Graduate Degree Program in Bioengineering

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This digital collection includes theses and dissertations from the Graduate Degree Program in Bioengineering.

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Open Access
A comparison of tri-polar concentric ring electrodes to disc electrodes for decoding real and imaginary finger movements
(Colorado State University. Libraries, 2019) Alzahrani, Saleh Ibrahim, author; Anderson, Charles W., advisor; Vigh, Jozsef, committee member; Rojas, Don, committee member; Abdel-Ghany, Salah, committee member
The electroencephalogram (EEG) is broadly used for diagnosis of brain diseases and research of brain activities. Although the EEG provides a good temporal resolution, it suffers from poor spatial resolution due to the blurring effects of volume conduction and signal-to-noise ratio. Many efforts have been devoted to the development of novel methods that can increase the EEG spatial resolution. The surface Laplacian, which is the second derivative of the surface potential, has been applied to EEG to improve the spatial resolution. Tri-polar concentric ring electrodes (TCREs) have been shown to estimate the surface Laplacian automatically with better spatial resolution than conventional disc electrodes. The aim of this research is to study how well the TCREs can be used to acquire EEG signals to decode real and imaginary finger movements. These EEG signals will be then translated into finger movements commands. We also compare the feasibility of discriminating finger movements from one hand using EEG recorded from TCREs and conventional disc electrodes. Furthermore, we evaluated two movement-related features, temporal EEG data and spectral features, in discriminating individual finger from one hand using non-invasive EEG. To do so, movement-related potentials (MRPs) are measured and analyzed from four TCREs and conventional disc electrodes while 13 subjects performed either motor execution or motor imagery of individual finger movements. The tri-polar-EEG (tEEG) and conventional EEG (cEEG) were recorded from electrodes placed according to the 10-20 International Electrode Positioning System over the motor cortex. Our results show that the TCREs achieved higher spatial resolution than conventional disc electrodes. Moreover, the results show that signals from TCREs generated higher decoding accuracy compared to signals from conventional disc electrodes. The average decoding accuracy of five-class classification for all subjects was of 70.04 ± 7.68% when we used temporal EEG data as feature and classified it using Artificial Neural Networks (ANNs) classifier. In addition, the results show that the TCRE EEG (tEEG) provides approximately a four times enhancement in the signal-to-noise ratio (SNR) compared to disc electrode signals. We also evaluated the interdependency level between neighboring electrodes from tri-polar, disc, and disc with Hjorth's Laplacian method in time and frequency domains by calculating the mutual information (MI) and coherence. The MRP signals recorded with the TCRE system have significantly less mutual information (MI) between electrodes than the conventional disc electrode system and disc electrodes with Hjorth's Laplacian method. Also, the results show that the mean coherence between neighboring tri-polar electrodes was found to be significantly smaller than disc electrode and disc electrode with Hjorth's method, especially at higher frequencies. This lower coherence in the high frequency band between neighboring tri polar electrodes suggests that the TCREs may record a more localized neuronal activity. The successful decoding of finger movements can provide extra degrees of freedom to drive brain computer interface (BCI) applications, especially for neurorehabilitation.
Open Access
A drug eluting, osseointegrative phospholipid coating for orthopedic implants
(Colorado State University. Libraries, 2011) Prawel, David A., author; James, Susan P., advisor; Popat, Ketul C., advisor; Kipper, Matt J., committee member; Ryan, Stewart D., committee member
Millions of implant surgeries are performed each year. Titanium is commonly used for implantable metallic devices, especially total hip and knee replacements. However, titanium implants are far from perfect. Although the absolute failure rate is not particularly high, the case-by-case direct and human cost of each device implant failure is tremendous. Cementless titanium implant devices, although preferred by surgeons, frequently fail due to loosening of the device, often as a result of poor integration of naturally forming bone with the metallic implant, and by infection. Phospholipids are naturally occurring substances that are shown to enhance integration of new bone with implants, and to help reduce inflammation, a common precursor to infection. In addition, numerous studies have shown phospholipids to be effective drug delivery agents. To date, dip and drip coating techniques for applying phospholipid coatings have been used on titanium. Both coating techniques are easy to perform, but result in coatings too thick and non-conformal for in vivo use. Electro-spraying (E-spray) is a method of atomizing a liquid by means of electrical forces. E-spraying provides the advantage of being able to create coatings with relatively high efficiencies because the electrical charge difference "carries" the liquid source material, which also provides good control of coating morphology, especially on rough and intricately shaped surfaces. Other advantages of this technique are low cost and easy setup. In our work, the E-spraying technique was successfully adapted to apply thin, conformal, consistent coatings of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) to small, flat, commercially pure titanium plates. DOPS coatings were E-sprayed, then loaded with gentamicin sulfate (GS), a popular antibiotic used in treatment of osteomyelitis. An elution study was completed to assess drug delivery capabilities of the coatings. This work demonstrated that elution profile could be modified by changing E-spray parameters. Rat marrow stromal cells were harvested, and seeded onto the test coatings. Mesenchymal stem cells (MSCs) were selected from the general cell population, successfully cultured and differentiated into osteoblasts. Cytotoxicity of the coatings, along with cell viability, cell differentiation, biomineralization activity, cell morphology and early osseogenesis markers were evaluated at multiple time points in dual multi-week studies. DOPS coatings were found to be non-cytotoxic, and cell viability and biomineralization were higher on DOPS-coated surfaces and gentamicin-loaded coatings than on plain titanium samples. At the two week time point, excessive delamination of the coatings occurred in the cell growth environment. Research was undertaken to identify and test techniques to enhance coating retention. Surface chemistry was modified by passivation and pretreatment with calcium-chloride, and cholesterol was added to the DOPS E-spray. A repeated elution study demonstrated that elution profile could be modified as a result of changes in coating chemistry. An additional MSC cell study was completed to reconfirm the effects of enhanced coating chemistry on the cytotoxicity, cell viability and biomineralization. Cell morphology was re-evaluated at all time points via SEM imaging. Hydroxyapatite formation was confirmed. Preliminary osseogenesis biomarkers were also measured, showing deposition of osteocalcin and osteopontin, important protein precursors to normal bone growth, on enhanced coatings. This work demonstrates the viability of electro-sprayed DOPS coatings on titanium orthopedic implant material, and the enhanced osseogenic characteristics of these coatings. We also demonstrated that DOPS coatings can carry and release an antibiotic over time at clinically relevant dosages, and that this release profile can be engineered by modifications to E-spray process parameters, surface chemistry and E-sprayed material formulation.
Open Access
Aging related co-contraction effects on balance recovery using the ankle strategy
(Colorado State University. Libraries, 2010) Mixco, Anthony, author; Reiser, Raoul Frederick, II, advisor; Tracy, Brian L., advisor; Greene, David Paul, committee member
Aging typically leads to an increased risk for falls. Fall risk has been associated with, amongst other variables, a decline in muscle strength and power. An accepted model to assess one's ability to recover balance is with a perturbation of quiet stance through the release of a horizontal tether pulling at the waist. PURPOSE: To examine ankle muscle co-contraction levels in young and older adults during static held maximum voluntary contraction (MVC), rate of force development (RFD), as well as prior to and during an induced perturbation. METHODS: 18 healthy adult women (9 young: 22.3+3.3 yrs and 9 older: 75.2+4.2 yrs) with no history of falls or injury performed a minimum of 3 trials each of isometric MVC where force was ramped to and held at maximum and RFD where force was built as quickly as possible from rest for both plantar flexion (PF) and dorsiflexion (DF). The perturbation trials were then performed where they recovered balance using the ankle strategy. Surface electromyography (sEMG) data were analyzed to compare co-contraction activity between groups. RESULTS: Height, body weight, and foot length were similar between groups (p>0.05). After normalizing for height, weight, and foot length, there were no differences between groups for muscle strength characteristics for PF and DF isometric trials (p>0.05). sEMG parameters were normalized using the PF MVC for the gastrocnemius (GAS) and soleus (SOL) and the DF MVC for the tibialis anterior (TA). No sEMG differences were found between groups for PF MVC, PF RFD, and DF RFD for all muscles (p>0.05). DF MVC showed significantly more co-contracting GAS activity for the young (young: 25.8+8.6%, older: 11.8+3.1%), (p=0.001) but not for SOL (young: 37.3+12.2%, older: 34.5+12.8%) or TA activity (young 66.9+9.1, older: 66.31+7.2%), (p>0.05). Perturbation sEMG showed no significant interactions or main effects for GAS and SOL between time and age (p>0.05). TA activity did show a significant interaction (p=0.001). Simple main effects at each time point comparison between the young and older showed significantly more TA activity in the older adults at 300-400ms after load release (young: 23.5+5.8%, older: 38.7+9.4%), (p=0.001) but nowhere else (p>0.098). Additionally the electromechanical delay (EMD) between torque and onset of muscle activity approached significance for the GAS (young: 0.064+0.009%, older: 0.071+0.01%), (p=0.053). CONCLUSIONS: As expected the older adults showed more TA antagonistic co-contraction during balance recovery. Unexpectedly the young adults showed more GAS activity during DF MVC without a significant difference in force output. The approaching significance of GAS EMD also shows evidence of age-related neuromuscular changes taking place. However because of the lack of difference in strength and rate of force development between the two age groups the additional antagonistic torque produced by the older adults may be compromising the balance recovery process. In less physically capable older adults, the differences seen in TA activity and EMD may cause problems in recovering balance from perturbations.
Open Access
Antibacterial effects of sputter deposited silver-doped hydroxyapatite thin films
(Colorado State University. Libraries, 2011) Trujillo, Nathan Anthony, author; Popat, Ketul, advisor; Williams, John, advisor; Reynolds, Melissa, committee member; Crans, Debbie, committee member
Over recent years, researchers have studied innovative ways to increase the lifespan of orthopedic implants in order to meet the soaring demand of hip and knee replacements. Since many of these implants fail as a result of loosening, wear, and inflammation caused by repeated loading on the joints, coatings such as hydroxyapatite (HAp) on titanium with a unique topography have been shown to improve the interface between the implant and the natural tissue. Other serious problems with long-term or ideally permanent implants are bacterial colonization. It is important to prevent initial bacterial colonization as existing colonies have potential to become encased in an extracellular matrix polymer (biofilm) which is resistant to antibacterial agents. The following work considers the potential of etching using plasma based ion implantation and ion beam sputter deposition to produce hydroxyapatite thin films on etched titanium doped with silver as an antibacterial component. Plasma-based ion implantation was used to examine the effects of pre-etching on plain titanium. Topographical changes to the titanium samples were examined and compared via scanning electron microscopy. It was determined that plasma-based ion implantation at -700eV could etch titanium to produce similar topography as ion beam etching in a shorter processing time. Hydroxyapatite and silver-doped hydroxyapatite thin films were then sputter deposited on titanium substrates etched at -700eV. For silver-doped films, two concentrations of silver (~0.5wt% and ~1.5wt%) were used. Silver concentrations in the film were determined using energy dispersive x-ray spectroscopy. Film thicknesses were determined by measuring the surface profile using contact profilometry. Staphylococcus epidermidis (SE) and Pseudomonas aeruginosa (PA) adhesion studies were performed on plain titanium, titanium coated with hydroxyapatite, titanium coated with ~0.5 wt% silver-doped hydroxyapatite, and titanium coated with ~1.5wt% silver-doped hydroxyapatite. It was discovered during the study that the films were delaminating from the samples thus killing bacteria in suspension. Release studies performed in addition to adhesion confirmed that the silver-doped films prevented SE and PA bacterial growth in suspension. To prevent delamination, the films were annealed by heat treatment in air at a temperature of 600°C. X-ray diffraction confirmed the presence of a crystalline hydroxyapatite phase on each sample type. Films were immersed in PBS at 37°C and remained in incubation for four weeks to determine there was no delamination or silver leaching.
Open Access
Application of passive flow control to mitigate the thromboembolic potential of bileaflet mechanical heart valves: an in-vitro study
(Colorado State University. Libraries, 2014) Forléo, Márcio Henrique, author; Dasi, Lakshmi Prasad, advisor; James, Susan, committee member; Orton, Christopher, committee member; Dinenno, Frank, committee member
Implantation of a bileaflet mechanical heart valve (BMHV) continues to be associated with risk of thromboembolic complications despite anti-coagulation therapy. Mechanical heart valves have been the gold standard in valve heart replacement since the 1950s with BMHVs currently still being the valve of choice for younger patients. Given that a large body of literature points to thromboembolic complications due to poor hemodynamics, improvements to the hemodynamic performance of BMHVs are needed. In this study, we explore the concept of passive flow controls that have been widely used in aerospace industry as a novel approach towards improving BMHV design. Passive flow control elements are small features on solid surfaces, such as vortex generators (VGs), that alter flow to achieve desired performance. The specific aims of this study are (1) develop a methodology to evaluate thromboembolic potential (TEP) of BMHVs using in-vitro particle image velocimetry technique, (2) quantify the efficacy of rectangular VGs distributed on BMHV leaflets to reduce TEP, and (3) quantify the hemodynamic performance impact of rectangular VGs. An in-vitro pulsatile flow loop along with Particle Image Velocimetry (PIV) flow visualization technique was developed, validated, and utilized to acquire time-resolved velocity fields and shear stress loading: Lagrangian particle tracking analysis of the upstream and downstream flow during diastole and systole enabled the calculation of predicted shear stress history and exposure times corresponding to platelets. This information was then used in numerical models of blood damage to predict the TEP of test heart valves using established platelet activation and platelet lysis parameters. BMHV leaflets were constructed using 3D printing technology with VGs based on micro-CT scans of a model BMHV leaflet. Two configurations were constructed: co-rotating VGs and counter-rotating VGs. Co-rotating VGs consist of single features 1mm tall and 2.8mm long spaced equally apart (5mm) at an angle of attack of 23 degrees. Counter-rotating VGs consist of mirrored feature pairs 1mm from each other with the same dimensions as the co-rotating VGs. The leaflets were tested using the methodology described above to elucidate their effect on the TEP of the BMHV compared to the control leaflets. For systolic flow downstream of the valve, we report a decrease in the average platelet activation and average platelet lysis TEP (both normalized by the average exposure time) largely in the central jet, with the vortex generator equipped leaflets compared to the control leaflets at a p-value of 0.05. However, for diastolic flow upstream of the valve, we report an increase in the average platelet lysis TEP and average platelet activation TEP (both normalized by the average exposure time) largely in the regurgitant jet zone with the vortex generator equipped leaflets compared to the control leaflets at a p-value of 0.05. Also, steady and pulsatile flow experiments were conducted to calculate the transvalvular pressure drop across the model BMHV with control leaflets (no VGs) and leaflets containing VGs to calculate effective orifice area (EOA), which is an index of valve performance and is related to the degree to which the valve obstructs blood flow. We report a significant increase in EOA values for valves with leaflets containing passive flow control elements in both steady and pulsatile flow experiments compared to the control leaflets. Under steady flow, the co-rotating VGs configuration had the best EOA value compared to the control leaflet and counter-rotating vortex generator configuration. However, under pulsatile conditions, the counter-rotating VGs configuration had the best EOA value compared to the control leaflet and co-rotating vortex generator configuration. PIV measurements highlight the delay in flow separation caused by the VGs and corroborate the increased pulsatile flow EOA values. This study shows that the TEP of BMHVs can be accurately evaluated using in-vitro PIV techniques and that there is room for improvement in BMHV design using passive flow control elements. With optimization of passive flow control configuration and design, it is possible to further decrease the TEP of BMHVs while increasing their hemodynamic performance; thus creating a safer, more efficient BMHV.
Open Access
Are lines of arrested growth in bone indicative of seasonal metabolic suppression in bears?
(Colorado State University. Libraries, 2016) Hinrichs, Jason, author; Donahue, Seth, advisor; Norrdin, Robert, committee member; Popat, Ketul, committee member
Large hibernators such as bears have seasonal metabolic suppression, hibernation (Tøien et al. 2012). During hibernation bear's activity is very low; to the point most other animals would exhibit disuse bone resorption. However bears do not exhibit disuse bone resorption during this time (McGeeLawrence et al. 2008). Are lines of arrested growth (LAGs) in bone indicative of seasonal metabolic suppression in bears? Through the use of toluidine blue stain light microscopy slides and backscattered scanning electron microscopy images (SEM), LAGs were counted and correlated with age. LAGs have a strong correlation with age. This is indicative of LAGs formation once per year, during set hibernation cycles. LAGs are metabolic markers, in bears with set hibernation cycles. These metabolic markers could be used to identify the specific time in which there is metabolic suppression, in bears. This identification could be used in the future to track blood serum and other chemical markers in an attempt to understand bear's natural resistance to disuse bone resorption. Bears ability to not exhibit disuse bone resorption could be biomimetically studied, in an attempt to adapt this protection to humans. Since humans experience disuse osteoporosis (extended bed rest and spaceflight) and osteoporosis (older population specifically women).
Open Access
Biomechanics of transapical mitral valve implantation
(Colorado State University. Libraries, 2014) Koenig, Evan Kienholz, author; Dasi, Lakshmi Prasad, advisor; Prasad, Ashok, committee member; Orton, Christopher, committee member
Heart disease is the number one killer in the United States. Within this sector, valve disease plays a very important role: Approximately 6% of the entire population has either prolapse or stenosis of the mitral valve and this percentage only increases when looking only at the elderly population. Transapical mitral valve implantation has promised to be a potential therapy for high-risk patients presenting with MR; however it is unclear what the best method of securing a valve within the mitral annulus may be to provide a safe and efficient valve replacement. The objective of this research is to study and understand the underlying biomechanics of fixation of transapical mitral valves within the native mitral annulus. Two different transapical mitral valve prosthesis designs were tested: One valve design has a portion of the leaflets atrialized such that it has a shorter stent height and the valve itself sits within the native annulus, the other design is not atrialized and protrudes further into the left ventricle. The valves were implanted in a left heart simulator to assess leaflet kinematics and hemodynamics using high speed imagery and particle image velocimetry techniques. An in vitro passive beating heart model was then used to assess the two different fixation methods (namely, anchored at the apex vs. anchored at the annulus) with respect to paravalvular regurgitation. Leaflet kinematics and hemodynamics revealed proper leaflet coaptation and acceptable pressure gradients and inflow fillings; however, both designs yielded elevated turbulence stresses within the ventricle. At 60 beats per minute, leaflet opening and closing times were both under 0.1 seconds, max Reynolds shear stresses were between 40 and 60 N/m2 and maximum velocities were approximately 1.4 m/s. Assessment of the different fixation methods during implantation revealed the superiority of the atrialized valve when anchored at the annulus (p<0.05), but showed no such comparison during tethered implantation. In addition to the results of statistical testing, observations show that the importance of the relationship between ventricular stent height and fixation method compared with native anatomy plays an important role in overall prosthesis function regardless of implantation method.
Open Access
Biopolymer nanomaterials for growth factor stabilization and delivery
(Colorado State University. Libraries, 2014) Place, Laura Walker, author; Kipper, Matt J., advisor; James, Susan, committee member; Popat, Ketul C., committee member; Miller, Benjamin, committee member
Biopolymers are useful in tissue engineering due to their inherent biochemical signals, including interactions with growth factors. There are six biopolymers used in this work, the glycosaminoglycans (GAGs), heparin (Hep), chondroitin sulfate (CS), and hyaluronan (HA), chitosan (Chi), a GAG-like molecule derived from arthropod exoskeletons, a Chi derivative N,N,N¬-trimethyl chitosan (TMC), and an extracellular matrix (ECM)-derived material, demineralized bone matrix (DBM). The direct delivery of growth factors is complicated by their instability. GAG side chains of proteoglycans stabilize growth factors. GAGs also regulate growth factor-receptor interactions at the cell surface. The majority of proteoglycan function is derived from its GAG side chain composition. Here we report the development of nanoparticles, proteoglycan-mimetic graft copolymers, incorporation of nanoparticles into electrospun nanofibers, and processing methods for electrospinning demineralized bone matrix to fabricate bioactive scaffolds for tissue engineering. The nanoparticles were found to show similar size, composition, and growth factor binding and stabilization as the proteoglycan aggrecan. We use basic fibroblast growth factor (FGF-2) as a model heparin-binding growth factor, demonstrating that nanoparticles can preserve its activity for more than three weeks. Graft copolymers were synthesized with either CS or Hep as the side chains at four different grafting densities. Their chemistry was confirmed via ATR-FTIR and proton NMR. They were shown to increase in effective hydrodynamic diameter with grafting density, resulting in a size range from 90-500 nm. Graft copolymers were tested for their ability to deliver FGF-2 to cells. The CS conditions and the Hep 1:30 performed equally as well as when FGF-2 was delivered in solution. Preliminary dynamic mechanical testing demonstrated that hydrogels containing the copolymers exhibit changes in compressive modulus with cycle frequency. Two electrospinning techniques were developed, using an emulsion and a coaxial needle, for incorporating growth factor into electrospun nanofibers. We bound FGF-2 to aggrecan-mimetic nanoparticles for stabilization throughout electrospinning. The two techniques were characterized for morphology, nanoparticle and FGF-2 incorporation, cytocompatibility, and FGF-2 delivery. We demonstrated that both techniques result in nanofibers within the size range of collagen fiber bundles and dispersion of PCNs throughout the fiber mat, and exhibit cytocompatibility. We determined via ELISA that the coaxial technique is superior to the emulsion for growth factor incorporation. Finally, FGF-2 delivery to MSCs from coaxially electrospun nanofibers was assessed using a cell activity assay. We developed a novel method for tuning the nanostructure of DBM through electrospinning without the use of a carrier polymer. This work surveys solvents and solvent blends for electrospinning DBM. The effects of DBM concentration and dissolution time on solution viscosity are reported and correlated to observed differences in fiber morphology. We also present a survey of techniques to stabilize the resultant fibers with respect to aqueous environments. Glutaraldehyde vapor treatment is successful at maintaining both macroscopic and microscopic structure of the electrospun DBM fibers. Finally, we report results from tensile testing of stabilized DBM nanofiber mats, and preliminary evaluation of their cytocompatibility. The DBM nanofiber mats exhibit good cytocompatibility toward human dermal fibroblasts (HDF) in a 4-day culture.
Open Access
Characterization of osseointegrative phosphatidylserine and cholesterol orthopaedic implant coatings
(Colorado State University. Libraries, 2013) Rodgers, William Paul, author; James, Susan, advisor; Popat, Ketul, committee member; Ehrhart, Nicole, committee member; De Long, Susan, committee member
Total joint arthroplasties/replacements are one of the most successful surgeries available today for improving patients’ quality of life. By 2030 in the US, demand for primary total hip and knee arthroplasties are expected to grow by 174% and 673% respectively to a combined total of over 4 million procedures performed annually, driven largely by an ageing population and an increased occurrence of obesity. Current patient options for load-bearing bone integrating implants have significant shortcomings. Nearly a third of patients require a revision surgery before the implant is 15 years old, and those who have revision surgeries are at an increased risk of requiring additional reoperations. A recent implant technology that has shown to be effective at improving bone to implant integration is the use of phosphatidylserine (DOPS) coatings. These coatings are challenging to analyze and measure due to their highly dynamic, soft, rough, thick, and optically diffractive properties. Previous work had difficulty investigating pertinent parameters for these coating’s development due in large part to a lack of available analytical techniques and a dearth of understanding of the micro- and nano-structural configuration of the coatings. This work addresses the lack of techniques available for use with DOPS coatings through the development of original methods of measurement, including the use of scanning white light interferometry and nanoindentation. These techniques were then applied for the characterization of DOPS coatings and the study of effects from several factors: 1. the influence of adding calcium and cholesterol to the coatings, 2. the effect of composition and roughness on aqueous contact angles, and 3. the impact of ageing and storage environment on the coatings. This project lays a foundation for the continued development and improvement of DOPS coatings, which have the promise of significantly improving current patient options for bone integrating implants. Using these newly developed and highly repeatable quantitative analysis methods, this study sheds light on the microstructural configuration of the DOPS coatings and elucidates previously unexplained phenomena of the coatings. Cholesterol was found to supersaturate in the coatings at high concentration and phase separate into an anhydrous crystalline form, while lower concentrations were found to significantly harden the coatings. Morphological and microstructural changes were detected in the coatings over the course of as little as two weeks that were dependent on the storage environment. The results and understanding gained pave the path for focused future research effort. Additionally, the methods and techniques developed for the analysis of DOPS coatings have a broader application for the measurement and analysis of other problematic biological materials and surfaces.
Open Access
Characterizing the diffusional behavior and trafficking pathways of Kv2.1 using single particle tracking in live cells
(Colorado State University. Libraries, 2013) Weigel, Aubrey, author; Krapf, Diego, advisor; Tamkun, Michael, committee member; Bamburg, James, committee member; Bartels, Randy, committee member
Studying the diffusion pattern of membrane components yields valuable information regarding membrane structure, organization, and dynamics. Single particle tracking serves as an excellent tool to probe these events. We are investigating of the dynamics of the voltage gated potassium channel, Kv2.1. Kv2.1 uniquely localizes to stable, micro-domains on the cell surface where it plays a non-conducting role. The work reported here examines the diffusion pattern of Kv2.1 and determines alternate functional roles of surface clusters by investigating recycling pathways using single particle tracking in live cells. The movement of Kv2.1 on the cell surface is found to be best modeled by the combination of a stationary and non-stationary process, namely a continuous time random walk in a fractal geometry. Kv2.1 surface structures are shown to be specialized platforms involved in trafficking of Kv channels to and from the cell surface in hippocampal neurons and transfected HEK cells. Both Kv2.1 and Kv1.4, a non-clustering membrane protein, are inserted and retrieved from the plasma membrane at the perimeter of Kv2.1 clusters. From the distribution of cluster sizes, using a Fokker-Planck formalism, we find there is no evidence of a feedback mechanism controlling Kv2.1 domain size on the cell surface. Interestingly, the sizes of Kv2.1 clusters are rather governed by fluctuations in the endocytic and exocytic machinery. Lastly, we pinpoint the mechanism responsible for inducing Kv2.1 non-ergodic dynamics: the capture of Kv2.1 into growing clathrin-coated pits via transient binding to pit proteins.
Open Access
Data analysis and predictive modeling for synthetic and naturally occurring biological switches
(Colorado State University. Libraries, 2016) Schaumberg, Katherine A., author; Prasad, Ashok, advisor; Medford, June, advisor; Shipman, Patrick, committee member; Antunes, Mauricio, committee member; Krapf, Diego, committee member
Biological switches are biochemical network motifs responsible for determining the chemical state of cells, and are a key part of every biological system. The impact of these biological switches on cell behavior is broad. For example, many diseases such as cancer are thought to be caused by a misregulation of the bio-chemical state in a cell or group of cells. Also cell fates in differentiating stem cells are controlled by biological switches. Because of their general importance the synthetic biology community has also constructed synthetic biological switches in living organisms. While there are different kinds of possible switches, in my thesis I study switches capable of stably generating two unique molecular states, also called bi-stable switches. Here these switches are studied from two perspectives. In Chapters 1-4 I present theoretical and experimental work on analysis of specific circuits that act like biological switches. In Chapter 5 I employ a data mining perspective to identify gene expression signatures of switches that are sensitive to cytotoxic cancer drugs. This dissertation starts with a computational analysis of the effect of leaky promoter expression on bi-stable biological switches. In several biological and synthetic systems gene transcription is never completely off, even when repressed. This residual expression is referred to here as leaky expression. Bi-stable systems would be expected to have some amount of leaky expression in their off state. However, the impact of leaky expression on the functioning and properties of biological switches has not been well studied. To help fill this gap we conducted a theoretical analysis of leaky expression’s effect on biological switches. Two switches, a positive feedback and negative inhibition-based switch were studied. We found that the different circuit topologies showed different advantages in terms of their ability to handle leaky expression. Next this dissertation describes work done in collaboration with the Medford lab at Colorado State University, to construct and characterize a library of genetic plant parts. These parts would later be used in construction of perhaps the first synthetic bi-stable toggle switch in a plant. As part of this study, experiments were designed and conducted for finding the nature of the experimental noise associated with the assays used to test these plant parts. A mathematical normalization was developed to estimate quantitative information on the performance of each part. Validation experiments were done to assess the usefulness of this method for predicting the behavior of stably transformed plants from higher throughput transient assays. In the end a library of over one hundred quantitatively characterized plant parts in both Arabidopsis and Sorghum was constructed. The quantitative parameters of this library of genetic parts were then used in combination with a probabilistic bootstrap method we developed to predict optimal part combinations for construction of a bi-stable switch in Arabidopsis. The dissertation concludes with a study of biological networks in cancer cells from a data mining perspective. A large amount of data exists in the public domain on the sensitivity of cancer cell lines to cytotoxic drugs. Some cancers appear to be in a "sensitive state" while others are in a "resistant state". We would like to be able to know the gene expression signatures of these two states in order to predict cancer drug sensitivity from gene expression data. As a first step towards this goal we assessed the repeatability of predictions between the two standard databases of cancer cell lines, the NCI60 and the GDSC. This lead to identification of a preprocessing method needed to combine data from multiple databases. This was then followed up with the development of a comparative analysis platform. This platform was used to test the accuracy of models designed to predict drug sensitivity, when different model construction methods were used.
Open Access
Design and fabrication of a flow chamber for the study of cell adhesion and hemocompatibilty in dynamic conditions
(Colorado State University. Libraries, 2011) Migita, Kevin, author; Popat, Ketul, advisor; Dasi, Lakshmi Prasad, advisor; Prasad, Ashok, committee member
Cell adhesion is a well characterized condition of both biomaterial and tissue engineering research. It plays a role in biocompatibility and the proliferation, differentiation and viability of seeded cells. With respect to hemocompatibility, platelet adhesion and subsequent activation is a driving factor in the failure of blood contacting medical devices. Platelets aggregates are vital components in the wound healing and foreign body responses and display various forms of adhesion based on blood flow. However, the study of platelet adhesion on implantable tissue engineering scaffolds under dynamic conditions is very limited, particularly with directional flow. A flow chamber which incorporates a tissue engineering scaffold or functionalized biomaterial was designed and fabricated for investigation of flow patterns and cellular adhesion in response to dynamic conditions on these surfaces. The device utilizes a combination of aspects from both tissue engineering bioreactors and microfluidics platforms to result in a flow chamber which provides the directional flow of a perfused flow bioreactor with the advantages of controlling chamber shape and real time monitoring presented by Polydimethylsiloxane microfluidics chambers. Results of fluid flow study in the chamber modeled for laminar and shear gradient simulated flow show the ability of the device to manipulate flow patterns. Dynamic and static studies of platelet adhesion to poly-(ε-caprolactone) flat and electrospun nanofiber surfaces utilizing the flow chamber provide insight into the hemocompatibility of tissue engineering scaffolds in a dynamic flow setting.
Open Access
Determing the efficacy of Poloxamer 188 (P188) in meniscal damage prevention
(Colorado State University. Libraries, 2016) Remley, Katherine, author; Donahue, Tammy, advisor; James, Sue, committee member; Easley, Jeremiah, committee member
Meniscal injuries compose 15% of all knee injuries, most often sports-related. Due to the meniscus's avascular nature, healing is difficult and injury often results in a partial meniscectomy. Research has shown meniscectomies drastically decrease contact area between the femur and tibia and increase strains experienced by the meniscus. This additional strain predisposes the knee to developing post-traumatic osteoarthritis. Poloxamer 188 (P188) is a non-ionic, amphillic surfactant that may have the ability to prevent cell death through selective insertion into the cell membrane. This in turn may prevent damaged cell signaling and reduce overall tissue degradation. While P188 has been studied and shown promise in mitigating cell death in cartilage, the effects of P188 on the meniscus are unknown. To investigate the effects of P188 on the meniscus, the goals of this project were to: 1) create a user friendly graphical user interface for a custom bioreactor capable of displacement control for precise loading of meniscal explants to physiological and supraphysiological strains; 2) determine the efficacy of P188 in mitigating meniscal tissue damage through cell viability, mechanical data and histological analysis.
Open Access
Development of a hierarchical electrospun scaffold for ligament replacement
(Colorado State University. Libraries, 2018) Pauly, Hannah Marie, author; Haut Donahue, Tammy L., advisor; Easley, Jeremiah, committee member; Kelly, Daniel J., committee member; Palmer, Ross, committee member; Popat, Ketul C., committee member
The anterior cruciate ligament (ACL) is a dense collagenous structure that connects the femur to the tibia and is vital for joint stability. The ACL possesses complex time-dependent viscoelastic properties and functions primarily to prevent excessive translations and rotations of the tibia relative to the femur. It is estimated that 400,000 ACL tears occur in the United States annually and the monetary burden of these injuries and their subsequent treatment is approximately $1 billion annually. After injury allografts and autografts are commonly implanted to reconstruct the torn ACL in an attempt to restore joint stability, prevent pain, and limit damage to surrounding tissues. However surgical reconstructions fail to completely restore knee functionality or prevent additional injury and regardless of intervention technique radiographic osteoarthritis is present in 13% of patients 10 years after ACL rupture. Drawbacks to traditional treatments for ACL ruptures motivate the development of a synthetic ACL replacement. Tissue engineering is the use of a scaffold, cells, and signaling molecules to create a replacement for damaged tissue. The goal of this work is to develop a polymer scaffold that can be utilized as a replacement for the ACL. A tissue engineered ACL replacement should replicate the hierarchical structure of the native ACL, possess reasonable time zero mechanical properties, and promote the deposition of de novo collagenous tissue in vitro. Additionally, the scaffold should be implantable using standard surgical techniques and should maintain in situ tibiofemoral contact mechanics. Thus, four specific aims are proposed: 1) Fabricated and characterize an aligned 3-dimensional electrospun scaffold for ACL replacement. 2) Assess the in vitro behavior of ovine bone marrow-derived stems cells seeded on the scaffold in the presence of conjugated growth factor. 3) Evaluate the performance of the electrospun scaffold using uniaxial mechanical testing. 4) Assess the effect of the electrospun scaffold on ovine stifle joint contact mechanics. Development of a tissue engineered ACL replacement that mimics the structure and function of the native ACL would provide a novel treatment to improve outcomes of ACL injuries.
Open Access
Development of a novel block copolymer hydrogel for meniscal replacement
(Colorado State University. Libraries, 2018) Fischenich, Kristine Marie, author; Haut Donahue, Tammy, advisor; Bailey, Travis, advisor; Easley, Jeremiah, committee member; Palmer, Ross, committee member; Goodrich, Laurie, committee member
Menisci are C-shaped fibrocartilaginous tissues responsible for distributing tibial-femoral contact pressure and are crucial for maintaining healthy joints and preventing osteoarthritis. Meniscal damage can be caused by age-related degradation, obesity, overuse from athletic activities, and trauma. Due to their primarily avascular nature, once damaged there is limited healing capacity and surgical intervention is often required. Limited technologies exist to replace damaged menisci, and standard treatment is to leave asymptomatic damage alone or perform partial meniscectomies, however, these treatment options lead to increased risk of OA. Attempts at tissue engineered meniscal scaffolds, and replacements have had mixed results due to design limitations and inability to recapitulate native tissue's material properties, shape, and pressure distribution. This project strives to create an artificial meniscus from a polystyrene-polyethylene oxide diblock copolymer. It is hypothesized that this hydrogel can be tuned to have material properties similar to those of the native meniscus. Furthermore, it is hypothesized this hydrogel can be molded into a 3D meniscal construct, implanted into the joint, and have similar pressure distribution properties as the native meniscus. Thus, the aims of this project are: 1) Mechanical comparison of a polystyrene-polyethylene oxide diblock copolymer TPE hydrogel to native meniscal tissue. 2) Develop a 3D meniscal construct which can be implanted into an ovine model and assess load distribution properties including contact area, mean pressure, and max pressure in both the medial and lateral hemijoints. If the goals of this project are met, there would exist a 3D TPE hydrogel construct that mimics the mechanical and functional properties of the native human meniscus. This meniscal replacement could provide a revolutionary addition to the field of osteoarthritis and meniscal injury.
Open Access
Development of an scAAVIGF-I gene therapeutic vector for the enhancement of cartilage repair
(Colorado State University. Libraries, 2014) Hemphill, Daniel, author; Goodrich, Laurie, advisor; McIlwraith, C. Wayne, advisor; Samulski, R. Jude, committee member; Slayden, Ric, committee member; James, Sue, committee member
In this work, we hypothesize scAAVIGF-I can be efficiently engineered and used as a gene therapeutic vector to transduce cartilage and synovium and elicit biochemical changes consistent with cartilage repair mechanisms. Here we show joint tissues are permissive to serotype specific, efficient AAV transduction. This is in agreement with previous in vitro and in vivo studies demonstrating AAV as an efficient vector for gene therapy. Interestingly, we show a clinically relevant occurrence of pre-existing, serotype specific AAV neutralization. After creation of an optimized scAAVIGF-I vector, we show transduction of chondrocytes results in significantly increased IGF-I levels that translate to biochemical changes in glycosaminoglycan and proteoglycan content. NextGen RNA transcriptome sequencing and PCR analysis revealed significant differential regulation of genes in pathways including cartilage biology and cell death, but not significant in other pathways associated with IGF-I treatment such as Wnt and Akt regulation. The problem addressed by this research is ultimately answered in that scAAVIGF-I can both efficiently transduce joint tissue and increase IGF-I concentrations sufficiently to cause biochemical changes indicative of healthy cartilage, and the pathways associated with upregulation of IGF-I gene therapy are revealed.
Open Access
Development of microsystems for point-of-use microorganism detection
(Colorado State University. Libraries, 2018) Wang, Lei, author; Dandy, David S., advisor; Tobet, Stuart A., committee member; Henry, Chuck S., committee member; Geiss, Brian J., committee member; Bailey, Travis S., committee member; Marchese, Anthony J., committee member
To view the abstract, please see the full text of the document.
Open Access
Development of paper-based analytical devices for particulate metals in welding fume
(Colorado State University. Libraries, 2015) Cate, David M., author; Henry, Charles S., advisor; Volckens, John, advisor; Dandy, David, committee member; Peel, Jennifer, committee member; Lear, Kevin, committee member
Exposure to metal-containing particulate matter places a tremendous burden on human health. Studies show that exposures lead to cardiovascular disease, asthma, flu-like illnesses, other respiratory disorders, and to increased morbidity. Individuals who work in occupations such as metalworking, construction, transportation, and mining are especially susceptible to unsafe exposures because of their proximity to the source of particle generation. Despite the risk to worker health, relatively few are routinely monitored for their exposure due to the time-intensive and cost-prohibitive analytical methods currently employed. The current paradigm for chemical speciation of workplace pollution is outdated and inefficient. Paper-based microfluidic devices, a new type of sensor technology, are poised to overcome issues associated with chemical analysis of particulate matter, specifically the cost and timeliness of exposure assessment. Paper sensors are designed to manipulate microliter liquid volumes and because flow is passively driven by capillary action, analysis costs are very low. The objective of this work was to develop new technology for rapidly measuring Ni, Cu, Fe, and Cr in welding fume using easy-to-use paper devices. This dissertation covers the development of two techniques for quantifying metal concentration: spot integration and distance-based detection. Metal concentrations as low as 0.02 ppm are reported. A method for controlling reagent deposition as well as a new interface for multiplexed detection of metals, is discussed.
Open Access
Effect of bone geometry on stress distribution patterns in the equine metacarpophalangeal joint
(Colorado State University. Libraries, 2012) Easton, Katrina L., author; Kawcak, Chris, advisor; McIlwraith, Wayne, committee member; Puttlitz, Christian, committee member; James, Susan, committee member; Shelburne, Kevin, committee member; Heyliger, Paul, committee member
Catastrophic injury of the equine metacarpophalangeal joint is of major concern for both the equine practitioner and the American public. It is one of the major reasons for retirement and sometimes euthanasia of Thoroughbred racehorses. The most common type of catastrophic injury is fracture of the proximal sesamoid bones and lateral condyle of the third metacarpal bone. Many times these injuries are so disastrous that there is no possibility of fixing them. Even in the injuries that are able to be fixed, complications arising from the fracture such as support limb laminitis may ultimately lead to the demise of the horse. Therefore, prevention of these types of injuries is key. In order to decrease the incidence of injury, it is important to understand the risk factors and pathogenesis of disease that leads to them. This project was established to create a finite element model of the equine metacarpophalangeal joint in order to investigate possible risk factors, namely bone geometry, and its effect on the stress distribution pattern in the joint. The first part of the study involved in vitro experiments in order to provide a comprehensive dataset of ligament, tendon, and bone strain and pressure distribution in the joint with which to validate the finite element model. Eight forelimbs from eight different horses were tested on an MTS machine to a load equivalent to that found at the gallop. Beyond providing data for validation, the study was the first to the author's knowledge to measure surface contact pressure between the distal condyles of the third metacarpal bone and the proximal sesamoid bones. A pressure distribution pattern that could lead to an area of high tension in the area of the parasagittal groove was found. This result could help explain the high incidence of lateral condylar fractures that initiate in this location. The second part of this study focused on the development and validation of a finite element model of the metacarpophalangeal joint. A model was created based on computed tomography (CT) data. It included the third metacarpal bone, the proximal phalanx, the proximal sesamoid bones, the suspensory ligament, medial and lateral collateral ligaments, medial and lateral collateral sesamoidean ligaments, medial and lateral oblique sesamoidean ligaments, and the straight sesamoidean ligament. The mesh resolution was varied to create three models to allow for convergence. The converged model was then validated using data from the previous part of the study as well as data from the literature. The result was a finite element model containing 121,533 nodes, 112,633 hexahedral elements, and 10 non-linear springs. The final section of this study used the converged and validated finite element model to study the effect of varying bone geometry. The model was morphed based on CT data from three horses: control, lateral condylar fracture, and contralateral limb to lateral condylar fracture. There was an area similar between all three groups of increased stress in the palmar aspect of the parasagittal grooves where fractures are thought to initiate. Other results showed distinct differences in the stress distribution pattern between the three groups. Further investigation into these differences may help increase the understanding of a horse's predisposition to injury. In conclusion, this study has shown that joint geometry plays a role in the stress distribution patterns found in the equine metacarpophalangeal joint. The differences in these patterns between the three groups may help explain the increased risk of a catastrophic injury for some horses. Further studies are warranted to better define the parameters leading to these changes.
Open Access
Effect of rear wheel suspension on tilt-in-space wheelchair shock and vibration attenuation
(Colorado State University. Libraries, 2017) Hischke, Molly, author; Reiser, Raoul F., II, advisor; Gilkey, David, committee member; Tracy, Brian, committee member
Suspension systems are designed to reduce shock and vibration exposure. Prior to the QuadshoX LLC suspension kit (Fort Collins, CO), manual tilt-in-space wheelchairs did not have rear wheel suspension available for use. Furthermore, it was anticipated that rear wheel diameter would have an independent effect on shock and vibration transmitted to the wheelchair. The aim of this study was to investigate the shock and vibration reducing capabilities of the newly available aftermarket rear wheel suspension system and wheel diameter for manual tilt-in-space wheelchairs. Ten healthy non-wheelchair users volunteered for the study (7 men, 3 women: age 22.1±3.36 yrs, height 1.75±0.067 m, weight 73.9±8.87 kg (mean±SD)). Subjects were pushed by the same trained investigator over four different obstacles while using a Quickie IRIS® Tilt-in-Space manual wheelchair (Sunrise Medical, Phoenix, AZ) with two different diameter solid wheels, (0.381 m and 0.508 m), Primo Cheng Shin Tires (Cheng Shin Rubber, Yuanlin, Taiwan). Surfaces included a/an 1) exterior door threshold, 2) truncated domes, 3) 2 cm descent, and 4) 2 cm ascent. The subjects traversed the obstacles with the wheelchair as manufactured, and followed ~ 2 weeks later with the QuadshoX suspension kit installed. A tri-axial accelerometer, (Model339A31, PCB Piezotronics, Depew, NY), was mounted to the rear of the wheelchair seat pan with signals sampled at 2000 Hz. Peak resultant accelerations were analyzed from surface 1, 3-4, root mean square (RMS) resultant accelerations were analyzed from surface 2, and vibration dose value (VDV) and total power were analyzed from all surfaces 1-4. Unweighted and ISO 2631-1 frequency weighted (FW) accelerations were analyzed. The use of suspension decreased the un-weighted peak acceleration at the rear wheel when it impacted the door threshold, and when the rear wheel traversed the 2 cm descent and ascent (p=0.043, p=0.001, p=0.001, respectively) and FW peak accelerations at the rear wheel when it impacted and left the door threshold, and when the rear wheel descended 2 cm (p=0.049, p= 0.001, p= 0.005, respectively). With suspension, RMS and total VDV significantly decreased 14% and 10- 22% respectively (p=0.011, p=0.004). There were no significant differences between the rigid and suspended chair in total vibration power in frequency octaves most harmful in human exposure (4 – 12 Hz). The results of wheel diameter were not evaluated because there were significant differences in time spent over the obstacles between the two diameters (door threshold p= 0.018, truncated domes p= 0.028, 2 cm descent p= 0.029, 2 cm ascent p = 0.024). However, there were not differences in time spent over the obstacles between rigid and suspended conditions (p ≥ 0.064). The results indicate the aftermarket rear wheel suspension reduces some aspects of shock and vibration exposure, specifically at the rear wheel. While low back pain, neck pain, discomfort, and muscle fatigue correlate with shock and vibration exposure there is no set threshold of reduction in shock and vibration exposure to decrease the health risks with exposure. Considering how much time tilt-in-space users spend in their wheelchairs, we expect the observed reductions in shock and vibration with the use of the aftermarket rear wheel suspension may decrease the health risks, such as pain and muscle fatigue.