Toshinori Fujie

Microwrinkled Conducting Polymer Interface for Anisotropic Multicellular Alignment

Surfaces with controlled micro and nanoscale topographical cues are useful as smart scaffolds and biointerfaces for cell culture. Recently, use of thin-film and surface wrinkling is emerging as a rapid unconventional method for preparing topographically patterned surfaces, especially suited for the production of smart patterns over large area surfaces. On the other hand, there is an increasing interest in employing conducting polymers as soft, biocompatible, conductive biointerfaces or as parts of bioelectronic devices. A novel convenient and versatile method is presented for producing anisotropic topographical cues at the micro- and nanoscale on conducting polymer surfaces. Micro and nanowrinkles were formed during the heat-shrinking process of a thermo-retractable polystyrene substrate. Surface wrinkling was due to the mismatch between the mechanical properties of a conducting polymer ultrathin film and the substrate. Various geometries of wrinkled structures were prepared, demonstrating the tunability of topography depending on the thickness of the conductive film. A method for patterning the conductive properties of the wrinkled substrates was also presented. Such surfaces acted as smart scaffolds for the functional alignment of cells, envisioning their electrical stimulation. Cell adhesion and proliferation were evaluated, comparing different topographies, and a preferential anisotropic alignment of C2C12 murine skeletal muscle cells along wrinkles was demonstrated. The observed trends were also confirmed concerning the formation of aligned myotubes in C2C12 differentiation stage. Furthermore, improved results in terms of aligned and mature myotube formation were obtained by co-culturing C2C12 cells with a fibroblasts feeder layer. The combination of living cells and tunable conductive nanowrinkles will represent a unique tool for the development of innovative biomedical devices.

Boron Nitride Nanotube-Mediated Stimulation of Cell Co-Culture on Micro-Engineered Hydrogels

In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves) polyacrylamide substrate, showing a precisely tuned Young’s modulus (∼ 14 kPa) and a small thickness (∼ 12 µm). We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric nanostructures (i.e., boron nitride nanotubes) activated by ultrasounds, thus exploiting the ability of boron nitride nanotubes to convert outer mechanical waves (such as ultrasounds) in intracellular electrical stimuli, by exploiting the direct piezoelectric effect. We demonstrated that nanotubes were internalized by muscle cells and localized in both early and late endosomes, while they were not internalized by the underneath fibroblast layer. Muscle cell differentiation benefited from the synergic combination of topographical, mechanical, chemical and nanoparticle-based stimuli, showing good myotube development and alignment towards a preferential direction, as well as high expression of genes encoding key proteins for muscle contraction (i.e., actin and myosin). We also clarified the possible role of fibroblasts in this process, highlighting their response to the above mentioned physical stimuli in terms of gene expression and cytokine production. Finally, calcium imaging-based experiments demonstrated a higher functionality of the stimulated co-cultures.

Myotube formation on gelatin nanofibers – Multi-walled carbon nanotubes hybrid scaffolds

Engineering functional muscle tissue requires the formation of densely packed, aligned, and mature myotubes. To enhance the formation of aligned myotubes with improved contractibility, we fabricated aligned electrospun gelatin multi-walled carbon nanotubes (MWNTs) hybrid fibers that were used as scaffolds for the growth of myoblasts (C2C12). The MWNTs significantly enhanced myotube formation by improving the mechanical properties of the resulting fibers and upregulated the activation of mechanotransduction related genes. In addition, the fibers enhanced the maturation of the myotubes and the amplitude of the myotube contractions under electrical stimulation (ES). Such hybrid material scaffolds may be useful to direct skeletal muscle cellular organization, improve cellular functionality and tissue formation.

Dual therapeutic action of antibiotic-loaded nanosheets for the treatment of gastrointestinal tissue defects.

An ultra-thin polymer film (nanosheet) composed of polysaccharides (i.e., polysaccharide nanosheet) provides sufficient adhesiveness, flexibility and robustness to act as an effective wound dressing. We have recently demonstrated the sealing effect of a nanosheet on a murine cecal puncture. Nevertheless, a small percentage of bacteria penetrated the nanosheet because of its ultra-thin structure. Here, we have developed an antibiotic-loaded nanosheet to inhibit bacterial penetration and investigated its therapeutic efficacy using a model of a murine cecal puncture. Tetracycline (TC) was sandwiched between a poly(vinylacetate) (PVAc) layer and the polysaccharide nanosheet (named PVAc-TC-nanosheet). Under physiological conditions, TC was released from the nanosheet for 6 h. Microscopic observation between the interface of the PVAc-TC-nanosheet and bacteria demonstrated how its potent anti-microbial effect was achieved. In vivo studies show that overlapping therapy with the PVAc-TC-nanosheet (thickness: 177 nm) significantly increases mouse survival rate after cecal puncture as well as suppressing an increase in the intraperitoneal bacterial count and leukocyte count.

Hydrodynamic Transformation of a Freestanding Polymer Nanosheet Induced by a Thermoresponsive Surface

Freestanding quasi-two-dimensional ultrathin films (e.g., 41 nm thick polymer nanosheets) were produced, on which stimuli-responsive 47 nm thick polymer brushes were constructed by atom transfer radical polymerization (ATRP) of poly(N-isopropylacrylamide). The resulting surfaces of the multilayered polysaccharide ultrathin films were evaluated by ellipsometry, IR imaging, in situ variable-temperature atomic force microscopy (AFM), and contact angle measurements. The morphological transformation of the freestanding polymer nanosheet bearing thermoresponsive polymer brushes was observed macroscopically through reversible structural color changes at the air-water interface. The dynamic shape change of the nanosheet was also monitored with the addition of a surfactant such as sodium n-dodecylsulfate to reduce the hydrophobicity of the surface. It was then demonstrated that the highly flexible freestanding polymer nanosheet is capable of acting as a unique platform for inducing stimuli-responsive behavior in nanomaterials.

Development of fibrinogen γ‐chain peptide‐coated, adenosine diphosphate‐encapsulated liposomes as a synthetic platelet substitute

Summary.  Background: The dodecapeptide HHLGGAKQAGDV (H12), corresponding to the fibrinogen γ‐chain carboxy‐terminal sequence (γ400‐411), is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated H12‐coated nanoparticles (polymerized albumin or liposome) as platelet function‐supporting synthetic products. Objectives: To strengthen the hemostatic ability of H12‐coated particles as a platelet substitute, we exploited installation of a drug delivery function by encapsulating adenosine diphosphate (ADP) into liposomes [H12‐(ADP)‐liposomes]. Methods and results: Via selective interaction with activated platelets through GPIIb/IIIa, H12‐(ADP)‐liposomes were capable of augmenting agonist‐induced platelet aggregation by releasing ADP in an aggregation‐dependent manner. When intravenously injected into rats, liposomes were readily targeted to sites of vascular injury as analyzed on computed tomography. In fact, comparable to fresh platelets, liposomes exhibited considerable hemostatic ability for correcting prolonged bleeding time in a busulphan‐induced thrombocytopenic rabbit model. In addition, the liposomes showed no activating or aggregating effects on circulating platelets in normal rabbits. Conclusion: H12‐(ADP)‐liposome may thus offer a promising platelet substitute, being made with only synthetic materials and exerting hemostatic functions in vivo via reinforcement of primary thrombus formation by residual platelets in thrombocytopenia at sites of vascular injury, but not in circulation.

Free-Standing Poly(L-lactic acid) Nanofilms Loaded with Superparamagnetic Nanoparticles

Freely suspended nanocomposite thin films based on soft polymers and functional nanostructures have been widely investigated for their potential application as active elements in microdevices. However, most studies are focused on the preparation of nanofilms composed of polyelectrolytes and charged colloidal particles. Here, a new technique for the preparation of poly(l-lactic acid) free-standing nanofilms embeddidng superparamagnetic iron oxide nanoparticles is presented. The fabrication process, based on a spin-coating deposition approach, is described, and the influence of each production parameter on the morphology and magnetic properties of the final structure is investigated. Superparamagnetic free-standing nanofilms were obtained, as evidenced by a magnetization hysteresis measurement performed with a superconducting quantum interference device (SQUID). Nanofilm surface morphology and thickness were evaluated by atomic force microscopy (AFM), and the nanoparticle dispersion inside the composites was investigated by transmission electron microscopy (TEM). These nanofilms, composed of a biodegradable polyester and remotely controllable by external magnetic fields, are promising candidates for many potential applications in the biomedical field.

Evaluation of substrata effect on cell adhesion properties using freestanding poly(L-lactic acid) nanosheets.

Investigation of the interactions between cells and material surfaces is important not only for the understanding of cell biology but also for the development of smart biomaterials. In this study, we investigated the substrate-related effects on the interaction between cell and polymeric ultrathin film (nanosheet) by modulating the mechanical properties of the nanosheet with a metal substrate or mesh. A freestanding polymeric nanosheet with tens-of-nanometers thickness composed of poly(L-lactic acid) (PLLA nanosheet) was fabricated by combination of a spin-coating technique and a water-soluble sacrificial layer. The freestanding PLLA nanosheet was collected on a stainless steel mesh (PLLA-mesh) and subsequently used for cell adhesion studies, comparing the results to the ones on a control SiO(2) substrate coated with an ultrathin layer of PLLA (PLLA-substrate). The adhesion of rat cardiomyocytes (H9c2) was evaluated on both samples after 24 h of culture. The PLLA-mesh with the tens-of-nanometers thick nanosheets induced an anisotropic adhesion of H9c2, while H9c2 on the PLLA-substrate showed an isotropic adhesion independent from the nanosheet thickness. Interestingly, an increment in the nanosheet thickness in the PLLA-mesh samples reduced the cellular anisotropy and led to a similar morphology to the PLLA-substrate. Considering the huge discrepancy of Youngs modulus between PLLA nanosheet (3.5-4.2 GPa) and metal substrate (hundreds of GPa), cell adhesion was mechanically regulated by the Youngs modulus of the underlying substrate when the thickness of the PLLA nanosheet was tens of nanometers. Modulation of the stiffness of the polymeric nanosheet by utilizing a rigid underlying material will allow the constitution of a unique cell culture environment.

Micropatterned Polymeric Nanosheets for Local Delivery of an Engineered Epithelial Monolayer

Like a carpet for cells, micropatterned polymeric nanosheets are developed toward local cell delivery. The nanosheets direct morphogenesis of retinal pigment epithelial (RPE) cells and allow for the injection of an engineered RPE monolayer through syringe needles without the loss of cell viability. Such an ultrathin carrier has the promise of a minimally invasive delivery of cells into narrow tissue spaces.

Roll to roll processing of ultraconformable conducting polymer nanosheets

Thin and compliant conductive materials and electronic devices that are able to stand as free-standing membranes or to conform to surfaces are relevant for the development of human-device interfaces and unperceivable skin-contact personal health monitoring systems. In this work, a roll-to-roll (R2R) process for the preparation of conductive polymer nanosheets on large areas has been developed in view to move such technology towards real-world applications. R2R conductive nanosheets are obtained as free-standing structures through release from a temporary substrate and then transferred in conformal contact with any target surface with arbitrary shape, curvature and surface topography (including biological tissue such as skin). A specific high-conductivity formulation of PEDOT:PSS has been optimized for skin-contact applications, by making use of butylene glycol (BG) as a dopant: a dermatologically approved ingredient. The R2R nanosheets were tested as unperceivable surface electromyography electrodes able to record muscle-electrical activity. The present R2R process has advantageous properties such as continuous, high throughput printing on large area rolls, cost-effectiveness, speed of execution and use of industry-ready/mass-scale manufacturing technology.