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Gene transfection is important in biotechnology and is used to modify cells intrinsically. It can be conducted in cell suspension or after cell adhesion, where the efficiency is dependent on many factors such as the type of nanocarrier used and cell division processes. Anchor-dependent cells are sensitive to the substrate they are attached to and adapt their behavior accordingly, including plasmid trafficking during gene transfection. Previously, it was shown in our group that the cytoskeleton is an essential factor in influencing gene transfection in skeletal myoblasts using nanogrooves as a substrate. In this study, the effect of the cytoskeleton on gene transfection efficiency of skeletal myoblasts was studied using various nanopillars and nanocarriers. Nanopillars with different diameters (200-1000 nm) and depths (200 or 400 nm) were fabricated using colloidal self-assembly and reactive ion etching. All surfaces were treated with oxygen plasma or polydopamine (PD) to further control cell morphology. Plasmid DNA was delivered into cells using jetPRIME or Lipofectamine 3000 nanocarriers. After screening hundreds of images, two distinguishable F-actin distributions were found, i.e., cells with or without a perinuclear actin cap (pnAC). Cells attached to nanopillars, especially the deep pillars, had a smaller spreading area, shorter F-actin, more 3D-like cell nuclei, and a lower percentage of pnAC, which lead to a higher gene transfection efficiency using jetPRIME. On the other hand, cells attached to the shallow nanopillars or flat surfaces had a larger spreading area, longer F-actin, more 2D-like cell nuclei, and a higher percentage of pnAC that facilitates gene transfection using Lipofectamine. The effects of cell density, cytoskeleton (cytoD), and focal adhesions (RGD) on gene transfection were also studied, and the results were consistent with our hypothesis that F-actin distribution is one of the critical factors in gene transfection. In conclusion, pnAC plays a vital role in the intracellular trafficking of nanocarrier/plasmid complexes and this study provides new insights into gene transfection in anchor-dependent cells. STATEMENT OF SIGNIFICANCE: This study provides a new perspective in gene transfection using attached cells where perinuclear actin cap (pnAC) is an essential factor involved in transfection efficiency. A series of nanopillars were used to harness cell and cytoskeleton morphology. Two distinguishable cytoskeletal structures were found including cells with or without pnAC. 2D-like cells with pnAC facilitate gene delivery using liposome-based nanocarriers, while 3D-like cells without pnAC benefit gene delivery using cationic polymer-based nanocarriers. This study reveals the importance of the cytoskeleton during gene transfection that is beneficial in tissue transfection., Competing Interests: Declaration of Interest Statement There is no conflict of interest in this study., (Copyright © 2020. Published by Elsevier Ltd.)

It is well known that the surface chemistry of biomaterials is important for both initial cell attachment and the downstream cell response. Surface chemistry gradients are a new format that allows the screening of the subtleties of cell-surface interactions in high throughput. In this study, two surface chemical gradients were fabricated using diffusion control during plasma polymerization via a tilted mask. Acrylic acid (AA) plasma polymer gradients were coated on a uniform 1,7-octadiene (OD) plasma polymer layer to generate OD-AA plasma polymer gradients, whilst diethylene glycol dimethyl ether (DG) plasma polymer gradients were coated on a uniform AA plasma polymer layer to generate AA-DG plasma polymer gradients. Gradient surfaces were characterized by X-ray photoelectron spectroscopy, infrared microscopy mapping, profilometry, water contact angle (WCA) goniometry and atomic force microscopy. Cell attachment density and differentiation into osteo- and adipo-lineages of rat-bone-marrow mesenchymal stem cells (rBMSCs) was studied on gradients. Cell adhesion after 24 h culture was sensitive to the chemical gradients, resulting in a cell density gradient along the substrate. The slope of the cell density gradient changed between 24 and 6 days due to cell migration and growth. Induction of rBMSCs into osteoblast- and adipocyte-like cells on the two plasma polymer gradients suggested that osteogenic differentiation was sensitive to local cell density, but adipogenic differentiation was not. Using mixed induction medium (50% osteogenic and 50% adipogenic medium), thick AA plasma polymer coating (>40 nm thickness with ∼11% COOH component and 35° WCA) robustly supported osteogenic differentiation as determined by colony formation and calcium deposition. This study establishes a simple but powerful approach to the formation of plasma polymer based gradients, and demonstrates that MSC behavior can be influenced by small changes in surface chemistry., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Cell patterning is an important tool for biomedical research. In this work, we modified a technique combining mussel-inspired surface chemistry and microcontact printing (μCP) to modulate surface chemistry for cell patterning. Polymerized dopamine on poly(dimethylsiloxane) stamps was transferred to several cell-unfavorable substrates via μCP. Since cells only attached to the polydopamine (PDA)-imprinted areas, cell patterns were formed on a variety of cell-unfavorable surfaces. The stability of PDA imprints was proved under several harsh conditions. The cell affinity of PDA was modulated by co-deposition with several poly(ethylene imine) (PEI)-based copolymers, such as PEI, PEI-g-PEG (poly(ethylene glycol)) and PEI-g-galactose. The imprints of PDA/PEI-g-PEG provide the formation of cell patterns on cell-favorable substrates. Neuronal PC12 cells were patterned via imprinting of PDA/PEI, while HepG2/C3A cells were arranged on the imprint of PDA/PEI-g-galactose. Finally, co-culture of HepG2/C3A cells and L929 fibroblasts was accomplished by our micropatterning approach. This study demonstrated this simple and economic technique provides a powerful tool for development of functional patterned substrates for cell patterning. This technique should profit the preparation of cell patterns to study fundamental cell biology and to apply to biomedical engineering such as cell-based biosensors, diagnostic devices and tissue engineering., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Substrate stiffness is emerging as an effective tool for the regulation of cell behaviours such as locomotion, proliferation and differentiation. In order to explore the potential application of this biophysical tool, material platforms displaying lateral and continuously graded stiffness are advantageous since they allow the systematic exploration of adherent cell response to substrate stiffness and the tuning of the material to elicit the desired cell behaviour. Here, we demonstrate a simple approach towards the fabrication of polydimethylsiloxane (PDMS) stiffness gradients (with an indentation modulus of 190 kPa-3.1 MPa across a 12 mm distance) by means of a temperature gradient during curing. We then apply these stiffness gradients to the screening of osteogenic differentiation in rat mesenchymal stem cells (rMSCs). Our proof-of-principle results show that mineralization of rMSCs is strongly dependent on the PDMS substrate stiffness, but is also influenced by the display of extracellular matrix proteins preadsorbed on the gradients. This screening capability holds tremendous potential for the design of improved implant materials and tissue engineering scaffolds., (Copyright © 2011 Acta Materialia Inc. All rights reserved.)

A surface modification technique based on poly(dopamine) deposition developed from oxidative polymerization of dopamine is known to promote cell adhesion to several cell-resistant substrates. In this study this technique was applied to articular cartilage tissue engineering. The adhesion and proliferation of rabbit chondrocytes were evaluated on poly(dopamine)-coated polymer films, such as polycaprolactone, poly(L-lactide), poly(lactic-co-glycolic acid) and polyurethane, biodegradable polymers that are commonly used in tissue engineering. Cell adhesion was significantly increased by merely 15 s of dopamine incubation, and 4 min incubation was enough to reach maximal cell adhesion, a 1.35-2.69-fold increase compared with that on the untreated substrates. Cells also grew much faster on the poly(dopamine)-coated substrates than on untreated substrates. The increase in cell affinity for poly(dopamine)-coated substrates was demonstrated via enhancement of the immobilization of serum adhesive proteins such as fibronectin. When the poly(dopamine)-coating technique was applied to three-dimensional (3-D) polyurethane scaffolds, the proliferation of chondrocytes and the secretion of glycosaminoglycans were increased compared with untreated scaffolds. Our results show that the deposition of a poly(dopamine) layer on 3-D porous scaffolds is a simple and promising strategy for articular cartilage tissue engineering, and may be applied to other types of tissue engineering., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The topographic and mechanical characteristics of engineered tissue constructs, simulating native tissues, should benefit tissue engineering. Previous studies reported that surface topography and substrate rigidity provide biomechanical cues to modulate cellular responses such as alignment, migration and differentiation. To fully address this issue, the present study aimed to examine the influence of nanogrooved substrates with different stiffnesses on the responses of rat cardiomyocytes. Nanogrooved substrates (450nm in groove/ridge width; 100 or 350nm in depth) made of polystyrene and polyurethane were prepared by imprinting from polydimethylsiloxane molds. The morphology and orientation of cardiomyocytes attached to the substrates were found to be influenced mainly by the nanogrooved structures, while the contractile function of the cells was regulated by the coupled effect of surface topography and substrate stiffness. The distribution of intracellular structural proteins such as vinculin and F-actin showed that the surface topography and substrate stiffness regulated the organization of the actin cytoskeleton and focal adhesion complexes, and consequently the contractile behavior of the cardiomyocytes. The beating rates of the cultured cardiomyocytes were dependent on both the surface topography and the substrate stiffness. The study provides insights into the interaction between cardiomyocytes and biomaterials, and benefits cardiac tissue engineering., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Non-fouling coatings play a critical role in many biomedical applications, such as diagnostic assay materials, biosensors, blood contacting devices and other implants. In the present work we have developed a facile, one step deposition method based on dopamine polymerization for preparation of non-fouling and biotinylated surfaces for biomedical applications. Poly(ethylene imine)-graft-poly(ethylene glycol) co-polymer (PEI-g-PEG) was mixed with an alkaline dopamine solution and then deposited onto different substrates. The dopamine coatings formed by this method were characterized by X-ray photoelectron spectroscopy (XPS), and the results indicated successful deposition of PEG. The resultant dopamine coatings formed on tissue culture polystyrene by this method revealed successful deposition of PEG, as shown by XPS. PEI-g-PEG/dopamine deposition for 2h inhibited the adsorption of serum proteins and the attachment of fibroblasts, suggesting that PEG molecules were immobilized in a sufficient density on the surface of the coating. Furthermore, co-deposition of PEI-g-PEG and PEI-g-biotin in alkaline dopamine solutions provided a cell-resisting background surface, at the same time providing accessible biotin molecules. We have demonstrated that the surface can be used for the selective binding of avidin, followed by the binding of Arg-Gly-Asp-Ser-biotin and enhanced cell attachment by specific cell-ligand interactions. In conclusion, our one step immobilization method provides a simple tool to fabricate surfaces with controllable cell affinity., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Layer-by-layer deposition of polyelectrolyte multilayer (PEM) thin films has recently been applied to biomaterial applications. This simple and versatile technique provides a wide variety of potential utilization by insertion of biomolecules such as cell adhesion peptides. In this work dual peptides containing RGD (a cell-binding domain) and LHRRVKI (a heparin-binding domain) were immobilized onto polystyrene by the PEM technique and the effects on osteoblast cell culture were investigated. These peptides were conjugated to the amino groups of poly(allylamine hydrochloride) and then adsorbed onto the top of a 10 layer poly(allylamine hydrochloride)/poly(acrylic acid) film assembled at either pH 2.0 or pH 6.5. Osteoblasts, isolated from neonatal rat calvariae, were then seeded and cultured on the peptide-conjugated surfaces. We found that the cells adhered and grew better on the RGD-conjugated PEM films. The osteoblasts exhibited a better differentiated phenotype on the pH 2.0 films than the pH 6.5 films with respect to calcium deposition. The incorporation of LHRRVKI did not support cell adhesion, growth and matrix mineral deposition. Our results showed that the efficacy of RGD conjugation on osteoblast behavior was affected by the base PEM film.

It is known that cellular behavior is affected by nano-patterned topography. For example, many cell types tend to align and extend along the direction of nano-grooves/ridges structures. In this study, we investigated the impact of nano-grooves/ridges on hepatocyte morphology and functions. HepG2/C3A (C3A) cells were cultured on nano-grooved silicon or polystyrene substrata with various widths (from 100 to 500 nm) and depths (from 100 to 380 nm). Nano-grooved substrates induced dramatic changes in C3A cell morphology. The cells formed spheroids on the flat substrates, while C3A cells spread and grew confluently with elongated and aligned morphology along the nano-grooves/ridges. Albumin synthesis was enhanced on the nano-grooved silicon substrates compared to the flat surface, and was decreased with increasing groove depths. Urea conversion on the shallow grooves (400 nm wide and 100 nm deep) remained at the same level of that on the flat surfaces, but was decreased on the deeper grooves. We found that the functions of hepatocytes were enhanced on the substrates with shallow grooves. The nano-grooved substrates may be applied as in vitro culture systems of hepatocytes for both diagnostic and therapeutic applications.