Bioinspired Molecular Scalpel for Two-Dimensional Metal-Organic Nanosheet: Design Strategies and Recent Progress.

Ultrathin two-dimensional (2D) metal-organic nanosheets (MONs) have attracted continued attention in the field of advanced functional materials. Their nanoscale thickness, high surface-to-volume ratio, and abundant accessible active sites, are superior advantages compared with their 3D bulk counterparts. Bioinspired molecular scalpel strategy is a promising method for the creation of 2D MONs, and may solve the current shortcomings of MONs synthesis. This review aims to provide a state-of-the-art overview of molecular scalpel strategies and share the results of current development to provide a better solution for MONs synthesis. Different types of molecular scalpel strategies have been systematically summarized. Both mechanisms, advantages and limitations of multiform molecular scalpel strategies have been discussed. Besides, the challenges to be overcome and the question to be solved are also introduced.

Annealing High Aspect Ratio Microgels into Macroporous 3D Scaffolds Allows for Higher Porosities and Effective Cell Migration.

Growing millimeter-scaled functional tissue remains a major challenge in the field of tissue engineering. Therefore, microporous annealed particles (MAPs) are emerging as promising porous biomaterials that are formed by assembly of microgel building blocks. To further vary the pore size and increase overall MAP porosity of mechanically stable scaffolds, rod-shaped microgels with high aspect ratios up to 20 are chemically interlinked into highly porous scaffolds. Polyethylene glycol based microgels (width 10 µm, lengths up to 200 µm) are produced via in-mold polymerization and covalently interlinked into stable 3D scaffolds via epoxy-amine chemistry. For the first time, MAP porosities can be enhanced by increasing the microgel aspect ratio (mean pore sizes ranging from 39 to 82 µm, porosities from 65 to 90%). These porosities are significantly higher compared to constructs made from spherical or lower aspect ratio rod-shaped microgels. Rapid filling of the pores by either murine or primary human fibroblasts is ensured as cells migrate and grow extensively into these scaffolds. Overall, this study demonstrates that highly porous, stable macroporous hydrogels can be achieved with a very low partial volume of synthetic, high aspect ratio microgels, leading to large empty volumes available for cell ingrowth and cell-cell interactions.

Strong, Healable, Stimulus-Responsive Fluorescent Elastomers Based on Assembled Borate Dynamic Nanostructures.

Self-healing materials integrated with robust mechanical property and fascinating functions synchronously hold great prospects in many applications, but it still remains a grand challenge. Here, a bottom-up assembly method of preparing borate dynamic nanostructures (BDN) with controllable morphologies and interfacial crosslinks is proposed, from which a robust self-healing elastomer is fabricated. The BDN is optimized to construct dense and strong interfacial boronic easter crosslinks, endowing the elastomer with outstanding stretchability (2050%), high strength (17.9 MPa) as well as healing efficiency (77.1%). Moreover, the elastomer also exhibits pH stimulus-responsive fluorescence property and excellent functional repairability, enabling its potential application in intelligent material fields such as information encoding and encryption. This study demonstrates a general approach to produce self-healable functional materials with robust mechanical properties, and defines a rich platform for exploring various functional nanostructured materials.

Inducing Microscale Structural Order in Phage Nanofilament Hydrogels with Globular Proteins.

Biological hydrogels play important physiological roles in the body. These hydrogels often contain ordered subdomains that provide mechanical toughness and other tissue-specific functionality. Filamentous bacteriophages are nanofilaments with a high aspect ratio that can self-assemble into liquid crystalline domains that could be designed to mimic ordered biological hydrogels and can thus find applications in biomedical engineering. We have previously reported hydrogels of pure cross-linked liquid crystalline filamentous phage formed at very high concentrations exhibiting a tightly packed microstructure and high stiffness. In this work, we report a method for inducing self-assembly of filamentous phage into liquid crystalline hydrogels at concentrations that are several orders of magnitude below that of lyotropic liquid crystal formation, thus creating structural order but a less densely packed microstructure. Hybrid hydrogels of M13 phage and bovine serum albumin (0.25 w/v%) were formed and shown to adsorb up to 16× their weight in water. Neither component gelled on its own at the low concentrations used, suggesting synergistic action between the two components in the formation of the hydrogel. The hybrid hydrogels exhibited repetitive self-healing under physiological conditions and at room temperature, autofluorescence in three channels, and antibacterial activity toward Escherichia coli host cells. Furthermore, the hybrid hydrogels exhibited a more than 2× higher ability to pack water compared to BSA-only hydrogels and 2× lower compression modulus compared to tightly packed M13-only hydrogels, suggesting that our method could be used to create hydrogels with tunable mechanical properties and pore structure through the addition of globular proteins, while maintaining bioactivity and microscale structural order.

Cell mimicry as a bottom-up strategy for hierarchical engineering of nature-inspired entities.

Artificial biology is an emerging concept that aims to design and engineer the structure and function of natural cells, organelles, or biomolecules with a combination of biological and abiotic building blocks. Cell mimicry focuses on concepts that have the potential to be integrated with mammalian cells and tissue. In this feature article, we will emphasize the advancements in the past 3-4 years (2017-present) that are dedicated to artificial enzymes, artificial organelles, and artificial mammalian cells. Each aspect will be briefly introduced, followed by highlighting efforts that considered key properties of the different mimics. Finally, the current challenges and opportunities will be outlined. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Programming Surface-Enhanced Raman Scattering of DNA Origami-templated Metamolecules.

DNA origami holds an unprecedented capability on assembling metallic nanoparticles into designer plasmonic metamolecules of emerging properties, including surface-enhanced Raman scattering (SERS). SERS metamolecules were produced by positioning nanoparticles in close proximity to each other on a DNA origami template for Raman enhancement. In earlier reports, SERS metamolecules were generally assembled into clusters containing small number of nanoparticles (2, 3, or 4) and thus had limited programmability over SERS. Herein, we expanded the structural complexity of SERS metamolecules by increasing the number of nanoparticles and by arranging them into sophisticated configurations. DNA origami hexagon tile was used as the assembling template to fabricate clusters consisting of 6, 7, 12, 18, and 30+ metallic nanoparticles. Programmable SERS was realized via controlling the size, number, or spatial arrangement of nanoparticles. We believe this method offers a general platform for fabricating sophisticated nanodevices with programmable SERS that may be applied to a variety of fields including plasmonics, nanophotonics, and sensing.

Biopolymer-Based Microcarriers for Three-Dimensional Cell Culture and Engineered Tissue Formation.

The concept of three-dimensional (3D) cell culture has been proposed to maintain cellular morphology and function as in vivo. Among different approaches for 3D cell culture, microcarrier technology provides a promising tool for cell adhesion, proliferation, and cellular interactions in 3D space mimicking the in vivo microenvironment. In particular, microcarriers based on biopolymers have been widely investigated because of their superior biocompatibility and biodegradability. Moreover, through bottom-up assembly, microcarriers have opened a bright door for fabricating engineered tissues, which is one of the cutting-edge topics in tissue engineering and regeneration medicine. This review takes an in-depth look into the recent advancements of microcarriers based on biopolymers-especially polysaccharides such as chitosan, chitin, cellulose, hyaluronic acid, alginate, and laminarin-for 3D cell culture and the fabrication of engineered tissues based on them. The current limitations and potential strategies were also discussed to shed some light on future directions.

Advanced Bottom-Up Engineering of Living Architectures.

Bottom-up tissue engineering is a promising approach for designing modular biomimetic structures that aim to recapitulate the intricate hierarchy and biofunctionality of native human tissues. In recent years, this field has seen exciting progress driven by an increasing knowledge of biological systems and their rational deconstruction into key core components. Relevant advances in the bottom-up assembly of unitary living blocks toward the creation of higher order bioarchitectures based on multicellular-rich structures or multicomponent cell-biomaterial synergies are described. An up-to-date critical overview of long-term existing and rapidly emerging technologies for integrative bottom-up tissue engineering is provided, including discussion of their practical challenges and required advances. It is envisioned that a combination of cell-biomaterial constructs with bioadaptable features and biospecific 3D designs will contribute to the development of more robust and functional humanized tissues for therapies and disease models, as well as tools for fundamental biological studies.

All-optical reconfigurable chiral meta-molecules.

Chirality is a ubiquitous phenomenon in the natural world. Many biomolecules without inversion symmetry such as amino acids and sugars are chiral molecules. Measuring and controlling molecular chirality at a high precision down to the atomic scale are highly desired in physics, chemistry, biology, and medicine, however, have remained challenging. Herein, we achieve all-optical reconfigurable chiral meta-molecules experimentally using metallic and dielectric colloidal particles as artificial atoms or building blocks to serve at least two purposes. One is that the on-demand meta-molecules with strongly enhanced optical chirality are well-suited as substrates for surface-enhanced chiroptical spectroscopy of chiral molecules and as active components in optofluidic and nanophotonic devices. The other is that the bottom-up-assembled colloidal meta-molecules provide microscopic models to better understand the origin of chirality in the actual atomic and molecular systems.

One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cells.

Here, we introduce a one-pot method for the bottom-up assembly of complex single- and multicompartment synthetic cells. Cellular components are enclosed within giant unilamellar vesicles (GUVs), produced at the milliliter scale directly from small unilamellar vesicles (SUVs) or proteoliposomes with only basic laboratory equipment within minutes. Toward this end, we layer an aqueous solution, containing SUVs and all biocomponents, on top of an oil-surfactant mix. Manual shaking induces the spontaneous formation of surfactant-stabilized water-in-oil droplets with a spherical supported lipid bilayer at their periphery. Finally, to release GUV-based synthetic cells from the oil and the surfactant shell into the physiological environment, we add an aqueous buffer and a droplet-destabilizing agent. We prove that the obtained GUVs are unilamellar by reconstituting the pore-forming membrane protein α-hemolysin and assess the membrane quality with cryotransmission electron microscopy (cryoTEM), fluorescence recovery after photobleaching (FRAP), and zeta-potential measurements as well as confocal fluorescence imaging. We further demonstrate that our GUV formation method overcomes key challenges of standard techniques, offering high volumes, a flexible choice of lipid compositions and buffer conditions, straightforward coreconstitution of proteins, and a high encapsulation efficiency of biomolecules and even large cargo including cells. We thereby provide a simple, robust, and broadly applicable strategy to mass-produce complex multicomponent GUVs for high-throughput testing in synthetic biology and biomedicine, which can directly be implemented in laboratories around the world.

Dielectrophoresis-assisted creation of cell aggregates under flow conditions using planar electrodes.

We present a microfluidic platform allowing dielectrophoresis-assisted formation of cell aggregates of controlled size and composition under flow conditions. When specific experimental conditions are met, negative dielectrophoresis allows efficient concentration of cells towards electric field minima and subsequent aggregation. This bottom-up assembly strategy offers several advantages with respect to the targeted application: first, dielectrophoresis offers precise control of spatial cell organization, which can be adjusted by optimizing electrode design. Then, it could contribute to accelerate the establishment of cell-cell interactions by favoring close contact between neighboring cells. The trapping geometry of our chip is composed of eight electrodes arranged in a circle. Several parameters have been tested in simulations to find the best configurations for trapping in flow. Those configurations have been tested experimentally with both polystyrene beads and human embryonic kidney cells. The final design and experimental setup have been optimized to trap cells and release the created aggregates on demand.

Clickable Microgel Scaffolds as Platforms for 3D Cell Encapsulation.

While microporous scaffolds are increasingly used for regenerative medicine and tissue repair applications, the most common techniques to fabricate these scaffolds use templating or top-down fabrication approaches. Cytocompatible bottom-up assembly methods afford the opportunity to assemble microporous systems in the presence of cells and create complex polymer-cell composite systems in situ. Here, microgel building blocks with clickable surface groups are synthesized for the bottom-up fabrication of porous cell-laden scaffolds. The facile nature of assembly allows for human mesenchymal stem cells to be incorporated throughout the porous scaffold. Particles are designed with mean diameters of ≈10 and 100 µm, and assembled to create varied microenvironments. The resulting pore sizes and their distribution significantly alter cell morphology and cytoskeletal formation. This microgel-based system provides numerous tunable properties that can be used to control multiple aspects of cellular growth and development, as well as providing the ability to recapitulate various biological interfaces.

Fabrication of elastomeric silk fibers.

Methods to generate fibers from hydrogels, with control over mechanical properties, fiber diameter, and crystallinity, while retaining cytocompatibility and degradability, would expand options for biomaterials. Here, we exploited features of silk fibroin protein for the formation of tunable silk hydrogel fibers. The biological, chemical, and morphological features inherent to silk were combined with elastomeric properties gained through enzymatic crosslinking of the protein. Postprocessing via methanol and autoclaving provided tunable control of fiber features. Mechanical, optical, and chemical analyses demonstrated control of fiber properties by exploiting the physical cross-links, and generating double network hydrogels consisting of chemical and physical cross-links. Structure and chemical analyses revealed crystallinity from 30 to 50%, modulus from 0.5 to 4 MPa, and ultimate strength 1-5 MPa depending on the processing method. Fabrication and postprocessing combined provided fibers with extensibility from 100 to 400% ultimate strain. Fibers strained to 100% exhibited fourth order birefringence, revealing macroscopic orientation driven by chain mobility. The physical cross-links were influenced in part by the drying rate of fabricated materials, where bound water, packing density, and microstructural homogeneity influenced cross-linking efficiency. The ability to generate robust and versatile hydrogel microfibers is desirable for bottom-up assembly of biological tissues and for broader biomaterial applications.

Wave-Tunable Lattice Equivalents toward Micro- and Nanomanipulation.

The assembly of micro- and nanomaterials is a key issue in the development of potential bottom-up construction of building blocks, but creating periodic arrays of such materials in an efficient and scalable manner still remains challenging. Here, we show that a cymatic assembly approach in which micro- and nanomaterials in a liquid medium that resonate at low-frequency standing waves can be used for the assembly in a spatially periodic and temporally stationary fashion that emerges from the wave displacement antinodes of the standing wave. We also show that employing a two-dimensional liquid, rather than a droplet, with a coffee-ring effect yields a result that exhibits distinct lattice equivalents comprising the materials. The crystallographic parameters, such as the lattice parameters, can be adjusted, where the parameters along the x- and y-axes are controlled by the applied wave frequencies, and the one along z-axis is controlled by a transparent layer as a spacer to create three-dimensional crystal equivalents. This work represents an advancement in assembling micro- and nanomaterials into macroscale architectures on the centimeter-length scale, thus establishing that a standing wave can direct micro- and nanomaterial assembly to mimic plane and space lattices.

A biological approach to assembling tissue modules through endothelial capillary network formation.

To create functional tissues having complex structures, bottom-up approaches to assembling small tissue modules into larger constructs have been emerging. Most of these approaches are based on chemical reactions or physical interactions at the interface between tissue modules. Here we report a biological assembly approach to integrate small tissue modules through endothelial capillary network formation. When adjacent tissue modules contain appropriate extracellular matrix materials and cell types that support robust endothelial capillary network formation, capillary tubules form and grow across the interface, resulting in assembly of the modules into a single, larger construct. It was shown that capillary networks formed in modules of dense fibrin gels seeded with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs); adjacent modules were firmly assembled into an integrated construct having a strain to failure of 117 ± 26%, a tensile strength of 2208 ± 83 Pa and a Young's modulus of 2548 ± 574 Pa. Under the same culture conditions, capillary networks were absent in modules of dense fibrin gels seeded with either HUVECs or MSCs alone; adjacent modules disconnected even when handled gently. This biological assembly approach eliminates the need for chemical reactions or physical interactions and their associated limitations. In addition, the integrated constructs are prevascularized, and therefore this bottom-up assembly approach may also help address the issue of vascularization, another key challenge in tissue engineering.