Critical adhesion areas of cells on micro-nanopatterns.

Cell adhesion to extracellular matrices (ECM) is critical to physiological and pathological processes as well as biomedical and biotechnological applications. It has been known that a cell can adhere on an adhesive microisland only over a critical size. But no publication has concerned critical adhesion areas of cells on microislands with nanoarray decoration. Herein, we fabricated a series of micro-nanopatterns with different microisland sizes and arginine-glycine-aspartate (RGD) nanospacings on a nonfouling poly(ethylene glycol) background. Besides reproducing that nanospacing of RGD, a ligand of its receptor integrin (a membrane protein), significantly influences specific cell adhesion on bioactive nanoarrays, we confirmed that the concept of critical adhesion area originally suggested in studies of cells on micropatterns was justified also on the micro-nanopatterns, yet the latter exhibited more characteristic behaviors of cell adhesion. We found increased critical adhesion areas of human mesenchymal stem cells (hMSCs) on nanoarrayed microislands with increased RGD nanospacings. However, the numbers of nanodots with respect to the critical adhesion areas were not a constant. A unified interpretation was then put forward after combining nonspecific background adhesion and specific cell adhesion. We further carried out the asymptotic analysis of a series of micro-nanopatterned surfaces to obtain the effective RGD nanospacing on unpatterned free surfaces with densely grafted RGD, which could be estimated nonzero but has never been revealed previously without the assistance of the micro-nanopatterning techniques and the corresponding analysis. Electronic Supplementary Material: Supplementary materials and methods (details of fabrication of micro-nanopatterns), and supplementary results (selective adhesion or localization of hMSCs on nanoarrayed microislands with non-fouling background, calculation of critical number of integrin-ligand binding N*, etc.) are available in the online version of this article at 10.1007/s12274-021-3711-6.

H∞ Static Output Feedback for Low-Frequency Networked Control Systems With a Decentralized Event-Triggered Scheme.

This paper investigates an H∞ static output feedback for a low-frequency (LF) networked control system (NCS). A decentralized event-triggered scheme (DETS) is proposed to reduce the network communication loads. The LF NCS with the DETS is modeled as a time-delay system with an LF constraint. With this model, H∞ performance analysis is developed via an LF integral quadratic constraint method and a generalized Kalman-Yakubovich-Popov lemma. Furthermore, a detailed controller design algorithm is presented to obtain an H∞ static output feedback controller for the LF NCS with the DETS via a two-stage approach. The numerical results are provided to demonstrate the effectiveness and superiority of the proposed techniques.

Double-Loop Stability for High Frequency Networked Control Systems Subject to Actuator Saturation.

This paper generalizes stability for a high frequency networked control system (NCS) subject to actuator saturation via integral quadratic constraints. A delta operator system with a high frequency constraint is used to model the high frequency NCS. Double-loop stability is treated via outer-loop and interloop feedback configurations for the high frequency NCS. Stability criteria are derived with the high frequency constraint and actuator saturation by a generalized Kalman-Yakubovich-Popov lemma. Numerical results are provided to demonstrate the effectiveness of the proposed techniques in this paper.

Identification and characterization of G-quadruplex formation within the EP0 promoter of pseudorabies virus.

EP0 is an important early gene that modulates the life cycle of pseudorabies virus (PRV). A guanine-rich sequence overlapping with three Sp1 binding sites is located upstream of the transcription start site (TSS) in the EP0 promoter. Using native polyacrylamide gel electrophoresis (PAGE) and circular dichroism (CD), we verified that the G-rich region in the EP0 promoter forms an intramolecular parallel G-quadruplex (G4) in the presence of K+ ions. Further dimethyl sulphate (DMS) footprinting and Taq polymerase stop assays indicates the potential polymorphic folding of G4. In addition, a small chemical ligand, pyridostatin (PDS), promotes and stabilizes the formation of G4. Interestingly, based on the results of electrophoretic mobility shift assays (EMSA), the Sp1 protein bound to G4-bearing DNA with more affinity than DNA lacking the G4 structure. According to the luciferase reporter assay, G4 negatively regulates the EP0 promoter activity. These results demonstrate that Sp1 and G4 cooperate to regulate EP0 promoter activity.

Fabrication of RGD micro/nanopattern and corresponding study of stem cell differentiation.

Micropatterns of gold (Au) nanoarrays on inorganic and polymeric substrates were fabricated by combining block copolymer micelle nanolithography to obtain gold nanoarrays on glass, photolithography plus hydrofluoric acid (HF) etching to generate microislands, and transfer lithography to shift the gold micro/nanopatterns from glass to a bioinert poly(ethylene glycol) (PEG) hydrogel surface. Further the modification of the gold nanodots via cell-adhesive arginine-glycine-aspartate (RGD) ligands was carried out to achieve peptide micro/nanopatterns. Whereas the micro/nanopatterns of noble metals could be useful in various applications, the peptide micro/nanopatterns especially enable persistent cell localization on adhesive micropatterns of RGD nanoarrays on the background of potently nonfouling PEG hydrogels, and thus offer a powerful tool to investigate cell-material interactions on both molecular and cellular levels. As a demonstration, we cultured human mesenchymal stem cells (hMSCs) on micro/nanopatterns with RGD nanoarrays of nanospacings 46 and 95 nm, and with micropans of side lengths 35 and 65 µm (four groups in total). The osteogenic and adipogenic differentiation of hMSCs was conducted, and the potential effect of RGD nanospacing and the effect of cell spreading size on cell differentiation were decoupled for the first time. The results reveal that RGD nanospacing, independent of cell spreading size, acts as a strong regulator of cell tension and stem cell differentiation, which cannot be concluded unambiguously based on either merely micropatterns or nanopatterns.

Adhesion, proliferation, and differentiation of mesenchymal stem cells on RGD nanopatterns of varied nanospacings.

The present report is an extension of our preceding publication in Biomaterials (2013) entitled "Effect of RGD nanospacing on differentiation of stem cells." Cell-adhesive peptide arginine-glycine-aspartate (RGD) was nanopatterned on a non-fouling poly(ethylene glycol) (PEG) hydrogel, and mesenchymal stem cells (MSCs) derived from rat bone marrow were cultured on the patterned surfaces at nanospacings from 37 to 124 nm. Cell adhesion parameters such as spreading areas varied with RGD nanospacings significantly. The differences were well observed at both the first and eighth days, which confirmed the persistence of this nanospacing effect on our nanopatterns. The proliferation rate also varied with the nanospacings. Osteogenic and adipogenic inductions were undertaken, and a significant influence of RGD nanospacing on stem cell differentiation was found. The effect on differentiation cannot be simply interpreted by differences in cell adhesion and proliferation. We further calculated the fractions of single, coupled, and multiple cells on those nanopatterns, and ruled out the possibility that the extent of cell-cell contact determined the different differentiation fractions. Accordingly, we reinforced the idea that RGD nanospacing might directly influence stem cell differentiation.

Effect of RGD nanospacing on differentiation of stem cells.

Nanopatterns of a cell-adhesive peptide arginine-glycine-aspartate (RGD) on a persistently non-fouling poly(ethylene glycol) hydrogel were prepared, and behaviours of mesenchymal stem cells (MSCs) on patterns of five RGD nanospacings from 37 to 124 nm were examined under a full level of serum for eight days. Besides cell adhesion, osteogenic and adipogenic inductions of MSCs from rat bone marrow were observed in corresponding media. We not only confirmed the nanospacing dependence of cell spreading previously reported in other cell types (non-stem cells) such as less spreading in the case of nanospacings larger than the critical 70 nm, but also found the effect of RGD nanospacing on lineage commitments of stem cells. Both osteogenic and adipogenic inductions resulted in higher differentiation extents on patterns of large nanospacings than of small nanospacings. Under co-induction in the mixed osteogenic/adipogenic media, osteogenesis was predominant over adipogenesis on patterns of large RGD nanospacings, although a less cell spreading itself was beneficial not for osteogenesis but for adipogenesis according to previous studies without nanopatterns. The effect of RGD nanospacing on lineage commitments of stem cells is unexpected and cannot be interpreted via the cell spreading effect. Thus, the differentiation of stem cells might be regulated inherently by nanospacing of bioactive ligands on the material surfaces.

Control of cell nucleus shapes via micropillar patterns.

We herein report a material technique to control the shapes of cell nuclei by the design of the microtopography of substrates to which the cells adhere. Poly(D,L-lactide-co-glycolide) (PLGA) micropillars or micropits of a series of height or depth were fabricated, and some surprising self deformation of the nuclei of bone marrow stromal cells (BMSCs) was found in the case of micropillars with a sufficient height. Despite severe nucleus deformation, BMSCs kept the ability of proliferation and differentiation. We further demonstrated that the shapes of cell nuclei could be regulated by the appropriate micropillar patterns. Besides circular and elliptoid shapes, some unusual nucleus shapes of BMSCs have been achieved, such as square, cross, dumbbell, and asymmetric sphere-protrusion.

Critical areas of cell adhesion on micropatterned surfaces.

The adhesive area is important to modulate cell behaviors on a substrate. This paper aims to semi-quantitatively examine the existence of the characteristic areas of cell adhesion on the level of individual cells. We prepared a series of micropatterned surfaces with adhesive microislands of various sizes on an adhesion-resistant background, and cultured cells of MC3T3-E1 (osteoblast), BMSC (bone mesenchymal stem cell) or NIH3T3 (fibroblast) on those modeled surfaces. We have defined seven characteristic areas of an adhesive microisland and confirmed that they are meaningful to describe cell adhesion behaviors. Those parameters are (1) the critical adhesion area from apoptosis to survival denoted as A∗ or A(c1), (2) the critical area from adhesion of a single cell to adhesion of multiple cells (A(c2)), (3) the basic area for one more cell to adhere (A(Δ)), (4) and (5) the characteristic areas of a microisland most probably occupied by one cell (A(peak1) and two cells (A(peak2)), (6) and (7) the characteristic areas of a microisland occupied by one cell (A(N1)) or two cells (A(N2)) on average. Besides the introduction of those basic parameters, the present paper demonstrates how to determine them experimentally. We further discussed the relationship between those characteristic areas and the spreading area on a non-patterned adhesive surface.