Every 34 seconds, someone in the United States suffers from a heart attack. Most heart attacks are caused by atherosclerotic build up in the coronary arteries, occluding normal blood flow. Balloon angioplasty procedures in combination with a metal stent often result in successful restoration of normal blood flow. However, bare metal stents often lead to restenosis and other complications. To compensate for this problem, industry has created drug-eluting stents to promote healing of the artery wall post stenting. These stents are continually advancing toward better drug-eluting designs and methods, resulting in a need for fast and reliable pre-clinical testing modalities. Dr. Kristen Cardinal recently developed a tissue engineered blood vessel mimic, with the goal of testing intravascular devices. However, the scaffold component of this model exhibits several physiological limitations that must be addressed to create a truly biomemtic BVM. The current model uses expanded poly(terafluorethylene) [ePTFE] or poly(lactic-go-glycolide) [PLGA] as the choice material for the scaffold. EPTFE has several advantages as it is a widely recognized biomaterial. However, ePTFE is very expensive and lacks native mechanical properties. PLGA is another polymer that is created in-house to produce a uniquely tailored scaffold for use in the BVM; resulting in a cheaper alternative scaffold material. However, PLGA again lacks the necessary native mechanical properties to properly mimic an in-vivo artery. The creation of a biological scaffold will provide a unique biomimetic material to most accurately recapitulate the artery in-vitro. Decellularization is the process of removing all cellular components from a tissue, leaving an acellular structure of extracellular matrix. Understanding the clinical problem and the potential of the BVM, the aim of this thesis is to develop the decellularization process for the creation of a biologic scaffold as a replacement to the non-physiologic polymer scaffolds for the BVM. The first phase of this thesis was to develop and optimize an acceptable protocol for the decellularization of porcine arteries. The use of a 0.075% sodium dodecyl sulfate detergent was sufficient for complete removal of all vascular cell types, without significant degradation to the scaffold wall. In the second phase of this thesis, the decellularized scaffolds were mechanically tested to ensure retention of their native properties. The longitudinal and radial properties of the scaffold were found to be similar to the native artery, indicating the decellularized scaffold improves several physiologically aspects when compared to a polymer scaffold. These mechanical attributes improve the testing environment when evaluating sent deployment or new balloon angioplasty devices; as the decellularized scaffold has an phsyiolgical compliance. The final phase of this thesis examined the cellular adhesion capacities of the scaffold through recellularization with human umbilical vein endothelial cells (hUVECS). Fluorescent microscopy analysis suggests uniform attachment of cells along the length of the scaffold creating a monolayer. These results indicate this new scaffold type can develop an endothelium to complete the ideal, most physiologically relevant BVM system. Further optimization of the decellularization procedures could enhance the ability of the scaffold to be cultured for long-term interaction with intravascular devices.

PREPARATION AND CHARACTERIZATION OF ELECTROSPUN POLY(D,L-LACTIDE-CO-GLYCOLIDE) SCAFFFOLDS FOR VASCULAR TISSUE ENGINEERING AND THE ADVANCEMENT OF AN IN VITRO BLOOD VESSEL MIMIC Tiffany Richelle Peña Currently, an estimated 1 in every 3 adult Americans are affected by one or more cardiovascular complications. The most common complication is coronary artery disease, specifically atherosclerosis. Outcomes of balloon angioplasty treatments have been significantly improved with the addition of drug eluting stents to the process. Although both bare metal and drug eluting stents have greatly increased the effectiveness of angioplasty and decreased the occurrence of restenosis, several complications still exist. For this reason, the stent industry is continually advancing toward better stent and drug-eluting designs, deployment methods, and adjuvant drug therapies, necessitating fast, reliable pre-clinical test methods. Recently, advancements in tissue engineering have led to the development of an in vitro blood vessel mimic (BVM) and the feasibility of evaluating cellular response to intravascular device implantation has been demonstrated. There are several physiological and scalability limitations of the current BVM model that must be addressed before effective use of the model can be initiated. The limiting aspect addressed in this thesis is the use of expanded poly(tetrafluorethylene) [ePTFE] scaffolding for the development of the BVM. There are several disadvantages and limitations to ePTFE including high cost and non-native mechanical properties. The ability to produce and tailor scaffolds in-house would greatly impact the scalability, cost effectiveness, and control over scaffold properties for BVM optimization. Also, in-house fabrication will open up further avenues of research into optimum scaffold design for better cellular responses when cultured in vitro. Electrospinning is a relatively simple and economical method of creating tissue engineering constructs with micro-architecture similar to the native extracellular matrix. Based on the clinical problem and the potential for the BVM, the aim of this thesis is to employ electrospinning for the development of poly(D,L-lactide-co-glycolide) [PLGA] vascular scaffolds as a replacement to ePTFE for the BVM. After primary literature review, PLGA was determined an advantageous polymer for tissue engineering vascular scaffolds and electrospinning based on evidence of adequate endothelial cell attachment, mechanical properties similar to the native vessels, controlled degradation, and good biocompatibility. The first phase of this thesis was to develop an acceptable protocol for the fabrication of electrospun PLGA scaffolds by varying solution concentration, flow rate and applied voltage. Electrospun solutions of 15 wt% PLGA in CHCl3 resulted in continuous un-beaded fibers of 5-6 microns and tensile properties (3-5 MPa) similar to the native vessel. The optimum protocol for electrospinning 15 wt% PLGA incorporated a flow rate of 5.5 ml/hr and an applied voltage of 12,000 V. In the second phase of this thesis, final protocol PLGA scaffolds were cultured in vitro with human umbilical vein endothelial cells (HUVECs) up to 6 days. Fluorescent microscopy and SEM analysis suggest the porous nature of the scaffolds was conducive to sub-luminal cellular penetration. Although results were not optimal for developing an endothelium for the ideal BVM design, the potential of using electrospinning for in-house production of scaffolds for tissue engineering was established. Further optimization of the electrospinning protocol to develop nano-sized structural features could enhance the ability to form an intimal lining of endothelial cells for the next generation BVM design.

Nanotechnology and its potential for biomedical applications has become an area of increasing interest over the last few decades. Specifically, ultrasmall nanoparticles, ranging in size from 5 to 50 nm, are highly sought after for their physical and chemical properties and their ability to be easily transmitted though the bloodstream. By adjusting the material properties, size, surface potential, morphology, surface modifications, and more, of nanoparticles, it is possible to tailor them to a specific use in biomedical areas such as drug and gene delivery, biodetection of pathogens or proteins, and tissue engineering. The aim of this study was to fabricate ultrasmall poly-(lactic-co-glycolic acid) nanoparticles (PLGA NPs) using a quick and easy nanoprecipitation method1, with some modifications, for general use in various biomedical areas. Nanoprecipitation of two solutions – PLGA dissolved in acetonitrile and aqueous poly(vinyl alcohol) (PVA) – at varying concentrations produced ultrasmall nanoparticles that range in size, on average, from 10 to 30 nm. By the data collected from this study, a selection method can be used to choose a desired PLGA nanoparticle size given a potential biomedical application. The desired nanoparticle can be fabricated using specific concentrations of the two nanoprecipitation solutions. Size of the ultrasmall PLGA NPs was characterized by dynamic light scattering (DLS) and confirmed by transmission electron microscopy (TEM). Spherical morphology of the PLGA NPs was also proved by TEM. By generalizing the ultrasmall PLGA NP fabrication process, the idea is that these NPs will be able to be used in various biomedical applications depending on the goal of the furthered study. As an example of potential application, ~15 to 20 nm PLGA NPs were consistently fabricated for use as virus-like particle (VLP) scaffolds. Following formation, PLGA NPs were introduced to modified human papillomavirus (HPV) protein during protein refolding and assembly into virus-like particles (VLPs) via buffer exchange. The size of the VLPs was monitored with and without PLGA nanoparticles present in solution during the refolding process and TEM images were collected to confirm encapsulation. / Master of Science / Nanotechnology, the manipulation of materials on an atomic or molecular scale, and its potential for biomedical applications has become an area of increasing interest over the last few decades. Nanoparticles, spherical or non-spherical entities of sizes approximately one-billionth of a meter, have been used to solve a wide variety of biomedical problems. For reference, a human hair is about 80,000 to 100,000 nm in size and the nanoscale typically ranges in size from 1 to 1000 nm. This size range is not visible to the naked eye, so methods of analysis via scientific equipment becomes paramount. Specifically, this study aims to fabricate ultrasmall nanoparticles, ranging in size from 5 to 50 nm, which are highly sought after for their physical and chemical properties and their ability to easily travel though the bloodstream. By adjusting the material properties, size, shape, surface charge, surface modifications, and more, of nanoparticles, it is possible to tailor them to a specific use in biomedical areas such as drug delivery, detection of viruses, and tissue engineering. The specific aim of this study was to fabricate ultrasmall poly-(lactic-co-glycolic acid) nanoparticles (PLGA NPs), a type of polymer, using a quick and easy nanoprecipitation method1, with some modifications. Nanoprecipitation occurs by combining two liquid solutions – PLGA and aqueous poly(vinyl alcohol) (PVA) – which interact chemically to form a solid component – a polymer nanoparticle. These two solutions, at varying concentrations, produced ultrasmall nanoparticles that range in size, on average, from 10 to 30 nm. Data collected from this study can be used to select a desired nanoparticle size given a potential application. The desired nanoparticle can be fabricated using specific concentrations of the two nanoprecipitation solutions. By generalizing the ultrasmall PLGA NP fabrication process, the idea is that these NPs can be used for a variety of biomedical applications depending on the goal of the furthered study. Two PLGA NP example applications are tested for in this work – in DNA loading and in encapsulation of virus-like particles (VLPs), which are synthetically produced proteins that can be neatly folded to resemble a virus. These VLPs can be used to as an alternative to live vaccines and they can be designed to stimulate the immune system. Positive initial results from this study confirm the potential of these nanoparticles to have a wide impact on the biomedical field depending on specific tailoring to a given application.

Yes / Articular cartilage is a vascular tissue with limited repair capabilities, leaving an afflicted person in extreme pain. The tissue experiences numerous forces throughout its lifetime. This study focuses on development of a novel hydrogel composed of chitosan and β-glycerophosphate for articular cartilage repair. The aim of this study was to investigate the mechanical properties and swelling behaviour of a novel hydrogel composed of chitosan and β-glycerophosphate for cartilage repair. The mechanical properties were measured for compression forces. Mach-1 mechanical testing system was used to obtain storage and loss modulus for each hydrogel sample to achieve viscoelastic properties of fabricated hydrogels. Two swelling tests were carried out to compare water retaining capabilities of the samples. The hydrogel samples were made of five different concentrations of β-glycerophosphate cross-linked with chitosan. Each sample with different β-glycerophosphate concentration underwent sinusoidal compression forces at three different frequencies -0.1Hz, 0.316Hz and 1Hz. The result of mechanical testing was obtained as storage and loss modulus. Storage modulus represents the elastic component and loss modulus represents the viscosity of the samples. The results obtained for 1Hz were of interest because the knee experiences frequency of 1Hz during walking.

Drug discovery contains many strategies, one of which is the privileged scaffold strategy. This strategy incorporates a similar molecular framework within a collection of drug-like compounds in order to target various receptors. These scaffolds are useful to drug discovery scientists since they assist in developing libraries as well as demonstrating selectivity to a target. Oxazolidinones are 5-membered heterocyclic compound containing an oxygen, a nitrogen, and a carbonyl within the ring system. In this present study, the oxazolidinone structure was utilized as a privileged scaffold to target serotonin receptor 7 (5-HT7), mutated BRAF kinase (BRAFV6000E), Bruton’s tyrosine kinase (BTK), and Cyclin-dependent protein kinase 4 and 6 (CDK4/6). Aryl piperazines and piperidines were integrated as another privileged scaffold to support the selectivity towards 5-HT7, while aminopyrimidines were employed to increase binding against the kinases. The 5-HT7 oxazolidinone series was successfully synthesized and analyzed against 5-HT7; however, the three kinase oxazolidinone series were not successfully synthesized.Candidemia is the most common bloodstream infection in the U.S. and is associated with high patient mortality rates. Due to prolonged and/or repeated clinical use of current antifungal agents, drug-resistant fungi have become an emerging problem. There is a need for new antifungals to assist in overcoming drug resistant fungi. In the second project outlined in this work, a series of ketoconazole analogs were designed and successfully synthesized. The ketoconazole analogs exhibited antifungal activity; however, no clear trends were observed in this series. Overall, the series exhibited less CYP3A4 inhibition than the parent compound, ketoconazole. / Pharmaceutical Sciences

No description available.

The iliotibial band (ITB) is a suitable scaffold for anterior cruciate ligament (ACL) reconstruction, providing a sufficient mechanical resistance to loading. Hence, ITB-derived fibroblasts attract interest for ligament tissue engineering but have so far not been characterized. This present study aimed at characterizing ITB fibroblasts before, during, and after emigration from cadaveric ITB explants to decipher the emigration behavior and to utilize their migratory capacity for seeding biomaterials. ITB and, for comparison, ACL tissues were assessed for the content of alpha smooth muscle actin (αSMA) expressing fibroblasts and degeneration. The cell survival and αSMA expression were monitored in explants used for cell isolation, monolayer, self-assembled ITB spheroids, and spheroids seeded in polyglycolic acid (PGA) scaffolds. The protein expression profile of targets typically expressed by ligamentocytes (collagen types I–III, elastin, lubricin, decorin, aggrecan, fibronectin, tenascin C, CD44, β1-integrins, vimentin, F-actin, αSMA, and vascular endothelial growth factor A [VEGFA]) was compared between ITB and ACL fibroblasts. A donor- and age-dependent differing percentage of αSMA positive cells could be detected, which was similar in ITB and ACL tissues despite the grade of degeneration being significantly higher in the ACL due to harvesting them from OA knees. ITB fibroblasts survived for several months in an explant culture, continuously forming monolayers with VEGFA and an increased αSMA expression. They shared their expression profile with ACL fibroblasts. αSMA decreased during the monolayer to spheroid/scaffold transition. Using self-assembled spheroids, the migratory capacity of reversible myofibroblastic ITB cells can be utilized for colonizing biomaterials for ACL tissue engineering and to support ligament healing.

Bakgrund: Prevalensen av ledbroskskador ökar idag globalt och det finns ingen fullt kurativ kirurgisk behandlingsmetod för att återställa ledens normala funktion vid olika ledbroskskador. Detta tillsammans med att ledbrosk har en låg inneboende läkningsförmåga medför att nya behandlingstekniker behöver utvecklas, en lovande sådan är matrix-assisterad autolog kondrocytimplantation (MAKI), där tredimensionella polymermatriser, ”scaffolds”, används för att stimulera kondrocytintegration och kondrogenes. En utmaning är att framställa scaffolds med optimal komposition för detta. Polymeren chitosan har visats sig kunna ha de efterfrågade egenskaperna. Syfte: Syftet med studien var att analysera scaffoldstruktur och integration av kondrocyter i chitosan-baserade 3D-scaffolds med olika andel chitosan, cellulosa och iso-vanillin. Metoder: Kondrocyter respektive broskfragment odlades under olika tidsintervall i scaffolds med olika komposition. Snitt från odlingarna färgades in med hematoxylin-eosin, DAPI och falloidin för att kunna undersöka scaffoldstrukturen respektive identifiera förekomst av celler i scaffoldsen. Snitten analyserades i ljus- och fluorescensmikroskop och representativa bilder togs. Resultat: Scaffoldstrukturen skiljde sig mellan alla scaffolds, där låg molekylvikt av chitosan gav en mer kompakt och sammanhängande struktur än chitosan av hög molekylvikt. Celler kunde inte identifieras med DAPI i någon av scaffoldsen odlade med kondrocyter eller broskfragment. Celler sågs med falloidin i scaffolds odlade med kondrocyter under en kortare tidsperiod. Slutsats: Resultaten kan inte bekräfta att chitosan-baserade scaffolds kan främja kondrocytintegration. Scaffoldsen som användes i denna studie var möjligtvis för porösa för att bidra till tillräcklig celladhesion. Skillnad på scaffoldstruktur kunde ses men ingen slutsats kring hur struktur påverkar kondrocytintegration kunde dras.

No description available.

In search of better scaffolding materials for in vitro culture of chondrocytes, the combination of chitosan (similar to glycosoaminoglycans) and gelatin (denatured collagen) was tested due to its resemblance to cartilage extra-cellular matrix (ECM). Porous scaffolds were fabricated from chitosan gelatin blends (1:1, 2:1, and 3:1). The response of chondrocytes to them was evaluated from the amount of sulphated GAG and collagen type 2 secreted after 3 and 5 weeks. The effect due to static (transwell inserts) and dynamic (rotating bioreactor) culture methods was analyzed. Results indicate that 1:1 chitosan gelatin blends showed the best chondro-conductive potential. The rotating bioreactor facilitated better cell distribution across scaffold but did not show higher ECM secretion compared to transwell culture after 3 weeks. Gelatin leeched out by dissolution in culture media and left an open and interconnected chitosan network. Chitosan gelatin scaffolds show a potential for use in cartilage tissue engineering applications