Keratins are a family of proteins found within human hair, skin and nails, as well as a broad variety of animal tissue. Prior research suggests hydrogel constructs of keratin and keratin derivatives exhibit several mechanical and biological properties that support their use for tissue engineering and regenerative medicine applications. Microparticle formulations of these hydrogels are an intriguing delivery vehicle for drugs and cellular payloads for tissue engineering purposes due to the ability to exploit size, surface area, loading potential and importantly, non-invasive delivery (i.e. injection) of cells and biologics.
Here we examine the water-in-oil emulsion synthesis procedure to produce keratin microparticles using an oxidized keratin derivative, keratose (KOS). Analyses of particle size, microstructure, and other characterization techniques were performed. Drug loading characteristics, release kinetics, and feasibility of use in two different microparticles was subsequently investigated, first using a model-drug and later testing an antibiotic payload on bacterial cultures to validate antibacterial applications. A suspension culture technique was developed to load bone marrow-derived mesenchymyal stromal cells (BM-MSCs), testing the capacity to maintain viability and express key protein-based factors in cell growth and development. Finally, we tested the in vitro effects of cell-loaded microparticles on the L6 skeletal muscle cell line to determine potentially beneficial outcomes for skeletal muscle tissue regeneration.
Largely spherical particles with a porous internal structure were obtained, displaying hydrogel properties and forming viscoelastic gels with small differences between synthesis components (solvents, crosslinkers), generating tailorable properties. The uniquely fibrous microstructure of KOS particles may lend them to applications in rapid drug release or other payload delivery wherein a high level of biocompatibility is desired. Data showed an ability to inhibit bacterial growth in the emulsion-generated system, and thereby demonstrated the potential for a keratin-based microparticle construct to be used in wound healing applications. Dense cell populations were loaded onto particles. Particles maintained cell viability, even after freeze-thaw cycling, and provided a material substrate that supported cell attachment through the formation of focal adhesions. Finally, in vitro studies show that both KOS and BM-MSCs support varying aspects of skeletal muscle development, with combinatorial treatments of cell-loaded particles conferring the greatest growth responses. / Doctor of Philosophy / Keratins and keratin hydrogels may exhibit several properties that support their use for tissue engineering and regenerative medicine applications. Microparticle formulations of these hydrogels are an intriguing delivery vehicle for payloads for tissue engineering purposes. Here we examine the water-in-oil emulsion synthesis procedure to produce keratin microparticles that were analyzed based on drug loading characteristics. A suspension culture technique was developed to load bone marrow-derived mesenchymyal stromal cells (BM-MSCs). Finally, we tested these products to determine potentially beneficial outcomes for skeletal muscle tissue regeneration. Particles with a porous structure were obtained. The microstructure of these particles may lend them to applications in drug release or other payload delivery. Data showed an ability to load and unload specific drug payloads. Dense cell populations were loaded onto particles. Finally, studies show that both keratin and BM-MSCs support skeletal muscle development, with combinatorial treatments of cell-loaded particles conferring the greatest growth responses.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/89345 |
| Date | 02 May 2019 |
| Creators | Thompson, Marc Aaron |
| Contributors | Department of Biomedical Engineering and Mechanics, Van Dyke, Mark, He, Jia-Qiang, Gourdie, Robert G., Goldstein, Aaron S., Lee, Yong Woo |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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