The selective modulation of endothelial cell mobility on RGD peptide containing surfaces by YIGSR peptides

The ability of the biomimetic peptides YIGSR, PHSRN and RGD to selectively affect adhesion and migration of human microvascular endothelial cells (MVEC) and vascular smooth muscle cells (HVSMC) was evaluated. Cell mobility was quantified by time-lapse video microscopy of single cells migrating on peptide modified surfaces. Polyethylene glycol (PEG) hydrogels modified with YIGSR or PHSRN allowed only limited adhesion and no spreading of MVEC and HVSMC. However, when these peptides were individually combined with the strong cell binding peptide RGD in PEG hydrogels, the YIGSR peptide was found to selectively enhance the migration of MVEC by 25% over that of MVEC on RGD alone (p<0.05). No corresponding effect was observed for HVSMC. This suggests that the desired response of specific cell types to tissue engineering scaffolds could be optimized through a combinatory approach to the use of biomimetic peptides.     

Hepatocyte behavior on synthetic glycopolymer matrix: inhibitory effect of receptor-ligand binding on hepatocyte spreading

The interaction of carbohydrate-based polymers with asialoglycoprotein receptors (ASGPRs) on the surface of hepatocytes has been used to design hepatocyte adhesion matrices. Therefore, we have characterized the interaction of ASGPR on the surface of hepatocytes with glycopolymer-coated surfaces. Since ASGPRs bound to glycopolymer surfaces escape from internalization and degradation, they were quantified by western blot analysis. The amount of hepatocyte ASGPRs that initially adhered to the glycopolymer surface was proportional to the concentration of the coated glycopolymer. We found that the initial adhesion of hepatocytes to the glycopolymer surface was enhanced by interactions with ASGPR, whereas interactions with ASGPR inhibited the post-adhesion process, a cell adhesion phenomenon that occurs following the initial adhesion. Furthermore, hepatocytes are much more spread on glycopolymer surfaces with lower coating density. Taken together, we suggest that the post-adhesion process triggered hepatocyte spreading on glycopolymer surfaces, and ASGPR-carbohydrate interactions act negatively on the post-adhesion mechanism as well as on hepatocyte spreading on glycopolymer surfaces depending on the density of coated glycopolymers.     

The biological response of poly(L-lactide) films modified by different biomolecules: role of the coating strategy

The interactions between the surface of synthetic scaffolds and cells play an important role in tissue engineering applications. To improve these interactions, two strategies are generally followed: surface coating with large proteins and surface grafting with small peptides. The proteins and peptides more often used and derived from the extracellular matrix, are fibronectin, laminin, and their active peptides, RGD and SIKVAV, respectively. The aim of this work was to compare the effects of coating and grafting of poly(L-lactide) (PLLA) films on MRC5 fibroblast cells. Grafting reactions were verified by X-ray photoelectron spectroscopy. Cell adhesion and proliferation on coated and grafted PLLA surfaces were measured by cell counting. Vinculin localization and distribution were performed on cell cultured on PLLA samples using a fluorescence microscopy technique. Finally, western blot was performed to compare signals of cell adhesion proteins, such as vinculin, Rac1, and RhoA, as well as cell proliferation, such as PCNA. These tests showed similar results for fibronectin and laminin coated PLLA, while RGD grafting is more effective compared with SIKVAV grafting. Considering the overall view of these results, although coating and grafting can both be regarded as effective methods for surface modification to enhance cell adhesion and proliferation on a biomaterial, RGD grafted PLLA show better cell adhesion and proliferation than coated PLLA, while SIKVAV grafted PLLA show similar adhesion but worse proliferation. These data verified different biological effects depending on the surface modification method used.     

Enhancing growth human endothelial cells on Arg-Gly-Asp (RGD) embedded poly (epsilon-caprolactone) (PCL) surface with nanometer scale of surface disturbance

To explore the application of PCL for the engineering of soft tissues, the PCL surface was first embedded in an amphiphilic moiety and then grafted with RGD peptide to enhance the growth rate of human endothelial cells (HUVEC) on the surface. To graft cell-adhesive peptide RGD on the PCL surface, the PCL surface was first etched by the selected solvent with only nanometer-scale of surface disturbance, and simultaneously embedded with DSPE-PEG [di-stearoyl-phosphatidyl-ethanolamine-methoxy-poly (ethylene glycol)] moiety. Then the PCL-PEG surface was photochemically grafted by GRGD to form PCL-PEG-RGD surface. PCL and the modified surfaces were characterized by surface morphology, surface disturbance, contact angles, ATR-FTIR functional group analysis, and the growth rate of HUVEC. The surface disturbances of PCL and the modified surfaces were examined by atomic force microscope (AFM) and presented by the topography and a roughness parameter, Ra. The Ra values were 16.4 +/- 3.0, 34.8 +/- 1.6, and 12.8 +/- 0.3 nm (n = 3) for PCL, PCL-PEG, and PCL-PEG-RGD surfaces, respectively. The topographies of the surfaces and Ra values indicated that the PCL modified technique developed by this study resulted in only nanometer scale of surface disturbance. In addition to reducing surface disturbances, reducing contact angle from 73.7 degrees +/- 0.4 (n = 3) for the PCL surface to 56.9 degrees +/- 4.0 (n = 3) for the PCL-PEG surface, and the ATR-FTIR transmission spectra at 1660 cm(-1) for shoulder of amine I of PCL-PEG-RGD surface both confirmed the successful modification of PCL surfaces. HUVECs adhered well and grew on the PCL-PEG-RGD surface after 36 h incubation, whereas other surfaces did not support growth. Moreover, the viability for the relative growth rate of HUVECs on the PCL-PEG-RGD surface analyzed by MTT assay showed 8.5 times greater growth than that of the unmodified one. In conclusion, a PCL-PEG-RGD surface for enhancing the growth rate of HUVECs has been prepared by a new technique that caused only a nanometer-scale of surface disturbance. This technique and the PCL-PEG-RGD surface could be further applied to engineer soft tissues.     

Suppression of apoptosis by enhanced protein adsorption on polymer/hydroxyapatite composite scaffolds

Bone tissue engineering is a promising alternative to bone grafting. Scaffolds play a critical role in tissue engineering. Composite scaffolds made of biodegradable polymers and bone mineral-like inorganic compounds have been reported to be advantageous over plain polymer scaffolds by our group and others. In this study, we compared cellular and molecular events during the early periods of osteoblastic cell culture on poly(l-lactic acid)/hydroxyapatite (PLLA/HAP) composite scaffolds with those on plain PLLA scaffolds, and showed that PLLA/HAP scaffolds improved cell survival over plain PLLA scaffolds. Most cells (MC3T3-E1) on PLLA/HAP scaffolds survived the early culture. In contrast, about 50% of the cells initially adhered to the plain PLLA scaffolds were detached within the first 12h and showed characteristics of apoptotic cell death, which was confirmed by TUNEL staining and caspase-3 activation. To investigate the mechanisms, we examined the adsorption of serum protein and adhesion molecules to the scaffolds. The PLLA/HAP scaffold adsorbed more than 1.4 times of total serum protein and much greater amounts of serum fibronectin and vitronectin than pure PLLA scaffolds. Similarly, significantly larger amounts of individual adhesion proteins and peptides (fibronectin, vitronectin, RGD, and KRSR) were adsorbed on the PLLA/HAP scaffolds than on the PLLA scaffolds, which resulted in higher cell density on the PLLA/HAP scaffolds. Furthermore, beta1 and beta3 integrins and phosphorylation of Fak and Akt proteins in the cells on the PLLA/HAP scaffolds were significantly more abundent than those on PLLA scaffolds, which suggest that enhanced adsorption of serum adhesion proteins to PLLA/HAP scaffolds protect the cells from apoptosis possibly through the integrin-FAK-Akt pathway. These results demonstrate that biomimetic composite scaffolds are advantageous for bone tissue engineering.     

Galactosylated chitosan as a synthetic extracellular matrix for hepatocytes attachment

Galactose moiety as the hepatocyte anchorage was covalently coupled with chitosan for the development of synthetic extracellular matrix. Hepatocytes adhesion to galactosylated chitosan (GC)-coated polystyrene (PS) dish became as high as 94.7% after 2 h incubation whereas the hepatocytes adhesion to chitosan-coated PS dish was 69.1%, indication of galactose-specific recognition between GC molecules and asialoglycoprotein receptors of hepatocytes. The DNA synthesis of the hepatocytes adhered to GC-coated dish was increased in the presence of epidermal growth factor (EGF) at low concentration of GC (0.05 microg/ml) whereas the DNA synthesis of the hepatocytes adhered to GC-coated dish was decreased in the presence of EGF at high concentration of GC (5 microg/ml). The spreading shapes of the hepatocytes adhered to the surface in the presence of EGF at low concentration of GC (0.05 microg/ml) were enhanced than in the absence of EGF. The hepatocytes adhered to the surface at high concentration of GC (5 microg/ml) showed round shapes and exhibited many spheroid formation after 24 h in the presence of EGF.     

RGD-grafted thermoreversible polymers to facilitate attachment of BMP-2 responsive C2C12 cells

The purpose of this study was to design thermoreversible biomaterials for enhanced adhesion of bone morphogenetic protein-2 (BMP-2)-responsive cells. Peptides containing the arginine-glycine-aspartic acid (RGD) sequence were conjugated to N-isopropylacrylamide (NiPAM) polymers via amine-reactive N-acryloxysuccinimide (NASI) groups. In monolayer cultures, the adhesion of BMP-2-responsive C2C12 cells to RGD-grafted NiPAM/NASI surfaces was significantly higher than adhesion on ungrafted NiPAM/NASI surfaces. Although the morphology of cells adhered to RGD-grafted NiPAM/NASI surfaces was comparable to cells adhered on tissue culture polystyrene (TCPS), long-term cell growth was limited on the NiPAM/NASI surfaces, even for RGD-grafted surfaces. Treatment of C2C12 cells with recombinant BMP-2 induced dose-dependent osteoblastic differentiation as assessed by alkaline phosphatase (ALP) activity. In the absence of BMP-2, cells cultured on NiPAM/NASI polymers (either grafted with RGD peptide or not) expressed significantly higher levels of ALP activity than the cells cultured on TCPS, indicating that the polymer surfaces induced some osteoblastic activity in C2C12 cells without the need for BMP-2. We conclude that NiPAM-based thermoreversible biomaterials, despite their limited ability to support cell growth, allowed an enhanced expression of the chosen osteogenic marker (ALP) by C2C12 cells in vitro.     

Hepatocyte performance on different crystallographic faces of rutile

The influence of crystallographic orientation of polished rutile single crystal surfaces of the (100), (110) and (111) orientation on hepatocyte performance was tested in cell culture over 3 days. Cell adhesion was observed on the titanium dioxide surfaces and their performance was measured by means of cell number attached (protein mass), cell viability (neutral red assays) and metabolic activity (thiazolyl tetrazolium bromide assay). Titanium dioxide displays no cytotoxic effects on hepatocytes, and shows a performance in the range of standard collagen-coated tissue culture polystyrene dish. The number of hepatocytes adhered on the different rutile surfaces were not significantly different to those on dense rutile polycrystalline ceramic. These findings suggest that hepatocytes do not recognize the specific differences of differently orientated rutile crystal surfaces.     

Adhesive and mechanical properties of hydrogels influence neurite extension

Photopolymerizable polyethylene glycol (PEG) hydrogels conjugated with bioactive ligands were examined for their use as scaffolds in peripheral nerve regeneration applications. The bioactivity and mechanical properties of PEG hydrogels can be tailored through the integration of bioactive factors (adhesion ligands, proteolytic sites, growth factors) and the alteration of PEG concentrations, respectively. For peripheral nerve regeneration, it will be important to determine the type and concentration of the bioactive molecules required to improve neurite extension. In this study, cell adhesion ligands (RGDS, IKVAV, and YIGSR) were covalently attached to PEG hydrogels. Both the type and concentration of cell adhesion ligand used affected neurite extension. Extension from PC12 cells was greater on hydrogels with RGDS incorporated than IKVAV, and the optimal concentration for each ligand was different. Cells adhered to but did not extend neurites on hydrogels with YIGSR. Cells did not adhere to hydrogels containing RGES. Furthermore, different combinations of these ligands affected neurite extension to different degrees. The mechanical properties of the hydrogels also significantly affected neurite extension. PC12 cells grown on more flexible hydrogels exhibited the greatest degree of neurite extension. PEG hydrogels have thus been developed with varying biochemical and mechanical properties that may enhance nerve regeneration.     

Endothelialization of microporous YIGSR/PEG-modified polyurethaneurea

Bioactive polyurethaneurea modified with polyethylene glycol (PEG) and the endothelial cell-adhesive peptide YIGSR was synthesized and fabricated into microporous scaffolds. This material has shown appropriate mechanical properties for vascular graft applications, resists platelet adhesion, and promotes endothelialization. In the current study, microporous scaffolds were formed by a gasfoaming and salt-leaching method. The scaffolds showed highly interconnected open pores throughout the matrices, with porosity of approximately 78% and pore sizes of 20-200 microm. The peptide modified scaffolds showed superior mechanical properties over peptide-free scaffolds (tensile strength, 1.4 +/- 0.03 versus 0.19 +/- 0.01 MPa; p < 0.01). Bovine aortic endothelial cells were seeded on the scaffolds, and cell attachment, proliferation, extracellular matrix production, and migration were investigated. Histological and scanning electron microscopy analysis showed that few cells adhered on peptide-free scaffolds, whereas confluent endothelial cell monolayers formed along the pores in peptide-modified scaffolds. DNA content, hydroxyproline production, and cell migration were also significantly greater in peptide-modified scaffolds.     

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.     

Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials.

The attachment, spreading, spreading rate, focal contact formation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) were investigated on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR). This approach was used to provide substrates that were adhesive to cells even in the absence of serum proteins and with no prior pretreatment of the surface with proteins of the cell adhesion molecule (CAM) family. This approach was used to dramatically enhance the cell-adhesiveness of substrates that were otherwise cell-nonadhesive and to improve control of cellular interactions with cell-adhesive materials by providing stably bound adhesion ligands. Glycophase glass was examined as a model cell-nonadhesive substrate prior to modification, and polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) were examined as representative materials for biomedical applications. The peptides were surface-coupled by their N-terminal amine to surface hydroxyl moieties using tresyl chloride chemistry. Prior to peptide grafting, the PET and PTFE were surface hydroxylated to yield PET-OH and PTFE-OH. The PET-OH was less cell-adhesive and the PTFE-OH was much more cell-adhesive than the native polymers. Radioiodination of a C-terminal tyrosine residue was used to quantify the amount of peptide coupled to the surface, and these amounts were 12.1 pmol/cm2 on glycophase glass, 139 fmol/cm2 on PET-OH, and 31 fmol/cm2 on PTFE-OH. Although the glycophase glass did not support adhesion or spreading even in the presence of serum, the RGD- and YIGSR-grafted glycophase glass did support adhesion and spreading, even when the only serum protein that was included was albumin. Although PET and PTFE-OH supported adhesion when incubated in serum-supplemented medium, neither of these materials supported adhesion with only albumin present, indicating that cell adhesion is mediated by adsorbed CAM proteins. When these materials were peptide-grafted, however, extensive adhesion and spreading did occur even when only albumin was present. Since the peptide grafting is quite easily controlled and is temporally stable, while protein adsorption is quite difficult to precisely control and is temporally dynamic, peptide grafting may be advantageous over other approaches employed to improve long-term cell adhesion to biomaterials.     

The interaction between bone marrow stromal cells and RGD-modified three-dimensional porous polycaprolactone scaffolds

We previously established a simple method to immobilize the Arg-Gly-Asp (RGD) peptide on polycaprolactone (PCL) two-dimensional film surfaces that significantly improved bone marrow stromal cell (BMSC) adhesion to these films. The current work extends this modification strategy to three-dimensional (3D) PCL scaffolds to investigate BMSC attachment, cellular distribution and cellularity, signal transduction and survival on the modified PCL scaffold compared to those on the untreated ones. The results demonstrated that treatment of 3D PCL scaffold surfaces with 1,6-hexanediamine introduced the amino functional groups onto the porous PCL scaffold homogenously as detected by a ninhydrin staining method. Followed by the cross-linking reaction, RGDC peptide was successfully immobilized on the surface of PCL scaffold. Although the static seeding method used in this study caused heterogeneous cell distribution, the RGD-modified PCL scaffold still demonstrated the improved BMSC attachment and cellular distribution in the scaffold. More importantly, the integrin-mediated signal transduction FAK–PI3K–Akt pathway was significantly up-regulated by RGD modification and a subsequent increase in cell survival and growth was found in the modified scaffold. The present study introduces an easy method to immobilize RGD peptide on the 3D porous PCL scaffold and provides further evidence that modification of 3D PCL scaffolds with RGD peptides elicits specific cellular responses and improves the final cell–biomaterial interaction.     

Effect of RGD secondary structure and the synergy site PHSRN on cell adhesion, spreading and specific integrin engagement

The relationship between the form of cell adhesion, ligand presentation, and cell receptor function was characterized using model Langmuir-Blodgett supported films, containing lipid-conjugated peptide ligands, in which isolated variables of the ligand presentation were systematically altered. First, the conformation of an adhesive Arginine-Glycine-Aspartic acid (RGD) peptide was varied by synthesizing linear and looped RGD peptide-containing amphiphiles and subsequently measuring the impact on the function of human umbilical vein endothelial cells. Secondly, the contribution of non-contiguous ligands to cellular engagement was assessed using multi-component biomimetic films. The peptide amphiphiles were composed of fibronectin-derived headgroups--GRGDSP, and its synergy site Pro-His-Ser-Arg-Asn (PHSRN)--attached to hydrocarbon tails. The peptide amphiphiles were diluted using polyethylene glycol (PEG) amphiphiles, where PEG inhibited non-specific cell adhesion. Cells adhered and spread on GRGDSP/PEG systems in a dose-dependent manner. The presentation of GRGDSP influenced integrin cell surface receptor specificity. Results demonstrated that beta1-containing integrins mediated adhesion to the linear GRGDSP presentation to a greater extent than did the alphavbeta3 integrin, and looped GRGDSP preferentially engaged alphavbeta3. GRGDSP/PHSRN/PEG mixtures that closely mimicked the RGD-PHSRN distance in fibronectin, enhanced cell spreading over their two-component analogues. This study demonstrated that controlling the microenvironment of the cell was essential for biomimetics to modulate specific binding and subsequent signaling events.     

Tissue anti-adhesion potential of ibuprofen-loaded PLLA-PEG diblock copolymer films

This study was designed to evaluate the effect of polyethylene glycol (PEG) and nonsteroidal anti-inflammatory drug (ibuprofen) on the prevention of postsurgical tissue adhesion. For this, poly(L-lactic acid) (PLLA)-PEG diblock copolymers were synthesized by ring opening polymerization of L-lactide and methoxy polyethylene glycol (Mw 5000) of different compositions. The synthesized copolymers were characterized by gel permeation chromatography and 1H-nuclear magnetic resonance spectroscopy. PLLA-PEG copolymer films were prepared by solvent casting. The prepared copolymer films were more flexible and hydrophilic than the control PLLA film, as investigated by the measurements of glass transition temperature, water absorption content, and water contact angle. The drug release behavior from the ibuprofen (10 wt%)-loaded copolymer films was examined by high performance liquid chromatography. It was observed that the drug was released gradually up to about 40% of total loading amount after 20 days, depending on PEG composition; more drug release from the films with higher PEG compositions. In vitro cell adhesions on the copolymer films with/without drug were compared by the culture of NIH/3T3 mouse embryo fibroblasts on the surfaces. For in vivo evaluation of tissue anti-adhesion potential, the copolymer films with/without drug were implanted between the cecum and peritoneal wall defects of rats and their tissue adhesion extents were compared. It was observed that the ibuprofen-containing PLLA-PEG films with high PEG composition (particularly PLLA113-PEG113 film with PEG composition, 50 mol%) were very effective in preventing cell or tissue adhesion on the film surfaces, probably owing to the synergistic effects of highly mobile, hydrophilic PEG and anti-inflammatory drug, ibuprofen.     

Biocompatibility of poly(epsilon-caprolactone)/poly(ethylene glycol) diblock copolymers with nanophase separation

In this study, we prepared diblock copolymers of poly(epsilon-caprolactone) (PCL) and poly(ethylene glycol) (PEG) by aluminum alkoxide catalysts. The biological responses to the spin cast surface of different PCL/PEG diblock copolymers were investigated in vitro. Our results showed that surface hydrophilicity improved with the increased PEG segments in diblock copolymers and that bacteria adhesion was inhibited by increased PEG contents. PCL-PEG 23:77 showed nanotopography on the surface. The number of adhered endothelial cells, platelets and monocytes on diblock copolymer surfaces was inhibited in PCL-PEG 77:23 and enhanced in PCL-PEG 23:77. Nevertheless, the platelet and monocyte activation on PCL-PEG 23:77 was reduced. PCL-PEG 23:77 had better cellular response as well as lower degree of platelet and monocyte activation. The current study was the first one to demonstrate that surface nanotopography could influence not only cell adhesion and growth but also platelet and monocyte activation.     

Cellular response to oxygen containing biomedical polymers modified by Ar and He implantation

Ion beam modification is an attractive way to adapt the response of a biopolymer surface with the view to modifying cellular processes. In this work we performed Ar and He implantations into three oxygen-containing biomedical polymers: polycaprolactone (PCL), poly(ethylene glycol) (PEG) and poly(methyl methacrylate) (PMMA). An ion energy of 25keV was selected on the basis of singularities observed in simulated implantations. The implantations were carried out with fluences of 5x10(13) cm(-2) considering also the ion current density as a source of differentiated damage. The modification of the polymer structure and composition was assayed by Fourier transform infrared spectroscopy, which confirmed the selectivity of the ion current density in producing polymer film damage. Biomedical assays denoted lack of structural stability on the PMMA surfaces. Surface analysis of proteins adsorbed from fetal bovine serum on ion-beam-modified PEG were realized by quartz-crystal microbalance with dissipation, which supported the film stabilization and anti-fouling behaviour of the films. On the other hand, protein adsorption studies on micropatterned PCL surfaces were performed by time-of-flight secondary ion mass spectroscopy and revealed a clear enhancement of protein immobilization in ion-beam-modified areas. The response of human mesenchymal stem cells to the surfaces was observed to depend on the biopolymer characteristics, showing adhesion inhibition onto He-modified PEG and specially enhanced colonization onto He-irradiated PCL.     

Platelet and cell interactions on gold sputter-deposited polymeric surfaces

Surface treatment as gold sputter-deposited treatment onto various polymeric surfaces has been investigated to improve the cell-, tissue- and blood-compatibility. Surface treated samples were characterized by measurement of contact angle goniometer and electron spectroscopy for chemical analysis (ESCA). The contact angles on the gold-coated polymeric surfaces decreased from 95-65 degrees to around 50 degrees, i.e., increased hydrophilicity due to incorporation of gold thin layer. From the results of ESCA analysis of the modified polymeric surfaces, surface modification by the gold-sputter method was successfully performed. Morphology of the adhered platelets on the gold-coated polymeric surfaces showed lesser activating than control, and the number of adhered platelets surface modified samples decreased with decreasing water contact angle. Fibroblast cell adhesion and growth on the gold-coated polymeric surfaces were more active than those of control. It seems that surface wettability and surface chemistry of gold play important roles for platelet adhesion and cell adhesion, spreading and growth.     

Selective adhesion of astrocytes to surfaces modified with immobilized peptides.

Under serum-free conditions, rat skin fibroblasts, but not cortical astrocytes, selectively adhered to glass surfaces modified with the integrin-ligand peptide RGDS. In contrast, astrocytes, but not fibroblasts, exhibited enhanced adhesion onto substrates modified with KHIFSDDSSE, a peptide that mimics a homophilic binding domain of neural cell adhesion molecule (NCAM). Astrocyte and fibroblast adhesion onto substrates modified with the integrin ligands IKVAV and YIGSR as well as the control peptides RDGS and SEDSDKFISH were similar to that observed on aminophase glass (reference substrate). This study is the first to demonstrate the use of immobilized KHIFSDDSSE in selectively modulating astrocyte and fibroblast adhesion on material surfaces, potentially leading to materials that promote specific functions of cells involved in the response(s) of central nervous system tissues to injury. This information could be incorporated into novel biomaterials designed to improve the long-term performance of the next generation of neural prostheses.     

Enhancement of the growth of human endothelial cells by surface roughness at nanometer scale

This study investigated whether a nanometer scale of surface roughness could improve the adhesion and growth of human endothelial cells on a biomaterial surface. Different molecular weights or chain lengths of polyethylene glycol (PEG) were mixed and then grafted to a polyurethane (PU) surface, a model smooth surface, to form a nanometer (nm) scale of roughness for PU-PEG surfaces (PU-PEG(mix)) while PEG with a molecular weight of 2000 was also grafted to PU to form PU-PEG(2000) for comparison. In addition, the concept was tested on cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) that was photochemically grafted to PU-PEG(mix) and PU-PEG(2000) surfaces (e.g., PU-PEG(mix)-GRGD and PU-PEG(2000)-GRGD surfaces, respectively). To prepare GRGD-grafted PU-PEG(mix) and PU-PEG(2000) surface, 0.025M of GRGD-SANPAH (N-Succinimidyl-6-[4'-azido-2'-nitrophenylamino]-hexanoate) solutions was grafted to PU-PEG(mix) and PU-PEG(2000) by surface adsorption of the peptide and subsequent ultraviolet (UV) irradiation for photoreaction. The grafting efficiencies for GRGD to PU-PEG(mix) and PU-PEG(2000) surfaces were about 67% for both surfaces, semi-quantitatively analyzed by an HPLC. The surface roughness, presented with a roughness parameter, R(a), and the topography of the tested surfaces were both measured and imaged by an atomic force microscope (AFM). Among the R(a) values of the films, PU was the smoothest (e.g., R(a)=1.53+/-0.20 nm, n=3) while PU-PEG(mix) was the roughest (e.g., R(a)=39.79+/-10.48 nm, n=4). Moreover, R(a) values for PU-PEG(mix) and PU-PEG(mix)-GRGD surfaces were about 20 nm larger than those for PU-PEG(2000) and PU-PEG(2000)-GRGD, respectively, which were consistent with the topographies of the films. Human umbilical vein endothelial cells (HUVECs) were adhered and grown on the tested surfaces after 36 h of incubation. Among the films, HUVEC's adhesion on the surface of PU-PEG(mix)-GRGD was the densest while that on the surface of PU-PEG(2000) was the sparsest. Also, the adhesion and growth of HUVECs for the roughness surfaces were statistically significantly better than that of smooth surface for both GRGD grafted and un-grafted surfaces, respectively. The viability for the growth of HUVECs on the tested surfaces analyzed by MTT assay also confirmed the efficacy of the increased surface roughness.In conclusion, increased surface roughness of biomaterial surfaces even at 10-10(2) nm scale could enhance the adhesion and growth of HUVECs on roughness surfaces that could be useful for applications of tissue engineering.