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.

On-Demand Tunability of Microphase Separation Structure of 3D Printing Material by Reversible Addition/Fragmentation Chain Transfer Polymerization.

Controlling the phase-separated structure of polymer alloys is a promising method for tailoring the properties of polymers. However, controlling the morphology of phase-separated structures is challenging. Recently, phase-separated structures have been fabricated via 3D printing; however, only a few methods that enable on-demand control of phase separation have been reported. In this study, laser-scanning stereolithography, a vat photopolymerization method, is used to form a phase-separated structure via polymerization-induced microphase separation by varying the scanning speed and using macro-reversible addition/fragmentation chain transfer (macro-RAFT) agents with different average molar masses, along with multiarmed macro-RAFT agents; such structures were used to fabricate 3D-printed parts. Various phase-separated morphologies including sea-island and reverse sea-island were achieved by controlling the laser scanning speed and RAFT type. Heterogeneous structures with different material properties were also achieved by simply changing the laser scanning speed. As the deformation due to shrinkage in the process of cleaning 3D-printed parts depends on the laser scanning speed, shape correction was introduced to suppress the effect of shrinkage and obtain the desired shape.

Cryopreservation of engineered hair follicle germs for hair regenerative medicine.

Hair regenerative medicine must involve practical procedures, such as cryopreservation of tissue grafts. This can aid in evaluating tissue safety and quality, as well as transportation to a clinic and multiple transplants. Hair follicle germs (HFGs), identified during in vivo development, are considered effective tissue grafts for hair regenerative medicine. However, to the best of our knowledge, methods for cryopreserving HFGs have not been explored yet. This study investigated the efficacy of slow vitrification methods for freezing HFGs. Cryoprotectants such as dimethyl sulfoxide (DMSO) and carboxylated poly-l-lysine were used for vitrification. The results indicate that DMSO vitrification yielded the most efficient de novo hair regeneration in mouse skin, comparable to that of non-cryoprotected HFGs. A microfinger was fabricated to scale up the cryopreservation method, considering that thousands of tissue grafts were required per patient in clinical practice. The microfinger can be used for a series of processes, holding the HFG, replacing it with a cryopreservation solution, freezing it in liquid nitrogen, thawing it in a warm medium, and transplanting it into the skin. Although de novo hair regeneration by HFGs cryopreserved using microfingers was reduced by approximately 20 % compared to those cryopreserved using flat plates for fertilized eggs, it exceeded 50 %. These findings demonstrate that vitrification with DMSO and microfingers could be a useful approach for the cryopreservation of tissue grafts in hair regenerative medicine for hair loss.

Gelatin acrylamide with improved UV crosslinking and mechanical properties for 3D biofabrication.

Photocrosslinkable gelatin has attracted increasing interest in the field of biofabrication, with the most studied and widely used photocrosslinkable gelatin being gelatin methacrylate (GelMa). However, the 3D fabrication of GelMa has presented several limitations and challenges, primarily due to its slow crosslinking speed. It is generally known that acryl-based functional groups have faster reaction kinetics than methacryl-base groups. However, gelatin acrylamide (GelAc) has not been widely investigated, largely due to its increased complexity of synthesis relative to GelMA. In this study, we developed a novel synthesis method for GelAc. By varying the reaction ratio of reagents, GelAc with a degree of substitution from 20% to 95% was produced. The UV crosslinking properties of GelAc was studied, demonstrating significantly faster crosslinking kinetics than GelMa, especially at lower concentrations and low photoinitiator concentrations. The swelling ratio and mechanical properties of the crosslinked GelAc hydrogel were also characterized, and biocompatibility experiments conducted via both surface seeding and hydrogel encapsulation of cells, with good cell viability observed. The application of GelAc for 3D biofabrication was demonstrated by 3D printing. GelAc can be a useful material for the fabrication of 3D conduits for tissue engineering applications.

Bone beads enveloped with vascular endothelial cells for bone regenerative medicine.

The transplantation of pre-vascularized bone grafts is a promising strategy to improve the efficacy of engraftment and bone regeneration. We propose a hydrogel microbead-based approach for preparing vascularized and high-density tissue grafts. Mesenchymal stem cell-encapsulated collagen microgels (2 µL), termed bone beads, were prepared through spontaneous constriction, which improved the density of the mesenchymal stem cells and collagen molecules by more than 15-fold from the initial day of culture. Constriction was attributed to cell-attractive forces and involved better osteogenic differentiation of mesenchymal stem cells than that of spheroids. This approach was scalable, and ∼2000 bone beads were prepared semi-automatically using a liquid dispenser and spinner flask. The mechanical stimuli in the spinner flask further improved the osteogenic differentiation of the mesenchymal stem cells in the bone beads compared with that in static culture. Vascular endothelial cells readily attach to and cover the surface of bone beads. The in vitro assembly of the endothelial cell-enveloped bone beads resulted in microchannel formation in the interspaces between the bone beads. Significant effects of endothelialization on in vivo bone regeneration were shown in rats with cranial bone defects. The use of endothelialized bone beads may be a scalable and robust approach for treating large bone defects. STATEMENT OF SIGNIFICANCE: A unique aspect of this study is that the hMSC-encapsulated collagen microgels were prepared through spontaneous constriction, leading to the enrichment of collagen and cell density. This constriction resulted in favorable microenvironments for the osteogenic differentiation of hMSCs, which is superior to conventional spheroid culture. The microgel beads were then enveloped with vascular endothelial cells and assembled to fabricate a tissue graft with vasculature in the interspaces among the beads. The significant effects of endothelialization on in vivo bone regeneration were clearly demonstrated in rats with cranial bone defects. We believe that microgel beads covered with vascular endothelial cells provide a promising approach for engineering better tissue grafts for bone-regenerative medicine.

Fabrication of Flexible Wiring with Intrinsically Conducting Polymers Using Blue-Laser Microstereolithography.

Recently, flexible devices using intrinsically conductive polymers, particularly poly(3,4-ethylenedioxythiophene) (PEDOT), have been extensively investigated. However, most flexible wiring fabrication methods using PEDOT are limited to two-dimensional (2D) fabrication. In this study, we fabricated three-dimensional (3D) wiring using the highly precise 3D printing method of stereolithography. Although several PEDOT fabrication methods using 3D printing systems have been studied, few have simultaneously achieved both high conductivity and precise accuracy. In this study, we review the post-fabrication process, particularly the doping agent. Consequently, we successfully fabricated wiring with a conductivity of 16 S cm-1. Furthermore, flexible wiring was demonstrated by modeling the fabricated wiring on a polyimide film with surface treatment and creating a three-dimensional fabrication object.

3D-Printed Micro-Tweezers with a Compliant Mechanism Designed Using Topology Optimization.

The development of handling technology for microscopic biological samples such as cells and spheroids has been required for the advancement of regenerative medicine and tissue engineering. In this study, we developed micro-tweezers with a compliant mechanism to manipulate organoids. The proposed method combines high-resolution microstereolithography that uses a blue laser and topology optimization for shape optimization of micro-tweezers. An actuation system was constructed using a linear motor stage with a force control system to operate the micro-tweezers. The deformation of the topology-optimized micro-tweezers was examined analytically and experimentally. The results verified that the displacement of the tweezer tip was proportional to the applied load; furthermore, the displacement was sufficient to grasp biological samples with an approximate diameter of several hundred micrometers. We experimentally demonstrated the manipulation of an organoid with a diameter of approximately 360 µm using the proposed micro-tweezers. Thus, combining microstereolithography and topology optimization to fabricate micro-tweezers can be potentially used in modifying tools capable of handling various biological samples.

Simple autofocusing method by image processing using transmission images for large-scale two-photon lithography.

We propose a simple autofocusing technique that can be introduced into conventional two-photon lithography systems without additional devices. Autofocusing is achieved by image processing using transmission images of photopolymerized voxels. The signal-to-noise ratio of transmission images was improved by optimal low-pass filtering to detect voxels in them. The focal point was detected with an accuracy of about 250 nm from the difference images. Further, we demonstrated mass-fabrication of a 5 × 5 spiral square array with an area of 900 × 900 µm2 using this method. The method has potential application in constructing low-cost, compact and versatile two-photon lithography apparatus.

Multi-scale laser direct writing of conductive metal microstructures using a 405-nm blue laser.

A multi-scale direct writing method for metal microstructures is proposed and demonstrated. In this study, metal structures were created in a gelatin matrix containing silver nitrate by photoreduction using a 405-nm blue laser. The influence of concentrations of materials in the sample solution was evaluated by measuring the conductivity of the fabricated microstructures. The fabrication line width could be controlled by changing the laser scanning speed. A network structure was also observed, which possibly helps in increasing the microstructure's conductivity. Finally, we demonstrated multi-scale drawing by using objective lenses with different numerical apertures. Our method can result in new possibilities for conductive metal direct writing.

Additive Manufacturing of Micromanipulator Mounted on a Glass Capillary for Biological Applications.

In this study, a three-dimensional (3D) micromanipulator mounted on a glass capillary is developed for handling biological samples, such as multicellular spheroids and embryos. To fabricate the micromanipulator, we developed an additive manufacturing system based on high-resolution microstereolithography using a 405-nm blue laser. The fabrication system makes it possible to fabricate 3D microstructures on a glass capillary with 2.5 µm lateral resolution and 25 µm layer thickness. We also demonstrated the capture and release of a spheroid with the micromanipulator fabricated using our additive manufacturing system. We showed that spheroids can be easily handled by a simple operation with minimal damage using a cage-like multiple finger structure. Additive manufacturing of tailor-made micromanipulators mounted on a glass capillary will be useful in biological and tissue engineering research.

Tailored cell sheet engineering using microstereolithography and electrochemical cell transfer.

Postoperative adhesion and occlusion remain a serious issue associated with various surgeries, including endoscopic surgery, in which proliferated fibrous tissues stick to adjacent tissues and often cause severe complications. Cell sheet engineering has emerged as an effective approach not only for cell transplantation but also for the treatment of postoperative adhesion and occlusion. However, as the tissues in the body, such as middle ear and small intestine, and typical operative sites are non-flat and spatially complicated, tailored cell sheets with three-dimensional (3D) configurations may lead to widespread use of this approach. In the present study, we used microstereolithography, biocompatible gold plating, and electrochemical cell detachment to achieve this purpose. Various objects with dimensions ranging from millimeter- to micrometer-scale were fabricated with photocurable resin using lab-made equipment for microstereolithography. To coat the fabricated objects with a thin gold layer, conventional cyanide-based gold plating was unusable because it severely damaged almost all cells. Electroless non-cyanide gold plating we prepared was cytocompatible and suitable for electrochemical cell detachment. Cell sheets on the gold-plated substrate could be directly transplanted into a mouse intraperitoneally using electrochemical cell detachment. We further demonstrated that cell sheets grown on gold-coated 3D objects were rapidly detached along with the desorption of electroactive-oligopeptide monolayer and transferred to a surrounding hydrogel. This approach may provide a promising strategy to prepare and directly transplant tailor-made cell sheets with suitable configurations.

Preparation of hair beads and hair follicle germs for regenerative medicine.

Hair regenerative medicine is a promising approach for hair loss, during which autologous follicular stem cells are transplanted into regions of hair loss to regenerate hairs. Because cells transplanted as a single cell suspension scarcely generate hairs, the engineering of three-dimensional (3D) tissues before transplantation has been explored to improve this process. Here, we propose an approach to fabricate collagen-enriched cell aggregates, named hair beads (HBs), through the spontaneous constriction of cell-encapsulated collagen drops. Mouse embryonic mesenchymal cells or human dermal papilla cells were encapsulated in 2-µl collagen microgels, which were concentrated >10-fold in volume during 3 days of culture. Interestingly, HB constriction was attributed to attraction forces driven by myosin II and involved the upregulation of follicular genes. Single HBs with epithelial cells seeded in U-shaped microwells formed dumbbell-like structures comprising respective aggregates (named bead-based hair follicle germs, bbHFGs), during 3 days of culture. bbHFGs efficiently generated hair follicles upon intracutaneous transplantation into the backs of nude mice. Using an automated spotter, this approach was scalable to prepare a large number of bbHFGs, which is important for clinical applications. Therefore, this could represent a robust and practical approach for the preparation of germ-like tissues for hair regenerative medicine.

3D Shape Reconstruction of 3D Printed Transparent Microscopic Objects from Multiple Photographic Images Using Ultraviolet Illumination.

We propose and demonstrate a simple, low-cost, three-dimensional (3D) shape acquisition method for transparent 3D printed microscopic objects. Our method uses ultraviolet (UV) illumination to obtain high-contrast silhouette images of transparent 3D printed polymer objects. Multiple silhouette images taken from different viewpoints make it possible to reconstruct the 3D shape of this transparent object. A 3D shape acquisition system consisting of a UV light-emitting diode, charge-coupled device camera and a rotation stage was constructed and used to successfully reconstruct the 3D shape of a transparent bunny model produced using micro-stereolithography. In addition, 3D printed pillar array models, with different diameters on the order of several hundred micrometers, were reconstructed. This method will be a promising tool for the 3D shape reconstruction of transparent 3D objects on both the micro- and macro-scale by changing the imaging lens.

Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine.

Hair follicle morphogenesis is triggered by reciprocal interactions between hair follicle germ (HFG) epithelial and mesenchymal layers. Here, we developed a method for large-scale preparation of HFGs in vitro via self-organization of cells. We mixed mouse epidermal and mouse/human mesenchymal cells in suspension and seeded them in microwells of a custom-designed array plate. Over a 3-day culture period, cells initially formed a randomly distributed single cell aggregate and then spatially separated from each other, exhibiting typical HFG morphological features. These self-sorted hair follicle germs (ssHFGs) were shown to be capable of efficient hair-follicle and shaft generation upon intracutaneous transplantation into the backs of nude mice. This finding facilitated the large-scale preparation of approximately 5000 ssHFGs in a microwell-array chip made of oxygen-permeable silicone. We demonstrated that the integrity of the oxygen supply through the bottom of the silicone chip was crucial to enabling both ssHFG formation and subsequent hair shaft generation. Finally, spatially aligned ssHFGs on the chip were encapsulated into a hydrogel and simultaneously transplanted into the back skin of nude mice to preserve their intervening spaces, resulting in spatially aligned hair follicle generation. This simple ssHFG preparation approach is a promising strategy for improving current hair-regenerative medicine techniques.

Development of a High-Density Microplasma Emission Source for a Micro Total Analysis System.

To achieve a highly sensitive and onsite analysis of a small amount samples, a microplasma-based micro total analysis systems (µ-TAS) device was developed. A dielectric barrier discharge (DBD) that can generate a stable plasma at atmospheric pressure was generated in a microchip and used as the plasma source. The use of DBD suppresses the temperature rise of the electrodes and enables operation for long times because of a reduction of the electrode damage due to suppression of the current via dielectric interposing between the electrodes. It is expected that the analytical system can be miniaturized because helium plasma is generated in the microchannel contained in the microchip. Emissions from gaseous Cl, Br, and I were analyzed using the plasma source, and it was found that the detection limits for these analytes were 0.22, 0.18, and 0.14 ppm, respectively. The calibration curves for gaseous Cl, Br, and I were also plotted obtaining correlation coefficients of 0.975, 0.955 and 0.986, respectively, and showing good linearity for the developed plasma source.

Single-anchor support and supercritical CO2 drying enable high-precision microfabrication of three-dimensional structures.

In high-precision two-photon microfabrication of three-dimensional (3-D) polymeric microstructures, supercritical CO(2) drying was employed to reduce surface tension, which tends to cause the collapse of micro/nano structures. Use of supercritical drying allowed high-aspect ratio microstructures, such as micropillars and cantilevers, to be fabricated. We also propose a single-anchor supporting method to eliminate non-uniform shrinkage of polymeric structures otherwise caused by attachment to the substrate. Use of this method permitted frame models such as lattices to be produced without harmful distortion. The combination of supercritical CO(2) drying and the single-anchor supporting method offers reliable high-precision microfabrication of sophisticated, fragile 3-D micro/nano structures.

Optically driven micropump with a twin spiral microrotor.

An optically driven micropump that employs viscous drag exerted on a spinning microrotor with left- and right-handed spiral blades on its rotational axis has been developed using two-photon microfabrication. It was demonstrated that the twin spiral microrotor provides a higher rotation speed than a single spiral microrotor. The rotation speed reached 560 rpm at a laser power of 500 mW. The twin spiral microrotor was also applied to a viscous micropump with a U-shaped microchannel. To pump fluid, the twin spiral microrotor located at the corner of the U-shaped microchannel was rotated by focusing a laser beam. The flow field inside the U-shaped microchannel was analyzed using the finite element method (FEM) based on the Navier-Stokes equation to optimize the shape of the microchannel. It was confirmed that the rotation of the twin spiral microrotor generated a unidirectional laminar flow. Finally, a tandem micropump using two twin spiral microrotors was driven by a dual optical trapping system using a spatial light modulation technique.

Femtosecond laser direct writing of metallic microstructures by photoreduction of silver nitrate in a polymer matrix.

Continuous silver microstructures were produced by three-dimensional (3-D) direct laser writing using a femtosecond-pulsed laser beam with polyvinylpyrrolidone (PVP) films containing silver ions. The lines drawn by scanning a tightly focused laser beam ranged from 200 nm to 1.7 microm. Using a sample solution of high density of silver nitrate, a continuous silver line with a resistivity of 3.48 x 10(-7) ohms m was produced. Not only 3-D microstructures such as pyramidal models but also hybrid microstructures comprising polymer and silver lines were demonstrated. The 3-D direct laser writing of metallic microstructures has potential for application to 3-D electrical wiring of electronic devices and MEMS devices.