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 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.

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.

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.

Inflammation-sensitive in situ smart scaffolding for regenerative medicine.

To cope with the rapid evolution of the tissue engineering field, it is now essential to incorporate the use of on-site responsive scaffolds. Therefore, it is of utmost importance to find new 'Intelligent' biomaterials that can respond to the physicochemical changes in the microenvironment. In this present report, we have developed biocompatible stimuli responsive polyaniline-multiwalled carbon nanotube/poly(N-isopropylacrylamide), (PANI-MWCNT/PNIPAm) composite nanofiber networks and demonstrated the physiological temperature coordinated cell grafting phenomenon on its surface. The composite nanofibers were prepared by a two-step process initiated with an assisted in situ polymerization followed by electrospinning. To obtain a smooth surface in individual nanofibers with the thinnest diameter, the component ratios and electrospinning conditions were optimized. The temperature-gated rearrangements of the molecular structure are characterized by FTIR spectroscopy with simultaneous macromolecular architecture changes reflected on the surface morphology, average diameter and pore size as determined by scanning electron microscopy. The stimuli responsiveness of the nanofibers has first been optimized with computational modeling of temperature sensitive components (coil-like and globular conformations) to tune the mechanism for temperature dependent interaction during in situ scaffolding with the cell membrane. The nanofiber networks show excellent biocompatibility, tested with fibroblasts and also show excellent sensitivity to inflammation to combat loco-regional acidosis that delay the wound healing process by an in vitro model that has been developed for testing the proposed responsiveness of the composite nanofiber networks. Cellular adhesion and detachment are regulated through physiological temperature and show normal proliferation of the grafted cells on the composite nanofibers. Thus, we report for the first time, the development of physiological temperature gated inflammation-sensitive smart biomaterials for advanced tissue regeneration and regenerative medicine.

Ultrastructural analysis of the decellularized cornea after interlamellar keratoplasty and microkeratome-assisted anterior lamellar keratoplasty in a rabbit model.

The decellularized cornea has received considerable attention for use as an artificial cornea. The decellularized cornea is free from cellular components and other immunogens, but maintains the integrity of the extracellular matrix. However, the ultrastructure of the decellularized cornea has yet to be demonstrated in detail. We investigated the influence of high hydrostatic pressure (HHP) on the decellularization of the corneal ultrastructure and its involvement in transparency, and assessed the in vivo behaviour of the decellularized cornea using two animal transplantation models, in relation to remodelling of collagen fibrils. Decellularized corneas were prepared by the HHP method. The decellularized corneas were executed by haematoxylin and eosin and Masson's trichrome staining to demonstrate the complete removal of corneal cells. Transmission electron microscopy revealed that the ultrastructure of the decellularized cornea prepared by the HHP method was better maintained than that of the decellularized cornea prepared by the detergent method. The decellularized cornea after interlamellar keratoplasty and microkeratome-assisted anterior lamellar keratoplasty using a rabbit model was stable and remained transparent without ultrastructural alterations. We conclude that the superior properties of the decellularized cornea prepared by the HHP method were attributed to the preservation of the corneal ultrastructure.

The outermost surface properties of silk fibroin films reflect ethanol-treatment conditions used in biomaterial preparation.

Silk fibroin has attracted interest as a biomaterial, given its many excellent properties. Cell attachment to silk substrates is usually weaker than to standard culture dishes, and cells cultured on silk films or hydrogels typically form spheroids and micro-aggregates. However, too little is known about the higher order structures and behavior of fibroin under different conditions to explain the features of silk fibroin as a culture substrate. For instance, different biomaterial surfaces, with distinct effects on cell culture, can be achieved by varying the conditions of crystallization by alcohol immersion. Here, we show that treatment of fibroin film with <80% ethanol results in a jelly-like, hydrated hydrogel as the outermost surface layer; fibroblasts preferably aggregate, rather than attach individually to such a hydrogel surface, and therefore aggregate into spheroids. In contrast, a fibroin film treated with >90% ethanol has a harder surface than the <80% ethanol-treated fibroin, to which individual cells prefer to attach (and then expand on the surface), rather than to aggregate. We discuss the influence of alcohol concentration on the surface properties, based on surface analysis of the films. The surface analysis involved assessment of static and dynamic contact angles, zeta potential, changes in crystallinity and microscopic morphology of electrospun fibers, and texture changes of the outermost surface at a nanometer-scale captured by a scanning probe microscope.

Corneal Regeneration by Deep Anterior Lamellar Keratoplasty (DALK) Using Decellularized Corneal Matrix.

The purpose of this study is to demonstrate the feasibility of DALK using a decellularized corneal matrix obtained by HHP methodology. Porcine corneas were hydrostatically pressurized at 980 MPa at 10°C for 10 minutes to destroy the cells, followed by washing with EGM-2 medium to remove the cell debris. The HHP-treated corneas were stained with H-E to assess the efficacy of decellularization. The decellularized corneal matrix of 300 µm thickness and 6.0 mm diameter was transplanted onto a 6.0 mm diameter keratectomy wound. The time course of regeneration on the decellularized corneal matrix was evaluated by haze grading score, fluorescein staining, and immunohistochemistry. H-E staining revealed that no cell nuclei were observed in the decellularized corneal matrix. The decellularized corneal matrices were opaque immediately after transplantation, but became completely transparent after 4 months. Fluorescein staining revealed that initial migration of epithelial cells over the grafts was slow, taking 3 months to completely cover the implant. Histological sections revealed that the implanted decellularized corneal matrix was completely integrated with the receptive rabbit cornea, and keratocytes infiltrated into the decellularized corneal matrix 6 months after transplantation. No inflammatory cells such as macrophages, or neovascularization, were observed during the implantation period. The decellularized corneal matrix improved corneal transparency, and remodelled the graft after being transplanted, demonstrating that the matrix obtained by HHP was a useful graft for corneal tissue regeneration.

Comprehensive genetic analysis of early host body reactions to the bioactive and bio-inert porous scaffolds.

To design scaffolds for tissue regeneration, details of the host body reaction to the scaffolds must be studied. Host body reactions have been investigated mainly by immunohistological observations for a long time. Despite of recent dramatic development in genetic analysis technologies, genetically comprehensive changes in host body reactions are hardly studied. There is no information about host body reactions that can predict successful tissue regeneration in the future. In the present study, porous polyethylene scaffolds were coated with bioactive collagen or bio-inert poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) and were implanted subcutaneously and compared the host body reaction to those substrates by normalizing the result using control non-coat polyethylene scaffold. The comprehensive analyses of early host body reactions to the scaffolds were carried out using a DNA microarray assay. Within numerous genes which were expressed differently among these scaffolds, particular genes related to inflammation, wound healing, and angiogenesis were focused upon. Interleukin (IL)-1ß and IL-10 are important cytokines in tissue responses to biomaterials because IL-1ß promotes both inflammation and wound healing and IL-10 suppresses both of them. IL-1ß was up-regulated in the collagen-coated scaffold. Collagen-specifically up-regulated genes contained both M1- and M2-macrophage-related genes. Marked vessel formation in the collagen-coated scaffold was occurred in accordance with the up-regulation of many angiogenesis-inducible factors. The DNA microarray assay provided global information regarding the host body reaction. Interestingly, several up-regulated genes were detected even on the very bio-inert PMB-coated surfaces and those genes include inflammation-suppressive and wound healing-suppressive IL-10, suggesting that not only active tissue response but also the inert response may relates to these genetic regulations.

Vascular-inducing poly(glycolic acid)-collagen nanocomposite-fiber scaffold.

For regenerative medicine with scaffolds, the immediate cellularization of the scaffold accompanied by angiogenesis inside is an important event. Such the aim is generally pursued by combining basic fibroblast growth factor (b-FGF) or vascular endothelial growth factor (VEGF) with the scaffold. In this study, we produced the nanocomposite nanofiber composed of poly(glycolic acid), PGA, and collagen to accomplish the recruitment of host cells and peripheral blood vessels without the bio-derived matter like growth factors. Structural analysis revealed that the fiber has the sheath-core like structure in which the surface region is abundant in PGA and the core region is abundant in collagen. This peculiar fibrous structure probably contributes the fragility of the fiber under the swelling in body fluid. The results of the animal experiment demonstrated that the PGA-collagen nanofiber sponge was entirely populated and vascularized within 5 days after the implantation. We hypothesized that the early fragmentation of the implanted fibrous sponge accelerated the host's inflammation reaction by phagocytized by macrophage, which followed by the recruitment of the fibroblasts and endothelial cells from the host tissue. Designing the suitable nanoscale structure of materials makes cellularization and vascularization of the scaffold possible without bio-derived factors.

Efficacy of a poly glycolic acid (PGA)/collagen composite nanofibre scaffold on cell migration and neovascularisation in vivo skin defect model.

Application of tissue engineering currently provides promising therapeutic options in the fields of plastic surgery and wound management. The ability of scaffold material for cell proliferation and differentiation is the key for tissue engineering. This study has developed a novel nanofibre composed of poly glycolic acid (PGA) and collagen, both of which have their own respective beneficial properties. This study aimed to estimate the in vivo efficiency of the PGA/collagen nanofibre on granulation histology and its ability to induce neovascularisation. The electrospinning technique produced the PGA/collagen nanofiber with a diameter of 500 nm and weight mixing ratio of 40%. The skin defects on the mouse model were covered with PGA/collagen or a commercially available collagen matrix (n = 9). The PGA/collagen group histologically showed significantly higher cell density and a fine microstructure with greater number of migrating cells as compared to collagen matrix. Then, both materials were applied to the microcirculatory angiogenesis model. The PGA/collagen group (n = 8) revealed significantly higher functional capillary density on days 5 and 7 after application. The findings substantiated the fact that our material had a superior ability regarding cellular migration and induction of neovascularisation compared with the elementary collagen matrix product. This better result might be attributed to the nano-size effect of fine structure and the incorporation of PGA, which has been associated with enhanced angiogenesis.

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.

Embossed-carving processing of cytoskeletons of cultured cells by using focused ion beam technology.

The focused ion beam (FIB) technology has drawn considerable attention in diverse research fields. FIB can be used to mill samples at the nanometer scale by using an ion beam derived from electrically charged liquid gallium (Ga). This powerful technology with accuracy at the nanometer scale is now being applied to life science research. In this study, we show the potential of FIB as a new tool to investigate the internal structures of cells. We sputtered Ga(+) onto the surface or the cross section of animal cells to emboss the internal structures of the cell. Ga(+) sputtering can erode the cell surface or the cross section and thus emboss the cytoskeletons quasi-3 dimensionally. We also identified the embossed structures by comparing them with fluorescent images obtained via confocal laser microscopy because the secondary ion micrographs did not directly provide qualitative information directly. Furthermore, we considered artifacts during the FIB cross sectioning of cells and propose a way to prevent undesirable artifacts. We demonstrate the usefulness of FIB to observe the internal structures of cells.

Fabrication of conducting electrospun nanofibers scaffold for three-dimensional cells culture.

Electrospinning is a versatile method to fabricate nanofibers of a range of polymeric and composite materials suitable as scaffolds for tissue engineering applications. In this study, we report the fabrication and characterization of polyaniline-carbon nanotube/poly(N-isopropyl acrylamide-co-methacrylic acid) (PANI-CNT/PNIPAm-co-MAA) composite nanofibers and PNIPAm-co-MAA nanofibers suitable as a three-dimensional (3D) conducting smart tissue scaffold using electrospinning. The chemical structure of the resulting nanofibers was characterized with FTIR and ¹H NMR spectroscopy. The surface morphology and average diameter of the nanofibers were observed by SEM. Cellular response of the nanofibers was studied with mice L929 fibroblasts. Cell viability was checked on 7 th day of cell culture by double staining the cells with calcein-AM and PI dye. PANI-CNT/PNIPAm-co-MAA composite nanofibers were shown the highest cell growth and cell viability as compared to PNIPAm-co-MAA nanofibers. Cell viability in the composite nanofibers was obtained in order of 98% that indicates the composite nanofibers provide a better environment as a 3D scaffold for the cell proliferation and attachment suitable for tissue engineering applications.

Detection of p53 gene point mutation using sequence-specific molecularly imprinted PoPD electrode.

An amperometric sequence-specific molecularly imprinted single-stranded oligodeoxyribonucleotide (ss-ODN) biosensor was fabricated and characterised in this study. Using ss-ODN as the template and o-phenylenediamine as the functional monomer, the ODN biosensor was fabricated by an electropolymerisation process on an indium-tin oxide (ITO) coated glass substrate. The template ss-ODN was washed out of the ss-ODN/poly(o-phenylenediamine)(PoPD)/ITO electrode using sterilised basic ethanol-water. The resulting ss-ODN imprinted PoPD/ITO electrode was characterised using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and cyclic voltammetry (CV). The amperometric responses, i.e., Δi as a function of the target ss-ODN concentration was studied. The biosensor using ss-ODN imprinted PoPD/ITO as the working electrode showed a linear Δ current response to the target ss-ODN concentration within the range of 0.01-300 fM. The biosensor showed a sensitivity of 0.62 µA/fM, with a response time of 14s. The present novel molecularly imprinted ss-ODN biosensor could greatly benefit in terms of cost-effectiveness, storage stability, ultra sensitivity and selectivity together with the potential for improved commercial genetic sensors.