Browsing by Author "Popat, Ketul, advisor"
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Item Open Access Antibacterial effects of sputter deposited silver-doped hydroxyapatite thin films(Colorado State University. Libraries, 2011) Trujillo, Nathan Anthony, author; Popat, Ketul, advisor; Williams, John, advisor; Reynolds, Melissa, committee member; Crans, Debbie, committee memberOver recent years, researchers have studied innovative ways to increase the lifespan of orthopedic implants in order to meet the soaring demand of hip and knee replacements. Since many of these implants fail as a result of loosening, wear, and inflammation caused by repeated loading on the joints, coatings such as hydroxyapatite (HAp) on titanium with a unique topography have been shown to improve the interface between the implant and the natural tissue. Other serious problems with long-term or ideally permanent implants are bacterial colonization. It is important to prevent initial bacterial colonization as existing colonies have potential to become encased in an extracellular matrix polymer (biofilm) which is resistant to antibacterial agents. The following work considers the potential of etching using plasma based ion implantation and ion beam sputter deposition to produce hydroxyapatite thin films on etched titanium doped with silver as an antibacterial component. Plasma-based ion implantation was used to examine the effects of pre-etching on plain titanium. Topographical changes to the titanium samples were examined and compared via scanning electron microscopy. It was determined that plasma-based ion implantation at -700eV could etch titanium to produce similar topography as ion beam etching in a shorter processing time. Hydroxyapatite and silver-doped hydroxyapatite thin films were then sputter deposited on titanium substrates etched at -700eV. For silver-doped films, two concentrations of silver (~0.5wt% and ~1.5wt%) were used. Silver concentrations in the film were determined using energy dispersive x-ray spectroscopy. Film thicknesses were determined by measuring the surface profile using contact profilometry. Staphylococcus epidermidis (SE) and Pseudomonas aeruginosa (PA) adhesion studies were performed on plain titanium, titanium coated with hydroxyapatite, titanium coated with ~0.5 wt% silver-doped hydroxyapatite, and titanium coated with ~1.5wt% silver-doped hydroxyapatite. It was discovered during the study that the films were delaminating from the samples thus killing bacteria in suspension. Release studies performed in addition to adhesion confirmed that the silver-doped films prevented SE and PA bacterial growth in suspension. To prevent delamination, the films were annealed by heat treatment in air at a temperature of 600°C. X-ray diffraction confirmed the presence of a crystalline hydroxyapatite phase on each sample type. Films were immersed in PBS at 37°C and remained in incubation for four weeks to determine there was no delamination or silver leaching.Item Open Access Biomimetic and antimicrobial surfaces for orthopedic implants(Colorado State University. Libraries, 2021) Wigmosta, Tara, author; Kipper, Matt, advisor; Popat, Ketul, advisor; Giess, Brian, committee member; DeLong, Susan, committee member; Schenkel, Alan, committee memberThe number of total knee and hip replacement surgeries is expected to continue to rise in the United States. As such, the number of revision surgeries is also expected to rise. The two most common causes of failure for these implants is aseptic loosening, caused by incomplete osseointegration, and infection. Therefore, preventing infection while increasing the osteogenic properties of the surfaces used in orthopedic implants could reduce the number of revision surgeries. It is the goal of this work to create nanostructured surfaces that both increase mineralization and antimicrobial properties of titanium surfaces commonly used in orthopedic implants. To accomplish this, chitosan/heparin polyelectrolyte multilayers (PEMs), with the addition of either bone morphogenetic protein 2 (BMP-2) or gentamicin, were adsorbed onto titania nanotubes. BMP-2 has been used in clinical applications to increase osseointegration in spinal fusions, and gentamicin is effective against the most common pathogens found in infected orthopedic implants. Both heparin and chitosan are biocompatible and have antimicrobial properties. BMP-2 has a binding site for heparin that increases BMP-2's half-life in vitro. The first chapter summarizes the motivation and previous strategies used to increase osseointegration and antimicrobial properties of nanostructured biomimetic orthopedic implant surfaces. The first chapter concludes with a shift in hypothesis testing, outlining three different hypotheses: 1) surface modification(s) increase cytocompatibility and the osteogenic properties of mammalian bone cells; 2) surface modification(s) reduce bacterial adhesion, proliferation, and infection rate, without decreasing cytocompatibility; and 3) surface modification(s) provide a favorable environment in which mammalian cells can beat bacterial cells and colonize the surface first, thus increasing the osteogenic and antimicrobial properties of the surface. The testing of these hypotheses are explored in chapters 2 through 4. The second chapter explores hypothesis 1) by testing if BMP-2 released from chitosan/heparin PEM coated titania nanotubes surfaces induce an osteogenic response from rat bone marrow cells. Chapter 3 explores hypothesis 2) by testing if iota-carrageenan/chitosan and pectin/chitosan PEMs have antimicrobial properties against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), and support rat bone marrow cell adhesion and proliferation. The last chapter explores hypothesis 3) by testing if gentamicin released from titania nanotubes coated with chitosan/heparin PEMs influences the "race to the surface" in favor of mammalian cells.Item Open Access Design and fabrication of a flow chamber for the study of cell adhesion and hemocompatibilty in dynamic conditions(Colorado State University. Libraries, 2011) Migita, Kevin, author; Popat, Ketul, advisor; Dasi, Lakshmi Prasad, advisor; Prasad, Ashok, committee memberCell adhesion is a well characterized condition of both biomaterial and tissue engineering research. It plays a role in biocompatibility and the proliferation, differentiation and viability of seeded cells. With respect to hemocompatibility, platelet adhesion and subsequent activation is a driving factor in the failure of blood contacting medical devices. Platelets aggregates are vital components in the wound healing and foreign body responses and display various forms of adhesion based on blood flow. However, the study of platelet adhesion on implantable tissue engineering scaffolds under dynamic conditions is very limited, particularly with directional flow. A flow chamber which incorporates a tissue engineering scaffold or functionalized biomaterial was designed and fabricated for investigation of flow patterns and cellular adhesion in response to dynamic conditions on these surfaces. The device utilizes a combination of aspects from both tissue engineering bioreactors and microfluidics platforms to result in a flow chamber which provides the directional flow of a perfused flow bioreactor with the advantages of controlling chamber shape and real time monitoring presented by Polydimethylsiloxane microfluidics chambers. Results of fluid flow study in the chamber modeled for laminar and shear gradient simulated flow show the ability of the device to manipulate flow patterns. Dynamic and static studies of platelet adhesion to poly-(ε-caprolactone) flat and electrospun nanofiber surfaces utilizing the flow chamber provide insight into the hemocompatibility of tissue engineering scaffolds in a dynamic flow setting.Item Open Access Evaluation of osteogenic design factors in electrospun poly(ε-caprolactone) nanofiber scaffolds(Colorado State University. Libraries, 2010) Ruckh, Timothy T., author; Popat, Ketul, advisor; James, Susan, committee member; Kipper, Matt, committee member; Ryan, Stewart, committee memberBiodegradable bone tissue scaffolds have the potential to impact patients with numerous ailments. Starting with fabrication techniques that produce nano-scale features, the ability to manipulate architecture, alter surface chemistry, and deliver biological molecules allows for the design of elegant and highly effective bone scaffolds. This work aimed to develop a porous, nanofiber scaffold with osteogenic design features the capability to deliver an antibiotic molecule from within the nanofibers. Two osteogenic design factors with unique mechanisms of action were selected; hydroxyapatite nanoparticles and oleic acid. Hydroxyapatite (HAp) is the primary inorganic phase of natural bone tissue and has been used to more closely mimic the extracellular environment of synthetic bone tissue scaffolds. Oleic acid (OLA) is an ω-9 fatty acid with suspected osteogenic effects due to activation of peroxisome proliferator-activator receptors (PPARs). In separate in vitro evaluations, OLA significantly increased osteoblast phenotypic behaviors and led to differential expression of the three PPAR isoforms, suggesting that the OLA is activating its anticipated receptor. HAp produced mixed results by inducing a small increase in alkaline phosphatase activity, but decreasing expression levels of bone matrix proteins. An in vivo evaluation of biocompatibility revealed that neither design factor increased the inflammatory response over control nanofiber scaffolds in paravertebral muscle pouches. However, both factors separately increased new osteoid production. Scaffolds with both HAp and OLA elicited the greatest osteogenic response in vivo, suggesting positive synergy between the two design factors. Finally, rifampicin (RIF), an antibiotic molecule was loaded into the nanofibers, and its release into static bacterial culture was effective in inhibiting bacterial population growth for both a Gram-positive and Gram-negative bacterial strain, separately. Overall, these nanofiber scaffolds were demonstrated to be effective carriers of soluble (OLA, RIF) and insoluble signals (HAp) which can modulate cell behaviors. Future work will aim to incorporate additional osteogenic features into the scaffolds and to develop multiple antibiotic release mechanisms from the nanofibers.Item Open Access Hemocompatibility of polymeric materials for blood-contacting applications(Colorado State University. Libraries, 2015) Woodbury, Jodi Marie, author; Popat, Ketul, advisor; Dasi, Prasad, committee member; Reynolds, Melissa, committee memberHemocompatibility of a biomaterial plays a vital role in the overall success of the biomaterial in the body. Every implanted biomaterial tends to cause an immune response by the host tissue. The intensity of said response depends on many factors, including the properties of the material itself. In this study, we have assessed the hemocompatibility of expanded polytetrafluoroethylene (ePTFE), linear low-density polyethylene (LLDPE) and polyethylene terephthalate (PET); 3 potential materials for blood-contacting applications. The surface morphology was characterized using scanning electron microscopy (SEM), and surface wettability was characterized using contact angle goniometry. The cytotoxicity was investigated using lactate dehydrogenase (LDH) assay. The adsorption of key blood serum proteins was evaluated using micro-bicinchoninic acid (micro-BCA) assay. The results were visualized using SEM. Platelet adhesion and activation was investigated using live cell staining and SEM. Whole blood clotting kinetics were evaluated using a hemolysis assay and the results visualized using SEM. The results indicate that none of these materials are cytotoxic. Protein adsorption was highest on PET, and platelet adhesion was significantly higher on PET. However, the percentage of activated platelets and whole blood clotting kinetics was comparable on all materials. This work successfully creates a baseline against which the hemocompatibility of modified ePTFE, LLDPE and PET can be measured.Item Open Access Influence of polymeric nanowire topography on the differentiation of adipose-derived stem cells(Colorado State University. Libraries, 2014) Trujillo, Nathan Anthony, author; Popat, Ketul, advisor; Williams, John, committee member; Dasi, Lakshmi Prasad, committee member; Kipper, Matt, committee memberConsidering the many advances in tissue engineering, there are still significant challenges associated with restructuring, repairing, or replacing damaged tissue in the body. Recently biodegradable synthetic scaffolds have shown to be a promising alternative. However, based on the application, it is essential that the scaffold possess specific surface properties that promote cell-scaffold interactions and aid in extracellular matrix deposition. In this work, we present a novel solvent-free, template-synthesis technique for creating substrate-bound nanowire scaffolds from polycaprolactone, a biocompatible and biodegradable polymer. Nanowire surfaces were also fabricated from polycaprolactone that included 1 wt% hydroxyapatite nanoparticles for osteogenic studies. The fundamental concept behind successful synthetic tissue-engineered scaffolds is to promote progenitor cell migration, adhesion, proliferation, and induce differentiation, extracellular matrix synthesis, and finally integration with host tissue. Recently, lipoaspirate tissue has been identified as a viable alternative source for mesenchymal stem cells because it contains a supportive stroma that can easily be isolated. Adipose derived stem cells can differentiate into a variety of mesodermal lineages including the osteogenic, chondrogenic, and adipogenic phenotypes. The results indicated that during the growth period i.e., initial 7 days of culture, the nanowire surfaces supported adhesion, proliferation, and viability of the cells in addition to morphological changes. Osteogenic, chondrogenic, and adipogenic differentiation potential of adipose derive stem cells was evaluated with and without differentiation supplements to determine the influence of nanowire architecture on mechanotrasnduction. It was determined that nanowire topography stimulated the expression of osteogenic marker proteins, osteocalcin and osteopontin, as well as mineralization and alkaline phosphatase activity.Item Open Access Investigating the osteogenic potential of multipotent mesenchymal stromal cells through the use of DNA microarray technology and biomaterial nanotopography(Colorado State University. Libraries, 2011) Berger, Dustin, author; Prasad, Ashok, advisor; Popat, Ketul, advisor; Deluca, Jennifer, committee memberTo view the abstract, please see the full text of the document.Item Open Access Investigation of adipose-derived mesenchymal stem cells interaction with electrospun demineralized bone matrix nanofiber scaffolds(Colorado State University. Libraries, 2016) Yaprak Akgul, Selin, author; Kipper, Matt, advisor; Popat, Ketul, advisor; Bailey, Travis, committee member; Ehrhart, Nicole, committee memberNanofiber demineralized bone matrix (DBM) scaffolds were fabricated by electrospinning, and their ability to support cell adhesion and cell viability of murine adipose-derived mesenchymal stem cells (AD-MSCs) for short-term in culture media was investigated. Poly (ε-caprolactone) (PCL) scaffolds were used as control surfaces. Live cell stain calcein-AM and CellTiter 96® Non-Radioactive Cell Proliferation assays were used for cell adhesion and cell proliferation, respectively. DBM scaffolds supported greater cell adhesion compared to PCL nanofiber scaffolds. For cell viability, the two types of scaffolds behaved similarly. The results led to further research on DBM scaffolds. The ability to support osteoblastic differentiation of AD-MSCs for long-term (three weeks) in osteogenic differentiation media was also investigated. Both PCL scaffolds and DBM scaffolds seeded with no cells were used as control surfaces. The total protein content of viable AD-MSCs on the scaffolds was assessed by bicinchoninic acid (BCA) assay. Nanofiber scaffolds displayed increased levels of alkaline phosphatase (ALP) activity for the first week for all cases. ALP activity dropped after one week. Scanning electron microscopy (SEM) and alizarin calcium staining techniques were used to examine mineralization patterns qualitatively on DBM and PCL nanofiber scaffolds. DBM scaffolds deposited more calcium mineral than PCL scaffolds during three-week experiments. Mineralization was quantified by energy-dispersive X-ray spectroscope (EDS). After three weeks of culture, EDS revealed high calcium and phosphorus deposition on DBM scaffolds compared to PCL controls. The DBM scaffolds exhibited increased mineralization over three weeks, both with and without cells. These results demonstrate that the adhesion, proliferation, and osteogenic differentiation of AD-MSCs were influenced by DBM scaffolds.Item Open Access Multifunctional nanowire scaffolds for neural tissue engineering applications(Colorado State University. Libraries, 2012) Bechara, Samuel Leo, author; Popat, Ketul, advisor; Tobet, Stuart, committee member; Legare, Marie, committee member; Rollin, Bernard, committee member; Sladek, John, committee memberUnlike other regions of the body, the nervous system is extremely vulnerable to damage and injury because it has a limited ability to self-repair. Over 250,000 people in the United States have spinal cord injuries and due to the complicated pathophysiology of such injuries, there are few options available for functional regeneration of the spinal column. Furthermore, peripheral nerve damage is troublingly common in the United States, with an estimated 200,000 patients treated surgically each year. The current gold standard in treatment for peripheral nerve damage is a nerve autograft. This technique was pioneered over 45 years ago, but suffers from a major drawback. By transecting a nerve from another part of the body, function is regained at the expense of destroying a nerve connection elsewhere. Because of these issues, the investigation of different materials for regenerating nervous tissue is necessary. This work examines multi-functional nanowire scaffolds to provide physical and chemical guidance cues to neural stem cells to enhance cellular activity from a biomedical engineering perspective. These multi-functional scaffolds include a unique nanowire nano-topography to provide physical cues to guide cellular adhesion. The nanowires were then coated with an electrically conductive polymer to further enhance cellular activity. Finally, nerve growth factor was conjugated to the surface of the scaffolds to provide chemical cues for the neural stem cells. The results in this work suggest that these multifunctional nanowire scaffolds could be used in vivo to repair nervous system tissue.Item Open Access Plasma processing for nanostructured topographies(Colorado State University. Libraries, 2012) Riedel, Nicholas Alfred, author; Williams, John, advisor; Popat, Ketul, advisor; Radford, Donald, committee member; Reynolds, Melissa, committee memberPlasma and directed ion interactions with materials have been widely observed to create complex surface patterns on a micro- and nano- scale. Generally, these texturizations are byproducts of another intended application (such as a feature formation on a sputtering target) and patterning is considered inconsequential or even detrimental. This work examined the possibility of using these phenomena as primary methods for producing beneficial topographies. Specifically, investigations focused on the use of helium plasma exposure and directed ion etching to create nanostructured surfaces capable of affecting biological interactions with implanted materials. Orthogonal argon ion etching and low energy helium plasma texturization of titanium were considered for use on orthopedic and dental implants as a means of increasing osteoblast activity and bone attachment; and oblique angle etching was evaluated for its use in creating topographies with cell deterrent or anti-thrombogenic properties. In addition, the helium driven evolution of surface features on 6061 aluminum alloy was characterized with respect to ion energy and substrate temperature. These surfaces were then considered for ice phobic applications.Item Open Access Sputter deposited hydroxyapatite thin films to enhance osseointegration(Colorado State University. Libraries, 2010) Riedel, Nicholas Alfred, author; Williams, John D., advisor; Popat, Ketul, advisor; Prieto, Amy L. (Amy Lucia), committee memberAs the demand for hip and knee replacements continues to grow, researchers look to increase the operational lifetimes of these implants. Many of these implants fail as a result of aseptic loosening caused from repeated loading of these joints. It is thought that implant life could be extended by improving the interface between the implant and natural tissue. To this effect, hydroxyapatite coatings have been demonstrated to improve implant to bone bonding and allow a more natural integration of the metallic substrates. This work explores the potential of using ion beam etching and sputter deposition to produce a hydroxyapatite thin film with a unique surface topography that would potentially enhance osseointegration. First, the effects of ion etching bare titanium were evaluated. Three ion energies (300 eV, 700 eV, and 1100 eV) were used to etch either as-received or polished substrates. Topographical changes were examined by scanning electron microscopy. Rat mesenchymal stem cells were differentiated to osteoblasts to test the biocompatibility of the surfaces with bone cells. It was found that ion etching the titanium increases cellular activity, and an ion energy of 700 eV appears to create the most beneficial topography. Hydroxyapatite thin films were then sputter deposited on titanium substrates etched at 700 eV. After the coatings were deposited, some of the hydroxyapatite films were re-etched in efforts to induce a unique topography. It was found that the hydroxyapatite coatings improved short term cell response but degraded over the course of the culture. Further investigation showed the as-sputtered coatings were amorphous. To prevent degradation of the coatings, annealed films were then prepared by heat treating at 600 °C for 2 hours. X-ray diffraction was used to confirm the presence of a crystalline hydroxyapatite phase. Films were immersed in culture media for four weeks, showing no signs of degradation. Ion etching performed on the substrates post annealing yielded a unique topography in the hydroxyapatite film. A final study was conducted evaluating the MSC response to the annealed and post-anneal etched films. It was found that the post-anneal etched hydroxyapatite coating had the highest cellular activity, indicating that this preparation may be an effective means to enhance osseointegration on medical implants.Item Open Access Titania nanotube arrays: interfaces for implantable devices(Colorado State University. Libraries, 2012) Smith, Barbara Symie, author; Popat, Ketul, advisor; Gonzalez-Juarrero, Mercedes, committee member; Prasad, Ashok, committee member; Dasi, Lakshmi Prasad, committee member; Dow, Steven, committee memberFor the 8-10% of Americans (20-25 million people) that have implanted biomedical devices, biomaterial failure and the need for revision surgery are critical concerns. The major causes for failure in implantable biomedical devices promoting a need for re- implantation and revision surgery include thrombosis, post-operative infection, immune driven fibrosis and biomechanical failure. The successful integration of long-term implantable devices is highly dependent on the early events of tissue/biomaterial interaction, promoting either implant rejection or a wound healing response (extracellular matrix production and vasculature). Favorable interactions between the implant surface and the respective tissue are critical for the long-term success of any implantable device. Recent studies have shown that material surfaces which mimic the natural physiological hierarchy of in vivo tissue may provide a possible solution for enhancing biomaterial integration, thus preventing infection and biomaterial rejection. Titania nanotube arrays, fabricated using a simple anodization technique, provide a template capable of promoting altered cellular functionality at a hierarchy similar to that of natural tissue. This work focuses on the fabrication of immobilized, vertically oriented and highly uniform titania nanotube arrays to determine how this specific nano-architecture affects skin cell functionality, hemocompatibility, thrombogenicity and the immune response. The results in this work identify enhanced dermal matrix production, altered hemocompatibility, reduced thrombogenicity and a deterred immune response on titania nanotube arrays. This evidences promising implications with respect to the use of titania nanotube arrays as beneficial interfaces for the successful implantation of biomedical devices.