Influence of poly(n-isopropylacrylamide)-CNT-polyaniline three-dimensional electrospun microfabric scaffolds on cell growth and viability.

This study investigates the effect on: (1) the bulk surface and (2) the three-dimensional non-woven microfabric scaffolds of poly(N-isopropylacrylamide)-CNT-polyaniline on growth and viability of cells. The poly(N-isopropylacrylamide)-CNT-polyaniline was prepared using coupling chemistry and electrospinning was then used for the fabrication of responsive, non-woven microfabric scaffolds. The electrospun microfabrics were assembled in regular three-dimensional scaffolds with OD: 400-500 µm; L: 6-20 cm. Mice fibroblast cells L929 were seeded on the both poly(N-isopropylacrylamide)-CNT-polyaniline bulk surface as well as non-woven microfabric scaffolds. Excellent cell proliferation and viability was observed on poly(N-isopropylacrylamide)-CNT-polyaniline non-woven microfabric matrices in compare to poly(N-isopropylacrylamide)-CNT-polyaniline bulk and commercially available Matrigel™ even with a range of cell lines up to 168 h. Temperature dependent cells detachment behavior was observed on the poly(N-isopropylacrylamide)-CNT-polyaniline scaffolds by varying incubation at below lower critical solution temperature of poly(N-isopropylacrylamide). The results suggest that poly(N-isopropylacrylamide)-CNT-polyaniline non-woven microfabrics could be used as a smart matrices for applications in tissue engineering.

Transient charge-masking effect of applied voltage on electrospinning of pure chitosan nanofibers from aqueous solutions.

The processing of a polyelectrolyte (whose functionality is derived from its ionized functional groups) into a nanofiber may improve its functionality and yield multiple functionalities. However, the electrospinning of nanofibers from polyelectrolytes is imperfect because polyelectrolytes differ considerably from neutral polymers in their rheological properties. In our study, we attempt to solve this problem by applying a voltage of opposite polarity to charges on a polyelectrolyte. The application of this 'countervoltage' can temporarily mask or screen a specific rheological property of the polyelectrolyte, making it behave as a neutral polymer. This approach can significantly contribute to the development of new functional nanofiber materials.

A novel shortened electrospun nanofiber modified with a 'concentrated' polymer brush.

We report the fabrication of shortened electrospun polymer fibers with a well-defined concentrated polymer brush. We first prepared electrospun nanofibers from a random copolymer of styrene and 4-vinylbenzyl 2-bromopropionate, with number-average molecular weight Mn=105 200 and weight-average molecular weight Mw=296 700 (Mw/Mn=2.82). The fibers had a diameter of 593±74 nm and contained initiating sites for surface-initiated atom transfer radical polymerization (SI-ATRP). Then, SI-ATRP of hydrophilic styrene sodium sulfonate (SSNa) was carried out in the presence of a free initiator and the hydrophobic fibers. Gel permeation chromatography confirmed that Mn and Mw/Mn values were almost the same for free polymers and graft polymers. Mn agreed well with the theoretical prediction, and Mw/Mn was relatively low (<1.3) in all the examined cases, indicating that this polymerization proceeded in a living manner. Using the values of the graft amount measured by Fourier transform infrared spectroscopy, the surface area, and Mn, we calculated the graft density σ as 0.22 chains nm-2. This value was nearly equal to the density obtained on silicon wafers (σ=0.24 chains nm-2), which is categorized into the concentrated brush regime. Finally, we mechanically cut the fibers with a concentrated poly(SSNa) brush by a homogenizer. With increasing cutting time, the fiber length became shorter and more homogenous (11±17 µm after 3 h). The shortened fibers exhibited excellent water dispersibility owing to the hydrophilic poly(SSNa) brush layer.

In vitro and in vivo blood compatibility of concentrated polymer brushes.

Concentrated polymer brushes (CPBs) and semi-dilute polymer brushes (SDPBs) of poly(2-hydroxyethyl methacrylate), poly(2-hydroxyethyl acrylate), poly[poly(ethylene glycol)methyl ether methacrylate] (PPEGMA) and poly(2-methoxyetyl acrylate) were prepared on silica particles and silicon wafers by surface-initiated atom transfer radical polymerization (SI-ATRP). In order to evaluate in vitro blood compatibility, plasma protein adsorption on the brushes was quantified with a BCA protein assay, and the adsorbed proteins on the brushes were identified using high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). All four CPBs displayed much less protein adsorption than their corresponding SDPBs. Interestingly, the number and type of identified proteins differed on the brushes. Platelet adhesion was then examined on the brushes, whereby CPBs suppressed platelet adhesion to a greater extent than the corresponding SDPBs, although platelet activation was observed on all surfaces. As a result, the CPBs of PPEGMA prevented platelet adhesion the most. After screening the polymers by in vitro evaluation, CPBs of PPEGMA were then grafted on a catheter by SI-ATRP. The catheter with the CPBs was implanted into the jugular vein of a rabbit. The in vivo assessment after three weeks of implantation confirmed that the CPBs caused little coagulation or inflammation, whereas the pristine catheter exhibited inflammation and encapsulation.

Preparation of a Cage-Type Polyglycolic Acid/Collagen Nanofiber Blend with Improved Surface Wettability and Handling Properties for Potential Biomedical Applications.

Electrospun biobased polymeric nanofiber blends are widely used as biomaterials for different applications, such as tissue engineering and cell adhesion; however, their surface wettability and handling require further improvements for their practical utilization in the assistance of surgical operations. Therefore, Polyglycolic acid (PGA) and collagen-based nanofibers with three different ratios (40:60, 50:50 and 60:40) were prepared using the electrospinning method, and their surface wettability was improved using ozonation and plasma (nitrogen) treatment. The effect on the wettability and the morphology of pristine and blended PGA and collagen nanofibers was assessed using the WCA test and SEM, respectively. It was observed that PGA/collagen with the ratio 60:40 was the optimal blend, which resulted in nanofibers with easy handling and bead-free morphology that could maintain their structural integrity even after the surface treatments, imparting hydrophilicity on the surface, which can be advantageous for cell adhesion applications. Additionally, a cage-type collector was used during the electrospinning process to provide better handling properties to (PGA/collagen 60:40) blend. The resultant nanofiber mat was then incorporated with activated poly (α,ß-malic acid) to improve its surface hydrophilicity. The chemical composition of PGA/collagen 60:40 was assessed using FTIR spectroscopy, supported by Raman spectroscopy.

In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen.

Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 °C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhibited a typical D-periodic unit of ∼67 nm, irrespective of volume ratio of both collagens. Differential scanning calorimetry revealed only one endothermic peak for the fibrils derived from collagen mixture or from each collagen solution, indicating that the resultant collagen fibrils were hybrids of type I fish scale collagen and type I porcine collagen.

Dual-functional liposomes for curcumin delivery and accelerating silk fibroin hydrogel formation.

The administration of a drug-loaded implantable hydrogel at the tumor site after surgical resection is a viable approach to prevent the local recurrence or metastasis. Dimyristoyl glycerophosphorylglycerol (DMPG)-based liposomes were developed for inducing the rapid gelation of silk fibroin (SF) and delivering an anticancer drug, curcumin. Curcumin was loaded in the liposomes and the stability of curcumin was enhanced. The gelation time of liposome-induced SF hydrogels ranged from 3 min to more than 6 h. The biological activity of liposome-SF hydrogels was evaluated in vitro using L929 fibroblasts and MDA-MB-231 breast cancer cells. The release of curcumin can inhibit the growth of cancer cells. Both cells cultured on the surface of the hydrogels loaded with curcumin displayed low cell survival due to the combination of low cell attachment and cytotoxicity of curcumin. Liposome-SF hydrogels show potential as a sealant administered at the tumor site to eliminate residual cancer cells after tumor removal.

Endothelial cell distributions and migration under conditions of flow shear stress around a stent wire.

BACKGROUND: Blood vessels are constantly exposed to flow-induced stresses, and endothelial cells (ECs) respond to these stresses in various ways. OBJECTIVE: In order to facilitate endothelialization after endovascular implantation, cell behaviors around a metallic wire using a flow circulation system are observed. METHODS: A parallel flow chamber was designed to reproduce constant shear stresses (SSs) on cell surfaces and to examine the effects of a straight bare metal wire on cell monolayers. Cells were then exposed to flow for 24 h under SS conditions of 1, 2, and 3 Pa. Subsequently, cell distributions were observed on the plate of the flow chamber and on the surface of the bare metal wire. Flow fields inside the flow chamber were analyzed using computational fluid dynamics under each SS condition. RESULTS: After 24 h, ECs on the bottom plate were concentrated toward the area of flow reattachment. The matching of higher cell density and CFD result suggests that flow-induced stimuli have an influence on EC distributions. CONCLUSION: Typical cell concentration occurs on dish plate along the vortexes, which produces large changes in SSs on cell layer.

In vivo evaluation of a novel scaffold for artificial corneas prepared by using ultrahigh hydrostatic pressure to decellularize porcine corneas.

PURPOSE: To evaluate the stability and biocompatibility of artificial corneal stroma that was prepared by using ultrahigh hydrostatic pressurization treatment to decellularize corneas. METHODS: The porcine cornea was decellularized by two methods, a detergent method and an ultrahigh hydrostatic pressure (UHP) method. Either 1% w/v Triton X-100 or sodium dodecyl sulfate (SDS) was used for the detergent method, and 10,000 atmospheres (atm; 7.6x10(6) mmHg) was applied to the cornea for 10 min at 10 degrees C by a high-pressure machine for the UHP method. Hematoxylin-eosin staining was performed to confirm the removal of the corneal cells, and then decellularized porcine corneal stroma was implanted into rabbit corneal pockets. After eight weeks, the rabbit eyes were enucleated to examine the tissue compatibility of the implanted stroma. RESULTS: Complete decellularization was confirmed only in corneas treated by the UHP method, and little inflammation was seen when they were implanted into the rabbit corneal pockets. CONCLUSIONS: Porcine corneal stroma completely decellularized by the UHP method has extremely high biocompatibility and is a possible corneal scaffold for an artificial cornea.

Simple Fabrication of PVA-ZnS Composite Films with Superior Photocatalytic Performance: Enhanced Luminescence Property, Morphology, and Thermal Stability.

Poly(vinyl alcohol) (PVA)-ZnS composite films were prepared by varying the composition of PVA ranging from 1-5 wt % through a simple solvent casting method. The photocatalytic enactment of the composites was evaluated along with the investigations of their photoluminescence (PL), optical transparency, morphology, and thermal properties. The firm interaction between the ZnS and PVA was confirmed by Fourier transform infrared, UV-vis, and PL spectroscopies. PVA-ZnS composites showed enhanced luminescence property than PVA. The composites exhibited very good optical transparency regardless of the amount of PVA addition. The thermogravimetric analysis data indeed exhibited better thermal stability of the composites. The glass transition temperature (T g), melting temperature (T m), enthalpy of melting (ΔH m), and crystallinity were evaluated for such composites. The composites demonstrated morphological variations depending on the amount of PVA addition, although the particle size of ZnS remained similar in the nanometer range (50-120 nm) for all composite samples. The prepared composite films exhibited superior photocatalytic performance in the degradation of methylene blue compared with the bare ZnS and PVA. This study may give a new insight into the fabrication of PVA-ZnS photocatalysts for the treatment of organic pollutants.

Re-epithelialization and remodeling of decellularized corneal matrix in a rabbit corneal epithelial wound model.

This study investigated the in vivo correlation between re-epithelialization and remodeling of a decellularized corneal matrix prepared by a high-hydrostatic pressure (HHP) method in rabbits. Decellularized corneal matrices were transplanted in a 6-mm-diameter recipient corneal interlamellar pocket with a 2 mm epithelial defect. The time course of graft status in rabbits was examined daily for 6 months by biomicroscopy and scored for clarity and re-epithelialization, after which the rabbits were sacrificed for histological analysis. Fluorescein staining revealed that the corneal epithelial cells had migrated onto the decellularized corneal matrix. Histological analysis revealed that the implanted decellularized corneal matrix was completely integrated with the recipient rabbit cornea and the stratified corneal epithelia consisting of multiple layers were regenerated, similar to that in the normal cornea. The recipient keratocytes infiltrated into the decellularized corneal matrix at 6 months after the operation and the decellularized corneal matrix was gradually remodeled into the recipient tissue. Transmission electron microscopy revealed that the ultrastructure of the decellularized corneal matrix was rearranged, similar to the normal cornea. These findings suggest that the decellularized corneal matrix serves as a template for remodeling. The decellularized corneal matrix obtained through HHP is a useful graft for corneal tissue regeneration.

Quantitative analysis of cell adhesion on aligned micro- and nanofibers.

In this study, we quantitatively analyzed the affinity of cell adhesion to aligned nanofibers composed of composites of poly(glycolic acid) (PGA) and collagen. Electrospun composite fibers were fabricated at various PGA/collagen weight mixing ratio (7, 18, 40, 67, and 86%) to generate fibers that ranged in diameter from 10 mum to 500 nm. Scanning electron microscopy (SEM) observation revealed that the PGA/collagen fibers were long and uniformly aligned, irrespective of the PGA/collagen weight mixing ratio. In addition, it was observed that a significantly higher number of NIH3T3 fibroblasts adhered to nanofibers with smaller diameters in comparison to fibers with larger diameters. The highest affinity of cell adhesion was observed in the PGA/collagen fibers with diameter of 500 nm and PGA/collagen weight mixing ratio of 40%. Furthermore, the adherent cells were more elongated on fibers with smaller diameters. Thus, based on the results here, PGA/collagen composite fibers are suitable for tissue culture studies and provide an attractive material for tissue engineering applications.

Bone regeneration on a collagen sponge self-assembled peptide-amphiphile nanofiber hybrid scaffold.

The objective of this study was to create a novel approach to promote bone induction through sustained release of growth factor from a 3-dimensional (3D) hybrid scaffold. Peptide-amphiphile (PA) was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. Collagen sponge was reinforced by incorporation of poly(glycolic acid) (PGA) fiber. A 3D network of nanofibers was formed by mixing basic fibroblast growth factor (bFGF) suspensions with dilute aqueous solutions of PA. A hybrid scaffold was fabricated by combination of self-assembled PA nanofibers and collagen sponge reinforced with incorporation of PGA fibers. The in vitro release profile of bFGF from hybrid scaffold was investigated, and ectopic bone formation induced by the released bFGF was assessed after subcutaneous implantation of hybrid scaffold into the backs of rats. Homogeneous bone formation was histologically observed throughout the hybrid scaffolds, in marked contrast to collagen sponge-incorporated bFGF. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high compared with collagen sponge incorporated with bFGF. The combination of bFGF incorporated in a collagen sponge self-assembled PA nanofiber hybrid scaffold is a promising procedure to improve bone regeneration.

Bone regeneration through controlled release of bone morphogenetic protein-2 from 3-D tissue engineered nano-scaffold.

The objective of the present study was to enhance ectopic bone formation through the controlled release of bone morphogenetic protein-2 (BMP-2) from an injectable three dimensional (3-D) tissue engineered nano-scaffold. We demonstrate that a 3-D scaffold can be formed by mixing of peptide-amphiphile (PA) aqueous solution with BMP-2 suspension. A 3-D network of nanofibers was formed by mixing BMP-2 suspensions with dilute aqueous solutions of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. In vivo release profile of BMP-2 from 3-D network of nanofibers was investigated. In addition, ectopic bone formation induced by the released BMP-2 was assessed in a rat model using histological and biochemical examinations. It was demonstrated that the injection of an aqueous solution of PA together with BMP-2 into the back subcutis of rats, resulted in the formation of a transparent 3-D hydrogel at the injected site and induced significant homogeneous ectopic bone formation around the injected site, in marked contrast to BMP-2 injection alone or PA injection alone. The combination of BMP-2-induced bone formation is a promising procedure to improve tissue regeneration.

An attempt to construct the stroma of cornea using primary cultured corneal cells.

The number of patients currently awaiting corneal transplantation has resulted in the need to develop an artificial corneal replacement. In this study, we aimed to construct the corneal stroma using non-transformed corneal cells and a perfusion cell culture method. Corneal cells isolated from chicken embryos or rabbit and were embedded in the alkaline solubilized collagen gels crosslinked by TSG (Pentaerythritol polyethyleneglycol ether tetrasuccinimidyl glutarate). During culture, the majority of cells migrated from inside of the gel. The chicken and rabbit cells changed their morphology and stratified structures were constructed within the gels. These microstructures were similar to the natural corneal tissue. TEM analysis was performed to confirm the nano-microstructure of the constructs. Contrary to expectation, the cornea-like nanostructure of collagen fibrils was not observed within the gels. Further study including for example, such as the addition of dynamic stress or co-culture with endothelial cells, are therefore required in order to produce artificial constructs with the same superstructure as natural corneal tissue.

Micro-patterned nanofibrous biomaterials.

Nanofibrous materials made from bioabsorbable and biocompatible polymers have promising applications as tissue-engineered scaffolds. Genetic analysis of human umbilical vein endothelial cells (HUVEC) that attached to Poly(glycolide) (PGA) nanofibrous materials prepared via electrospinning methods demonstrated high expression of Integrin v and VEGF receptor genes, which are known angiogenesis markers. In order to improve the function of the PGA nanofibrous materials for tissue engineering applications, we used a micro-patterned template instead of a flat collector in the electrospinning process. "Micro-patterned nanofibrous material" demonstrated uniformly sized dents with diameters of 200 micrometers and depths of 36 micrometers. The dents were regularly spaced, with a 250 micrometer space between two dents. These sizes are similar to that of the template. We will discuss further applications of this designable micro-patterned nanofibrous biomaterial.

Physicochemical characterization of densely packed poly(ethylene glycol) layer for minimizing nonspecific protein adsorption.

Modification of the surface with densely packed poly(ethylene glycol) (PEG) brush layer was studied to improve the protein repellent ability of the surface. A PEG-brushed layer was constructed on a gold substrate using a PEG possessing a mercapto group at the chain end. The density of the PEG brushed layer substantially increased with repetitive adsorption/rinse cycles of the PEG on the gold substrate, allowing dramatic reduction of nonspecific protein adsorption. Notably, formation of a short, filler layer of PEG (2 kDa) in the preconstructed longer PEG brushed layer (5 kDa) achieved high density brush and almost complete prevention of nonspecific protein adsorption. On the other hand, surface modification with only long PEG chain (5 kDa) showed lower PEG brush density regardless of repetitive immobilization. Detailed characterization of the PEGylated surface was done from the physicochemical (QCM, contact angle, and SPR) as well as the biological (protein adsorption) point of view to highlight the relation between the PEG brush density and the protein repellent ability. Densely packed PEG surface which showed great protein repellent ability, presented in this study, suggests promising utility as engineered biomaterials including high-throughput screening and clinical diagnostics.

Amniotic membrane immobilized poly(vinyl alcohol) hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium.

Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis.

Ectopic bone formation in collagen sponge self-assembled peptide-amphiphile nanofibers hybrid scaffold in a perfusion culture bioreactor.

The objective of this study was to enhance ectopic bone formation in a three-dimensional (3-D) hybrid scaffold in combination with bioreactor perfusion culture system. The hybrid scaffold consists of two biomaterials, a hydrogel formed through self-assembly of peptide-amphiphile (PA) with cell suspensions in media, and a collagen sponge reinforced with poly(glycolic acid) (PGA) fiber incorporation. PA was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. A 3-D network of nanofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. Osteogenic differentiation of mesenchymal stem cells (MSC) in the hybrid scaffold was greatly influenced by the perfusion culture method compared with static culture method. When the osteoinduction activity of hybrid scaffold was studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds when perfusion culture was used compared with static culture method. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture method. We conclude that combination of MSC-seeded hybrid scaffold and the perfusion method was promising to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.

Enhanced angiogenesis through controlled release of basic fibroblast growth factor from peptide amphiphile for tissue regeneration.

In the present study, we hypothesized that a novel approach to promote vascularization would be to create injectable three-dimensional (3-D) scaffolds with encapsulated growth factor that enhance the sustained release of growth factor and induce the angiogenesis. We demonstrate that a 3-D scaffold can be formed by mixing of peptide-amphiphile (PA) aqueous solution with basic fibroblast growth factor (bFGF) suspension. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH(2) terminus of the peptide. A 3-D network of nanofibers was formed by mixing bFGF suspensions with dilute aqueous solutions of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. In vitro and in vivo release profile of bFGF from 3-D network of nanofibers was investigated while angiogenesis induced by the released bFGF was assessed. When aqueous solution of PA was subcutaneously injected together with bFGF suspension into the back of mice, a transparent 3-D hydrogel was formed at the injected site and induced significant angiogenesis around the injected site, in marked contrast to bFGF injection alone or PA injection alone. The combination of bFGF-induced angiogenesis is a promising procedure to improve tissue regeneration.