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Browsing Theses and Dissertations by Author "Almodovar Montanez, Jorge Luis, author"

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    Polysaccharide-based nanostructures for growth factor delivery and mesenchymal stem cell activation
    (Colorado State University. Libraries, 2011) Almodovar Montanez, Jorge Luis, author; Kipper, Matt J., advisor; Bailey, Travis S., committee member; Kisiday, John D., committee member; Prasad, Ashok, committee member
    Mesenchymal stem cells (MSCs) are very promising in tissue engineering and regenerative medicine because of their ability to differentiate into different type of cells including bone and cartilage. MSCs differentiation can be modulated using both chemical (i.e. proteins) and physical cues (ie. topography). This thesis presents work performed evaluating polysaccharide-based nanostructures for growth factor delivery and MSCs activation. Different polysaccharide-based nanostructures were developed and characterized including polyelectrolyte multilayers (PEMs) and electrospun nanofibers. On flat gold-coated glass surfaces, PEMs were constructed using the polycations chitosan and N,N,N -trimethyl chitosan, and the polyanions hyaluronan, chondroitin sulfate, and heparin. An exhaustive spectroscopic study was performed on all of the PEMs pairs to investigate the effects of polyelectrolyte charge density on thickness, swelling, composition, and ion-pairing. The results demonstrated that hydrophilicity and swelling are reduced when one polyelectrolyte is strong and the other is weak, while ion pairing is increased. The stability of adsorbed proteins to PEMs was also investigated using IR spectroscopy. Construction of PEMs and adsorption of basic fibroblast growth factor (FGF-2) was evaluated on heparin chitosan PEMs constructed on gold-coated glass, tissue culture polystyrene (TCPS), and titanium. In vitro testing of the FGF-2-loaded PEM constructed on TCPS and titanium was performed using ovine bone marrow-derived MSCs. It was noted that FGF-2 activity is enhanced, with regards to MSCs proliferation, when delivered from PEMs compared to delivery in solution. Chitosan nanofibers were successfully electrospun from a trifluoroacetic acid and dichloromethane solution. A new technique was developed to modify electrospun chitosan nanofibers with polyelectrolyte multilayers using N,N,N -trimethyl chitosan and heparin. Controlled release of bioactive FGF-2, complexed with heparin-chitosan polyelectrolyte complex nanoparticles, from electrospun chitosan nanofiber mats was achieved with zero order kinetics over a period of 27 days. When the nanofibers are further modified with a single PEM bilayer (PEM, composed of N,N,N -trimethyl chitosan and heparin), the release is completely prevented. The mitogenic activity of the released FGF-2 was also evaluated, with respect to the proliferation of ovine bone marrow-derived MSCs. The effect on osteogenic differentiation of bone marrow-derived ovine and equine MSCs seeded on electrospun chitosan nanofibers versus flat TCPS was investigated. The effect of dexamethasone on osteogenic differentiation was also investigated. We found that we can successfully grow and maintain both equine and ovine MSCs on electrospun chitosan nanofibers. Also, both MSCs exhibit higher differentiation markers (alkaline phosphatase activity) when cultured on chitosan nanofibers compared to flat TCPS surfaces. This work demonstrates new systems for stabilizing and controlling the delivery of heparin-binding growth factors for the activation of bone marrow-derived MSCs, using polysaccharide-based nanomaterials. These novel materials have potential applications in musculoskeletal tissue regeneration.