Biomedical Engineering Department Theses and Dissertations

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Adipose stromal cells enhance keratinocyte survival and migration in vitro, and graft revascularization in mouse wound healing model
(2013-12-11) Knowles, Kellen Alexander; Berbari, Edward J.; March, Keith Leonard, 1963-; Ji, Julie
In the US, more than 1 million burn injuries are reported annually. About 45,000 injuries due to fires and burns result in hospitalization and ten percent of these result in death every year. Advances in burn treatment have led to a reduction in mortality rate over the last decades. Since more patients are surviving the initial resuscitation phase even with very large areas of skin being burned away, wound care has become increasingly important to ensure continued patient survival and improvement. While currently a common treatment for third degree burn wounds, skin grafts have several drawbacks. The availability of donor sites for autografts may be limited, especially in incidences of extensive skin loss. The rejection associated with the use of allografts and xenografts may render them inadequate or undesirable. Even if a suitable graft is found, poor retention due to infection, hematoma, and low vascularity at the recipient site are other drawbacks associated with the use of skin grafts as a primary treatment for severe burn wounds. As such, research has been done into alternative treatments, which include but are not limited to artificial skin, cell therapy, and growth factor application. We propose the delivery of adipose derived stem cells (ASC) in combination with endothelial progenitor cells (EC) via Integra Dermal Regenerative Template (DRT) to promote faster graft vascularization and thus faster healing of wounds. Integra DRT is an acellular skin substitute that consists of a dermal layer composed of bovine collagen and chondroitin-6-sulfate glycosaminoglycan, and an "epidermal" layer, which consists of silicone polymer. This silicone layer is removed after the collagen matrix is adequately vascularized (usually takes 2-3 weeks), and then a thin layer autograft is applied to the top of the neo-dermis. ASC are derived from the stromal-vascular fraction (SVF) of adipose tissue and are a readily available, pluripotent, mesenchymal cell known to promote angiogenesis. They are being explored as a treatment for a myriad of diseases and conditions, including wound healing. In combination with ECs, they form stable microvessel networks in vitro and in vivo. In our work, we found that ASC+EC form stable microvessel networks when cultured on Integra DRT. Also, ASC and ASC+EC conditioned media promoted both survival and migration of human epidermal keratinocytes compared to control medium. In a full thickness wound healing model, using healthy NSG mice, the ASC+EC case showed a significantly higher rate of wound closure compared to control. Based on best linear unbiased estimates (BLUE), the difference between the healing rates of ASC alone treatment and the Control treatment group is -0.45 +/- 0.22 mm²/day (p=0.041), which is not less than 0.025 and thus not statistically significant (Bonferroni Adjusted). However, the BLUE for the difference between the ASC+EC group and the Control group healing rates is -0.55 +/- 0.28 mm²/day (p = 0.017<0.025, Bonferroni Adjusted), which is statistically significant. Histology revealed a significantly higher number of vessels compared to control in both ASC alone and ASC+EC case. CD31 staining revealed the presence of human vessels in ASC+EC treatment scaffolds. We conclude that the combination of ASC and EC can be used to accelerate healing of full-thickness wounds when delivered to site of the wound via Integra. This result is especially compelling due to the fact that the mice used were all healthy. Thus our treatment shows an improvement in healing rate even compared to normal wound healing.
Analysis of Heart Rate Variability During Focal Parasympathetic Drive of the Rat Baroreflex
(2020-05) Bustamante, David J.; Schild, John; Yoshida, Ken; Salama, Paul
Autonomic control of the heart results in variations in the intervals between heart beats, known as heart rate variability. One of the defining components of autonomic control is the baroreflex, a negative feedback controller that balances heart rate and blood pressure. The baroreflex is under constant command from the branches of the autonomic nervous system. To better understand how the autonomic nervous system commands the baroreflex, a baroreflex reflexogenic animal protocol was carried out. Heart rate variability analysis and baroreflex sensitivity were used to quantify the neural control of the heart. This thesis reconfirmed the existence of sexually dimorphic properties in the baroreflex through the use of heart rate variability analysis and baroreflex sensitivity. It was discovered that there are many caveats to utilizing heart rate variability analysis, which have to be addressed both in the experimental protocol and the signal processing technique. Furthermore, it was suggested that the slope method for quantifying baroreflex sensitivity also has many caveats, and that other baroreflex sensitivity methods are likely more optimal for quantifying sustained activation of the baroreflex. By utilizing various heart rate variability signal processing algorithms to assess autonomic tone in Sprague-Dawley rats during rest and sustained electrical activation of the baroreflex, the null hypothesis was rejected.
Analysis of the Bioelectric Impedance of the Tissue-Electrode Interface Using a Novel Full-Spectrum Approach
(2014-01-15) Sempsrott, David Robert; Yoshida, Ken; Salama, Paul; Berbari, Edward J.
Non-invasive surface recording of bioelectric potentials continues to be an essential tool in a variety of research and medical diagnostic procedures. However, the integrity of these recordings, and hence the reliability of subsequent analysis, diagnosis, or recommendations based on the recordings, can be significantly compromised when various types of noise are allowed to penetrate the recording circuit and contaminate the signals. In particular, for bioelectric phenomena in which the amplitude of the biosignal is relatively low, such as muscle activity (typically on the order of millivolts) or neural traffic (microvolts), external noise may substantially contaminate or even completely overwhelm the signal. In such circumstances, the tissue-electrode interface is typically the primary point of signal contamination since its impedance is relatively high compared to the rest of the recording circuit. Therefore, in the recording of low-amplitude biological signals, it is of paramount importance to minimize the impedance of the tissue-electrode interface in order to consistently obtain low-noise recordings. The aims of the current work were (1) to complete the development of a set of tools for rapid, simple, and reliable full-spectrum characterization and analytical modeling of the complex impedance of the tissue-electrode interface, and (2) to characterize the interfacial impedance and signal-to-noise ratio (SNR) at the surface of the skin across a variety of preparation methods and determine a factor or set of factors that contribute most effectively to the reduction of tissue-electrode impedance and noise contamination during recording. Specifically, we desired to test an initial hypothesis that surface abrasion is the principal determining factor in skin preparation to achieve consistently low-impedance, low-noise recordings. During the course of this master’s study, (1) a system with portable, battery-powered hardware and robust acquisition/analysis software for broadband impedance characterization has been achieved, and (2) the effects of skin preparation methods on the impedance of the tissue-electrode interface and the SNR of surface electromyographic recordings have been systematically quantified and compared in human subjects. We found our hypothesis to be strongly supported by the results: the degree of surface abrasion was the only factor that could be correlated to significant differences in either the interfacial impedance or the SNR. Given these findings, we believe that abrasion holds the key to consistently obtaining a low-impedance contact interface and high-quality recordings and should thus be considered an essential component of proper skin preparation prior to attachment of electrodes for recording of small bioelectric surface potentials.
Analysis of the efficacy of EPIONE therapies to treat phantom limb pain
(2017-03) Comoglio, Caleb C.; Yoshida, Ken
The primary objectives of this thesis are (1) to discuss the current understanding of phenomena associated with, proposed mechanisms of, and suggested treatments for amputation related pain, (2) to describe the software developed for analyzing results of a clinical study for the treatment of phantom limb pain (PLP), (3) to discuss the methods for a multi-center trial by the EPIONE consortium along with presenting preliminary results, and (4) to discuss the methods and results of a case study involving a new therapy modality for alleviating PLP. Each objective has been expanded into a chapter as described below. Chapter 1 serves as a literature review introducing the topic of amputation, associ- ated phenomena, and proposed mechanisms. The chapter also discusses the currently available treatments and the instruments used to measure PLP. Key topics include the definition of PLP, the prevalence of PLP, current treatment options for PLP, and experimental measurement of PLP. The final objective of this chapter is to introduce topics related to the investigation paradigm utilized for the studies following in Chapter 4 and Chapter 5. Therefore, a minor emphasis has been put on surface electrical stimulation (SES) and operant conditioning. As with any multi-center clinical study, coordination is key. Chapter 2 introduces the common clinical protocol (CCP) and methods of analysis for the clinical trials conducted by the EPIONE consortium. In order to analyze results in an automated fashion, a software tool was developed. This tool, the EPIONE Extraction Program (EEP) along with its extension the Group Analysis Module (GAM), is the focus of Chapter 3. A high-level overview of the requirements, process flow, and software testing are described. This chapter also discusses the methods of analysis for several self-report instruments used to determine effect size in Chapter 4 and Chapter 5. The outputs of the software tools make up the results presented and described in these chapters. In addition to the details included in Chapter 3, supplemental information is available in Appendix A and Appendix B, which are the detailed User Guides for the EEP and GAM. Chapter 4 reviews the pilot study data conducted by the EPIONE consortium. The primary and two secondary instruments used for analysis are discussed. This chapter provides a brief overview of results from the group. Each clinical site used slightly different variations of a common clinical protocol to better understand what effectively drives alleviation of PLP and to allow comparison of results. The work done at Indiana University - Purdue University Indianapolis (IUPUI) represents a small part of several other universities involved in the EPIONE consortium. Chapter 5 focuses on a case study at IUPUI with a more in-depth review of data collected throughout the study period. Using SES, we seek to reverse cortical reorganization by giving meaningful stimuli through existing circuitry. In this chapter the present work is discussed by introducing a case study in detail with an analysis of psychophysical data.
Application of pulse width modulation to a Western blotting device
(2016) TruongVo, ThucNhi; Yokota, Hiroki
One of the critical steps in a current Western blot technique is a blotting process, which in general requires one electrophoretic gel for every protein species to be analyzed. In most cases, multiple protein species are analyzed simultaneously and thus it is necessary for a scientist to run multiple gels. In order to make it possible to analyze multiple protein species from a single gel, a novel blotting device, BlotMan, was employed in this study. Designed by Dr. Chien’s group (YC Bioelectric), BlotMan uses pulse width modulation (PWM) for applying a protein size-dependent voltage during a blotting process. In this study, the differential average voltage profile, depending on protein size (e.g. 17 kDa to 140 kDa), was built and enabled BlotMan to transfer all protein species in equal efficiency regardless of the protein size. Furthermore, Blot- Man consists of a user-friendly, custom-made interface box, which can be remotely controlled by a smart phone. BlotMan’s capability was evaluated using standard protein markers, as well as protein samples that were isolated from chondrosarcoma cells (SW1353) and breast cancer cells (MDA-MB-213). The experimental results revealed that BlotMan was capable of generating 5 blotting membranes from a single gel simultaneously. Protein species such as c-Src, eukaryotic translation initiation factor 2 alpha (eIF2α) and its phosphorylated form (p-eIF2α), lamin B, and β-actin were successfully detected. It is also demonstrated that compared to a regular constant voltage, PWM signals improved transfer efficiency and a signal-to-noise ratio. In conclusion, this study demonstrated that BlotMan was able to facilitate Western blotting analysis by generating multiple blotting membranes from a single gel with an improved signal-to-noise ratio. Further analysis is recommended for understanding the mechanism of PWMts action on transfer efficiency and noise reduction.
Application of quantitative analysis in treatment of osteoporosis and osteoarthritis
(2013-11-08) Chen, Andy Bowei; Yokota, Hiroki, 1955-; Na, Sungsoo; Schild, John H.
As our population ages, treating bone and joint ailments is becoming increasingly important. Both osteoporosis, a bone disease characterized by a decreased density of mineral in bone, and osteoarthritis, a joint disease characterized by the degeneration of cartilage on the ends of bones, are major causes of decreased movement ability and increased pain. To combat these diseases, many treatments are offered, including drugs and exercise, and much biomedical research is being conducted. However, how can we get the most out of the research we perform and the treatment we do have? One approach is through computational analysis and mathematical modeling. In this thesis, quantitative methods of analysis are applied in different ways to two systems: osteoporosis and osteoarthritis. A mouse model simulating osteoporosis is treated with salubrinal and knee loading. The bone and cell data is used to formulate a system of differential equations to model the response of bone to each treatment. Using Particle Swarm Optimization, optimal treatment regimens are found, including a consideration of budgetary constraints. Additionally, an in vitro model of osteoarthritis in chondrocytes receives RNA silencing of Lrp5. Microarray analysis of gene expression is used to further elucidate the mode of regulation of ADAMTS5, an aggrecanase associated with cartilage degradation, by Lrp5, including the development of a mathematical model. The math model of osteoporosis reveals a quick response to salubrinal and a delayed but substantial response to knee loading. Consideration of cost effectiveness showed that as budgetary constraints increased, treatment did not start until later. The quantitative analysis of ADAMTS5 regulation suggested the involvement of IL1B and p38 MAPK. This research demonstrates the application of quantitative methods to further the usefulness of biomedical and biomolecular research into treatment and signaling pathways. Further work using these techniques can help uncover a bigger picture of osteoarthritis's mode of action and ideal treatment regimens for osteoporosis.
Biomechanical and morphological characterization of common iliac vein remodeling: Effects of venous reflux and hypertension
(2014) Brass, Margaret Mary; Kassab, Ghassan S. (Ghassan Sleewa), 1965-; Na, Sungsoo; Berbari, Edward J.
The passive properties of the venous wall are important in the development of venous pathology. Increase in venous pressure due to retrograde flow (reflux) and obstruction of venous flow by intrinsic and extrinsic means are the two possible mechanisms for venous hypertension. Reflux is the prevailing theory in the etiology of venous insufficiency. The objective of this thesis is to quantify the passive biomechanical response and structural remodeling of veins subjected to chronic venous reflux and hypertension. To investigate the effects of venous reflux on venous mechanics, the tricuspid valve was injured chronically in canines by disrupting the chordae tendineae. The conventional inflation-extension protocol in conjunction with intravascular ultrasound (IVUS) was utilized to investigate the passive biomechanical response of both control common iliac veins (from 9 dogs) and common iliac veins subjected to chronic venous reflux and hypertension (from 9 dogs). The change in thickness and constituent composition as a result of chronic venous reflux and hypertension was quantified using multiphoton microscopy (MPM) and histological evaluation. Biomechanical results indicate that the veins stiffened and became less compliant when exposed to eight weeks of chronic venous reflux and hypertension. The mechanical stiffening was found to be a result of a significant increase in wall thickness (p < 0.05) and a significant increase in the collagen to elastin ratio (p < 0.05). After eight weeks of chronic reflux, the circumferential Cauchy stress significantly reduced (p < 0.05) due to wall thickening, but was not restored to control levels. This provided a useful model for development and further analysis of chronic venous insufficiency and assessment of possible intervention strategies.
Building a Tensegrity-Based Computational Model to Understand Endothelial Alignment Under Flow
(2021-12) Al-Muhtaseb, Tamara; Ji, Julie; Na, Sungsoo; Tovar, Andres
Endothelial cells form the lining of the walls of blood vessels and are continuously subjected to mechanical stimuli from the blood flow. Microtubule-organizing center (MTOC), also known as centrosome is a structure found in eukaryotic cells close to the nucleus. MTOC relocates relative to the nucleus when endothelial cells are exposed to shear stress which determines their polarization, thus it plays a critical role in cell migration and wound healing. The nuclear lamina, a mesh-like network that lies underneath the nuclear membrane, is composed of lamins, type V intermediate filament proteins. Mutations in LMNA gene that encodes A-type lamins cause the production of a mutant form of lamin A called progerin and leads to a rare premature aging disease known as Hutchinson-Gilford Progeria Syndrome (HGPS). The goal of this study is to investigate how fluid flow affects the cytoskeleton of endothelial cells. This thesis consists of two main sections; computational mechanical modeling and laboratory experimental work. The mechanical model was implemented using Ansys Workbench software as a tensegrity-based cellular model in order to simulate the state of an endothelial cell under the effects of induced shear stress from the blood fluid flow. This tensegrity-based cellular model - composed of a plasma membrane, cytoplasm, nucleus, microtubules, and actin filaments - aims to understand the effects of the fluid flow on the mechanics of the cytoskeleton. In addition, the laboratory experiments conducted in this study examined the MTOC-nuclear orientation of endothelial cells under shear stress with the presence of wound healing. Wild-type lamin A and progerin-expressing BAECs were studied under static and sheared conditions. Moreover, a custom MATLAB code was utilized to measure the MTOC-nuclear orientation angle and classification. Results demonstrate that shear stress leads to different responses of the MTOC orientation between the wild-type and progerin-expressing cells around the vertical wound edge. Future directions for this study involve additional experimental work together with the improved simulation results to confirm the MTOC orientation relative to the nucleus under shear stress.
Coating of Polyvinylchloride for Reduced Cell / Bacterial Adhesion and Antibacterial Properties
(2019-05) Almousa, Rashed Abdulaziz R.; Xie, Dong; Na, Sungsoo; Li, Jiliang
A Polyvinylchloride surface was modified by coating a biocompatible, hydrophilic and antibacterial polymer by a mild surface modification method. The surface was first activated and then functionalized, followed by coating with polymer. The surface functionality was evaluated using cell adhesion, bacterial adhesion and bacterial viability for polymers with antibacterial properties. 3T3 mouse fibroblast cells were used for cell adhesion, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus were used for bacterial adhesion in the first study, Pseudomonas aeruginosa and Staphylococcus aureus were used for bacterial adhesion and antibacterial activity in the second study. Chapter 2 reports how we synthesized, immobilized and evaluated a novel hydrophilic polymer with anti-fouling properties onto surface of polyvinylchloride via an effective and mild surface coating technique. The polyvinylchloride surface was first activated by azidation as well as amination, and then tethering a newly synthesized hydrophilic and biocompatible polyvinylpyrrolidone having pendent reactive succinimide functionality onto the surface. Results show that the coated hydrophilic polymer significantly reduced the 3T3 fibroblast cell adhesion as well as the adhesion of the three bacterial species. Chapter 3 reports how we prepared, immobilized and evaluated an antibacterial and anti-fouling polymer onto polyvinylchloride surface following an efficient and simple method of surface modification. The surface coated with a terpolymer constructed with N-vinylpyrrolidone, 3,4-Dichloro-5-hydroxy-2(5H)-furanone derivative and succinimide residue was evaluated with cell adhesion, bacterial adhesion and bacterial viability. Surface adhesion was evaluated with 3T3 mouse fibroblast cells and two bacterial species. Also, antibacterial activity was evaluated by bacterial viability assay with the two bacterial species. Results showed that the polymer-modified polyvinylchloride surface exhibited significantly decreased 3T3 fibroblast cell adhesion and bacterial adhesion. Furthermore, the modified polyvinylchloride surfaces exhibited significant antibacterial functions by inhibiting bacterial growth with bactericidal activity. Altogether, we have successfully modified the surface of polyvinylchloride using a novel efficient and mild surface coating technique. The first hydrophilic polymer-coated polyvinylchloride surface significantly reduced cell adhesion as well as adhesion of three bacterial species. The second hydrophilic and antibacterial polymer-coated polyvinylchloride surface demonstrated significant antibacterial functions by inhibiting bacterial growth and killing bacteria in addition to significantly reduced 3T3 fibroblasts and bacterial adhesions.
A Comparative Analysis of Local and Global Peripheral Nerve Mechanical Properties During Cyclical Tensile Testing
(2022-05) Doering, Onna Marie; Yoshida, Ken; Wallace, Joseph; Goodwill, Adam
Understanding the mechanical properties of peripheral nerves is essential for chronically implanted device design. The work in this thesis aimed to understand the relationship between local deformation responses to global strain changes in peripheral nerves. A custom-built mechanical testing rig and sample holder enabled an improved cyclical uniaxial tensile testing environment on rabbit sciatic nerves (N=5). A speckle was placed on the surface of the nerve and recorded with a microscope camera to track local deformations. The development of a semi-automated digital image processing algorithm systematically measured local speckle dimension and nerve diameter changes. Combined with the measured force response, local and global strain values constructed a stress-strain relationship and corresponding elastic modulus. Preliminary exploration of models such as Fung and 2-Term Mooney-Rivlin confirmed the hyperelastic nature of the nerve. The results of strain analysis show that, on average, local strain levels were approximately five times smaller than globally measured strains; however, the relationship was dependent on global strain magnitude. Elastic modulus values corresponding to ~9% global strains were 2.070 ± 1.020 MPa globally and 10.15 ± 4 MPa locally. Elastic modulus values corresponding to ~6% global strains were 0.173 ± 0.091 MPa globally and 1.030 ± 0.532 MPa locally.

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