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Browsing by Author "Ehrhart, Nicole, committee member"

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    Characterization of osseointegrative phosphatidylserine and cholesterol orthopaedic implant coatings
    (Colorado State University. Libraries, 2013) Rodgers, William Paul, author; James, Susan, advisor; Popat, Ketul, committee member; Ehrhart, Nicole, committee member; De Long, Susan, committee member
    Total joint arthroplasties/replacements are one of the most successful surgeries available today for improving patients’ quality of life. By 2030 in the US, demand for primary total hip and knee arthroplasties are expected to grow by 174% and 673% respectively to a combined total of over 4 million procedures performed annually, driven largely by an ageing population and an increased occurrence of obesity. Current patient options for load-bearing bone integrating implants have significant shortcomings. Nearly a third of patients require a revision surgery before the implant is 15 years old, and those who have revision surgeries are at an increased risk of requiring additional reoperations. A recent implant technology that has shown to be effective at improving bone to implant integration is the use of phosphatidylserine (DOPS) coatings. These coatings are challenging to analyze and measure due to their highly dynamic, soft, rough, thick, and optically diffractive properties. Previous work had difficulty investigating pertinent parameters for these coating’s development due in large part to a lack of available analytical techniques and a dearth of understanding of the micro- and nano-structural configuration of the coatings. This work addresses the lack of techniques available for use with DOPS coatings through the development of original methods of measurement, including the use of scanning white light interferometry and nanoindentation. These techniques were then applied for the characterization of DOPS coatings and the study of effects from several factors: 1. the influence of adding calcium and cholesterol to the coatings, 2. the effect of composition and roughness on aqueous contact angles, and 3. the impact of ageing and storage environment on the coatings. This project lays a foundation for the continued development and improvement of DOPS coatings, which have the promise of significantly improving current patient options for bone integrating implants. Using these newly developed and highly repeatable quantitative analysis methods, this study sheds light on the microstructural configuration of the DOPS coatings and elucidates previously unexplained phenomena of the coatings. Cholesterol was found to supersaturate in the coatings at high concentration and phase separate into an anhydrous crystalline form, while lower concentrations were found to significantly harden the coatings. Morphological and microstructural changes were detected in the coatings over the course of as little as two weeks that were dependent on the storage environment. The results and understanding gained pave the path for focused future research effort. Additionally, the methods and techniques developed for the analysis of DOPS coatings have a broader application for the measurement and analysis of other problematic biological materials and surfaces.
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    Detection of bovine respiratory pathogens using real-time PCR and bead-based technologies
    (Colorado State University. Libraries, 2024) Holmes, Joey, author; Pabilonia, Kristy, advisor; Mayo, Christie, advisor; Ehrhart, Nicole, committee member
    The global cattle industry suffers financial losses of $900 million USD annually from infections caused by respiratory pathogens in the bovine respiratory disease complex (BRD). Accurate and timely detection of BRD pathogens provides cattle producers with a diagnosis so they can institute patient care and prevent pathogen spread. We sought to implement Luminex xTAG technology to detect four pathogens that cause BRD - bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), bovine herpes virus-1 (BHV-1), and Mycoplasma bovis (M. bovis). We compared singleplex real-time polymerase chain reaction (real-time PCR) to a newly developed xTAG testing protocol. Nucleic acids were extracted from 28 bovine lung samples that previously tested positive on PCR for each of the viral pathogens: BRSV (5), BVDV (5), BHV-1 (5), and M. bovis (5). All samples for BRSV and BHV-1 were detected on xTAG with a mean fluorescent index (MFI) well above 10,000 while detection of BVDV is limited to an MFI of 10,000 and M. bovis is detected inconsistently by xTAG. Lungs from six co-infected animals that tested positive for two BRD pathogens were tested on xTAG and real-time PCR side-by-side, revealing similar findings to the single positive lungs where BHV-1 and BRSV targets are more detectable than BVDV and M. bovis. Spiked pools of all pathogens resulted in MFI decreases as the number of pathogens per sample increases. With proper optimization, Luminex xTAG may be utilized in the veterinary diagnostic setting to circumvent issues with multiplex real-time PCR while maintaining high standards of diagnostic testing.
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    Evaluation of parathyroid hormone and zoledronic acid in promoting bone healing after stereotactic radiation therapy for local control of osteosarcoma in an orthotopic rat model
    (Colorado State University. Libraries, 2014) Curtis, Ryan C., author; VandeWoude, Sue, advisor; Donahue, Seth, advisor; Custis, Jamie, committee member; Ehrhart, Nicole, committee member; Ehrhart, EJ, committee member
    Clinical studies using definitive-intent stereotactic radiation therapy (SRT) for the local treatment of canine osteosarcoma (OSA) have achieved similar median survival times in patients as the current standard of care (amputation and adjuvant chemotherapy). Despite this, there remains an unacceptably high risk of pathologic fracture following radiation treatment. Zoledronic acid (ZA) and parathyroid hormone (PTH) are therapeutic candidates for decreasing this fracture risk post-irradiation. Due to differing mechanisms, we hypothesized that the combined treatment with ZA and PTH would significantly improve bone healing more than ZA or PTH treatment alone. Using an orthotopic model of canine osteosarcoma in athymic rats, we evaluated bone healing following clinically-relevant doses of radiation therapy (12Gy x 3 fractions, 36 Gy total). Groups included 36 Gy SRT only, 36 Gy SRT plus ZA , 36 Gy SRT plus ZA and PTH, 36 Gy SRT plus PTH, and 36 Gy SRT plus localized PTH treatment. Our study showed significant increases in bone volume and polar moments of inertia within the region of interest (distal femoral metaphysis) 8 weeks after radiation in the combined (ZA/PTH) treatment group as compared to radiation treatment alone. Histomorphometric analysis revealed evidence of active mineralization at study endpoint as well as successful tumor-cell kill across all treatment groups. This work provides further evidence for the expanding potential indications for ZA and PTH therapy, including post-irradiated bone disease due to canine osteosarcoma.
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    Identifying novel molecular mechanisms of healthspan using multi-omics
    (Colorado State University. Libraries, 2023) Smith, Meghan Elizabeth, author; LaRocca, Tom, advisor; Hamilton, Karyn, committee member; Broussard, Josiane, committee member; Ehrhart, Nicole, committee member
    An important goal in research on aging is to extend healthspan, the period of life spent healthy and disease-free. Next-generation sequencing and other emerging bioinformatics technologies (e.g., RNA-seq/transcriptomics, epigenetic profiling, and proteomics) have made it possible to broadly profile potential molecular mediators of aging, and perhaps identify therapeutic targets. The studies in this dissertation focus on using transcriptomics and complementary "multi-omics" strategies to characterize novel cellular mechanisms of aging, and to determine their relevance to systemic/functional health in humans. With the guidance of my mentoring team, I completed three studies in which I identified novel mediators of healthspan-related exercise training responsiveness, age-related inflammation, and cognitive/motor function decline in middle-aged and older adults. One particularly novel focus among these studies was the role of non-coding repetitive RNAs (derived from transposable elements) in healthspan. Transposable elements have been linked to known mechanisms of aging, and this topic is reviewed at the start of this dissertation to provide perspective on their role in the context of research on aging biology. Collectively, my findings represent new ideas for targetable genes and proteins that may influence human healthspan.
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    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 member
    Nanofiber 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.
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    Penetrative osseointegrative phospholipid coatings on 3D titanium lattice structures
    (Colorado State University. Libraries, 2012) Hudson, Hannah Katherine, author; James, Susan, advisor; Prawel, David, committee member; Ehrhart, Nicole, committee member
    Titanium is a commonly used material for implantable metallic devices though these devices still have many issues. The cost of implant surgery and the likely revision surgery that will follow is high. Cementless implants frequently fail due to aseptic loosening of the device, typically as a result of poor osseointegration. Phospholipids are naturally occurring substances that have been used to enhance new bone growth and integration of this bone with the implants. Electrospraying (e-spraying) is a method that uses electrical forces to drive source material to a target conductor. It typically has very high efficiency because it uses electrical charge to carry the material. This process also provides good control of coating morphology as this can be effected by the parameters used to e-spray. In our work the E-spraying technique was used to apply coatings of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) to Ti-6V-4Al porous lattice structures. These lattice structures are created using Electron Beam Melting (EBM). This manufacturing process is an additive process, part of the solid free form fabrication group, a subset of rapid prototyping. EBM enables precise control of complex geometries. When e-spraying these lattice structures can become Faraday cages when an electric field is applied to them. A Faraday cage is a conductor that becomes an equipotential surface when an electric field is applied and thus in its interior lacks an electric field. The exclusion of an internal electric field can inhibit to the e-spray process which relies on field lines to carry material to the target. In our work the Faraday cage effect was observed in two conditions, one in which the lattice structures were externally, circumferentially insulated and one in which the lattices were not insulated. Three different porosity lattices, with different pore sizes, were tested and all became Faraday cages when insulated and only the lowest porosity lattice became a strong Faraday cage when not insulated. The lattices that did coat did not exhibit conformal and uniform coatings when the Faraday cage effect was present. E-spray parameter variation was not able to mitigate the Faraday cage effect nor was it able to affect the morphology of the coatings. The surface topography of the structures is important for preferential cell adhesion and can be controlled using acid etching to modify the surface. In attempt to coat titanium lattice structures with a phospholipid coating this work discovered the Faraday cage effect as it relates to the electrospraying of phospholipids. It currently defines the limitations of the e-spray process as well as outlines what has been tried to mitigate the Faraday cage effect and discover how the Faraday cage effect changes coating morphology. In the future continuing work on mitigating the Faraday cage effect will be done as well as combining the e-spray process with one that uses a mechanical force to accelerate particles.
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    Phosphatidylserine and antibiotic coatings for allograft bone
    (Colorado State University. Libraries, 2014) Tait, Douglas, author; James, Susan, advisor; Ehrhart, Nicole, committee member; Deines, Susan, committee member
    Osteosarcoma is the most common type of primary bone tumor in humans. Treatment usually involves both surgical resection of the tumor and chemotherapy. Limb sparing often necessitates the use of massive bone allografts, however patients on anticancer drug regimens are at increased risk of infection, non-union and mechanical failure. The purpose of this work was to develop and test antibiotic eluting phospholipid coatings for massive bone allografts which may be useful in revision surgery for patients with osteomyelitis infection. This project was motivated by previous research performed on coatings of the phospholipid 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and the antibiotic Gentamicin Sulfate (GS) applied to metallic implants. The potential of these coatings to combat infection and enhance osseointegration was evaluated in vivo using massive femoral allografts in a murine model with a well established osteomyelitis infection. Phospholipid coatings were applied to decellularized mouse femur segments using an electrospray method. Antibiotic was incorporated between two DOPS layers. The presence of both DOPS and GS was verified by examining the treated allografts with a scanning electron microscope (SEM). Allografts were then prepared and implanted into 50 mice in seven different treatment groups. In four of these treatment groups the mice were deliberately infected with osteomyelitis one week prior to allograft implantation using a genetically modified bioluminescent strain of Staphylococcus aureus that enabled tracking of the infection in vivo. Mice were sacrificed at 28 days post allograft implantation and allografts were evaluated histologically. After completing the in vivo portion of the study, the antibiotic eluting characteristics of the coatings were analyzed in vitro with a total sink elution method and antibiotic in the eluent was quantified using an agar diffusion test. Results showed that mice receiving antibiotic coated allografts displayed significantly reduced infection up to fifteen days post allograft implantation. Measurable infection remained until the end of the study however and none of the infected mice exhibited any osseointegration with the allograft. These results were most likely due to the severity of the osteomyelitis infection and the rapid elution of the antibiotic from the allografts, as confirmed by the in vitro elution study. Osseointegration was observed in the uninfected mice however no statistically significant differences were found between the DOPS coated treatment groups and the uncoated control group. This was attributed to the small sample size of the uninfected groups and the small number of histological sections, and was perhaps exacerbated by inconsistent host-graft apposition. Further research is therefore necessary to validate the potential of DOPS/GS allograft coatings to fight infection and enhance osseointegration.