A jet impingement investigation of osteoblastic cell adhesion

When designing dental and orthopedic implants, it is important to consider phenomena occurring at the microscopic level, particularly at the bone-implant interface. The presence of hard tissue at this interface is essential to implant viability. The integrity of this tissue-biomaterial interface is dependent on appropriate osteoblast functions (adhesion, matrix deposition, etc.) in the immediate area. Researchers have modified various materials with cell-adhesive peptides with the ultimate goal of controlling osteoblast functions. This study used microjet impingement to compare the strength of adhesion of osteoblastic cells (at varying populations) and fibroblasts to peptide-modified substrates in the presence and absence of fetal bovine serum. In the presence of the serum, there was no significant difference in cellular adhesion strength between substrates. In the absence of serum, all cells tested adhered more strongly to underlying substrates, and the strength of cellular adhesion was greater on modified surfaces than on plain glass surfaces. In the absence of serum, second-passage osteoblastic cells generally adhered to substrates more strongly than first-passage osteoblastic cells; fibroblasts adhered similarly to second-passage osteoblastic cells. Fundamental studies such as the present increase the understanding of cell adhesion to various substrates--knowledge that may be ultimately useful in creating an optimal bone-implant interface.     

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.     

GRGDSP peptide-bound silicone membranes withstand mechanical flexing in vitro and display enhanced fibroblast adhesion

Mechanobiological studies of cardiac tissue require devices that allow forces to be exerted on cells in vitro. Silicone elastomer is often used in these devices because it is flexible and transparent, permitting optical imaging of the cells. However, native untreated silicone is hydrophobic and is unsuitable for cell culture. Peptides covalently bound to silicone surfaces are examined here for the enhancement of cellular adhesion during in vitro dynamic flexing. A procedure is described for the chemical modification of medical grade silicone membranes with covalently bound GRGDSP peptides. The conditions for mechanical studies of cardiac cell cultures are then duplicated and it is demonstrated that the peptide layers survive 48 h of mechanical flexing in vitro. Specifically, mechanical flexing in vitro of the 30 pmol/cm2 peptide-modified silicone membranes has no significant effect on the amount of peptides that remains bound to the surface. Cardiac fibroblasts display enhanced adhesion to these peptide-bound silicone membranes for at least 24 h of growth, compared with native silicone or tissue culture polystyrene. The effects of serum versus serum-free media on fibroblast growth are also examined.     

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.     

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.     

Cell adhesion and proliferation on hydrophilic dendritically modified surfaces

Dendritically modified, or "dendronized" surfaces are generated by modification of a substrate with perfectly branched polymers, known as dendrimers. Here, such dendronized surfaces were prepared by initial chemisorption of poly(ethylene glycol)-mono-thiol (HS-PEG(650)-OH) onto gold-coated silicon wafers, followed by divergent synthesis of aliphatic polyester dendrons, generation 1-4, starting from the terminal PEG OH- group. The adhesion and proliferation of human corneal epithelial cells (HCEC) and mouse 3T3 fibroblasts (M-3T3) as model cells on these hydroxyl-terminated dendronized surfaces were investigated. In addition, the effect of covalently attaching PEG mono-methyl ether (PEG-OMe) chains (M(n)=2000Da) to the peripheral hydroxyl groups of G1- and G2-dendronized surfaces on adhesion and proliferation of the same cell lines was studied. Little or no HCEC adhesion was noted on gold surfaces modified with PEG mono-thiol (HO-PEG-SH) in serum-free medium. These cells showed a greater affinity for the dendronized surfaces compared to the control Au surfaces at early incubation stages (1 day). At longer incubation times, HCEC proliferation increased exponentially on the dendronized surfaces. However, when G1- and G2-dendronized surfaces were modified with PEG-OMe chains, adhesion of both HCEC and M-3T3 cells was significantly reduced. Cell studies with M-3T3 fibroblasts, carried out in serum-containing medium, showed that cell attachment was diminished for the PEG-grafted Au surfaces compared to the control Au and G1-G4 dendronized surfaces.     

Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition

The goal of the current study was to evaluate matrix protein synthesis by cells cultured on materials that had been modified with cell adhesion ligands. We examined the effects of surface peptide density and of peptides with different affinities on the extracellular matrix production of smooth muscle cells, endothelial cells and fibroblasts. While initial adhesion was greatest on the higher density peptide surfaces, all cell types exhibited decreased matrix production on the more highly adhesive surfaces. Similarly, when different peptides were evaluated, matrix production was the lowest on the most adhesive surface and highest on the least adhesive surface. These results suggest that extracellular matrix synthesis may be regulated, to some extent, by signal transduction initiated by adhesion events. This may pose limitations for use of bioactive materials as tissue engineering scaffolds, as matrix production is an important aspect of tissue formation. However, it may be possible to increase matrix production on highly adhesive surfaces using exogenous factors. TGF-beta was shown to increase matrix production by both smooth muscle cells and endothelial cells.     

Interactions of corneal epithelial cells and surfaces modified with cell adhesion peptide combinations

In order to facilitate the adhesion of corneal epithelial cells to a poly dimethyl siloxane (PDMS) substrate ultimately for the development of a synthetic keratoprosthesis, PDMS surfaces were modified by covalent attachment of combinations of cell adhesion and synergistic peptides derived from laminin and fibronectin. Peptides studied included YIGSR and its synergistic peptide PDSGR from laminin and the fibronectin derived RGDS and PHSRN. Surfaces were modified with combinations of peptides determined by an experimental design. Peptide surface densities, measured using 125-I labeled tyrosine containing analogs, were on the order of pmol/cm2. Surface density varied as a linear function of peptide concentration in the reaction solution, and was different for the different peptides examined. The lowest surface density at all solution fractions was obtained with GYRGDS, while the highest density was consistently obtained with GYPDSGR. These results provide evidence that the surfaces were modified with multiple peptides. Water contact angles and XPS results provided additional evidence for differences in the chemical composition of the various surfaces. Significant differences in the adhesion of human corneal epithelial cells to the modified surfaces were noted. Statistical analysis of the experimental adhesion results suggested that solution concentration YIGSR, RGDS, and PHSRN as well as the interaction effect of YIGSR and PDSGR had a significant effect on cell interactions. Modification with multiple peptides resulted in greater adhesion than modification with single peptides only. Surface modification with a control peptide PPSRN in place of PHSRN resulted in a decrease in cell adhesion in virtually all cases. These results suggest that surface modification with appropriate combinations of cell adhesion peptides and synergistic peptides may result in improved cell surface interactions.     

Controlling osteopontin orientation on surfaces to modulate endothelial cell adhesion

Osteopontin (OPN) is an important extracellular matrix protein that has been shown to impact wound healing, inflammation, and the foreign body reaction, and has been identified as a potential surface component for engineered biomaterials. OPN contains the arginine-glycine-aspartic acid (RGD) moiety that has been shown to mediate cell adhesion through interactions with integrins. In its preferred orientation and conformation on a surface, the functional domains of OPN will be presented to cells to the greatest extent. However, control of protein orientation and conformation is still challenging. In this work, we investigated OPN adsorption and cell adhesion to the OPN layer on self-assembled monolayers (SAMs) of alkanethiols terminated with various functional groups and on a gold surface. The four SAM terminal groups studied were --CH3, --OH, --NH2, and --COOH, representing hydrophobic, hydrophilic but neutral, positively charged, and negatively charged surfaces, respectively. Surface plasmon resonance biosensor and atomic force microscopy were used to characterize the adsorption of OPN on these surfaces. An in vitro cell adhesion assay of bovine aortic endothelial cells was performed to test the functionality of OPN on various SAMs. Surface plasmon resonance results showed that the amount of protein adsorbed on the --NH2 surface is close to a monolayer and similar to that on the --COOH surface, consistent with the atomic force microscopy results. However, based on cell adhesion experiments, both cell count and average cell spreading area on the --NH2 surface are much higher than those on the --COOH surface. From these results, it is suggested that the orientation and conformation of OPN on a positively charged --NH2 surface is more favorable for cell adhesion and spreading than on a negatively charged --COOH surface. The surface coverage of bovine aortic endothelial cells on the surfaces studied decreased in the following order: --NH2 > Au > --CH3 > --COOH > --OH whereas the mean cell spreading area decreased in the following order: --NH2 > Au > --CH3 > --COOH. Our studies show that surface properties will alter OPN behavior on surfaces, thus influencing cell interactions.     

Controlling the orientation of bone osteopontin via its specific binding with collagen I to modulate osteoblast adhesion

Osteopontin (OPN) is an important matricellular protein that modulates cell functions. It is potentially an excellent surface-coating component for engineered biomaterials. It is believed that in its preferred orientation and conformation on a surface, the functional domains of OPN such as the arginine-glycine-aspartic acid (RGD) motif will be presented to cells to the greatest extent. Previously, the authors demonstrated that OPN orientation could be modulated by surface charge. In this work, the authors attempt to control the orientation/conformation of bone OPN via its specific interactions with type I collagen. Surface plasmon resonance was used to confirm the specific binding between bone OPN and collagen I. A radiolabeled OPN adsorption assay was used to determine the amount of adsorbed OPN on tissue culture polystyrene (TCPS) surfaces with or without collagen I as an interlayer. An in vitro cell adhesion assay using osteoblast MC3T3-E1 was performed to compare the functionality of collagen-bound OPN and adsorbed OPN on TCPS. With the same amount of OPN on the surfaces, the number of cells adhered to collagen-bound OPN is significantly higher than to OPN alone on TCPS. A cell inhibition assay using soluble GRGDSP peptides showed that a higher GRGDSP concentration was needed to completely block osteoblast adhesion to collagen-bound OPN than to OPN directly on TCPS. Enhanced cell adhesion and higher blocking peptide concentration suggest that collagen-bound bone OPN has a preferable orientation/conformation for cell adhesion compared with OPN alone on TCPS. Thus, the specific binding of OPN to collagen I may naturally orient OPN, thus influencing osteoblast adhesion.     

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.     

Platelet inhibition and endothelial cell adhesion on elastin-like polypeptide surface modified materials

Platelet adhesion and activation are important early markers of biomaterial blood compatibility, while surfaces that promote enhanced endothelial cell adhesion and eNOS expression are strategic targets for long term vascular graft applications. Materials surface modified with fluorinated surface modifiers, containing peptides inspired from elastin cross-linking domains, have been used for the cross-linking of elastin-like polypeptide 4 (ELP4) macromolecules onto polyurethane surfaces. In the present study, ELP4 modified polyurethanes were evaluated in vitro to assess platelet adhesion, microparticle formation and bulk platelet activation following blood-material interactions. Reduced platelet adhesion and bulk platelet activation were observed following contact between reconstituted human blood and the ELP4 materials, relative to the uncoated base polyurethane controls. ELP4 modified materials also promoted endothelial cell adhesion and retention over a period of one week and showed that the endothelial cells exhibited an organized actin cytoskeleton and enhanced endothelial nitric oxide synthase (eNOS) expression relative to the control surfaces. These results indicate that polyurethane elastomers modified with ELP4 covalently bound to fluorinated surface modifiers provide a promising approach for endowing synthetic elastomers with both reduced blood platelet activation properties and enhanced endothelial cell adhesion for potential use in vascular graft applications.     

Improved endothelial cell adhesion and proliferation on patterned titanium surfaces with rationally designed, micrometer to nanometer features

Previous in vitro studies have demonstrated increased vascular endothelial cell adhesion on random nanostructured titanium (Ti) surfaces compared with conventional (or nanometer smooth) Ti surfaces. These results indicated for the first time the potential nanophase metals have for improving vascular stent efficacy. However, considering the structural properties of the endothelium, which is composed of elongated vascular endothelial cells aligned with the direction of blood flow, it has been speculated that rationally designed, patterned nano-Ti surface features could further enhance endothelial cell functions by promoting a more native cellular morphology. To this end, patterned Ti surfaces consisting of periodic arrays of grooves with spacings ranging from 750 nm to 100 microm have been successfully fabricated in the present study by utilizing a novel plasma-based dry etching technique that enables machining of Ti with unprecedented resolution. In vitro rat aortic endothelial cell adhesion and growth assays performed on these substrates demonstrated enhanced endothelial cell coverage on nanometer-scale Ti patterns compared with larger micrometer-scale Ti patterns, as well as controls consisting of random nanostructured surface features. Furthermore, nanometer-patterned Ti surfaces induced endothelial cell alignment similar to the natural endothelium. Since the re-establishment of the endothelium on vascular stent surfaces is critical for stent success, the present study suggests that nanometer to submicrometer patterned Ti surface features should be further investigated for improving vascular stent efficacy.     

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.     

Enhancing the neuronal interaction on fluoropolymer surfaces with mixed peptides or spacer group linkers

Embryonic hippocampal neurons cultured on surface modified fluoropolymers showed enhanced interaction and neurite extension. Poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) film surfaces were aminated by reaction with a UV-activated mercury ammonia system yielding FEP-[N/O]. Laminin-derived cell-adhesive peptides (YIGSR and IKVAV) were coupled to FEP surface functional groups using tresyl chloride activation. Embryonic (E18) hippocampal neurons were cultured in serum-free medium for up to 1 week on FEP film surfaces that were modified with either one or both of GYIGSR and SIKVAV or GGGGGGYIGSR and compared to control surfaces of FEP-[N/O] and poly(L-lysine)/laminin-coated tissue culture polystyrene. Neuron-surface interactions were analyzed over time in terms of neurite outgrowth (number and length of neurites), cell adhesion and viability. Neurite outgrowth and adhesion were significantly better on peptide-modified surfaces than on either FEP or FEP-[N/O]. Cells on the mixed peptide (GYIGSR/SIKVAV) and the spacer group peptide (GGGGGGYIGSR) surfaces demonstrated similar behavior to those on the positive PLL/laminin control. The specificity of the cell-peptide interaction was demonstrated with a competitive assay where dissociated neurons were incubated in media containing peptides prior to plating. Cell adhesion and neurite outgrowth diminished on all surfaces when hippocampal neurons were pre-incubated with dissolved peptides prior to plating.     

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.     

Effects of defensin and lactoferrin on functional activity of endothelial cells <Emphasis Type="Italic">in vitro</Emphasis>

We studied the effects of antibacterial peptides and proteins (defensins and lactoferrins) on functional activity of endothelial cells in vitro: proliferative activity and adhesion of human endothelial ECV-304 cells to the matrix were evaluated. α-Defensin (NP-2) from rabbit neutrophils, total α-defensin (HNP 1-3) from human neutrophils, and lactoferrins from porcine neutrophils and human milk were studied. Defensins stimulated and lactoferrin in doses of 1–10 μg/ml inhibited proliferation and adhesion of endothelial cell. The stimulatory effect of defensins on proliferation and adhesion was reproduced in fibroblast culture. Lactoferrins did not modify proliferation of fibroblasts, but suppressed their adhesion. These data suggest that antibiotic proteins and peptides are prospective objects for the creation of drugs regulating angiogenesis. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 9, pp. 306–309, September, 2007     

Human endothelial cell interaction with biomimetic surfactant polymers containing Peptide ligands from the heparin binding domain of fibronectin

Biomimetic materials that mimic the extracellular matrix (ECM) provide a means to control cellular functions such as adhesion and growth, which are vital to successful engineering of tissue-incorporated biomaterials. Novel "ECM-like" biomimetic surfactant polymers consisting of a poly(vinyl amine) backbone with pendant cell-adhesive peptides derived from one of the heparin-binding domains of fibronectin were developed to improve endothelial cell adhesion and growth on vascular biomaterials. Heparin-binding peptide (HBP) sequences, alone and in combination with RGD peptides, were examined for their ability to promote human pulmonary artery endothelial cell (HPAEC) adhesion and growth (HBP1, WQPPRARI; HBP2, SPPRRARVT; HBP1:RGD; and HBP2:RGD) and compared with cell adhesion and growth on fibronectin and on negative control polymer surfaces in which alanines were substituted for the positively charged arginine residues in the two peptides. The results showed that HPAECs adhered and spread equally well on all HBP-containing polymers and the positive fibronectin control, showing similar stress fiber and focal adhesion formation. However, the HBP alone was unable to support long-term HPAEC growth and survival, showing a loss of focal adhesions and cytoskeletal disorganization by 24 h after seeding. With the addition of RGD, the surfaces behaved similarly or better than fibronectin. The negative control polymers showed little to no initial cell attachment, and the addition of soluble heparin to the medium reduced initial cell adhesion on both the HBP2 and HBP2:RGD surfaces. These results indicate that the HBP surfaces promote initial HPAEC adhesion and spreading, but not long-term survival.     

Stimulated endothelial cell adhesion and angiogenesis with laminin-5 modification of expanded polytetrafluoroethylene

Biomedical implants often exhibit poor clinical performance due to the formation of a periimplant avascular fibrous capsule. Surface modification of synthetic materials has been evaluated to accelerate the formation of functional microcirculation in association with implants. The current study used a flow-mediated protein deposition system to modify expanded polytetrafluoroethylene (ePTFE) with a laminin-5-rich conditioned growth medium and with medium from which laminin-5 had been selectively removed. An in vitro model of endothelial cell adherence determined that laminin-5 modification resulted in significantly increased adhesion of human microvessel endothelial cells to ePTFE. In vivo studies evaluating the periimplant vascular response to laminin-5-treated samples indicated that absorption of laminin-5-rich conditioned medium supported accelerated neovascularization of ePTFE implants. A flow system designed to treat porous implant materials facilitates laminin-5 modification of commercially available ePTFE, resulting in increased endothelial cell adhesion in vitro and increased vascularization in vivo.     

Improved cell adhesion by plasma-induced grafting of L-lactide onto polyurethane surface

Lactide-grafted polyurethanes were prepared by exposing the polyurethane films to argon plasma discharge, followed by grafting L-lactide onto the plasma-treated surface. The modified surfaces were characterized by measuring the static contact angle and by electron spectroscopy for chemical analysis (ESCA). The water contact angle of polyurethanes was decreased by L-lactide grafting, indicating hydrophilicity of the modified surface. Grafting also increased the O/C atomic ratio and C(C=O)/Ctotal percentage on the surfaces as detected by ESCA. The grafted surfaces showed enhanced attachment and growth in both 3T3 fibroblast and human umbilical vein endothelial cell culture tests. Platelet adhesion to the modified surfaces was also reduced in vitro. L-Lactide monomers grafted onto polyurethane substrates could therefore be useful in facilitating endothelial cell seeding process in small vascular graft applications.