No description available.

No description available.

No / Streptococcus mutans was shown to produce two extracellular proteases capable of degrading both gelatin and collagen-like substrates. These enzymes have molecular masses of 52 and 50 kDa when analysed by SDS-PAGE. Both enzymes were inhibited by EDTA, but not by a range of other inhibitors with different specificities, indicating that they are metalloproteases. The activity of EDTA-inactivated enzymes could be restored by the addition of manganese and zinc. The identical inhibition and restoration profiles of the two enzymes suggest that one of the proteases may be a degradation product of the other.

The local release of complexed siRNA from biomaterials enables targeted therapy of specific cells and tissues. This thesis focused on gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA) as a drug delivery system for siRNA. The siRNA-loaded cGM were aggregated with SaOS-2 cells or human mesenchymal stem cells (hMSC) to microtissues and stimulated with osteogenic supplements. Cell survival and tissue formation in microtissues could be improved by incorporating cGM in spheroid cultures. We observed hydroxyapatite deposition in the particles in dependence of medium and cell type. Osteogenic stimulation with BMP-2 and simultaneous silencing of BMP-2 antagonist chordin accelerated matrix mineralization of the microtissues. Higher cross-linking degree of cGM positively influenced chordin silencing and alkaline phosphatase (ALP) activity as a marker for osteogenic differentiation. These higher cross-linked cGM mineralized in an osteogenic medium within 8–9 days, in presence and absence of cells. The effects of pre-differentiated and chordin-silenced microtissues were investigated by simulation of in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). Increased ALP activity and osteoprotegerin (OPG) secretion were observed after 14 days compared to co-cultures with siRNA-free controls. These results indicate that the pre-differentiated and silenced microtissues can induce osteogenic differentiation of surrounding unstimulated cells. Using the microtissue approach with siRNA complexed with tyrosine-modified low molecular weight polyethyleneimine (P10Y/P5Y) as transfection reagent was not successful. The results of this thesis indicate that the pre-differentiation of microtissues with BMP-2 in combination with chordin silencing stimulates and enhances osteogenic differentiation of other stem cells. As a combination of biomaterial, RNAi, and autologous cells, microtissues could be a promising approach to regenerating bone defects.:Chapter I: Controlled release of siRNA for bone tissue engineering Chapter II: Microtissues from mesenchymal stem cells and siRNA-loaded cross-linked gelatin microparticles for bone regeneration Chapter III: Mineralizing gelatin microparticles as cell carrier and drug delivery system for siRNA for bone tissue engineering Chapter IV: Tyrosine-modified polyethylenimines for siRNA transfection in microtissues Chapter IV: Final discussion

No / The most important issue during synthesis of nanoparticles (NPs) is to avoid particle agglomeration and adhesion. There have been several attempts to use special substances such as organic surfactants, polymers and stable ligands for this purpose. In this study, silver NPs were synthesised with and without gelatin macromolecules, as a green natural biopolymer, which resulted in NPs with varying shapes and sizes. The effect of morphological characteristics on the antibacterial and antifungal properties of the synthesised NPs were studied, by comparing Gram-negative (Escherichia coli) versus Gram-positive (Staphylococcus aureus) bacteria as well as fungi (Candida albicans) by calculation of minimal inhibition concentration value. The results corresponded well with the assumptions on the effects of shape and size on the antibacterial and antifungal properties of the studied NPs.

The proteins wheat gluten and gelatin were tested for use in biocomposites and soft actuating materials, respectively. In Chapter II, the self-assembly mechanism of trypsin hydrolyzed wheat gluten (THWG) into rigid β-sheets was applied to an aqueous polyvinyl alcohol (PVA) environment. Aqueous PVA was used in order to determine the effects of an aqueous environment other than pure water on THWG self-assembly kinetics and to realize the potential use of THWG as a nanofiller in polymer matrices. THWG was able to self-assemble into anisotropic spikes and agglomerates of spikes called "pompons" through hydrophobic interactions. THWG self-assembly kinetics were retarded in aqueous PVA solutions compared to water, with the highest molecular weight PVA solution showing the slowest self-assembly kinetics. Chapters III and IV explore the potential of gelatin hydrogels for use in soft actuators. A gelatin bilayer system was designed where an active layer swelled more than a passive layer to cause the system to bend/actuate in response to an environmental stimulus. In Chapter III, gelatin layers were chemically crosslinked to different degrees with glutaraldehyde to achieve bilayer bending when placed in water. Curvature of the bilayer system was found to be dependent on the difference in volume swell ratio between the two layers. It was determined that maximum bending occurred when the passive layer swelled to 60% of the swelling of the active layer. Addition of pre-gelatinized starch to the active layer increased layer swelling and bilayer curvature. Treating the starch containing bilayer with -amylase returned the bilayer to its original shape. In Chapter IV, a pH responsive gelatin bilayer was constructed using Type A and Type B gelatin. Type A and Type B gelatin gels had different chemical properties and swelled to different volumes based on the gel solution pH. Bilayers constructed from Type A and Type B gelatin exhibited different degrees of bending when placed in various pH solutions with maximum curvature occuring at pH 10. A cyclic actuator could be formed when the bent bilayers were placed in a minimum of 0.01M NaCl solution. Placement in salt solution resulted in the unbending of the bilayer. Overall, this work demonstrated the various applications of proteins as functional and green materials. / Doctor of Philosophy / The majority of plastics consist of synthetic polymers derived from oil that cannot be broken down by the environment (i.e., not biodegradable). Research is underway to develop sustainable, biodegradable materials. Proteins are a biological polymer that have a wide range of chemical, structural, and functional properties; for this reason they are an excellent source material for use in the design of environmental friendly materials. In Chapter II, the ability of wheat gluten protein to self-assemble into rigid, nanosized structures is used to explore the potential of the protein to be used as a biodegradable nanofiller. A nanofiller is added to various materials in order to improve the overall mechanical properties of the material. Wheat gluten is self-assembled in an aqueous polymer environment. The results show that the polymer environment stunts or slows down the self-assembly rate of the protein compared to a pure water environment. Nanometer sized spikes form in the polymer solutions, indicating wheat gluten could be used as a nanofiller in certain materials. Chapters III and IV explore the use of gelatin proteins for applications in soft robotics. Soft robots and their moveable parts, called soft actuators, are deformable and respond to changes in the environment such as pH, light, temperature, etc. For this reason, soft robots are considerable adaptable compared to traditional rigid robots. Designing a soft actuator from gelatin gels would result in a “smart” material that is biocompatible and biodegradable. A gelatin soft actuator is created using a bilayer design in which one layer of the bilayer swells more than the other layer causing the entire system to bend/actuate. Depending on how the bilayer system was fabricated, bending could be achieved based on stimuli such as the presence of water, the presence of a substrate and enzyme, and changes in pH. Overall, this dissertation demonstrates the extraordinary potential for the use of proteins in designing sustainable materials.

Gelatin methacrylate (GelMA) is a ubiquitous biocompatible photopolymer used in tissue engineering and regenerative medicine due to its cost-effective synthesis, tunable mechanical properties, and cellular response. Biotechnology applications utilizing GelMA have ranged from developing cell-laden hydrogel networks to cell encapsulation and additive manufacturing (3D printing). However, extrusion based 3D printing is the most common technique used with GelMA. Mask projection microstereolithography (MPµSL or µSL) is an advanced 3D printing technique that can produce geometries with high resolution, high complexity, and feature sizes unlike extrusion based printing. There are few biomaterials available for µSL applications, so 3D printing GelMA using µSL would not only add to the repertoire materials, but also demonstrate the advantages of µSL over other 3D printing techniques. A novel GelMA resin was tested with µSL to create a porous scaffold with a height and print time that has not been displayed in the literature before for a scaffold of this size. The resin consists of GelMA, deionized water, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, photoinitiator), and 2-Hydroxy-4-methoxybenzophenone-5-sulfonic acid (sulisobenzone, UV blocker) and can be processed at room temperature. Four resins were tested (w/w %) and characterized for µSL printing: 20% GelMA 0.5% UV blocker, 20% GelMA 1.0% UV blocker, 30% GelMA 0.5% UV Blocker, and 30% GelMA 1.0% UV blocker. Swell testing, working curve, photo-rheology, photo-DSC (dynamic scanning calorimetry), 3D printing, and cell culture tests were performed and results showed that 30% GelMA 1.0% UV blocker had the best 3D print fidelity among resin compositions. / Master of Science / Three dimensional (3D) printing is a widely used technology to rapidly produce structures with varying degrees of complexity. 3D printing of biological components is of interest because as the world population increases, there is a lack of donors available to compensate for organ loss and tissue replacement. 3D printing offers a solution to great custom scaffolds and structures that mimic physiological geometry and properties. One printing technique is known as microstereolithography, or µSL, which uses a projector-like system to pattern ultraviolet (UV) light in specific arrangements to generate complex geometries and 3D parts. Gelatin is a material of interest for this technology because gelatin is derived from collagen, which is the most abundant protein found in the body. Gelatin can be modified so that it is reactive with UV light, and can be processed with µSL to generate 3D structures. In this work, gelatin was modified into the form of gelatin methacrylate (GelMA) in order to develop and test resin formulations for use with µSL. Four different resins were tested and characterized and the results indicated that one GelMA resin produced prints with greater fidelity and resolution than other formulations. This resin has been identified for potential applications in tissue engineering and 3D printed organ development.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xviii, 279 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

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

A novel dendritic wound dressing was designed and characterized for its potential to treat chronic wounds. Comprised of gelatin, dendrimer, synthetic polymer and antibiotics, the dressing was electrospun to mimic the natural extracellular matrix (ECM). Gelatin is biocompatible, biodegradable, non-toxic, and easily available. The antibiotic, doxycycline, has the ability to inhibit matrix metalloproteinases. Matrix metalloproteinases, which occur in excess in chronic wounds, degrade the reconstituted ECM. Starburst™ polyamidoamine (PAMAM) dendrimer G3.5, which provides a versatile and structurally controlled architecture to construct nanomedicine, was covalently bonded to the gelatin backbone and electrospun into nanofibers with gelatin, doxycycline and stabilizing polymers. The proposed gelatin/dendrimer hybrid provides a bacterial free environment and mimics the ECM to promote wound healing. The development of this new polymeric matrix is an important step in advancing the use of bioactive nanofibers with targeted and controlled drug delivery as a wound dressing.