Polymer microarrays: identification of substrates for phagocytosis assays

A polymer microarray of 120 polyurethanes was used to identify polymers that promoted the adhesion of bone marrow dendritic cells (BMDC). Identified polymers were coated onto glass cover slips and shown to be efficient substrates for the immobilisation of these primary cells, which underwent efficient phagocytosis while still presumably maintaining their immature state.     

Differential regulation of endothelial cell adhesion, spreading, and cytoskeleton on low-density polyethylene by nanotopography and surface chemistry modification induced by argon plasma treatment

Plasma treatment of polymer surfaces can modify the nanoscale roughness, wettability, and oxygen surface functionalities. However, how these modifications regulate cell behavior is not well understood. The objective of this investigation was to examine adhesion, spreading, and cytoskeleton of vascular endothelial cells seeded on low-density polyethylene surfaces modified by Ar plasma. In the absence of serum, adhesion and spreading of the cells and actin filament assembly were enhanced by high-energy Ar plasma-induced hydrophilicity and formation of C-O groups at the surface. Although serum increased cell adhesion and spreading on untreated surfaces for a relatively short period, this behavior was not stable for a long time. In contrast to the untreated polymer surfaces, serum suppressed cell adhesion and spreading on the plasma-treated surfaces. The preadsorption of albumin from the bovine serum on the polymer surfaces inhibited cell adhesion and spreading. Results demonstrate the differential effects of Ar plasma-induced surface modifications on endothelial cell behavior and provide insight into complex interactions among polymer surfaces, adsorbed proteins, and cells. The findings of this study have significant implications in surface engineering for vascular repair.     

The influence of protein adsorption on interactions of cultured human endothelial cells with polymers

A systematic study of the effects of polymer surface properties on the interaction with human endothelial cells (HEC) may lead to the development of small-diameter vascular grafts. HEC, suspended in culture medium containing 20% serum adhered and spread onto moderately wettable polymers such as TCPS (tissue culture polystyrene). Reduced or no adhesion of HEC was observed upon the hydrophobic polymers PETP (polyethyleneterephthalate, Dacron) and FEP (fluoroethylenepropylene copolymer, Teflon). Polymers precoated with the proteins albumin (Alb), high density lipoprotein (HDL), and immunoglobulin G (IgG) inhibited the adhesion of HEC, whereas fibronectin (Fn) coatings promoted cell adhesion. Endothelialization of PETP and FEP only occurred after precoating of these materials with Fn. The adsorption of Fn, Alb, HDL, and IgG from solutions of different serum concentrations onto TCPS, PETP, and FEP was related to the adhesion of HEC. Serum Fn only adsorbed onto TCPS, with the maximum at 0.1% serum concentration. Maximal cell adhesion onto TCPS was also observed after pretreatment with a solution containing 0.1% serum. The cell adhesion inhibiting proteins Alb and HDL preferentially adsorbed at higher serum concentrations. Desorption of these proteins and exchange for, e.g., cellular Fn may result in cell spreading and proliferation of HEC upon TCPS.     

Preparation of a protein micro-array using a photo-reactive polymer for a cell-adhesion assay

A protein micro-array, called a "cell chip" was constructed by using a photo-reactive polymer for a cell-adhesion assay. Various amounts of albumin or fibronectin were covalently immobilized on a polystyrene dish using a micro-spotter with a dip pen. First, poly(acrylic acid) carrying azidophenyl groups was synthesized as the photo-reactive polymer. Secondly, the aqueous solution of a photo-reactive polymer (several nanoliters) was cast using the dip pen of the micro-spotter and dried in air. Subsequently, aqueous solutions of proteins were cast on the same place using the micro-spotter. After drying, the dish was irradiated with ultraviolet light. Finally, the immobilization was confirmed by staining with a dye. The immobilization was stable even after washing with Tween-20. The protein-immobilized area depended on the manipulation of the micro-spotter and the size of the dip pen. Subsequently, cell adhesion on the photo-immobilized protein micro-array was investigated. The adhesion behavior of cells depended on the kind of immobilized proteins and the kind of cells. The protein micro-array will be useful for cell diagnosis and for the selection of biomaterials to regulate cell behavior.     

Oriented Schwann cell growth on micropatterned biodegradable polymer substrates

This paper investigates the influence of substrate-mediated chemical and physical guidance on the growth and alignment of Schwann cells in vitro. Novel techniques were developed to fabricate microgrooves with adsorbed proteins on biodegradable polymer substrates made of poly(D,L-lactic acid). Compression molding and solvent-casting were used to transfer micropatterns from quartz and silicon substrates onto biodegradable polymer films. Laminin was selectively adsorbed onto the grooves and rat sciatic Schwann cells were seeded on the substrates. Laminin was found to improve adhesion of Schwann cells on the substrates. The microgrooves were found to cause the Schwann cells to align along the direction of the grooves. The groove width influenced Schwann cell alignment the most, while groove depth did not seem to play a significant role. The degradation of the grooves in the solvent cast films was much slower than those in the compression-molded films, making them the preferred substrates for Schwann cell culture.     

Temperature-responsive glass coverslips with an ultrathin poly(N-isopropylacrylamide) layer

A temperature-responsive cross-linked polymer gel was covalently grafted onto glass coverslips by electron beam irradiation. The grafted thickness and amount of polymer as well as the surface wettability increased with the initial monomer concentration. When the monomer concentration was 5 wt.%, the grafted polymer density was 0.84microgcm(-2), and cells adhered and spread on the surface at 37 degrees C, but detached at 20 degrees C. In contrast, when the monomer concentration was 35 wt.%, the polymer density was 1.28microgcm(-2), and the surfaces were cell repellent even at 37 degrees C. These results show a remarkable contrast to those obtained from temperature-responsive polymer-grafted tissue culture polystyrene dishes, since various types of cells showed temperature-dependent cell adhesion/detachment when the grafted density was around 2microgcm(-2) on these surfaces. We discuss the possible molecular mechanisms underlying this discrepancy.     

Fibronectin facilitates adhesion of K562 leukemic cells normally growing in suspension to cationic surfaces.

The role of protein adsorption in the forced adhesive growth of K562 leukemic cells onto a cationic surface composed of polylysine was investigated. Numerous studies have demonstrated that adhesion in anchorage-dependent cells is mediated in vitro by adsorption of serum proteins [particularly proteins of the extracellular matrix (ECM) such as fibronectin and vitronectin] present in the growth medium. Specifically, adhesion has been shown to occur when ECM proteins attach to the substratum and act as ligands for specific receptors located on the surface of cells. K562 cells are human erythroleukemic cells that normally grow in suspension. These cells are not involved in the same cell adhesion processes as anchorage-dependent cells and do not need to be attached to ECM proteins in order to survive and grow. Thus, with these systems, it is possible to better determine the role of protein adsorption in the adhesion of cells, growing in suspension such as blood cells, onto charged surfaces. The results presented show that adhesion of K562 cells onto the positively charged polylysine surface in the presence of serum is mediated through specific interactions between fibronectin receptors present on K562 cells and fibronectin adsorbed onto that cationic surface. Specifically, determination of cell adhesion under different experimental conditions indicates that nonspecific charge interactions do not take place directly between the cells and polylysine, but rather take place between polylysine and fibronectin, which adsorbs onto the cationic polymer. In addition, flow cytometric analyses reveal that only fibronectin receptors are present on these cells and, consequently, only fibronectin can be responsible for the actual adhesion of these cells onto the cationic surface. In view of the data presented, the possibility should be considered that ECM components adsorbed onto surfaces with specific charges and/or belonging to certain functional groups are involved in structural and functional modifications in cells. These cells grow in suspension and are normally not involved in adhesion phenomena, though these components should be considered. These considerations should be made especially when designing biomaterials that can modulate the response of cells growing in suspension, such as blood cells, and also in tissue engineering of blood substitutes.     

Boronate-containing polymer brushes: characterization, interaction with saccharides and mammalian cancer cells

Boronate-containing polymer brushes were synthesized by free radical copolymerization of N,N-dimethylacrylamide (DMAA) and N-acryloyl-m-phenylboronic acid (NAAPBA) (9:1) on the surface of 3-mercaptopropyl-silylated glass plates and capillaries. The brushes were characterized with time-of-flight secondary ion mass-spectrometry (ToF SIMS), atomic force microscopy and contact angle measurements. Fructose caused a well-expressed drop spreading on the surface of copolymer-grafted glass, due to the strong interaction with the boronate groups. Sedimentation of murine hybridoma cells M2139 or human myeloid leukemia cells KG1 onto the DMAA-NAAPBA copolymer-grafted glass plates from 10 mM phosphate buffer solution (pH 8.0) resulted in the cell adhesion. The adhered M2139 and KG1 cells could be quantitatively detached from the grafted plates with 0.1 M fructose, which competed with cell surface carbohydrates for binding to the boronates. Evaluation of the binding strength between M2139 cells and the copolymer brush was performed by exposure of the adhered cells to a shear stress. Detachment of a fraction of 18% of the adhered M2139 cells was obtained at a shear force of 1400-2800 pN/cell generated by the running phosphate buffer (pH 8.0), whereas the remaining adhered cells (70%) could be detached with 0.1 M fructose dissolved in the same buffer. Possible applications of the boronate-containing polymer brushes to affinity cell separation can be based upon the facile recovery of the attached cells.     

Inhibition of fibroblast cell adhesion on substrate by coating with 2-methacryloyloxyethyl phosphorylcholine polymers

Fibroblast adhesion and growth behavior were examined on various polymers coated on a poly(ethylene telephthalate) (PET) substrate. The polymers are poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate copolymer (PMB)s with different MPC unit compositions, and poly(2-hydroxyethyl methacrylate). Surface analysis by dynamic contact angle measurement revealed that the mobility of the polymer chain on the PET substrate depended on the MPC unit composition, but there was no significant difference between the PMBs with 3-10 mol% MPC units and poly(HEMA). Fibronectin adsorption on the polymer surface from a cell culture medium was determined by immunoassay. The adsorbed fibronection was evenly distrubuted in every polymer, however, the amount was reduced with an increase in the MPC unit composition in the PMB. This result suggested that the MPC unit could weaken the interaction between the polymer surface and proteins. When fibroblast L-929 cells, were cultured on the polymers, the cells adhered and the number of cells increased on not only the hydrophobic poly(BMA) but also on the hydrophilic poly(HEMA). However, the number of cells that adhered on the PMB surface decreased with an increase in the MPC unit composition. This was a result of the fibronectin adsorption behavior. Thus, it could be concluded that since the PMB could suppress cell adhesion proteins e.g. fibronectin, the PMB showed excellent cell adhesive resistance properties.     

Inhibition of fibroblast cell adhesion on substrate by coating with 2-methacryloyloxyethyl phosphorylcholine polymers

Fibroblast adhesion and growth behavior were examined on various polymers coated on a poly(ethylene telephthalate) (PET) substrate. The polymers are poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylatel copolymer (PMB)s with different MPC unit compositions, and poly(2-hydroxyethyl methacrylate). Surface analysis by dynamic contact angle measurement revealed that the mobility of the polymer chain on the PET substrate depended on the MPC unit composition, but there was no significant difference between the PMBs with 3-10 mol% MPC units and poly(HEMA). Fibronectin adsorption on the polymer surface from a cell culture medium was determined by immunoassay. The adsorbed fibronection was evenly distrubuted in every polymer, however, the amount was reduced with an increase in the MPC unit composition in the PMB. This result suggested that the MPC unit could weaken the interaction between the polymer surface and proteins. When fibroblast L-929 cells, were cultured on the polymers, the cells adhered and the number of cells increased on not only the hydrophobic poly(BMA) but also on the hydrophilic poly(HEMA). However, the number of cells that adhered on the PMB surface decreased with an increase in the MPC unit composition. This was a result of the fibronectin adsorption behavior. Thus, it could be concluded that since the PMB could suppress cell adhesion proteins e.g. fibronectin, the PMB showed excellent cell adhesive resistance properties.     

Tailoring of surfaces with ultrathin polymer films for survival and growth of neurons in culture.

We have studied the adhesion, growth, and survival of dissociated cells of rat cerebellum onto glass surfaces derivatized with covalently bound ultrathin polymer layers. The surface coatings were prepared by growing polymer chains directly at the surfaces of solid substrates by using self-assembled monolayers of radical chain initiators. Methacrylate and acrylamide polymers with different polarities were covalently attached to the surfaces and the adhesion behaviour of the neuronal cells to these surfaces was studied. Best adhesion and neurite outgrowth properties were found using a positively charged polymer (poly(methacryloyl oxypropyl (trimethyl ammonium) bromide)). This 'grafting from' method for the generation of surface modifications can be used for tailoring the composition of the surface of the substrates. Our experiments demonstrate the potential of these layers for the design of polymeric surface coatings for long term stability of neuronal culture.     

Targeted cell adhesion on selectively micropatterned polymer arrays on a poly(dimethylsiloxane) surface

Herein, we introduce the fabrication of polymer micropattern arrays on a chemically inert poly(dimethylsiloxane) (PDMS) surface and employ them for the selective adhesion of cells. To fabricate the micropattern arrays, a mercapto-ester—based photocurable adhesive was coated onto a mercaptosilane—coated PDMS surface and photopolymerized using a photomask to obtain patterned arrays at the microscale level. Robust polymer patterns, 380 μm in diameter, were successfully fabricated onto a PDMS surface, and cells were selectively targeted toward the patterned regions. Next, the performance of the cell adhesion was observed by anchoring cell adhesive linker, an RGD oligopeptide, on the surface of the mercapto-ester—based adhesive-cured layer. The successful anchoring of the RGD linker was confirmed through various surface characterizations such as water contact angle measurement, XPS analysis, FT-IR analysis, and AFM measurement. The micropatterning of a photocurable adhesive onto a PDMS surface can provide high structural rigidity, a highly–adhesive surface, and a physical pathway for selective cell adhesion, while the incorporated polymer micropattern arrays inside a PDMS microfluidic device can serve as a microfluidic platform for disease diagnoses and high-throughput drug screening.     

Modulating bone cells response onto starch-based biomaterials by surface plasma treatment and protein adsorption

The effect of oxygen-based radio frequency glow discharge (rfGD) on the surface of different starch-based biomaterials (SBB) and the influence of proteins adsorption on modulating bone-cells behavior was studied. Bovine serum albumin, fibronectin and vitronectin were used in single and complex protein systems. RfGD-treated surfaces showed to increase in hydrophilicity and surface energy when compared to non-modified SBB. Biodegradable polymeric blends of cornstarch with cellulose acetate (SCA; 50/50wt%), ethylene vinyl alcohol (SEVA-C; 50/50wt%) and polycaprolactone (SPCL; 30/70wt%) were studied. SCA and SCA reinforced with 10% hydroxyapatite (HA) showed the highest degree of modification as result of the rfGD treatment. Protein and control solutions were used to incubate with the characterized SBB and, following this, MG63 osteoblast-like osteosarcoma cells were seeded over the surfaces. Cell adhesion and proliferation onto SCA was found to be enhanced for non-treated surfaces and on SCA+10%HA no alteration was brought up by the plasma modification. Onto SCA surfaces, BSA, FN and VN single solutions improved cell adhesion, and this same effect was found upscaled for ternary systems. In addition, plasma treated SEVA-C directed an increase in both adhesion and proliferation comparing to non-treated surfaces. Even though adhesion onto treated and untreated SPCL was quite similar, plasma modification clearly promoted MG63 cells proliferation. Regarding MG63 cells morphology it was shown that onto SEVA-C surfaces the variation of cell shape was primarily defined by the protein system, while onto SPCL it was mainly affected by the plasma treatment.     

Role of endothelial cell-substrate contact area and fibronectin-receptor affinity in cell adhesion to HEMA/EMA copolymers.

The objective of this study was to examine the effect of substrate hydrophobicity on cell-substrate contact area and the affinity between adsorbed fibronectin (Fn) and its receptor. Homo- and copolymer films of hydrophobic ethyl methacrylate (EMA) and hydrophilic hydroxyethyl methacrylate (HEMA) were spun-cast onto glass slides. Bovine aortic endothelial cells (BAEC) were plated for 2 h in serum-free medium onto polymers preadsorbed with Fn. Cells were fixed, labeled, and examined by total internal reflection fluorescence microscopy (TIRFM) to determine the topography of the basal surface as a function of distance from the substrate. Phase contrast microscopy was used to examine the total projected area of adherent cells. The cumulative contact area was greatest on cells attached to surfaces prepared from 0% HEMA and lowest on surfaces with the highest HEMA content. An equilibrium adhesion model used these data together with the critical force for detachment and the Fn density (Burmeister et al., J Biomed Mater Res 1996;30:13-22) to determine the affinity between Fn and its receptor and the bond strength. The affinity and force per bond decreased with increasing HEMA content. These results indicate that differences in the strength of endothelial cell adhesion to polymers are influenced by the conformation of the adsorbed adhesion proteins.     

Oriented astroglial cell growth on micropatterned polystyrene substrates

In an effort to develop a permissive environment for neural stem cell differentiation, directional growth of astrocytes has been achieved on polymer substrates in vitro. Manipulating a combination of physical and chemical cues, astrocyte adhesion and alignment in vitro were examined. To provide physical guidance, micropatterned polymer substrates of polystyrene (PS) were fabricated. Laminin was selectively adsorbed onto the grooves of the patterned surface. Rat type-1 astrocytes were seeded onto the micropatterned PS substrates, and the effects of substrate topography and the adsorption of laminin to the PS substrates on the behavior and morphology of the astrocytes were explored. The astrocytes were found to align parallel to the micropatterned grooves at initial seeding densities of approximately 7500, 13,000, and 20,000 cells/cm(2) due to the effects of the physical and chemical guidance mechanisms. Adsorbing laminin in the microgrooves of the micropatterned PS substrates improved cell adhesion and spreading of cytoskeletal filaments significantly. At these initial seeding densities, over 85% astrocyte alignment in the direction of the grooves was achieved on the micropatterned PS substrates with laminin adsorbed in the grooves. This combination of guidance cues has the potential to provide a permissive substrate for in vivo regeneration within the central nervous system.     

Blood compatible aspects of poly(2-methoxyethylacrylate) (PMEA)--relationship between protein adsorption and platelet adhesion on PMEA surface

Platelet adhesion and spreading is suppressed when a poly(2-methoxyethylacrylate) (PMEA) surface is used, compared with other polymer surfaces. To clarify the reason for this suppression, the relationship among the amount of the plasma protein adsorbed onto PMEA, its secondary structure and platelet adhesion was investigated. Poly(2-hydroxyethylmethacrylate) (PHEMA) and polyacrylate analogous were used as references. The amount of protein adsorbed onto PMEA was very low and similar to that absorbed onto PHEMA. Circular dichroism (CD) spectroscopy was applied to examine changes in the secondary structure of the proteins after adsorption onto the polymer surface. The conformation of the proteins adsorbed onto PHEMA changed considerably, but that of proteins adsorbed onto PMEA differed only a little from the native one. These results suggest that low platelet adhesion and spreading are closely related to the low degree of the denaturation of the protein adsorbed onto PMEA. PMEA could be developed as a promising material to produce a useful blood-contacting surface for medical devices.     

An assessment of the strength of NG108-15 cell adhesion to chemically modified surfaces

The strength of adhesion of NG108-15 cells to glass substrates modified with adsorbed proteins (laminin and poly-ornithine) or modified with covalently bound peptides (tri-ornithine and Tyr-Ile-Gly-Ser-Arg) was quantitatively assessed, by determining the shear stresses necessary to denude the cells from substrates using a spinning disk device. The shear stresses required to detach NG108-15 cells from glass modified with either adsorbed poly-ornithine or with both poly-ornithine and laminin were significantly (P < 0.05) higher than the shear stresses required to detach the cells from plain glass substrates. Covalent surface modifications resulted in higher strengths of NG108-15 adhesion than were exhibited on surfaces modified with adsorbed proteins. NG108-15 cell adhesion strength was maximal on surfaces covalently modified with only amine groups (without any peptides or proteins). These results indicate that general (i.e., not necessarily receptor-specific) surface modification strategies, which increase the net surface charge of a substrate, will elicit strong adhesion of NG108-15 cells.     

Platelet adhesion onto polyetherurethane urea derivatives: effect of cytoskeleton proteins of the platelet

Adhesion and activation of platelets upon adhesion onto synthetic polymers were investigated with reference to participation of the cytoskeleton proteins. Platelets were treated with cytoskeleton breakers, and then the adhesion of platelets onto polyetherurethane urea derivatives and serotonin release from adhered platelets were investigated. In the adhesion onto glass, platelets were strongly stimulated and accompanied rearrangement of the cytoskeleton system, and also serotonin release involved the action of the cytoskeleton system. On the other hand, platelets were not strongly stimulated upon adhering onto polyetherurethane urea derivatives. The platelet adhesion onto cationic polymers exceptionally accompanied the rearrangement of the cytoskeleton system. The participation of the cytoskeleton in platelet adhesion onto polyetherurethane urea derivatives was influenced by the presence of plasma proteins. It was found that protein layers deposited on the material surface play an important role in platelet adhesion.     

聚合物表面生物修饰对肌腱细胞黏附特性的影响

为了探讨增强肌腱细胞与聚合物材料黏附力学特性的措施 ,采用生物可降解聚合物—乳酸与羟基乙酸共聚物 85 / 15 ,制成透光的薄膜 ,在膜表面裱衬聚赖氨酸的基础上 ,表面裱衬细胞外基质 ( I型胶原蛋白、纤维粘连蛋白 ,以及相应的抗体 )和生长因子 (类胰岛素生长因子 1) ,接种转化人胚肌腱细胞后 ,利用微吸管实验技术测定转化人胚肌腱细胞与聚合物薄膜的黏附力。结果显示 :在聚合物薄膜表面裱衬纤维粘连蛋白或 I型胶原蛋白 ,可明显提高转化人胚肌腱细胞与聚合物薄膜的黏附力 ( P<0 .0 5 ) ,但若在此基础上进一步分别复合裱衬纤维粘连蛋白抗体或 I型胶原蛋白抗体 ,则引起转化人胚肌腱细胞与聚合物薄膜的黏附力明显下降 ( P<0 .0 5 ) ;肌腱细胞对聚合物薄膜的黏附力与细胞外基质蛋白 (纤维粘连蛋白或 I型胶原蛋白 )的裱衬浓度有很好的依赖性 ;I型胶原蛋白和纤维粘连蛋白介导转化人胚肌腱细胞与聚合物薄膜的特异性黏附作用 ;二者复合裱衬浓度达到一定比例时 ,可产生协同作用 ,增强黏附效果 ;这种特异性黏附作用可被相应的抗体分子所抑制 ;生长因子对转化人胚肌腱细胞有明显的促黏附作用。提示 ,材料表面生物修饰可促进转化人胚肌腱细胞与聚合物的黏附作用 ,这对构建组织工程化肌腱具有重要的指导意义     

Controlled polymerization chemistry to graft architectures that influence cell-material interactions

Acrylate monomers were photografted from polymer substrates to create cell responsive chemically and biologically active surfaces that manipulate cell response. Three monomers, polyethylene glycol monoacrylate (MW 375 g/mol) (PEG375A), a monomeric extra-cellular matrix protein, and a cell-cleavable fluorescent monomer, were spatially photopatterned from a base substrate. The base substrate consisted of a dithiocarbamate (DTC) functionalized urethane diacrylate/tri(ethylene glycol)diacrylate copolymer and was shown to non-specifically support NIH 3T3 fibroblast cell adhesion. The DTC-containing polymer was further modified by grafting PEG375A to demonstrate selective blocking of cell-material interactions. Next, acrylated collagen type I was patterned onto polymer substrates to further promote specific cell interactions (i.e. by presenting cell-adhesive moieties). Hydrophilic PEG375A grafted patterns were shown to prevent 3T3 fibroblast adhesion to polymer in spatially grafted regions, while biologically active acrylated collagen type I promoted cell-surface interactions. Collagen type I was grafted at varying densities (0-7.5 pmol/grafted square), and the extent of cell adhesion and spreading were evaluated for each of these graft densities using fluorescence microscopy. Finally, methacrylated carboxyfluorescein diacetate (CFDA) was synthesized and photografted onto a cell-adhesive substrate as a cell sensing mechanism. The acetate groups found in the structure of CFDA cleave in the presence of cells. This cell-responsive substrate results in fluorescence indicative of acetate-group cleavage associated with cell interactions that occurs in patterned regions on polymer surfaces. Collectively, the results herein show the utility and application of a spatially and temporally controlled photografting process for designing cell responsive polymer surfaces.