Using SERS Tags to Image the Three‐Dimensional Structure of Complex Cell Models

Methods to image complex 3D cell cultures are limited by issues such as fluorophore photobleaching and decomposition, poor excitation light penetration, and lack of complementary techniques to verify the 3D structure. Although it remains insufficiently demonstrated, surface‐enhanced Raman scattering (SERS) imaging is a promising tool for the characterization of biological complex systems. To this aim, a controllable 3D cell culture model which spans nearly 1 cm² in surface footprint is designed. This structure is composed of fibroblasts containing SERS‐encoded nanoparticles (i.e., SERS tags), arranged in an alternating layered structure. This “sandwich” type structure allows monitoring of the SERS signals in the z‐axis and with mm dimensions in the xy‐axis. Taking advantage of correlative microscopy techniques such as electron microscopy, it is possible to corroborate nanoparticle positioning and distances in z‐depths of up to 150 µm. This study reveals a proof‐of‐concept method for detailed 3D SERS imaging of a complex, dense 3D cell culture model.     

A Strategy for the Construction of Controlled,Three‐Dimensional,Multilayered, Tissue‐Like Structures

Differentiated cells make up tissues and organs, and communicate within a complex, three dimensional (3D) environment. The spatial arrangement of cellular interactions is difficult to recapitulate in vitro. Here, a simple and rapid method for stepwise formation of 2D multicellular structures through the biotin‐streptavidin (SA) interaction and further construction of controlled, 3D, multilayered, tissue‐like structures by using the stress‐induced rolling membrane (SIRM) technique is reported. The biotinylated cells connect with the SA‐coated adherent cells to form a bilayer. The bilayer of two types of cells on the SIRM is transformed into 3D tubes, in which two types of cells can directly interact and communicate with each other, mimicking the in vivo conditions of tubular structures such as blood vessel. This method has the potential to recapitulate functional tubular structures for tissue engineering.     

Highly Efficient Self‐Healable and Dual Responsive Cellulose‐Based Hydrogels for Controlled Release and 3D Cell Culture

To face the increasing demand of self‐healing hydrogels with biocompatibility and high performances, a new class of cellulose‐based self‐healing hydrogels are constructed through dynamic covalent acylhydrazone linkages. The carboxyethyl cellulose‐graft‐dithiodipropionate dihydrazide and dibenzaldehyde‐terminated poly(ethylene glycol) are synthesized, and then the hydrogels are formed from their mixed solutions under 4‐amino‐DL‐phenylalanine (4a‐Phe) catalysis. The chemical structure, as well as microscopic morphologies, gelation times, mechanical and self‐healing performances of the hydrogels are investigated with ¹H NMR, Fourier transform infrared spectroscopy, atomic force microscopy, rheological and compression measurements. Their gelation times can be controlled by varying the total polymer concentration or 4a‐Phe content. The resulted hydrogels exhibit excellent self‐healing ability with a high healing efficiency (≈96%) and good mechanical properties. Moreover, the hydrogels display pH/redox dual responsive sol‐gel transition behaviors, and are applied successfully to the controlled release of doxorubicin. Importantly, benefitting from the excellent biocompatibility and the reversibly cross‐linked networks, the hydrogels can function as suitable 3D culture scaffolds for L929 cells, leading to the encapsulated cells maintaining a high viability and proliferative capacity. Therefore, the cellulose‐based self‐healing hydrogels show potential applications in drug delivery and 3D cell culture for tissue engineering.     

Three‐Dimensional Printing of Elastomeric,Cellular Architectures with Negative Stiffness

Three‐dimensional printing of viscoelastic inks to create porous, elastomeric architectures with mechanical properties governed by the ordered arrangement of their sub‐millimeter struts is reported. Two layouts are patterned, one resembling a “simple cubic” (SC)‐like structure and another akin to a “face‐centered tetragonal” (FCT) configuration. These structures exhibit markedly distinct load response with directionally dependent behavior, including negative stiffness. More broadly, these findings suggest the ability to independently tailor mechanical response in cellular solids via micro‐architected design. Such ordered materials may one day replace random foams in mechanical energy absorption applications.     

New TDOA‐Based Three‐Dimensional Positioning Method for 3GPP LTE System

Recently, mobile positioning enhancement has attracted much attention in the 3rd generation partnership project long‐term evolution system. In particular, for urban canyon environments, the need for three‐dimensional (3D) positioning has increased to enable the altitude of users to be measured. For several decades, several time difference of arrival (TDOA‐) based 3D positioning methods have been studied; however, they are only available when at least four evolved Node Bs (eNBs) exist nearby or when all eNBs have the same height. Therefore, in this paper, we propose a new 3D positioning method that estimates the 3D coordinates of a user using three types of two‐dimensional (2D) TDOAs. However, the give inaccurate results owing to the undefined axis of the 2D coordinate plane. Therefore, we propose a novel derivation of the hyperbola equation, which includes the undefined axis coordinate in the 2D hyperbola equation. Then, we propose an interaction algorithm that mutually supplies the undefined axis coordinate of users among 2D TDOAs. By performing extensive simulations, we verify that the proposed method is the only solution applicable by using three eNBs with different heights.     

Large‐Scale Fabrication of Three‐Dimensional Surface Patterns Using Template‐Defined Electrochemical Deposition

A new strategy to achieve large‐scale, three‐dimensional (3D) micro‐ and nanostructured surface patterns through selective electrochemical growth on monolayer colloidal crystal (MCC) templates is reported. This method can effectively create large‐area (>1 cm²), 3D surface patterns with well‐defined structures in a cost‐effective and time‐saving manner (<30 min). A variety of 3D surface patterns, including semishells, Janus particles, microcups, and mushroom‐like clusters, is generated. Most importantly, our method can be used to prepare surface patterns with prescribed compositions, such as metals, metal oxides, organic materials, or composites (e.g., metal/metal oxide, metal/polymer). The 3D surface patterns produced by our method can be valuable in a wide range of applications, such as biosensing, data storage, and plasmonics. In a proof‐of‐concept study, we investigated, both experimentally and theoretically, the surface‐enhanced Raman scattering (SERS) performance of the fabricated silver 3D semishell arrays.     

Controlled Fabrication of Multitiered Three‐Dimensional Nanostructures in Porous Alumina

We present the fabrication of multitiered branched porous anodic alumina (PAA) substrates consisting of an array of pores branching into smaller pores in succeeding tiers. The tiered three‐dimensional structure is realized by sequentially stepping down the anodization potential while etching of the barrier layer is performed after each step. We establish the key processing parameters that define the tiered porous structure through systematically designed experiments. The characterization of the branched PAA structures reveals that, owing to constriction, the ratio of interpore distance to the anodization potential is smaller than that for pristine films. This ratio varies from 1.8 to 1.3 nm V^?1 depending on the size of the preceding pores and the succeeding tier anodization potential. Contact angle measurements show that the multitiered branched PAA structures exhibit a marked increased in hydrophilicity over two‐dimensional PAA films.     

Free‐Standing Cell Sheet Assembled with Ultrathin Extracellular Matrix as an Innovative Approach for Biomimetic Tissues

Current artificial tissue‐substitutes have limited clinical applications due to unmatched complex combination of cells and extracellular matrix (ECM) as seen in native tissues. From a developmental perspective, the construction of effective biomimetic tissues is from the bottom (one‐dimensional nanoparticles or two‐dimensional membranes) up (three‐dimensional scaffolds or more complex composite). In a hierarchical architecture, each sub‐structure can be assembled in a flexible way with specific regulators and cells, which overcomes the deficiency of one‐for‐all scaffold. Here, a cell‐compatible cell‐lined layered nano‐membrane is developed. Bioactive molecules are mounted on a nano‐membrane and later released to its lined cell sheet. The cell‐lined membrane is in a free‐standing form to regulate cellular functions. The major advantage of this methodology is to provide a versatile approach to construct biomimetic tissues for clinical applications.     

Highly Conductive Nanocomposites with Three‐Dimensional,Compactly Interconnected Graphene Networks via a Self‐Assembly Process

Polymer‐based materials with high electrical conductivity are of considerable interest because of their wide range of applications. The construction of a 3D, compactly interconnected graphene network can offer a huge increase in the electrical conductivity of polymer composites. However, it is still a great challenge to achieve desirable 3D architectures in the polymer matrix. Here, highly conductive polymer nanocomposites with 3D compactly interconnected graphene networks are obtained using a self‐assembly process. Polystyrene (PS) and ethylene vinyl acetate (EVA) are used as polymer matrixes. The obtained PS composite film with 4.8 vol% graphene shows a high electrical conductivity of 1083.3 S/m, which is superior to that of the graphene composite prepared by a solvent mixing method. The electrical conductivity of the composites is closely related to the compact contact between graphene sheets in the 3D structures and the high reduction level of graphene sheets. The obtained EVA composite films with the 3D graphene structure not only show high electrical conductivity but also exhibit high flexibility. Importantly, the method to fabricate 3D graphene structures in polymer matrix is facile, green, low‐cost, and scalable, providing a universal route for the rational design and engineering of highly conductive polymer composites.     

High Performance Three‐Dimensional Chemical Sensor Platform Using Reduced Graphene Oxide Formed on High Aspect‐Ratio Micro‐Pillars

The sensing performance of chemical sensors can be achieved not only by modification or hybridization of sensing materials but also through new design in device geometry. The performance of a chemical sensing device can be enhenced from a simple three‐dimensional (3D) chemiresistor‐based gas sensor platform with an increased surface area by forming networked, self‐assembled reduced graphene oxide (R‐GO) nanosheets on 3D SU8 micro‐pillar arrays. The 3D R‐GO sensor is highly responsive to low concentration of ammonia (NH₃) and nitrogen dioxide (NO₂) diluted in dry air at room temperature. Compared to the two‐dimensional planar R‐GO sensor structure, as the result of the increase in sensing area and interaction cross‐section of R‐GO on the same device area, the 3D R‐GO gas sensors show improved sensing performance with faster response (about 2%/s exposure), higher sensitivity, and even a possibly lower limit of detection towards NH₃ at room temperature.     

Resolving the Three‐Dimensional Microstructure of Polymer Electrolyte Fuel Cell Electrodes using Nanometer‐Scale X‐ray Computed Tomography

The electrodes of a polymer electrolyte fuel cell (PEFC) are composite porous layers consisting of carbon and platinum nanoparticles and a polymer electrolyte binder. The proper composition and arrangement of these materials for fast reactant transport and high electrochemical activity is crucial to achieving high performance, long lifetimes, and low costs. Here, the microstructure of a PEFC electrode using nanometer‐scale X‐ray computed tomography (nano‐CT) with a resolution of 50 nm is investigated. The nano‐CT instrument obtains this resolution for the low‐atomic‐number catalyst support and binder using a combination of a Fresnel zone plate objective and Zernike phase contrast imaging. High‐resolution, non‐destructive imaging of the three‐dimensional (3D) microstructures provides important new information on the size and form of the catalyst particle agglomerates and pore spaces. Transmission electron microscopy (TEM) and mercury intrusion porosimetry (MIP) is applied to evaluate the limits of the resolution and to verify the 3D reconstructions. The computational reconstructions and size distributions obtained with nano‐CT can be used for evaluating electrode preparation, performing pore‐scale simulations, and extracting effective morphological parameters for large‐scale computational models.     

Three‐Dimensional SiCN Ceramic Microstructures via Nano‐Stereolithography of Inorganic Polymer Photoresists

We report a newly synthesized inorganic polymer photoresist with a high ceramic yield by the functionalization of polyvinylsilazane (KiON VL20) with 2‐isocyanatoethyl methacrylate via linkage or insertion reaction routes. The chemistry of the synthesis and the pyrolytic conversion as well as the mechanical evaluation were investigated by using various analytical instruments. We show for the first time that this photosensitive resin is a novel precursor for the fabrication of complex 3D SiCN ceramic microstructures with a 210 nm resolution via a two‐photon absorbed crosslinking process and subsequent pyrolysis at 600 °C under a nitrogen atmosphere. Moreover, the dimensional deformation during pyrolysis was significantly reduced by adding silica nanoparticles as a filler. In particular, the ceramic microstructures containing 40 wt % silica nanoparticles exhibited a relatively isotropic shrinkage owing to its sliding free from the substrate during pyrolysis.     

Regulation of Stem Cell Fate in a Three‐Dimensional Micropatterned Dual‐Crosslinked Hydrogel System

Micropatterning technology is a powerful tool for controlling the cellular microenvironment and investigating the effects of physical parameters on cell behaviors, such as migration, proliferation, apoptosis, and differentiation. Although there have been significant developments in regulating the spatial and temporal distribution of physical properties in various materials, little is known about the role of the size of micropatterned regions of hydrogels with different crosslinking densities on the response of encapsulated cells. In this study, a novel alginate hydrogel system that can be micropatterned three‐dimensionally is engineered to create regions that are crosslinked by a single mechanism or dual mechanisms. By manipulating micropattern size while keeping the overall ratio of single‐ to dual‐crosslinked hydrogel volume constant, the physical properties of the micropatterned alginate hydrogels are spatially tunable. When human adipose‐derived stem cells (hASCs) are photoencapsulated within micropatterned hydrogels, their proliferation rate is a function of micropattern size. Additionally, micropattern size dictates the extent of osteogenic and chondrogenic differentiation of photoencapsulated hASC. The size of 3D micropatterned physical properties in this new hydrogel system introduces a new design parameter for regulating various cellular behaviors, and this dual‐crosslinked hydrogel system provides a new platform for studying proliferation and differentiation of stem cells in a spatially controlled manner for tissue engineering and regenerative medicine applications.     

Mesocylindrical Aluminosilica Monolith Biocaptors for Size‐Selective Macromolecule Cargos

Immobilization of biological macromolecules, such as protein, onto solid supports is an important method for diagnostic assays andgenetechnology. This present study reports the size‐selective adsorption/removal of virtual proteins that have different shapes, sizes, functions, and properties, such as insulin, cytochrome c, lysozyme, myoglobin, β‐lactoglobin, α‐amylase, hemoglobin, and myosin in aqueous water using mesobiocaptor monoliths. To prevent large proteins from adsorbing and remaining attached to adsorbent surfaces, large, open, cylindrical‐pored, three‐dimensional cubic aluminosilica mesostructures with large aluminum contents and micrometer‐sized monolith particles were fabricated. The unique physical properties and the surface functionality of the mesobiocaptors enhance protein adsorption characteristics in terms of loading capacity and quantity of the sample, ensuring a higher concentration of adsorbed proteins, interior pore diffusivity, and encapsulation in a short period. Thermodynamic studies indicate that protein adsorption into the mesobiocaptor pores is favorable and spontaneous. Theoretical models were used to investigate the major driving forces for the most optimal performance of the protein adsorption. The geometrical findings point to key factors, such as surface energy, intermolecular forces, charge distribution, hydrophobicity, and electrostatic interaction, which might control the adsorption into the interior large, open cylindrical mesobiocaptor cavities (sized 3–16 nm) without aggregation of these proteins on the exterior surfaces of monoliths. Indeed, the availability of adsorption of single proteins from mixtures based on size‐ and shape‐selective separation opens new avenues of research in encapsulation of proteins and bioanalysis.     

High‐Density Hotspots Engineered by Naturally Piled‐Up Subwavelength Structures in Three‐Dimensional Copper Butterfly Wing Scales for Surface‐Enhanced Raman Scattering Detection

Very recently, wing scales of natural Lepidopterans (butterflies and moths) manifested themselves in providing excellent three dimensional (3D) hierarchical structures for surface‐enhanced Raman scattering (SERS) detection. But the origin of the observed enormous Raman enhancement of the analytes on 3D metallic replicas of butterfly wing scales has not been clarified yet, hindering a full utilization of this huge natural wealth with more than 175 000 3D morphologies. Herein, the 3D sub‐micrometer Cu structures replicated from butterfly wing scales are successfully tuned by modifying the Cu deposition time. An optimized Cu plating process (10 min in Cu deposition) yields replicas with the best conformal morphologies of original wing scales and in turn the best SERS performance. Simulation results show that the so‐called “rib‐structures” in Cu butterfly wing scales present naturally piled‐up hotspots where electromagnetic fields are substantially amplified, giving rise to a much higher hotspot density than in plain 2D Cu structures. Such a mechanism is further verified in several Cu replicas of scales from various butterfly species. This finding paves the way to the optimal scale candidates out of ca. 175 000 Lepidopteran species as bio‐templates to replicate for SERS applications, and thus helps bring affordable SERS substrates as consumables with high sensitivity, high reproducibility, and low cost to ordinary laboratories across the world.     

Morphology‐Controlled Growth of Large‐Area Two‐Dimensional Ordered Pore Arrays

A solution‐dipping template strategy for large‐area synthesis of morphology‐controlled, ordered pore arrays is reported. The morphology of the pore array can easily be controlled by concentration of the precursor solution and treatment conditions. With decrease of the concentration from a high level to a very low level nanostructured complex (pore–hole, and pore–particle) arrays, through‐pore arrays, and even ring arrays can, in turn, be obtained. The pore size is adjustable over a large range by changing the diameter of the template's latex spheres. This synthesis route is universal and can be used for various metals, semiconductors and compounds on any substrate. Such structures may be useful in applications such as energy storage or conversion, especially in integrated next‐generation nanophotonics devices, and biomolecular labeling and identification.     

4D Corneal Tissue Engineering: Achieving Time‐Dependent Tissue Self‐Curvature through Localized Control of Cell Actuators

While tissue engineering is widely used to construct complex tridimensional biocompatible structures, researchers are now attempting to extend the technique into the fourth dimension. Such fourth dimension consists in the transformation of 3D materials over time, namely, by changing their shape, composition, and/or function when subjected to specific external stimuli. Herein, producing a 4D biomaterial with an internal mechanism of stimulus, using contractile cells as bio‐actuators to change tissue shape and structure, is explored. Specifically, producing cornea‐shaped, curved stromal tissue equivalents via the controlled, cell‐driven curving of collagen‐based hydrogels. This is achieved by modulating the activity of the bio‐actuators in delimited regions of the gels using a contraction‐inhibiting peptide amphiphile. The self‐curved constructs are then characterized in terms of cell and collagen fibril reorganization, gel stiffness, cell phenotype, and the ability to sustain the growth of a corneal epithelium in vitro. Overall, the results show that the structural and mechanical properties of self‐curved gels acquired through a 4D engineering method are more similar to those of the native tissue, and represent a significant improvement over planar 3D scaffolds. In this perspective, the study demonstrates the great potential of cell bio‐actuators for 4D tissue engineering applications.     

Micropatterned Down‐Converting Coating for White Bio‐Hybrid Light‐Emitting Diodes

White hybrid light‐emitting diodes (WHLEDs) are considered as a solid approach toward environmentally sustainable lighting sources that meet the “Green Photonics” requirements. Here, WHLEDs with protein‐based down‐converting coatings, i.e., Bio‐WHLEDs, are demonstrated and exhibit worthy white color quality, luminous efficiency, and stability values. The coatings feature a multilayered cascade‐like architecture with thicknesses of 1–3 mm. This limits the efficiency due to the low optical transmittance. Thus, submillimeter coatings, where the location of the proteins is well‐defined, are highly desired. It is in this context where the thrust of this work sets in. Here, a straightforward way to design microstructured single‐layer coatings, in which the proteins are placed at our command by using 3D printing, is presented. Based on comprehensive spectroscopic and rheological investigations, the optimization of the matrix and the plotting to prepare different micropatterns, i.e., lines, open‐grids, and closed‐grids, is rationalized. The latter are applied to prepare Bio‐WHLEDs with ≈5‐fold enhancement of the luminous efficiency compared to the reference devices with a cascade‐like coating, without losing stability and color quality. As such, this work shows a new route to exploit proteins for optoelectronics, setting a new avenue of research into the emerging field of Bio‐WHLEDs.     

Designing Microgels for Cell Culture and Controlled Assembly of Tissue Microenvironments

Micrometer‐sized hydrogels, termed microgels, are emerging as multifunctional platforms that can recapitulate tissue heterogeneity in engineered cell microenvironments. The microgels can function as either individual cell culture units or can be assembled into larger scaffolds. In this manner, individual microgels can be customized for single or multicell coculture applications, or heterogeneous populations can be used as building blocks to create microporous assembled scaffolds that more closely mimic tissue heterogeneities. The inherent versatility of these materials allows user‐defined control of the microenvironments, from the order of singly encapsulated cells to entire 3D cell scaffolds. These hydrogel scaffolds are promising for moving towards personalized medicine approaches and recapitulating the multifaceted microenvironments that exist in vivo.