Formation of Polyion Complex Aggregate Formed from a Cationic Block Copolymer and Anionic Polysaccharide.

Block copolymers (PmMn; P20M101 and P100M98) comprising poly(2-(methacryloyloxy)ethylphosphorylcholine) (PMPC, P) containing biocompatible phosphorylcholin pendants and cationic poly((3-acryloylaminopropyl) trimethylammonium chloride) (PMAPTAC, M) were synthesized via a controlled radical polymerization method. The degrees of polymerization of the PMPC and PMAPTAC segments are denoted by subscripts (PmMn). The mixture of cationic PmMn and anionic sodium chondroitin sulfate C (CS) with the pendant anionic carboxylate and sulfonate groups formed polyion complex (PIC) aggregates in phosphate-buffered saline. A charge-neutralized mixture of P20M101 with CS formed P20M101/CS PIC vesicles with a hydrodynamic radius (Rh) of 97.2 nm, zeta potential of ca. 0 mV, and aggregation number (Nagg) of 23,044. PMPC shells covered the surface of the PIC vesicles. The mixture of P100M98 and CS formed PIC spherical micelles with the PIC core and hydrophilic PMPC shells. The Rh, zeta potential, and Nagg of the PIC micelles were 26.4 nm, ca. 0 mV, and 404, respectively. At pH < 4, the carboxylate anions in CS were protonated. Thus, the charge balance in the PIC micelles shifted to decrease the core density owing to the electrostatic repulsions of the excess cations in the core. The PIC micelles dissociated at a NaCl concentration ≥0.6 M owing to the charge screening effect. The positively charged PIC micelles with excess P100M98 can encapsulate anionic dyes owing to electrostatic interaction.

The pH responsiveness of fluorescein loaded in polysaccharide composite films.

Stimuli-responsive materials have been used in biomedical applications. Composite films fabricated using polyion complexes comprising anionic and cationic polysaccharides exhibited loading and release abilities for water-soluble molecules, the release ability of which depended on the solution pH. However, the interactions between polysaccharides and loaded molecules in the film have not been evaluated. In this study, polysaccharide composite films loaded with fluorescein (FL) as a probe molecule were fabricated and the film properties, FL ionization, and release behaviour of FL were investigated. FL loading did not significantly affect the mechanical and morphological properties of the films. The release behaviour of FL was determined by the pH of the solution as well as the electrostatic interaction between polysaccharides and FL ionic structures in FL-loaded films. Furthermore, the ionic structure change of FL that remained in the film was suppressed due to interactions with polysaccharides, such as through hydrogen bonding. Additionally, the pH responsiveness of FL in the film in the dried state was evaluated. The result shows that polysaccharide composite films were swollen because of air moisture and that the diffusion of molecules inside the film accelerated. These findings are useful to understand the properties of the loaded molecules such as ionic state and diffusiveness in the films made of polyion complexes.

Preparation of Polymer-Immobilized Polyimide Films Using Hot Pressing and Titania Coatings.

Recent studies have revealed that polymer molecules at film surfaces exhibit unique physical properties compared to those in bulk. On the other hand, such a topic has not been extensively focused for the cases of rigid polymers such as polyimide (PI). This study investigated whether hot pressing could induce the immobilization of other polymers, poly(4-vinylphenol) (PVP), on PI film surfaces. Results supported the immobilization of PVP on the PI film surfaces, and the increase of hot-press temperature resulted in the increase of the immobilization amount of PVP. The mechanism of immobilization is discussed considering the effects of hot pressing on the interactions between PVP and PI at the interfaces of their films. Sol-gel titania coatings were further conducted to the obtained PVP-immobilized PI films. The effect of PVP immobilization on formability and the adhesion of titania layers on the film surfaces were evaluated. These results demonstrate that hot pressing of other polymers is a useful approach for the surface modification of PI films, particularly introducing certain functional groups, and indicate that the polymer immobilization mechanism might be correlated with the surface physical properties of PI films.

Noninvasive Fingerprinting-Based Tracking of Replicative Cellular Senescence Using a Colorimetric Polyion Complex Array.

A fingerprint-based sensing approach was used to characterize in vitro cellular senescence. Secretion profiles of cultured human fibroblasts in different senescent stages were transformed into colorimetric enzyme-activity fingerprints by applying cell culture media to a polyion complex array. Analysis of the obtained fingerprints using pattern recognition methods, such as linear discriminant analysis and hierarchical clustering analysis, revealed that the polyion complex array allows the noninvasive tracking of the replicative senescence progress even in those stages where a conventional marker such as senescence-associated ß-galactosidase is negative. This fingerprint-based approach should thus offer an effective way for the routine monitoring or screening of in vitro cell senescence studies.

Effect of protein adsorption layers and solution treatments on hydroxyapatite deposition on polystyrene plate surfaces in simulated body fluids.

We have developed a method to functionalize polystyrene (PS) cell culture plates with hydroxyapatite (HAp) via protein adsorption layers such as human serum albumin (HSA) in simulated body fluids (SBFs). In order to investigate the versatility the method, in this study the effect of protein adsorption layers on HAp deposition on PS plate surfaces in SBF was evaluated. Pretreatments with alternate soaking process (ASP) using solutions containing calcium ions and phosphate ions followed by incubation with SBF for 24 h resulted in HAp deposition on PS plates with adsorption layers of HSA, type I collagen, hen egg white lysozyme, and poly L-glutamic acid, an acidic protein analogue: the deposition behaviors were correlated with adsorption ability and charge state of proteins. We also demonstrated that commercially available tissue culture-treated PS (TCPS) were directly coated with bone-like HAp using the same ASP and SBF processes. Human mesenchymal stem cells adhered and stretched on the HAp-coated TCPS plates in a similar manner to the case of the HAp-coated PS plates prepared via HSA adsorption layers. The results indicate that the present methods are useful for preparing bone-like HAp-coated cell culture plates that can be utilized function of adsorbed proteins and that are obtainable conveniently and at low-cost.

Hot-Press-Assisted Adhesions between Polyimide Films and Titanium Plates Utilizing Coating Layers of Silane Coupling Agents.

The development of low material-consuming adhesion techniques for different kinds of materials such as polymers and metals is important for the realization of sustainable societies. This study demonstrates that coating layers, expected to be formed as self-assembled monolayers, of silane coupling agents can act as adhesion layers at the polymer film-metal plate interfaces. Polyimide films were alkaline hydrolyzed to generate carboxy groups on their surfaces, whereas titanium plate surfaces were treated with the aminosilanes to form their coating layers thereon. These modified surfaces were placed in contact with each other and then hot pressed, which resulted in adhesion between them. An examination of the adhesion strength using lap shear tests and surface characterization of the prepared surfaces using X-ray photoelectron spectroscopy and other techniques indicated the formation of ionic bonds and/or amide bonds between the carboxy groups of the PI film surfaces and the amino groups immobilized on the titanium plate surfaces. The activation of the carboxy groups using N-hydroxysuccinimide resulted in adhesion obtaining a water-resistant property, which supported the increase in amide bond formation. On the basis of the results, the adhesion mechanism and the possible breaking points upon the breaking of adhesions are proposed.

Preparation of polysaccharide-apatite hybrid microtubes using layer-by-layer assembly and biomimetic mineralization process.

Organic-inorganic hybrid microtubes were prepared that consisted of polysaccharide inner layers and hydroxyapatite (HAp) outer layers. Poly(methyl methacrylate) (PMMA) fibers containing small amounts of polyethyleneimine (PEI) were used as templates for the layer-by-layer (LbL) assembly of chondroitin sulfate C and chitosan. HAp layers were then deposited on polysaccharide layer-coated fibers using biomimetic processes. PMMA-PEI fiber templates were removed by immersing the samples in chloroform. Examination of the resulting materials using various physical characterizations such as scanning electron microscopy, FT-IR spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction studies supported the successful formation of the hybrid microtubes as designed. The results also showed that incorporation of PEI into PMMA fiber matrices was effective in inducing HAp deposition. The present procedure can be applied to the preparation of various hybrid microtubes consisting of biocompatible organic inner layers formed using LbL assemblies and HAp outer layers. Some of these hybrids have potential applications in regenerative medicine or tissue engineering.

Preparation of Mechanically Anisotropic Polysaccharide Composite Films Using Roll-Press Techniques.

Natural polysaccharides are biocompatible and biodegradable; therefore, they can be used as feedstock for biodegradable structural materials and biomaterials. In this study, anisotropic polysaccharide composite films consisting of chondroitin sulfate C (CS) and chitosan (CHI) were fabricated from their polyion complex (PIC) gels by roll-press techniques. The obtained films (CS/CHI films) were thin and transparent, similar to the composite films prepared by hot-press techniques. The roll-press conditions were optimized, and it was observed that the molecular weight of CHI did not significantly affect the formability of the films, whereas the roll temperature and rolling speed were important. The tensile tests of the roll-pressed films revealed that the mechanical strength of the films in the mechanical direction (MD) was approximately 5 times higher than that in the transverse direction (TD), indicating that the roll-press techniques imparted mechanical anisotropy to the films. Additionally, the films shrank in the MD and expanded in the TD after immersion in aqueous solutions, followed by drying. Such anisotropic shrinking and expanding properties indicate that these films can be used as shape-memory materials.

Detection of Influenza Virus by Agglutination of Microparticles Immobilized a Mixed Glycan Receptor Produced from Cells.

The hemagglutination inhibition (HAI) assay is one of the detection methods for influenza virus (IFV) under global influenza surveillance, which uses freshly prepared animal red blood cells (RBCs). Here, we demonstrate that a mixed glycan-modified polystyrene microparticle, which can be chemically prepared in advance, can replace animal RBCs in the HAI assay. A mixture of azide-conjugated glycans containing sialyl- and sulfated-lactose moieties was produced from Madin-Darby canine kidney (MDCK) cells, which are used for IFV isolation, and then immobilized on the surface of a polystyrene microparticle using click chemistry. Human HA and IFV were detected with high sensitivity when using the mixed glycan-immobilized particle.

Effects of surface carboxylic acid groups of cerasomes, morphologically stable vesicles having a silica surface, on biomimetic deposition of hydroxyapatite in body fluid conditions.

Biomimetic mineralization of supramolecular scaffolds consisting of biomolecules or their analogues has received much attention recently from the viewpoint of creation of novel biomaterials. This study investigated biomimetic deposition of hydroxyapatite (HAp) on cerasomes, morphologically stable organic-inorganic hybrid vesicles. Scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction studies revealed that the pristine cerasomes induced heterogeneous nucleation of HAp when they were immersed in 1.5SBF, a solution having 1.5 times higher ion concentration than that of a simulated body fluid (SBF). The HAp deposition was further accelerated when dicarboxylic and monocarboxylic acid groups were displayed on cerasome surfaces. These carboxylic acid groups are expected to enhance calcium ion binding to the cerasome surface, causing an increase of HAp nucleation sites. At lower surface concentrations on the cerasome surface, the dicarboxylic acid group is apparently more effective for HAp deposition than the monocarboxylic acid group. The resultant HAp-cerasome hybrids are useful as biocompatible materials having unique properties deriving from the lipid bilayer structure of the cerasomes.

Polyethyleneimine-induced astaxanthin accumulation in the green alga Haematococcus pluvialis by increased oxidative stress.

The unicellular green microalga Haematococcus pluvialis accumulates large amounts of the red ketocarotenoid astaxanthin under stress conditions such as nitrogen deficiency. In this study, we discovered an astaxanthin accumulation in H. pluvialis cells by the addition of a synthetic cationic polymer, polyethyleneimine (PEI), into the cell culture. With an increase in PEI amount, amount of astaxanthin accumulation was increased. To investigate the mechanism for the accumulation of astaxanthin by the addition of PEI in H. pluvialis cells, we measured a localization of PEI in the cells and a production of reactive oxygen species. PEI was internalized in the cells through the negatively-charged cell walls, leading to excessive production of reactive oxygen species in the cells. Thus, the increased oxidative stress by cellular uptake of PEI resulted in the acceleration of astaxanthin accumulation in H. pluvialis.

Preparation of Cell-Paved and -Incorporated Polysaccharide Hollow Fibers Using a Microfluidic Device.

Cellular constructs having hollow tubular structures are expected to be used as artificial blood vessels. We have recently demonstrated that water-insoluble polyion complexes (PICs) were formed from water-soluble polysaccharides with opposite charges at the interface of coaxial flows, which resulted in the formation of hollow fibers. In this study, both inside- and outside-cell-laden chondroitin sulfate C (CS)/chitosan (CHI) hollow fibers were prepared by utilizing a microfluidic device and modification with cell adhesive molecules. Loading of type I collagen (COL) and surface modification with fibronectin and gelatin using layer-by-layer assembly techniques improved the adhesion and spreading of fibroblast cells to/on the surface of CS/CHI hollow fibers. On the other hand, by suspending mesenchymal stem cells (MSCs) in the core flow solution, cells were successfully loaded in the walls of the hollow fibers. As the culture time extended, cells trapped in the PIC structures constituting the wall of the hollow fibers migrated to the interface between the hollow fibers and the medium: cells adhered to and stretched "on" the lumen surfaces in the COL-loaded fibers. In contrast, for the case of unmodified hollow fibers, it was difficult for cells to adhere to the lumen surfaces. Therefore, cell aggregates were formed "in" the lumen. Results of the live/dead assay and MTT assay clearly demonstrated that MSCs possessed certain levels of cell viability and proliferated for up to 10 days, especially for the cases of COL-loaded hollow fibers. On the basis of these results, the utility of the present hollow fibers in the formation of cellular constructs corresponding to blood vessels is also discussed.

Determination of surface area and porosity of small, nanometer-thick films by quartz crystal microbalance measurement of gas adsorption.

The Brunauer-Emmett-Teller (BET) surface area of 15 nm-thick films made of TiO2/polyelectrolyte bilayer was determined by quartz crystal microbalance (QCM) measurement of N2 and Ar adsorption isotherms at 77 K. The measurements were carried out using a home-built vacuum chamber that includes built-in 9 MHz QCM and cryostat units. As little as 1 ng of the adsorbed gas could be detected, and the BET surface area of a flat Au film (ca. 0.5 cm2) on an oscillator was determined within an experimental error of +/-5%. The titania/polymer composite film gives N2 and Ar adsorption isotherms consisting of a less-pronounced type-I curve and a break at around p/p0 = 0.7. This behavior is ascribed to the presence of irregular micropores and 6 nm phi-mesopores in the composite film. An analysis of the isotherms shows that the porosity of the composite film is about 12%, which is much smaller than that of bulk titania gel powder. The greater density appears to be related to the reported superior properties (robustness and resistance to electrical breakdown) of the organic/inorganic multilayer film. We conclude that the QCM-based, high-precision measurement of gas adsorption is a powerful tool for investigation of the detailed morphology of nanometer-thick films.

Angle-controlled arrangement of single-walled carbon nanotubes solubilised by 8-quinolinol metal chelate derivatives on mica.

We solubilised SWNTs of short length using a mechanochemical high-speed vibration milling (HSVM) through formation of complexes between the SWNTs and chelate complexes; the mixture formed a network structure on mica.

Utilization of Proteins and Peptides to Create Organic-Hydroxyapatite Hybrids.

BACKGROUND: Various types of proteins play important roles in the biomineralization of hydroxyapatite (HAp, Ca10(PO4)6(OH)2). The resulting organic-HAp nanohybrids have highlyorganized hierarchical structures that show unique morphological, structural, and mechanical properties. By mimicking the biomineralization process, organic-HAp hybrid materials have been created by utilizing proteins and peptides. OBJECTIVES: In this review, firstly the roles of proteins in HAp mineralization in vivo are briefly explained. Recent progresses in the creation of organic-HAp hybrids through the utilization of proteins and peptides are then described. RESULTS: Roles of collagen and amelogenin on the formation of bones and teeth were explained. Then, recent advances, including those by the authors, in the creation of organic-HAp hybrids through the utilization of these proteins, their derivatives, and synthetic peptides, including engineering- isolated ones, were reviewed. CONCLUSION: Organic-HAp hybrid materials have been intensively created by utilizing proteins and peptides. Among them, engineering-isolated or rationally designed peptides and their derivatives represent future promising building components for organic-HAp hybrids with precise hierarchical structures. Not only the excellent functions of the resultant hybrids materials, but also the creation of materials by biomimetic synthetic processes at a low cost and environmental burden are important for sustainable industrial development.

Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells in Electrospun Silica Nonwoven Fabrics.

Silica nonwoven fabrics (SNFs) with enough mechanical strength are candidates as implantable scaffolds. Culture of cells therein is expected to affect the proliferation and differentiation of the cells through cell-cell and cell-SNF interactions. In this study, we examined three-dimensional (3D) SNFs as a scaffold of mesenchymal stem cells (MSCs) for bone tissue engineering applications. The interconnected highly porous microstructure of 3D SNFs is expected to allow omnidirectional cell-cell interactions, and the morphological similarity of a silica nanofiber to that of a fibrous extracellular matrix can contribute to the promotion of cell functions. 3D SNFs were prepared by the sol-gel process, and their mechanical properties were characterized by the compression test and rheological analysis. In the compression test, SNFs showed a compressive elastic modulus of over 1 MPa and a compressive strength of about 200 kPa. These values are higher than those of porous polystyrene disks used for in vitro 3D cell culture. In rheological analysis, the elastic modulus and fracture stress were 3.27 ± 0.54 kPa and 25.9 ± 8.3 Pa, respectively. Then, human bone marrow-derived MSCs were cultured on SNFs, and the effects on proliferation and osteogenic differentiation were evaluated. The MSCs seeded on SNF proliferated, and the thickness of the cell layer became over 80 µm after 14 days of culture. The osteogenic differentiation of MSCs on SNFs was induced by the culture in the commercial osteogenic differentiation medium. The alkaline phosphatase activity of MSCs on SNFs increased rapidly and remained high up to 14 days and was much higher than that on two-dimensional tissue culture-treated polystyrene. The high expression of RUNX2 and intense staining by alizarin red s after differentiation supported that SNFs enhanced the osteogenic differentiation of MSCs. Furthermore, permeation analysis of SNFs using fluorescein isothiocyanate-dextran suggested a sufficient permeability of SNFs for oxygen, minerals, nutrients, and secretions, which is important for maintaining the cell viability and vitality. These results suggested that SNFs are promising scaffolds for the regeneration of bone defects using MSCs, originated from highly porous and elastic SNF characters.

Control of cell adhesion and proliferation utilizing polysaccharide composite film scaffolds.

We have developed polysaccharide composite films made of anionic polysaccharides and chitosan (CHI) by utilizing hot press techniques. In order to demonstrate the versatility of these films as cell scaffolds, the present study investigated the adhesion and proliferation of fibroblasts on composite films prepared by using various kinds of anionic polysaccharides and that were modified with proteins. Cells were spread on heparin/CHI and alginic acid/CHI films and grew well, whereas those on chondroitin sulfate C (CS)/CHI and hyaluronic acid/CHI films were round in shape. The differences in adhesion and proliferation behaviors of cells could be explained by the differences in the biochemical function of the anionic polysaccharides and the physical properties of the films such as morphology, storage modulus, ζ-potentials, and swelling ratios. Among them, the number of cells on CS/CHI films remained almost unchanged. The mechanisms underlying growth suppression on CS/CHI films were investigated by using an integrin stimulator, the TNIIIA2 peptide, and platelet-derived growth factor-B. It was indicated that the growth suppression was due to the lack of fibronectin-integrin growth signaling. The surface modification of CS/CHI films with fibronectin promoted the adhesion and proliferation of cells. These results show that the chemical and physical properties of the polysaccharide composite films, which resulted from the chemical species of anionic polysaccharides or surface modifications of the films, can modulate cell adhesion and proliferation properties thereon.

Preparations of self-supporting nanofilms of metal oxides by casting processes.

We recently developed a solution-based method for the preparation of self-supporting (free-standing) titania films with thicknesses less than a micrometre. In this study, we demonstrated its wide applicability to other metal oxides. Film fabrication by spin casting was employed for preparation of self-supporting nanofilms (<100 nm thickness) from various combinations of metal oxides, polymer underlayers, and polymer middle layers. All the metal alkoxides we examined gave self-supporting ultrathin films of AlO, NbO, ZrO, SiO and LaO. It was found that the photoresist polymer underlayer employed for ready film detachment could be replaced by other commercial polymers. The polymer underlayer could be omitted when a cast acrylic sheet was used as the solid substrate. The importance of the PVA middle layer was confirmed by successful formation of uniform large metal oxide films compared to the case of other polymers. This is attributed to a high population of the surface hydroxyl group that facilitates efficient development of lateral metal oxide network within the film. Admixing of organic carboxylic acids in the casting solution provided similarly uniform metal oxide nanofilms. These results revealed that the spin casting method is applicable to a wide variety of metal oxides and polymer layers.We also developed the flow casting method for film fabrication and its usefulness was discussed. These newer approaches were compared with the surface sol-gel process which we previously described. Among the three casting methods, the spin casting method was most effective for obtaining uniform large nanofilms of metal oxides by simple operation, although it was limited to flat surfaces.

Surface functionalization of polymer substrates with hydroxyapatite using polymer-binding peptides.

Material-binding peptides are used as non-covalent bond linkers for surface functionalization because they bind to materials under mild conditions without affecting the properties of the materials and are functionalized by conjugating with other molecules. In the present study, the surface functionalization of polyetherimide (PEI) with hydroxyapatite (HAp) was examined using two types of PEI-binding peptides conjugated with other sequences. One peptide consisted of PEI-binding peptide p1 (TGADLNT) and a triasparate sequence for the biomimetic mineralization of HAp in simulated body fluids (SBFs), while the other consisted of p1 and HAp-binding peptide (HABP, CMLPHHGAC) for the immobilization of HAp and amorphous calcium phosphate (ACP) nanoparticles. The results obtained revealed deposits of HAp on PEI films treated with the peptide consisting of p1 and triasparate. HAp and ACP nanoparticles were immobilized on PEI films treated with peptides consisting of p1 and HABP, and immersion of the resultant substrates in SBFs completely covered the surfaces of PEI films with HAp.

Surface functionalization of tissue culture polystyrene plates with hydroxyapatite under body fluid conditions and its effect on differentiation behaviors of mesenchymal stem cells.

The surfaces of polystyrene (PS) cell culture plates were functionalized with hydroxyapatite (HAp) under body fluid conditions utilizing protein adsorption layers and a pretreatment with an alternate soaking process (ASP) using solutions containing calcium and phosphate ions. Adsorption layers of human serum albumin (HSA) formed on the surface of each well of commercial 24-well PS plates by solution processes. CaCl2 and K2HPO4 solutions were alternately added to the wells, the plates were incubated to form the precursors, and this was followed by the addition of simulated body fluid (SBF) and a further incubation for 24h. These treatments resulted in the surfaces of the PS cell culture plates being completely covered with bone-like HAp. The coating of PS plates with HAp promoted the adhesion of mesenchymal stem cells (MSCs) and maintained cell growth that was as fast as that on tissue culture-treated PS (TCPS) plates. Osteogenic differentiation was greater, whereas adipogenic and chondrogenic differentiation was less in the culture on HAp-coated PS plates than in that on TCPS plates. The present method is useful for preparing HAp-coated PS plates at clean benches without the need for any expensive apparatus. HAp coated on PS plates by this method was a bone-like apatite with high bioactivity; therefore, the present HAp-coated PS plates are promising materials for assays of bone-related cells in the bone remodeling process.