Development of 3D scaffolds for directional angiogenic network formation

The objective of this study is to investigate the possibility to produce surface modified polymeric fibres with the capability of both directional endothelial cell (EC) patterning and inducing angiogenesis in a 3D cell culture system. This study was conducted in three steps as following: 1) surface modification and characterization of materials, and more specifically, polymeric fibre surfaces, involving a multilayer, surface modification approach, using plasma polymer deposition methods, dextran and certain bioactive compounds grafting, to induce predictable biological responses. 2) In vitro evaluation of the surface modified polymer fibres towards EC behaviour in a 2D cell culture system. 3) In vitro evaluation of surface modified, polymer fibres towards EC behaviour (angiogenesis) in three 3D cell culture systems. Initially, the surfaces of 100-?m diameter poly (ethylene terephtalate) (PET) or polytetrafluoroethylene (PTFE) fibres (as monofilaments) were coated, utilising a multilayered-surface modification strategy, performed in three steps. These substrates were initially coated, using the radiofrequency glow discharge deposition technique, with a thin, polymeric interfacial bonding layer, produced via n-hepthylamine plasma polymer (HApp) or an acetaldehyde plasma polymer (AApp), having amine and aldehyde groups, respectively. Carboxy-methyl-dextran (CMD) was then covalently immobilized, using water-soluble carbodiimide chemistry (EDC/NHS), onto the surface amine groups, present either on the HApp-coated or on AApp-PEI-coated substrate surfaces. In the last step, GRGDS peptides were covalently immobilized onto the CMD-coated fibre surfaces. The second research theme involves the in vitro evaluation of surface modified substrates towards EC behaviour. For this purpose, human umbilical vein ECs (HUVECs) were seeded and grown on surfaces to evaluate the cell responses such as cell adhesion, spreading, cytoskeleton reorganization and cell orientation. Some alternative substrates were also examined in order to further characterize the cell behaviour. The cell behaviour was related with the surface physicochemical properties of the test modified surfaces. On CMD-coated substrates, cell adhesion was reduced, in contrast the amine-, aldehyde- and GRGDS-coated substrates promoted cell attachment and spreading and actin filaments and focal adhesions formations. Conversely, the reduced cell adhesion on GRGES (negative control), demonstrates that the increased EC adhesion, on GRGDS-grafted surfaces were attributed to specific biological responses of cell surface integrins towards the RGD ligands present on the surfaces. Cell adhesion was enhanced as the GRGDS solution concentration was increased from 0.1 mg/ml to 1 mg/ml. In comparison with"flat" substrates, fibre curvature promoted cell orientation along the fibre axis. In the third step, surface modified PET polymer fibres were evaluated towards"angiogenesis" in in vitro 3D tissue construct models, using three different methods of cell seeding. The result showed that angiogenesis process occurred when either HUVEC pre-coated fibres were embedded in fibrin gel, or HUVECs and cell-free fibres were sandwiched together between two layers of fibrin gel. These results suggest that the physical effect of the fibres, in conjunction with the surface chemistry, promotes in vitro EC attachment, induces angiogenesis and enhances directional angiogenic structures formation in the fibrin-based models. By prolonging the incubation period, the number of angiogenic structures increased and a network was formed, in which angiogenic structures interconnected to each other, from one fibre to another, following an optimal fibre spacing ranging from 200 to 600 [mu]m. These results demonstrate that through the use of a fibrous polymeric material that is surface coated with cell adhesive materials, in particular with extracellular matrix components such as RGD peptide or gelatin, it becomes possible to both enhance and direct the angiogenic process. Therefore, the two main goals of this study which were (i) to establish the feasibility of pre-vascularizing an in vitro tissue construct, and (ii) to influence the guidance of microvessel growth in a pre-determined direction, using phenomena known as"contact guidance" by means of polymer fibers and as"signaling molecules" by means of ligand-integrin interactions were largely achieved."--Résumé abrégé par UMI

A Facile Approach for the Mass Production of Submicro/Micro Poly (Lactic Acid) Fibrous Mats and Their Cytotoxicity Test towards Neural Stem Cells

Despite many of the studies being conducted, the electrospinning of poly (lactic acid) (PLA), dissolved in its common solvents, is difficult to be continuously processed for mass production. This is due to the polymer solution droplet drying. Besides, the poor stretching capability of the polymer solution limits the production of small diameter fibers. To address these issues, we have examined the two following objectives: first, using an appropriate solvent system for the mass production of fibrous mats with fine-tunable fiber diameters; second, nontoxicity of the mats towards Neural Stem Cell (NSC). To this aim, TFA (trifluoroacetic acid) was used as a cosolvent, in a mixture with DCM (dichloromethane), and the solution viscosity, surface tension, electrical conductivity, and the continuity of the electrospinning process were compared with the solutions prepared with common single solvents. The binary solvent facilitated PLA electrospinning, resulting in a long lasting, stable electrospinning condition, due to the low surface tension and high conductivity of the binary-solvent system. The fiber diameter was tailored from nano to micro by varying effective parameters and examined by scanning electron microscopy (SEM) and image-processing software. Laminin-coated electrospun mats supported NSC expansion and spreading, as examined using AlamarBlue assay and fluorescent microscopy, respectively

A Facile Approach for the Mass Production of Submicro/Micro Poly (Lactic Acid) Fibrous Mats and Their Cytotoxicity Test towards Neural Stem Cells

Despite many of the studies being conducted, the electrospinning of poly (lactic acid) (PLA), dissolved in its common solvents, is difficult to be continuously processed for mass production. This is due to the polymer solution droplet drying. Besides, the poor stretching capability of the polymer solution limits the production of small diameter fibers. To address these issues, we have examined the two following objectives: first, using an appropriate solvent system for the mass production of fibrousmats with fine tunable fiber diameters; second, nontoxicity of the mats towards Neural Stem Cell (NSC). To this aim, TFA(trifluoroacetic acid) was used as a cosolvent, in a mixture with DCM(dichloromethane), and the solution viscosity, surface tension, electrical conductivity, and the continuity of the electrospinning process were compared with the solutions prepared with common single solvents. The binary solvent facilitated PLA electrospinning, resulting in a long lasting, stable electrospinning condition, due to the low surface tension and high conductivity of the binary-solvent system. The fiber diameter was tailored from nano to micro by varying effective parameters and examined by scanning electron microscopy (SEM) and image-processing software. Laminin-coated electrospun mats supported NSC expansion and spreading, as examined using AlamarBlue assay and fluorescent microscopy, respectively

Development of 3D scaffolds for directional angiogenic network formation

The objective of this study is to investigate the possibility to produce surface modified polymeric fibres with the capability of both directional endothelial cell (EC) patterning and inducing angiogenesis in a 3D cell culture system. This study was conducted in three steps as following: 1) surface modification and characterization of materials, and more specifically, polymeric fibre surfaces, involving a multilayer, surface modification approach, using plasma polymer deposition methods, dextran and certain bioactive compounds grafting, to induce predictable biological responses. 2) In vitro evaluation of the surface modified polymer fibres towards EC behaviour in a 2D cell culture system. 3) In vitro evaluation of surface modified, polymer fibres towards EC behaviour (angiogenesis) in three 3D cell culture systems. Initially, the surfaces of 100-?m diameter poly (ethylene terephtalate) (PET) or polytetrafluoroethylene (PTFE) fibres (as monofilaments) were coated, utilising a multilayered-surface modification strategy, performed in three steps. These substrates were initially coated, using the radiofrequency glow discharge deposition technique, with a thin, polymeric interfacial bonding layer, produced via n-hepthylamine plasma polymer (HApp) or an acetaldehyde plasma polymer (AApp), having amine and aldehyde groups, respectively. Carboxy-methyl-dextran (CMD) was then covalently immobilized, using water-soluble carbodiimide chemistry (EDC/NHS), onto the surface amine groups, present either on the HApp-coated or on AApp-PEI-coated substrate surfaces. In the last step, GRGDS peptides were covalently immobilized onto the CMD-coated fibre surfaces. The second research theme involves the in vitro evaluation of surface modified substrates towards EC behaviour. For this purpose, human umbilical vein ECs (HUVECs) were seeded and grown on surfaces to evaluate the cell responses such as cell adhesion, spreading, cytoskeleton reorganization and cell orientation. Some alternative substrates were also examined in order to further characterize the cell behaviour. The cell behaviour was related with the surface physicochemical properties of the test modified surfaces. On CMD-coated substrates, cell adhesion was reduced, in contrast the amine-, aldehyde- and GRGDS-coated substrates promoted cell attachment and spreading and actin filaments and focal adhesions formations. Conversely, the reduced cell adhesion on GRGES (negative control), demonstrates that the increased EC adhesion, on GRGDS-grafted surfaces were attributed to specific biological responses of cell surface integrins towards the RGD ligands present on the surfaces. Cell adhesion was enhanced as the GRGDS solution concentration was increased from 0.1 mg/ml to 1 mg/ml. In comparison with"flat" substrates, fibre curvature promoted cell orientation along the fibre axis. In the third step, surface modified PET polymer fibres were evaluated towards"angiogenesis" in in vitro 3D tissue construct models, using three different methods of cell seeding. The result showed that angiogenesis process occurred when either HUVEC pre-coated fibres were embedded in fibrin gel, or HUVECs and cell-free fibres were sandwiched together between two layers of fibrin gel. These results suggest that the physical effect of the fibres, in conjunction with the surface chemistry, promotes in vitro EC attachment, induces angiogenesis and enhances directional angiogenic structures formation in the fibrin-based models. By prolonging the incubation period, the number of angiogenic structures increased and a network was formed, in which angiogenic structures interconnected to each other, from one fibre to another, following an optimal fibre spacing ranging from 200 to 600 [mu]m. These results demonstrate that through the use of a fibrous polymeric material that is surface coated with cell adhesive materials, in particular with extracellular matrix components such as RGD peptide or gelatin, it becomes possible to both enhance and direct the angiogenic process. Therefore, the two main goals of this study which were (i) to establish the feasibility of pre-vascularizing an in vitro tissue construct, and (ii) to influence the guidance of microvessel growth in a pre-determined direction, using phenomena known as"contact guidance" by means of polymer fibers and as"signaling molecules" by means of ligand-integrin interactions were largely achieved."--Résumé abrégé par UMI

Nano-niosomes as nanoscale drug delivery systems: an illustrated review.

The field of nanochemistry research has shown a great progress in the developing of novel nanocarriers as potential drug delivery systems. Niosome is a class of molecular cluster formed by self-association of non-ionic surfactants in an aqueous phase. The unique structure of niosome presents an effective novel drug delivery system (NDDS) with ability of loading both hydrophilic and lipophilic drugs. Numerous research articles have been published in scientific journals, reporting valuable results of individual case studies in this context. However, surveying and discussing the recent, rapidly growing reported studies along with their theoretical principals is required for the fully understanding and exploring the great potential of this approach. To this aim, we have provided an illustrated and comprehensive study from the view of a supramolecular chemist, interested in the synthesizing and studying chemical aggregates on the nanoscale for the development of nanotechnological clusters including niosomes. First, a connectional review of the molecular structure and physicochemical properties of niosome forming non-ionic surfactants and additive agents have been discussed. Second, a systematic survey of niosome preparation and loading methods, administration routes, characterization of niosomes, their toxicity studies and mechanism of drug release; used in recent articles have been performed

Formulation and Characterization of Bovine Serum Albumin-Loaded Niosome.

Niosomal vesicle, as a unique novel drug delivery system, is synthesized by non-ionic surfactants. Both hydrophilic and lipophilic drugs and also biomacromolecular agents, such as peptides and proteins can be encapsulated in this vesicular particle. Regarding polypeptide-based component loading, and delivery potential of the niosome, some valuable studies have been conducted in recent years. However, exploring the full potential of this approach requires fine tuned optimization and characterization approaches. Therefore, this study was conducted to achieve the following two goals. First, formulation and optimization of bovine serum albumin (BSA) load and release behavior as a function of cholesterol (CH) to sorbitan monostearate (Span 60) molar ratio. Second, investigating a cost- and time-effective polypeptide detecting method via methyl orange (MO) dye. To this aim, BSA-loaded niosomes were prepared by reversed-phase evaporation technique. The effect of CH to Sorbitan monostearate (Span 60) molar ratio on noisome entrapment efficiency (EE%) and release profile of BSA was studied using a ultraviolet (UV) spectrophotometer technique (NanoDrop 2000/2000c).Niosome with a 60% CH content showed the highest BSA EE% and release behavior. Then, BSA was dyed using MO in an acidic solution and used in BSA-niosome formulation. The MO-colored protein, loaded into the vesicles, was successfully assessed by an inverted light microscope, in order to observe the protein location in the vesicle. The results obtained in this study can be useful for various applications in different fields, including pharmaceutical, cosmetics, and drug delivery in biomedical and tissue engineering