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Bioengineering Program

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Bone tissue engineering utilizing adult stem cells in biologically functionalized hydrogels

(Georgia Institute of Technology, 2013-04-09) Dosier, Christopher R.

Repair of large bone defects remains a clinical challenge for orthopedic surgeons. Current treatment strategies such as autograft and allograft are limited by the amount of available tissue in the case of the former, and failure of revascularization effecting engraftment in the case of the latter. Tissue engineering offers an alternative approach to this challenging clinical problem. The general principle of tissue engineering for bone regeneration prescribes delivery of osteoinductive factors to induce an endogenous response within the host to repair a defect that will not normally heal. One such tissue engineering approach is cell based therapy and this is attractive in the cases of patients with a lack of endogenous osteoprogenitors cells due to volumetric loss of tissue/ageing. Stem cell therapy has emerged as a possible alternative to current treatment modalities, however many challenges to clinical translation remain. Central to these challenges for bone tissue engineering are lingering questions of which cells to use and how to effectively deliver those cells. The goal of this thesis was to elucidate more effective ways to enhance bone repair utilizing adult stem cells. First, we investigated adipose derived stem cells (ADSCs) as a viable cell source for bone tissue engineering. Upon isolation, adipose derived stem cells are a heterogeneous population of multipotent cells predisposed to adipogenic differentiation. We developed an enrichment protocol that demonstrated the osteogenic potential of ADSCs can be enhanced in a dose dependent manner with resveratrol, which had been demonstrated to up-regulate Runx-2 expression. This enrichment strategy produced an effective method to enhance the osteogenic potential of ADSCs while avoiding cell sorting and gene therapy techniques, thus bypassing the use of xenogenic factors to obtain an enriched source of osteoprogenitor cells. This protocol was also used to investigate differences between human and rat ADSCs and demonstrated that rat ADSCs have a higher osteogenic potential than human ADSCs in vitro. The second major thrust of this thesis was to develop an injectable hydrogel system to facilitate bone formation in vivo. Both a synthetic and a naturally based polymer system was investigated, the results of which demonstrated that the naturally based alginate hydrogel was a more effective vehicle for both cell viability in vitro and bone formation in vivo. Our results also demonstrated that despite the ability to increase the osteogenic potential of ADSCs in vitro with resveratrol treatment, this was insufficient to induce bone formation in vivo. However, the inclusion of bone marrow mesenchymal stem cells (BMMSCs) in BMP-2 functionalized alginate hydrogels resulted in significantly greater mineralization than acellular hydrogels. Finally, the effect of timing of delivery of therapeutics to a non-healing segmental bone defect in the femur was investigated. We hypothesized that delivery of biologics after the initial inflammation response caused by injury to the host tissue would result in greater regeneration of tissue in terms of newly formed bone. Contrary to our initial hypothesis, these experiments demonstrated that delayed implantation of therapeutics has a detrimental effect on the overall healing response. It was, however, demonstrated that the inclusion of BMMSCs results in greater bone volume regenerated in the defect site over acellular hydrogels. In conclusion, this work has rigorously investigated the use of adipose derived stem cells for bone tissue engineering, and further produced an injectable hydrogel system for stem cell based bone tissue engineering. This work also demonstrated that the inclusion of adult stem cells, specifically BMMSCs, can enhance the regeneration response in a non-healing bone defect model relative to acellular hydrogel.
Effects of the mechanical microenvironment on early avian morphogenesis

(Georgia Institute of Technology, 2013-04-08) Henkels, Julia Ann

The objective of this work is to investigate the elastic modulus of gastrula-stage avian embryos and the effect of substrate stiffness on presumptive precardiac cell fate. Our overall hypothesis is that the mechanical microenvironment, specifically, tissue modulus and substrate stiffness, influences gastrulation and cardiac induction. Large-scale morphogenetic movements during early embryo development are driven by complex changes in biochemical and biophysical factors. Current models for amniote primitive streak morphogenesis and gastrulation take into account numerous genetic pathways but largely ignore the role of mechanical forces. Here, we used atomic force microscopy (AFM) to obtain for the first time precise biomechanical properties of the early avian embryo. Our data reveal that the primitive streak is significantly stiffer than neighboring regions of the epiblast, and that it is stiffer than the pre-primitive streak epiblast. To test our hypothesis that these changes in mechanical properties are due to a localized increase of actomyosin contractility, we inhibited actomyosin contractility via the Rho kinase (ROCK) pathway using the small-molecule inhibitor Y-27632. Our results using several different assays show the following: 1) primitive streak formation was blocked; 2) the time-dependent increase in primitive streak stiffness was abolished; and 3) convergence of epiblast cells to the midline was inhibited. Taken together, our data suggest that actomyosin contractility is necessary for primitive streak morphogenesis, and specifically, ROCK plays a critical role. To better understand the underlying mechanisms of this fundamental process, future models should account for the findings presented in this study. As presumptive cardiac cells traverse the course of differentiation into cardiac myocytes during cardiogenesis, the sequence, magnitude, and spatiotemporal map of biomechanical and biochemical signals has not been fully explored. There have been many studies detailing the induction of cardiogenesis on a variety of substrates and extracellular matrix (ECM) proteins, but none have completed a rigorous study of the effects of substrate stiffness on the induction of precardiac cells prior to the onset of cardiac gene expression (smooth muscle alpha actin [SMAA] at stage 5.) We investigate the effects of the mechanical environment on precardiac cell behaviors in an in vitro setting to elucidate the effect of substrate stiffness and inducing factors on precardiac tissue and the potential connection between them. The cells in the anterior portion of the primitive streak are fated to form the heart, and we show differing levels of SMAA expression on substrates of differing moduli, which suggests that substrate stiffness may play a role in cardiac differentiation. We cannot determine the physical mechanisms during morphogenesis without understanding the response of precardiac cells to changes in their mechanical environment.
Computational modeling reveals new control mechanisms for lignin biosynthesis

(Georgia Institute of Technology, 2012-08-16) Lee, Yun

Lignin polymers provide natural rigidity to plant cell walls by forming complex molecular networks with polysaccharides such as cellulose and hemicellulose. This evolved strategy equips plants with recalcitrance to biological and chemical degradation. While naturally beneficial, recalcitrance complicates the use of inedible plant materials as feedstocks for biofuel production. Genetically modifying lignin biosynthesis is an effective way to generate varieties of bioenergy crops with reduced recalcitrance, but certain lignin-modified plants display undesirable phenotypes and/or unexplained effects on lignin composition, suggesting that the process and regulation of lignin biosynthesis is not fully understood. Given the intrinsic complexities of metabolic pathways in plants and the technical hurdles in understanding them purely with experimental methods, the objective of this dissertation is to develop novel computational tools combining static, constraint-based, and dynamic, kinetics-based modeling approaches for a systematic analysis of lignin biosynthesis in wild-type and genetically engineered plants. Pathway models are constructed and analyzed, yielding insights that are difficult to obtain with traditional molecular and biochemical approaches and allowing the formulation of new, testable hypotheses with respect to pathway regulation. These model-based insights, once they are verified experimentally, will form a solid foundation for the rational design of genetic modification strategies towards the generation of lignin-modified crops with reduced recalcitrance. More generically, the methods developed in this dissertation are likely to have wide applicability in similar studies of complex, ill-characterized pathways where regulation occurring at the metabolic level is not entirely known.
Development of virtual mitral valve leaflet models from three-dimensional echocardiography

(Georgia Institute of Technology, 2012-07-05) Icenogle, David A.

Mitral valve (MV) disease is responsible for approximately 2,581 deaths and 41,000 hospital discharges each year in the US. Mitral regurgitation (MR), retrograde blood from through the MV, is often an indicator of MV disease. Surgical repair of MVs is preferred over replacement, as it is correlated with better patient quality of life. However, replacement rates are still near 40% because MV surgical repair expertise is not spread across all hospitals. In addition, 15-80% of surgical repair patients have recurrent MR within 10 years. Quantitative patient-specific models could aid these issues by providing less experienced surgeons with additional information before surgery and a quantitative map of patient valve changes after surgery. Real-time 3D echocardiography (RT3DE) can provide high quality 3D images of MVs and has been used to generate quantitative models previously. However, there is not currently an efficient, dynamic, and validated method that is fast enough to use in common practice. To fill this need, a tool to generate quantitative 3D models of mitral valve leaflets from RT3DE in an efficient manner was created. Then an in vitro echocardiography correction scheme was devised and a dynamic, in vitro validation of the tool was performed. The tool demonstrated that it could generate dynamic, complex MV geometry accurately and more efficiently than current methods available. In addition, the ability for mesh interpolation techniques to reduce segmentation time was demonstrated. The tool generated by this study provides a method to quickly and accurately generate MV geometry that could be applied to dynamic patient specific geometry to aid surgical decisions and track patient geometry changes after surgery.
Impaired signaling in senescing T cells: investigation of the role of reactive oxygen species using microfluidic platforms and computational modeling

(Georgia Institute of Technology, 2012-06-21) Rivet, Catherine-Aurélie

The goal of cancer immunotherapies is to boost the immune system's ability to detect tumor antigens and mount an effective anti-tumor immune response. Currently, adoptive T cell transfer therapy (ACT), the administration of ex vivo expanded autologous tumor-specific T cells, is one of the most promising immunotherapies under development; however, its efficacy has been limited so far with a mere 10% complete remission rate in the most successful clinical trials. The prolonged ex vivo culture process is a potential reason for this ineffectiveness because the transfused cells may reach replicative senescence and immunosenescence prior to patient transfer. The objective of this thesis is to offer two approaches towards an improvement of treatment efficacy. First, we generated a 'senescence metric' from the identification of biomarkers that can be used in the clinic towards predicting age and responsiveness of ex vivo expanded T cells. The second approach is to understand at the molecular level the changes that occur during ex vivo expansion to devise improved ACT protocols. In particular, we focused on the shift towards a pro-oxidizing environment and its potential effects on calcium signaling. The combined development and application of microfluidic technologies and computational models in this thesis facilitated our investigations of the phenotypic and signaling changes occurring in T cells during the progression towards immunosenescence. Our findings of altered T cell properties over long term culture provide insight for the design of future cancer immunotherapy protocols.
Controlling the microenvironment of human embryonic stem cells: maintenance, neuronal differentiation, and function after transplantation

(Georgia Institute of Technology, 2011-11-14) Drury-Stewart, Danielle Nicole

Precise control of stem cell fate is a fundamental issue in the use of human embryonic stem (hES) cells in the context of cell therapy We examined three ways in which the microenvironment can be controlled to alter hES cell behavior, providing insight into the best conditions for maintenance of pluripotency and neural differentiation in developmental and therapeutic studies. We first examined the effects of polydimethylsiloxane (PDMS) growth surfaces on hES cell survival and maintenance of pluripotency. Lightly cured, untreated PDMS was shown to be a poor growth surface for hES cells. Some of the adverse effects caused by PDMS could be mitigated with increased curing or UV treatment of the surface, but neither modification provided a growth surface that supported pluripotent hES cells as well as polystyrene. This work provides a basis for further optimizing PDMS for hES cell culture, moving towards the use of microdevices in establishing precise control over stem cell fate. The second study explored the use of an easily constructed diffusion-based device to grow hES cells in culture on a defined, physiologic oxygen (O₂) gradient. We observed greater hES cell survival and higher levels of pluripotency markers in the lower O₂ regions of the gradient. The greatest benefit was observed at O₂ levels below 5%, narrowing the potential optimal range of O₂ for the maintenance of pluripotent hES cells. Finally, we developed a small molecule-mediated adherent and feeder-free neural differentiation protocol that reduced the cost and time scale for in vitro differentiation of neural precursors and functional neurons from human pluripotent cells. hES cell-derived neural precursors transplanted into a murine model of focal ischemic stroke survived, improved neurogenesis, and differentiated into neurons. Transplant also led to a more consistent and measurable sensory recovery after stroke as compared to untransplanted controls. This protocol represents a potentially translatable method for the generation of CNS progenitors from human pluripotent stem cells.
Bio-functionalized peg-maleimide hydrogel for vascularization of transplanted pancreatic islets

(Georgia Institute of Technology, 2011-11-08) Phelps, Edward Allen

Type 1 diabetes affects one in every 400-600 children and adolescents in the US. Standard therapy with exogenous insulin is burdensome, associated with a significant risk of dangerous hypoglycemia, and only partially efficacious in preventing the long term complications of diabetes. Pancreatic islet transplantation has emerged as a promising therapy for type 1 diabetes. However, this cell-based therapy is significantly limited by inadequate islet supply (more than one donor pancreas is needed per recipient), instant blood-mediated inflammatory reaction, and loss of islet viability/function during isolation and following implantation. In particular, inadequate revascularization of transplanted islets results in reduced islet viability, function, and engraftment. Delivery of pro-vascularization factors has been shown to improve vascularization and islet function, but these strategies are hindered by insufficient and/or complex release pharmacokinetics and inadequate delivery matrices as well as technical and safety considerations. We hypothesized that controlled presentation of angiogenic cues within a bioartificial matrix could enhance the vascularization, viability, and function of transplanted islets. The primary objective of this dissertation was to enhance allogenic islet engraftment, survival and function by utilizing synthetic hydrogels as engineered delivery matrices. Polyethylene glycol (PEG)-maleimide hydrogels presenting cell adhesive motifs and vascular endothelial growth factor (VEGF) were designed to support islet activities and promote vascularization in vivo. We analyzed the material properties and cyto-compatibility of these engineered materials, islet engraftment in a transplantation model, and glycemic control in diabetic subjects. The rationale for this project is to establish novel biomaterial strategies for islet delivery that support islet viability and function via the induction of local vascularization.
Incorporation of protease-sensitive biomaterial degradation and tensile strain for applications in ligament-bone interface tissue engineering

(Georgia Institute of Technology, 2011-11-02) Yang, Peter J.

The interface between tendon/ligament and bone tissue is a complex transition of biochemical, cellular, and mechanical properties. Investigating computational and tissue engineering models that imitate aspects of this interface may supply critical design parameters for designing future tissue replacements to promote increased biochemical and mechanical integration between tendon/ligament and bone. Strategies for modeling this tissue have typically focused on the development of heterogeneous structures to create gradients or multiphasic materials that mimic aspects of the transition. However, further work is required to elucidate the role of specific mechanical and material stimuli in recapitulating features of the tendon/ligament-bone insertion. In particular, in constructs that exhibit variation in both mechanical and biochemical properties, the interplay of mechanical, material, and chemical signals can complicate understanding of the particular factors at work in interface formation. Thus, the overall goal of this dissertation was to provide insight into the role of mechanical strain and scaffold degradability on cell behavior within heterogeneous biomaterials. Specifically, a method for determining cell vertical position within a degradable gel through a laminated interface was developed. A computational model was created to examine possible variation in local mechanical strain due to heterogeneity in mechanical properties and different interface geometries. Finally, the influence of biomaterial degradability on changes in encapsulated human mesenchymal stem cell morphology under response to cyclic mechanical strain was explored. Together, these studies provide insight into mechanical and material design considerations when devising tissue engineering strategies to regenerate the tendon/ligament-bone interface.
Rational design and synthesis of drug delivery platforms for treating diseases associated with intestinal inflammation

(Georgia Institute of Technology, 2011-08-29) Wilson, David Scott

Over 500 million people worldwide suffer from disease associated with intestinal inflammation, including gastric cancer, inflammatory bowel disease, h. pylori infections, and numerous viral and bacterial infections. Although potentially effective therapeutics exist for many of these pathologies, delivery challenges thwart their clinical viability. The objective of this work was to develop drug delivery platforms that could target toxic immunomodulatory therapeutics to diseased intestinal tissues. To meet this objective, we developed an oral delivery vehicle for siRNA and an NF-κB inhibiting nanoparticle that reduces drug-resistance. Small interfering RNA (siRNA) represents a promising treatment strategy for numerous gastrointestinal (GI) diseases; however, the oral delivery of siRNA to inflamed intestinal tissues remains a major challenge. In this presentation, we describe a delivery vehicle for siRNA, termed thioketal nanoparticles (TKNs), that can orally deliver siRNA to sites of intestinal inflammation, and thus inhibit gene expression in diseased intestinal tissue. Using a murine model of ulcerative colitis, we demonstrate that orally administered TKNs loaded with TNFα-siRNA (TNFα-TKNs) diminish TNFα messenger RNA (mRNA) levels in the colon and protect mice from intestinal inflammation. Activation of nuclear factor-κB (NF-κB) results in the expression of numerous prosurvival genes that block apoptosis, thus mitigating the efficacy of chemotherapeutics. Paradoxically, all conventional therapeutics for cancer activate NF-κB, and in doing so initiate drug resistance. Although adjuvant strategies that block NF-κB activation could potentiate the activity of chemotherapeutics in drug resistant tumors, clinical evidence suggests that current adjuvant strategies also increase apoptosis in non-malignant cells. In this presentation, we present a nanoparticle, formulated from a polymeric NF-κB-inhibiting prodrug, that target the chemotherapeutic irinotecan (CPT-11) to solid tumors, and thus abrogates CPT-11-mediated drug resistance and inhibits tumor growth. In order to maximize the amount of NF-κB inhibitor delivered to tumors, we synthesized a novel polymeric prodrug, termed PCAPE, that releases the NF-κB inhibitor caffeic acid phenethyl ester (CAPE) as its major degradation product. Using a murine model of colitis-associated cancer, we demonstrate that when administered systemically, CPT-11-loaded PCAPE-nanoparticles (CCNPs) are three time more effective than a cocktail of the free drugs at reducing both tumor multiplicity and tumor size.
Novel nanocarriers for invasive glioma

(Georgia Institute of Technology, 2011-07-08) Munson, Jennifer Megan

The invasive nature of glioblastoma (GBM) represents a significant challenge to the standard of care and contributes to poor clinical outcomes. Invasion of tumors into healthy brain restricts chemotherapeutic access and complicates surgical resection. The central hypothesis of the thesis is that an effective anti-invasive agent can enhance the standard chemotherapeutic response in invasive brain tumors. Through a screen of novel compounds, a new anti-invasive small molecule, Imipramine Blue (IB), was identified. This triphenylmethane compound inhibits invasion of highly invasive glioma in vitro and in vivo. To elicit a response in vivo, Imipramine Blue was liposomally encapsulated to yield better delivery to tumor. Using this formulation, it is shown that IB attenuates invasion of glioma in vivo leading to a more compact tumor in an aggressively invasive rodent glioma model. Further, it is shown that this novel compound binds NADPH oxidases and alters expression of actin regulatory elements to elicit this anti-invasive activity. To test our hypothesis that anti-invasive therapy coupled with chemotherapy will enhance efficacy, nano-IB therapy was followed by liposomally encapsulated doxorubicin (DXR) chemotherapy. Additionally, a co-encapsulated formulation of IB and DXR was developed and tested in vivo. This combination therapy significantly enhanced survival compared to IB or DXR alone, resulting in long-term survival in the syngeneic invasive rat astrocytoma model RT2. It was seen that sequential treatment was more effective than the co-encapsulated treatment indicating a benefit of pre-treating the tumor with the anti-invasive. This thesis demonstrates that novel anti-invasive IB mediated 'containment' of diffuse glioma significantly enhances the efficacy of DXR chemotherapy compared to chemotherapy or anti-invasive therapy alone.