymer microparticle (MP) system to control the local microenvironment of lymph nodes (LNs). LNs are key sites in the development of immune responses. LNs are composed of different microdomains that coordinate immune cell interactions such as germinal centers (GCs), where B cells develop. These MPs are loaded with myelin self-antigen (MOG35-55) and an mTOR inhibitor, rapamycin (rapa). The MPs are designed to be too large to passively diffuse from the LNs; instead, they slowly degrade releasing encapsulated immune cues to immune cells within the lymph node (LN). Our previous work demonstrates this treatment approach induces antigen-specific tolerance in a preclinical model of MS, but the role of APCs – including DCs and B cells - has not been elucidated. This dissertation reveals that MP treatment alters key LN structural components responsible for interactions between cells in GCs. In addition, MPs alter interactions between B cells/DCs and T cells, as measured by presentation of encapsulated antigen and inhibition of T cell costimulatory molecules by encapsulated rapa. These changes inhibit myelin-specific T cell proliferation and promote regulatory T cells. Finally, B cells from MOG/rapa and MOG MP treated lymph nodes transfer myelin-specific efficacy to mice induced with EAE. These findings illustrate how LN and cellular processes can be regulated by MPs to promote myelin-specific tolerance informing the development of myelin-specific immunotherapies for MS. Together, this body of work provides insight into how biomaterials can be designed to exploit native LN and immune cell functions in the design of next-generation approaches to safely induce myelin-specific tolerance during MS or other autoimmune diseases.Item INSTRUMENTATION AND AUTOMATION FOR STIMULATED BRILLOUIN SPECTROSCOPY(2023) Frank, Eric; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The use of Brillouin spectroscopy for noninvasive probing of the mechanical properties of biologically relevant materials shows great promise. Stimulated Brillouin scattering (SBS) spectroscopy has the potential to significantly improve measurement speed and resolution by amplifying the scattered signal resonantly. However, current SBS spectrometers have been limited by fundamental and practical constraints in detection parameters. Here, we develop and demonstrate a novel LabVIEW-automated SBS instrumentation scheme in which a number of instruments that otherwise operate independently are automatized and synchronized from a singular LabVIEW program with emphasis on the user interface. Additionally, localization theory, originating from fluorescence-based super resolution microscopy techniques, is applied to the acquisition of SBS spectra, and experimentally demonstrated using this instrumentation scheme, resulting in spectra being acquired an order of magnitude faster while maintaining performances in terms of signal to noise ratio (SNR) and measurement precision.Item Assessment of Mechanical Cues to Enhance the Clinical Translation of Extracellular Vesicles(2022) Kronstadt, Stephanie Marie; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mesenchymal stem cells (MSCs) are a common source for cell-based therapies due to their innate regenerative properties. However, these cells often die shortly after injection and, if they do survive, run the risk of forming tumors. Cell-secreted nanoparticles known as extracellular vesicles (EVs) have been identified as having therapeutic effects similar to those of their parental cells without the safety risks. Specifically, MSC EVs have emerged as a promising therapeutic modality in a multitude of applications, including autoimmune and cardiovascular diseases, cancer, and wound healing. Despite this promise, low levels of naturally occurring EV cargo may necessitate repeated doses to achieve clinical benefit, countering the advantages of EVs over MSCs. The current techniques to combat low EV potency (e.g., loading external molecules or using chemicals) are not agreeable to large-scale manufacturing techniques and would substantially increase the regulatory burden associated with EV translation. Fortunately, mechanical cues within the microenvironment have potential to overcome these translational barriers as they can alter EV therapeutic effects but are also cost-effective and can be precisely manipulated in a reproducible manner. The goal of this project is to understand how these cues impact MSC EV secretion and physiological effects. We showed that flow-derived shear stress applied to MSCs seeded within a 3D-printed scaffold (i.e., the bioreactor) can significantly upregulate EV production (EVs/cell) while maintaining the in vitro pro-angiogenic effects of MSC EVs. Interestingly, we demonstrated that MSC EVs generated using the bioreactor system significantly improved wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Furthermore, for the first time, we showed that mechanical confinement of MSCs within micropillars could augment MSC EV production and bioactivity. Lastly, we demonstrated that soft substrates composed of various polydimethylsiloxane (PDMS) formulations could increase MSC EV production and activity as well. Through the work performed here, we have laid the groundwork to elucidate the relationship between cell mechanobiology and EV activity that will ultimately enable an adaptable and scalable EV therapeutic platform.Item Strategies for small RNA loading into extracellular vesicles(2022) Pottash, Alex; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Small RNAs are an exciting class of therapeutics with significant untapped therapeutic potential, due to their ability to affect cell behavior at the RNA level. However, delivery of RNA is a challenge due to its size and labile nature. Extracellular vesicles (EVs) are promising as delivery vehicles due to their natural role as physiological intercellular microRNA transporters, and research has shown that EVs have significant advantages compared to competing technologies such as lipid nanoparticles. Specifically, EVs more readily transport through biological barriers, deliver RNA more efficiently, and are less immunogenic. However, intrinsic microRNA content in EVs is low and thus active small RNA loading strategies are needed to enable therapeutic use. Consequently, a variety of small RNA loading methods for EVs have been developed. These include endogenous and exogenous approaches. Exogenous approaches, in which EVs are loaded directly, have been shown to enable loading of hundreds to thousands of small RNAs per EV, but they are not readily amenable to scalable production processes. Endogenous approaches, in which EVs are loaded by upstream manipulation of the producer cell, are compatible with large scale EV production, but loading by these approaches is inconsistent and has scarcely been quantitatively analyzed. The work in this dissertation is focused on enabling small RNA therapeutics via EV delivery. The lack of an ideal small RNA loading approach for EVs is addressed by tackling important issues of both endogenous and exogenous loading. First, the loading capacity of several common endogenous loading methods was optimized and quantitatively analyzed. Additionally, new approaches to endogenous small RNA loading involving genetic manipulation of the RNA structure and the microRNA cellular processing pathway were developed and evaluated. Finally, exogenous loading via sonication was applied to enable delivery of a novel microRNA combination that was identified via a rational selection process. This combination of miR-146a, miR-155, and miR-223 was found to have potentially synergistic anti-inflammatory activity, and EV-mediated delivery of the combination opens the possibility for therapeutic application in inflammatory diseases and conditions such as sepsis. Overall, this work both improves understanding of current techniques for small RNA loading into EVs and opens new opportunities for advanced strategies, bringing EV-based small RNA therapeutics closer to clinical application.Item A NANOCOMPOSITE HYDROGEL FOR STROMAL CELL-DERIVED FACTOR-1 ALPHA DELIVERY AND MODULATION OF MACROPHAGE PHENOTYPE FOR SKIN TISSUE REGENERATION(2021) Yu, Justine Ruth; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chronic, non-healing skin wounds arising as a sequela of underlying disease are often difficult to treat clinically, susceptible to infection, and may severely reduce a patient’s quality of life. Tissue engineered constructs may be employed to aid in wound closure and skin regeneration, but no single skin substitute is currently capable of fully restoring normal skin structure and physiological function. One critical factor directing wound healing progression and the resulting functional outcome is the host inflammatory response. Circulating monocytes migrate to wound sites and differentiate into macrophages, which further polarize to pro- or anti-inflammatory phenotypes depending on microenvironmental properties including extracellular matrix composition and local cytokine gradients. In chronic wounds, polarization is predominantly pro-inflammatory, resulting in the secretion of cytokines that impede healing. Studies have identified stromal cell-derived factor-1 alpha (SDF-1α) as a potent chemokine that recruits mesenchymal stem cells (MSCs) and macrophages, modulating their phenotype to promote the secretion of anti-inflammatory cytokines. We endeavor to fabricate a tissue engineered hydrogel-based biomaterial that can sustain the release of SDF-1α to initiate pro-healing effects at chronic wound sites. In the first aim of this project, we develop and characterize a nanocomposite hydrogel system capable of releasing SDF-1α and exerting bioactive effects on MSCs. This demonstrates its capability to controllably release the chemokine over time at physiologically relevant levels. In the second aim, we study this hydrogel’s effects on macrophage migration and phenotype both in vitro as well as in vivo using wild type and diabetic murine models. We show that our material allows macrophages primarily of the anti-inflammatory phenotype to infiltrate wounded tissue, and subsequently demonstrate its ability to stimulate skin tissue formation and vascularization so as to improve the rate of healing. The findings described in this dissertation detail the successful development of a nanocomposite hydrogel delivery system for immunomodulatory and wound healing applications, which may support the future development of clinical wound dressings, skin substitutes, and other immune-informed strategies for tissue regeneration applications.