Theses and Dissertations

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Open Access
Interaction between membrane and protein properties on flux decline during sterile microfiltration
(Colorado State University. Libraries, 2010) Cutler, Hailey, author; Wickramasinghe, Sumith Ranil, advisor; Kipper, Matt J., committee member; Venayayamoorthy, S. Karan, committee member
Microfiltration is widely used in industry to filter out particulate matter that contaminates or slows down the performance of the membrane. In the biopharmaceutical industry in particular, bacteria, microorganisms and viruses are filtered out using sterile microfiltration. Numerous studies have been conducted to further the understanding of flux decline due to protein fouling. Many times the operating conditions, the type of membrane and type of protein all interact to have an effect on protein fouling and flux decline. Normal-flow microfiltration experiments were conducted using uncoated polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF) membranes, and PTFE and PVDF membranes coated with polyvinyl alcohol (PVA). Feed streams consisted of lysozyme, β-lactoglobulin and ovalbumin. The pH values of the solution were set at the isoelectric point of each of the proteins (11.0, 5.8, and 4.7 respectively). The experiments were operated with a feed pressure of 2 or 10 psi. Each of the proteins was tested at 0.1 and 2 g/L with uncoated PTFE. No flux decline was seen using 0.1 g/L, so 2 g/L was focused on for PVA coated PTFE, PVA coated PVDF and uncoated PVDF membranes. Protein fouling of the membrane was investigated by determining the variation of permeate flux versus filtrate volume and by analysis of Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectra and Field Emission Scanning Electron Microscopy (FESEM) images of unfouled membranes and membranes after microfiltration. Results indicate that the greatest amount of fouling occurs with ovalbumin. The order of most to least fouling was found to be ovalbumin> β-lactoglobulin>lysozyme. Fouling was more severe at the higher protein concentration (2 g/L) and feed pressure (10 psi) and seen only when filtering the solution through uncoated and PVA coated PTFE. Flux decline under these conditions was analyzed using classical pore blockage models. In general, flux decline was found to be caused by complete pore blocking. In the case of ovalbumin filtered through PVA coated PTFE, the flux decline was first caused by pore blockage and then later transitioned to cake filtration. The proteins which showed significant fouling conditions were looked at more closely by pre-filtering the protein solution. The goal of pre-filtration was to decrease any protein aggregates present in solution. This pre-filtration step was conducted with 0.2, 0.45 and 1 μm diameter pore sizes. The flux decline when pre-filtering the feed solution with 1 μm pores was equivalent to the filtration experiments without pre-filtration. The only significant decrease in flux was present when pre-filtering with the 1μm pores. Additional experiments were conducted using hemoglobin (Hb) at 2 g/L and 10 psi operating conditions. Previous literature had shown that using 1 μm pre-filtration, there was severe flux decline for uncoated and PVA coated PTFE. To follow up on these experiments, Hb was pre-filtered using 0.2 and 0.45 μm pre-filtration membranes and then filtered through uncoated and PVA coated PTFE. These experiments resulted in no flux decline. The Hb experiments verified the results from the ovalbumin and β-lactoglobulin experiments. All together, these results indicate that there is an interaction among membrane properties, protein properties, operating conditions and pre-filtration characteristics that determine whether fouling occurs and to what extent.
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
Evaluation of osteogenic design factors in electrospun poly(ε-caprolactone) nanofiber scaffolds
(Colorado State University. Libraries, 2010) Ruckh, Timothy T., author; Popat, Ketul, advisor; James, Susan, committee member; Kipper, Matt, committee member; Ryan, Stewart, committee member
Biodegradable bone tissue scaffolds have the potential to impact patients with numerous ailments. Starting with fabrication techniques that produce nano-scale features, the ability to manipulate architecture, alter surface chemistry, and deliver biological molecules allows for the design of elegant and highly effective bone scaffolds. This work aimed to develop a porous, nanofiber scaffold with osteogenic design features the capability to deliver an antibiotic molecule from within the nanofibers. Two osteogenic design factors with unique mechanisms of action were selected; hydroxyapatite nanoparticles and oleic acid. Hydroxyapatite (HAp) is the primary inorganic phase of natural bone tissue and has been used to more closely mimic the extracellular environment of synthetic bone tissue scaffolds. Oleic acid (OLA) is an ω-9 fatty acid with suspected osteogenic effects due to activation of peroxisome proliferator-activator receptors (PPARs). In separate in vitro evaluations, OLA significantly increased osteoblast phenotypic behaviors and led to differential expression of the three PPAR isoforms, suggesting that the OLA is activating its anticipated receptor. HAp produced mixed results by inducing a small increase in alkaline phosphatase activity, but decreasing expression levels of bone matrix proteins. An in vivo evaluation of biocompatibility revealed that neither design factor increased the inflammatory response over control nanofiber scaffolds in paravertebral muscle pouches. However, both factors separately increased new osteoid production. Scaffolds with both HAp and OLA elicited the greatest osteogenic response in vivo, suggesting positive synergy between the two design factors. Finally, rifampicin (RIF), an antibiotic molecule was loaded into the nanofibers, and its release into static bacterial culture was effective in inhibiting bacterial population growth for both a Gram-positive and Gram-negative bacterial strain, separately. Overall, these nanofiber scaffolds were demonstrated to be effective carriers of soluble (OLA, RIF) and insoluble signals (HAp) which can modulate cell behaviors. Future work will aim to incorporate additional osteogenic features into the scaffolds and to develop multiple antibiotic release mechanisms from the nanofibers.
Open Access
Evaluating filtration membranes and detection systems for use with virus surrogates
(Colorado State University. Libraries, 2010) Stump, Emily D., author; Wickramasinghe, Sumith Ranil, advisor; Quackenbush, Sandra Lynn, committee member; Kipper, Matt, committee member; Pellegrino, John, committee member
Virus filtration membranes are used to provide size exclusion removal of viruses during the purification of biopharmaceutical products. This viral clearance is required by regulatory agencies to ensure the safety of patients by preventing contamination of product by adventitious or endogenous virus. Viral clearance studies are often laborious, expensive, and require highly trained personnel. Detection and quantification of virus using standard assays has restrictions in terms of limit of detection, extraneous contamination and false positives. Moreover, biosafety for personnel and the environment is always a concern when working with live virus. In order to avoid the hazards of live, adventitious virus, bacteriophages have been used previously as virus surrogates (Aranha-Creado & Brandwein, 1999). While the health threat associated with using live viruses is eliminated using bacteriophages as surrogates, the detection systems and quantitative assays are still laborious and difficult. Development of a non-biological system to simulate and quantify virus particles could reduce the time taken to perform viral clearance tests; reduce development costs; reduce the risk to personnel performing the tests; and lead to more reliable data, since a non-biological system will reduce variability in assays. Here we develop a prototype of a novel, gigantic magnetoresistive (GMR) detection system for magnetic virus surrogates. In addition, we investigate various polymeric membranes for their ability to reject virus. Results will be used as a benchmark for evaluating the behavior of a future, superparamagnetic virus surrogate. GMR-based technology has increasingly been on the rise since the 2007 Nobel Prize in physics was awarded to Albert Fert and Peter Grünberg for its discovery. GMR sensors show potential for being extremely sensitive, inexpensive, and flexible devices for use in biodetection assays. Compared to the current magnetic detection technology of the superconducting quantum interference device (SQUID), which requires complex instrumentation and qualified users, GMR technologies can be fabricated in such a manner so as to be applied to lab-on-chip systems. Here we discuss the sensor design and fabrication. Initial measurements indicate that 104 iron oxide nanoparticles, approximately 20nm in diameter, can be detected in 0.5μl of solution. Various virus filters as well as ultrafiltration membranes were challenged with feed streams spiked with high concentrations of minute virus of mice (MVM) in the presence and absence of 1% bovine serum albumin (BSA) (w/v). Changes in permeate flux with filtrate volume were determined in conjunction with changes in rejection of parvovirus. Decrease in permeate flux resulting from fouling of BSA was evaluated for its effect on virus rejection. The results, which compare the performance of virus filtration and similar ultrafiltration membranes, provide insights into the comparison of live virus with future virus surrogates.
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
Kinematic and kinetic analysis of canine thoracic limb amputees at a trot
(Colorado State University. Libraries, 2011) Jarvis, Sarah, author; Reiser, Raoul, advisor; Worley, Deanna, committee member; Haussler, Kevin, committee member
Most dogs appear to adapt well to the removal of a thoracic limb, but clinically there is a particular subset of dogs that still have problems with gait that seem to be unrelated to age, weight, or breed. The purpose of this study was to objectively characterize biomechanical changes in gait associated with amputation of a thoracic limb. Sixteen amputees and 24 control dogs of various breeds with similar stature and mass greater than 14 kg were recruited and participated in the study. Dogs were trotted across three in-series force platforms as spatial kinematic and ground reaction force data were recorded during the stance phase. Ground reaction forces, impulses, and stance durations were computed as well as stance widths, stride lengths, limb and spinal joint angles. Kinetic results show that thoracic limb amputees have increased stance times and vertical impulses. The remaining thoracic limb and pelvic limb ipsilateral to the side of amputation compensate for the loss of braking, and the ipsilateral pelvic limb also compensates the most for the loss of propulsion. The carpus, and ipsilateral hip and stifle joints are more flexed during stance, and the T1, T13, and L7 joints experience significant differences in spinal motion in both the sagittal and horizontal planes throughout the gait cycle stance phases. The spine, carpus, and ipsilateral hip and stifle joints are of most concern when considering the biomechanical impact that a thoracic limb amputation may have for a given dog.
Open Access
Investigating the osteogenic potential of multipotent mesenchymal stromal cells through the use of DNA microarray technology and biomaterial nanotopography
(Colorado State University. Libraries, 2011) Berger, Dustin, author; Prasad, Ashok, advisor; Popat, Ketul, advisor; Deluca, Jennifer, committee member
To view the abstract, please see the full text of the document.
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
Kinematic and kinetic analysis of canine pelvic limb amputees at a trot
(Colorado State University. Libraries, 2011) Hogy, Sara, author; Reiser, Raoul, advisor; Worley, Deanna, committee member; Haussler, Kevin, committee member
Osteosarcoma is the most common form of bone tumors in dogs. Treatment options include palliative or curative-intent options. Of the curative-intent treatments, the most common is amputation, due to lower cost and the ability to perform the procedure on all osteosarcoma patients irrespective of tumor location, as opposed to limb-sparing options which can only be used when the tumor is located at the distal radius or ulna of the thoracic limb. Overall, dogs with amputations adjust well to the loss of the limb; however, there still remains a subset of patients which do not. In this study, the ground reaction force kinetics and joint angular kinematics of pelvic limb amputees and four-legged dogs were compared to identify compensation strategies adopted by amputees after the loss of a pelvic limb. It was hypothesized that there would be increased flexion and extension of all limbs within the amputees, as well as increased spinal motion. In addition, it was hypothesized that there would be decreased vertical impulses in all limbs of amputees, as well as, decreased propulsion forces within the thoracic limbs of amputees, as compared to controls. The four-legged control population consisted of 24 dogs and the pelvic limb amputee population consisted of 12 dogs. Both populations had dogs of varying breeds. Ground reaction force data were captured using three serial force platforms while dogs were trotted down an over-ground walkway. Concurrently, joint angular kinematic data were captured by motion capture software using retroflective markers affixed to bony landmarks along the limbs and axial skeleton. Peak ground reaction forces and impulses were slightly different between pelvic limb amputees and four-legged dogs. Pelvic limb amputees had increased peak braking forces in the contralateral thoracic limb and increased peak propulsion in both the ipsilateral thoracic limb and remaining pelvic limb. In addition, amputees had increased peak vertical force and propulsion impulse in the remaining pelvic limb. Time to peak braking force was significantly decreased in all limbs of the amputees, while time to peak propulsion ground reaction force was increased in all limbs of the amputees. Limb kinematics of pelvic limb amputees were very similar to the kinematics of four-legged dogs. The only compensatory strategy adopted within the limbs of the amputee was increased range of motion of the hock joint within the remaining pelvic limb. However, the pelvic limb amputees had various spinal compensatory changes within the sagittal plane. Amputees had increased regional spinal motion about both the T1 and T13 markers and increased extension about the L7 marker, compared to four-legged controls. The motion of the spine in the horizontal plane varied only in the regional angular motion about the L7 marker. Overall, ground reaction force kinetic and joint angular kinematic gait analysis of pelvic limb amputees showed that there are various compensation strategies adopted by pelvic limb amputees to adjust for the loss of a limb. Combined, these compensation strategies allow for successful adaptation to a three-legged gait pattern after the removal of a pelvic limb. Clinically, this information will be valuable for determining factors related to adaptive strategies with pelvic limb amputees. This information can also be used to create a set of quantitative measures needed to classify canine amputees into adapted or poorly adapted gait parameters.
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
In vivo efficacy of antibiotic-eluting phospholipid coated implants
(Colorado State University. Libraries, 2011) Triffo, Thomas, author; James, Susan P., advisor; Criswell, Marvin E., committee member; Ehrhart, Nicole P., committee member
Implant-associated infection can be a serious problem for patients that receive orthopedic implants, such as hip and knee replacements. This is a common cause for early implant loosening, which requires revision surgeries and results in an even greater risk of infection. To address this issue, our lab has developed a novel electrospraying technique for applying phospholipid coatings to orthopedic implants. These coatings consist of two layers of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), with antibiotic loaded in between layers. In vitro tests were performed to evaluate how modifications to these coatings affect coating retention, based on a clinically relevant test, and antibiotic elution from these coatings. Coating retention tests were performed by inserting implants through segments of mouse bone and then examining the implants under SEM. Antibiotic elution was performed using a total sink elution combined with OPA assay for detection of antibiotic. These results showed that the coatings that were retained the most and eluted antibiotic slowest were samples that were pre-treated with calcium and were electrosprayed with a mixture of 6:1 DOPS-to-cholesterol. This coating was selected to be used in an in vivo study to determine the efficacy of the coatings in treating osteomyelitis. Osteomyelitis was induced in a murine model using genetically modified bacteria, which allowed tracking of the infection prior to sacrificing the animals via bioluminescent imaging, a technique that makes use of genetically modified bacteria producing luciferin and luciferase which causes emission of photons. It was observed that antibiotic-eluting implants cleared the infection faster than implants without antibiotic during a 4 week study. Also, no kidney damage was observed based on creatinine, blood urea nitrogen, and urine protein tests. Histology confirmed observations from the bioluminescent imaging. These results show that our antibiotic-eluting implant coatings were able to reduce infection in vivo without resulting in adverse effects. Bioluminescent imaging showed significant reduction of emission of photons, p < 0.05, in the antibiotic loaded group compared to the control samples. The results also suggest that the implants exhausted their supply of antibiotic at the end of the study, and in future studies a greater amount of antibiotic will be loaded onto the implants.
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
Multifunctional nanowire scaffolds for neural tissue engineering applications
(Colorado State University. Libraries, 2012) Bechara, Samuel Leo, author; Popat, Ketul, advisor; Tobet, Stuart, committee member; Legare, Marie, committee member; Rollin, Bernard, committee member; Sladek, John, committee member
Unlike other regions of the body, the nervous system is extremely vulnerable to damage and injury because it has a limited ability to self-repair. Over 250,000 people in the United States have spinal cord injuries and due to the complicated pathophysiology of such injuries, there are few options available for functional regeneration of the spinal column. Furthermore, peripheral nerve damage is troublingly common in the United States, with an estimated 200,000 patients treated surgically each year. The current gold standard in treatment for peripheral nerve damage is a nerve autograft. This technique was pioneered over 45 years ago, but suffers from a major drawback. By transecting a nerve from another part of the body, function is regained at the expense of destroying a nerve connection elsewhere. Because of these issues, the investigation of different materials for regenerating nervous tissue is necessary. This work examines multi-functional nanowire scaffolds to provide physical and chemical guidance cues to neural stem cells to enhance cellular activity from a biomedical engineering perspective. These multi-functional scaffolds include a unique nanowire nano-topography to provide physical cues to guide cellular adhesion. The nanowires were then coated with an electrically conductive polymer to further enhance cellular activity. Finally, nerve growth factor was conjugated to the surface of the scaffolds to provide chemical cues for the neural stem cells. The results in this work suggest that these multifunctional nanowire scaffolds could be used in vivo to repair nervous system tissue.
Open Access
New Frank-Starling based contractility and ventricular stiffness indices: clinically applicable alternative to Emax
(Colorado State University. Libraries, 2013) Tan, Haiming, author; Dasi, Lakshimi Prasad, advisor; Popat, Ketul, committee member; Orton, Chris, committee member
Heart disease is the #1 cause of death in the United States with congestive heart failure (CHF) being a leading component. Load induced CHF, i.e. CHF in response to chronic pressure or volume overload, may be classified either as systolic failure or diastolic failure, depending on the failure mode of the pumping chamber. To assess the severity of systolic failure, there exist clinical indices that quantify chamber contractility, namely: ejection fraction, (dP/dt), Emax (related to the rate of pressure rise in the pumping chamber), and Emax (related to the time-dependent elastance property of the ventricle). Unfortunately, these indices are plagued with limitations due to inherent load dependence or difficulty in clinical implementation. Indices to assess severity of diastolic failure are also limited due to load dependence. The goal of this research is to present (1) a new framework that defines a new contractility index, Tmax, and ventricular compliance 'a', based on Frank-Starling concepts that can be easily applied to human catheterization data, and (2) discusses preliminary findings in patients at various stages of valve disease. A lumped parameter model of the pumping ventricle was constructed utilizing the basic principles of the Frank-Startling law. The systemic circulation was modeled as a three element windkessel block for the arterial and venous elements. Based on the Frank-Starling curve, the new contractility index, Tmax and ventricular compliance 'a' were defined. Simulations were conducted to validate the load independence of Tmax and a computed from a novel technique based on measurements corresponding to the iso-volumetric contraction phase. Recovered Tmax and 'a' depicted load independence and deviated only a few % points from their true values. The new technique was implemented to establish the baseline Tmax and 'a' in normal human subjects from a retrospective meta-data analysis of published cardiac catheterization data. In addition, Tmax and 'a' was quantified in 12 patients with a prognosis of a mix of systolic and diastolic ventricular failure. Statistical analysis showed that Tmax was significantly different between the normal subjects group and systolic failure group (p<0.019) which implies that a decrease in Tmax indeed predicts impending systolic dysfunction. Analysis of human data also shows that the ventricular compliance index 'a' is significantly different between the normal subjects and concentric hypertrophy (p < 0.001). This research has presented a novel technique to recover load independent measures of contractility and ventricular compliance from standard cardiac catheterization data.
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
Mesenchymal stem cell rescue for bone formation following stereotactic radiotherapy of osteosarcoma
(Colorado State University. Libraries, 2013) Schwartz, Anthony L., author; Ehrhart, Nicole, advisor; Ryan, Stewart, committee member; James, Susan, committee member; Goodrich, Laurie, committee member; Custis, Jamie, committee member
Background: Osteosarcoma (OSA) is the most common form of primary bone cancer in dogs and humans. Curative-intent treatment options include amputation, radiation therapy or surgical limb salvage for local tumor control combined with adjuvant chemotherapy for prevention or delay of metastatic disease. Stereotactic radiotherapy (SRT) delivers high dose per fraction radiation to a defined tumor volume with relative sparing of surrounding normal tissues. It has been successfully used as a non-surgical limb salvage procedure to achieve local tumor control of spontaneous OSA in dogs. The most common complication observed with this treatment is pathologic fracture of the irradiated bone. Mesenchymal stem cells (MSCs) are multipotent stem cells that have the capability to differentiate into many cell types including bone. The ability of MSCs to differentiate into bone suggests that they should be investigated as a potential therapy to regenerate bone in SRT treated bone. Methods: In experiments described herein, we developed an orthotopic model of canine osteosarcoma in athymic rats and evaluated the ability of SRT to achieve local tumor control. We then evaluated the ability of MSCs to regenerate bone after SRT treatment of OSA. Results: We demonstrated that the canine OSA cell line reliably engrafted in the rat femur. We characterized progression in order to create a reproducible model in which to replicate a clinical scenario to test MSC behavior following SRT of OSA. Two weeks after OSA cell inoculation was identified as the time period when the same clinical characteristics were observed as in canine OSA cases and was chosen to be an appropriate time for SRT treatment. The optimal SRT protocol to achieve local tumor control while minimizing acute radiation effects was determined to be 3 fractions of 12 Gy delivered on consecutive days. MSCs administered either intravenously or intraosseously 2 weeks after SRT revealed no new bone formation; however, decreased tumor necrosis was observed after MSC treatment. Conclusion: The results herein describe the characterization of an orthotopic rat model of canine OSA. This model was useful for the evaluation of different dose and fractionation SRT protocols along with combination adjuvant therapies that may be clinically relevant for canine or human OSA. The administration of MSCs following SRT did not induce new bone growth. The lack of efficacy is most likely due to the radiation-induced alterations to the bone microenvironment that resulted in conditions poorly suited to MSC survival and/or differentiation.
Open Access
Tissue electrophoresis for generation of porcine acellular dermal matrices
(Colorado State University. Libraries, 2013) Duran, Celso, Jr., author; Orton, Christopher, advisor; Dasi, Lakshmi Prasad, committee member; James, Susan P., committee member
Background: Acellular dermal matrices have several applications including treatment of burns, reconstructive surgery, and treatment of chronic ulcers. Xenogeneic acellular dermal matrices have the advantage of increased availability compared to matrices derived from human cadavers (i.e. allogeneic dermal matrices), however they have a higher potential for generating an inflammatory response in the recipient. One approach to creating an acellular dermal matrix is through chemical and detergent-based processes collectively known as decellularization. Concerns regarding the completeness of soluble protein and antigen removal associated with current detergent-based decellularization treatments have been raised. The aim of this study was to compare the efficacy of a standard detergent-based decellularization and a novel electrophoresis-based method at removing soluble protein and protein antigens. Hypothesis: I hypothesized that tissue electrophoresis would enhance the removal of soluble proteins and protein antigens from porcine dermis compared to a standard detergent-based decellularization protocol. Methods: Skin was harvested from 6 pig cadavers. A portion of skin from each pig was assigned to four treatment groups: 1. Epidermis removal without sodium dodecyl sulfate (SDS) (positive or untreated control) 2. Epidermis removal with 0.5% SDS (epidermis removal control) 3. Epidermis removal with 0.5% SDS and standard 0.5% SDS decellularization treatment with a 6 h passive diffusion washout period 4. Epidermis removal with 0.5% SDS and Tissue Electrophoresis-based decellularization (0.5% SDS, 2% agarose gel, and 0.5 Amp) for 6 h The completeness of soluble protein and antigen removal was evaluated by SDS-PAGE and immunoblot analysis, respectively. Rabbit anti-porcine and human IgG serums were the primary antibodies for immunoblot analysis. Results: Tissue electrophoresis decellularization increased removal of soluble proteins from porcine dermis when compared to standard passive detergent-based decellularization, based on SDS-PAGE analysis. Antigen removal, based on immunoblot analysis, was increased compared to untreated dermis, but was not significantly different between standard detergent-based and tissue electrophoresis-based decellularization treatments. Conclusion: Tissue electrophoresis enhances removal of soluble proteins from porcine dermis compared to standard detergent-based decellularization. This enhanced removal of soluble proteins may translate into reduced inflammatory response to xenogeneic acellular dermal matrices implanted into humans. Optimization of electrophoretic parameters may further increase the efficiency of tissue electrophoresis as a decellularization method.
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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.
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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
Hyaluronic acid enhancement of expanded polytetrafluoroethylene for small diameter vascular grafts
(Colorado State University. Libraries, 2014) Lewis, Nicole R., author; James, Susan P., advisor; Popat, Ketul C., committee member; Bailey, Travis, committee member
Cardiovascular disease is the leading cause of mortality and morbidity in the United States and other developed countries. In the United States alone, 8 million people are diagnosed with peripheral arterial disease per year and over 250,000 patients have coronary bypass surgery each year. Autologous blood vessels are the standard graft used in small diameter (<6mm) arterial bypass procedures. Synthetic small diameter grafts have had limited success. While polyethylene (Dacron) and expanded polytetrafluoroethylene (ePTFE) are the most commonly used small diameter synthetic vascular graft materials, there are significant limitations that make these materials unfavorable for use in the low blood flow conditions of the small diameter arteries. Specifically, Dacron and ePTFE grafts display failure due to early thrombosis or late intimal hyperplasia. With the shortage of tissue donors and the limited supply of autologous blood vessels available, there is a need for a small diameter synthetic vascular graft alternative. The aim of this research is to create and characterize ePTFE grafts prepared with hyaluronic acid (HA), evaluate thrombogenic potential of ePTFE-HA grafts, and evaluate graft mechanical properties and coating durability. The results in this work indicate the successful production of ePTFE-HA materials using a solvent infiltration technique. Surface interactions with blood show increased platelet adhesion on HA-modified surfaces, though evidence may suggest less platelet activation and erythrocyte lysis. Significant changes in mechanical properties of HA-modified ePTFE materials were observed. Further investigation into solvent selection, uniformity of HA, endothelialization, and dynamic flow testing would be beneficial in the evaluation of these materials for use in small diameter vascular graft bypass procedures.