Cultivation and Transplantation of Three-Dimensional Skins with Laser-Processed Biodegradable Membranes.

For the treatment of irreversible, extensive skin damage, artificial skins or cultured skins are useful when allogeneic skins are unavailable. However, most of them lack vasculature, causing delayed perfusion and hence delay or failure in engraftment of the tissues. We previously developed a prevascularized three-dimensional (3D) cultured skin based on the layer-by-layer cell coating technique (LbL-3D skin), in which cells are seeded and laminated on a porous polymer membrane for medium supply to the thick cultured tissue. Recent animal studies have demonstrated that LbL-3D skin can achieve rapid perfusion and high graft survival after transplantation. However, there were practical issues with separating LbL-3D skins from the membranes before transplantation and the handling separated LbL-3D skins for transplantation. To address these problems, in this study, we examined the use of biodegradable porous polymer membranes that enabled the transplantation of LbL-3D skins together with the membranes, which could be decomposed after transplantation. Thin films made from poly (lactic-co-glycolic acid) (PLGA) were irradiated with femtosecond laser pulses to create micro through-holes, producing porous membranes. We designed and fabricated culture inserts with the PLGA membranes and cultivated LbL-3D skins with 2 × 106 neonatal normal human dermal fibroblasts and 1 × 104 human umbilical vein endothelial cells in the dermis of 20 cell layers and 1 × 105 neonatal human epidermal keratinocytes in the epidermis. Histological analyses revealed that the skins cultured on the PLGA membranes had thickness of about 400 µm and that there were no defects in the quality of the skins cultured on the PLGA membranes when compared with those cultured on the conventional (nonbiodegradable) commercial membranes. The cultured LbL-3D skins were then transplanted together with the PLGA membranes onto full-thickness excisional wounds in mice. At 7 days posttransplantation onto a mouse, the tissues above and below the membrane were connected through the holes with collagen-positive fibers that appeared to migrate from both the host and donor sides, and favorable reepithelization was observed throughout the transplanted skin region. However, insufficient engraftment was observed in some cases. Thus, further optimization of the membrane conditions would be needed to improve the transplantation outcome.

Simultaneous laser-based graphitization and microstructuring of bamboo for supercapacitors derived from renewable resources.

Utilizing renewable resources for electrodes realizes the sustainable fabrication of a supercapacitor with high environmental friendliness. Laser-based graphitization of biomass has been emerging as a promising technique for patterning the electrodes of a supercapacitor with renewable resources. Herein, simultaneous patterning and microstructuring of laser-induced graphene (LIG) on a renewable biomass resource, bamboo, by a laser-based graphitization technique was demonstrated. By irradiating femtosecond laser pulses onto bamboo, graphitization and microstructuring were both induced simultaneously, forming conductive structures with high surface area. Furthermore, LIG patterned on bamboo by our method was used as the electrodes of supercapacitors. NaCl was selected as the electrolyte for the fabrication of supercapacitors. The proposed method realizes the fabrication of environmentally-friendly supercapacitors comprised of all renewable biomass resources.

Laser-based molecular delivery and its applications in plant science.

Lasers enable modification of living and non-living matter with submicron precision in a contact-free manner which has raised the interest of researchers for decades. Accordingly, laser technologies have drawn interest across disciplines. They have been established as a valuable tool to permeabilize cellular membranes for molecular delivery in a process termed photoinjection. Laser-based molecular delivery was first reported in 1984, when normal kidney cells were successfully transfected with a frequency-multiplied Nd:YAG laser. Due to the rapid development of optical technologies, far more sophisticated laser platforms have become available. In particular, near infrared femtosecond (NIR fs) laser sources enable an increasing progress of laser-based molecular delivery procedures and opened up multiple variations and applications of this technique.This review is intended to provide a plant science audience with the physical principles as well as the application potentials of laser-based molecular delivery. The historical origins and technical development of laser-based molecular delivery are summarized and the principle physical processes involved in these approaches and their implications for practical use are introduced. Successful cases of laser-based molecular delivery in plant science will be reviewed in detail, and the specific hurdles that plant materials pose will be discussed. Finally, we will give an outlook on current limitations and possible future applications of laser-based molecular delivery in the field of plant science.

Tailored Structure and Antibacterial Properties of Silica-Coated Silver Nanoplates by Pulsed Laser Irradiation.

We coated triangular-shaped silver nanoparticles, a type of anisotropic nanoplate (NPL), with silica (i.e., prepared Ag@SiO2 NPLs). When we irradiated Ag@SiO2 NPLs with nanosecond-pulsed laser light for 10 s, the triangular shape changed to spherical because of the photothermal effect. A high laser power exposed the silver core, and the particles exhibited strong antimicrobial activity. In contrast, at a moderate laser power, the silica layer crystallized, and the particles' antimicrobial activity decreased. Thus, a combination of Ag@SiO2 NPLs and an appropriately tuned power of pulsed laser irradiation facilitated a decreased or an increased antimicrobial activity.

Laser Direct Writing of Graphene Quantum Dots inside a Transparent Polymer.

Graphene quantum dots (GQDs) have emerged as a promising new class of environmentally friendly quantum dots with unique properties. However, the limitations of synthesis and patterning methods have hindered GQDs from displaying their true potentials to date. Here, we demonstrate the simultaneous synthesis and patterning of GQDs for the first time inside a transparent polymer, polydimethylsiloxane (PDMS), using femtosecond laser pulses. By focusing and scanning femtosecond laser pulses, arbitrary fluorescent patterns such as a concealed fluorescent QR code can be readily patterned without pre- and/or post-treatment. In addition, the proposed method is applied to the fabrication of fluorescent three-dimensional structures inside a transparent polymer via multiphoton interactions. The proposed method realizes single-stepped and spatially selective patterning of GQDs directly inside polymer substrates and expands the possibilities of GQDs for applications in novel flexible three-dimensional optoelectrical devices.

Microfabrication of cellulose nanofiber-reinforced hydrogel by multiphoton polymerization.

The mechanical strength of hydrogel microstructures is crucial for obtaining the desired flexibility, robustness, and biocompatibility for various applications such as cell scaffolds and soft microrobots. In this study, we demonstrate the fabrication of microstructures composed of cellulose nanofibers (CNFs) and poly(ethylene glycol) diacrylate (PEGDA) hydrogels by multiphoton polymerization. The stress of the fabricated microstructure during tensile testing increased with an increase in the CNF concentration, indicating that the mechanical strength of the microstructure was enhanced by using CNFs as fillers. Moreover, the swelling ratio of the microstructure increased with increasing CNF concentration in the PEGDA hydrogel. Our results show the potential of the technique for the microfabrication of advanced cell scaffolds and soft microrobots with the desired mechanical strength.

Fabrication of Hollow Channels Surrounded by Gold Nanoparticles in Hydrogel by Femtosecond Laser Irradiation.

The fabrication of hollow channels surrounded by gold nanoparticles in poly(ethylene glycol) diacrylate (PEGDA) is demonstrated. The absorption spectra show that gold nanoparticles were formed at the periphery of the focus by reduction of gold ions. The microscope observation and Raman spectroscopy analyses indicate that the center of the channels were void of PEGDA, which can be attributed to the femtosecond laser-induced degradation of the hydrogel. Since both the hydrogel and gold nanoparticles are biocompatible, this technique of fabricating hollow channels surrounded by gold nanoparticles is promising for tissue engineering, drug screening, and lab-on-a-chip devices.

Myoblast adhesion and proliferation on biodegradable polymer films with femtosecond laser-fabricated micro through-holes.

Controlling cell adhesion and cell differentiation is necessary to fabricate a tissue with arbitrary properties for tissue engineering applications. A substrate with a porous structure as a cell scaffold allows the diffusion of the cell culture medium through the scaffold. In this work, we show that the femtosecond laser fabricated micro through-holes in biodegradable polymer films, enhance myoblast adhesion, and accelerates proliferation and differentiation. ChR2-C2C12 and UT-C2C12 cells were seeded on the films with micro through-holes each fabricated by a single femtosecond laser pulse. Cell adhesion was enhanced on films with holes fabricated by laser irradiation. In addition, cell proliferation was accelerated on films with micro through-holes that penetrate the film, compared to on films with micro craters that do not penetrate the film. On films with arrays consisting of micro through-holes, cells aligned along the arrays and cell fusion was enhanced, indicating the acceleration of cell differentiation.

Synthesis of silicon carbide nanocrystals and multilayer graphitic carbon by femtosecond laser irradiation of polydimethylsiloxane.

Laser-based modification of polymer materials has been emerging as a versatile and efficient technique to simultaneously form and pattern electrically conductive materials. Recently, it has been revealed that native polydimethylsiloxane (PDMS) can be modified into electrically conductive structures using femtosecond laser irradiation; however, the details regarding the structures formed by this method have yet to be revealed. In this work, structures were fabricated by focusing and scanning femtosecond laser pulses onto the surface of PDMS. Raman Spectroscopy and Transmission Electron Microscopy (TEM) analyses revealed the formation of silicon carbide (SiC) nanocrystals, as well as multilayer graphitic carbon, in the modified regions of PDMS. The state of the formed material differed depending on the distance from the focal spot, suggesting that photo-thermal effects contributed to the degradation of PDMS into conductive material. Electrical conductivity measurements, in addition to Raman results, indicated that the amount of disorder in the formed graphitic carbon contributes to the electrical conductivity of the fabricated structures.

Fabrication of a Monolithic Lab-on-a-Chip Platform with Integrated Hydrogel Waveguides for Chemical Sensing.

Hydrogel waveguides have found increased use for variety of applications where biocompatibility and flexibility are important. In this work, we demonstrate the use of polyethylene glycol diacrylate (PEGDA) waveguides to realize a monolithic lab-on-a-chip device. We performed a comprehensive study on the swelling and optical properties for different chain lengths and concentrations in order to realize an integrated biocompatible waveguide in a microfluidic device for chemical sensing. Waveguiding properties of PEGDA hydrogel were used to guide excitation light into a microfluidic channel to measure the fluorescence emission profile of rhodamine 6G as well as collect the fluorescence signal from the same device. Overall, this work shows the potential of hydrogel waveguides to facilitate delivery and collection of optical signals for potential use in wearable and implantable lab-on-a-chip devices.

Spatially-targeted laser fabrication of multi-metal microstructures inside a hydrogel.

The spatially-targeted fabrication of bimetallic microstructures coexisting in the supporting hydrogel is demonstrated by multi-photon photoreduction. Microstructures composed of gold and silver were fabricated along a predefined trajectory by taking advantages of the hydrogel's ionic permeability. Different resonant wavelengths of optical absorption were obtained for gold, silver, and their bimetallic structures. Transmission electron microscopy and energy dispersive X-ray analysis revealed that the optical properties are attributable to the formation of bimetallic structure consisted of core-shell nanoparticles. The fabrication of dissimilar metal structures within hydrogel is a promising technique for optically driven actuators in soft robotics and sensing applications by allowing for site-selective optical properties.

Photoacoustic diagnosis of pharmacokinetics and vascular shutdown effects in photodynamic treatment with indocyanine green-lactosome for a subcutaneous tumor in mice.

Indocyanine green lactosome (ICG-lactosome) is an attractive new-generation agent for photodynamic therapy (PDT) that is characterized by a near-infrared excitation wavelength and high stability in the bloodstream. Fluorescence imaging has been used to examine its pharmacokinetics in vivo, but no depth-resolved information can be obtained with this method. In this study, we applied photoacoustic (PA) imaging to visualize the depth distribution of ICG-lactosome in a mouse subcutaneous tumor model. With this method, the depth distribution of blood vessels can also be visualized, enabling detection of vascular shutdown effects due to PDT. We performed PA imaging of both the distributions of ICG-lactosome and blood vessels in a tumor before and after PDT, and we found that PA signals originating from ICG-lactosome were greatly increased at 18 h after drug injection but rapidly decreased after PDT. These results indicate efficient accumulation of ICG-lactosome and rapid photobleaching due to the PDT reaction in the tumor, respectively. After PDT, PA amplitudes of hemoglobin were significantly decreased, being attributable to vascular shutdown effects. These results show the usefulness of PA imaging for monitoring not only photosensitizer accumulation and bleaching but also vascular responses in PDT with ICG-lactosome. This method can be applied to the diagnosis of many types of PDT processes.

Surface-Enhanced Raman Spectroscopy (SERS) of Mancozeb and Thiamethoxam Assisted by Gold and Silver Nanostructures Produced by Laser Techniques on Paper.

Advanced gold (Au) and silver (Ag) nanostructures were produced by laser techniques on printer paper substrate. Surface-enhanced Raman spectroscopy (SERS) analyses of the fungicide mancozeb (Dithane DG) and insecticide thiamethoxam (Aktara 25 BG) in quantities smaller than usually applied in agricultural medicine were performed for the first time assisted by the structures fabricated. The investigations and results show an easy alternative and cheap way to detect small amounts or residue of harmful environmental pollutants, which has a direct bearing on food quality and thus on human health.

Femtosecond Laser-Based Modification of PDMS to Electrically Conductive Silicon Carbide.

In this paper, we experimentally demonstrate femtosecond laser direct writing of conductive structures on the surface of native polydimethylsiloxane (PDMS). Irradiation of femtosecond laser pulses modified the PDMS to black structures, which exhibit electrical conductivity. Fourier-transform infrared (FTIR) and X-ray diffraction (XRD) results show that the black structures were composed of ß-silicon carbide (ß-SiC), which can be attributed to the pyrolysis of the PDMS. The electrical conductivity was exhibited in limited laser power and scanning speed conditions. The technique we present enables the spatially selective formation of ß-SiC on the surface of native PDMS only by irradiation of femtosecond laser pulses. Furthermore, this technique has the potential to open a novel route to simply fabricate flexible/stretchable MEMS devices with SiC microstructures.

Gold nanoparticle-mediated laser stimulation induces a complex stress response in neuronal cells.

Stimulation of neuronal cells generally resorts to electric signals. Recent advances in laser-based stimulation methods could present an alternative with superior spatiotemporal resolution. The avoidance of electronic crosstalk makes these methods attractive for in vivo therapeutic application. In particular, nano-mediators, such as gold nanoparticles, can be used to transfer the energy from a laser pulse to the cell membrane and subsequently activate excitable cells. Although the underlying mechanisms of neuronal activation have been widely unraveled, the overall effect on the targeted cell is not understood. Little is known about the physiological and pathophysiological impact of a laser pulse targeted onto nanoabsorbers on the cell membrane. Here, we analyzed the reaction of the neuronal murine cell line Neuro-2A and murine primary cortical neurons to gold nanoparticle mediated laser stimulation. Our study reveals a severe, complex and cell-type independent stress response after laser irradiation, emphasizing the need for a thorough assessment of this approach's efficacy and safety.

Shrinkable silver diffraction grating fabricated inside a hydrogel using 522-nm femtosecond laser.

The integration of metal microstructures and soft materials is promising for the realization of novel optical and biomedical devices owing to the flexibility and biocompatibility of the latter. Nevertheless, the fabrication of three-dimensional metal structures within a soft material is still challenging. In this study, we demonstrate the fabrication of a silver diffraction grating inside a biocompatible poly(ethylene glycol) diacrylate (PEGDA) hydrogel by using a 522-nm femtosecond laser via multi-photon photoreduction of silver ions. The optical diffraction pattern obtained with the grating showed equally spaced diffraction spots, which indicated that a regular, periodic silver grating was formed. Notably, the distance between the diffraction spots changed when the water content in the hydrogel was reduced. The grating period decreased when the hydrogel shrank owing to the loss of water, but the straight shapes of the line structures were preserved, which demonstrated the optical tunability of the fabricated structure. Our results demonstrate the potential of the femtosecond laser-based photoreduction technique for the fabrication of novel tunable optical devices as well as highly precise structures.

Laser-assisted fabrication of gold nanoparticle-composed structures embedded in borosilicate glass.

We present results on laser-assisted formation of two- and three-dimensional structures comprised of gold nanoparticles in glass. The sample material was gold-ion-doped borosilicate glass prepared by conventional melt quenching. The nanoparticle growth technique consisted of two steps - laser-induced defect formation and annealing. The first step was realized by irradiating the glass by nanosecond and femtosecond laser pulses over a wide range of fluences and number of applied pulses. The irradiation by nanosecond laser pulses (emitted by a Nd:YAG laser system) induced defect formation, expressed by brown coloration of the glass sample, only at a wavelength of 266 nm. At 355, 532 and 1064 nm, no coloration of the sample was observed. The femtosecond laser irradiation at 800 nm also induced defects, again observed as brown coloration. The absorbance spectra indicated that this coloration was related to the formation of oxygen deficiency defects. After annealing, the color of the irradiated areas changed to pink, with a corresponding well-defined peak in the absorbance spectrum. We relate this effect to the formation of gold nanoparticles with optical properties defined by plasmon excitation. Their presence was confirmed by high-resolution TEM analysis. No nanoparticle formation was observed in the samples irradiated by nanosecond pulses at 355, 532 and 1064 nm. The optical properties of the irradiated areas were found to depend on the laser processing parameters; these properties were studied based on Mie theory, which was also used to correlate the experimental optical spectra and the characteristics of the nanoparticles formed. We also discuss the influence of the processing conditions on the characteristics of the particles formed and the mechanism of their formation and demonstrate the fabrication of structures composed of nanoparticles inside the glass sample. This technique can be used for the preparation of 3D nanoparticle systems embedded in transparent materials with potential applications in the design of new optical components, such as metamaterials and in plasmonics.

Effects of pulsed laser irradiation on gold-coated silver nanoplates and their antibacterial activity.

Gold-coated silver nanoplates, when subjected to pulsed laser irradiation, changed their shape from triangular to spherical, accompanied by a shift of their extinction spectra. The simple single crystal structure of the silver nanoplates changed to multiple small crystal domains. The ratio of silver to gold of the particles also changed from 22 : 1 to 4.5 : 1, enabling more silver to be released. As a result, the antibacterial activity of the gold-coated silver nanoplates was significantly increased after pulsed laser irradiation.

Intracellular localization and delivery of plasmid DNA by biodegradable microsphere-mediated femtosecond laser optoporation.

Micro-/nanosphere-mediated femtosecond laser cell perforation is one of the high throughput technologies used for macro-molecule-delivery into multiple cells. We have demonstrated the delivery of plasmid-DNA/liposome complexes into cells using biodegradable polymer microspheres and a femtosecond laser and investigated the intracellular localization of the complexes by delivering fluorescence-labeled plasmid-DNA/liposome complexes into cells. The utilization of liposomes increases the number of complexes delivered into the cytoplasm by laser illumination, which contributed to the increased transfection rate. In the experiment involving polystyrene (PS) microspheres of different diameters, the fluorescence of the complexes was detected in the nucleus as well as cytoplasm after laser illumination for PS microspheres of 3.0 µm diameter. The direct delivery of complexes into the nucleus is probably attributed to the enhancement of the nuclear membrane permeability by the enhanced optical field obtained close to the nucleus. These revelations on the intracellular localization of foreign DNA would provide effective laser-based transfection. Picture: Intranuclear delivery of plasmid-DNA/liposome complexes by utilizing dielectric microspheres and a femtosecond laser.