Browsing by Author "Kipper, Matt J., advisor"
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Item Open Access A study of protective clothing to understand nanoparticle exposure and surface contamination(Colorado State University. Libraries, 2021) Maksot, Aigerim, author; Kipper, Matt J., advisor; Tsai, Candace S., advisor; Li, Yan V., committee memberIn this study, we investigated engineered nanoparticle (ENP) release associated with the contamination of personal protective clothing during the human activities of the worker wearing the ENP-contaminated protective clothing and evaluated the relative ENP retention to each used fabric type. The release of ENPs as airborne nanoparticles can cause inhalation exposure, which is the route of exposure of most concern to cause adverse health effects. The methods used were associated with four different fabric materials of contaminated laboratory coats (cotton, polypropylene, polyester/cotton blend, and Tyvek®) and three ENPs (Al2O3, carbon black and CNT). Two types of tests were performed: contamination and release experiments under two different durations (30 minutes and 6 hours of release processes). The magnitude of contamination and particle release were investigated in this study by measuring the number concentration increase and the weight change on fabric pieces. This study simulated real-life occupational exposure scenarios and was performed in cleanroom environments to investigate the effect of background aerosols on the measurements. Concentrations were measured using particle spectrometers for diameters from 10 nm to 10 μm. Collected aerosol particles and contaminated fabric surfaces were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and elemental carbon analysis. The magnitude of particle release from contaminated lab coat fabric was found to vary by the type of fabric material; cotton fabric showed the highest level of particle release, followed by polyester cotton, Tyvek® and polypropylene. Moreover, Tyvek® fabric was determined as the best fabric for trapping Al2O3 and carbon black ENPs indicating less resuspension of particles and highest mass change per unit mass after aerosolization and release processes. Two dominant forces responsible for ENP adhesion on the surface of the fabric were theoretically calculated to be van der Waals force and capillary force. To sum up, Tyvek® fabric is considered the most reliable fabric against ENPs, but not durable enough to wear for the long term compared with other fabrics.Item Open Access Biopolymer nanomaterials for growth factor stabilization and delivery(Colorado State University. Libraries, 2014) Place, Laura Walker, author; Kipper, Matt J., advisor; James, Susan, committee member; Popat, Ketul C., committee member; Miller, Benjamin, committee memberBiopolymers are useful in tissue engineering due to their inherent biochemical signals, including interactions with growth factors. There are six biopolymers used in this work, the glycosaminoglycans (GAGs), heparin (Hep), chondroitin sulfate (CS), and hyaluronan (HA), chitosan (Chi), a GAG-like molecule derived from arthropod exoskeletons, a Chi derivative N,N,N¬-trimethyl chitosan (TMC), and an extracellular matrix (ECM)-derived material, demineralized bone matrix (DBM). The direct delivery of growth factors is complicated by their instability. GAG side chains of proteoglycans stabilize growth factors. GAGs also regulate growth factor-receptor interactions at the cell surface. The majority of proteoglycan function is derived from its GAG side chain composition. Here we report the development of nanoparticles, proteoglycan-mimetic graft copolymers, incorporation of nanoparticles into electrospun nanofibers, and processing methods for electrospinning demineralized bone matrix to fabricate bioactive scaffolds for tissue engineering. The nanoparticles were found to show similar size, composition, and growth factor binding and stabilization as the proteoglycan aggrecan. We use basic fibroblast growth factor (FGF-2) as a model heparin-binding growth factor, demonstrating that nanoparticles can preserve its activity for more than three weeks. Graft copolymers were synthesized with either CS or Hep as the side chains at four different grafting densities. Their chemistry was confirmed via ATR-FTIR and proton NMR. They were shown to increase in effective hydrodynamic diameter with grafting density, resulting in a size range from 90-500 nm. Graft copolymers were tested for their ability to deliver FGF-2 to cells. The CS conditions and the Hep 1:30 performed equally as well as when FGF-2 was delivered in solution. Preliminary dynamic mechanical testing demonstrated that hydrogels containing the copolymers exhibit changes in compressive modulus with cycle frequency. Two electrospinning techniques were developed, using an emulsion and a coaxial needle, for incorporating growth factor into electrospun nanofibers. We bound FGF-2 to aggrecan-mimetic nanoparticles for stabilization throughout electrospinning. The two techniques were characterized for morphology, nanoparticle and FGF-2 incorporation, cytocompatibility, and FGF-2 delivery. We demonstrated that both techniques result in nanofibers within the size range of collagen fiber bundles and dispersion of PCNs throughout the fiber mat, and exhibit cytocompatibility. We determined via ELISA that the coaxial technique is superior to the emulsion for growth factor incorporation. Finally, FGF-2 delivery to MSCs from coaxially electrospun nanofibers was assessed using a cell activity assay. We developed a novel method for tuning the nanostructure of DBM through electrospinning without the use of a carrier polymer. This work surveys solvents and solvent blends for electrospinning DBM. The effects of DBM concentration and dissolution time on solution viscosity are reported and correlated to observed differences in fiber morphology. We also present a survey of techniques to stabilize the resultant fibers with respect to aqueous environments. Glutaraldehyde vapor treatment is successful at maintaining both macroscopic and microscopic structure of the electrospun DBM fibers. Finally, we report results from tensile testing of stabilized DBM nanofiber mats, and preliminary evaluation of their cytocompatibility. The DBM nanofiber mats exhibit good cytocompatibility toward human dermal fibroblasts (HDF) in a 4-day culture.Item Open Access Dynamics of protein interactions with new biomimetic interfaces: toward blood-compatible biomaterials(Colorado State University. Libraries, 2019) Hedayati, Mohammadhasan, author; Kipper, Matt J., advisor; Krapf, Diego, committee member; Reynolds, Melissa, committee member; Bailey, Travis, committee memberNonspecific blood protein adsorption on the surfaces is the first event that occurs within seconds when a biomaterial comes into contact with blood. This phenomenon may ultimately lead to significant adverse biological responses. Therefore, preventing blood protein adsorption on biomaterial surfaces is a prerequisite towards designing blood-compatible artificial surfaces.Item Open Access Engineering a biomimetic periosteum on cortical bone allografts for the reconstruction of critical-sized bone defects in mice(Colorado State University. Libraries, 2017) Romero, Raimundo, author; Kipper, Matt J., advisor; Ehrhart, Nicole P., advisor; Kisiday, John D., committee member; Schenkel, Alan R., committee memberLoad bearing bone allografts suffer from clinical failure due to low allograft-host tissue integration. Removal of the periosteum, a thin tissue layer on bone with a high regenerative capacity, is responsible for bone allografts' decreased clinical performance. This interdisciplinary project addressed this problem by creating multiple engineered periostea on mice bone allografts. Using a polysaccharide biomaterial, chitosan, tissue engineering scaffolds constructed on these bone allografts were modified with the glycosaminoglycan, heparin, and a chitosan derivative, trimethyl chitosan, to create periostea with different scaffold morphologies yet similar surface chemistries. Cell instructive cues such as growth factors fibroblast growth factor-2 (FGF-2) and transforming growth factor-β1 (TGF-β1) were adsorbed onto the engineered periostea and found to release up to 14 and 7 days in-vitro, respectively. Engineered allografts were found to support adipose-derived stem cell (ASC) adhesion and proliferation. FGF-2 and TGF-β1 delivered from the engineered allografts to ASC supported an osteoprogenitor phenotype in ASC and did not inhibit alkaline phosphatase and receptor activator of nuclear factor-kappaB (RANKL) protein expression. From in vitro results, the nanofiber engineered periosteum was found to be the most cytocompatible scaffold and was selected for subsequent implantation in a pre-clinical mouse critical-sized femoral defect model. We assessed the engineered periosteum's efficacy on modulating allograft healing and incorporation. We confirmed the engineered allografts successfully delivered ASC, FGF-2, and TGF-β1 to the femur defect and found ASC persisted in the femur defect for at least 7 days, similar to other reports in the literature. At week 6, microcomputed tomography results of excised femurs showed no statistical difference in new bone volume formation between experimental groups. However, treatment groups containing ASC showed a trend of at least 24% more bone volume compared to their respective cell-free controls suggesting possible therapeutic effects of ASC. Union ratio and histological analysis both confirmed the nanofiber engineered periosteum did not degrade at 6 weeks and inhibited allograft incorporation. Subsequent relative gene expression experiments showed that ASC maintained an undifferentiated phenotype in response to FGF-2 and TGF-β1 delivered from chitosan nanofibers. Overall, this project developed a novel polysaccharide-based engineered periosteum for delivering growth factors and progenitor cells to a bone defect for regenerative medicine applications.Item Open Access Engineering nanostructured polysaccharide-based polyelectrolyte complexes(Colorado State University. Libraries, 2009) Boddohi, Soheil, author; Kipper, Matt J., advisorThe overall goal of this dissertation is to demonstrate how the structure and composition of polysaccharide-based materials might be tuned at the nanometer length scale. Nanostructured biomaterials are promising candidates for biomedical engineering applications. Among all biomaterials, polysaccharides have shown great potential because of their many biochemical functions and their complex nanoscale structure in biological contexts. The nanoscale structure of polysaccharides is an important property that controls their biochemical and biological functions, in a variety of tissues. Therefore, in this dissertation, the nanoscale assembly of polysaccharides-based polyelectrolytes using polyelectrolyte multilayers (PEMs), polyelectrolyte complex nanoparticles (PCNs), and combinations of these two nanostructures was investigated. These new nanostructured surface coatings are being further developed by the Kipper research group as means of stabilizing and delivering therapeutic proteins, and as bioactive surface coatings for stem cell engineering. Thus the ability to tune their structure and composition is an important contribution of the current work.Item Open Access 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 memberMesenchymal 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.Item Open Access Tuning interfacial biomolecule interactions with massively parallel nanopore arrays(Colorado State University. Libraries, 2021) Wang, Dafu, author; Kipper, Matt J., advisor; Snow, Chris D., advisor; Bailey, Travis S., committee member; Stasevich, Tim J., committee memberThis project studied interfacial interactions of macromolecules with nanoporous materials, with an ultimate goal of exploiting these interactions in functional biomaterials. We quantified interaction forces and energies for guest molecules threaded into the pores of protein crystals via nano-mechanical atomic force microscopy (AFM) pulling experiments. We demonstrated that both double-stranded DNA and poly(ethylene glycol) are rapidly absorbed within porous protein crystals, where they presumably bind to the inner "wall" surfaces of the protein crystal nanopores. These "guest" molecules can be retrieved from the "host" crystal by chemically modified AFM tips, enabling precise measurements of the adhesion forces and interaction energies. Based on these experiments, machine learning approaches were developed to classify hundreds of thousands of individual force-distance curves obtained in the AFM experiments. Furthermore, we showed that the interactions between protein crystal "hosts" and "guest" macromolecules can be used to modulate cell behavior, by presenting cell adhesion ligands tethered to different lengths of macromolecules that thereby modulate the maximum traction force cells can apply before rupturing bonds tethering the adhesion ligand to the porous protein crystal interior. This method affords the opportunity to create biomaterials that store an internal reservoir of cell-specific signals that can be presented to independently modulate the behavior of different cell populations in a single material. In the first chapter, some recent advancements, and methodologies of measuring interfacial biomolecule interactions are reviewed and compared. The reviewed technics include atomic force microscopy, fluorescence recovery after photobleaching, the total internal reflection fluorescence, confocal microscopy, and optical tweezers. Furthermore, this chapter interduces the application of machine learning to assist the interfacial biomolecule interaction studies, especially the AFM measurements. This chapter further prospects of the future of interfacial biomolecule interactions studies. In the second chapter, the methodologies of probing and observing the surface of highly porous Camphylobacter Jejuni formed protein crystals (CJ protein crystals) by high-resolution AFM are introduced. Throughout this chapter, the morphologies of CJ protein crystals are comprehensively investigated by AFM and have been discussed in this chapter. In the third chapter, for the first time, the interactions of DNA with porous protein crystals are quantitatively measured by high-resolution AFM and chemical force microscopy. The surface structure of protein crystals with unusually large pores was observed in liquid via high-resolution AFM. Force-distance (F-D) curves were also obtained using AFM tips modified to present or capture DNA. The interactions of DNA molecules with protein crystals to be quantitatively studied while revealing the morphology of the protein crystal surface in detail, in buffer, reveals how a new protein-based biomaterial can be used to bind DNA guest molecules. In the fourth chapter, strategies of machine learning are introduced which pioneered the use of machine learning to classify and cluster the interaction patterns between DNA and protein crystals, enabling us to process thousands of F-D curves collected by AFM. Finally, in the fifth chapter, we quantitatively measure and take advantage of the interaction between poly(ethylene glycol) (PEG)-arginine-glycine-aspartic acid (RGD) complex and nanoporous protein crystals to understand how non-covalent surface presentation of peptide adhesion ligands can influence cell behavior. Through AFM, F-D curves of interactions between PEG-RGD and host protein crystals were obtained for the first time. Furthermore, a strategy is developed that enables us to design surfaces that non-covalently present multiple different ligands to cells with tunable adhesive strength for each ligand, and with an internal reservoir to replenish the precisely defined crystalline surface.