Browsing by Author "Lee, Yong Woo"
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- 3-D Bio-inspired Microenvironments for In Vitro Cell MigrationHosseini, Seyed Yahya (Virginia Tech, 2015-10-21)Cancer metastasis is the leading cause of death related to cancer diseases. Once the cancer cells depart the primary tumor site and enter the blood circulation, they spread through the body and will likely initiate a new tumor site. Therefore, understanding the cell migration and stopping the spread in the initial stage is the utmost of importance. In this dissertation, we have proposed a 3-D microenvironment that (partially) mimics the structures, complexity and circulation of human organs for cell migration studies. We have developed the tools to fabricate 3-D complex geometries in PDMS from our previously developed single-mask, single-etch technology in silicon. In this work, 3-D patterns are transferred from silicon structures to glass following anodic bonding and high temperature glass re-flow processes. Silicon is etched back thoroughly via wet etching and the glass is used as master device to create 3-D PDMS structures for use in dielectrophoresis cell sorting applications. Furthermore, this work has been modified to fabricate 3-D master devices in PDMS to create 3-D structures in collagen hydrogels to mimic native tissue structures. We have studied the interaction of endothelial cells with model geometries of blood vessels in collagen hydrogel at different concentrations to mimic the biomechanical properties of tissues varying from normal to tumor under the growth factor stimulation. Finally, we have designed and fabricated a silicon-based transmigration well with a 30um-thick membrane and 8um pores. This platform includes a deep microfluidic channel on the back-side sealed with a glass wafer. The migratory behavior of highly metastatic breast cancer cells, MDA-MB-231, is tested under different drug treatment conditions. This versatile platform will enable the application of more complex fluidic circulation profile, enhanced integration with other technologies, and running multiple assays simultaneously.
- 3D Coiling at the Protrusion Tip: New Perspectives on How Cancer Cells Sense Their Fibrous SurroundingsMukherjee, Apratim (Virginia Tech, 2021-05-24)Cancer metastasis, the spread of cancer from the primary site to distant regions in the body, is the major cause of cancer mortality, accounting for almost 90% of cancer related deaths. During metastasis, cancer cells from the primary tumor initially probe the surrounding fibrous tumor microenvironment (TME) prior to detaching and subsequently migrating towards the blood vessels for further dissemination. It has widely been acknowledged that the biophysical cues provided by the fibrous TME greatly facilitate the metastatic cascade. Consequently, there has been a tremendous wealth of work devoted towards elucidating different modes of cancer cell migration. However, our knowledge of how cancer cells at the primary tumor site initially sense their fibrous surroundings prior to making the decision to detach and migrate remains in infancy. In part, this is due to the lack of a fibrous in vitro platform that allows for precise, repeatable manipulation of fiber characteristics. In this study, we use the non-electrospinning, Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with exquisite control on fiber dimensions and network architecture and use these networks to investigate how single cancer cells biophysically sense fibers mimicking in vivo dimensions. Using high spatiotemporal resolution imaging (63x magnification/1-second imaging interval), we report for the first time, that cancer cells sense individual fibers by coiling (i.e. wrapping around the fiber axis) at the tip of a cell protrusion. We find that coiling dynamics are mediated by both the fiber curvature and the metastatic capacity of the cancer cells with less aggressive cancer cells showing diminished coiling. Based on these results, we explore the possibility of using coiling in conjunction with other key biophysical metrics such as cell migration dynamics and forces exerted in the development of a genetic marker independent, biophysical predictive tool for disease progression. Finally, we identify the membrane curvature sensing Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) as a key regulator of protrusive activity with IRSp53 knockout (KO) cells exhibiting significantly slower protrusion dynamics and diminished coil width compared to their wild-type (WT) counterparts. We demonstrate that the hindered protrusive activity ultimately translates to impaired contractility, alteration in the nucleus shape and slower migration dynamics, thus highlighting the unique role of IRSp53 as a signal transducer – linking the protrusive activity at the cell membrane to changes in cytoskeletal contractility. Overall, these findings offer novel perspectives to our understanding of how cancer cells biophysically sense their fibrous surroundings. The results from this study could ultimately pave the way for elucidating the precise fiber configurations that either facilitate or hinder cancer cell invasion, allowing for the development of new therapeutics in the long term that could inhibit the metastatic cascade at a relatively nascent stage and yield a more promising prognosis in the perennial fight against cancer.
- Attributes of Astrocyte Response to Mechano-Stimulation by High-Rate OverpressureHlavac, Nora (Virginia Tech, 2018-11-29)Blast neurotrauma represents a significant mode of traumatic injury to the brain. The incidence of blast neurotrauma is particularly high amongst military combat personnel and can be debilitating and endure clinically for years after injury is sustained. Mechanically, blast represents a unique and complex loading paradigm associated with compressive shock waves that propagate out from an explosive event and interact with the head and other organs through high-rate loading. When subjected to such insult, brain cells undergo characteristic injury responses which include neuroinflammation, oxidative stress, edema and persistent glial activation. These features of the injury have emerged as important mediators of the chronic brain damage that results from blast. Astrocytes have emerged as a potential therapeutic target because of their ubiquitous roles in brain homeostasis, tissue integrity and cognitive function. This glial subtype has a characteristic reactive response to mechanical trauma of various modes. In this work, custom in vitro injury devices were used to characterize functional models of astrocyte reactivity to high-rate insult to study mechano-stimulation mechanisms associated with the reactive phenotype. The working hypothesis was that high-rate overpressure exposure would cause metabolic aberrations, cell junction changes, and adhesion signal transduction activation, all of which would contribute to astrocyte response and reactivity. Astrocyte cultures were exposed to a 20 psi high-rate overpressure scheme using an underwater explosion-driven device. Astrocytes experienced dynamic energetic fluctuations in response to overpressure which were followed by the assumption of a classically defined reactive phenotype. Results indicated specific roles for cationic transduction, cell junction dynamics (gap junction and anchoring junctions) and downstream signal transduction mechanisms associated with adhesion alterations in onset of the astrocyte reactive phenotype. Investigation into adhesion signaling regulation by focal adhesion kinase in 2D and 3D cultures was also explored to better understand cellular reactivity as a function of extracellular environment. Additionally, another underwater in vitro device was built to study combination effects from overpressure and fluid shear associated with insult. Overall, the combined studies offer multiple mechanisms by which to explore molecular targets for harnessing astrocytes' potential for repair after traumatic injury to the brain.
- Bioactive Poly(Lactic-co-Glycolic Acid)-Calcium Phosphate Scaffolds for Bone Tissue RegenerationPopp, Jenni Rebecca (Virginia Tech, 2009-03-27)Bone is currently the second most transplanted tissue, second only to blood. However, significant hurdles including graft supply and implant failure continue to plague researchers and clinicians. Currently, standard clinical procedures include autologous and allogeneic grafting. Autologous grafts may achieve functional repair; yet, they are available in limited supply and are associated with donor site morbidity. Allogeneic grafts are available in greater supply, but have a higher risk of infection. To overcome the disadvantages of current grafts, tissue engineering has become a major focus for the regeneration of bone. The goal of tissue engineering is to use a multidisciplinary approach to create biomimetic constructs that stimulate osteogenic regeneration to heal bone defects and restore tissue function. Biodegradable scaffolds are used in tissue engineering strategies as an interim template for tissue regeneration. The scaffold architecture provides mechanical support for cell attachment and tissue regeneration. Biocompatible poly(lactic-co-glycolic acid) (PLGA) has been processed through a number of techniques to create porous 3D architectures. Hydroxyapatite (HAP) and tricalcium phosphate have been used in conjunction with polymer scaffolds due to their osteoconductivity and biocompatibility, but they often lack osteoinductivity and are resistant to biodegradation. Conversely, amorphous calcium phosphate (ACP) is a mineral that solubilizes under aqueous conditions, releasing calcium and phosphate ions, which have been postulated to enhance osteoblast differentiation and mineralization. Controlled dissolution can be achieved by stabilizing ACP with divalent cations such as zinc or copper. Furthermore, incorporation of such osteogenic ACPs within a biodegradable PLGA scaffold could enhance the osteoconductivity of the scaffold while providing calcium and phosphate ions to differentiating osteoprogenitor cells, thereby stimulating osteogenesis when implanted in vivo. In this research, the effect of zinc on the differentiation of osteoprogenitor cells was investigated. Zinc supplementation of the culture media had no stimulatory effect on cell proliferation or differentiation. ACPs were synthesized using zirconium (ZrACP) and zinc (ZnACP) as stabilizers to achieve sustained ion release. Elevated concentrations suggested sustained ion release over the course of 96 hours and enhanced solubility of ZrACP and ZnACP. X-ray diffraction analysis showed a conversion of ZrACP to a semi-crystalline material after 96 hours, but ZnACP showed no conversion after 96 hours. Composite scaffolds were fabricated by incorporating HAP, zirconium-stabilized ACP (ZrACP), or zinc-stabilized ACP (ZnACP) into a sintered PLGA microsphere matrix and then characterized to determine the effect of the minerals on the in vitro differentiation of MC3T3-E1 cells. Scanning electron microscopy revealed a porous microsphere matrix with calcium phosphate powders distributed on the surface of the microspheres. Measurements of mechanical properties indicated that incorporation of 0.5 wt% calcium phosphates resulted in a 30% decrease in compressive modulus. When cells were cultured in the scaffolds, composite ACP scaffolds stimulated proliferation and ALP activity, while HAP scaffolds stimulated osteoblast gene expression. Overall, the results of this work indicate the addition of calcium phosphate minerals to PLGA scaffolds supported cell growth and stimulated osteogenic differentiation, making the scaffolds a promising alternative for bone tissue regeneration.
- Biomedical research application of a novel double-layer parallel-plate flow chamberLee, Won Hee (Virginia Tech, 2007-05-09)Since integrity and functions of vascular endothelial cells are greatly affected by shear stress, a variety of in vitro systems to subject endothelial cells under precisely controlled fluid conditions has been developed. Complicated designs of the conventional flow devices, however, have impeded such implementation. In the present study, we designed and developed a novel parallel-plate flow chamber (PPFC). It consists of multiple layers of different materials to adjust the required geometries of the chamber and provide a wide span of biomedical research applications. Because the chamber stacks separate layers to constitute the flow channel, different pieces can be easily removed or replaced. Moreover, the multilayer design only requires 2D cutting, which is easier and faster to manufacture. It is also capable of accepting up to four glass slides facing each other so that the flow within the channel is exclusively formed by endothelial cells. Furthermore, it minimizes the pressure loss across the chamber while maximizing the effective area of endothelial cells up to 96 cm2. Results from mathematical analysis and dye injection experiments showed that a uniform magnitude of shear stress is applied throughout the entire surface of endothelial cells. In addition, the morphological changes and attenuated gene expression of pro-inflammatory mediators were observed in endothelial cells exposed to the physiologically relevant shear stress. These findings indicate that our newly designed PPFC can provide a better in vitro system for versatile applications of biomedical research. The reperfusion of blood flow occurred in a number of conditions such as stroke and organ transplantation immensely augments tissue injury and can cause more severe damage than prolonged ischemia. The injuries caused by cessation and reperfusion of blood flow are closely related to the inflammatory reactions involving in endothelium-leukocyte cascade responding to a shear stress exerted by the flow. Shear stress is also known to play an important role in human chronic diseases including atherosclerosis, neurological disorders, and cancer metastasis. Therefore, it is important to investigate the transmission of mechanical stimuli such as shear stress to various complex endothelial cell signaling pathways which process as a whole is often referred as mechanotransduction. Shear stress-mediated signaling pathways have been known to trigger endothelial cell responses and contribute to the pathophysiology of human vascular diseases. The present study was designed to apply the novel PPFC to biomedical research, especially ischemia/reperfusion injury. The changes in mRNA and protein expression of inflammatory mediators in endothelial cells were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RBE4 and HMEC-1 cells were either maintained in continuous laminar flow condition (Normal Flow) or subjected to 1 h of flow cessation followed by reperfusion of flow (Ischemia/Reperfusion) for 24 h. Ischemia/Reperfusion significantly up-regulated expression of inflammatory mediators, such as IL-6, MCP-1, ICAM-1, VCAM-1, and E-selectin, in microvascular endothelial cells. Furthermore, antioxidant pyrrolidine dithiocarbamate (PDTC) significantly attenuated ischemia/reperfusion-induced overexpression of pro-inflammatory mediators. These data indicates that our newly designed PPFC provide a better in vitro system for versatile applications of biomedical research.
- Bursts of Bipolar Microsecond Pulses Inhibit Tumor GrowthSano, Michael B.; Arena, Christopher B.; Bittleman, Katelyn Rose; DeWitt, Matthew R.; Cho, Hyung J.; Szot, Cchristopher S.; Saur, Dieter; Cissell, James M.; Robertson, John L.; Lee, Yong Woo; Davalos, Rafael V. (Nature Publishing Group, 2015-10-13)Irreversible electroporation (IRE) is an emerging focal therapy which is demonstrating utility in the treatment of unresectable tumors where thermal ablation techniques are contraindicated. IRE uses ultra-short duration, high-intensity monopolar pulsed electric fields to permanently disrupt cell membranes within a well-defined volume. Though preliminary clinical results for IRE are promising, implementing IRE can be challenging due to the heterogeneous nature of tumor tissue and the unintended induction of muscle contractions. High-frequency IRE (H-FIRE), a new treatment modality which replaces the monopolar IRE pulses with a burst of bipolar pulses, has the potential to resolve these clinical challenges. We explored the pulse-duration space between 250 ns and 100 μs and determined the lethal electric field intensity for specific H-FIRE protocols using a 3D tumor mimic. Murine tumors were exposed to 120 bursts, each energized for 100 μs, containing individual pulses 1, 2, or 5 μs in duration. Tumor growth was significantly inhibited and all protocols were able to achieve complete regressions. The H-FIRE protocol substantially reduces muscle contractions and the therapy can be delivered without the need for a neuromuscular blockade. This work shows the potential for H-FIRE to be used as a focal therapy and merits its investigation in larger pre-clinical models.
- Cell-Fiber Interactions: A New Route to Mechano-Biological Investigations in Developmental and Disease BiologySheets, Kevin Tyler (Virginia Tech, 2014-11-03)Cells in the body interact with a predominantly fibrous microenvironment and constantly adapt to changes in their neighboring physiochemical environment, which has implications in developmental and disease biology. A myriad of in vitro platforms including 2D flat and 3D gel substrates with and without anisotropy have demonstrated cellular alterations to subtle changes in topography. Recently, our work using suspended fibers as a new in vitro biological assay has revealed that cells are able to sense and respond to changes in fiber curvature and structural stiffness as evidenced by alterations to cytoskeleton arrangement, including focal adhesion cluster lengths and nucleus shape indices, leading to altered migration speeds. It is hypothesized that these behaviors occur due to modulation of cellular inside-out forces in response to changes in the external fibrous environment (outside-in). Thus, in this study, we investigate the role of fiber curvature and structural stiffness in force modulation of single cells attached to suspended fibers. Using our previously reported non-electrospinning Spinneret based Tunable Engineered Parameters (STEP) fiber manufacturing platform, we present our findings on single cell inside-out and outside-in forces using fibers of three diameters (250 nm, 400 nm and 800 nm) representing a wide range of structural stiffness (3-45 nN/μm). To investigate cellular adaptability to external perturbation, we present the development of a first-of-its-kind force measurement 'nanonet' platform capable of investigating cell adhesion forces in response to symmetric and non-symmetric (injury model) loading. Our combined findings are multi-fold: (i) Cells on suspended fibers are able to form focal adhesion clusters approximately four times longer than those on flat substrates, which gives them potential to double their migration speeds, (ii) Nanonets as force probes show that the contractility-based inside-out forces are nearly equally distributed on both sides of the cell body, and that overall force magnitudes are dependent on fiber structural stiffness, and (iii) External perturbation can evenly (symmetric) or unevenly (non-symmetric) distribute forces within the cell, and the resulting bias causes diameter-dependent outside-in adhesion force response. Finally, we demonstrate the power of the developed force measurement platform by extending our studies to cell-cell junctional forces as well as single-cell disease models including cancer and aortic aneurysm.
- Cellular and Molecular Mechanisms of ASDLee, Yong Woo (2012-10-12)Yong Woo Lee describes the role of inflammation in disease and the pathophysiological mechanisms of ASD, and suggests multidisciplinary autism research into the efficacy of anti-oxidant and anti-inflammatory drugs as potential treatment for autism.
- Chronic Cerebral Hypoperfusion Induces Alterations of Matrix Metalloproteinase-9 and Angiopoietin-2 Levels in the Rat HippocampusKim, Min-Soo; Choi, Bo-Ryoung; Lee, Yong Woo; Kim, Dong-Hee; Han, Ye Sun; Jeon, Won Kyung; Han, Jung-Soo (2018-08)Angiogenic factors contribute to cerebral angiogenesis following cerebral hypoperfusion, and understanding these temporal changes is essential to developing effective treatments. The present study examined temporal alterations in angiogenesis-related matrix metalloproteinase-9 (MMP-9) and angiopoietin-2 (ANG-2) expression in the hippocampus following bilateral common carotid artery occlusion (BCCAo). Male Wistar rats (12 weeks of age) were randomly assigned to sham-operated control or experimental groups, and expression levels of MMP-9 and ANG-2 were assessed after BCCAo (1 week, 4 weeks, and 8 weeks), using western blotting. Protein expression increased 1 week after BCCAo and returned to control levels at 4 and 8 weeks. In addition, immunofluorescence staining demonstrated that the MMP-9- and ANG-2-positive signals were primarily observed in the NeuN-positive neurons with very little labeling in non-neuronal cells and no labeling in endothelial cells. In addition, these cellular locations of MMP-9-and ANG-2-positive signals were not altered over time following BCCAo. Other angiogenic factors such as vascular endothelial growth factor and hypoxia-inducible factor did not differ from controls at 1 week; however, expression of both factors increased at 4 and 8 weeks in the BCCAo group compared to the control group. Our findings increase understanding of alterations in angiogenic factors during the progression of cerebral angiogenesis and are relevant to developing effective temporally based therapeutic strategies for chronic cerebral hypoperfusion-associated neurological disorders such as vascular dementia.
- Corticosteroid-Encapsulated Nanoparticles in Thermoreversible Gels for the Amelioration of Choroidal Neovascularization in Age-Related Macular DegenerationHirani, Anjali A. (Virginia Tech, 2015-04-30)Age-related macular degeneration (AMD) is one of the leading causes of blindness in adults over the age of 60. Currently, at least 11 million patients in the United States have some form of macular degeneration and this number is projected to grow as the population ages. The more severe form of the disease – neovascular (wet) AMD, is characterized by intraocular neovascularization, inflammation, and retinal damage; however, the disease progression can be deterred through intraocular injections of anti-angiogenic agents. The complications and burden that arise from repetitive injections as well as the difficulty posed by targeting the posterior segment of the eye make this an interesting territory for the development of novel drug delivery systems. New methods for drug delivery are being investigated exploring the use of nanoparticles and other polymeric materials. The goal of this project is to study the potential use of poly(lactide-co-glycolic acid)-polyethylene glycol (PLGA-PEG) nanoparticles in thermoreversible gels as localized sustained intraocular drug delivery. We prepared stable and reproducible corticosteroid-encapsulated nanoparticles in thermoreversible gels to inhibit vascular endothelial growth factor (VEGF) overexpression characteristic of neovascular AMD. We characterized the drug delivery system by obtaining size, shape, and drug encapsulation data. We also demonstrated that the polymer could be injected into the vitreous as a solution and transition to a gel phase based on the temperature difference between regular indoor environment and the vitreous body. The drug delivery system was tested on human retinal pigment epithelial cells (ARPE-19), for cytotoxicity, uptake and VEGF expression. We also examined the drug delivery system's ability to mitigate the disease progression in a mouse model of choroidal neovascularization (CNV). The effect on blood vessel area was shown and the changes in the mRNA expression of angiogenesis mediators were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). These results indicate that the proposed drug delivery systems has the promise to be developed for retinal diseases, involving CNV, including neovascular AMD. Further studies are warranted in developing this promising intraocular drug delivery system for wet AMD and similar ophthalmic diseases.
- Cytotoxicity and Cellular Uptake of Cellulose NanocrystalsDong, Shuping; Hirani, Anjali A.; Colacino, Katelyn R.; Lee, Yong Woo; Roman, Maren (2012-09-21)There is growing evidence that filamentous nanoparticles offer advantages over spherical ones in drug delivery applications. The purpose of this study was to assess the potential of rod-like, plant-derived cellulose nanocrystals (CNCs) for nanomedical uses. Besides a nonspherical morphology, their facile bioconjugation, surface hydrophilicity and small size render CNCs promising drug carriers. The cytotoxicity of CNCs against nine different cell lines (HBMEC, bEnd.3, RAW 264.7, MCF-10A, MDA-MB-231, MDA-MB-468, KB, PC-3 and C6) was determined by MTT and LDH assay. CNCs showed no cytotoxic effects against any of these cell lines in the concentration range and exposure time studied (0–50 µg/mL and 48 h, respectively). Cellular uptake of fluorescein-50 - isothiocyanate-labeled CNCs by these cell lines, quantified with a fluorescence microplate reader, was minimal. The lack of cytotoxicity and the low nonspecific cellular uptake support our hypothesis that CNCs are good candidates for nanomedical applications.
- The Design and Assembly of 3D Liver Mimetic Cellular ArchitecturesKim, Yeonhee (Virginia Tech, 2010-09-07)We report the assembly of three-dimensional (3D) liver sinusoidal mimics comprised of primary rat hepatocytes, human or rat liver sinusoidal endothelial cells denoted as hLSECs and rLSECs respectively, and an intermediate chitosan-hyaluronic acid (HA) polyelectrolyte multilayer (PEM). The height of the PEMs ranged from 30-55nm and exhibited a shear modulus of ~ 100kPa. Primary rat hepatocytes coated with 5 and 15 PE layers exhibited stable urea and albumin production over a seven day period and these values were either comparable or superior to that in a collagen sandwich (CS). Hepatocyte-PEM-hLSEC liver mimics exhibited stable urea production and increasing albumin secretion over the culture period in comparison to hepatocyte-LSEC samples. In the 3D liver mimics, hLSEC phenotype was maintained and verified by the uptake of acetylated low-density lipoprotein (AcLDL). A sixteen-fold increase in CYP1A1/2 activity was observed for hepatocyte-PEM-10,000 hLSEC samples, thereby, suggesting that interactions between hepatocytes and hLSECs play a key role in enhancing hepatic phenotypes in in vitro cultures. As the first step towards elucidating key signaling pathways involved in cell-cell communications, global genome-wide transcriptional profiles of primary hepatocytes cultured in CS and hepatocyte monolayers (HMs) were performed over an eight-day period using DNA microarray measurements and Gene Set Enrichment Analysis (GSEA) in order to derive biologically meaningful information at the level of gene sets. The gene expression in CS cultures steadily diverged from that in HMs. Gene sets up-regulated in CS are those linked to liver metabolic and synthetic functions, such as lipid, fatty acid, alcohol and carbohydrate metabolism, urea production, and synthesis of bile acids. Monooxygenases such as CYP enzymes were significantly up-regulated starting on day 3 in CS cultures. These results serve as a baseline for further investigation into the systems biology of engineered liver tissues. 3D hepatic constructs were also assembled with primary rat hepatocytes and rLSECs, and a chitosan-HA PEM. In these hepatic models, the phenotype of hepatocytes and rLSECs were maintained. rLSEC phenotype was verified over a twelve-day period through immunostaining with the sinusoidal endothelial-1 (SE-1) antibody. In contrast, rLSECs cultured as monolayers lost their phenotype within 3 days. A two-fold increase in albumin production was observed only in the 3D liver models. rLSEC-PEM-hepatocyte cultures exhibited three- to six-fold increased CYP1A1/2 and CYP3A enzymatic activity. Well-defined bile canaliculi were observed in only 3D hepatic constructs. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte constructs can be used as liver models for future studies.
- The Design of Three-Dimensional Multicellular Liver Models Using Detachable, Nanoscale Polyelectrolyte MultilayersLarkin, Adam Lyston (Virginia Tech, 2012-08-29)We report the design and assembly of three-dimensional (3D) multi-cellular liver models comprised of primary rat hepatocytes, liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs). LSECs and KCs in the liver model were separated from hepatocytes by a nanoscale, detachable, optically transparent chitosan and hyaluronic acid (HA) polyelectrolyte multilayer (PEM) film. The properties of the PEM were tuned to mimic the Space of Disse found in liver. The thickness of the detachable PEM was 650 to 1000 nm under hydrated conditions. The Young's modulus of the PEM was approximately 42 kPa, well within the range of modulus values reported for bulk liver. The 3D liver models comprised of all three cell types and a detachable PEM exhibited stable urea production and increased albumin secretion over a 12 day culture period. Additionally, the 3D liver model maintained the phenotype of both LSECs and KCs over the 12 day culture period, verified by CD32b and CD163 staining, respectively. Additionally, CYP1A1 enzyme activity increased significantly in the 3D liver models. The number of hepatocytes in the 3D liver model increased by approximately 60% on day 16 of culture compared to day 4 indicating. Furthermore, only the 3D hepatic model maintained cellular compositions virtually identical to those found in vivo. DNA microarray measurements were conducted on the hepatocyte fractions of the 3D liver mimic to obtain insights into hepatic processes. Gene sets up-regulated in the 3D liver model were related to proliferation, migration, and deposition of extracellular matrix, all functions observed in regenerating hepatocytes. Taken together, these results suggest that inter-cellular signaling between the different cell types in the 3D liver model led to increased hepatic functions. To the best of our knowledge, this is the first study where three of the major hepatic cell types have been incorporated into a model that closely mimics the structure of the sinusoid. These studies demonstrate that the multi-cellular liver models are physiologically relevant. Such models are very promising to conduct detailed investigations into hepatic inter-cellular signaling.
- Development and Validation of Human Body Finite Element Models for Pedestrian ProtectionPak, Wansoo (Virginia Tech, 2019-10-21)The pedestrian is one of the most vulnerable road users. According to the World Health Organization (WHO), traffic accidents cause about 1.34 million fatalities annually across the world. This is the eighth leading cause of death across all age groups. Among these fatalities, pedestrians represent 23% (world), 27% (Europe), 40% (Africa), 34% (Eastern Mediterranean), and 22% (Americas) of total traffic deaths. In the United States, approximately 6,227 pedestrians were killed in road crashes in 2018, the highest number in nearly three decades. To protect pedestrians during Car-to-Pedestrian Collisions (CPC), subsystem impact tests, using impactors corresponding to the pedestrian's head and upper/lower leg were included in regulations. However, these simple impact tests cannot capture the complex vehicle-pedestrian interaction, nor the pedestrian injury mechanisms, which are crucial to understanding pedestrian kinetics/kinematics responses in CPC accidents. Numerous variables influence injury variation during vehicle-pedestrian interactions, but current test procedures only require testing in the limited scenarios that mostly focus on the anthropometry of the 50th percentile male subject. This test procedure cannot be applied to real-world accidents nor the entire pedestrian population due to the incredibly specific nature of the testing. To better understand the injury mechanisms of pedestrians and improve the test protocols, more pre-impact variables should be considered in order to protect pedestrians in various accident scenarios. In this study, simplified finite element (FE) models corresponding to 5th percentile female (F05), 50th percentile male (M50), and 95th percentile male (M95) pedestrians were developed and validated in order to investigate the kinetics and kinematics of pedestrians in a cost-effective study. The model geometries were reconstructed from medical images and exterior scanned data corresponding to a small female, mid-sized male, and tall male volunteers, respectively. These models were validated based on post mortem human surrogate (PMHS) test data under various loading including valgus bending at knee joint, lateral/anterior-lateral impact at shoulder, pelvis, thorax, and abdomen, and lateral impact during CPC. Overall, the kinetic/kinematic responses predicted by the pedestrian FE models showed good agreement against the corresponding PMHS test data. To predict injuries from the tissue level up to the full-body, detailed pedestrian models, including sophisticated musculoskeletal system and internal organs, were developed and validated as well. Similar validations were performed on the detailed pedestrian models and showed high-biofidelic responses against the PMHS test data. After model development and validation, the effect of pre-impact variables, such as anthropometry, pedestrian posture, and vehicle type in CPC impacts were investigated in different impact scenarios. The M50-PS model's posture was modified to replicate pedestrian gait posture. Five models were developed to demonstrate pedestrian posture in 0, 20, 40, 60, and 80 % of the gait cycle. In a sensitivity study, the 50th percentile male pedestrian simplified (M50-PS) model in gait predicted various kinematic responses as well as the injury outcomes in CPC impact with different vehicle type. The pedestrian FE models developed in this work have the capability to reproduce the kinetic/kinematic responses of pedestrians and to predict injury outcomes in various CPC impact scenarios. Therefore, this work could be used to improve the design of new vehicles and current pedestrian test procedures, which eventually may reduce pedestrian fatalities in traffic accidents.
- Development of a Nanoparticle Vaccine Delivery System with Polymeric Oral Adjuvants for PoultryCary, Jewel Maria (Virginia Tech, 2019-09-06)Development of new vaccination technology has been hindered by a lack of new adjuvants to enable development of protective immunity using different vaccine delivery methods. A vaccine delivery system using oral adjuvants would be applicable across species for both individual and mass vaccination in both the medical and veterinary fields. We sought to create an oral nanoparticle (NP) vaccine delivery system that is easy to produce and uses polymers as oral adjuvants with killed virus. Our hypothesis was gelatin and chitosan would enhance viral uptake and stimulate immune cells to produce protective immunity. This would allow the safer killed form of each virus to be used in place of modified live (MLV) viruses and avoid undesirable side effects like immunosuppression. The research objectives were to 1. Fabricate and characterize gelatin NPs encapsulating inert materials of similar size and shape to the viruses of interest for fabrication proof-of-concept 2. Modify the NP delivery system to minimize immune cell cytoxicity for the vaccine delivery application 3. Fabricate and characterize FPV and HEV viral nanoparticles' stability, cellular uptake/infectivity, and released viruses' ability to replicate 4. Compare the abilities of the killed HEV nanovaccine, killed HEV with loose gelatin and chitosan polymers (no nanoparticle), and a live HEV commercial vaccine to induce textit{in vivo} seroconversion, protective immunity, and side effects during clinical and challenge studies in turkeys We proved our hypothesis to be correct in addition to demonstrating matching the encapsulation material size to empty NP size leads to preferred encapsulated NP formulation parameters, chitosan stabilizes the NP formulation bypassing the need for cytotoxic crosslinkers, and paraformaldehyde is able to kill virus prior to vaccine formulation while still preserving virus morphology sufficiently for immune cell recognition. This development constitutes one of the first novel adjuvants discoveries in 70 years and opens the door for conversion of injectable vaccines to oral delivery across species.
- Effect of Mechanical Environment on the Differentiation of Bone Marrow Stromal Cells for Functional Bone Tissue EngineeringKavlock, Katherine Dulaney (Virginia Tech, 2009-03-27)Bone is the second most transplanted tissue after blood and the need for bone graft materials continues to rise at an average annual growth rate of over 18%. An engineered bone substitute consisting of a bone-like extracellular matrix deposited on the internal pores of a resorbable biomaterial scaffold is postulated to stimulate normal bone remodeling when implanted in vivo. Part one of this engineering strategy, the deposition of bone-like extracellular matrix, can be achieved by the directed differentiation of progenitor cells such as bone marrow stromal cells (BMSCs). Part two of the engineering strategy, the biomaterial scaffold, can be fabricated with the appropriate mechanical properties using a synthetic polymer system with tunable properties like polyurethanes. Finally, BMSCs seeded within the biomaterial scaffold can be cultured in a perfusion flow bioreactor to stimulate osteoblastic differentiation and the deposition of bioactive factors. Using the three-part engineering strategy described, I hypothesize that the extracellular matrix produced by BMSCs can be modulated by two stimuli: the stiffness of the scaffold and perfusion flow. First, I propose that culturing BMSCs on polyurethane scaffolds with increasing stiffness will increase markers of osteoblastic differentiation. Secondly, I suggest that mechanically stimulating BMSCs with novel perfusion strategies will also increase markers of osteoblastic differentiation. In aim 1, a family of segmented degradable poly(esterurethane urea)s (PEUURs) were synthesized. The modulus of the PEUUR materials was systematically increased from 0.18 to 0.80 MPa by systematically increasing the molecular weight of the poly(ε-caprolactone) (PCL) soft segment from 1425 to 2700 Da. BMSCs were cultured on both rigid polymer films and on porous foam scaffolds to dissociate the effect of variation in polymer chemistry from the effect of scaffold modulus on cell phenotype. These studies demonstrated changes in osteoblastic differentiation as measured by prostaglandin E2 production, alkaline phosphatase activity (ALP) activity, and osteopontin gene expression. However, the increased levels of these phenotypic markers on the PCL 2700 material could not be attributed to scaffold chemistry or modulus. Instead, the differences may be related to polymer crystallinity or surface topography. In aim 2, novel dynamic perfusion strategies were used to investigate the influence of frequency on osteoblastic differentiation. BMSCs were seeded on porous foam scaffolds and exposed to both steady perfusion and pulsatile perfusion at 0.017, 0.050, and 0.083 Hz frequencies. The data presented here demonstrated that while some markers of osteoblastic phenotype such as ALP activity are enhanced by 0.05 Hz pulsatile flow over continuous flow, they are insensitive to frequency at low frequencies. Therefore, future studies will continue to investigate the effect of a larger range of frequencies. Additionally, fluid flow has also been shown to stimulate the deposition of bioactive factors such as BMP-2 and VEGF-A, and these growth factors are known to significantly enhance healing in bone defect models. Therefore, we plan to investigate the effect of dynamic flow strategies on the deposition of these bioactive factors. We propose that an engineered bone graft material containing a bone-like extracellular matrix and producing these growth factors will show more rapid formation of bone when implanted in vivo.
- Effects of acute ingestion of different fats on oxidative stress and inflammation in overweight and obese adults.Peairs, Abigail D.; Rankin, Janet L. Walberg; Lee, Yong Woo (2011-11-07)Background Studies show that obese individuals have prolonged elevations in postprandial lipemia and an exacerbated inflammatory response to high fat meals, which can increase risk for cardiovascular diseases. As epidemiological studies indicate an association between type of fat and circulating inflammatory markers, the purpose of this study was to investigate the acute effect of different fat sources on inflammation and oxidative stress in overweight and obese individuals. Methods Eleven overweight and obese subjects consumed three high fat milkshakes rich in monounsaturated fat (MFA), saturated fat (SFA), or long-chain omega 3 polyunsaturated fat (O3FA) in random order. Blood samples collected at baseline, 1, 2, 4, and 6 hours postprandial were analyzed for markers of inflammation (soluble intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor- α (TNF-α), and C-reactive protein (CRP)), oxidative stress (8-epi-prostaglandin-F2α (8-epi) and nuclear factor-κB (NF-κB)), and metabolic factors (glucose, insulin, non-esterified free fatty acids, and triglycerides (TG)). Results O3FA enhanced NF-kB activation compared to SFA, but did not increase any inflammatory factors measured. Conversely, SFA led to higher ICAM-1 levels than MFA (p = 0.051), while MFA increased TG more than SFA (p < 0.05). CRP increased while TNF-α and 8-epi decreased with no difference between treatments. Conclusions While most of the inflammatory factors measured had modest or no change following the meal, ICAM-1 and NF-κB responded differently by meal type. These results are provocative and suggest that type of fat in meals may differentially influence postprandial inflammation and endothelial activation.
- The Effects of Carbohydrate and Quercetin on Team Sport Athletic Performance and Exercise-Induced Inflammation and Oxidative StressAbbey, Elizabeth Lea (Virginia Tech, 2009-03-31)Over 270 million people play soccer worldwide, and its popularity grows every day. In team sport exercise, fatigue may result from numerous factors including limited fuel, depleted energy stores and production of compounds that promote an inflammatory response. While inflammation is an essential mechanism for repairing damaged muscle tissue with exercise, prolonged inflammation leads to increased production of reactive oxygen species that can damage cell membranes, muscle, and signaling proteins. To prevent this response and improve performance, athletes are increasingly looking to nutritional interventions. Carbohydrate and antioxidant supplementation have both shown evidence of producing an ergogenic effect and attenuating inflammation and oxidative stress with prolonged endurance exercise. Less is known about how these interventions may influence intermittent, high-intensity exercise characteristic of soccer. In particular, this exercise presents a unique challenge in that opportunities for nutrient intake are limited to pre-game and half-time. In our first study, we had 10 male collegiate soccer players perform a 90-min. soccer-simulation test, that we developed, which was followed by a progressive shuttle run (PSR) test to exhaustion. They consumed a honey-sweetened beverage (H), a sports drink (S), or a placebo (P) before and half-way through the protocol. Both H and S provided 1.0 g·kg⁻¹ carbohydrate and ~17.6 mL·kg⁻¹ total volume for each trial. Overall, the test resulted in increased fatigue and production of inflammatory markers and antioxidant capacity. There was no significant difference between treatments for any performance measure. Mean times for a high intensity run and rating of perceived exertion increased with time, and there was an overall decrease in PSR time compared to baseline (-22.9%). There was a rise in glucose (15.6%), IL-6 (548%), IL-1ra, IL-10 (514%) and ORAC (15%) post-test but no change in cortisol. Insulin was significantly lower by 1 h-post. IL-1ra levels increased post-test for H (25.8%), S (65.5%), and P (63.9%), but the change for H was less than the other treatments. No treatment effects for the other blood measures were observed. The lack of an ergogenic effect of carbohydrate on soccer performance calls into question the benefit of supplementation at a frequency typical of a regulation soccer match in highly trained athletes with adequate energy stores. Since acute carbohydrate ingestion in the first study did not attenuate some markers of inflammation (e.g. IL-6), we chose to focus on an alternative theory for the rise in inflammatory markers with strenuous exercise in our second study. One aspect of soccer, repeated sprinting, results in increased ROS production partially through the activation of the enzyme xanthine oxidase (XO). Quercetin, a flavonol in plants that has shown some ergogenic effects with endurance exercise, inhibits XO in vitro. The effect of quercetin on team sport exercise had not been studied. We gave recreationally active males a commercial sports drink (S) or S + 500 mg of quercetin (Q) 2x/d for 1 wk prior to a repeated sprint test (RST). Sprint times increased (5.9%) for both treatments as did plasma XO activity (47%), IL-6 (77%), and uric acid (25%) from pre-test to post-test. Q supplementation did not attenuate plasma XO activity or IL-6 and actually increased one calculated index of fatigue, percent fatigue decrement (5.1%- Q and 3.8%- P). These findings add to the growing body of literature that quercetin supplementation does not attenuate exercise-induced inflammation and oxidative stress in vivo. Collectively, this research has practical implications for sports drink companies who are exploring the use of flavonoid compounds in product formulation. Specifically, they should reconsider adding quercetin to their beverages if they are marketing to team sport athletes. Also, soccer players should be made aware that, at ingestion frequencies typical of a soccer match, they may not expect a significant performance benefit from acute carbohydrate supplementation.
- Effects of Febuxostat on Autistic Behaviors and Computational Investigations of Diffusion and PharmacokineticsSimmons, Jamelle Marquis (Virginia Tech, 2019-02-06)Autism spectrum disorder (ASD) is a lifelong disability that has seen a rise in prevalence from 1 in 150 children to 1 in 59 between 2000 and 2014. Patients show behavioral abnormalities in the areas of social interaction, communication, and restrictive and repetitive behaviors. As of now, the exact cause of ASD is unknown and literature points to multiple causes. The work contained within this dissertation explored the reduction of oxidative stress in brain tissue induced by xanthine oxidase (XO). Febuxostat is a new FDA approved XO-inhibitor that has been shown to be more selective and potent than allopurinol in patients with gout. The first study developed a computational model to calculate an effective diffusion constant (Deff) of lipophilic compounds, such as febuxostat, that can cross endothelial cells of the blood-brain barrier (BBB) by the transcellular pathway. In the second study, male juvenile autistic (BTBR) mice were treated with febuxostat for seven days followed by behavioral testing and quantification of oxidative stress in brain tissue compared to controls. Results of the first study showed that the lipophilic tracer chosen, as a substitute for febuxostat, could be modeled under the assumption of passive diffusion while experimental controls did not fit this model. The second study revealed no significant differences between BTBR mice that received febuxostat or the drug vehicle in both behavioral testing and quantification of oxidative stress in brain tissue. In the final study, of the four models proposed, one model was selected as the most plausible that considered transport into the CNS. As there is currently no literature surrounding tissue and organ ADME for febuxostat the final proposed model would need to be updated as new information becomes available.
- Effects of Keratin Biomaterial Therapeutics on Cellular and Inflammatory Mechanisms in Injury and Disease ModelsWaters, Michele (Virginia Tech, 2018-06-11)Keratins are fibrous structural proteins found in human hair that have been used to develop bioactive and biocompatible constructs for a wide variety of tissue engineering and healthcare applications. Their ubiquity, capacity for self-assembly, ease of use and versatility in blended materials, and ability to modulate cell behavior and promote tissue ingrowth have made keratins well-suited for the development of regenerative therapies. In particular, keratins have demonstrated bioactivity in both in-vivo and in-vitro studies, by altering immune and stem cell phenotype and function and promoting an anti-inflammatory/wound healing environment. This work seeks to build on previous research by investigating the ability of low and high molecular weight keratins to augment anti-inflammatory primary macrophage phenotypes and examining the influence of keratin biomaterials on cellular and inflammatory mechanisms in two models of injury and disease. Rodent models of blast induced neurotrauma (BINT) and severe osteoporosis were used to inform the development of 2D and 3D in-vitro models of macrophage/endothelial cell injury and osteogenic differentiation respectively. Keratin biomaterials exhibited some potential to alter macrophage and endothelial cell dynamics following blast, specifically by promoting anti-inflammatory (M2c-like) macrophage polarization and diminishing endothelial cell injury responses (i.e. endothelial glycocalyx shedding). A more clinically relevant model of osteoporosis found that stem cells harvested from older, osteoporotic animals demonstrated limited proliferative and bone differentiation potential compared to healthy cells. However, 3D constructs (especially keratin-based materials) were able to enhance calcification and osteogenic gene expression of diseased cells. These results highlight the complexity of macrophage phenotypic switching and cellular dynamics in these systems. However, keratin-based therapeutics may prove useful for facilitating tissue regeneration and limiting detrimental inflammatory and cellular responses in various models of injury and disease.