Evaluation of the immunotoxicity potential of nanomaterials using THP-1 cells.

With the expansion of nanomaterials (NMs) usage, concerns about their toxicity are increasing, and the wide variety of NMs makes it difficult to assess their toxicity. Therefore, the development of a high-throughput, accurate, and certified method to evaluate the immunotoxicity of NMs is required. In this study, we assessed the immunotoxicity potential of various NMs, such as nanoparticles of silver, silica, and titanium dioxide, using the human Cell Line Activation Test (h-CLAT) at the cellular level. After exposure to silver nanoparticle dispersions, the expression levels of CD86 and CD54 increased, suggesting the activation of antigen-presenting cells (APCs) by silver nanoparticles. Quantification of silver ions eluted from silver nanoparticles and the activation of APCs by silver ions suggested that it was due to the release of silver ions. Silica nanoparticles also increased the expression of CD86 and/or CD54, and their activation ability correlated with the synthesis methods and hydrodynamic diameters. The ability of titanium dioxide to activate APCs differed depending on the crystal type and hydrodynamic diameter. These results suggest a potential method to evaluate the immunotoxicity potential of various NMs based on their ability to activate APCs using human monocytic THP-1 cells. This method will be valuable in assessing the immunotoxicity potential and elucidating the immunotoxic mechanisms of NMs.

Microfabrication of Gelatin Methacrylate/Hydroxyapatite Composites by Utilizing Alternate Soaking Process.

To construct a complex three-dimensional (3D) structure mimicking bone microstructure, hydrogel models of polymerized gelatin methacrylate (pGelMA) were fabricated by using stereolithography and modified with hydroxyapatite (HAp) via an alternate soaking process (ASP) using a solution of calcium and phosphate ions. Fabricated pGelMA line models whose widths were designed as 100, 300, and 600 µm were modified with HAp by ASP by changing the immersion time and number of cycles. After ASP, all of the line models with widths of 100, 300, and 600 µm were successfully modified with HAp, and large amounts of HAp were covered with the fabricated models by increasing both the immersion time and the number of cycles in ASP. HAp was observed near the surface of the line model with a width of 600 µm after ASP at an immersion time of 10 s, while the entire model was modified with HAp using ASPs for longer immersion times. The adhesion and spread of mesenchymal stem cells (MSCs) on the pGelMA-HAp discs depended on the ASP conditions. Moreover, the HAp modification of 3D pyramid models without alteration of the microstructure was also conducted. This two-step fabrication method of first fabricating frameworks of hydrogel models by stereolithography and subsequently modifying the fabricated models with HAp will lead to the development of 3D cell culture systems to support bone grafts or to create biological niches, such as artificial bone marrow.

Three-dimensional co-culture model employing silica nonwoven fabrics to enhance cell-to-cell communication of paracrine signaling between hepatocytes and fibroblasts.

The realization that soluble factors secreted by heterotypic cells play an importanta role in paracrine signaling, which facilitates intercellular communication, enabled the development of physiologically relevant co-culture models for drug screening and the engineering of tissues, such as hepatic tissues. The most crucial issues confronting the use of conventional membrane inserts in segregated co-culture models that are used to study paracrine signaling between heterotypic cells have been identified as long-term viability and retention of cell-specific functions, especially when isolated primary cells are used. Herein, we present an in vitro segregated co-culture model consisting of a well plate incubated with rat primary hepatocytes and normal human dermal fibroblasts which were segregated using a membrane insert with silica nonwoven fabric (SNF) on it. SNF, which mimics a physiological environment much more effectively than a two-dimensional (2D) one, promotes cell differentiation and resultant paracrine signaling in a manner that is not possible in a conventional 2D culture, owing to high mechanical strength generated by its inorganic materials and interconnected network structure. In segregated co-cultures, SNF clearly enhanced the functions of hepatocytes and fibroblasts, thereby showing its potential as a measure of paracrine signaling. These results may advance the understanding of the role played by paracrine signaling in cell-to-cell communication and provide novel insights into the applications of drug metabolism, tissue repair, and regeneration.

Specific ion effects on the aggregation of polysaccharide-based polyelectrolyte complex particles induced by monovalent ions within Hofmeister series.

Polysaccharide-based polyelectrolyte complex (PEC) particles have been utilized as carriers for drug delivery systems (DDS) and as building components for material development. Despite their versatility, the aggregation mechanism of PEC particles in the presence of salts remains unclear. To clarify the aggregation mechanism, the specific ion effects of monovalent salts within the Hofmeister series on the aggregation behavior of PEC particles composed of chitosan and chondroitin sulfate C, which are often used as DDS carriers and materials, were studied. Here, we found that weakly hydrated chaotropic anions promoted the aggregation of positively charged PEC particles. The hydrophobicity of the PEC particles was increased by these ions. Strongly hydrated ions such as Cl- are less likely to accumulate in these particles, whereas weakly hydrated chaotropic ions such as SCN- are more likely to accumulate. Molecular dynamics simulations suggested that the hydrophobicity of PECs might be strengthened by ions due to changes in intrinsic and extrinsic ion pairs and hydrophobic interactions. Based on our results, it is expected that the control of surface hydrophilicity or hydrophobicity is an effective approach for controlling the stability of PEC particles in the presence of ions.

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.

Surface modification of silica nonwoven fabrics for osteogenesis of bone marrow-derived mesenchymal stem cells.

Silica nonwoven fabrics (SNFs) with high mechanical strength and porosity are known to exhibit high cell proliferation and osteogenic differentiation potential of mesenchymal stem cells (MSCs) by morphologically mimicking the extracellular matrix (ECM). To further improve the osteoinductive ability of SNFs, it could be effective to increase the interaction between MSCs and ECM components because exogenous ECM components seem to modulate the fate of MSCs differentiation. In this study, we developed immobilization methods for ECM components, such as collagen, fibronectin, and chondroitin sulphate C on SNFs, to improve cell-matrix interactions and examined their suitability for bone tissue regeneration. Collagen and fibronectin were immobilized via physical adsorption and chondroitin sulphate C was also immobilized by the layer-by-layer method combined with chitosan on SNF surfaces to maintain the high porosity of SNFs. The treated SNFs were characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. In osteogenic differentiation culture, modified SNFs showed significantly increased expression of osteogenic differentiation marker genes compared to unmodified SNFs. These results suggest that the present methods improve cell-matrix interactions and enhance the cellular functions of MSCs. We are convinced that these simple modification techniques for ECM components are effective in functionalizing various 3D fabric scaffolds possessing hydrophilic groups.

Analysis of the aggregation mechanism of chondroitin sulfate/chitosan particles and fabrication of hydrogel cell scaffolds.

In this study, the aggregation mechanism of polyion complex (PIC) particles from chitosan (CHI) and chondroitin sulfate C (CS) in phosphate-buffered saline (PBS) was analyzed, and a novel method for the fabrication of hydrogels via aggregation was developed. The PBS induced a decrease in the ζ-potential of the CS/CHI PIC particles, increase in their diameter, and aggregation in a concentration-dependent manner. The hydrogels prepared by mixing CS/CHI PIC particle dispersion and PBS showed the PIC components, with porous structure, high swelling ratio (161.4 ± 13.3%), and high storage moduli (26.2 ± 1.4 kPa). By mixing PBS with suspended adhesive cells and CS/CHI PIC particle dispersion, hydrogels with high cell-loading efficiency were successfully synthesized. The loaded cells within the hydrogels exhibited high viability, uniform distribution, and formation of cell aggregates. These results indicate that CS/CHI-based hydrogels have a potential application as three-dimensional scaffolds for cell culture in tissue engineering.

Hydroxyapatite Particles from Simulated Body Fluids with Different pH and Their Effects on Mesenchymal Stem Cells.

Bone-like hydroxyapatite (HAp) has been prepared by biomimetic synthesis using simulated body fluid (SBF), mimicking inorganic ion concentrations in human plasma, or 1.5SBF that has 1.5-times higher ion concentrations than SBF. In this study, the controllable preparations of HAp particles from 1.5SBF with different pH values were examined. The particles obtained as precipitates from 1.5SBF showed different morphologies and crystallinities depending on the pH of 1.5SBF. Micro-sized particles at pH 7.4 of 1.5SBF had a higher Ca/P ratio and crystallinity as compared with nano-sized particles at pH 8.0 and pH 8.4 of 1.5SBF. However, a mixture of micro-sized and nano-sized particles was obtained from pH 7.7 of 1.5SBF. When Ca2+ concentrations in 1.5SBF during mineralization were monitored, the concentration at pH 7.4 drastically decreased from 12 to 24 h. At higher pH, such as 8.0 and 8.4, the Ca2+ concentrations decreased during pH adjustment and slightly decreased even after 48 h. In this investigation at pH 7.7, the Ca2+ concentrations were higher than pH 8.0 and 8.4.Additionally, cytotoxicity of the obtained precipitates to mesenchymal stem cells was lower than that of synthetic HAp. Controllable preparation HAp particles from SBF has potential applications in the construction of building components of cell scaffolds.

Heterogeneous Ganglioside-Enriched Nanoclusters with Different Densities in Membrane Rafts Detected by a Peptidyl Molecular Probe.

The specific features of the lateral distribution of gangliosides play key roles in cell-cell communications and the onset of various diseases related to the plasma membrane. We herein demonstrated that an artificial peptide identified from a phage-displayed library is available as a molecular probe for specific ganglioside nanoclustering sites in caveolae/membrane rafts on the cell surface. Atomic force microscopy studies indicated that the peptide specifically binds to the highly enriched monosialoganglioside GM1 nanodomains of reconstituted lipid bilayers composed of GM1, sphingomyelin, cholesterol, and unsaturated phospholipids. The ganglioside-containing area recognized by the peptide on the surface of PC12 cells was part of the area recognized by the cholera toxin B subunit, which has high affinity for GM1. Furthermore, the peptide bound to the cell surface after a treatment with methyl-ß-cyclodextrin (MßCD), which disrupts membrane rafts by removing cholesterol. The present results indicate that there are heterogeneous ganglioside clusters with different ganglioside densities in caveolae/membrane rafts, and the peptidyl probe selectively recognizes the high-density ganglioside nanodomain that resists the MßCD treatment. This peptidyl probe will be useful for obtaining information on the lipid organization of the cell membrane and will help clarify the mechanisms by which the lateral distribution of gangliosides affects biological functions and the onset of diseases.

Cell Scaffolds for Bone Tissue Engineering.

Currently, well-known surgical procedures for bone defects are classified into four types: (1) autogenous bone graft transplantation, (2) allogeneic bone graft transplantation, (3) xenogeneic bone graft transplantation, and (4) artificial bone graft transplantation. However, they are often risky procedures and related to postoperative complications. As an alternative, tissue engineering to regenerate new bone often involves the use of mesenchymal stem cells (MSCs), derived from bone marrow, adipose tissues, and so on, which are cultured into three-dimensional (3D) scaffolds to regenerate bone tissue by osteoinductive signaling. In this manuscript, we provide an overview of recent treatment of bone defects and the studies on the creation of cell scaffolds for bone regeneration. Bone regeneration from bone marrow-derived mesenchymal stem cells using silica nonwoven fabric by the authors' group were provided. Potential application and future direction of the present systems were also described.

Fabrication and Characterization of Polysaccharide Composite Films from Polyion Complex Particles.

Biomaterials made of natural polysaccharides have attracted much attention due to the fact of their excellent properties, such as high biocompatibility and biodegradability, and their specific biological functions based on their chemical structures. This study demonstrates that polysaccharide composite films can be fabricated from polyion complexes (PICs) with their particles used as building components. Dispersion of PIC particles prepared by mixing, centrifugation, and re-dispersion of dilute solutions of cationic and anionic polysaccharides were cast, dried, and formed into films several micrometers thick. These films were homogenous and water insoluble. It was revealed that the component anionic polysaccharides affected the film's properties such as the swelling behavior and mechanical characteristics. Adhesion of NIH3T3 cells (integrin: high, CD44: lack or weak) and A549 cells (integrin: high, CD44: high) to the composite films were examined. Both NIH3T3 and A549 cells adhered to heparin/chitosan (HEP/CHI) film because HEP has an affinity for integrin through fibronectin. However, A549 cells adhered to chondroitin sulfate (CS)/CHI and hyaluronic acid (HYA)/CHI films, whereas NIH3T3 cells did not, because both CS and HYA have affinity for CD44. These results indicated that the biological functions of anionic polysaccharides were maintained on the surface of the composite films. It was also possible to fabricate films composed of three kinds of polysaccharides: one cationic polysaccharide and two kinds of anionic polysaccharides. These results show that the properties of films composed of three kinds of polysaccharides may be controllable depending on the anionic polysaccharide composition rates.

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.

Improvement of Hepatic Functions by Spheroids Coculture with Fibroblasts in 3D Silica Nonwoven Fabrics.

In order to realize organ-on-a-chip as an effective tool for regenerative medicine and drug development, tissue-mimic cell culture methods which promote liver-specific function for long period have been developed. We have previously demonstrated that coculture of hepatocyte spheroids on fibroblasts using micropatterned substrate improved the hepatic functions due to the heterotypic cell-cell interactions and paracrine signaling from each other. In addition, hepatocyte function cultured as monolayer was also promoted in separately coculture with fibroblasts cultured as monolayer, and it is more improved in separately coculture with fibroblasts in 3D silica nonwoven fabrics. In this study, separately coculture of hepatocyte spheroids with fibroblasts cultured on 3D silica nonwoven fabrics was estimated for further improvement of hepatocyte functions. The hepatic function cocultured with fibroblast was more promoted than mono spheroids culture. The functional enhancement was significantly most improved in separately coculture with fibroblast in 3D silica nonwoven fabrics. Thus, these results were suggested that 3D culture of fibroblasts in 3D silica nonwoven fabrics increased the heterotypic secretion of paracrine factors, and it is essential for improved hepatic performance.

N-Hydroxysuccinimide Bifunctionalized Triblock Cross-Linker Having Hydrolysis Property for a Biodegradable and Injectable Hydrogel System.

The design of biocompatible, degradable, and injectable hydrogel has been attractive for achievement of safe and efficient tissue engineering. Herein, we designed a N-hydroxysuccinimide (NHS) ester-terminated ABA triblock copolymer composed of poly(ethylene glycol) (PEG) as hydrophilic A segments and poly(dl-lactide) (PLA) as B segment having hydrolysis property (NHS-PEG-b-PLA-b-PEG-NHS) to be a cross-linker of polymer segments having amine groups for facile construction of injectable and degradable hydrogel. The PLA domain, which is widely accepted hydrolyzable segments, is inherently hydrophobic and simple introduction of the NHS group on the ends of PLA would not have high reactivity in aqueous milieu to construct injectable hydrogel. Thus, in this design, hydrophilic PEG was introduced as A segments to increase the reactivity of NHS groups at the ends of linkers by increasing the mobility. To demonstrate the property as a cross-linker for constructing degradable and injectable hydrogel, carboxylmethyl chitosan (CH), which is a polymer segment having amine groups, and NHS-PEG-b-PLA-b-PEG-NHS solutions were mixed to form the hydrogel (CH/PEG-PLA-PEG) under physiological condition. The formation of CH/PEG-PLA-PEG hydrogel proceeded within minute-order period after mixing the solutions, suggesting NHS-PEG-b-PLA-b-PEG-NHS is applicable to the cross-linker for construction of injectable hydrogel system with time-dependent gelation property. Degradation of the obtained CH/PEG-PLA-PEG hydrogel was observed, whereas that of CH/PEG, which was prepared from NHS-PEG-NHS and CH, was not observed, appealing the degradation property of the CH/PEG-PLA-PEG hydrogel based on hydrolysis of the PLA domain. Furthermore, chondrocytes embedded in CH/PEG-PLA-PEG hydrogels promoted collagen synthesis compared to CH/PEG. These demonstrations indicate the designed NHS-PEG-b-PLA-b-PEG-NHS is a promising cross-linker to construct the injectable and degradable hydrogel and eventually promote hydrogel performance as a tissue regeneration scaffold.

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.

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.

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.

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.

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.

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.