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Ovalbumin-Based Scaffolds Reinforced with Cellulose Nanocrystals for Bone Tissue EngineeringGlaesemann, Benjamin Paul 04 August 2011 (has links)
In the field of tissue engineering, a major area of study is developing bone scaffolds that will provide support for osteoblasts. Despite many advances in recent years there is still a significant need for new bio-based 3-D porous scaffolds that possess sufficient initial mechanical properties to prevent immediate failure upon implantation. Ovalbumin (OVA), a glycoprotein from chicken egg whites, has been use to fabricate biodegradable, porous hydrogel bone scaffolds that promote osteoblast attachment and proliferation.
Although ovalbumin scaffolds encourage bioactivity and are naturally resorbed into the body after bone regeneration, they are also very fragile. Extremely stiff cellulose nanocrystals (CNCs), derived from wood pulp, can be utilized to reinforce these scaffolds while improving biocompatibility. When chemically modified to incorporate surface amine groups, cellulose nanocrystals become capable of covalently crosslinking with the OVA matrix for improved mechanical resilience.
Three concentrations (2, 5, 10 wt. %) of CNCs were incorporated and crosslinked to form nanocomposite scaffolds then were compared to pure OVA scaffolds. After fabrication, pore size morphology was compared between each CNC loading using SEM. The images revealed that the 10 wt. % CNC concentration doubled the pore compared to pure OVA scaffolds. Under high magnification, the CNCs were incorporated into the pore walls, providing a contoured surface. AFM was applied to analyze the topography of OVA with CNCs present. The surfaces laden with CNCs had a higher mean surface roughness, but was insufficient to impact cell behavior.
Compression testing was carried out on both Instron and DMA machines to demonstrate any reinforcing effect provided by the CNCs. While the compressive modulus remained constant, the elastic limit and strain increased with CNC loading, indicating a change in the resilience of the reinforced scaffolds. With a MTT Assay, it was shown that MC3T3-E1 preosteoblasts significantly increase in metabolic activity on 2 wt. % films and scaffolds, an indication of proliferation. All scaffolds had a net increase in metabolic activity suggesting overall biocompatibility for OVA scaffolds and those incorporating CNCs. Overall, the 5 wt. % scaffolds had the highest mechanical strength and had a positive cell response. / Master of Science
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Creation of Ovalbumin Based Scaffolds for Bone Tissue RegenerationFarrar, Gabrielle 02 June 2009 (has links)
Bio-based materials are a viable alternative to synthetic materials for tissue engineering. Although many bio-based materials have been used, Ovalbumin (OA) has not yet been researched to create 3D structures that promote cellular responses.
Micro-porous scaffolds are a promising construct for bone tissue regeneration; therefore OA crosslinked with three different concentrations (10%, 15% and 20%) of glutaraldehyde (GA) was used in this research. After fabrication, a porous morphology was observed using SEM. Average pore sizes were found to be comparable to scaffolds previously shown to promote cellular response. A TNBS assay determined percent crosslinking in the scaffolds, however there was no significant difference in percent crosslinking despite differing GA concentrations used. Possible explanations include an excess of GA was used.
Using DSC, a glass transition temperature (Tg) was found for control indicating the scaffolds are amorphous. Average dry and wet compressive strengths were also found. As expected, differing GA concentrations had no significant effect on Tg and average compressive strengths due to an excess used. Scaffolds were mechanically tested at 37°C with no significant difference found; therefore these scaffolds can be used in the body.
It was shown through cell studies that MC3T3-E1 pre-osteoblast cells significantly increased in number on the 10% and 15% scaffolds, therefore cell proliferation occurred. Because of a positive cellular response, 10% GA scaffolds were used for differentiation studies that showed an increase in osteocalcin at 21 days and alkaline phosphatase levels for scaffolds cultured for 14 days. Overall OA scaffolds have shown to be a promising 3D construct for bone tissue regeneration. / Master of Science
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Sintering Additives For Nanocrystalline Titania And Processing Of Porous Bone Tissue Engineering ScaffoldsMenon, Arun 01 January 2009 (has links)
Titania (Titanium dioxide, TiO2) has been researched as a promising biomaterial due to its excellent biocompatibility. However, the main limitation of titania is its poor mechanical properties which limit its use in many load-bearing applications. In this thesis report, the properties of titania were improved by doping with small quantities of MgO, ZnO and SiO2 as sintering additives. Nanocrystalline powder was selected, as it possesses outstanding properties over conventional coarse-grained powders due to reduced grain size. Nanocrystalline anatase powder of size 5-15 nm was synthesized via a simple sol-gel technique. Small quantities of dopants were introduced into pure titania powder, through homogeneous mixing. The doped powder compositions were compacted uniaxially and sintered at 1300°C and 1500°C, separately, in air. The effects of sintering cycle and temperature on the microstructure, densification and mechanical properties of the sintered structures were studied. Mg doped structures recorded maximum sintered density of 3.87 g.cm-3. Phase analysis was carried out using powder XRD technique using Cu K[alpha] radiation. Microstructural analysis was performed using Scanning electron microscopy. The mechanical properties were assessed by evaluating hardness and biaxial flexural strength (ASTM F-394) of the structures. Results showed 12% increase in hardness and 18% increase in biaxial flexural strength in structures doped with ZnO and SiO2, respectively. Further, simulated body fluid maintained at 36.5°C was used to study the bioactivity and degradation behavior of the structures. The second part of the work aimed in the processing of porous titania scaffolds using polyethylene glycol as the pore-former. The green structures were sintered at 1400°C and 1500°C, separately in air and their properties have been studied. Microstructural analysis was carried out using Scanning electron microscope (SEM). Porosity was evaluated using the immersion technique. Vickers hardness and biaxial flexural tests were used to carry out the mechanical characterization. Further, the biomechanical/biodegradation behavior of the structures was assessed in simulated body fluid (SBF). Biodegradation and change in biomechanical properties as a function of time were studied in terms of weight change, change in Vickers hardness and biaxial flexural strength. The mechanical properties of porous titania scaffolds doped separately with MgO and ZnO have also been studied to investigate the influence of these additives on the properties of porous structures. The Vickers hardness and biaxial flexural strength were seen to improve with the addition of these sintering additives.
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Effects of Therapeutic Radiation on Polymeric ScaffoldsCooke, Shelley L. 16 January 2014 (has links)
High levels of ionizing radiation are known to cause degradation and/or cross-linking in polymers. Lower levels of ionizing radiation, such as x-rays, are commonly used in the treatment of cancers. Material characterization has not been fully explored for polymeric materials exposed to therapeutic radiation levels. This study investigated the effects of therapeutic radiation on three porous scaffolds: polycaprolactone (PCL), polyurethane (PU) and gelatin.
Porous scaffolds were fabricated using solvent casting and/or salt leaching techniques. Scaffolds were placed in phosphate buffered saline (PBS) and exposed to a typical cancer radiotherapy schedule. A total dose of 50 Gy was broken into 25 dosages over a three-month period. PBS was collected over time and tested for polymer degradation through high performance liquid chromatography (HPLC) and bicinchoninic acid (BCA) protein assay. Scaffolds were characterized by changes in microstructure using Scanning Electron Microscopy (SEM), and crystallization using Differential Scanning Calorimetry (DSC). Additionally, gelatin ε-amine content was analyzed using Trinitrobenzene Sulfonic Acid Assay (TNBSA). Gelatin scaffolds immersed in PBS for three months without radiation served as a control.
Each scaffold responded differently to radiation. PCL showed no change in molecular weight or microstructure. However, the degree of crystallinity decreased 32% from the non-irradiated control. PU displayed both changes in microstructure and a decrease in crystallinity (85.15%). Gelatin scaffolds responded the most dramatically to radiotherapy. Samples were observed to swell, yet maintain shape after exposure. As gelatin was considered a tissue equivalent, further studies on tissues are needed to better understand the effects of radiotherapy. / Master of Science
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Novel Stereolithographic Manufacture of Biodegradable Bone Tissue ScaffoldsCooke, Malcolm Norman 02 July 2004 (has links)
No description available.
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Synthesis, characterization, and biological evaluation of gelatin-based scaffoldsTronci, Giuseppe January 2010 (has links)
This work presents the development of entropy-elastic gelatin based networks in the form of films or scaffolds. The materials have good prospects for biomedical applications, especially in the context of bone regeneration. Entropy-elastic gelatin based hydrogel films with varying crosslinking densities were prepared with tailored mechanical properties. Gelatin was covalently crosslinked above its sol gel transition, which suppressed the gelatin chain helicity. Hexamethylene diisocyanate (HDI) or ethyl ester lysine diisocyanate (LDI) were applied as chemical crosslinkers, and the reaction was conducted either in dimethyl sulfoxide (DMSO) or water. Amorphous films were prepared as measured by Wide Angle X-ray Scattering (WAXS), with tailorable degrees of swelling (Q: 300-800 vol. %) and wet state Young’s modulus (E: 70 740 kPa). Model reactions showed that the crosslinking reaction resulted in a combination of direct crosslinks (3-13 mol.-%), grafting (5-40 mol.-%), and blending of oligoureas (16-67 mol.-%).
The knowledge gained with this bulk material was transferred to the integrated process of foaming and crosslinking to obtain porous 3-D gelatin-based scaffolds. For this purpose, a gelatin solution was foamed in the presence of a surfactant, Saponin, and the resulting foam was fixed by chemical crosslinking with a diisocyanate. The amorphous crosslinked scaffolds were synthesized with varied gelatin and HDI concentrations, and analyzed in the dry state by micro computed tomography (µCT, porosity: 65±11–73±14 vol.-%), and scanning electron microscopy (SEM, pore size: 117±28–166±32 µm).
Subsequently, the work focused on the characterization of the gelatin scaffolds in conditions relevant to biomedical applications. Scaffolds showed high water uptake (H: 630-1680 wt.-%) with minimal changes in outer dimension. Since a decreased scaffold pore size (115±47–130±49 µm) was revealed using confocal laser scanning microscopy (CLSM) upon wetting, the form stability could be explained. Shape recoverability was observed after removal of stress when compressing wet scaffolds, while dry scaffolds maintained the compressed shape. This was explained by a reduction of the glass transition temperature upon equilibration with water (dynamic mechanical analysis at varied temperature (DMTA)). The composition dependent compression moduli (Ec: 10 50 kPa) were comparable to the bulk micromechanical Young’s moduli, which were measured by atomic force microscopy (AFM). The hydrolytic degradation profile could be adjusted, and a controlled decrease of mechanical properties was observed. Partially-degraded scaffolds displayed an increase of pore size. This was likely due to the pore wall disintegration during degradation, which caused the pores to merge.
The scaffold cytotoxicity and immunologic responses were analyzed. The porous scaffolds enabled proliferation of human dermal fibroblasts within the implants (up to 90 µm depth). Furthermore, indirect eluate tests were carried out with L929 cells to quantify the material cytotoxic response. Here, the effect of the sterilization method (Ethylene oxide sterilization), crosslinker, and surfactant were analyzed. Fully cytocompatible scaffolds were obtained by using LDI as crosslinker and PEO40 PPO20-PEO40 as surfactant. These investigations were accompanied by a study of the endotoxin material contamination. The formation of medical-grade materials was successfully obtained (<0.5 EU/mL) by using low-endotoxin gelatin and performing all synthetic steps in a laminar flow hood. / Diese Arbeit beschreibt die Entwicklung Entropie-elastischer Gelatine-basierter Netzwerke als Filme und Scaffolds. Mögliche Anwendungen für die entwickelten Materialien liegen im biomedizinischen Bereich, insbesondere der Knochenregeneration.
Im ersten Schritt der Arbeit wurden Entropie-elastische, Gelatine-basierte Hydrogel-Filme entwickelt, deren mechanische Eigenschaften durch die Veränderung der Quervernetzungsdichte eingestellt werden konnten. Dazu wurde Gelatine in Lösung oberhalb der Gel-Sol-Übergangstemperatur kovalent quervernetzt, wodurch die Ausbildung helikaler Konformationen unterdrückt wurde. Als Quervernetzer wurden Hexamethylendiisocyanat (HDI) oder Lysindiisocyanat ethylester (LDI) verwendet, und die Reaktionen wurden in Dimethylsulfoxid (DMSO) oder Wasser durchgeführt. Weitwinkel Röntgenstreuungs Spektroskopie (WAXS) zeigte, dass die Netzwerke amorph waren. Der Quellungsgrad (Q: 300-800 vol. %) und der Elastizitätsmodul (E: 70 740 kPa) konnten dabei durch die systematische Veränderung der Quervernetzungsdichte eingestellt werden. Die Analyse der Quervernetzungsreaktion durch Modellreaktionen zeigte, dass die Stabilisierung der Hydrogele sowohl auf kovalente Quervernetzungen (3-13 mol.-%) als auch auf Grafting von (5-40 mol.-%) und Verblendung mit Oligoharnstoffen (16-67 mol.-%) zurückgeführt werden kann.
Die Erkenntnisse aus dem Umgang mit dem Bulk-Material wurden dann auf einen integrierten Prozess der Verschäumung und chemischen Quervernetzung transferiert, so dass poröse, dreidimensionale Scaffolds erhalten wurden. Dafür wurde eine wässrige Gelatinelösung in Gegenwart eines Tensids, Saponin, verschäumt, und durch chemische Quervernetzung mit einem Diisocyanat zu einem Scaffold fixiert. Die Scaffolds hergestellt mit unterschiedlichen Mengen HDI und Gelatine, wurden im trockenen Zustand mittels Mikro Computertomographie (µCT, Porosität: 65±11–73±14 vol.-%) und Rasterelektronenmikroskopie (SEM, Porengröße: 117±28–166±32) charakterisiert.
Anschließend wurden die Scaffolds unter Bedingungen charakterisiert, die für biomedizinische Anwendungen relevant sind. Die Scaffolds nahmen große Mengen Wasser auf (H: 630 1680 wt.-%) bei nur minimalen Änderungen der äußeren Dimensionen. Konfokale Laser Scanning Mikroskopie zeigte, dass die Wasseraufnahme zu einer verminderten Porengröße führte (115±47–130±49 µm), wodurch die Formstabilität erklärbar ist. Eine Formrückstellung der Scaffolds wurde beobachtet, wenn Scaffolds im nassen Zustand komprimiert wurden und dann entlastet wurden, während trockene Proben in der komprimierten Formen blieben (kalte Deformation). Dieses Entropie-elastische Verhalten der nassen Scaffolds konnte durch die Verminderung der Glasübergangstemperatur des Netzwerks nach Wasseraufnahme erklärt werden (DMTA). Die zusammensetzungsabhängigen Kompressionsmoduli (Ec: 10 50 kPa) waren mit den mikromechanischen Young’s moduli vergleichbar, die mittels Rasterkraftmikroskopie (AFM) gemessen wurden. Das hydrolytische Degradationsprofil konnte variiert werden, und während des Abbaus kam es nur zu kontrolliert-graduellen Änderungen der mechanischen Eigenschaften. Während der Degradation konnte ein Anstieg der mittleren Porengröße beobachtet werden, was durch das Verschmelzen von Poren durch den Abbau der Wände erklärt werden kann.
Die Endotoxinbelastung und die Zytotoxizität der Scaffolds wurden untersucht. Humane Haut-Fibroblasten wuchsen auf und innerhalb der Scaffolds (bis zu einer Tiefe von 90 µm). Indirekte Eluat-Tests mit L929 Mausfibroblasten wurden genutzt, um die Zytotoxizität der Materialien, insbesondere den Einfluss des Quervernetzertyps und des Tensids, zu bestimmen. Vollständig biokompatible Materialien wurden erzielt, wenn LDI als Quervernetzer und PEO40 PPO20-PEO40 als Tensid verwendet wurden. Durch den Einsatz von Gelatine mit geringem Endotoxin-Gehalt, und die Synthese in einer Sterilarbeitsblank konnten Materialien für medizinische Anwendungen (Endotoxin-Gehalt < 0.5 EU/mL) hergestellt werden.
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