Thermo-responsive gelatin-functionalized PCL film surfaces for improvement of cell adhesion and intelligent recovery of gene-transfected cells

Efficient local gene transfection on a tissue scaffold is dependent on good cell-adhesion characteristics. In this work, the thermo-responsive gelatin-functionalized polycaprolactone (PCL) films were proposed for improvement of cell adhesion and intelligent recovery of gene-transfected cells. Functional copolymer brushes (PCL-g-P(NIPAAm-co-MAAS)) were first prepared via surface-initiated ATRP of N-isopropylacrylamide (NIPAAm) and methacrylic acid sodium salt (MAAS) from the initiator-funcationalized PCL surfaces. The pendant carboxyl end-groups of the PCL-g-P(NIPAAm-co-MAAS) surface were subsequently coupled with gelatin via carbodiimide chemistry to produce the thermo-responsive gelatin-functionalized PCL surface. The thermo-responsive gelatin-functionalized PCL film surface can improve cell adhesion and proliferation above the LCST of P(NIPAAm) without destroying cell detachment properties at lower temperatures. The dense transfected cells can be recovered simply by lowering culture temperature. The thermo-responsive gelatin-functionalized PCL films are potentially useful as intelligent adhesion modifiers for directing cellular functions within tissue scaffolds.     

Temperature-responsive stationary phase utilizing a polymer of proline derivative for hydrophobic interaction chromatography using an aqueous mobile phase

A new method of chromatography is proposed, utilizing a thermo-responsive polymer carrying an amino acid ester residue for the stationary phase of high-performance liquid chromatography (HPLC). We have been investigating the new concept of chromatography, a temperature-responsive chromatography, using temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm)-modified surface for HPLC with a constant aqueous media as the mobile phase. In this study, we designed and synthesized thermo-responsive poly(acryloyl-L-proline methyl ester) and its copolymer with N-isopropylacrylamide (NIPAAm). Homopolymers of acryloyl-L-proline methyl ester and copolymer were prepared by the reaction of radical telomerization. These polymers underwent a reversible phase transition from water-soluble forms into aggregates by changing the temperature, similar to PNIPAAm. The surface properties and functions of stationary phases modified with poly(acryloyl-L-proline methyl ester) were controlled by the external temperature. In the chromatographic system, we separated steroids and amino acids with a variety of hydrophobicities using a sole aqueous mobile phase. In contrast to a PNIPAAm-modified surface, a poly(acryloyl-L-proline methyl ester)-modified surface showed a greater affinity for hydrophobic amino acids.     

Potential‐Responsive Surfaces for Manipulation of Cell Adhesion,Release, and Differentiation

In living systems, interfacial molecular interactions control many biological processes. New stimuli‐responsive strategies are desired to provide versatile model systems that can regulate cell behavior in vitro. Described here are potential‐responsive surfaces that control cell adhesion and release as well as stem cell differentiation. Cell adhesion can be modulated dynamically by applying negative and positive potentials to surfaces functionalized with tailored monolayers. This process alters cell morphology and ultimately controls behavior and the fate of the cells. Cells can be detached from the electrode surface as intact clusters with different geometries using electrochemical potentials. Importantly, morphological changes during adhesion guide stem cell differentiation. The higher accessibility of the peptide under a positive applied potential causes phenotypic changes in the cells that are hallmarks of osteogenesis, whereas lower accessibility of the peptide promoted by negative potentials leads to adipogenesis.     

Potential‐Responsive Surfaces for Manipulation of Cell Adhesion,Release, and Differentiation

In living systems, interfacial molecular interactions control many biological processes. New stimuli‐responsive strategies are desired to provide versatile model systems that can regulate cell behavior in vitro. Described here are potential‐responsive surfaces that control cell adhesion and release as well as stem cell differentiation. Cell adhesion can be modulated dynamically by applying negative and positive potentials to surfaces functionalized with tailored monolayers. This process alters cell morphology and ultimately controls behavior and the fate of the cells. Cells can be detached from the electrode surface as intact clusters with different geometries using electrochemical potentials. Importantly, morphological changes during adhesion guide stem cell differentiation. The higher accessibility of the peptide under a positive applied potential causes phenotypic changes in the cells that are hallmarks of osteogenesis, whereas lower accessibility of the peptide promoted by negative potentials leads to adipogenesis.     

Thermo-responsive polymer brush-grafted porous polystyrene beads for all-aqueous chromatography

Poly(N-isopropylacrylamide) (PIPAAm) brush-grafted porous polystyrene beads with variable grafted polymer densities were prepared using surface-initiated atom transfer radical polymerization (ATRP) for applications in thermo-responsive chromatography. Utilization of these grafted beads as a stationary phase in aqueous chromatographic analysis of insulin provides a graft density-dependent analyte retention behavior. The separations calibration curve on PIPAAm-grafted polystyrene was obtained using pullulan standards and exhibited inflection points attributed to analyte diffusion into bead pores and partitioning into grafted PIPAAm brush surfaces. Presence of these inflection points supports a separation mechanism where insulin penetrates pores in polystyrene beads and hydrophobically interacts with PIPAAm brushes grafted within the pores. Control of PIPAAm brush graft density on polystyrene facilitates effective aqueous phase separation of peptides based on thermally modulated hydrophobic interactions with grafted PIPAAm within stationary phase pores. These results indicated that PIPAAm brush-grafted porous polystyrene beads prepared by surface-initiated ATRP was effective stationary phase of thermo-responsive chromatography for aqueous phase peptide separations.     

Mussel adhesive protein mimetic polymers for the preparation of nonfouling surfaces

A new biomimetic strategy for modification of biomaterial surfaces with poly(ethylene glycol) (PEG) was developed. The strategy exploits the adhesive characteristics of 3,4-dihydroxyphenylalanine (DOPA), an important component of mussel adhesive proteins, to anchor PEG onto surfaces, rendering the surfaces resistant to cell attachment. Linear monomethoxy-terminated PEGs were conjugated either to a single DOPA residue (mPEG-DOPA) or to the N-terminus of Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (mPEG-MAPD), a decapeptide analogue of a protein found in Mytilus edulis adhesive plaques. Gold and titanium surfaces were modified by adsorption of mPEG-DOPA and mPEG-MAPD from solution, after which surface analysis by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectroscopy confirmed the presence of immobilized PEG on the surface. The ability of modified surfaces to resist cell attachment was examined by culturing 3T3 fibroblasts on the surfaces for up to 14 days. Quantitative image analysis revealed that cell adhesion to mPEG-DOPA and mPEG-MAPD modified surfaces decreased by as much as 98% compared to control surfaces. Modified Ti surfaces exhibited low cell adhesion for up to 2 weeks in culture, indicating that the nonfouling properties of mPEG-DOPA and mPEG-MAPD treated surfaces persist for extended periods of time. This strategy paradoxically exploits the strong fouling characteristics of MAP analogues for antifouling purposes and may be broadly applied to medical implants and diagnostics, as well as numerous nonmedical applications in which the minimization of surface fouling is desired.     

Tailoring the trajectory of cell rolling with cytotactic surfaces

Cell separation technology is a key tool for biological studies and medical diagnostics that relies primarily on chemical labeling to identify particular phenotypes. An emergent method of sorting cells based on differential rolling on chemically patterned substrates holds potential benefits over existing technologies, but the underlying mechanisms being exploited are not well characterized. In order to better understand cell rolling on complex surfaces, a microfluidic device with chemically patterned stripes of the cell adhesion molecule P-selectin was designed. The behavior of HL-60 cells rolling under flow was analyzed using a high-resolution visual tracking system. This behavior was then correlated to a number of established predictive models. The combination of computational modeling and widely available fabrication techniques described herein represents a crucial step toward the successful development of continuous, label-free methods of cell separation based on rolling adhesion.     

Particle-collision and porogen-leaching technique to fabricate polymeric porous scaffolds with microscale roughness of interior surfaces

A facile technique is herein reported to fabricate three-dimensional(3D) polymeric porous scaffolds with interior surfaces of a topographic microstructure favorable for cell adhesion.As demonstration,a well-known biodegradable polymer poly(lactide-co-glycolide)(PLGA) was employed as matrix.Under the porogen-leaching strategy,the large and soft porogens of paraffin were modified by colliding with small and hard salt particles,which generated micropits on the surfaces of paraffin spheres.The eventual PLGA scaffolds after leaching the modified porogens had thus interior surfaces of microscale roughness imprinted by those micropits.The microrough scaffolds were confirmed to benefit adhesion of bone marrow stromal cells(BMSCs) of rats and meanwhile not to hamper the proliferation and osteogenic differentiation of the cells.The insight and technique might be helpful for biomaterial designing in tissue engineering and regenerative medicine.     

Micropatterning gradients and controlling surface densities of photoactivatable biomolecules on self-assembled monolayers of oligo(ethylene glycol) alkanethiolates

Background: Bioactive molecules that are covalently immobilized in patterns on surfaces have previously been used to control or study cell behavior such as adhesion, spreading, movement or differentiation. Photoimmobilization techniques can be used, however, to control not only the spatial pattern of molecular immobilization, termed the micropattern, but also the surface density of the molecules — a characteristic that has not been previously exploited.Results: Oligopeptides containing the bioactive Arg-Gly-Asp cell-adhesion sequence were immobilized upon self-assembled monolayers of an oligo(ethylene glycol) alkanethiolate in patterns that were visualized and quantified by autoradiography. The amount and pattern of immobilized peptide were controlled by manipulating the exposure of the sample to a LIV lamp or a laser beam. Patterns of peptides, including a density gradient, were used to control the location and number of adherent cells and also the cell shape.Conclusions: A photo immobilization technique for decorating surfaces with micropatterns that consist of variable densities of bioactive molecules is described. The efficacy of the patterns for controlling cell adhesion and shape has been demonstrated. This technique is useful for the study of cell behavior on micropatterns.     

Photopatterned polymer brushes promoting cell adhesion gradients

The ability to spatially control cellular adhesion in a continuous manner on a biocompatible substrate is an important factor in designing new biomaterials for use in wound healing and tissue engineering applications. In this work, a novel method of engineering cell-adhesive RGD-ligand density gradients to control specific cell adhesion across a substrate is presented. Polymer brushes exhibiting spatially defined gradients in chain density are created and subsequently functionalized with RGD to create ligand density gradients capable of inducing cell adhesion on an otherwise weakly adhesive substrate. Cell studies indicate that these ligand-functionalized surfaces are noncytotoxic, with cellular adhesion increasing with RGD-ligand density across the gradient brush surface.     

Thermo-responsive stick-slip behavior of advancing water contact angle on the surfaces of poly(N-isopropylacrylamide)-grafted polypropylene membranes

Wettability of a solid surface is highly important to its practical application,especially for the surface that shows thermoresponsive properties.In this paper,we describe a thermo-responsive stick-slip behavior of water droplets on the surfaces of poly(N-isopropylacrylamide)(PNIPAM)-grafted polypropylene membranes.Field emission scanning electron microscope(FESEM) images elucidate that the morphology of PNIPAM-grafted membrane surface is thermo-responsive,i.e.,the surface becomes rougher above the lower cr...     

Cell adhesion and surface properties of polystyrene surfaces grafted with poly(N-isopropylacrylamide)

Cell adhesion plays a key role in various aspects of biological and medical sciences. In this study, poly(Nisopropylacrylamide) (PNIPAM) was grafted on polystyrene surfaces using different solvents under UV radiation. Moreover, the relation between surface roughness and cell adhesion were evaluated by gravimetric, SEM, AFM, contact angle measurement and cellular analyses. The gravimetric analysis clearly indicated an increase in the grafting by adding 10% methanol to water. The study of surface topography by AFM images showed an increase in the surface roughness and as a result of which, a decrease in wettablity was observed. At 37 °C, epithelial cells were well attached and proliferated on the grafted surfaces, while these cells were spontaneously detached below 32 °C in the absence of any enzymes. Moreover, MTT assay and SEM images indicated good cell viability and an increase in cell adhesion caused by the roughness increase. The results of this study reveal the great potential of PNIPAM-grafted polystyrene for being used in the biomedical fields such as drug delivery systems, tissue engineering and cell separation.     

Control of cell detachment in a microfluidic device using a thermo-responsive copolymer on a gold substrate

The control of cell adhesion is crucial in many procedures in cellular biotechnology. A thermo-responsive poly(N-isopropylacrylamide)-poly(ethylene glycol)-thiol (PNIPAAm-PEG-thiol) copolymer was synthesized for the formation of self-assembled monolayers (SAM) that allow the control of adhesion of cells on gold substrates. The contact angle of water on these layers varies between 65 degrees at a temperature of 45 degrees C and 54 degrees at 25 degrees C. This behaviour is consistent with a transition of the polymer chains from an extended and highly hydrated to a collapsed coil-like state. At 37 degrees C, cultivated fibroblasts adhere and spread normally on this surface and detach by reducing the temperature below the lower critical solution temperature (LCST). Layers can repeatedly be used without loss of their functionality. In order to quantify the capability of the copolymer layer to induce cell detachment, defined shear forces are applied to the cells. For this purpose, the laminar flow in a microfluidic device is used. Our approach provides a strategy for the optimization of layer properties that is based on establishing a correlation between a functional parameter and molecular details of the layers.     

Poly(vinylpyrrolidone-b-styrene) block copolymers tethered surfaces for protein adsorption and cell adhesion regulation

Poly(vinylpyrrolidone-b-styrene) (PVP-b-PS) diblock copolymers tethered to glass surfaces were prepared, and the effects on protein adsorption and cellular behavior to the glass and the modified glass surfaces investigated. The PVP-b-PS grafting process was confirmed by water contact angle and XPS measurements. The results obtained for the water contact angles suggest that there are two phases that coexist on the PVP-b-PS block copolymer tethered surface, under aqueous conditions. Although the PVP-b-PS surface possessed, to some extent, a protein resistant property, following introduction of the PS segment to the end of tethered PVP, both fibrinogen and lysozyme adsorption were increased significantly. The PVP-b-PS modified surface, based on Western-blot analysis, appeared to have the greatest amount of surface bound vitronectin, however the conformation of the adsorbed vitronectin may have subsequently been affected by the surface tethered copolymer as was suggested by cell culture results. From these results, we proposed that protein adsorption and cell adhesion can be regulated by tuning the chemical compositions of diblock copolymers tethered to surfaces.     

Electrochemical evidence for asialoglycoprotein receptor – mediated hepatocyte adhesion and proliferation in three dimensional tissue engineering scaffolds

Asialoglycoprotein receptor (ASGPR) is one of the recognition motifs on the surface of hepatocytes, which promote their adhesion to extracellular matrix in liver tissue and appropriate artificial surfaces. ASGPR-mediated adhesion is expected to minimize trans-differentiation of hepatocytes in vitro that is generally observed in integrin-mediated adhesion. The aim of the present study is to verify the role of ASGPR in hepatocyte adhesion and proliferation in scaffolds for hepatic tissue engineering. Scanning Electrochemical Microscopy (SECM) is emerging as a suitable non-invasive analytical tool due to its high sensitivity and capability to correlate the morphology and activity of live cells. HepG2 cells and rat primary hepatocytes cultured in Polyvinyl alcohol (PVA)/Gelatin hydrogel scaffolds with and without galactose (a ligand for ASGPR) modification are studied using SECM. Systematic investigation of live cells cultured for different durations in scaffolds of different compositions (9:1 and 8:2 PVA:Gelatin with and without galactose) reveals significant improvement in cell–cell communication and proliferation on galactose incorporated scaffolds, thereby demonstrating the positive influence of ASGPR-mediated adhesion. In this work, we have also developed a methodology to quantify the respiratory activity and intracellular redox activity of live cells cultured in porous tissue engineering scaffolds. Using this methodology, SECM results are compared with routine cell culture assays viz., MTS ((1-Oxyl-2,2,5,5,-tetramethyl-Δ3-pyrroline-3-methyl) Methanethiosulfonate) and Albumin assays to demonstrate the better sensitivity of SECM. In addition, the present study demonstrates SECM as a reliable and sensitive tool to monitor the activity of live cells cultured in scaffolds for tissue engineering, which could be used on a routine basis.     

Effects of an avidin-biotin binding system on chondrocyte adhesion and growth on biodegradable polymers

Cell adhesion to a scaffold is a prerequisite for tissue engineering. Many studies have been focused on enhancing cell adhesion to synthetic materials that are used for scaffold fabrication. In this study, we applied an avidin-biotin binding system to enhance chondrocyte adhesion to biodegradable polymers. Biotin molecules were conjugated to the cell membrane of chondrocytes, and mediated cell adhesion to avidin-coated surfaces. We demonstrated that immobilization of biotin molecules to chondrocyte surfaces enhanced cell adhesion to avidin-coated biodegradable polymers such as poly(L-lactic acid), poly(D,L-lactic acid), and polycaprolactone, compared to the adhesion of normal chondrocytes to the same type of biodegradable polymer. The biotinylated chondrocytes still maintained their proliferation ability. This study showed the promise of applying the avidin-biotin system in cartilage tissue engineering. [diagram in text].     

Photocontrol of cell adhesion on amino-bearing surfaces by reversible conjugation of poly(ethylene glycol) via a photocleavable linker

Dynamic control of cell adhesion on substrates is a useful technology in tissue engineering and basic biology. This paper describes a method for the control of cell adhesion on amino-bearing surfaces by reversible conjugation of an anti-fouling polymer, poly(ethylene glycol) (PEG), via a newly developed photocleavable linker, 1-(5-methoxy-2-nitro-4-prop-2-ynyloxyphenyl)ethyl N-succinimidyl carbonate (1). This molecule has alkyne and succinimidyl carbonate at each end, which are connected by photocleavable 2-nitrobenzyl ester. Under this molecular design, the molecule crosslinked azides and amines, whose linkage cleaved upon application of near-UV light. By using aminosilanised glass and silicon as model substrates, we studied their reversible surface modification with PEG-azide (M(w) = 5000) based on contact angle measurements, ellipsometry, and AFM morphological observations. Protein adsorption and cell adhesion dramatically changed by PEGylation and the following irradiation, which can be used for cellular patterning. Also, the capability of the substrate to change cell adhesiveness by photoirradiation during cell cultivation was demonstrated by inducing cell migration. We believe this method will be useful for dynamic patterning of cells on protein-based scaffolds.     

Adhesion of nonplanar wrinkled surfaces

Topological patterns on polymer surfaces can significantly alter and control adhesion. In this study, the effect of surface wrinkles on a spherical surface on adhesion has been studied. Surface wrinkling induced by swelling of a crosslinked polydimethylsiloxane elastomer constrained by a stiff, thin surface layer (silicate) is used to produce topographic features of various length scales over a large curved area. By controlling the properties of the stiff layer and the applied strain conditions, surface wrinkles of varying amplitude and wavelength are obtained. The effect of wrinkle morphology on adhesion is quantified, and the results display a transition from enhancement of adhesion to decrease depending upon wrinkle dimensions. A simple phenomenological model is proposed that describes the change of adhesion behavior as a function of wrinkle morphology. Our results provide a critical understanding toward tuning the adhesion behavior of nonplanar surfaces consisting of periodic topographic structures. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2011     

Adsorption and conformation behavior of biotinylated fibronectin on streptavidin-modified TiO(X) surfaces studied by SPR and AFM

It is well-known that protein-modified implant surfaces such as TiO(2) show a higher bioconductivity. Fibronectin is a glycoprotein from the extracellular matrix (ECM) with a major role in cell adhesion. It can be applied on titanium oxide surfaces to accelerate implant integration. Not only the surface concentration but also the presentation of the protein plays an important role for the cellular response. We were able to show that TiO(X) surfaces modified with biotinylated fibronectin adsorbed on a streptavidin-silane self-assembly multilayer system are more effective regarding osteoblast adhesion than surfaces modified with nonspecifically bound fibronectin. The adsorption and conformation behavior of biotinylated and nonbiotinylated (native) fibronectin was studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Imaging of the protein modification revealed that fibronectin adopts different conformations on nonmodified compared to streptavidin-modified TiO(X) surfaces. This conformational change of biotinylated fibronectin on the streptavidin monolayer delivers a fibronectin structure similar to the conformation inside the ECM and therefore explains the higher cell affinity for these surfaces.     

Study of temperature-responsibility on the surfaces of a thermo-responsive polymer modified stationary phase

We investigated a thermo-sensitive polymer, poly(N-isopropylacrylamide) (PNIPAAm), which is the basis of an HPLC stationary phase. We prepared a PNIPAAm terminally-modified surface. In this study, we investigated the effect of PNIPAAm on the surface of a stationary phase on separation based on changes of the retention time with the temperature step gradient. As the temperature changed the surface property of the stationary phase switched from hydrophilic to hydrophobic. The retention on the polymer-modified stationary phase remarkably changed upon changing the temperature. Using a column packed with PNIPAAm-modified silica, the separation of steroids was carried out by changing the temperature. With increasing temperature, an increased interaction between solutes and PNIPAAm-grafted surfaces of the stationary phases was observed. A temperature-dependent resolution of steroids was achieved using only water as a mobile phase. The PNIPAAm-modified surface of the stationary phase exhibited temperature-controlled hydrophilic-hydrophobic changes. The drastic and reversible surface hydrophilic-hydrophobic property alteration for PNIPAAm terminally-grafted surfaces should be due to rapid changes in the polymer hydration state around the polymer's transition temperature. A solvent gradient elution-like effect could be achieved with a single mobile phase by programmed temperature changes during chromatographic runs. This system should be highly useful to control the function and property of the stationary phase for HPLC only by changing the temperature with an aqueous solvent.