Angiogenesis, the sprouting of new blood vessels from existing vasculature, is a complex biological process of interest to both the treatment of numerous pathologies and the creation of thick engineered tissues. In the context of tissue engineering, one potential solution to the diffusion limitation is to create a vascular network in vitro that can subsequently anastomose with the host after implantation, allowing the implantation of thicker, more complex tissues. In this study, the ability of endothelial cells to sprout and form stable vascular networks in 3-dimensional (3D) fibrin matrices was investigated as a function of matrix density in a prevascularized tissue model. The results demonstrate that while increasing matrix density leads to a nearly 7-fold increase in compressive stiffness, vascular sprouting is virtually eliminated in the most dense matrix condition. However, the addition of human mesenchymal stem cells (HMSCs) to the denser matrices reverses this effect, resulting in an up to a 7-fold increase in network formation. Although the matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MT1-MMP are all upregulated early on with the addition of HMSCs, MT1-MMP appears to play a particularly important role in the observed angiogenic response among these proteases. This study provides a means to design stiffer prevascularized tissues utilizing naturally derived substrates, and its results may yield new mechanistic insights into stem cell-based angiogenic therapies.

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Congenital mastoid cholesteatomas have a variable and nonspecific clinical presentation. Surgical challenges arise from the indolent nature of this clinical entity, which belies the extent of otologic involvement. Imaging with CT and magnetic resonance imaging are diagnostic, defines the extent of these lesions, and facilitates preoperative surgical planning.

Branched disulfide-containing poly(amido ethyleneimines) (SS-PAEIs) are biodegradable polymeric gene carrier analogs of the well-studied, non-degradable and often toxic branched polyethylenimines (bPEIs), but with distinct advantages for cellular transgene delivery. Clinical success of polycationic gene carriers is hampered by obscure design and formulation requirements. This present work reports synthetic and formulation properties for a graft copolymer of polyethylene glycol (PEG) and a branched SS-PAEI, poly(triethylentetramine/cystaminebisacrylamide) (p(TETA/CBA)). Several labs have previously demonstrated the advantages of PEG conjugation to gene carriers, but have also shown that PEG conjugation may perturb plasmid DNA (pDNA) condensation, thereby interfering with nanoparticle formation. With this foundation, our studies sought to mix various amounts of p(TETA/CBA) and p(TETA/CBA)-g-PEG2k to alter the relative amount of PEG in each formulation used for polyplex formation. The influence of different PEG/polycation amounts in the formulations on polymer/nucleic acid nanoparticle (polyplex) size, surface charge, morphology, serum stability and transgene delivery were studied. Polyplex formulations were prepared using p(TETA/CBA)-g-PEG2k, p(TETA/CBA), and mixtures of the two species at 10/90 and 50/50 volumetric mixture ratios (wt/wt %), respectively. As expected, increasing the amount of PEG in the formulation, adversely affects polyplex formation. However, optimal polymer mixtures could be identified using this facile approach to further clarify design and formulation requirements necessary to understand and optimize carrier stability and biological activity. This work demonstrates the feasibility to easily overcome typical problems observed when polycations are modified and thus avoids the need to synthesize multiple copolymers to identify optimal gene carrier candidates. This approach may be applied to other polycation-PEG preparations to alter polyplex characteristics for optimal stability and biological activity.

The pathogenesis of type-1 diabetes is complicated, and a clear, single mechanism has yet to be identified. Reports have indicated that the activating receptor NKG2D plays an important role in the development of disease. Exploiting a natural phenomenon observed in tumors, plasmid DNA encoding for a soluble ligand to NKG2D (sRAE-1γ) was isolated and engineered into a plasmid expression system. A polymeric gene delivery system was developed to deliver the soluble RAE-1 plasmid locally to the pancreatic islets for the prevention of type-1 diabetes. The bioreducible cationic polymer poly(cystamine bisacrylamide – diamino hexane) (p(CBA-DAH)) was modified with poly(ethylene glycol) (PEG) and the targeting peptide CHVLWSTRC, known to target the EphA2 and EphA4 receptors. The PEG serves to improve stability and tissue selectivity, while the peptide will target EphA2 and A4, overexpressed in the pancreatic microvasculature. The targeting polymer Eph-PEG-p(CBA-DAH) shows selective uptake by the target cell line, indicative of the targeting properties that will be seen in systemic administration. Using the delivery system, the therapeutic plasmid can be delivered to the pancreas, reduce interactions between the beta-cells and infiltrating NKG2D positive lymphocytes, and effectively protect beta-cells from autoimmune destruction and prevent type 1 diabetes.

The activating receptor NKG2D plays an important role in the development of type-1 diabetes. Exploiting a natural phenomenon observed in tumors, plasmid DNA encoding for a soluble ligand to NKG2D (sRAE-1γ) was isolated and engineered into a plasmid expression system. A polymeric gene delivery system was developed to deliver the soluble RAE-1 plasmid to the pancreatic islets. The bioreducible cationic polymer poly(cystamine bisacrylamide–diamino hexane) (p(CBA-DAH)) was modified with poly(ethylene glycol) (PEG) and the targeting peptide CHVLWSTRC, known to target the EphA2 and EphA4 receptors. We observed a higher uptake of the targeting polymer Eph-PEG-p(CBA-DAH) in the pancreas of NOD mice compared to non-targeting controls. To evaluate the efficacy of preventing diabetes, the Eph-PEG-p(CBA-DAH)/RAE-1 complex (polyplex) was intravenously injected into 6-week-old female NOD mice. Within 17 weeks blood glucose levels were stabilized in animals injected with polyplex, while those treated without therapeutic plasmid developed progressive hyperglycemia. Additionally, the degree of insulitis and the infiltration of CD8+ T-cells in the polyplex treated group were improved over the targeting polymer only treated group. The current study suggest that the therapy of the Eph-PEG-p(CBA-DAH) delivering therapeutic sRAE-1 gene may be used to protect β-cells from autoimmune destruction and prevent type-1 diabetes.

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