Cell Adhesion Studies Research Articles

Preparation and characterization of novel chitosan/gelatin membranes using chitosan hydrogel

Chitin and chitosan are novel biomaterials. The novel chitosan/gelatin membranes were prepared using the suspension of chitosan hydrogel mixed with gelatin. The prepared chitosan/gelatin membranes were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), mechanical, swelling, and thermal studies. The morphology of these chitosan/gelatin membranes was found to be very smooth and homogeneous. The XRD studies showed that the chitosan/gelatin membranes have good compatibility and interaction between the chitosan and gelatin. The stress and elongation of chitosan/gelatin membranes on wet condition showed excellent when the mixture ratio of gelatin was 0.50. The prepared chitosan/gelatin membranes showed good swelling, mechanical and thermal properties. Cell adhesion studies were also carried out using human MG-63 osteoblast-like cells. The cells incubated with chitosan/gelatin membranes for 24 h were capable of forming cell adhesion. Thus the prepared chitosan/gelatin membranes are bioactive and are suitable for cell adhesion suggesting that these membranes can be used for tissue-engineering applications. Therefore, these novel chitosan/gelatin membranes are useful for biomedical applications.

Effect of Nanoconfinement on the Collapse Transition of Responsive Polymer Brushes

Nanopatterned brushes of a thermo-responsive polymer, poly(2-(2-methoxyethoxy)ethyl methacrylate) (PMEO2MA), displaying a collapse temperature in the physiological range were synthesized for grafting diameters from a few micrometers down to 35 nm. The reversible collapse transition of the nanobrushes was studied in water as a function of their lateral confinement, down to ensembles of brushes containing only approximately 300 chains. The confinement results in a considerable broadening of the collapse transition and in an increase of the degree of vertical swelling, which can be explained by the internal structure of the nanodroplets derived from a theoretical model of dry nanobrushes. These results enable the rational design of responsive surfaces having a tunable topography engineered at the nanometer scale, which is of direct interest for the development of soft nanoactuators and new substrates for cell adhesion studies.

Biophysical Properties of Cadherin Bonds Do Not Predict Cell Sorting

Differential binding between cadherin subtypes is widely believed to mediate cell sorting during embryogenesis. However, a fundamental unanswered question is whether cell sorting is dictated by the biophysical properties of cadherin bonds, or by broader, cadherin-dependent differences in intercellular adhesion or membrane tension. This report describes atomic force microscope measurements of the strengths and dissociation rates of homophilic and heterophilic cadherin (CAD) bonds. Measurements conducted with chicken N-CAD, canine E-CAD, and Xenopus C-CAD demonstrated that all three cadherins cross-react and form multiple, intermolecular bonds. The mechanical and kinetic properties of the heterophilic bonds are similar to the homophilic interactions. The thus quantified bond parameters, together with previously reported adhesion energies were further compared with in vitro cell aggregation and sorting assays, which are thought to mimic in vivo cell sorting. Trends in quantified biophysical properties of the different cadherin bonds do not correlate with sorting outcomes. These results suggest that cell sorting in vivo and in vitro is not governed solely by biophysical differences between cadherin subtypes.

Characterization of an NbTaWZr alloy designed for magnetic resonance angiography compatible stents

The majority of stent materials are not fully compatible with magnetic resonance imaging due to their ferromagnetic or paramagnetic compositions. This leads to image artifact which can obscure clinical data in the vicinity of the stent. An Nb–28Ta–3.5W–1.3Zr alloy has been developed specifically to provide reduced magnetic susceptibility and therefore reduce image artifact. This study reports on initial surface characterization, corrosion behaviour, endothelial cell response and MR image performance. Surface analysis confirms the presence of a niobium oxide with some tantalum oxide also present. Electrochemical corrosion testing demonstrates the oxide to be stable with no evidence of film breakdown. Leaching of metallic ions during a 60-day immersion test shows low levels of release, comparable to cobalt–chromium L605. A short term endothelial cell adhesion study shows that the Nb–28Ta–3.5W–1.3Zr may be similar to stainless steel for supporting cell growth. The MR artifact assessment shows that the material has significantly reduced artifact compared to stainless steel. In summary, results from this initial study show that the Nb–28Ta–3.5W–1.3Zr meets many on the criteria expected of a stent material and that improved MR imaging behaviour is also obtained.

Biocompatibility implications of polypyrrole synthesis techniques

Polypyrrole (PPy) is an inherently conducting polymer that has shown great promise for biomedical applications within the nervous system. However, to effectively use PPy as a biomaterial implant, it is important to understand and reproducibly control the electrical properties, physical topography and surface chemistry of the polymer. Although there is much research published on the use of PPy in various applications, there is no systematic study linking the methodologies used for PPy synthesis to PPy's basic polymeric properties (e.g., hydrophilicity, surface roughness), and to the biological effects these properties have on cells. Electrochemically synthesized PPy films differ greatly in their characteristics depending on synthesis parameters such as dopant, substrate and thickness, among other parameters. In these studies, we have used three dopants (chloride (Cl), tosylate (ToS), polystyrene sulfonate (PSS)), two substrates (gold and indium tin oxide-coated glass), and a range of thicknesses, to measure and compare the biomedically important characteristics of surface roughness, contact angle, conductivity, dopant stability and cell adhesion (using PC-12 cells and Schwann cells). As predicted, we discovered large differences in roughness depending on the dopant used and the thickness of the film, while substrate choice had little effect. From contact angle measurements, PSS was found to yield the most hydrophilic material, most likely because of free charges from the long PSS chains exposed on the surface of the PPy. ToS-doped PPy films were tenfold more conductive than Cl- or PSS-doped films. X-ray photoelectron spectroscopy studies were used to evaluate dopant concentrations of PPy films stored in water and phosphate buffered saline over 14 days, and conductance studies over the same timeframe measured electrical stability. PSS proved to be the most stable dopant, though all films experienced significant decay in conductivity and dopant concentration. Cell adhesion studies demonstrated the dependence of cell outcome on film thickness and dopant choice. The strengths and weaknesses of different synthesis parameters, as demonstrated by these experiments, are critical design factors that must be leveraged when designing biomedical implants. The results of these studies should provide practical insight to researchers working with conducting polymers, and particularly PPy, on the relationships between synthesis parameters, polymeric properties and biological compatibility.

Surface Modification of Polyurethanes with Covalent Immobilization of Heparin

AbstractThrombus formation and blood coagulation is a major problem associated with blood contacting products such as catheters, vascular grafts, arteries, artificial hearts and heart valves. An intense research is being conducted towards the synthesis of new hemocompatible materials and modifications of surfaces with biological molecules. In this study, polyurethane (PU) films were synthesized in medical purity from diisocyanate and polyol without using any other ingredients and their surfaces were modified by covalent immobilization of heparin. Two types of heparin, unfractionated (UFH) and low molecular weight heparin (LMWH), were immobilized to investigate their effect on cell adhesion. The surface properties of the modified PUs were examined with ESCA, ATR‐FTIR and AFM. ESCA results demonstrated sulfur peaks indicating the presence of heparin and AFM results showed the alteration of surface structure after coating with heparin. Cell adhesion studies were conducted with heparinized whole human blood. The surfaces of the UFH immobilized films resulted in lesser red blood cell adhesion in comparison to LMWH demonstrating strong anti‐thrombogenic activity of the latter.

Engineering the 3D architecture and hydrophobicity of methyltrichlorosilanenanostructures

Three-dimensional nano-architectures with varying shape, morphologyand size were fabricated by the phase separation of methyltrichlorosilane(CH3SiCl3) on commerciallyavailable glass and SiO2 substrates. By changing the synthesis conditions,CH3SiCl3 nanostructures evolved from discrete to quasi-network or from fibrous to spherical forms.Individual nanofibers and nanospheres have diameters of 18–90 and 240–300 nm,respectively, while the film thicknesses could reach 320 nm. The possible mechanisms for thethree-dimensional growth of nanofibers and nanospheres are proposed. The resultantmorphologies exhibited two main energy states: Wenzel and Cassie–Baxter states.Moreover, superhydrophobic surfaces with both high contact angle and high hysteresisresulted from the growth of the nanostructures. The new approaches presented herein areimportant additions to the current range of surface modification methods and couldharness novel physical and chemical properties conducive to optimal performance inbiosensing, antistiction, droplet manipulation, drag reduction, protein adsorption, and celladhesion studies.

Increasing cell adhesion on plasma deposited fluorocarbon coatings by changing the surface topography

In designing new biomaterials, it is of outstanding importance to consider how cells respond to specific chemical and topographical features on the material surface. The behavior of most cell types in vivo is strictly related to specific chemical and topographical cues that characterize the extra cellular environment. In particular, during their lives cells react to topographical patterns such as those of the extracellular matrix (ECM), of micro and/or nanometric dimensions. The production of micrometric and/or nanometric features on artificial materials usually involves expensive and time-consuming methods of manufacturing, such as electron beam and colloidal lithography. In this article, different "Teflon-like" structured surfaces were deposited from tetrafluoroethylene (C(2)F(4))-fed plasmas, for the study of cell adhesion and growth. The reaction of different cell lines to different topographical features was evaluated and compared with cell behavior on flat samples with the same chemical composition. Cell adhesion was calculated from area covered by cells at different time of culture. Beside this, cell proliferation was determined with the MTT test. Cell morphology and filopodia interaction with the nanofeatures were also estimated by optical and scanning electron microscopy. A dramatic difference both in adhesion and growth was found between cells seeded on flat and rough surfaces with the density and spreading of adhered cells varying as a function of the roughness of coatings.

Multifunctional surfaces with discrete functionalized regions for biological applications.

In this paper we describe a method for creating multifunctional glass surfaces presenting discrete patches of different proteins on an inert PEG-functionalized background. Microcontact printing is used to stamp the substrate with octadecyltrichlorosilane to define the active regions. The substrate is then back-filled with PEG-silane {[[2-methoxypoly(ethyleneoxy)]propyl]trimethoxysilane} to define passive regions. A microfluidics device is subsequently affixed to the substrate to deliver proteins to the active regions, with as many channels as there are proteins to be patterned. Examples of trifunctional surfaces are given which present three terminating functional groups, i.e., protein 1, protein 2, and PEG. These surfaces should be broadly useful in biological studies, as patch size is well established to influence cell viability, growth, and differentiation. Three examples of cellular interactions with the surfaces are demonstrated, including the capture of cells from a single cell suspension, the selective sorting of cells from a mixed suspension, and the adhesion of cells to ligand micropatches at critical shear stresses. Within these examples, we demonstrate that the patterned immobilized proteins are active, as they retain their ability to interact with either antibodies in solution or receptors presented by cells. When appropriate (e.g., for E-selectin), proteins are patterned in their physiological orientations using a sandwich immobilization technique, which is readily accommodated within our method. The protein surface densities are highly reproducible in the patches, as supported by fluorescence intensity measurements. Potential applications include biosensors based on the interaction of cells or of marker proteins with protein patches, fundamental studies of cell adhesion as a function of patch size and shear stress, and studies of cell differentiation as a function of surface cues.

Novel poly(HEMA‐co‐METAC)/alginate semi‐interpenetrating hydrogels for biomedical applications: Synthesis and characterization

Novel p(HEMA-co-METAC)/alginate semi-interpenetrating hydrogels (semi-IPNs) were developed in the attempt to improve poly(hydroxyethylmethacrylate) biological response, extending its applications in the biomedical field. Materials with different alginate contents were synthesized by copolymerization of 2-hydroxyethylmethacrylate and 2-methacryloxy ethyltrimethyl ammonium chloride monomers in the presence of aqueous solutions of alginate and characterized with respect to p(HEMA-co-METAC) synthesized in the presence of water. Swelling studies in water revealed high values of water uptake (>100%) with marked differences in the swelling degree at increasing polysaccharide content. The effect of ionic strength and of pH on the swelling behavior of hydrogels was also investigated. Higher ionic strengths resulted in a minor swelling degree accordingly with hydrogels polyelectrolyte nature. The introduction of the natural ionizable polysaccharide into the network made the semi-IPNs swelling depending on pH values of the solutions. A biological characterization was performed in terms of protein absorption on hydrogel surfaces, cytotoxicity (ISO 10993-5) and cell adhesion and proliferation studies using both murine 3T3 and human fibroblasts. Hydrogels proved noncytotoxic; moreover, semi-IPN surfaces allowed cell attachment and proliferation, thus supporting their potential biomedical use.

Synergistic Microfluidic and Electrochemical Strategy to Activate and Pattern Surfaces Selectively with Ligands and Cells

We show a straightforward, flexible synergistic approach that combines microfluidics, electrochemistry, and a general immobilization strategy to activate regions of a substrate selectively for the precise immobilization of ligands and cells in patterns for a variety of cell-based assays and cell migration and cell adhesion studies. We develop microfluidic microchips to control the delivery of electrolyte solution to select regions of an electroactive hydroquinone SAM. Once an electrical potential is applied to the substrate, only the hydroquinone exposed to electrolyte solution within the microfluidic channels oxidizes to the corresponding quinone. The quinone form can then react chemoselectively with oxyamine-tethered ligands to pattern the surface. Therefore, this microfluidic/electrochemistry strategy selectively activates the surface for ligand patterning that exactly matches the channel design of the microfluidic channel. We demonstrate the ease of this system by first quantitatively characterizing the electrochemical activation and immobilization of ligands on the surface. Second, we immobilize a fluorescent dye to show the fidelity of the methodology, and third, we show the immobilization of biospecific cell adhesive peptide ligands to pattern cells. This is the first report that combines microfluidics/electrochemistry and a general electroactive immobilization strategy to pattern ligands and cells. We believe that this strategy will be of broad utility for applications ranging from fundamental studies of cell behavior to patterning molecules on a variety of materials for molecular electronic devices.

Implementation of maskless laser lithography using a Raman spectroscopy microprobe

A new use of a Raman spectroscopy microprobe is described. This kind of instrument uses an intense light source, usually a laser, to obtain chemical information from the scattered light sent to the sample through a microscope objective lens. A Raman microprobe is most often equipped with a motorized stage. The coupling of a focused laser light with the possibility of moving the stage under the very same light can be used to perform maskless photolithography as a laser writer. The versatility and resolution of this application are tested and described, producing different arrays of holes on photoresist. Holes as small as 1 mum are produced, thus demonstrating a lateral resolution sufficient for several applications. As an example of application, a pattern of a human protein, fibronectin, has been produced on a gold surface, which can then be used for the study of cell adhesion.

Iodine inhibits antiadhesive effect of PEG: Implications for tissue engineering

The success of a biomaterial implant may be affected by the surface chemistry's impact on protein adsorption. We have developed a series of poly(ethylene glycol) (PEG) containing, tyrosine-derived polycarbonates, which have been rendered radio-opaque by the iodination of tyrosine units in the copolymer backbone for use in resorbable biomedical implants including vascular stents and grafts. We tested the hypothesis that protein adsorption along with seeding, growth, and migration of human aortic smooth muscle cells (SMC) and human aortic endothelial cells (EC) will be modified by the presence of iodine and PEG within the polymer composition. Thin films of these polymers were prepared for the protein-material and cell-material interaction studies. Dot blot, SDS-PAGE, and XPS were used to evaluate relative protein adsorption. Cell adhesion and growth studies were performed using an MTS assay. Cell migration was evaluated using an injury model. The presence of PEG attenuated protein adsorption, cell adhesion, and growth. With the subsequent incorporation of iodine, protein adsorption markedly increased while the antiadhesive effect of PEG was counteracted by iodine for EC and SMC adhesion and SMC growth. Iodine incorporation into the polymer resulted in increased protein adsorption thus counteracting the effect of PEG.

Macromol. Biosci. 12/2007

Cover: A novel poly(lactic acid)‐based polymer containing pendent amine‐specific reactive groups was synthesized and cast into thin films, allowing for the covalent attachment of the cell adhesion peptide GRGDS to the surface. Subsequent cell adhesion studies with 3T3 fibroblasts showed increased cellular adhesion and spreading over unmodified films. Further details can be found in the article by E. L. Prime, J. J. Cooper‐White, and G. G. Qiao* on page 1272.

Conjugation of Bioactive Groups to Poly(lactic acid) and Poly[(lactic acid)‐co‐(glycolic acid)] Films

A novel PLA-based polymer containing reactive pendent ketone or hydroxyl groups was synthesized by the copolymerization of L-lactide with epsilon-caprolactone-based monomers. The polymer was activated with NPC, resulting in an amine-reactive polymer which was then cast into thin polymeric films, either alone or as part of a blend with PLGA, before immersion into a solution of the cell adhesion peptide GRGDS in PBS buffer allowed for conjugation of GRGDS to the film surfaces. Subsequent 3T3 fibroblast cell adhesion studies demonstrated an increase in cellular adhesion and spreading over films cast from unmodified PLGA. Hence the new polymer can be used to obtain covalent linkage of amine-containing molecules to polymer surfaces.

Biocompatibility of highly macroporous ceramic scaffolds: cell adhesion and morphology studies

Hydroxyapatite porous scaffolds can be used for tissue engineering applications since they can serve as templates for cell adhesion, proliferation and ultimately for tissue repair. One way to address this issue is to evaluate the cell adhesion using several characterization techniques namely, cytotoxicity assays and cell visualization. On the other hand, when using highly macroporous scaffolds some techniques may not be adequate for evaluation, such as MTT. In this work, cytotoxicity assay (MTS), scanning electron microscopy (SEM) and Confocal laser scanning microscopy (CLSM) were used to evaluate cell adhesion in highly macroporous hydroxyapatite scaffolds. It was possible to observe that some techniques were not suitable to evaluate cell adhesion. In addition, it was shown that for this kind of scaffolds, confocal laser scanning microscopy is a powerful tool for cell adhesion and proliferation evaluation.

Future Design of a New Keratoprosthesis. Physical and Biological Analysis of Polymeric Substrates for Epithelial Cell Growth

One of the main issues in the development of new biocolonizable materials is to understand the influence of the synthetic material on the biological response in terms of cellular adhesion, proliferation, and differentiation. In this study, we characterized different polymeric materials (with different hydrophobicity/hydrophilicity ratios and electrical charges) using dynamic-mechanical analysis, equilibrium water content, and surface energy. Cell adhesion, viability, morphology, and proliferation studies were conducted with these materials using a conjunctival epithelial cell line (IOBA-NHC). The biological data regarding physicochemical parameters of the materials were also correlated. When conjunctival epithelial cells were grown on poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymers, P(EA-co-HEA), samples with up to 20% hydrophilic groups on their polymeric chain showed adhesion, viability, and proliferation, although these three factors decreased as the hydrophilic group content increased. The poly(ethyl acrylate-co-methacrylic acid) 90/10 copolymer, P(EA-co-MAAc) 90/10, showed better results than poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymers and were even better than tissue control polystyrene (TCPS). This feature is explained by the presence of electrical charges on the surface of the poly(ethyl acrylate-co-methacrylic acid) 90/10 copolymer. The fact that the ionic groups are configured in domains structured in nanophases as happens in this copolymer improves cell adhesion even further.

Exploratory study of novel leukemia bone marrow stromal cell adhesion mediated drug resistance model

To construct cell adhesion mediated drug resistance (CAM-DR) model based on Acute lymphocyte leukemia bone marrow stromal cells(BMSC) for further studying drug resistance of leukemia. Firstly, we adhesively cultured Jurkat cell strain of human leukaemia lymphocyte with the matrix cell radiated by (60)Co to construct the model of CAM-DR, then evaluated the model in morph and construction by scanning electron microscope. The IC50 of DNR on Jurkat cell was examen by MTT and the concentration and distribution of DNR in the cell was detected by flow cytometry. When the Jurkat cells were cultured with BMSC for 24 h, we found that Jurkat cells had adhesioned the bone marrow stromal cell layer by parapodium and some of them had nichd the mesh consisted by the confluence of BMSC. At the point of 48 h, some Jurkat cells had migrated to underlayer of BMSC, and Jurkat cells were nichd the mesh of BMSC like nidi. The accumulation and distribution of DNR in the Jurkat cells were not affected in the model, but the reaction of Jurkat cells to DNR were significantly inhibited. The model of CAM-DR based on Acute lymphocyte leukemia bone marrow stromal cells was successfully built.

Cell adhesion study of the titanium alloys exposed to glow discharge

Titanium for biomedical application stems mainly from its advantageous bulk mechanical properties in combination with a high degree of biocompatibility that is largely attributable to their surface properties. This work is focused on the investigation of surface properties of treated titanium and cell adhesion to titanium treated in glow-discharge plasma.Pure titanium samples (grade 4) were exposed to low-energy ion irradiation in a specially constructed plasma generator, where materials were irradiated by ions of residual gases in vacuum. The ion energy was 1–10keV. The irradiation dose was maintained at 1017ionscm−2. The irradiation time varied from 5 to 60min.Rutherford backscattering spectrometry (RBS) was used for surface studies. RBS showed the presence of iron on the titanium surface that occurred from the cathode of plasma generator.In vitro biocompatibility test have been carried out with model cell lines (L929 mouse fibroblasts) to demonstrate that low-energy ion irradiation can favorably influence the surface of titanium for biomedical application. Scanning electron microscopy (SEM) was the main tool to demonstrate the cell attachment properties.

WITHDRAWN: Cell adhesion study of the titanium alloys exposed to glow discharge

WITHDRAWN: Cell adhesion study of the titanium alloys exposed to glow discharge