Browsing by Author "Ehrhart, Nicole P., committee member"
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Item Open Access In vivo efficacy of antibiotic-eluting phospholipid coated implants(Colorado State University. Libraries, 2011) Triffo, Thomas, author; James, Susan P., advisor; Criswell, Marvin E., committee member; Ehrhart, Nicole P., committee memberImplant-associated infection can be a serious problem for patients that receive orthopedic implants, such as hip and knee replacements. This is a common cause for early implant loosening, which requires revision surgeries and results in an even greater risk of infection. To address this issue, our lab has developed a novel electrospraying technique for applying phospholipid coatings to orthopedic implants. These coatings consist of two layers of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), with antibiotic loaded in between layers. In vitro tests were performed to evaluate how modifications to these coatings affect coating retention, based on a clinically relevant test, and antibiotic elution from these coatings. Coating retention tests were performed by inserting implants through segments of mouse bone and then examining the implants under SEM. Antibiotic elution was performed using a total sink elution combined with OPA assay for detection of antibiotic. These results showed that the coatings that were retained the most and eluted antibiotic slowest were samples that were pre-treated with calcium and were electrosprayed with a mixture of 6:1 DOPS-to-cholesterol. This coating was selected to be used in an in vivo study to determine the efficacy of the coatings in treating osteomyelitis. Osteomyelitis was induced in a murine model using genetically modified bacteria, which allowed tracking of the infection prior to sacrificing the animals via bioluminescent imaging, a technique that makes use of genetically modified bacteria producing luciferin and luciferase which causes emission of photons. It was observed that antibiotic-eluting implants cleared the infection faster than implants without antibiotic during a 4 week study. Also, no kidney damage was observed based on creatinine, blood urea nitrogen, and urine protein tests. Histology confirmed observations from the bioluminescent imaging. These results show that our antibiotic-eluting implant coatings were able to reduce infection in vivo without resulting in adverse effects. Bioluminescent imaging showed significant reduction of emission of photons, p < 0.05, in the antibiotic loaded group compared to the control samples. The results also suggest that the implants exhausted their supply of antibiotic at the end of the study, and in future studies a greater amount of antibiotic will be loaded onto the implants.Item Open Access The osteogenic characterization and cryopreservation of equine bone marrow derived mesenchymal stem cells with SCAAV-equine-BMP-2(Colorado State University. Libraries, 2019) Ball, Alyssa N., author; Goodrich, Laurie R., advisor; Donahue, Seth W., committee member; McIlwraith, C. Wayne, committee member; Ehrhart, Nicole P., committee member; Kawcak, Christopher E., committee member; Bark, David L., Jr., committee memberOptimizing the environment of complex bone healing and improving treatment of catastrophic bone fractures and segmental bone defects remains an unmet clinical need both human and equine veterinary medical orthopedics. Animal models of fracture repair often involve small rodents, as historically significant large animal models, like the dog, continue to gain favor as companion animals. This trend continues despite the well documented limitations in comparing fracture repair in humans, as few similarities exist. Study design, number of studies, and availability of funding also continues to limit large animal studies. Osteoinduction is often therapeutically targeted to incite new bone growth. Osteoinduction with recombinant BMP-2 (rhBMP-2) results in robust bone formation; although, long-term quality is scrutinized due to poor bone mineral quality. Gene therapy continues to gain popularity among researchers to augment bone healing, and with the approval of the first cell-based gene therapy treatment in South Korea, the clinical reputation of gene therapy is under scrutiny. Progenitor cell therapies and the content variation of patient-side treatments (e.g. PRP and BMAC) are being studied in humans, while the immunologic properties of autologous and allogeneic treatments are being studied in the horse. The objective of this Masters Thesis was to determine whether scAAV-equine-BMP-2 transduced cells would induce osteogenesis in equine bone marrow derived mesenchymal stem cells (BMDMSCs) in vitro, and if these cells could be cryopreserved in an effort to osteogenically prime them as an "off-the-shelf" gene therapeutic approach for fracture repair. Our study found that transgene expression is altered by cell expansion, as would be expected by a transduction resulting in episomal transgene expression, and that osteoinductive levels could still be achieved five days after recovery. Further, protein expression continues up to fourteen days after initial transduction. This is the first evidence that cryopreservation of genetically modified BMDMSCs would not alter the osteoinductive potential or clinical use of allogeneic donor cells in cases of equine fracture repair. Future directions should include in vivo pilot data aimed at elucidating whether or not these cells expedite clinical healing. Other genes and gene combinations (e.g. PTH 1-34 and BMP-2) could be introduced to the expression vector backbone and compared to the vector utilized in this study.