Polysaccharide-based nanostructures for growth factor delivery and mesenchymal stem cell activation

2011 Summer.Includes bibliographical references.Mesenchymal stem cells (MSCs) are very promising in tissue engineering and regenerative medicine because of their ability to differentiate into different type of cells including bone and cartilage. MSCs differentiation can be modulated using both chemical (i.e. proteins) and physical cues (ie. topography). This thesis presents work performed evaluating polysaccharide-based nanostructures for growth factor delivery and MSCs activation. Different polysaccharide-based nanostructures were developed and characterized including polyelectrolyte multilayers (PEMs) and electrospun nanofibers. On flat gold-coated glass surfaces, PEMs were constructed using the polycations chitosan and N,N,N -trimethyl chitosan, and the polyanions hyaluronan, chondroitin sulfate, and heparin. An exhaustive spectroscopic study was performed on all of the PEMs pairs to investigate the effects of polyelectrolyte charge density on thickness, swelling, composition, and ion-pairing. The results demonstrated that hydrophilicity and swelling are reduced when one polyelectrolyte is strong and the other is weak, while ion pairing is increased. The stability of adsorbed proteins to PEMs was also investigated using IR spectroscopy. Construction of PEMs and adsorption of basic fibroblast growth factor (FGF-2) was evaluated on heparin chitosan PEMs constructed on gold-coated glass, tissue culture polystyrene (TCPS), and titanium. In vitro testing of the FGF-2-loaded PEM constructed on TCPS and titanium was performed using ovine bone marrow-derived MSCs. It was noted that FGF-2 activity is enhanced, with regards to MSCs proliferation, when delivered from PEMs compared to delivery in solution. Chitosan nanofibers were successfully electrospun from a trifluoroacetic acid and dichloromethane solution. A new technique was developed to modify electrospun chitosan nanofibers with polyelectrolyte multilayers using N,N,N -trimethyl chitosan and heparin. Controlled release of bioactive FGF-2, complexed with heparin-chitosan polyelectrolyte complex nanoparticles, from electrospun chitosan nanofiber mats was achieved with zero order kinetics over a period of 27 days. When the nanofibers are further modified with a single PEM bilayer (PEM, composed of N,N,N -trimethyl chitosan and heparin), the release is completely prevented. The mitogenic activity of the released FGF-2 was also evaluated, with respect to the proliferation of ovine bone marrow-derived MSCs. The effect on osteogenic differentiation of bone marrow-derived ovine and equine MSCs seeded on electrospun chitosan nanofibers versus flat TCPS was investigated. The effect of dexamethasone on osteogenic differentiation was also investigated. We found that we can successfully grow and maintain both equine and ovine MSCs on electrospun chitosan nanofibers. Also, both MSCs exhibit higher differentiation markers (alkaline phosphatase activity) when cultured on chitosan nanofibers compared to flat TCPS surfaces. This work demonstrates new systems for stabilizing and controlling the delivery of heparin-binding growth factors for the activation of bone marrow-derived MSCs, using polysaccharide-based nanomaterials. These novel materials have potential applications in musculoskeletal tissue regeneration

Role of retinal pigment epithelium-derived exosomes and autophagy in new blood vessel formation

Autophagy and exosome secretion play important roles in a variety of physiological and disease states, including the development of age‐related macular degeneration. Previous studies have demonstrated that these cellular mechanisms share common pathways of activation. Low oxidative damage in ARPE‐19 cells, alters both autophagy and exosome biogenesis. Moreover, oxidative stress modifies the protein and genetic cargo of exosomes, possibly affecting the fate of surrounding cells. In order to understand the connection between these two mechanisms and their impact on angiogenesis, stressed ARPE‐19 cells were treated with a siRNA‐targeting Atg7, a key protein for the formation of autophagosomes. Subsequently, we observed the formation of multivesicular bodies and the release of exosomes. Released exosomes contained VEGFR2 as part of their cargo. This receptor for VEGF—which is critical for the development of new blood vessels—was higher in exosome populations released from stressed ARPE‐19. While stressed exosomes enhanced tube formation, exosomes became ineffective after silencing VEGFR2 in ARPE‐19 cells and were, consequently, unable to influence angiogenesis. Moreover, vessel sprouting in the presence of stressed exosomes seems to follow a VEGF‐independent pathway. We propose that abnormal vessel growth correlates with VEGFR2‐expressing exosomes release from stressed ARPE‐19 cells, and is directly linked to autophagy

Bilayer Electrospun Membranes For Treating Fracking Wastewater Via Membrane Distillation

A bilayer electrospun membranes for treating hydraulic fracking wastewater via membrane distillation, and more particularly to bilayer electrospun membranes having an omniphobic layer to prevent low-surface tension solution wicking and an oleophobic antifouling surface to prevent foulant depositing on the membrane. Nanoparticles are decorated on the omniphobic surface through electrochemical interaction, which is coated with a fluorine monomer on the nanoparticles. A zwitterionic co-polymer is grafted using self-assembly between hydroxy groups on the antifouling surface generated by alkaline treatment and anchor segment epoxy groups on zwitterionic co-polymer

Bilayer Electrospun Membranes For Treating Fracking Wastewater Via Membrane Distillation

A bilayer electrospun membranes for treating hydraulic fracking wastewater via membrane distillation, and more particularly to bilayer electrospun membranes having an omniphobic layer to prevent low-surface tension solution wicking and an oleophobic antifouling surface to prevent foulant depositing on the membrane. Nanoparticles are decorated on the omniphobic surface through electrochemical interaction, which is coated with a fluorine monomer on the nanoparticles. A zwitterionic co-polymer is grafted using self-assembly between hydroxy groups on the antifouling surface generated by alkaline treatment and anchor segment epoxy groups on zwitterionic co-polymer

Liquid Crystalline Collagen Assemblies as Substrates for Directed Alignment of Human Schwann Cells

Collagen is a key component of the extracellular matrix (ECM) and well-oriented domains of collagen are important for mimicking the local cell environment in vitro. While there has been significant attention directed towards the alignment of collagen, formation of large-scale oriented domains remains a key challenge. Type I collagen self-assembles to form liquid crystalline (LC) mesophases in acidic conditions at concentrations above 100 mg mL−1. The LC mesophase provides an efficient platform for large-scale alignment and patterning of collagen coated substrates. However, there still exist challenges related to solubilizing and processing of collagen at such high concentrations in order to replicate the native ECM. In this contribution, we report on centimeter-scale alignment in collagen-coated glass substrates using solutions that are well below the LC-forming concentrations. Importantly, we are also able to extend this method to macroscopic 3-D LC-collagen hydrogels with programmed anisotropy within them to create a mimic of the native ECM. We show that the orientation and aspect ratio of human Schwann cells are strongly coupled with the alignment of the collagen substrate/hydrogel. We use a simple model to estimate the critical magnetic field strength needed for a given concentration of collagen to permit macroscopic alignment-enabling guidance for future studies on alignment of collagen at high concentrations

Liquid Crystalline Assembly of Collagen for Deterministic Alignment and Spread of Human Schwann Cells

Collagen is a key component of the extracellular matrix and well-oriented domains of collagen are relevant for mimicking the local cell environment in vitro. While there has been significant attention directed towards the alignment of collagen, formation of large-scale oriented domains remains a key challenge. Type I collagen self-assembles to form liquid crystalline (LC) mesophases in acidic conditions at concentrations above 100 mg/ml. The LC mesophase provides an efficient platform for large-scale alignment and patterning of collagen coated substrates. However, there exist challenges related to solubilizing and processing of collagen at such high concentrations in order to replicate the native extra cellular matrix (ECM). In this contribution, we report on centimeter-scale alignment in collagen-coated glass substrates using solutions that are well below the LC-forming concentrations. Importantly, we are also able to extend this method to create a mimic of the native ECM via macroscopic 3-D collagen hydrogels with programmed anisotropy within them. We explain the formation of these uniform domains via shear-induced and magnetically-induced liquid crystallinity of the collagen solutions. We show that the orientation, spreading and aspect ratio of Human Schwann Cells (HSCs) all are strongly coupled with the alignment of the collagen substrate/hydrogel. We use a simple Metroplis-based model to reveal that a critical magnetic field strength exists for a given concentration of collagen, exceeding which, macroscopic alignment is permissible- enabling guidance for future studies on alignment of collagen at high concentrations

A Qualitative Ethnographic Case Study Exploring the Hispanic/Latinx Interpretations of Collegiate Aviation Safety Culture

Through a combination of ethnographic principles and a qualitative case study structure, this study strives to understand how Hispanic/ Latinx aviation students perceive the current aviation safety culture in their flight training program. Grounded in the reciprocal safety culture model, the researchers attempt to answer how does Hispanic/Latinx culture influence perceptions towards commitment to aviation safety? and what are Hispanic/Latinx students’ perceptions of their ability to influence aviation safety culture? Three major themes emerged from the data: behavioral signs of safety culture at the collegiate level, obstacles to a sound safety culture, and methods to improve the safety culture. Moreover, factors such as individualism, masculinity, access to economic resources, and language were prevalent in the findings on how Hispanic aviation students perceive their collegiate flight training safety culture. Future studies should explore the perception of different ethnic groups of aviation students at various geographical locations to identify any added layers of threat, behavioral attitudes, and safety compromises related to flight training

TPEA good practice guide: lessons for cross-border MSP from transboundary planning in the European Atlantic

This Good Practice Guide is the outcome of a project co-funded by the European Commission (DG Mare) called Transboundary Planning in the European Atlantic (TPEA), which ran from December 2012 to May 2014. The aim of the project was to demonstrate approaches to transboundary maritime spatial planning (MSP) in the European Atlantic region. This is one of a series of projects exploring the opportunities and challenges of carrying out cross-border MSP in Europe’s regional seas, making connections with integrated coastal management (ICM). TPEA focused on two pilot areas: one involving Portugal and Spain and the other Ireland and the United Kingdom. Despite distinct identities in the region relating to different traditions of planning and stages of MSP implementation, TPEA worked towards a commonly-agreed approach to transboundary MSP and developed principles of cross-border working which it is hoped will be of wider benefit. This guide presents these principles, illustrated with examples from the TPEA project

Biodegradable Microneedle Patch for Delivery of Meloxicam for Managing Pain in Cattle

Microneedle patches are a promising source for transdermal diffusion of macromolecules and are designed to painlessly penetrate the skin. In this study, a biodegradable chitosan microneedle patch to deliver meloxicam for managing pain in cattle was tested. The potential of reuse of the polymeric solution to fabricate the patches, optimization of fabrication, morphological analysis of the microneedle patch and analysis of preservation of the chemical composition after sterilization were evaluated. In-vitro analysis consisted of studying in-vitro penetration mechanical properties, compression testing analysis of microneedle patch, and in-vitro drug release analysis. In-vivo studies were performed to analyze the dissolution capability of the microneedle patch. Results regarding the physical characteristics, chemical composition, and mechanical properties confirmed that rheological properties of the chitosan solution, present significant differences over time, demonstrating that reusing the solution on the fourth day results in failure patches. Morphological characteristics and chemical composition studies revealed that the process of sterilization (ethylene oxide gas) needed for implanting the patches into the skin did not affect the properties of microneedle patches. In-vitro studies showed that approximately 33.02 ± 3.88% of the meloxicam was released over 7 days. A full penetration of the microneedles into the skin can be obtained by applying approximately 3.2 N. In-vivo studies demonstrated that microneedle patches were capable of swelling and dissolving, exhibiting a dissolution percentage of more than 50% of the original height of microneedle after 7 days. No abnormal tissue, swelling, or inflammation was observed in the implanted area. The results of this work show that chitosan biodegradable microneedle patches may be useful to deliver meloxicam to improve pain management of cattle with positive effects for commercial manufacturing

Prioritization of Candidate Biomarkers for Degenerative Aortic Stenosis through a Systems Biology-Based In-Silico Approach

Degenerative aortic stenosis is the most common valve disease in the elderly and is usually confirmed at an advanced stage when the only treatment is surgery. This work is focused on the study of previously defined biomarkers through systems biology and artificial neuronal networks to understand their potential role within aortic stenosis. The goal was generating a molecular panel of biomarkers to ensure an accurate diagnosis, risk stratification, and follow-up of aortic stenosis patients. We used in silico studies to combine and re-analyze the results of our previous studies and, with information from multiple databases, established a mathematical model. After this, we prioritized two proteins related to endoplasmic reticulum stress, thrombospondin-1 and endoplasmin, which have not been previously validated as markers for aortic stenosis, and analyzed them in a cell model and in plasma from human subjects. Large-scale bioinformatics tools allow us to extract the most significant results after using high throughput analytical techniques. Our results could help to prevent the development of aortic stenosis and open the possibility of a future strategy based on more specific therapies