Nondestructive Real‐Time Monitoring of Enhanced Stem Cell Differentiation Using a Graphene‐Au Hybrid Nanoelectrode Array

Stem cells have attracted increasing research interest in the field of regenerative medicine because of their unique ability to differentiate into multiple cell lineages. However, controlling stem cell differentiation efficiently and improving the current destructive characterization methods for monitoring stem cell differentiation are the critical issues. To this end, multifunctional graphene–gold (Au) hybrid nanoelectrode arrays (NEAs) to: (i) investigate the effects of combinatorial physicochemical cues on stem cell differentiation, (ii) enhance stem cell differentiation efficiency through biophysical cues, and (iii) characterize stem cell differentiation in a nondestructive real‐time manner are developed. Through the synergistic effects of physiochemical properties of graphene and biophysical cues from nanoarrays, the graphene‐Au hybrid NEAs facilitate highly enhanced cell adhesion and spreading behaviors. In addition, by varying the dimensions of the graphene‐Au hybrid NEAs, improved stem cell differentiation efficiency, resulting from the increased focal adhesion signal, is shown. Furthermore, graphene‐Au hybrid NEAs are utilized to monitor osteogenic differentiation of stem cells electrochemically in a nondestructive real‐time manner. Collectively, it is believed the unique multifunctional graphene‐Au hybrid NEAs can significantly advance stem‐cell‐based biomedical applications.     

Ligand Diffusion Enables Force-Independent Cell Adhesion via Activating α5β1 Integrin and Initiating Rac and RhoA Signaling

Cells reside in a dynamic microenvironment in which adhesive ligand availability, density, and diffusivity are key factors regulating cellular behavior. Here, the cellular response to integrin-binding ligand dynamics by directly controlling ligand diffusivity via tunable ligand–surface interactions is investigated. Interestingly, cell spread on the surfaces with fast ligand diffusion is independent of myosin-based force generation. Fast ligand diffusion enhances α5β1 but not αvβ3 integrin activation and initiates Rac and RhoA but not ROCK signaling, resulting in lamellipodium-based fast cell spreading. Meanwhile, on surfaces with immobile ligands, αvβ3 and α5β1 integrins synergistically initiate intracellular-force-based canonical mechanotransduction pathways to enhance cell adhesion and osteogenic differentiation of stem cells. These results indicate the presence of heretofore-unrecognized pathways, distinct from canonical actomyosin-driven mechanisms, that are capable of promoting cell adhesion.     

Impact of local versus global ligand density on cellular adhesion

α(v)β(3) integrin-mediated cell adhesion is crucially influenced by how far ligands are spaced apart. To evaluate the impact of local ligand density versus global ligand density of a given surface, we used synthetic micronanostructured cell environments with user-defined ligand spacing and patterns to investigate cellular adhesion. The development of stable focal adhesions, their number, and size as well as the cellular adhesion strength proved to be influenced by local more than global ligand density.     

Nanomaterials can dynamically steer cell responses to biological ligands

Traditional tissue regeneration approaches to activate cell behaviors on biomaterials rely on the use of extracellular-matrix-based or soluble growth-factor cues. In this article, a novel approach is highlighted to dynamically steer cellular phenomena such as cell motility based on nanoscale substratum features of biological ligands. Albumin-derived nanocarriers (ANCs) with variable nanoscale-size features are functionalized with fibronectin III9-10 matrix ligands, and their effects on primary human keratinocyte activation are investigated. The presentation of fibronectin fragments from ANCs significantly enhances cell migration as compared to free ligands at equivalent concentrations. Notably, cell migration is influenced by the size of the underlying ANCs even for variably sized ANCs covered in comparable levels of fibronectin fragment. For equivalent ligand concentrations, cell migration on the smaller-sized ANCs (30 and 50 nm) is significantly enhanced as compared to that on larger-sized ANCs (75 and 100 nm). In contrast, the enhancement of cell migration on nanocarriers is abolished by the use of immobilized, biofunctionalized ANCs, indicating that "dynamic" nanocarrier internalization events underlie the role of nanocarrier geometry on the differential regulation of cell migration kinetics. Uptake studies using fluorescent ANCs indicate that larger-sized ANCs cause delayed endocytic kinetics and hence could present barriers for internalization during the cell adhesion and motility processes. Motile cells exhibit diminished migration upon exposure to clathrin inhibitors, but not caveolin inhibitors, suggesting the role of clathrin-mediated endocytosis in facilitating cell migratory responsiveness to the nanocarriers. Overall, a monotonic relationship is found between the nanocarrier cytointernalization rate and the cell migration rate, suggesting the possibility of designing biointerfacial features for the dynamic control of cell migration. Thus, the functionalization of a mobile nanocarrier by a biorelevant ligand can be used to sensitize cellular motility activation to the adhesion ligands, and such nanocarrier interfaces can dynamically attune cell migration kinetics by modulating the uptake of the ligand-nanocarrier complex via nanocarrier size.     

Microfluidic Gradients Reveal Enhanced Neurite Outgrowth but Impaired Guidance within 3D Matrices with High Integrin Ligand Densities

The density of integrin‐binding ligands in an extracellular matrix (ECM) is known to regulate cell migration speed by imposing a balance of traction forces between the leading and trailing edges of the cell, but the effect of cell‐adhesive ligands on neurite chemoattraction is not well understood. A platform is presented here that combines gradient‐generating microfluidic devices with 3D protein‐engineered hydrogels to study the effect of RGD ligand density on neurite pathfinding from chick dorsal root ganglia‐derived spheroids. Spheroids are encapsulated in elastin‐like polypeptide (ELP) hydrogels presenting either 3.2 or 1.6 mM RGD ligands and exposed to a microfluidic gradient of nerve growth factor (NGF). While the higher ligand density matrix enhanced neurite initiation and persistence of neurite outgrowth, the lower ligand density matrix significantly improved neurite pathfinding and increased the frequency of growth cone turning up the NGF gradient. The apparent trade‐off between neurite extension and neurite guidance is reminiscent of the well‐known trade‐off between adhesive forces at the leading and trailing edges of a migrating cell, implying that a similar matrix‐mediated balance of forces regulates neurite elongation and growth cone turning. These results have implications in the design of engineered materials for in vitro models of neural tissue and in vivo nerve guidance channels.     

The design of cyclic AMP--recognizing oligopeptides and evaluation of its capability for cyclic AMP recognition using an electrochemical system

A novel 17-mer peptide ligand for cyclic AMP was designed using the amino acid sequences of essential subsites in various cyclic AMP-dependent protein kinase (protein kinase A) families. The Au disk electrode, which was modified with the designed 17-mer oligopeptide, responded to cyclic AMP but virtually did not respond to any other cyclic nucleotides using the ion channel sensor mechanism. On the other hand, a scrambled peptide, which had the same amino acid composition as and had an amino acid sequence different from the 17-mer oligopeptide, did not respond to any nucleotides. This indicates that the designed 17-mer peptide actually acted as a selective ligand for cyclic AMP. This ligand-designing strategy using peptide sequences in target-binding proteins may possibly be extended to the design of peptide ligands for other second messengers.     

Chirality and Surface Bonding Correlation in Atomically Precise Metal Nanoclusters

Chirality is ubiquitous in nature and occurs at all length scales. The development of applications for chiral nanostructures is rising rapidly. With the recent achievements of atomically precise nanochemistry, total structures of ligand-protected Au and other metal nanoclusters (NCs) are successfully obtained, and the origins of chirality are discovered to be associated with different parts of the cluster, including the surface ligands (e.g., swirl patterns), the organic–inorganic interface (e.g., helical stripes), and the kernel. Herein, a unified picture of metal–ligand surface bonding-induced chirality for the nanoclusters is proposed. The different bonding modes of M–X (where M = metal and X = the binding atom of ligand) lead to different surface structures on nanoclusters, which in turn give rise to various characteristic features of chirality. A comparison of Au–thiolate NCs with Au–phosphine ones further reveals the important roles of surface bonding. Compared to the Au–thiolate NCs, the Ag/Cu/Cd–thiolate systems exhibit different coordination modes between the metal and the thiolate. Other than thiolate and phosphine ligands, alkynyls are also briefly discussed. Several methods of obtaining chiroptically active nanoclusters are introduced, such as enantioseparation by high-performance liquid chromatography and enantioselective synthesis. Future perspectives on chiral NCs are also proposed.     

Polyelectrolyte Multilayer-Treated Electrodes for Real-Time Electronic Sensing of Cell Proliferation

We report on the use of polyelectrolyte multilayer (PEM) coatings as a non-biological surface preparation to facilitate uniform cell attachment and growth on patterned thin-film gold (Au) electrodes on glass for impedance-based measurements. Extracellular matrix (ECM) proteins are commonly utilized as cell adhesion promoters for electrodes; however, they exhibit degradation over time, thereby imposing limitations on the duration of conductance-based biosensor experiments. The motivation for the use of PEM coatings arises from their long-term surface stability as promoters for cell attachment, patterning, and culture. In this work, a cell proliferation monitoring device was fabricated. It consisted of thin-film Au electrodes deposited with a titanium-tungsten (TiW) adhesion layer that were patterned on a glass substrate and passivated to create active electrode areas. The electrode surfaces were then treated with a poly(ethyleneimine) (PEI) anchoring layer and subsequent bilayers of sodium poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). NIH-3T3 mouse embryonic fibroblast cells were cultured on the device, observed by optical microscopy, and showed uniform growth characteristics similar to those observed on a traditional polystyrene cell culture dish. The optical observations were correlated to electrical measurements on the PEM-treated electrodes, which exhibited a rise in impedance with cell proliferation and stabilized to an approximate 15 % increase as the culture approached confluency. In conclusion, cells proliferate uniformly over gold and glass PEM-treated surfaces, making them useful for continuous impedance-based, real-time monitoring of cell proliferation and for the determination of cell growth rate in cellular assays.     

Large-scale dendrimer-based uneven nanopatterns for the study of local arginine-glycine-aspartic acid （RGD） density effects on cell adhesion

Cell adhesion processes are governed by the nanoscale arrangement of the extracellular matrix （ECM）, being more affected by local rather than global concentrations of cell adhesive ligands. In many cell-based studies, grafting of dendrimers on surfaces has shown the benefits of the local increase in concentration provided by the dendritic configuration, although the lack of any reported surface characterization has limited any direct correlation between dendrimer disposition and cell response. In order to establish a proper correlation, some control over dendrimer surface deposition is desirable. Here, dendrimer nanopatterning has been employed to address arginine-glycine-aspartic acid （RGD） density effects on cell adhesion. Nanopatterned surfaces were fully characterized by atomic force microscopy （AFM）, scanning tunneling microscopy （STM） and X-ray photoelectron spectroscopy （XPS）, showing that tunable distributions of cell adhesive ligands on the surface are obtained as a function of the initial dendrimer bulk concentration. Cell experiments showed a clear correlation with dendrimer surface layout： Substrates presenting regions of high local ligand density resulted in a higher percentage of adhered cells and a higher degree of maturation of focal adhesions （FAs）. Therefore, dendrimer nano- patterning is presented as a suitable and controlled approach to address the effect of local ligand density on cell response. Moreover, due to the easy modification of dendrimer peripheral groups, dendrimer nanopatterning can be further extended to other ECM ligands having density effects on cells.     

Effect of Thiolated Ligands in Au Nanowire Synthesis

Thiolated ligands are seldom used as morphology‐directing reagent in the synthesis of Au nanostructures due to their low selectivity toward the different facets. Recently, we developed a thiolated ligands‐induced synthesis of nanowires where the selective Au deposition only occurs at the ligand‐deficient Au–substrate interface. Herein, the structural effect of thiolated ligands in this active surface growth is systematically investigated. It is revealed that their ability of rendering surface is closely related to the molecular structure. Ligands with aromatic backbones are capable of inducing nanowire formation, whereas those with aliphatic backbones cannot, likely because the former can pack better at short time scale of the rapid growth. The substituents of the ligands are critical for the colloidal stability of the final structure. It is further demonstrated that aromatic and aliphatic ligands could be mixed to turn on the continual lateral growth, leading to nanowires with tapered ends. The ligand generality in this growth mode also allows the creation of superhydrophobic surface, with the nanowire forest providing the nanoscale surface roughness and the hydrophobic ligand offering the surface property. These applications of the thiolated ligands in the nanosynthesis open a new approach for controlled synthesis of Au‐based nanostructures with various morphologies and properties.     

Self-Strengthening Adhesive Force Promotes Cell Mechanotransduction

The extracellular matrix (ECM) undergoes dynamic remodeling and progressive stiffening during tissue regeneration and disease progression. However, most of the artificial ECMs and in vitro disease models are mechanically static. Here, a self-strengthening polymer coating mimicking the dynamic nature of native ECM is designed to study the cellular response to dynamic biophysical cues and promote cell mechanical sensitive response. Spiropyran (SP) is utilized as dynamic anchor group to regulate the strength of cell adhesive peptide ligands. Benefiting from spontaneous thermal merocyanine-to-spiropyran (MC–SP) isomerization, the resulting self-responsive coating displays dynamic self-strengthening of interfacial interactions. Comparing with the static and all of the previous dynamic artificial ECMs, cells on this self-responsive surface remodel the weakly bonded MC-based coatings to activate α5β1 integrin and Rac signaling in the early adhesion stage. The subsequent MC-to-SP conversion strengthens the ligand–integrin interaction to further activate αvβ3 integrin and RhoA/ROCK signaling in the latter stage. This sequential process enhances cellular mechanotransduction as well as the osteogenic differentiation of mesenchymal stem cells (MSCs). It is worth emphasizing that the self-strengthening occurs spontaneously in the absence of any stimulus, making it especially useful for implanted scaffolds in regenerative medicine.     

Adaptive Liquid Interfacially Assembled Protein Nanosheets for Guiding Mesenchymal Stem Cell Fate

There is a growing interest in the development of dynamic adaptive biomaterials for regulation of cellular functions. However, existing materials are limited to two-state switching of the presentation and removal of cell-adhesive bioactive motifs that cannot emulate the native extracellular matrix (ECM) in vivo with continuously adjustable characteristics. Here, tunable adaptive materials composed of a protein monolayer assembled at a liquid–liquid interface are demonstrated, which adapt dynamically to cell traction forces. An ultrastructure transition from protein monolayer to hierarchical fiber occurs through interfacial jamming. Elongated fibronectin fibers promote formation of elongated focal adhesion structures, increase focal adhesion kinase activation, and enhance neuronal differentiation of stem cells. Cell traction force results in spatial rearrangement of ECM proteins, which feeds back to alter stem cell fate. The reported biomimetic adaptive liquid interface enables dynamic control of stem cell behavior and has potential translational applications.     

Mechanical regulation of histone modifications and cell plasticity

Cell plasticity is important in development and tissue remodeling. Cells can sense physical and chemical cues from their local microenvironment and transduce the signals into the nucleus to regulate the epigenetic state and gene expression, resulting in a change in cell phenotype. In this review, we highlight the role of mechanical cues in regulating stem cell differentiation and cell reprogramming through the modulation of histone modifications. The effects of various mechanical cues, including matrix stiffness, mechanical stretch, and shear stress, on cell fate during tissue regeneration and remodeling will be discussed. Taken together, recent work demonstrates that the alterations in histone modifications by mechanical stimuli can facilitate epigenetic changes during cell phenotypic switching, which has potential applications in the development of biomaterials and bioreactors for cell engineering.     

Bioorthogonal Click Chemistry‐Based Synthetic Cell Glue

Artificial methods of cell adhesion can be effective in building functional cell complexes in vitro, but methods for in vivo use are currently lacking. Here, a chemical cell glue based on bioorthogonal click chemistry with high stability and robustness is introduced. Tetrazine (Tz) and trans‐cyclooctene (TCO) conjugated to the cell surface form covalent bonds between cells within 10 min in aqueous conditions. Glued, homogeneous, or heterogeneous cell pairs remain viable and stably attached in a microfluidic flow channel at a shear stress of 20 dyn cm⁻². Upon intravenous injection of assembled Jurkat T cells into live mice, fluorescence microscopy shows the trafficking of cell pairs in circulation and their infiltration into lung tissues. These results demonstrate the promising potential of chemically glued cell pairs for various applications ranging from delivering therapeutic cells to studying cell–cell interactions in vivo.     

Ligand-free Au nanoclusters/g-C3N4 ultra-thin nanosheets composite photocatalysts for efficient visible-light-driven photocatalytic H2 generation

In this paper, we report the synthesis of ligand-free Au nanoclusters (NCs)/g-C₃N₄ ultra-thin nanosheets (NSs) composite via a facile wet-impregnation method with post-annealing. On the one hand, post-annealing was used for the exfoliation of multi-layered g-C₃N₄ to obtain ultra-thin NSs; on the other hand, after Au₂₅(Cys)₁₈ NCs were loaded, post-annealing was further adopted to remove the ligands to obtain clean surface on Au NCs. It is demonstrated that the loaded Au NCs were aggregating resistant by post-annealing. Constructing heterojunctions with appropriate inter-band structures between the ligand-free Au NCs and the ultra-thin g-C₃N₄ NSs, along with the mono-distribution of the Au NCs and their intimate contact with g-C₃N₄ NSs ensured the smooth interfacial charge transfer. As a result, the composite photocatalysts exhibited efficient visible-light-induced photocatalytic H₂ generation, mainly due to the local electric field enhancement induced by excitation of Au NCs under visible light and the improved charge separation in g-C₃N₄. This work provides a general strategy for the synthesis of noble metal NCs based composites with clean surface as the efficient photocatalysts for solar energy conversion.

Graphical Abstract

A stepwise post-annealing strategy is exploited to prepare g-C₃N₄ ultra-thin nanosheets modified with highly dispersed ligand-free Au nanoclusters for efficient photocatalytic hydrogen production.

     

Mesenchymal stem cell adhesion and spreading on nanostructured biomaterials

Bone marrow-derived human mesenchymal stem cells were seeded in serum-free media onto ion beam-deposited nanostructured metalloceramic (Ti-Cr-N) films and plasma-nitrided titanium disks, which were left uncoated as well as precoated with fetal bovine serum. Precoating the disks with serum appears to stimulate cell spreading on both the titanium nitride and metalloceramic materials for as little as 1 hour incubation time. The implication is that both of these materials can adsorb serum proteins in amounts sufficient to influence cell adhesion and spreading for potentially improved in vivo response of orthopedic and dental implants. The materials in this study may prove to exhibit enhanced biological and mechanical properties when compared to conventional micron-scale implant materials such as titanium or cobalt-chrome alloys.     

Ligand density-dependent influence of arginine–glycine–aspartate functionalized gold nanoparticles on osteogenic and adipogenic differentiation of mesenchymal stem cells

Extracellular matrix (ECM) plays a very important role in regulating cell function and fate. It is highly desirable to fabricate biomimetic models to investigate the role of ECM in stem cell differentiation. In this study, arginine–glycine–aspartate (RGD)-modified gold nanoparticles (Au NPs) with tunable surface ligand density were prepared to mimic the ECM microenvironment. Their effect on osteogenic and adipogenic differentiation of human mesenchymal stem cells (MSCs) was investigated. The biomimetic Au NPs were taken up by MSCs in a ligand density-dependent manner. The biomimetic NPs with a high RGD density had an inhibitive effect on the alkaline phosphatase (ALP) activity, calcium deposition, and osteogenic marker gene expression of MSCs. Their effect on oil droplet formation and adipogenic marker gene expression was negative when RGD density was low, while their effect was promotive when RGD density was high. The biomimetic Au NPs regulated the osteogenic and adipogenic differentiation of MSCs mainly through affecting the focal adhesion and cytoskeleton. This study highlights the roles of biomimetic NPs on stem cell differentiation that could provide a meaningful strategy in fabricating functional biomaterials for tissue engineering and biomedical applications.

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Cell adhesion and related phenomena on the surface-modified Au-deposited nerve microelectrode examined by total impedance measurement and cell detachment tests

This study investigated alkanethiolate self-assembled monolayers (SAMs) of varied chain lengths adsorbed upon novel Au-coated microelectrodes, of which the surface properties were quantitatively evaluated by surface characterization and 3T3 fibroblast cell adhesion, total impedance and cell detachment tests. Thin-film SAMs adsorbed upon Au/PI/Si provided a hydrophobic or passive surface with increased water contact angle and initial total impedance. From cell adhesion tests, we can observe that the film formed as a dense-packed spacer resulted in incomplete cell sealing of 3T3 cells upon the surface-modified microelectrode. Thus the decrease in cell coverage rate and in the slope in association with total impedance as a function of cell-surface reaction time can be found. To study the adhesion force of a comparable single cell attached upon varied modified surfaces, a cell detachment test using a triangular probe tip of a well defined cantilever was carried out in medium containing fibroblast cells. Overall, both the peak force and the work required to detach a comparable single cell from the anchoring domain corresponded well to the increased length of alkyl chains adsorbed upon Au/PI/Si. Both measurements on the SAM modified surfaces demonstrated much smaller values than those on the pristine Au/PI/Si surface. These results concluded that a cell-repulsive characteristic was clearly formed on the SAM modified microelectrode surface. The non-adhering properties of surface-modified microelectrodes should provide better sensitivity for neuromuscular stimulation as well as for the recording of infinitesimal neural signals in future applications of neural prostheses.     

Synthesis, characterization, and direct intracellular imaging of ultrasmall and uniform glutathione-coated gold nanoparticles

Gold nanoparticles (AuNPs) with core sizes below 2 nm and compact ligand shells constitute versatile platforms for the development of novel reagents in nanomedicine. Due to their ultrasmall size, these AuNPs are especially attractive in applications requiring delivery to crowded intracellular spaces in the cytosol and nucleus. For eventual use in vivo, ultrasmall AuNPs should ideally be monodisperse, since small variations in size may affect how they interact with cells and how they behave in the body. Here we report the synthesis of ultrasmall, uniform 144-atom AuNPs protected by p-mercaptobenzoic acid followed by ligand exchange with glutathione (GSH). Quantitative scanning transmission electron microscopy (STEM) reveals that the resulting GSH-coated nanoparticles (Au(GSH)) have a uniform mass distribution with cores that contain 134 gold atoms on average. Particle size dispersity is analyzed by analytical ultracentrifugation, giving a narrow distribution of apparent hydrodynamic diameter of 4.0 ± 0.6 nm. To evaluate the nanoparticles' intracellular fate, the cell-penetrating peptide TAT is attached noncovalently to Au(GSH), which is confirmed by fluorescence quenching and isothermal titration calorimetry. HeLa cells are then incubated with both Au(GSH) and the Au(GSH)-TAT complex, and imaged without silver enhancement of the AuNPs in unstained thin sections by STEM. This imaging approach enables unbiased detection and quantification of individual ultrasmall nanoparticles and aggregates in the cytoplasm and nucleus of the cells.