Browsing by Subject "Tissue engineering"

Now showing 1 - 8 of 8
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    The egress of fluid from the brain via arachnoid transport: foundational work for the tissue engineering of the arachnoid granulation
    (2011-12) Lam, Cornelius Hoktsim
    The arachnoid tissue is a critical component for the removal of cerebrospinal fluid (CSF) and other substances. Failure results in hydrocephalus, increased intracranial pressure, and buildup of toxic materials in the brain. The purpose of this thesis is to establish a foundation for a biomimetic arachnoid construct. First, we characterized arachnoid cell transport in culture and on three-dimensional collagen scaffolds. Arachnoid cells were harvested from rat brainstems and cultured onto bilayered bovine collagen scaffolds. Cells exhibited arachnoid cell phenotype (positive for vimentin, desmoplakin, and cytokeratin), readily penetrated the collagen scaffold, and doubled approximately every 2–3 days. The transepithelial electrical resistance for a monolayer of cells was 160 Ω∙cm2, and permeability of indigo carmine was 6.7+1.1X10- 6 cm/s. Hydraulic conductivity of the collagen construct was 6.39 mL/min/mmHg/cm2. Because of practical limitations of primary culture which include slow growth, early senescence, and poor reproducibility, we created two immortalized rat arachnoid cell lines using retroviral gene transfer of SV40 large T antigen (SV40 LTAg) either with or without human telomerase (hTERT). They stably expressed either SV40 LTAg alone, or SV40 LTAg and hTERT, and demonstrated high proliferative rate, contact inhibition at confluence, and stable expression of protein markers characteristic of native arachnoid cells for more than 160 passages. We subsequently used them to determine arachnoidal barrier properties and paracellular transport. Permeabilities of urea, mannitol, and inulin were 2.9+1.1X10-6, 0.8+.18x10-6, and 1.0+.29x10-6 cm/s respectively. Size differential permeability testing with dextran clarified the arachnoidal blood-CSF-barrier limit and established a rate of intracellular transport to be two orders of magnitude slower than paracellular transport in a polyester membrane diffusion chamber. The theoretical pore size for paracellular space was 11Å and the occupancy to length ratios were 0.8 and 0.72 cm-1 for urea and mannitol respectively. The monolayer permeability was not significantly different from an apical to basal direction or vice versa. Gap junction may have a role in barrier formation. Although up-regulation of claudin by dexamethasone did not significantly alter paracellular transport, increasing intracellular cAMP decreased mannitol permeability. Calcium modulated paracellular transport, but only selectively with the ion chelator, EDTA, and with disruption of intracellular stores. Without the neurovascular unit of the blood-brain-barrier, the blood-CSF-barrier at the arachnoid tissue is anatomically and physiologically different from the vascular based blood-brain-barrier. These studies provide a three dimensional architecture, a stable cellular substrate, and baseline blood-CSF-barrier properties for the establishment of a viable bioartificial arachnoid shunt.
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    Experimental studies of flow through deformable silicone and tissue engineered valves.
    (2009-12) Amatya, Devesh M.
    Annually, approximately 250,000 repair/replacement heart valve surgeries are performed world-wide. Currently the two options available for valve replacement are mechanical or bioprosthetic valves. Thrombosis (blood clots) and embolic events (movement of the clots through the blood vessels) have been linked with the mechanical valves, so that life-long anticoagulant therapy is required. Deterioration of the structural integrity, in part due to calcification, has been linked with bioprosthetic valves. The current paradigm is to replace a living, but incompetent valve with a non-living valve, be it mechanical or biological prosthetic. A living prosthetic valve grown with patient donor-based tissue engineering paradigm may be a possible solution. The primary objective of this study was to characterize the in vitro performance of the tissue engineered valve equivalents in a cardiovascular pulse duplicator and assess their potential for clinical use as valve replacement prostheses. A second objective was to conduct experiments under different flow conditions with synthetic silicone polymer valves of various geometries and materials similar in mechanical properties to those of the valve equivalents that are more amenable to experimental measurements of velocity and structural deformation using two-dimensional particle image velocimetry of high spatial resolution, three-dimensional velocimetry of volumetric measurements, and hot film anemometry of high temporal resolution. These measurements are needed to validate computational codes incorporating fluid-structure interaction and may be applied towards tissue engineered heart valve design and optimization. All of the silicone materials tested showed a neo-Hookean material response at engineering strains less than 0.5. The silicone linear elastic modulus was similar in order to the values measured in native aortic valve leaflets. The diaphragm valves with an orifice deformed to a concave shape with respect to the upstream flow for both steady and pulsatile flow conditions, along with orifice expansion at increasing flow rates. The orifice expansion (up to 75% increase in area) led to reduced pressure drops as compared with non-expanding or rigid diaphragm valves. A jet with significant inward radial velocity was present immediately downstream of the deformed diaphragm valves for both steady and pulsatile flows. This inward flow was associated with vena contracta. For low Reynolds number, laminar steady upstream flow conditions, the diaphragm valve supported the formation of relatively large scale vortices with passage frequency of St = 0.34. For pulsating flow, a leading vortex ring followed by a trailing jet was present during forward flow acceleration. Phase-averaged velocity measurements show lower fluctuations during the acceleration phase than during the deceleration phase of the flow. The deformation of the transparent bileaflet silicone valve in the pulsating flow showed leaflets deforming in similar concave state with respect to the upstream forward flow of systole and towards the lower pressures during diastole. The bileaflet silicone valve showed asymmetry in root deformation and a slot-like elliptical jet flow profile through the leaflets unlike the circular profile of the diaphragm valve. Downstream flow stagnation and recirculation were present during systole and areas of recirculation were present both upstream and downstream. These flow features were less organized for the latter during diastole. The tissue engineered valve equivalents harvested after development in the bioreactor and placed within a rigid housing were able to withstand pressures of ~50 mmHg, pressure drops of ~40 mmHg, and flow rates of ~25 L/min throughout the loading of the right ventricular cardiac cycle. The temporal pressures and flow signatures replicated right physiological conditions. The flow downstream indicated an elliptical jet during systole similar to the bileaflet silicone valve. The locations of tissue engineered valve equivalent failures were at the leaflet commissure and Dacron cuff-valve root interface.
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    Fundamentals of a systems biology approach to In Vitro tissue growth
    (2013-05) Beck, Richard Joseph
    Tissue engineering needs a paradigm shift in order to generate clinically useful products. The field has yet to regularly produce implantable tissue-engineered products. The conventional manner in which input stimuli are applied without consideration of current cellular activity level is certainly suboptimal. The objective of this line of research is to produce a method for rationally choosing input stimuli that drive the cells toward optimal tissue growth. Transient phosphorylation of signaling proteins after a perturbation in stimuli contains biological information concerning downstream tissue growth. The overall project aims to build a statistical model predictive of tissue growth via information of the upstream phosphoproteome minutes after a change in stimuli. The validity of such a statistical model can be tested based on its utility to direct tissue growth: stimuli will be chosen on the basis of which corresponding phosphoproteome profile(s) is predicted to yield the best downstream tissue growth; this can be directly compared to conventional tissue engineering methods. This doctoral project focused on obtaining sample types and tailoring methods appropriate for a systems biology and statistical approach, especially in regard to the label-free quantification of phosphopeptide enrichments. Neonatal human dermal fibroblasts (nhDF) were expanded to near confluence, at which point basal medium for tissue production was applied. After two days, nhDF were perturbed with basal medium supplemented with 1 or 10 ng/mL TGF-β1. Cells were harvested at 10, 20, or 30 minutes for intracellular proteins. Resultant protein lysates were digested to peptides via trypsin and enriched for phosphopeptides via Iron Immobilized Metal Affinity Chromatography (IMAC). Phosphopeptide enrichments were analyzed by tandem mass spectrometry. A total of 1689 peptides were both identified with phosphorylation and quantified using distinct algorithms. Under strict statistical tests, 22 of these peptides were found to differ between treatments/time. Corresponding downstream collagen deposition was also found to differ between treatments. These results indicate that the type of quantitative data needed for the overall project can be acquired. The methods developed can be used in finding a statistical relationship between tissue growth and upstream phosphoproteome profiles.
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    High-Throughput Method for Microfluidic Placement of Cells in Micropatterned Tissues
    (2013-04-20) Sevcik, Emily
    Recent studies have shown that cell shape and tissue structure can dictate functional behavior in engineered tissues (1). One method for controlling tissue structure in vitro is microcontact printing, where extracellular matrix proteins are patterned on a substrate to construct arrays of single cells or multicellular tissues. This technique is used to create tissues that mimic in vivo architecture which can be used to study tissue properties and disease mechanisms (2). Traditional seeding of cells on the substrate is imprecise, but our group has developed a microfluidic device for spatial control of cell seeding, which creates more replicable high-fidelity tissues. However, the current method is low-throughput and labor intensive. Here, we present a scalable system of multiple microfluidic devices for parallel cell seeding. This high-throughput, precise approach reduces experimental variation, making biochemical assays on single cell arrays possible in future work. We will use this system to create large arrays of single cells of various shapes for phenotypic studies and to create arrays of tissues with varying cellular organization. 1)Alford, P. W., Nesmith, A. P., Seywerd, J. N., Grosberg, A., & Parker, K. K. (2011). Vascular smooth muscle contractility depends on cell shape. Integrative Biology, 3(11), 1063-1070. 2)Ruiz, S. A., & Chen, C. S. (2007). Microcontact printing: A tool to pattern. Soft Matter, 3(2), 168-177.
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    Modulation of mesenchymal stem cell invasion into decellularized engineered tissues
    (2013-06) Weidenhamer, Nathan Kyle
    Mechanical forces play an important role in shaping the organization and alignment of the extracellular matrix (ECM) in developing and mature tissues, where the organization gives the tissue its unique functional properties. This dissertation will discuss research that is fundamental to the understanding of the interaction of cells with the extracellular matrix (ECM), a concept that is vital to creating functional engineered tissues, such as arteries and heart valves. The work presented is divided into two main focus areas; the influence of mechanical forces on the development of cell and ECM alignment and the influence of ECM alignment and culture conditions on cell invasion. The first focus area developed a novel method to create and stretch tubular cell sheets by seeding neonatal dermal fibroblasts (nHDF) onto a rotating silicone tube. Fibroblasts proliferated to create a confluent monolayer around the tube and a collagenous, isotropic tubular tissue over 4 weeks of static culture. These silicone tubes with overlying tubular tissue constructs were mounted into a cyclic distension bioreactor and subjected to cyclic circumferential stretch at 5% strain, 0.5 Hz for 3 weeks. Tissue subjected to cyclic stretch compacted axially over the silicone tube in comparison to static controls, leading to a circumferentially-aligned tissue with higher membrane stiffness and maximum tension. In a subsequent study, the tissue constructs were constrained against axial compaction during cyclic stretching. The resulting alignment of fibroblasts and collagen was perpendicular (axial) to the stretch direction (circumferential). When the cells were devitalized with sodium azide before stretching, similarly constrained tissue did not develop strong axial alignment. This work suggests that both mechanical stretching and mechanical constraints are important in determining tissue organization, and that this organization is dependent on an intact cytoskeleton. The second focus area explored the influence of ECM alignment and culture conditions on human mesenchymal stem cell (hMSC) invasion into decellularized tissues. These studies showed that the soluble factors insulin and ascorbic acid promote the invasion of hMSCs into decellularized engineered tissues. We speculate that this is due to an increase in motility and proliferation of hMSCs when exposed to these factors. Furthermore, hMSC invasion into aligned and non-aligned matrices was not different, although there was a difference in cell orientation between aligned and non-aligned matrices. Finally, we show that, regardless of culture conditions or ECM alignment, hMSCs appear to be differentiating toward a myofibroblast-like phenotype.
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    Tissue-engineered islet graft design and assessment
    (2012-10) Suszynski, Thomas Mark
    Diabetes mellitus is a metabolic disease characterized by an inability to maintain normoglycemia and is usually associated with an insufficient beta-cell mass. Islet transplantation (IT, a beta-cell replacement therapy) has exhibited promise in reversing the diabetic state, yet it remains an experimental therapy only among a small subset of patients and widespread availability remains elusive. A main reason for its limited use is the large islet dose required to produce insulin independence, often requiring tissue from multiple donor pancreata. Abundant evidence exists suggesting that acute and chronic islet loss and dysfunction occur in the post-IT period. The minimum islet dose could be reduced if the quality (potency, viability) of the islet product is improved. Since islets do not possess the cellular machinery to produce energy effectively in the absence of oxygen, strategies that improve oxygenation during the ischemic period (donor cardiac death to complete revascularization) are important to develop. Due to the many steps involved in the process (pancreas preservation, and islet isolation, purification, culture, assessment, transplant and engraftment), the quality of an islet preparation can change. Thus, it is critical to develop improved islet quality assessment tools that enable accurate and quantitative characterization of islet viability and potency, which can be performed prospectively, and which do not require much tissue for assessment or which rely on non-invasive methods. Furthermore, intrahepatic IT may not be the optimal approach, and tissue-engineered strategies for extrahepatic IT may be better. The work presented herein focuses on improving islet quality assessment, examining the oxygenation status of the intraportally transplanted islet, and developing a tissue-engineered strategy for the design and non-invasive assessment of islet graft viability - all with the hope that someday IT could be used to successfully treat many more diabetics.
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    Toward the Construction of a Vascularized, Hydrogel-Based Lymph Node Model for In Vitro and In Vivo Therapeutic Applications
    (2021-04) Harff, Caleb
    Clinical trials for drugs and vaccines often suffer from the use of culture or animal models that do not accurately recreate the microenvironment of human tissues, including the lymph nodes. Furthermore, insufficient immune function resulting from genetic deficits, cancer or auto-immune disease, and the loss of lymph nodes due to surgical resection or radiation therapy may benefit from the implantation of therapeutic immune cells. These needs could be served through the development of a biocompatible, vascularized, 3D hydrogel scaffold that supports leukocytes and recreates lymph node function by providing a biomimetic microenvironment. While other lymph node models exist, their complexity and function are limited in that they incorporate few of the bioactive molecules from the lymph node microenvironment and do not contain vasculature. Preliminary viability studies were performed to determine the optimal choice of hydrogel type and density for the 3D culture of peripheral blood mononuclear cells (PBMCs). Fibrin hydrogels were found to better maintain PBMC viability over three days compared to gelatin methacrylate hydrogels. The inclusion of ECM from decellularized porcine lymph nodes into fibrin hydrogels was met with technical challenges with regard to solubilization and peripheral blood cell (PBC) viability assessment, indicating that additional steps or different approaches were required. However, successful decellularization was demonstrated by the sufficient removal of DNA content as determined by DNA quantification and histological staining. Cytokine and growth factor analysis showed significant depletion of many of the analytes but retention of IFNα2, IL-3, IL-9, IL-13, IL-17A, and VEGF. To construct a perfusable model, pin molds were used to create channels that would undergo eventual endothelialization and angiogenesis. Few of these channels maintained structural integrity. While continued experimentation is required to implement all of the features desired for the construction of a biomimetic and functional human lymph node model, these results indicate that it is feasible to improve upon previous designs by incorporating decellularized ECM and introducing vascularization in fibrin hydrogels.