Controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces

A newly discovered class of cell resistant surfaces, specifically engineered polyelectrolyte multilayers, was patterned with varying densities of adhesion ligands to control attachment of mammalian cells and to study the effects of ligand density on cell activity. Cell adhesive patterns were created on cell resistant multilayer films composed of poly(acrylic acid) and polyacrylamide through polymer-on-polymer stamping of poly(allylamine hydrochloride) PAH and subsequent reaction of the amine functional groups with an adhesion ligand containing RGD (Arg-Gly-Asp). These cell patterns demonstrated great promise for long-term applications since they remained stable for over 1 month, unlike ethylene glycol functional surfaces. By changing the stamping conditions of PAH, it was possible to alter the number of available functional groups in the patterned regions, and as a result, control the ligand density. Cell spreading, morphology, and cytoskeletal organization were compared at four different RGD densities. The highest RGD density, approximately 152 000 molecules/microm2, was created by stamping PAH at a pH of 11.0. Lowering the stamping ink pH led to patterns with lower ligand surface densities (83 000 molecules/microm2 for pH 9.0, 53,000 molecules/ microm2 for pH 7.0, and 25 000 molecules/microm2 for pH 3.5). An increasing number of cells attached and spread as the RGD density of the patterns increased. In addition, more cells showed well-defined actin stress fibers and focal adhesions at higher levels of RGD density. Finally, we found that pattern geometry affected cytoskeletal protein organization. Well-formed focal adhesions and cell-spanning stress fibers were only found in cells on wider line patterns (at least 25 microm in width).     

Improved mesenchymal stem cell seeding on RGD-modified poly(L-lactic acid) scaffolds using flow perfusion

Arg-Gly-Asp (RGD) has been widely utilized to increase cell adhesion to three-dimensional scaffolds for tissue engineering. However, cell seeding on these scaffolds has only been carried out statically, which yields low cell seeding efficiencies. We have characterized, for the first time, the seeding of rat mesenchymal stem cells on RGD-modified poly(L-lactic acid) (PLLA) foams using oscillatory flow perfusion. The incorporation of RGD on the PLLA foams improves scaffold cellularity in a dose-dependent manner under oscillatory flow perfusion seeding. When compared to static seeding, oscillatory flow perfusion is the most efficient seeding technique. Cell detachment studies show that cell adhesion is dependent on the applied flow rate, and that cell attachment is strengthened at higher levels of RGD modification.     

Controlling cell attachment selectively onto biological polymer-colloid templates using polymer-on-polymer stamping

A new patterning approach using polymer-on-polymer stamping (POPS) has been developed to fabricate polymer-colloid templates for controlling selective cell attachment. In this paper, a polyamine surface patterned onto a poly(acrylic acid)/poly(allylamine hydrochloride) (PAA/PAH) cell resistant multilayer platform serves as a template for the deposition of close- or loose-packed colloidal particles. Peptides containing the RGD adhesion sequence were used to modify the PAH/colloid surface for specific cell attachment. Cell behavior was studied by varying colloidal packing array density, pattern geometry, and surface chemistry. It was found that loose-packed RGD-modified colloidal arrays enhance cell adhesion, as observed through the development of focal adhesion contacts and orientation of actin stress fibers, but close-packed colloidal arrays induce a rounded and nonadhesive cell morphology and yield a smaller number of attached cells. On loose-packed arrays, cells adjust their shapes to the pattern geometry when the stripe width is smaller than 50 microm and increase their extent of attachment when the concentration of surface RGD peptides is increased. This new biomaterials system allows the examination of cell behavior as a function of RGD surface distribution on the molecular to micrometer scale and reveals cellular response to different surface roughnesses.     

Cell adhesion and growth to Peptide-patterned supported lipid membranes

Lipid vesicles displaying RGD peptide amphiphiles were fused with glass coverslips to control the ability of these surfaces to support cell adhesion and growth. Cell adhesion was prevented on phosphatidylcholine bilayers in the absence of RGD, whereas cells adhered and grew in the presence of accessible RGD amphiphiles. This specific interaction between cells and RGD peptides was further explored in a concentration-dependent fashion by creating surface composition arrays using microfluidics. For the range of concentrations studied adhesion and growth were favored by increased peptide concentration, but this concentration dependence was found to diminish in the higher concentration regions of the array. Developing peptide composition gradients in a membrane environment is demonstrated as an effective method to screen biological probes for cell adhesion and growth.     

Combination of integrin-binding peptide and growth factor promotes cell adhesion on electron-beam-fabricated patterns

Understanding and controlling cell adhesion on engineered scaffolds is important in biomaterials and tissue engineering. In this report we used an electron-beam (e-beam) lithography technique to fabricate patterns of a cell adhesive integrin ligand combined with a growth factor. Specifically, micron-sized poly(ethylene glycol) (PEG) hydrogels with aminooxy- and styrene sulfonate-functional groups were fabricated. Cell adhesion moieties were introduced using a ketone-functionalized arginine-glycine-aspartic acid (RGD) peptide to modify the O-hydroxylamines by oxime bond formation. Basic fibroblast growth factor (bFGF) was immobilized by electrostatic interaction with the sulfonate groups. Human umbilical vein endothelial cells (HUVECs) formed focal adhesion complexes on RGD- and RGD and bFGF-immobilized patterns as shown by immunostaining of vinculin and actin. In the presence of both bFGF and RGD, cell areas were larger. The data demonstrate confinement of cellular focal adhesions to chemically and physically well-controlled microenvironments created by a combination of e-beam lithography and "click" chemistry techniques. The results also suggest positive implications for addition of growth factors into adhesive patterns for cell-material interactions.     

Response of Human Mesenchymal Stem Cells to Patterned and Randomly Oriented Poly(Vinyl Alcohol) Nano-fibrous Scaffolds Surface-Modified with Arg-Gly-Asp (RGD) Ligand

The aim of this study was to obtain a better insight of how nano-fibrous scaffolds can affect human mesenchymal stem cells responses. Therefore, in this study, using electrospinning technique, poly(vinyl alcohol) (PVA) nano-fibers with two different patterns were prepared. In the first structure, PVA nano-fibers were oriented randomly and in the second structure, nano-fibers were electrospun in such a way that a special pattern was obtained. In order to enhance their stability, scaffolds were cross-linked using glutaraldehyde vapor. RGD immobilization was used to improve cell adhesion properties of the scaffolds. SEM micrographs demonstrated that the cell adhesion was effectively enhanced after RGD immobilization and higher cell densities were observed on RGD-modified scaffolds. Randomly oriented nano-fibers showed better cell adhesion compared to patterned structure. Patterned structure also revealed slightly lower cell viability compared to random nano-fibers. Finally, it was assumed that randomly oriented nano-fibers provide a more favorable surface for cells.     

Self-assembly and bioactivity of a polymer/peptide conjugate containing the RGD cell adhesion motif and PEG

The self-assembly and bioactivity of the peptide–polymer conjugate DGRFFF–PEG3000 containing the RGD cell adhesion motif has been examined, in aqueous solution. The conjugate is designed to be amphiphilic by incorporation of three hydrophobic phenylalanine residues as well as the RGD unit and a short poly(ethylene glycol) (PEG) chain of molar mass 3000 kg mol⁻¹. Above a critical aggregation concentration, determined by fluorescence measurements, signals of β-sheet structure are revealed by spectroscopic measurements, as well as X-ray diffraction. At high concentration, a self-assembled fibril nanostructure is revealed by electron microscopy. The fibrils are observed despite PEG crystallization which occurs on drying. This suggests that DGRFFF has an aggregation tendency that is sufficiently strong not to be prevented by PEG crystallization. The adhesion, viability and proliferation of human corneal fibroblasts was examined for films of the conjugate on tissue culture plates (TCPs) as well as low attachment plates. On TCP, DGRFFF–PEG3000 films prepared at sufficiently low concentration are viable, and cell proliferation is observed. However, on low attachment surfaces, neither cell adhesion nor proliferation was observed, indicating that the RGD motif was not available to enhance cell adhesion. This was ascribed to the core–shell architecture of the self-assembled fibrils with a peptide core surrounded by a PEG shell which hinders access to the RGD unit.     

Tumor targeting by surface-modified protein microspheres

Protein microspheres have been used in the fields of biomedical imaging and drug delivery, but surface modification for cell targeting has been problematic. We have for the first time used an electrostatic adhesion approach to adhere arginine-glutamic acid-aspartic acid (RGD) containing peptides to the surface of protein microspheres for the purpose of targeting these vesicles to tumor cells. RGD sequences are recognized by integrin membrane receptors, which are overexpressed in various tumors. We have succeeded in modifying the surface of serum albumin core-shell microspheres, which have a fluorescent nonaqueous core by using several polylysine peptides containing the RGD sequence. Fluorescence microscopy reveals that these modified microspheres are selectively bound and taken up by HT29 human colon cancer cells in vitro.     

Engineering of Covalently Immobilized Gradients of RGD Peptides on Hydrogel Scaffolds: Effect on Cell Behaviour

Summary: The aim of this study has been to design a system for the preparation of Polyethylene-glycol (PEG) based hydrogels with a controlled spatial distribution of covalently immobilised RGD adhesion signals in order to control and guide cell response for tissue engineering application. Gradients of immobilised RGD peptides were characterized by confocal microscopy analysis. Moreover, the effect of RGD spatial distribution on cell behaviour was evaluated by using mouse embryo fibroblasts NIH3T3. In particular, we observed cell adhesion and migration of fibroblasts seeded on RGD gradient compared to cells on control hydrogels having an uniform distribution of RGD. Our data suggest that a linear gradient of covalently immobilised adhesion signals affects cell behaviour. In particular, cells feel RGD gradient and oriented themselves and move along gradient direction.     

Block Copolymer Brushes for Completely Decoupled Control of Determinants of Cell–Surface Interactions

The known determinants for cell–surface interactions, comprising biochemical cues, patterns, passivating functionality, and control of tether mechanical properties, are fully decoupled in tailored block copolymer brushes synthesized by surface‐initiated atom transfer radical polymerization. Exploiting sequential polymerization of a passivating underlying polyacrylamide (PAAm) block with defined cross‐linking followed by a second poly(acrylic acid) block, which can be conjugated with a selective adhesion peptide, hierarchically structured brushes that can be micro‐patterned by soft lithography were obtained. The interaction of NIH 3T3 fibroblasts and PaTu 8988t pancreatic tumor cells with brushes that differed only in the stiffness of the hidden PAAm block or only in the peptide ligand, while keeping all other parameters constant, revealing profound differences in cell adhesion and morphology. In particular, cells could only attach well to stiff RGD presenting brushes.     

Gradient immobilization of a cell adhesion RGD peptide on thermal responsive surface for regulating cell adhesion and detachment

Using surface initiated atomic transfer radical polymerization (ATRP) and an injection method, a poly(N-isopropylacrylamide)-b-poly(acrylic acid)-g-RGD (PNIPAAm-b-PAA-g-RGD) gradient surface was prepared. First, a thermoresponsive surface with a constant thickness of PNIPAAm was fabricated, onto which the AA monomers were block copolymerized using the PNIPAAm macromolecules as initiators. During this process, a continuous injection method was employed to yield a molecular weight gradient of PAA on the underlying uniform PNIPAAm layer. RGD peptide was finally covalently immobilized onto the PAA gradient by carbodiimide chemistry. In vitro culture of HepG2 cells showed that immobilization of the RGD peptide could accelerate cell attachment, while the thermoresponsive layer beneath could effectively release the cells by simply lowering temperature. Thus, the PNIPAAm-b-PAA-g-RGD gradient surface, combining the thermal response with cell affinity properties, can well regulate the cell adhesion and detachment, which may thus be useful for investigation of cell-substrate interactions with a smaller number of samples.     

Issues of ligand accessibility and mobility in initial cell attachment

The influence of lateral ligand mobility on cell attachment and receptor clustering has previously been explored for membrane-anchored molecules involved in cell-cell adhesion. In this study, we considered instead a cell binding motif from the extracellular matrix. Even though the lateral mobility of extracellular matrix ligands in membranes does not occur in vivo, we believe it is of interest for cell engineering in vitro. As is the case for cell-cell adhesion molecules, lateral mobility of extracellular matrix ligands could influence cell attachment and, subsequently, cell behavior in cell culture. In this paper, the accessibility and functionality of extracellular matrix ligands presented at surfaces were evaluated for the conditions of laterally mobile versus non-mobile ligands by studying ligand-antibody binding events and early cell attachment as a function of ligand concentration. We compare the initial attachment of rat-derived adult hippocampal progenitor (AHP) cells on laterally mobile, supported phospholipid bilayer membranes to non-mobile, poly-L-lysine-grafted-poly(ethylene glycol) (PLL-g-PEG) polymer films functionalized with a range of laminin-derived IKVAV-containing peptide densities. To this end, synthesis of a new PLL-g-PEG/PEG-IKVAV polymer is described. The characterization of available IKVAV peptides on both surface presentations schemes was explored by studying the mass uptake of anti-IKVAV antibodies using a combination of the surface-sensitive techniques quartz crystal microbalance with dissipation monitoring, surface plasmon resonance spectroscopy, and optical waveguide lightmode spectroscopy. IKVAV-containing peptides presented on laterally mobile, supported phospholipid bilayers and non-mobile PLL-g-PEG were recognized by the anti-IKVAV antibody in a dose-dependent manner, indicating that the amount of available IKVAV ligands increases proportionally with ligand density over the concentrations tested. Attachment of AHP cells to IKVAV-functionalized PLL-g-PEG and supported phospholipid bilayers followed a sigmoidal dependence on peptide concentration, with a critical concentration of approximately 3 pmol/cm2 IKVAV ligands required to support initial AHP cell attachment for both surface modifications. There appeared to be little influence of IKVAV peptide mobility on the initial attachment of AHP cells. Although the spread in the cell attachment data was larger for the PLL-g-PEG surface modification, this was reduced when observed after 24 h, indicating that the cells might need longer times to establish attachment strengths equivalent to those observed on peptide-functionalized supported lipid bilayers. The present study is a step toward understanding the influence of extracellular-matrix-derived ligand mobility on cell fate. Further analysis should focus on the systematic tuning of lateral ligand diffusion, as well as a comparison between the response of non-spreading cells (i.e., AHPs), versus spreading cells (i.e., fibroblasts).     

RGD改性聚醚氨酯及其内皮细胞相容性的研究

利用氢键稳定的溶液互穿技术对聚醚氨酯(PEU)进行改性.用ATR-FTIR对十八烷基-聚氧乙烯-4,4'-二苯甲烷二异氰酸酯-聚氧乙烯-十八烷基(MSPEO)与PEU共混膜表面进行研究,结果表明,MS-PEO中的氨基甲酸酯链段与PEU基材之间发生了氢键缔合的作用.通过水化处理PEO及十八烷基自发地富集在基材表面.根据氢键缔合和表面自迁移原理,设计了两种RGD改性聚醚氨酯的方法:(1)将含RGD端基的聚氧乙烯-4,4'-二苯甲烷二异氰酸酯-聚氧乙烯偶联物(MPEO-RGD)与PEU进行共混改性,利用RGD端基及PEO的自迁移特性获得RGD富集的表面;(2)将含甲磺酸酯端基的聚氧乙烯-4,4'-二苯甲烷二异氰酸酯-聚氧乙烯偶联物(MPEO-mesyl)与PEU共混成膜,并对膜片进行水化处理,使甲磺酸酯端基富集在PEU表面,浸泡于RGD的PBS溶液中,在膜片表面成功地原位接枝了RGD.对两种RGD改性方法获得的表面进行了内皮细胞的培养,结果表明,两种改性方法均大大提高了PEU的细胞相容性,其中方法(1)共混改性的表面细胞相容性略优于方法(2)的接枝改性表面.     

Engineering Photocleavable Protein-decorated Hydrogels to Regulate Cell Adhesion and Migration

Cell adhesion and migration play essential roles in tissue development and maintenance, and abnormal cell migration is involved in life-threatening diseases, including vascular disease, tumor formation, and metastasis. The advances in hydrogel-based 3D cell culture development facilitated the investigation of cell motility behavior, including cell-cell and cell-matrix adhesion and cell migration in a microenvironment more related to in vivo situations. Establishing advanced methods for these in vitro studies is thus necessary. Photo-sensitive proteins show advantages in remote and non-invasive regulation of hydrogels' properties, and thus are of great potential in regulating 3D cultured cells' behavior. In the presented study, we engineered photocleavable protein(PhoCl)-decorated hydrogels to regulate cell adhesion and migration of MDA-MB-231. The integrin-binding motif RGD was fused to the PhoCl and was decorated on the hydrogel. After being exposed to light at 405 nm, the PhoCl was cleaved and the RGD motif was released, resulting in detachment of the binding cells. The regulatory effect of the light illumination showed a time-dependent and cell density-dependent manner. Furthermore, the elimination of RGD by patterned light exposure completely suspended the cell migration to the corresponding region, suggesting a controllable regulation of the cell migration direction.     

Cellular Adhesion Tripeptide RGD Inhibits Growth of Human Ileocecal Adenocarcinoma Cells HCT-8 and Induces Apoptosis

The tripeptide,Arg-Gly-Asp(RGD)motif is an integrin-recognition site found in adhesive proteins present in extracellular matrices(ECM)and in the blood.HCT-8 cells were treated with cellular adhesion tripeptide RGD at various concentrations.MTT assay was performed to examine the growth and proliferation of HCT-8 cells after treatment with RGD for 48 h.Haematoxylin and Eosin(HE)staining and electromicroscope were used to observe the morphology of apoptotic cells.Survivin and flow cytometry were also used to analyze the HCT-8 apoptosis.Cellular adhesion tripeptide RGD significantly inhibits the growth and proliferation of HCT-8 cells in a dose-dependent manner and induces apoptosis of HCT-8.These results indicate that cellular adhesion tripeptide RGD inhibits the growth and proli-feration of tumor HCT-8 cell,probably by the aid of inducing apoptosis of HCT-8 cell.     

Protein adsorption and cell adhesion on RGD-functionalized silicon substrate surfaces

A method was developed to modify silicon surfaces with good protein resistance and specific cell attachment. A silicon surface was initially deposited using a block copolymer of N-vinylpyrrolidone (NVP) and 2-hydroxyethyl methacrylate (HEMA) (PVP-b-PHEMA) film through surface-initiated atom transfer radical polymerization and then further immobilized using a short arginine-glycine-aspartate (RGD) peptide. Our results demonstrate that the RGD-modified surfaces (Si-RGD) can suppress non-specific adsorption of proteins and induce the adhesion of L929 cells. The Si-RGD surface exhibited higher cell proliferation rates than the unmodified silicon surface. This research established a simple method for the fabrication of dual-functional silicon surface that combines antifouling and cell attachment promotion.     

Radio‐Analytical Determination of the Coating Efficiency of Cyclic RGD Peptides

Coating of artificial surfaces with RGD (= arginine‐glycine‐aspartate) peptides to enhance cell adhesion is an ongoing issue. Thereby, the physiological adhesion process to the extra‐cellular matrix (ECM) is mimicked by the peptide coating, leading to a strong cell‐surface contact, followed by spreading and proliferation of the cells. For comparable cell adhesion studies, it is important to know the density of the RGD peptides on the surface. Here, we present an approach to determine the amount of bound cyclic RGD peptide by radio labeling with ¹²⁵I of a tyrosine‐containing RGD peptide on different materials surfaces (poly(methyl methacrylate) (PMMA), titanium, and silicon). For all surfaces, the amount of bound peptides is in the range of pmol/cm¹.     

Cellular responses to patterned poly(acrylic acid) brushes

We use patterned poly(acrylic acid) (PAA) polymer brushes to explore the effects of surface chemistry and topography on cell-surface interactions. Most past studies of surface topography effects on cell adhesion have focused on patterned feature sizes that are larger than the dimensions of a cell, and PAA brushes have been characterized as cell repellent. Here we report cell adhesion studies for RBL mast cells incubated on PAA brush surfaces patterned with a variety of different feature sizes. We find that when patterned at subcellular dimensions on silicon surfaces, PAA brushes that are 30 or 15 nm thick facilitate cell adhesion. This appears to be mediated by fibronectin, which is secreted by the cells, adsorbing to the brushes and then engaging cell-surface integrins. The result is detectable accumulation of plasma membrane within the brushes, and this involves cytoskeletal remodeling at the cell-surface interface. By decreasing brush thickness, we find that PAA can be 'tuned' to promote cell adhesion with down-modulated membrane accumulation. We exemplify the utility of patterned PAA brush arrays for spatially controlling the activation of cells by modifying brushes with ligands that specifically engage IgE bound to high-affinity receptors on mast cells.     

The accumulation of phytoalexin in cucumber plant after stress

During the course of pathogens penetrating the plant cell, besides of chemical secretion, the pathogens may cause mechanical signal by the physical pressure on the plant cell. In the current study, we use the pressure as the stress signal to study the induction in plant resistance and the effect of accumulation of phytoalexin. We found that stress can induce the resistance in cucumber seeding significantly. Peptides contained RGD motif can specific block the adhesion between plant cell wall and plasma membrane. When breaking the plant cell wall and plasma membrane by using RGD peptides, the stress induction effect is almost absolutely eliminated. The results of assay with TLC and HPLC showed that stress stimulation could increase the accumulation of cucumber seeding phytoalexin. So, we can conclude that the accumulation of phytoalexin is one possible reason of improve the stress induced resistance. When block the adhesion between plant cell wall and plasma membrane by RGD, there are only part of accumulation of phytoalexin. The results suggest that stress induced resistance and accumulation of phytoalexin of plant is required for the adhesion of plant cell wall–plasma membrane.     

Physico-chemical and thermodynamic aspects of fibroblastic attachment on RGDS-modified chitosan membranes

In the present study, the cell attachment/spreading behaviour of L929 mouse fibroblasts on chitosan membranes was evaluated by using physico-chemical properties. For this purpose chitosan membranes were prepared and then photochemically modified with the cell adhesive peptide RGDS (Arg-Gly-Asp-Ser). The physico-chemical properties of unmodified (CHI) and RGDS-modified chitosan (CHI-RGDS) membranes were evaluated by calculating surface free energy (γ_sv) and interfacial free energy (γ_sw) values using captive bubble contact angle measurements and harmonic mean equation. The cell attachment experiments were performed both in 10% FBS containing and serum-free media with CHI and CHI-RGDS membranes. Eventually, it was not possible to predict a direct relationship between the change in physico-chemical properties and L929 cell attachment behaviour. The experimental results obtained from cell attachment agree with the theoretical prediction for the free energy of adhesion except for the cell attachment on CHI membrane in serum-free medium. Although a negative interfacial free energy of adhesion was calculated for CHI membrane in serum-free medium (ΔF_adh = −2.19 ergs/cm²), the cell attachment was poor (70%) compared to CHI-RGDS (90%) and none of the cells were spread on CHI surface to gain a fibroblastic morphology. Negative energy of adhesion was calculated for CHI and CHI-RGDS in 10% FBS medium, in which 100% of cells were attached on the membranes correlating with the thermodynamic approach. It can be suggested that, adsorption of serum proteins strongly affected the cell attachment meanwhile the presence of biosignal RGDS molecules triggered the cell spreading in serum medium.