Patterned poly(chlorotrifluoroethylene) guides primary nerve cell adhesion and neurite outgrowth

Central nervous system (CNS) neurons, unlike those of the peripheral nervous system, do not spontaneously regenerate following injury. Recently it has been shown that in the developing CNS, a combination of cell-adhesive and cell-repulsive cues guide growing axons to their targets. We hypothesized that by mimicking these guidance signals, we could guide nerve cell adhesion and neurite outgrowth in vitro. Our objective was to direct primary nerve cell adhesion and neurite outgrowth on poly(chlorotrifluoroethylene) (PCTFE) surfaces by incorporating alternating patterns of cell-adhesive (peptide) and nonadhesive (polyethylene glycol; PEG) regions. PCTFE was surface-modified with lithium PEG-alkoxide, demonstrating the first report of metal-halogen exchange with an alkoxide and PCTFE. Titanium and then gold were sputtered onto PEG-modified films, using a shadow-masking technique that creates alternating patterns on the micrometer scale. PCTFE-Au regions then were modified with one of two cysteine-terminated laminin-derived peptides, C-GYIGSR or C-SIKVAV. Hippocampal neuron cell-surface interactions on homogeneously modified surfaces showed that neuron adhesion was decreased significantly on PEG-modified surfaces and was increased significantly on peptide-modified surfaces. Cell adhesion was greatest on CGYIGSR surfaces while neurite length was greatest on CSIKVAV surfaces and PLL/laminin positive controls, indicating the promise of peptides for enhanced cellular interactions. On patterned surfaces, hippocampal neurons adhered and extended neurites preferentially on peptide regions. By incorporating PEG and peptide molecules on the surface, we were able to simultaneously mimic cell-repulsive and cell-adhesive cues, respectively, and maintain the biopatterning of primary CNS neurons for over 1 week in culture.     

Cellular control of tissue architectures using a three-dimensional tissue fabrication technique

Tissue engineering seeks to provide regenerated tissue architectures in vitro but has not yet successfully created thick, highly vascularized, multi-functional tissues replicating native structure. We describe a novel method to fabricate pre-vascularized tissue equivalents using multi-layered cultures combining micro-patterned endothelial cells as vascular pre-cursors with fibroblast monolayer sheets as tissue matrix. Stratified tissue equivalents are constructed by alternately layering fibroblast monolayer sheets with patterned endothelial cell sheets harvested from newly developed thermo-responsive micro-patterned surfaces alternating 20 microm-wide cell-adhesive lanes with 60 microm non-adhesive zones. Cell culture substrates covalently grafted with different thermo-responsive polymers permit spatial switching of cell adhesion and detachment using applied small temperature changes. Endothelial cell patterning fidelity was maintained within the multi-layer tissue constructs after assembly, leading to self-organization into microvascular-like networks after 5-day tissue culture. This novel technique holds promise for the study of cell-cell communications and angiogenesis in reconstructed, three-dimensional environments as well as for the fabrication of tissues with complex, multicellular architecture.     

Control of cell adhesion on poly(methyl methacrylate)

Keratoprostheses have been constructed from a wide variety of transparent materials, including poly(methyl methacrylate) (PMMA). However, the success of keratoprosthesis has been plagued by numerous shortcomings that include the weakening of the implant-host interface due to weak cell adhesion and opaque fibrous membrane formation over the inner surface of the implant due to fibroblast attachment. An effective solution requires a surface modification that would selectively allow enhanced cell attachment at the implant-host interface and reduced cell attachment over the interior surface of the implant. Here, we have developed a novel and simple peptide conjugation scheme to modify PMMA surfaces, which allowed for region-specific control of cell adhesion. This method uses di-amino-PEG, which can be grafted onto PMMA using hydrolysis or aminolysis method. PEG can resist cell adhesion and protein adsorption. The functionalization of grafted di-amino-PEG molecules with RGD peptide not only restored cell adhesion to the surfaces, but also enhanced cell attachment and spreading as compared to untreated PMMA surfaces. Long-term cell migration and micropatterning studies clearly indicated that PEG-PMMA surfaces with and without RGD conjugation can be used to differentiate cell adhesion and control cell attachment spatially on PMMA, which will have potential applications in the modification of keratoprostheses.     

Photolithographic patterning of polyethylene glycol hydrogels

A simple, inexpensive photolithographic method for surface patterning deformable, solvated substrates is demonstrated using photoactive poly(ethylene glycol) (PEG)-diacrylate hydrogels as model substrates. Photolithographic masks were prepared by printing the desired patterns onto transparencies using a laser jet printer. Precursor solutions containing monoacryloyl-PEG-peptide and photoinitiator were layered onto hydrogel surfaces. The acrylated moieties in the precursor solution were then conjugated in monolayers to specific hydrogel regions by exposure to UV light through the transparency mask. The effects of UV irradiation time and precursor solution concentration on the levels of immobilized peptide were characterized, demonstrating that bound peptide concentration can be controlled by tuning these parameters. Multiple peptides can be immobilized to a single hydrogel surface in distinct patterns by sequential application of this technique, opening up its potential use in co-cultures. In addition, 3D structures can be generated by incorporating PEG-diacrylate into the precursor solution. To evaluate the feasibility of using these patterned surfaces for guiding cell behavior, human dermal fibroblast adhesion on hydrogel surfaces patterned with acryloyl-PEG-RGDS was investigated. This patterning method may find use in tissue engineering, the elucidation of fundamental structure-function relationships, and the formation of immobilized cell and protein arrays for biotechnology.     

Val-ala-pro-gly, an elastin-derived non-integrin ligand: smooth muscle cell adhesion and specificity

The elastin-derived peptide val-ala-pro-gly (VAPG) may be useful as a biospecific cell adhesion ligand for smooth muscle cells. By grafting the peptide sequence into a hydrogel material, we were able to assess its effects on smooth muscle cell adhesion and spreading. These materials are photopolymerizable hydrogels based on acrylate-terminated derivatives of polyethylene glycol (PEG). Because of their high PEG content, these materials are highly resistant to protein adsorption and cell adhesion. However, PEG diacrylate derivatives can be mixed with adhesive peptide-modified PEG monoacrylate derivatives to facilitate cell adhesion. Following photopolymerization, PEG monoacrylate derivatives are grafted into the hydrogel network formed by the PEG diacrylate. This results in covalent immobilization of adhesive peptides to the hydrogel via a flexible linker chain. The resistance of PEG to protein adsorption makes it an ideal material for this model system since cell-material interactions are limited to biomolecules that are covalently incorporated into the material. In this case we were able to demonstrate that VAPG is specific for adhesion of smooth muscle cells. It also was shown that fibroblasts, endothelial cells, and platelets cannot adhere to VAPG. In addition, not only was smooth muscle cell adhesion dependent on ligand concentration, but also cell spreading increased with increasing ligand concentration.     

Length-scale mediated adhesion and directed growth of neural cells by surface-patterned poly(ethylene glycol) hydrogels

We engineered surfaces that permit the adhesion and directed growth of neuronal cell processes but that prevent the adhesion of astrocytes. This effect was achieved based on the spatial distribution of sub-micron-sized cell-repulsive poly(ethylene glycol) [PEG] hydrogels patterned on an otherwise cell-adhesive substrate. Patterns were identified that promoted cellular responses ranging from complete non-attachment, selective attachment, and directed growth at both cellular and subcellular length scales. At the highest patterning density where the individual hydrogels almost overlapped, there was no cellular adhesion. As the spacing between individual hydrogels was increased, patterns were identified where neurites could grow on the adhesive surface between hydrogels while astrocytes were unable to adhere. Patterns such as lines or arrays were identified that could direct the growth of these subcellular neuronal processes. At higher hydrogel spacings, both neurons and astrocytes adhered and grew in a manner approaching that of unpatterned control surfaces. Patterned lines could once again direct growth at cellular length scales. Significantly, we have demonstrated that the patterning of sub-micron/nano scale cell-repulsive features at microscale lengths on an otherwise cell-adhesive surface can differently control the adhesion and growth of cells and cell processes based on the difference in their characteristic sizes. This concept could potentially be applied to an implantable nerve-guidance device that would selectively enable regrowing axons to bridge a spinal-cord injury without interference from the glial scar.     

Immobilized RGD peptides on surface-grafted dextran promote biospecific cell attachment

Dextran has recently been investigated as an alternative to poly(ethylene glycol) (PEG) for low protein-binding, cell-resistant coatings on biomaterial surfaces. Although antifouling properties of surface-grafted dextran and PEG are quite similar, surface-bound dextran has multiple reactive sites for high-density surface immobilization of biologically active molecules. We recently reported nontoxic aqueous methods to covalently immobilize dextran on material surfaces. These dextran coatings effectively limited cell adhesion and spreading in the presence of serum-borne cell adhesion proteins. In this study we utilized the same nontoxic aqueous methods to graft cell adhesion peptides on low protein-binding dextran monolayer surfaces. Chemical composition of all modified surfaces was verified by X-ray photoelectron spectroscopy (XPS). Surface-grafted cell adhesion peptides stimulated endothelial cell, fibroblast, and smooth muscle cell attachment and spreading in vitro. In contrast, surface-grafted inactive peptide sequences did not promote high levels of cell interaction. Surface-grafted high affinity cyclic RGD peptides promoted cell type-dependent interactions. With dextran-based surface coatings, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in long-term biomaterial implants.     

Immobilization of cell-adhesive peptides to temperature-responsive surfaces facilitates both serum-free cell adhesion and noninvasive cell harvest

We have developed temperature-responsive cell culture surfaces to harvest intact cell sheets for tissue-engineering applications. Both cost and safety issues (e.g., prions, bovine spongiform encephalopathy) are compelling reasons to avoid use of animal-derived materials, including serum, in such culture. In the present study, synthetic cell-adhesive peptides are immobilized onto temperature-responsive polymer-grafted surfaces, and cell adhesion and detachment under serum-free conditions were examined. The temperature-responsive polymer poly(N-isopropylacrylamide) (PI-PAAm) was functionalized by copolymerization with a reactive comonomer having both a carboxyl group and an isopropylacrylamide group. These copolymers were covalently grafted onto tissue culture-grade polystyrene dishes. Synthetic cell-adhesive peptides were then immobilized onto these surfaces via carboxyl groups. Bovine aortic endothelial cells both adhered and spread on these surfaces even under serum-free conditions at 37 degrees C, similar to those in 10% serum-supplemented culture. Spread cells promptly detached from the surfaces on lowering culture temperatures below the lower critical solution temperature of the polymer, 32 degrees C. These surfaces would be useful for serumfree culture for tissue-engineering applications.     

Surface-immobilized dextran limits cell adhesion and spreading

Dextran has recently been investigated as an alternative to polyethylene glycol (PEG) for low protein-binding, cell-resistant coatings on biomaterial surfaces. Although anti-fouling properties of surface-grafted dextran and PEG are quite similar, the multivalent properties of dextran are advantageous when high-density surface immobilization of biologically active molecules to low protein-binding surface coatings is desired. The preferred methods of dextran immobilization for biomaterial applications should be simple with minimal toxicity. In this report, a method is described for covalent immobilization of dextran to material surfaces which involves low residual toxicity reagents in mild aqueous reaction conditions. 70 kDa MW dextran was immobilized on glass and polyethylene terephthalate (PET) surfaces. 3T3 fibroblast cell adhesion was compared on untreated, aminated, and dextran-coated materials. Dextran coatings effectively limited cell adhesion and spreading on glass and PET surfaces in the presence of serum-borne cell adhesion proteins. With dextran-based surface coatings, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in long-term biomaterial implants.     

Novel patterned cell coculture utilizing thermally responsive grafted polymer surfaces.

Here we demonstrate a novel cell coculture method without any apparent limitation in cell-type combinations that exploits thermally responsive polymer-grafted patterns to alter cell-cell and cell-surface interactions. Thermally responsive acrylamide polymer is first covalently patterned onto culture surfaces by masked electron beam irradiation. One cell type is then cultured to confluency at 37 degrees C. Reducing cell culture temperature below 32 degrees C selectively swells temperature sensitive polymer-grafted domains, detaching adherent cells only from these grafted patterns. Another cell type is then seeded over the same surface at 37 degrees C. These subsequently seeded cells adhere only to the now-exposed polymer-grafted domains. Initially seeded cells remaining adherent on nonpatterned surfaces and cells added in the second seeding are then cocultured at 37 degrees C in well-ordered patterns.     

A simple soft lithographic route to fabrication of poly(ethylene glycol) microstructures for protein and cell patterning

We present a simple, direct soft lithographic method to fabricate poly(ethylene glycol) (PEG) microstructures for protein and cell patterning. This lithographic method involves a molding process in which a uniform PEG film is molded with a patterned polydimethylsiloxane stamp by means of capillary force. The patterned surfaces created by this method provide excellent resistance towards non-specific protein and cell adsorption. The patterned substrates consist of two regions: the molded PEG surface that acts as a resistant layer and the exposed substrate surface that promotes protein or cell adsorption. A notable finding here is that the substrate surface can be directly exposed during the molding process due to the ability to control the wetting properties of the polymer on the stamp, which is a key factor to patterning proteins and cells.     

Analysis of the biological response of endothelial and fibroblast cells cultured on synthetic scaffolds with various hydrophilic/hydrophobic ratios: influence of fibronectin adsorption and conformation

In this study we developed polymer scaffolds intended as anchorage rings for cornea prostheses among other applications, and examined their cell compatibility. In particular, a series of interconnected porous polymer scaffolds with pore sizes from 80 to 110 microns were manufactured varying the ratio of hydrophobic to hydrophilic monomeric units along the polymer chains. Further, the effects of fibronectin precoating, a physiological adhesion molecule, were tested. The interactions between the normal human fibroblast cell line MRC-5 and primary human umbilical vein endothelial cells (HUVECs) with the scaffold surfaces were evaluated. Adhesion and growth of the cells was examined by confocal laser scanning microscopy. Whereas MRC-5 fibroblasts showed adhesion and spreading to the scaffolds without any precoating, HUVECs required a fibronectin precoating for adhesion and spreading. Although both cell types attached and spread on scaffold surfaces with a content of up to a 20% hydrophilic monomers, cell adhesion, spreading, and proliferation increased with increasing hydrophobicity of the substrate. This effect is likely due to better adsorption of serum proteins to hydrophobic substrates, which then facilitate cell adhesion. In fact, atomic force microscopy measurements of fibronectin on surfaces representative of our scaffolds revealed that the amount of fibronectin adsorption correlated directly with the hydrophobicity of the surface. Besides cell adhesion we also examined the inflammatory state of HUVECs in contact with the scaffolds. Typical patterns of platelet/endothelial cell adhesion molecule-1 expression were observed at intercellular boarders. HUVECs adhering on the scaffolds retained their proinflammatory response potential as shown by E-selectin mRNA expression after stimulation with lipopolyssacharide (LPS). The proinflammatory activation occurred in most of the cells, thus confirming the presence of a functionally intact endothelium. Little or no expression of the proinflammatory activation markers in the absence of LPS stimulation was observed for HUVECs growing on scaffolds with up to a 20% of hydrophilic component, whereas activation of these markers was observed after stimulation. In conclusion, scaffolds containing up to 20% hydrophilic monomers exhibited excellent cell compatibility toward human fibroblast cell line MRC-5 and human endothelial cells. Atomic force microscopy confirmed that adsorbed serum proteins such as fibronectin probably accounted for the positive correlation of HUVEC adhesion and surface hydrophobicity.     

Conditions of lateral surface confinement that promote tissue-cell integration and inhibit biofilm growth

Surfaces with cell adhesiveness modulated at micro length scales can exploit differences between tissue/bacterial cell size, membrane/wall plasticity, and adhesion mechanisms to differentially control tissue-cell/material and bacteria/material interactions. This study explores the short-term interactions of Staphylococcus aureus and osteoblast-like cells with surfaces consisting of cell-adhesive circular patches (1–5 μm diameter) separated by non-adhesive electron-beam patterned poly(ethylene glycol) hydrogel thin films at inter-patch distances of 0.5–10 μm. Osteoblast-like U2OS cells both bind to and spread on the modulated surfaces, in some cases when the cell-adhesive area comprises only 9% of the total surface and in several cases at least as well as on the continuously adhesive control surfaces. In contrast, S. aureus adhesion rates are 7–20 times less on the modulated surfaces than on the control surfaces. Furthermore, the proliferation of those bacteria that do adhere is inhibited by the lateral confinement imposed by the non-adhesive boundaries surrounding each patch. These findings suggest a new approach to create biomaterial surfaces that may promote healing while simultaneously reducing the probability of infection.     

Endothelial cell adhesion on polyurethanes containing covalently attached RGD-peptides.

Peptides based on cell-adhesive regions of fibronectin, Arg-Gly-Asp-Ser (RGDS), and vitronectin, Arg-Gly-Asp-Val (RGDV), were covalently bound to a polyurethane backbone via amide bonds. Nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopies were used to monitor the reactions. The amount of grafted peptide was determined by amino acid analysis. X-ray photoelectron spectroscopy (XPS) suggested the presence of the grafted peptide at the polymer-air interface in vacuo. Dynamic contact angle analysis showed that, in water, the peptide-grafted polyurethane surfaces were more polar than the underivatized polyurethane indicating enrichment of peptide groups at the surface. The attachment and spreading of human umbilical vein endothelial cells (HUVECs) on the underivatized and peptide-grafted polyurethanes was investigated. The GRGDSY- and GRGDVY-grafted substrates supported cell adhesion and spreading even without serum in the culture medium. The GRGDVY-grafted substrate supported a larger number of adherent cells and a higher extent of cell spreading than the GRGDSY-grafted substrate. These RGD-containing peptide-grafted polyurethane copolymers may be useful in providing an easily prepared cell-adhesive substrate for various biomaterial applications.     

Long-term stability of cell micropatterns on poly((3-(methacryloylamino)propyl)-dimethyl(3-sulfopropyl)ammonium hydroxide)-patterned silicon oxide surfaces

In this work, we compared the long-term stability and integrity of cell patterns on newly reported, zwitterionic poly((3-(methacryloylamino)propyl)dimethyl(3-sulfopropyl)ammonium hydroxide) (poly(MPDSAH)) films with those on widely used, poly(poly(ethylene glycol) methyl ether methacrylate) (poly(PEGMEMA)) ones. The micropatterns of both polymers were formed on a silicon oxide surface by a combination of micropattern generation of a photoresist, vapor deposition of a silane-based polymerization initiator, and surface-initiated, atom transfer radical polymerization (SI-ATRP) of each monomer, MPDSAH or PEGMEMA. The successful formation of the silane initiator SAMs, and poly(MPDSAH) and poly(PEGMEMA) micropatterns was confirmed by X-ray photoelectron spectroscopy (XPS) and imaging ellipsometry. Onto each substrate patterned with poly(MPDSAH) or poly(PEGMEMA), NIH 3T3 fibroblast cells were seeded, and the cell micropatterns were generated by the selective adhesion of cells on the cell-adhesive region of the patterned surfaces. The cell pattern formed on the poly(MPDSAH)-patterned surface was observed to have a superior ability of finely maintaining its original, line-shaped structure up to for 20 days, when compared with the cell pattern formed on the poly(PEGMEMA)-patterned surface. In order to verify the relationship between the integrity of the cell micropatterns and the stability of the underlying non-biofouling polymer layers, we also investigated the long-term stability of the polymer films themselves, immersed in the cell culture media, for one month, in the aid of ellipsometry, contact goniometry, and XPS.     

Sterically blocking adhesion of cells to biological surfaces with a surface-active copolymer containing poly(ethylene glycol) and phenylboronic acid.

Graft copolymers were designed that could spontaneously bind to biological surfaces and block subsequent recognition and adhesion at those surfaces. Phenylboronic acid (PBA) moieties in the polymer backbone provided binding to surfaces, forming reversible covalent complexes with cis-diols found in many biological molecules. Pendant poly(ethylene glycol) (PEG) side chains sterically protected those surfaces from subsequent interactions with other proteins and cells. The PEG and PBA grafting ratios on these poly-L-lysine-graft-(PEG;PBA) copolymers [PLL-g-(PEG;PBA)] were varied, and the polymers were tested in models relevant to undesirable wound-healing responses such as peritoneal adhesion formation and posterior capsule opacification. PLL-g-(PEG;PBA) polymers spontaneously coated tissue culture polystyrene and completely blocked rabbit lens epithelial cell adhesion to the surface over a wide range of PEG grafting ratios. PLL-g-(PEG;PBA)s with optimal grafting ratios were able to coat adsorbed serum proteins or extracellular matrices and block cell spreading on the surfaces at 4 h, although the effect was lost within 24 h. The polymer also enhanced the efficacy of surgical lysis of peritoneal adhesions in rats. The reversible covalent complexes formed by the PBA moieties on the copolymer backbone were more effective at binding biological surfaces than electrostatic interactions formed via a copolymer lacking the PBA moieties, that is, PLL-g-PEG.     

Spatially controlled bacterial adhesion using surface-patterned poly(ethylene glycol) hydrogels

We constructed surface-patterned hydrogels using low-energy focused electron beams to locally crosslink poly(ethylene glycol) (PEG) thin films on silanized glass substrates. Derived from electron-beam lithography, this technique was used to create patterned hydrogels with well-defined spatial positions and degrees of swelling. We found that cells of the bacterium Staphylococcus epidermidis adhered to and grew on the silanized glass substrates. These cells did not, however, adhere to surfaces covered by high-swelling lightly crosslinked PEG hydrogels. This finding is consistent with the cell-repulsiveness generally attributed to PEGylated surfaces. In contrast, S. epidermidis cells did adhere to surfaces covered by low-swelling highly crosslinked hydrogels. By creating precise patterns of repulsive hydrogels combined with adhesive hydrogels or with exposed glass substrate, we were able to spatially control the adhesion of S. epidermidis. Significantly, adhesive areas small enough to trap single bacterial cells could be fabricated. The results suggest that the lateral confinement imposed by cell-repulsive hydrogels hindered the cell proliferation and development into larger bacterial colonies.     

Nanofabrication for micropatterned cell arrays by combining electron beam-irradiated polymer grafting and localized laser ablation

Most methods reported for cell-surface patterning are generally based on photolithography and use of silicon or glass substrates with processing analogous to semiconductor manufacturing. Herein, we report a novel method to prepare patterned plastic surfaces to achieve cell arrays by combining homogeneous polymer grafting by electron beam irradiation and localized laser ablation of the grafted polymer. Poly(N-isopropylacrylamide) (PIPAAm) was covalently grafted to surfaces of tissue culture-grade polystyrene dishes. Subsequent ultraviolet ArF excimer laser exposure to limited square areas (sides of 30 or 50 microm) produced patterned ablative photodecomposition of only the surface region (approximately 100-nm depth). Three-dimensional surface profiles showed that these ablated surfaces were as smooth and flat as the original tissue culture-grade polystyrene surfaces. Time-of-flight secondary ion mass spectrometry analysis revealed that the ablated domains exposed basal polystyrene and were surrounded with PIPAAm-grafted chemistry. Before cell seeding, fibronectin was adsorbed selectively onto ablated domains at 20 degrees C, a condition in which the non-ablated grafted PIPAAm matrix remains highly hydrated. Hepatocytes seeded specifically adhered onto the ablated domains adsorbed with fibronectin. Because PIPAAm, inhibits cell adhesion and migration even at 37 degrees C when the grafted density is > 3 microg/cm2, all the cells were confined within the ablated domains. A 100-cell domain array was achieved by this method. This surface modification technique can be utilized for fabrication of cell-based biosensors as well as tissue-engineered constructs.     

Micropatterned surfaces modified with select peptides promote exclusive interactions with osteoblasts

Microcontact printing techniques were used to pattern circles (diameters 10. 50, 100, and 200 microm) of N1[3-(trimethoxysilyl)-propyl]diethylenetriamine (DETA) surrounded by octadecyltrichlorosilane (OTS) borders on borosilicate glass, a model substrate. The DETA regions were further modified by immobilization of either the cell-adhesive peptides Arginine-Glycine-Aspartic Acid-Serine (RGDS) and Lysine-Arginine-Serine-Arginine (KRSR) or the non-adhesive peptides Arginine-Aspartic Acid-Glycine-Serine (RDGS) and Lysine-Serine-Serine-Arginine (KSSR). After four hours under standard cell culture conditions but in the absence of serum, adhesion of either osteoblasts or fibroblasts on surfaces patterned with the non-adhesive peptides RDGS and KSSR was random and low. In contrast, both osteoblasts and fibroblasts adhered and formed clusters onto circles modified with the adhesive peptide RGDS, whereas only osteoblasts adhered and formed clusters onto the circles modified with KRSR, a peptide that selectively promotes adhesion of osteoblasts. These results provide evidence that patterning of select peptides can direct adhesion of specific cell lines exclusively to predetermined regions on material surfaces.     

Directed cell attachment by tropoelastin on masked plasma immersion ion implantation treated PTFE

The ability to generate cell patterns on polymer surfaces is critical for the detailed study of cellular biology, the fabrication of cell-based biosensors, cell separation techniques and for tissue engineering. In this study contact tape masking and steel shadow masks were used to exclude plasma immersion ion implantation (PIII) treatment from defined areas of polytetrafluoroethylene (PTFE) surfaces. This process enabled patterned covalent binding of the cell adhesive protein, tropoelastin, without employing chemical linking molecules. Tropoelastin coating rendered the untreated regions cell adhesive and the PIII-treated area non-adhesive, allowing very fine patterning of cell adhesion to PTFE surfaces. A blocking step, such as with BSA or PEG, was not required to prevent cell binding to the underlying PIII-treated regions as tropoelastin coating alone performed this blocking function. Although tropoelastin coated the entire PTFE surface, the cell binding C-terminus of tropoelastin was markedly less solvent exposed on the PIII-treated, hydrophilic regions. The differential exposure of the C-terminus correlated with the patterned distribution of tropoelastin-mediated cell adhesion. This new methodology specifically enables directed cell behavior on a polymer surface using a simple one-step treatment process, by modulating the adhesive activity of a single extracellular matrix protein.