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ItemInduced Pluripotent Stem Cell-based in vitro Modeling of the Osteogenesis and Chondrogenesis of Juvenile Osteochondritis Dissecans(Georgia Institute of Technology, 2019-05) Nations, Catriana C.The application of pluripotent stem-cell based in vitro models has become increasingly popular in medical research, especially for situations in which animal modeling is not sufficient to accurately describe the condition or for diseases where there is little research demonstrating the relationships between disease phenotype, pathological cellular mechanisms, and gene expression. Such is the case with Juvenile Osteochondritis Dissecans (JOCD), a degenerative bone disease that predominately affects the knee joints of children and progresses to early onset osteoarthritis. Previous research has involved the use of animal models or diseases similar to JOCD, but there has been little to no focus on the cellular mechanisms of this condition. Therefore, this study aimed to elucidate the cellular pathophysiology of JOCD as well as provide a test bed for future therapeutic interventions. We hypothesized that our iPSC in vitro models of JOCD would show protein dysfunction and accumulation in the rough endoplasmic reticulum as a hallmark of the disease, as previously shown in familial and equine OCD.
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ItemQuantification of Microvessel Fragments from Primary Isolation Using 3-D Confocal Microscopy(Georgia Institute of Technology, 2017-12) Rather, Matthew HoltAngiogenesis, or the migration, growth, and differentiation of endothelial cells to form new blood vessels, is an essential component in any tissue engineering project. A variety of studies with transgenic and gene-targeted mice have demonstrated the importance of angiogenesis in fracture healing, and have provided insights into regulatory processes governing fracture angiogenesis; however, patterns of microvessel development before implementation and their effect on bone growth have yet to be quantified. Here we search to find the best quantification parameters of microvessels through 3-D confocal microscopy as they grow in-vitro before being seeded in a scaffold and delivered to a bone defect. Quantification will be done through both Amira and ImageJ software as each program has its strengths and weaknesses in image quantification.
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ItemEffects of Surface Modification on the Osseointegration Properties of Polyetheretherketone(Georgia Institute of Technology, 2017-12) Johnson, Allison-FranPolyetheretherketone (PEEK) is a thermoplastic polymer with many clinical orthopedic applications. With an elastic modulus similar to bone, PEEK is a preferred implant model due to specific mechanical properties. The limitations of PEEK arise from its poor osseointegration which in a clinical setting may cause implant slippage or dislodgement. Recent methods to improve the osseointegration of PEEK have involved surface modification, including the change of surface structure as well as surface chemistry. The purpose of this study is to characterize various surface structures of PEEK so that their effectiveness at implantation may be evaluated. Surface porous, soda blasted, and smooth injected molded PEEK as well as titanium oxide coated PEEK were evaluated for surface roughness using LEXT imaging. These roughness values will then later be compared with implant integration for each sample. Based on the optimized surface structure of PEEK, future studies will begin to characterize the optimal surface coating or surface chemistry for PEEK implants so that the inert properties of PEEK may be overcome in the clinical setting by surface modification.
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ItemDecellularized cartilage microcarriers as a novel platform for chondrogenic expansion(Georgia Institute of Technology, 2017-05) Marr, ElizabethOsteoarthritis is a degenerative disease associated with the degradation of cartilage. One of the few therapies aimed at regenerating cartilage, autologous chondrocyte implantation (ACI), involves ex vivo expansion and re-implantation of patient derived chondrocytes. Chondrocytes have relatively slow proliferation rates, however, and rapidly de-differentiate during the ex vivo culture performed with ACI. The objective of this study was to develop microcarriers (µCs) that provide a microenvironment that more closely mimics the complex extracellular matrix of native cartilage for chondrocyte expansion and retention of phenotype. Porcine cartilage was isolated, lyophilized, milled, and sifted overnight to obtain µCs approximately 450 µm and 600 µm in diameter. Multiple decellularization procedures were tested and ultimately a series of chemical and enzymatic washes resulted in removal of more than 98% of the original DNA content. Preliminary seeding experiments performed with chondrogenic ATDC5 cells resulted in chondrogenic proliferation and viability over 7 days of culture on decellularized cartilage microcarriers (DC µCs). Primary human chondrocytes were then cultured on the DC µCs, commercially available gelatin CultiSpher®-G (CG µCs), or tissue culture polystyrene over 14 days. Chondrocyte-laden DC µCs contained significantly more GAGs than the CG µCs or plated tissue culture polystyrene (TCPS) chondrocytes at all time points. Ongoing experiments are evaluating changes in chondrogenic phenotype on µCs through histological, immunohistochemical, and gene expression analysis. DC µCs support chondrocyte proliferation, and although the rate of expansion is slower than CG µCs or TCPS, these microcarriers have GAG/DNA ratios more similar to that of native cartilage. This makes DC µCs a promising platform for chondrogenic expansion and suitable for direct implantation.
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ItemCellular Characterization of Microvascular Fragments and Stromal Vascular Fraction for the Treatment of Composite Bone-Muscle Defects(Georgia Institute of Technology, 2017-05) Wang, YuyanThe current treatment for critical-sized boned defects, bone morphogenetic protein 2 (BMP-2) loaded collagen sponge grafting, is often inadequate for composite bone-muscle defects, which heal more slowly with higher rates of non- or malunion. Due the additional muscle defect, there is a vacancy of vasculature around the bone defect, causing insufficient nutrient and cytokine delivery and thus hindering bone regeneration. Hypothesizing that increasing angiogenesis will improve regeneration, we have previously proposed to co-deliver microvascular fragments (MVF) or stromal vascular fraction (SVF), which have been shown to mature into microvascular networks in vitro and in vivo, with BMP-2 in a collagen sponge. However, in preliminary studies, collagen sponge co-loaded with BMP-2 and MVF showed augmented release of BMP-2 compared to those co-loaded with SVF or with BMP-2 only. Although they are both derived from the same adipose tissue source, we hypothesize that their cellular composition may differ and thus affect BMP-2 kinetics. Therefore, this project aims to characterize the cellular components of MVF and SVF using flow cytometry to elucidate the potential mechanism of differential BMP-2 release. It was found that MVF has a relatively lower percentage of mesenchymal stem cells (MSCs) and relatively higher percentage of mature endothelial cells (ECs) than SVF, suggesting a role for ECs in the augmented BMP-2 release from MVF-loaded collagen sponges.
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ItemSmall Animal Model of Osteochondritis Dissecans(Georgia Institute of Technology, 2016-07-18) Cobb, Destiny AugustJuvenile Osteochondritis Dissecans (JOCD) is a joint disorder that predominantly affects athletic children and adolescents. It is characterized by the formation of osteochondral loose bodies which cause pain and swelling in the affected area, limit the patient’s activity level, and in some cases requires surgery to fix or remove loose bodies depending on the stage to which the disorder has progressed. There is interest in developing preclinical animal models of the disorder to be used as a tool for developing regenerative therapeutics for treating the disorder. The objective of this project is to develop a small animal model of JOCD using Lewis rats. The model is being developed through a series of pilot studies with iterative modifications made to the procedure. The surgical procedure aims to mechanically and chemically induce an initial lesion just below that subchondral bone, which would have a secondary effect on the overlaying cartilage over time. By drilling into the left medial femoral condyle, we are to create a 1 mm deep and 1mm wide defect. The right leg serves as the contralateral control. Pilot studies have relied on an injection of various doses of monosodium iodoacetate (MIA, in saline), a glycolytic inhibitor, into the cauterized drilled defect to induce the initial necrosis. The current procedure is being modified to create a thermal insult. 3 weeks post-surgery, the animals are euthanized and the morphology and integrity of the articular cartilage, including the thickness, volume, and attenuation, of both the left and right femurs are examined using EPIC-μCT. This is followed by histological analysis. Additionally, diseased and healthy human biopsy cores provided by Children’s Healthcare of Atlanta are being analyzed in a similar manner to provide a standard by which the model can be compared and its efficacy evaluated.