VTechWorks Repository :: Browsing by Author "Rylander, M. Nichole"

Browsing by Author "Rylander, M. Nichole"

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Advancements in Irreversible Electroporation for the Treatment of Cancer
Arena, Christopher Brian (Virginia Tech, 2013-05-03)
Irreversible electroporation has recently emerged as an effective focal ablation technique. When performed clinically, the procedure involves placing electrodes into, or around, a target tissue and applying a series of short, but intense, pulsed electric fields. Oftentimes, patient specific treatment plans are employed to guide procedures by merging medical imaging with algorithms for determining the electric field distribution in the tissue. The electric field dictates treatment outcomes by increasing a cell's transmembrane potential to levels where it becomes energetically favorable for the membrane to shift to a state of enhanced permeability. If the membrane remains permeabilized long enough to disrupt homeostasis, cells eventually die. By utilizing this phenomenon, irreversible electroporation has had success in killing cancer cells and treating localized tumors. Additionally, if the pulse parameters are chosen to limit Joule heating, irreversible electroporation can be performed safely on surgically inoperable tumors located next to major blood vessels and nerves. As with all technologies, there is room for improvement. One drawback associated with therapeutic irreversible electroporation is that patients must be temporarily paralyzed and maintained under general anesthesia to prevent intense muscle contractions occurring in response to pulsing. The muscle contractions may be painful and can dislodge the electrodes. To overcome this limitation, we have developed a system capable of achieving non-thermal irreversible electroporation without causing muscle contractions. This progress is the main focus of this dissertation. We describe the theoretical basis for how this new system utilizes alterations in pulse polarity and duration to induce electroporation with little associated excitation of muscle and nerves. Additionally, the system is shown to have the theoretical potential to improve lesion predictability, especially in regions containing multiple tissue types. We perform experiments on three-dimensional in vitro tumor constructs and in vivo on healthy rat brain tissue and implanted tumors in mice. The tumor constructs offer a new way to rapidly characterize the cellular response and optimize pulse parameters, and the tests conducted on live tissue confirm the ability of this new ablation system to be used without general anesthesia and a neuromuscular blockade. Situations can arise in which it is challenging to design an electroporation protocol that simultaneously covers the targeted tissue with a sufficient electric field and avoids unwanted thermal effects. For instance, thermal damage can occur unintentionally if the applied voltage or number of pulses are raised to ablate a large volume in a single treatment. Additionally, the new system for inducing ablation without muscle contractions actually requires an elevated electric field. To ensure that these procedures can continue to be performed safely next to major blood vessels and nerves, we have developed new electrode devices that absorb heat out of the tissue during treatment. These devices incorporate phase change materials that, in the past, have been reserved for industrial applications. We describe an experimentally validated numerical model of tissue electroporation with phase change electrodes that illustrates their ability to reduce the probability for thermal damage. Additionally, a parametric study is conducted on various electrode properties to narrow in on the ideal design.
Anti-inflammatory Effects and Biodistribution of Cerium Oxide Nanoparticles
Hirst, Suzanne Marie (Virginia Tech, 2010-02-04)
Cerium oxide nanoparticles have the unique ability to accept and donate electrons, making them powerful antioxidants. Their redox nature is due to oxygen defects in the lattice structure, which are more abundant at the nanoscale. Reactive oxygen species (ROS) are pro-oxidants whose presence is increased during periods of inflammation in the body. ROS damage tissues and cellular function by stripping electrons from proteins, lipids, and DNA. We investigated the ability of nanoceria to quench ROS in vitro and in vivo, and examined the biodistribution and biocompatibility of nanoceria in murine models. Nanoceria was internalized in vitro by macrophages, is non-toxic at the concentrations we investigated, and proteins, mRNA, and oxidative markers of ROS were abated with nanoceria pretreatment in immune stimulated cells as measured by western blot, real time RT PCR, and Greiss assay respectively. In vivo, nanoceria was deposited in the spleen and liver, with trace amounts in the lungs and kidneys as determined by ICP-MS. Using IVIS in vivo imaging, it appeared that nanoceria deposition occurred in lymph tissue. Histology grades show no overt pathology associated with nanoceria deposition, although white blood cell (WBC) counts were generally elevated with nanoceria treatment. Nanoceria suspect particles were seen in lysosomes from kidney samples of IV injected mice in HRTEM images. Lastly, IV nanoceria treatment appears to reduce markers of oxidative stress in mice treated with carbon tetrachloride (CCl4) to induce ROS production. Taken together, our data suggest that nanoceria treatment has the potential to reduce oxidative stress.
Bioactive Poly(Lactic-co-Glycolic Acid)-Calcium Phosphate Scaffolds for Bone Tissue Regeneration
Popp, Jenni Rebecca (Virginia Tech, 2009-03-27)
Bone is currently the second most transplanted tissue, second only to blood. However, significant hurdles including graft supply and implant failure continue to plague researchers and clinicians. Currently, standard clinical procedures include autologous and allogeneic grafting. Autologous grafts may achieve functional repair; yet, they are available in limited supply and are associated with donor site morbidity. Allogeneic grafts are available in greater supply, but have a higher risk of infection. To overcome the disadvantages of current grafts, tissue engineering has become a major focus for the regeneration of bone. The goal of tissue engineering is to use a multidisciplinary approach to create biomimetic constructs that stimulate osteogenic regeneration to heal bone defects and restore tissue function. Biodegradable scaffolds are used in tissue engineering strategies as an interim template for tissue regeneration. The scaffold architecture provides mechanical support for cell attachment and tissue regeneration. Biocompatible poly(lactic-co-glycolic acid) (PLGA) has been processed through a number of techniques to create porous 3D architectures. Hydroxyapatite (HAP) and tricalcium phosphate have been used in conjunction with polymer scaffolds due to their osteoconductivity and biocompatibility, but they often lack osteoinductivity and are resistant to biodegradation. Conversely, amorphous calcium phosphate (ACP) is a mineral that solubilizes under aqueous conditions, releasing calcium and phosphate ions, which have been postulated to enhance osteoblast differentiation and mineralization. Controlled dissolution can be achieved by stabilizing ACP with divalent cations such as zinc or copper. Furthermore, incorporation of such osteogenic ACPs within a biodegradable PLGA scaffold could enhance the osteoconductivity of the scaffold while providing calcium and phosphate ions to differentiating osteoprogenitor cells, thereby stimulating osteogenesis when implanted in vivo. In this research, the effect of zinc on the differentiation of osteoprogenitor cells was investigated. Zinc supplementation of the culture media had no stimulatory effect on cell proliferation or differentiation. ACPs were synthesized using zirconium (ZrACP) and zinc (ZnACP) as stabilizers to achieve sustained ion release. Elevated concentrations suggested sustained ion release over the course of 96 hours and enhanced solubility of ZrACP and ZnACP. X-ray diffraction analysis showed a conversion of ZrACP to a semi-crystalline material after 96 hours, but ZnACP showed no conversion after 96 hours. Composite scaffolds were fabricated by incorporating HAP, zirconium-stabilized ACP (ZrACP), or zinc-stabilized ACP (ZnACP) into a sintered PLGA microsphere matrix and then characterized to determine the effect of the minerals on the in vitro differentiation of MC3T3-E1 cells. Scanning electron microscopy revealed a porous microsphere matrix with calcium phosphate powders distributed on the surface of the microspheres. Measurements of mechanical properties indicated that incorporation of 0.5 wt% calcium phosphates resulted in a 30% decrease in compressive modulus. When cells were cultured in the scaffolds, composite ACP scaffolds stimulated proliferation and ALP activity, while HAP scaffolds stimulated osteoblast gene expression. Overall, the results of this work indicate the addition of calcium phosphate minerals to PLGA scaffolds supported cell growth and stimulated osteogenic differentiation, making the scaffolds a promising alternative for bone tissue regeneration.
Biomedical research application of a novel double-layer parallel-plate flow chamber
Lee, Won Hee (Virginia Tech, 2007-05-09)
Since integrity and functions of vascular endothelial cells are greatly affected by shear stress, a variety of in vitro systems to subject endothelial cells under precisely controlled fluid conditions has been developed. Complicated designs of the conventional flow devices, however, have impeded such implementation. In the present study, we designed and developed a novel parallel-plate flow chamber (PPFC). It consists of multiple layers of different materials to adjust the required geometries of the chamber and provide a wide span of biomedical research applications. Because the chamber stacks separate layers to constitute the flow channel, different pieces can be easily removed or replaced. Moreover, the multilayer design only requires 2D cutting, which is easier and faster to manufacture. It is also capable of accepting up to four glass slides facing each other so that the flow within the channel is exclusively formed by endothelial cells. Furthermore, it minimizes the pressure loss across the chamber while maximizing the effective area of endothelial cells up to 96 cm2. Results from mathematical analysis and dye injection experiments showed that a uniform magnitude of shear stress is applied throughout the entire surface of endothelial cells. In addition, the morphological changes and attenuated gene expression of pro-inflammatory mediators were observed in endothelial cells exposed to the physiologically relevant shear stress. These findings indicate that our newly designed PPFC can provide a better in vitro system for versatile applications of biomedical research. The reperfusion of blood flow occurred in a number of conditions such as stroke and organ transplantation immensely augments tissue injury and can cause more severe damage than prolonged ischemia. The injuries caused by cessation and reperfusion of blood flow are closely related to the inflammatory reactions involving in endothelium-leukocyte cascade responding to a shear stress exerted by the flow. Shear stress is also known to play an important role in human chronic diseases including atherosclerosis, neurological disorders, and cancer metastasis. Therefore, it is important to investigate the transmission of mechanical stimuli such as shear stress to various complex endothelial cell signaling pathways which process as a whole is often referred as mechanotransduction. Shear stress-mediated signaling pathways have been known to trigger endothelial cell responses and contribute to the pathophysiology of human vascular diseases. The present study was designed to apply the novel PPFC to biomedical research, especially ischemia/reperfusion injury. The changes in mRNA and protein expression of inflammatory mediators in endothelial cells were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RBE4 and HMEC-1 cells were either maintained in continuous laminar flow condition (Normal Flow) or subjected to 1 h of flow cessation followed by reperfusion of flow (Ischemia/Reperfusion) for 24 h. Ischemia/Reperfusion significantly up-regulated expression of inflammatory mediators, such as IL-6, MCP-1, ICAM-1, VCAM-1, and E-selectin, in microvascular endothelial cells. Furthermore, antioxidant pyrrolidine dithiocarbamate (PDTC) significantly attenuated ischemia/reperfusion-induced overexpression of pro-inflammatory mediators. These data indicates that our newly designed PPFC provide a better in vitro system for versatile applications of biomedical research.
Computational and Experimental Modeling of the Bioheat Transfer Process of Perfusion in Tissue Applied to Burn Wounds
Al-Khwaji, Abdusalam (Virginia Tech, 2013-04-29)
A new mathematical model has been developed along with a new parameter estimation routine using surface temperature and heat flux measurements to estimate blood perfusion and thermal resistance in living tissue. Dynamic thermal measurements collected at the surface of the sensor before and after imposing a dynamic thermal cooling event are used with the model to estimate the blood perfusion, thermal resistance and core temperature. The Green\'s function based analytical solution does not require calculation of the whole tissue temperature distribution, which was not the case for the previous models. The result from the new model was proved to have better and more consistent results than previous models. The new model was validated to solve one of the unsolved biomedical problems which is the ability of detecting burn severity. The method was tested with a phantom perfusion system. The results matched known blood perfusion and thermal resistance values. The method was also tested with burns on animal models. Inflammation effects associated with the burns were studied using a newly developed term called the Burn Factor. This correlated with the severity of imposed burns. This work consists of three journal papers. The first paper introduces the mathematical model and its validation with finite-difference solutions. The second paper validates the physical aspects of the usage of the model with thermal measurement in detecting simulated burned layers and the associated perfusion. The third paper demonstrates the ability of the model to use thermal measurements to detect different burn severity of an animal model and to study the healing process.
Creation and Characterization of Several Polymer/Conductive Element Composite Scaffolds for Skeletal Muscle Tissue Engineering
Fischer, Kristin Mckeon (Virginia Tech, 2012-02-17)
After skeletal muscle damage, satellite cells move towards the injured area to assist in regeneration. However, these cells are rare as their numbers depend on the age and composition of the injured muscle. This regeneration method often results in scar tissue formation along with loss of function. Although several treatment methods have been investigated, no muscle replacement treatment currently exists. Tissue engineering attempts to create, repair, and/or replace damaged tissue by combining cells, biomaterials, and tissue-inducing substances such as growth factors. Electrospinning produces a non-woven scaffold out of biomaterials with fiber diameters ranging from nanometers to microns to create an extracellular-like matrix on which cells attach and proliferate. Our focus is on synthetic polymers, specifically poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), and poly(ε-caprolactone) (PCL). Skeletal muscle cells grown on electrospun scaffolds tend to elongate and fuse together thus, mimicking natural tissue. Electrical stimulation has been shown to increase the number of cells fused in culture and decreased the time needed in culture for cells to contract. Therefore, a conductive element was added to each scaffold, specifically polyaniline (PANi), gold nanoparticles (Au Nps), and multi-walled carbon nanotubes (MWCNT). Our project goal is to create a polymeric, conductive, and biocompatible scaffold for skeletal muscle regeneration. PANi and PDLA were mixed to form the following solutions 24% (83% PDLA/17% PANi), 24% (80% PDLA/20% PANi), 22% (75%PDLA/25% PANi), 29% (83% PDLA/17% PANi), and 29% (80% PDLA/20% PANi). Only the 75/25 electrospun scaffold was conductive and had a calculated conductivity of 0.0437 S/cm. Scaffolds with larger amounts of PANi were unable to be electrospun. PDLA/PANi scaffolds were biocompatible as primary rat skeletal muscle cells cultured in vitro did attach. However, the scaffolds shrunk, degraded easily, and became brittle. Although PDLA/PANi scaffolds were easily manufactured, our results indicate that this polymer mixture is not appropriate for skeletal muscle scaffolds. PLLA and Au Nps were electrospun together to form three composite scaffolds: 7% Au-PLLA, 13% Au-PLLA, and 21% Au-PLLA. These were compared to PLLA electrospun scaffolds. Measured scaffold conductivities were 0.008 ± 0.015 S/cm for PLLA, 0.053 ± 0.015 S/cm for 7% Au-PLLA, 0.076 ± 0.004 S/cm for 13% Au-PLLA, and 0.094 ± 0.037 S/cm for 21% Au-PLLA. It was determined via SEM with a Bruker energy dispersive x-ray spectrometer (EDS) that the Au Nps were not evenly distributed within the scaffolds as they had agglomerated. Rat primary muscle cells cultured on the three Au-PLLA scaffolds displayed low cellular activity. A second cell study was conducted to determine Au NPs toxicity. The results show that the Au Nps were not toxic to the cells and the low cellular activity may be a marker for myotube fusion. Elastic modulus and yield stress values for the three Au-PLLA scaffolds measured on days 0, 7, 14, 21, and 28 were much larger than skeletal muscle tissue. Due to the larger mechanical properties and Au Nps agglomeration, a third polymer and conductive element scaffold was investigated. PCL was chosen as the new synthetic polymer as it had a lower elastic modulus and high elongation. MWCNT were chosen as the conductive element as they disperse well within PCL when acid functionalized. A third component was added to the scaffold to help it move similar to skeletal muscle. Ionic polymer gels (IPG) are hydrogels that respond to an external stimulus such as temperature, pH, light, and electric field. A poly(acrylic acid)/poly(vinyl alcohol) (PAA/PVA) mixture is one type of IGP that responds to an electric field. The scaffolds were coaxially electrospun so that each fiber had a PCL-MWCNT interior with a PAA/PVA sheath. These scaffolds were compared to electrospun PCL and PCL-MWCNT ones. The addition of MWCNT to the PCL did increase scaffold conductivity. Actuation of the PCL-MWCNT-PAA/PVA scaffold occurred when 15V and 20V were applied. All three scaffolds had rat primary skeletal muscle cells attached but, more multinucleated cells with actin interaction were seen on PCL-MWCNT-PAA/PVA scaffolds. Once again the mechanical properties were greater than muscle, but because of its ability to actuate we believe the PCL-MWCNT-PAA/PVA scaffold has potential as a bioartificial muscle. Further characterization of the PCL-MWCNT-PAA/PVA included varying the ratios of PAA/PVA, smaller crosslinking times, and lower amounts of MWCNT. Four ratios, 83/17, 60/40, 50/50, and 40/60, were successfully coaxially electrospun with PCL and MWCNT. Overall, very few differences were seen between the four ratios in conductivity, cellular biocompatibility, actuation angular speed, and mechanical properties. The 83/17 and 40/60 ratios were chosen for additional investigation into mechanical properties and actuation. As the mechanical properties of the two types of scaffolds did not change significantly through degradation, lower PVA crosslinking times were tested. No significant effects were found and it was hypothesized that the evaporation of the solution played a role in the crosslinking process. The smaller MWCNT amount scaffolds also did not significantly affect the mechanical properties or the actuation angular speeds. More work into lowering the scaffold mechanical properties while increasing the actuation angular speed is necessary. Though the mechanical properties for the 83/17 and 40/60 scaffolds remained high compared to skeletal muscle, we also looked for differences in in vivo biocompatibility. Both scaffolds were implanted into the right vastus lateralis muscle of Sprague-Dawley rats. The left vastus lateralis muscle served as either the PBS injected sham surgery or an unoperated control. Biocompatibility was evaluated using enzymes, creatine kinase (CK) and lactate dehydrogenase (LDH), levels, fibrosis formation, inflammation, scaffold cellular infiltration, and neovascularization on days 7, 14, 21, and 28 post-implantation. Fibrotic tissue formation, inflammation, and elevated CK and LDH levels were observed initially but responses decreased during the four week study. Cells infiltrated the scaffolds and histological staining showed more fibroblasts than myogenic cells initially but over time, the fibroblasts decreased and myogenic cells increased. Neovascularization of both scaffolds was also recorded. PCL-MWCNT-PAA/PVA scaffolds were determined to be biocompatible, but some differences between the two types were noted. The 83/17 scaffolds caused less of a response from the body compared to the 40/60 scaffolds and had more myogenic cells attached. However, the 40/60 scaffolds had a larger number of blood vessels running through the scaffold. In conclusion, we have successfully fabricated a polymeric, conductive, and biocompatible scaffold that can actuate for skeletal muscle tissue engineering. Although our results are promising, more work is necessary to continue developing and refining the scaffold.
Design and nondestructive imaging of a bioengineered vascular graft endothelium
Whited, Bryce Matthew (Virginia Tech, 2013-02-01)
Cardiovascular disease is currently the leading cause of death in the U.S. that frequently requires bypass surgery using vascular grafts for treatment. Current limitations with fully synthetic grafts have led researchers to bioengineered alternatives that consist of a combination of vascular scaffolds and cells. A major challenge in creating a functional bioengineered vascular graft is development of a confluent endothelium on the lumen that is able to resist detachment under physiologic fluid flow. In addition, methodologies used to assess the growth and maturation of the endothelium in a noninvasive and dynamic manner are severely lacking. Therefore, the overall goal of this research is to advance the field of vascular tissue engineering by 1) creating methodologies to enhance EC adherence to a vascular graft and 2) development of a noninvasive and real-time imaging system capable of assessing the graft endothelium. To achieve these objectives, three separate studies were performed. In the first study, electrospun scaffold fiber diameter and alignment were systematically varied to determine their effect on endothelial cell (EC) morphology and adherence under fluid flow. ECs on uniaxially aligned nanofibers displayed elongated and aligned morphologies leading to higher adherence to the scaffolds under physiologic levels of fluid flow as compared to those on randomly oriented scaffolds. In the second study, a fiber optic based (FOB) imaging system was developed to image fluorescent ECs through a thick electrospun scaffold. Results demonstrated that the FOB imaging system was able to accurately visualize fluorescent ECs in a noninvasive manner through the thick and highly opaque scaffold. In the final study, the FOB imaging system was used to noninvasively quantify vascular graft endothelialization, EC detachment, and apoptosis through the vessel wall with greater imaging penetration depth than two-photon microscopy. Additionally, the FOB method was capable of continuously tracking EC migration and endothelialization of a bioengineered graft in a bioreactor. Overall, these results demonstrate that aligned scaffold topographies enhance EC adherence under fluid flow and the FOB imaging system is a promising tool to monitor endothelium development and response to fluid flow in a manner that has not previously been afforded using conventional imaging methods.
Design, Fabrication, and Characterization of Three Dimensional Complete Scaffolds for Bone Tissue Engineering
Andric, Tea (Virginia Tech, 2012-03-03)
Skeletal loss and bone deficiencies are major worldwide problem that is only expected to increase due to increase in aging population. As current standards in treatment autografts and allografts are not without drawbacks, there is a need for alternative bone grafts substitutes. The goal of this project was to utilize electrospinning and heat sintering techniques to create biodegradable full thickness three dimensional biomimetic polymeric scaffolds with macro and nano architecture similar to natural bone for bone tissue engineering. First we have investigated pretreatment with 0.1M NaOH and electrospinning gelatin/PLLA blends as means to increase overall mineral precipitation and distribution throughout the scaffolds when incubated in concentrated simulated body fluid (SBF)10XSBF. Mixture of 10% gelatin and PLLA resulted in the significantly higher degree of mineralization, increased mechanical properties, and scaffolds that supported cellular adhesion and proliferation. In the next step we applied heat sintering technique to fabricate 3D electrospun scaffolds that were used to evaluate effects of mineralization and fiber orientation on scaffold strength. Fiber orientation can make a slight difference in nanofibrous scaffold compressive mechanical properties, but this difference is not as profound as the difference seen with increased mineralization. We also developed a technique to fabricate scaffolds that mimic the organization of an osteon, the structural unit of cortical bone. Resulting scaffolds consisted of concentric layers of electrospun gelatin/PLLA nanofibers wrapped around microfiber core with diameters that ranged from 200-600µm. Individual osteon-like scaffolds were heat sintered to fabricate three dimensional scaffolds contained a system of channels running parallel to the length of the scaffolds, as found naturally in bone tissue. Finally we combined two previously fabricated structures, sintered electrospun sheets and individual osteon-like scaffolds, to create novel scaffolds that mimic dual structural organization of natural bone with cortical and trabecular regions. Mineralization for 24 hr significantly increased mechanical properties of the scaffolds, both yield stress and compressive modulus under physiological conditions. Both nonminerlized and mineralized scaffolds were found to support cellular attachment and proliferation over 28 days in culture, but scaffolds mineralized for 24hr were found to better support osteoblastic differentiation and mineral deposition.
Developing a Living Composite Ligament by Combining Prolotherapy and Nanoparticles as Treatment for Damaged Connective Tissue
Empson, Yvonne Marie (Virginia Tech, 2014-05-06)
Significant cost and debilitation results from connective tissue injury and disease every year. Prolotherapy is an effective medical treatment used to increase joint stability. However, most associated studies are retrospective or case studies, rather than comprehensive laboratory investigation originating with the cellular response to exposure to the proliferant solutions. As a parallel consideration, nanoparticles are being investigated for use in drug delivery and heat shock treatment of cancerous tissue due to their unique structural and thermal properties. The phenomenal strength and stiffness of carbon nanoparticles have been used for commercial purposes in composite materials, but investigation of biomedical applications is still fairly nascent. In an attempt to develop a non-surgical approach to supporting and healing damaged ligaments and tendons resulting from injury or disease by combining prolotherapy and the use of nanoparticles, the author presents studies investigating the cellular response to proliferative therapy solution as well as tendon and ligament tissue's mechanical and cellular response to exposure to nanoparticles. In the prolotherapy solution cell studies, the results suggested that there is an optimal dosage of the proliferant for in vitro studies, different responses between cell types, and a dosage-dependent response in cell viability and collagen production to the solution P2G in preosteoblasts. In the nanoparticle studies, cell populations tolerated nanoparticles at the levels tested, tendon mechanical properties were increased (stiffness significantly so), and bright field and transmission electron microscopic histological images were taken of connective tissue and carbon nanohorn interactions.
Development of a Hollow-Core Fiberoptic Microneedle Device for the Treatment of Invasive Bladder Cancer
Hood, Robert L. (Virginia Tech, 2011-08-11)
The hydraulic resistance characterization manuscript chronicles the early development of the hollow-core fiberoptic microneedle device (FMD). The study determined that for straight tubing with an inner bore of 150 ?m and a length greater than 50 mm long, Poiseuille's Law was shown to be accurate within 12% of experimental data for the pressure range of 69-517 kPa. Comparison between different needle design geometries indicated that tip diameters <55 ?m cause a significant increase in hydraulic resistance. Tubing length should be kept to a minimum and tip diameter should be kept above this threshold to reduce overall hydraulic resistance. The bladder treatment study describes the fabrication and testing of the FMD for treatment of invasive urothelial cell carcinomas (UCCs). Experiments investigating the fluid dispersal of single-walled carbon nanohorns (SWNHs) in the wall of inflated, healthy ex vivo bladders demonstrated that perfusion of 2 cm° on the bladder wall's surface can be achieved with a 5 minute infusion at 50 ?L/min. Irradiation of the SWNH perfused bladder wall tissue with a free space, 1064 nm laser at an irradiance of 0.95 W/cm° for 40 seconds yielded a 480% temperature increase relative to similar irradiation of a non-infused control. Co-delivery experiments demonstrated both SWNH and light delivery though a single hollow-core fiber to heat the bladder wall 33 °C with an irradiance of 400 W/cm°, demonstrating that the FMD can be used to achieve hyperthermia-based therapeutic effects via interstitial irradiation.
Dynamic Non-Destructive Monitoring of Bioengineered Blood Vessel Development within a Bioreactor using Multi-Modality Imaging
Gurjarpadhye, Abhijit Achyut (Virginia Tech, 2013-08-20)
Regenerative medicine involves formation of tissue or organ for replacement of a wounded or dysfunctional tissue. Healthy cells extracted from the patient are expanded and are seeded on a three-dimensional biodegradable scaffold. The structure is then placed in a bioreactor and is provided with nutrients for the cells, which proliferate and migrate throughout the scaffold to eventually form a desired to tissue that can be transplanted into the patient's body. Inability to monitor this complex process of regeneration in real-time makes control and optimization of this process extremely difficult. Histology, the gold standard used for tissue structural assessment, is a static technique that only provides "snapshots" of the progress and requires the specimen to be sacrificed. This inefficiency severely limits our understanding of the biological processes associated with tissue growth during the in vitro pre-conditioning phase. Optical Coherence Tomography (OCT) enables imaging of cross sectional structure in biological tissues by measuring the echo time delay of backreflected light. OCT has recently emerged as an important method to assess the structures of physiological, pathological as well as tissue engineered blood vessels. The goal of the present study is to develop an imaging system for non-destructive monitoring of blood vessels maturing within a bioreactor. Non-destructive structural imaging of tissue-engineered blood vessels cultured in a novel bioreactor was performed using free-space and catheter-based OCT imaging, while monitoring of the endothelium development was performed using a fluorescence imaging system that utilizes a commercial OCT catheter. The project included execution of three specific aims. Firstly, we developed OCT instrumentation to determine geometrical and optical properties of porcine and human skin in real-time. The purpose of the second aim was to assess structural development of tissue-engineered blood vessels maturing in a bioreactor. We constructed a novel quartz-based bioreactor that will permit free space and catheter-based OCT imaging of vascular grafts. The grafts were made of biodegradable PCL-collagen and seeded with multipotent mesenchymal cells. We imaged the maturing grafts over 30 days to assess changes in graft wall thickness. We also monitored change in optical properties of the grafts based on free-space OCT scanning. Finally, in order to visualize the proliferation of endothelial cells and development of the endothelium, we developed an imaging system that utilizes a commercial OCT catheter for single-cell-level imaging of the growing endothelium of a tissue-engineered blood vessel. We have developed two modules of an imaging system for non-destructive monitoring of maturing bioengineered vascular grafts. The first module provides the ability to non-destructively examine the structure of the grafts while the second module can track the progress of endothelialization. As both modules use the same endoscope for imaging, when operated in sequence, they will produce high-resolution, three-dimensional, structural details of the graft and two-dimensional spatial distribution of ECs on the lumen. This non-destructive, multi-modality imaging can be potentially used to monitor and assess the development of luminal bioengineered constructs such as colon or trachea.
Dynamic, Nondestructive Imaging of a Bioengineered Vascular Graft Endothelium
Whited, Bryce M.; Hofmann, Matthias C.; Lu, Peng; Xu, Yong; Rylander, Christopher G.; Wang, Ge; Sapoznik, Etai; Criswell, Tracy; Lee, Sang Jin; Soker, Shay; Rylander, M. Nichole (PLOS, 2013-04-09)
Bioengineering of vascular grafts holds great potential to address the shortcomings associated with autologous and conventional synthetic vascular grafts used for small diameter grafting procedures. Lumen endothelialization of bioengineered vascular grafts is essential to provide an antithrombogenic graft surface to ensure long-term patency after implantation. Conventional methods used to assess endothelialization in vitro typically involve periodic harvesting of the graft for histological sectioning and staining of the lumen. Endpoint testing methods such as these are effective but do not provide real-time information of endothelial cells in their intact microenvironment, rather only a single time point measurement of endothelium development. Therefore, nondestructive methods are needed to provide dynamic information of graft endothelialization and endothelium maturation in vitro. To address this need, we have developed a nondestructive fiber optic based (FOB) imaging method that is capable of dynamic assessment of graft endothelialization without disturbing the graft housed in a bioreactor. In this study we demonstrate the capability of the FOB imaging method to quantify electrospun vascular graft endothelialization, EC detachment, and apoptosis in a nondestructive manner. The electrospun scaffold fiber diameter of the graft lumen was systematically varied and the FOB imaging system was used to noninvasively quantify the affect of topography on graft endothelialization over a 7-day period. Additionally, results demonstrated that the FOB imaging method had a greater imaging penetration depth than that of two-photon microscopy. This imaging method is a powerful tool to optimize vascular grafts and bioreactor conditions in vitro, and can be further adapted to monitor endothelium maturation and response to fluid flow bioreactor preconditioning.
Effective Cancer Therapy Design Through the Integration of Nanotechnology
Fisher, Jessica Won Hee (Virginia Tech, 2008-07-25)
Laser therapies can provide a minimally invasive treatment alternative to surgical resection of tumors. However, therapy effectiveness is limited due to nonspecific heating of target tissue, leading to healthy tissue injury and extended treatment durations. These therapies can be further compromised due to heat shock protein (HSP) induction in tumor regions where non-lethal temperature elevation occurs, thereby imparting enhanced tumor cell viability and resistance to subsequent therapy treatments. Introducing nanoparticles (NPs), such as multi-walled nanotubes (MWNTs) or carbon nanohorns (CNHs), into target tissue prior to laser irradiation increases heating selectivity permitting more precise thermal energy delivery to the tumor region and enhances thermal deposition thereby increasing tumor injury and reducing HSP expression induction. This research investigates the impact of MWNTs and CNHs in untreated and laser-irradiated monolayer cell culture, tissue phantoms, and/or tumor tissue from both thermal and biological standpoints. Cell viability remained high for all unheated NP-containing samples, demonstrating the non-toxic nature of both the nanoparticle and the alginate phantom. Up-regulation of HSP27, 70 and 90 was witnessed in samples that achieved sub-lethal temperature elevations. Tuning of laser parameters permitted dramatic temperature elevations, decreased cell viability, and limited HSP induction in NP-containing samples compared to those lacking NPs. Preliminary work showed MWNT internalization by cells, which presents imaging and multi-modal therapy options for NT use. The lethal combination of NPs and laser light and NP internalization reveals these particles as being viable options for enhancing the thermal deposition and specificity of hyperthermia treatments to eliminate cancer.
Fiber array for optical imaging and therapeutics
(United States Patent and Trademark Office, 2019-03-05)
The present invention relates to the field of optical imaging and therapeutics. More particularly, embodiments of the present invention provide minimally-invasive Fiberoptic Microneedle Devices (FMDs) for light-based therapeutics, which physically penetrate tissue and deliver light directly into the target area below the skin surface. Embodiments of the invention enable depth-selective and deep photothermal therapeutics and include methods of treating cancer, methods of re-shaping or removing adipose tissue, and methods of delivering drugs or co-delivering drugs and energy to selected tissue.
Fiberoptic Microneedles for Transdermal Light Delivery
Kosoglu, Mehmet Alpaslan (Virginia Tech, 2011-10-19)
Shallow light penetration in tissue has been a technical barrier to the development of photothermal therapies for cancers in the epithelial tissues and skin. This problem can potentially be solved by utilizing minimally invasive probes to deliver light directly to target areas potentially > 2 mm deep within tissue. To develop this solution, fiber optic microneedles capable of delivering light for therapy were manufactured. We have manufactured fiberoptic microneedles by tapering silica-based optical fibers employing a melt-drawing process. These fiberoptic microneedles were 35 to 139 microns in diameter and 3 mm long. Some of the microneedles were manufactured to have sharper tips (tip diameter < 8 microns) by changing the heat source during the melt-drawing process. All of the microneedles were individually inserted into ex vivo porcine skin samples to demonstrate the feasibility of their application in human tissues. Skin penetration experiments showed that sharp fiber optic microneedles with a minimum average diameter of 73 microns and a maximum tip diameter of 8 microns were able to penetrate skin without buckling. Flat microneedles, which had larger tip diameters, required a minimum average diameter of 125 microns in order to penetrate through porcine skin samples. Force versus displacement plots showed that a sharp tip on a fiber optic microneedle decreased the skin's resistance during insertion. Also, the force acting on a sharp microneedle increased more steadily compared with a microneedle with a flat tip. Melt-drawn fiberoptic microneedles provided a means to mechanically penetrate dermal tissue and deliver light directly into a localized target area. We also described an alternate fiberoptic microneedle design with the capability of delivering more diffuse, but therapeutically useful photothermal energy using hydrofluoric acid etching of optical fibers. Microneedles etched for 10, 30, and 50 minutes, and an optical fiber control was compared for their ability to deliver diffuse light using three techniques. First, red light delivery from the microneedles was evaluated by imaging the reflectance of the light from a white paper. Second, spatial temperature distribution of the paper in response to near-IR light (1,064 nm, 1 W, CW) was recorded using infrared thermography. Third, ex vivo adipose tissue response during 1,064 nm, (5 W, CW) irradiation was recorded with bright field microscopy. Increasing etching time decreased microneedle diameter (from 125 to 33 microns), resulting in increased uniformity of red and 1,064 nm light delivery along the microneedle axis. For equivalent total energy delivery, microneedles with smaller diameters reduced carbonization in the adipose tissue experiments. However, thin fiberoptic microneedles designed to minimize tissue disruption and deliver diffuse therapeutic light are limited in their possible clinical application due to a lack of mechanical strength. Fiberoptic microneedles have been embedded in an elastomeric support medium (polydimethylsiloxane, PDMS) to mitigate this issue. The critical buckling force of silica microneedles with 55, 70, and 110 microns diameters and 3 mm length were measured with and without the elastomeric support in place (N = 5). Average increases in the mechanical strength for microneedles of 55, 70, and 110 microns diameters were measured to be 610%, 290%, and 33%, respectively. Aided by mechanical strengthening through an elastomeric support, microneedles with 55 microns diameter were able to repeatedly penetrate ex vivo porcine skin.
Flow Measurements in a Blood-Perfused Collagen Vessel Using X-Ray Micro-Particle Image Velocimetry
Antoine, Elizabeth E.; Buchanan, Cara; Fezzaa, Kamel; Lee, Wah-Keat; Rylander, M. Nichole; Vlachos, Pavlos P. (2013-11-18)
Blood-perfused tissue models are joining the emerging field of tumor engineering because they provide new avenues for modulation of the tumor microenvironment and preclinical evaluation of the therapeutic potential of new treatments. The characterization of fluid flow parameters in such in-vitro perfused tissue models is a critical step towards better understanding and manipulating the tumor microenvironment. However, traditional optical flow measurement methods are inapplicable because of the opacity of blood and the thickness of the tissue sample. In order to overcome the limitations of optical method we demonstrate the feasibility of using phase-contrast x-ray imaging to perform microscale particle image velocimetry (PIV) measurements of flow in blood perfused hydrated tissue-representative microvessels. However, phase contrast x-ray images significantly depart from the traditional PIV image paradigm, as they have high intensity background, very low signal-to-noise ratio, and volume integration effects. Hence, in order to achieve accurate measurements special attention must be paid to the image processing and PIV cross-correlation methodologies. Therefore we develop and demonstrate a methodology that incorporates image preprocessing as well as advanced PIV cross-correlation methods to result in measured velocities within experimental uncertainty.
High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction
Arena, Christopher B.; Sano, Michael B.; Rossmeisl, John H. Jr.; Caldwell, John L.; Garcia, Paulo A.; Rylander, M. Nichole; Davalos, Rafael V. (2011-11-21)
Background Therapeutic irreversible electroporation (IRE) is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death. Methods A combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE). A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for in vivo treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for in vivo experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation. Results No visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain. Conclusions H-FIRE is a feasible technique for non-thermal tissue ablation that eliminates muscle contractions seen in IRE treatments performed with unipolar electric pulses. Therefore, it has the potential to be performed clinically without the administration of paralytic agents.
Hydrodynamic Characterization of an Arterial Flow Bioreactor
Voigt, Elizabeth Elena (Virginia Tech, 2010-07-14)
An in vitro arterial flow bioreactor system for the generation of physiological flows in a biological environment was designed, constructed, and characterized. The design was based on models previously used to investigate the response of endothelial cells to shear. The model interfaces a bioreactor with flow elements to compose a flow loop that reproduces arterial flow conditions within the bioreactor. High-resolution (8.6 microns) time-resolved (4 ms) velocity field measurements within the bioreactor were obtained using Particle Image Velocimetry (PIV). Two physiological flows were considered, corresponding to medium human arteries at rest and exercise conditions: first, with an average Reynolds number of 150 and a Womersley parameter of 6.4, and second, with an average Reynolds number of 300 and a Womersley parameter of 9.0. Two cases were considered: first, using a smooth artery section, and second, with a confluent layer of human microvascular endothelial cells grown on the inner surface of the artery section. The instantaneous wall shear stress, time-averaged wall shear stress, and oscillatory shear index were computed from the velocity field measurements and compared for the cases with and without cells. These measurements were used to assess the value of the system for measurement of correlations between fluid dynamics and the response of biological tissue. It was determined that the flow present in such a system is not an accurate reproduction of physiological flow, and that direct measurement of the flow is necessary for accurate quantification of cellular response to fluid parameters.
Identification of Cell Biomechanical Signatures Using Three Dimensional Isotropic Microstructures
Nikkhah, Mehdi (Virginia Tech, 2010-12-03)
Micro and nanofabrication technologies have been used extensively in many biomedical and biological applications. Integration of MEMS technology and biology (BioMEMS) enables precise control of the cellular microenvironments and offers high throughput systems. The focus of this research was to develop three dimensional (3-D) isotropic microstructures for comprehensive analysis on cell-substrate interactions. The aim was to investigate whether the normal and cancerous cells differentially respond to their underlying substrate and whether the differential response of the cells leads to a novel label-free technique to distinguish between normal and cancerous cells. Three different generations of 3-D isotropic microstructures comprised of curved surfaces were developed using a single-mask, single-etch step process. Our experimental model included HS68 normal human fibroblasts, MCF10A normal human breast epithelial cells and MDA-MB-231 metastatic human breast cancer cells. Primary findings on the first generation of silicon substrates demonstrated a distinct adhesion and growth behavior in HS68 and MDA-MB-231 cells. MDA-MB-231 cells deformed while the fibroblasts stretched and elongated their cytoskeleton on the curved surfaces. Unlike fibroblasts, MDA-MB-231 cells mainly trapped and localized inside the deep microchambers. Detailed investigations on cytoskeletal organization, adhesion pattern and morphology of the cells on the second generation of the silicon substrates demonstrated that cytoskeletal prestress and microtubules organization in HS68 cells, cell-cell junction and cell-substrate adhesion strength in MCF10A cells, and deformability of MDA-MB-231 cells (obtained by using AFM technique) affect their behavior inside the etched cavities. Treatment of MDA-MB-231 cells with experimental breast cancer drug, SAHA, on the second generation of substrates, significantly altered the cells morphology, cytoarchitecture and adhesion pattern inside the 3-D microstructures. Third generation of silicon substrates was developed for comprehensive analysis on behavior of MDA-MB-231 and MCF10A cells in a co-culture system in response to SAHA drug. Formation of colonies of both cell types was evident inside the cavities within a few hours after seeding the cells on the chips. SAHA selectively altered the morphology and cytoarchitecture in MDA-MB-231 cells. Most importantly, the majority of MDA-MB-231 cells stretched inside the etched cavities, while the adhesion pattern of MCF10A cells remained unaltered. In the last part of this dissertation, using AFM analysis, we showed that the growth medium composition has a pronounced effect on cell elasticity. Our findings demonstrated that the proposed isotropic silicon microstructures have potential applications in development of biosensor platforms for cell segregation as well as conducting fundamental biological studies.
Improvements in fluidic device evaluation using particle image velocimetry
Raben, Jaime Melton Schmieg (Virginia Tech, 2013-09-09)
This work investigates flow measurement capabilities within meso- and micro-scaled medically relevant devices using particle image velocimetry (PIV). Medical devices can be particularly challenging to validate due to small length scales and complex geometries, which can reduce measurement accuracy by introducing noise and reducing available signal. Although the sources of such problems are often device specific, the effective outcome is a reduction in the signal-to-noise ratios (SNRs) of PIV images and correlations. This effort utilizes advanced PIV processing and post-processing techniques to establish protocols for achieving high accuracy PIV measurements in challenging flow environments. This investigation takes place within three wide-ranging medically related devices. First, channel flow in a microfluidic device is investigated to evaluate improvements in measurement accuracy gained using phase correlations in comparison to confocal microscopy. This work found substantial improvements in error with respect to the ensemble field for phase correlations while only moderate improvements were observed for confocal imaging with standard processing techniques. Secondly, an evaluation of stenting procedures was executed resulting in the first published PIV and computational fluid dynamics (CFD) joint study on bifurcating stents. This work analyzes steady flow in three bifurcation angles and four different single- and double-stenting procedures, which are clinically used in coronary bifurcations. Finally, a medical device analog was evaluated to develop a comprehensive CFD validation dataset, including a full uncertainty analysis for velocity and wall shear stress as well as estimates for pressure fields and relevant flow statistics including Reynolds stresses and dissipation.

Browsing by Author "Rylander, M. Nichole"

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