Browsing by Subject "tissue engineering"

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Computational and Experimental Comparison on the Effects of Flow-Induced Compression on the Permeability of Collagen Gels
(2020-08) Vidmar, Chris
Collagen is a fibrous material and is ubiquitous throughout the human body. It is a biphasic material, consisting of a solid fibrous matrix and interstitial fluid. Collagen is one of the primary components within the extracellular matrix, which plays a vital role in the physiological, mechanical, and transport functions of various systems and processes of the body. Understanding the mechanical and transport properties of collagen can help us understand the roles and processes of the extracellular matrix. Additionally, understanding these properties may lead to more rational design choices in tissue engineering, where the permeability of the biomaterial in tissue engineering is critical for the transport of nutrients. The underlying goal of this study is to experimentally determine and to develop a finite element model showing the effect of concentration and compression of collagen gels on permeability. In this study, two methods to determine the permeability of collagen gels was developed. With both methods, the interstitial fluid of collagen gels was expelled under a pressure load, resulting in compressed collagen gels that were denser than the starting gels. The permeability of collagen gels was determined using Darcy’s Law. In the vertical apparatus, a changing height of fluid pushed water through and compressed a collagen sample. In the horizontal apparatus, a syringe pump delivered water through three collagen concentrations (1.98 mg/mL, 3.5 mg/mL, 5 mg/mL) at a constant volumetric flow rate of 0.85 ml/min. Instantaneous permeability values were obtained at various points of compression and fitted to the α and M values of the strain-dependent Holmes-Mow permeability model where α and M are defined as intrinsic permeability parameters. A finite element model was developed to model the biphasic compression of the collagen gels using FEBio. A neo-Hookean material was used to model the solid matrix and Young’s modulus was changed to match the degree of compression. The vertical apparatus found a higher permeability compared to the horizontal apparatus. The vertical apparatus showed a permeability of 2.37 x 10-11 ± 2.4 x 10-11 m2. The initial permeability doubled as the collagen went from a starting concentration of 5 mg/mL to a starting concentration of 1.98 mg/mL. Each concentration compressed to a final concentration of about 12 mg/mL, resulting in no dependence on starting concentration for the permeability of compressed samples. The M values of the Holmes-Mow model increased from 2.4 to 5 with increasing concentration, while the α value decreased from 1.3 to 0.5 with increasing concentration. The Young’s modulus found by the finite element model increased from 200 to 3700 Pa with increasing initial collagen concentration. The Young’s modulus determined in the current study was similar to the short-time modulus of other published works.
Design of a Fibrin-Based Vascular Graft Seeded with Blood Outgrowth Endothelial Cells
(2010-11) Ahmann, Katherine
A clinical need for small-diameter vascular grafts exists, particularly in cases where coronary artery bypass surgery is the best treatment option, but the patient lacks a viable autologous graft due to repeat procedures or diseased vasculature. The comprehensive goal of this work was the in vitro fabrication and assessment of a completely biological, small-diameter vascular graft. Our approach uses tissue cells entrapped in a tubular fibrin gel with blood outgrowth endothelial cells (BOECs) seeded on the lumenal surface, in an attempt to recreate important properties of the medial and intimal layers of a native artery. Two major areas were examined to achieve this goal: 1) controlled fibrin degradation and its possible role in improving new matrix deposition, cellularity, and ultimately, mechanical properties and 2) seeding of BOECs on these small-diameter grafts to form a complete vessel and ensure thromboresistance. We hypothesized that controlling the rate of fibrin degradation could allow for improved matrix remodeling in fibrin-based constructs. To this end, we examined collagen and elastin deposition and cellularity in fibrin-based constructs grown in varied concentrations of the fibrinolysis inhibitor ε-aminocaproic acid (ACA). Decreasing the concentration of ACA led to increased fibrin degradation and better biochemical and mechanical properties. The byproducts of fibrin degradation, fibrin degradation products (FDPs), were shown to be physiological stimulators of collagen deposition, a fact that can be exploited to increase collagen deposition in fibrin-based vascular constructs. These fibrin-based constructs were then utilized as a substrate for seeding of BOECs, a novel endothelial cell expanded from circulating endothelial progenitor cells in peripheral blood. BOECs adhered to the bioartificial tissue and remained adherent under physiological shear stress. They also exhibited low expression of pro-inflammatory markers and reduced platelet binding compared to unseeded tissue. Exposure to shear stress decreased pro-inflammatory marker expression on TNF-α stimulated BOECs, increased endothelial nitric oxide synthase expression and nitric oxide production, and decreased platelet adhesion during whole blood flow. These outcomes indicate that BOECs are shear stress responsive and are functionally similar to mature endothelial cells in their response to shear stress and their ability to limit platelet binding to bioartificial vascular grafts. Together, these lines of research allow for the formation of a functional, small-diameter vascular graft, while elucidating key aspects of the remodeling process and BOEC phenotype.
Design of an Oxygen-Delivering Porous Chitosan Scaffold Encapsulated in a Calcium Alginate Hydrogel for Treatment of Hypoxic Wounds
(2018-07) Kollaja, Benjamin
Several oxygen-delivering wound dressings have been proposed in recent literature with the aim of improving healing outcomes of chronic wounds. Oxygen generation has been achieved in numerous ways, including the incorporation of peroxide salts and perfluorocarbons (PFCs) to provide oxygen-loading capacity. Here, we have designed a multilayer wound dressing composed of chitosan encapsulated in calcium alginate, incorporating calcium peroxide and PFCs to act as oxygen-generators and oxygen shuttles, respectively. We hypothesize that the combination of oxygen-generating CaO2 and oxygen-carrying PFCs will act synergistically to improve sustained oxygen delivery to the underlying wound and thus improve healing outcomes. Oxygen generation is quantified by fluorescence microscopy using aqueous tris(bipyridine)ruthenium (II) chloride in a closed flow system.
Development of Bi-layer Engineered Cardiac Tissues Containing Cardiomyocytes and Microvessels
(2016-08) Schaefer, Jeremy
The prevalence of coronary heart disease and myocardial infarction coupled with the limited availability of donor hearts to replace damaged tissue leaves many patients with insufficient treatment. Cardiac tissue engineering has emerged to attempt to fill this need, but engineered cardiac tissues are limited in size by a lack of vascularization. In this work, we aim to create a vascularized cardiac tissue through the combination of an established microvessel construct with a cardiomyocyte construct, and we investigate its use in a nude rat model of myocardial infarction. The results detailed in this dissertation indicate that the bi-layer construct not only improves contractile function of the cardiomyocytes, but also that the human microvessels can sprout into the cardiomyocyte layer and become perfused when implanted in vivo.
Development of Pre-Vascularized Tissues Containing Aligned and Perfusable Microvessels
(2016-05) Riemenschneider, Sonja
The single greatest restraint in tissue engineering is the inability to create and perfuse functional microvasculature in dense engineered tissues of physiological stiffness. Without active delivery of nutrients and oxygen, tissue size is diffusion-limited to thicknesses of around 400 µm, or much less for highly metabolic tissues. Thus, the creation of pre-vascularized tissues that have a high density of organized microvessels that could be perfused is a major goal of tissue engineering. The present work makes significant advances toward this goal. Tissue patches containing a high density of human microvessels that were either randomly oriented or aligned were placed acutely on rat hearts post-infarction and in both cases, inosculation occurred and perfusion of the transplanted human microvessels was maintained, proving the in vivo vascularization potential of these engineered tissues. In vitro, a high-throughput assay was developed to investigate optimal conditions for angiogenic sprouting, vasculogenic microvascular network formation, and inosculation of the sprouts and microvessels in 3D fibrin gels. Samples loaded with vascular endothelial growth factor and fibroblast growth factor exhibited enhanced angiogenic sprouting, and a hybrid medium culture regimen resulted in enhanced sprouting, well-developed microvascular networks, and inosculation of the microvessels and sprouts. These results showed potential for the in vitro perfusion of larger-scale microvascular tissues. An engineering strategy was developed to perfuse endothelialized microchannels that could form sprouts into fibrin gels containing a microvascular network. An in vitro perfusion bioreactor was designed and tested that enabled these microvascular tissues to be cultured, compacted, and aligned to form a dense network of microvessels that also contained perfusable microchannels with sprouts. Different microchannel seeding regimens and perfusion regimens were applied and it was determined which conditions ultimately led to microchannel endothelialization, sprouting, perfusion, and maintenance during gel compaction. While inosculation and perfusion of the microvessels has yet to be achieved, this work presents the building blocks for a potential strategy that could ultimately enable the perfusion of a dense, aligned microvascular network through anastomoses of sprouts and microvessels. Achievement of this goal would unlock a number of tissue engineering opportunities in the development of large engineered tissues for regenerative therapies.
Engineering functional muscle tissues and modeling muscular diseases using myogenic cells differentiated from human pluripotent stem cells or human fibroblasts
(2019-09) Xu, Bin
The advances in efficient generation of myogenic cells using human pluripotent stem cells (hPSCs) offer unlimited opportunities for translational applications, such as the study of muscle development and diseases, drug screening, and regenerative medicine. Functional muscle constructs tissue-engineered from these myogenic cells prove excellent tools for those applications. However, these myogenic cells are developmentally immature, and the protocol to derive them is time-consuming. In this thesis, we aim to model muscle diseases and to improve the maturity of muscles using hPSC-derived myogenic cells. We also develop transdifferentiation as an alternative method to obtain myogenic cells more quickly. We modeled Duchenne Muscular Dystrophy (DMD), a genetic disorder leading to muscle wasting and death. We fabricated nanogrooved substrate immobilized with muscle basement membrane mimicking materials and discovered that non-diseased and DMD muscles derived from hPSCs exhibit substantial differences in cell alignments on nanogrooved substrates when these substrates are functionalized with laminin. To improve the maturity of the muscles, we generated hPSC-derived muscle constructs in customized devices and discovered that addition of the endothelial growth medium-2 supplements in the first two weeks of differentiation leads to substantial increases in contractile forces. These constructs show wider myotubes and higher gene expression levels for skeletal muscle-specific myosin heavy chain isoforms, suggesting that a more mature differentiation stage of the cells. Those tissue-engineered constructs were also used to validate the screening of small molecules for enhancing the function and maturation during myogenic differentiation. We found a significant increase in contractile force generation when treated with a cocktail of four small molecules (SB431542, DAPT, Dexamethasone, and Forskolin). To explore an alternative approach to generating functional human muscles more quickly, we chose to transdifferentiate normal human dermal fibroblasts (NHDF) transduced with inducible MyoD. We demonstrated that myogenic transdifferentiation of NHDF could be enhanced by using small molecules CHIR99021 and DAPT when coupled with MyoD induction. We further proved that muscle constructs engineered from transdifferentiated NHDF can generate contractile forces in response to electrical stimuli after 2-week 3D culture. Temporal expression of MyoD in the first week boosts twitch and tetanic forces significantly, and small molecule (CHIR and DAPT) treatment could further improve force generation.
Feasibility of Pulmonary Airway Tissue Engineering and Repair Using a Cell Spraying Device and Decellularized Porcine Trachea
(2019-08) Rajendran, Vijay
Methods for tracheal repair and regeneration are necessary due to the limitations of tracheal resection and reconstruction for certain disorders such as tracheal stenosis, tracheomalacia, and tracheal tumors. Additionally, pulmonary injuries such as airway burns do not have effective treatment options aside from supportive care. The feasibility of a cell spraying device is investigated here as a system for applying human bronchial epithelial cells (HBECs) to decellularized porcine trachea matrices for creation of engineered grafts or as a minimally invasive method for delivering cells for wound healing. HBECs show viability greater than 90% after spraying onto cell culture media or tissue culture plastic. Similarly, one day after spraying onto decellularized trachea, viabilities are seen to be around 90%. Around day three, viabilities were slightly decreased to around 80%. After culturing for over one week, HBECs sprayed onto decellularized trachea displayed a basal cell marker (cytokeratin-5, CK5) and a club cell marker (uteroglobin). Markers for ciliated cells and goblet cells that are crucial for tracheal epithelium could not be found, but this needs to be investigated further. To validate the mechanical performance of the decellularized trachea, compressive resistance testing was performed before and after decellularization of tracheal rings. Results were generally inconclusive with high degrees of variability. A paired sample test conducted with 4 tracheas provided the most interesting results and showed that the decellularization process produced a significantly different compressive resistance compared to the native samples. In practice though this did not seem to be noticeable as the variability found within tracheal samples masked the difference. This would suggest that the decellularization process is not detrimental to the compressive resistance of trachea rings. Based on the results reported here, using a cell spraying device for engineering tracheal grafts and airway epithelial repair seems achievable.
Monitoring and improving oxygenation of organs, cells, and tissue engineered grafts
(2015-12) Weegman, Bradley
Oxygen is vital to the survival of many living things, and evolution has provided the human body with a complex cardiovascular system to ensure that all of the cells in the body are provided with adequate oxygen. Achieving adequate oxygen delivery remains of critical importance to the clinical management of many human diseases and has been the impetus for the development of many medical procedures and technologies. Despite much advancement in the understanding about oxygen delivery in the body, the current inability to attain life-sustaining levels of tissue oxygenation remains the major limitation for the emerging fields of cell, tissue, and organ replacement. There is a large body of research focused on developing methods to improve vascularization and oxygen supply for transplanted cells, tissues and organs, and this substantial challenge will require an interdisciplinary approach utilizing both engineering principles and a broad understanding of the physical science. The islet transplantation process can be divided into three critical steps: tissue procurement and preservation; isolation, culture and shipment; and graft transplantation and monitoring. To begin, whole organ oxygen consumption rate (WOOCR) measurements are presented for the assessment of organ viability, followed by the description of new techniques for improving the efficacy of pancreas cooling during procurement, and the use of hypothermic machine perfusion (HMP) to improve pancreas preservation. These methods can be used to qualify biological tissue products and to evaluate and improve organ procurement and preservation. Next, HMP combined with silicon-rubber-membrane (SRM) culture systems are presented as techniques to improve the quality of tissues isolated from juvenile porcine pancreata, and advanced nutrient supplementation with suspension culture systems are shown to improve β-cell expansion. Finally, 19F-MRS oximetry techniques are presented for non-invasive oxygen monitoring of tissue-engineered grafts (TEGs), and these techniques are further applied to develop, implement, and validate a novel method for oxygen delivery to an implanted tissue-engineered islet grafts.
Regenerating Pulmonary Vascular Endothelium of Tissue-Engineered Lungs using Endothelial Progenitor Cells and Acellular Scaffolds
(2021-10) Akinnola, Ifeolu
Terminal lung diseases damage the organ through substantial irreversible changes to its architecture, leading to a suboptimal level of function. For this patient population, lung transplantation is the primary curative method to re-obtaining their previous quality of life. Relative to other whole organs used for transplant, the acquisition of transplantable lungs is low, resulting in a large deficit of lungs available for patients on the transplant list. Ongoing efforts towards creating functional organs from acellular scaffolds has the potential to address the deficit of lungs. The regeneration of a functional vasculature within acellular organ and tissue scaffolds is a necessity for their overall longevity and function. Current efforts in engineering transplantable lungs with long-term functionality are hindered due to vascular related complications within the newly generated organ. In over to overcome these limitations, improvements in re-endothelialization of scaffolds and acellular tissue are required. The studies described in this thesis provide valuable insight towards characterization of pulmonary endothelial progenitors and cells for re-endothelization, understanding the interaction between endothelial progenitors and vessel-specific extracellular matrices, and the potential for using iPSC-derived angiogenic hemangioblasts for developing vascular endothelium. Within both in vitro culture and acellular murine lung matrices, we have successfully characterized a highly proliferative rat pulmonary endothelial progenitor capable of re-endothelialization of the vasculature without site preference and can be used as a model for identifying progenitors in humans that are suitable for tissue engineering and clinical applications. We continued our efforts by using label-free liquid chromatography-mass spectrometry (LC/MS) analysis of decellularized porcine pulmonary vessels to compare the diversity and abundance of various extracellular matrix (ECM) proteins. The data generated from our LC/MS analysis can be used for future quantitative proteomic analyses. The decellularized ECM from both pulmonary arteries and veins were both used to evaluate their effect on endothelial progenitor maturation, and we were able to detect increase expression of endothelial genes from cells cultured in ECM hydrogels. Finally, we developed a protocol to differentiate human iPSCs into angiogenic hemangioblasts. These differentiated cultures contained sub-populations of CD31+/VEGF2+ and VE-Cadherin+/CD73+ cells. The outcomes from this study will provide guidance for future experiments and insight on characterizing both endothelial progenitors for re-endothelialization and proteomic analysis of pulmonary vasculature ECM.
Studying vascular smooth muscle cell characteristics using an alternating-substrate modulus system
(2015-05-14) Becicka, Austin
Cardiovascular diseases resulting from atherosclerosis comprise the single biggest cause of death in the developed world1. In atherosclerosis, both local vessel stiffness and extracellular organization is perturbed. Vascular smooth muscle cell (VSMC) function is perturbed in atherosclerosis, but it is not clear if this is due to the evolving changes in extracellular mechanical stimuli. Here, we will build upon previous work using microfabrication techniques to determine the influence of extracellular matrix (ECM) mechanics and ECM organization on VSMC self-‐ organization. We will use our previously developed alternating-‐substrate modulus system combined with microcontact printing (MCP) to observe and quantify cell self-‐organization when cells are guided by both the stiffness of the underlying substrate and stamped lines of fibronectin protein. This study will provide insight on VSMC behavior with competing influences on cell self-‐organization and may help provide insight to cell characteristics that may be useful in developing atherosclerosis therapies.

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