Adhesion of corneal epithelial cells to cell adhesion peptide modified pHEMA surfaces

Epithelialization of a corneal implant is a desirable property. In this study we compared surface modification of poly (2-hydroxyethyl methacrylate) (pHEMA) with the cell adhesion peptides RGDS and YIGSR. Various parameters in the tresyl chloride activation and modification reactions were considered in order to maximize surface coverage with the peptide including tresyl chloride reaction solvent. tresyl chloride reaction time, tresyl chloride concentration, peptide concentration, and peptide reaction pH. Surface chemistry and corneal epithelial cell adhesion to the modified surfaces were examined. X-ray photoelectron spectroscopy data suggested that while peptide modification had occurred, surface coverage with the peptide was incomplete. Acetone was found to result in a higher fraction of nitrogen and surface bound carboxyl groups compared to dioxane and ether. Furthermore, corneal epithelial cell adhesion to the surfaces for which acetone was used for the activation reaction was significantly greater. Statistical analysis of the various samples suggests that lower peptide concentrations and higher tresyl chloride reaction times result in better cell adhesion. Furthermore, modification with YIGSR resulted in higher surface concentrations and better cell adhesion than modification with RGDS. Little or no cell adhesion was noted on the unmodified pHEMA controls. Protein adsorption results suggest that the differences in cell adhesion cannot be attributed to differences in serum protein adsorption from the culture medium. We conclude that YIGSR modified surfaces have significant potential for further development in corneal applications.     

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.     

Biofunctionalization of collagen for improved biological response: scaffolds for corneal tissue engineering

Residual dendrimer amine groups were modified with incorporate COOH group containing biomolecules such as cell adhesion peptides into collagen scaffolds. YIGSR, as a model cell adhesion peptide, was incorporated into both the bulk structure of the gels and onto the gel surface. The effects of the peptide modified collagen gels on corneal epithelial cell behavior were examined with an aim of improving the potential of these materials as tissue-engineering scaffolds. YIGSR was first chemically attached to dendrimers and the YIGSR attached dendrimers were then used as collagen crosslinkers, incorporating the peptide into the bulk structure of the collagen gels. YIGSR was also attached to the surface of dendrimer crosslinked collagen gels through reaction with excess amine groups. The YIGSR modified dendrimers were characterized by H-NMR and MALDI mass spectra. The amount of YIGSR incorporated into collagen gels was determined by (125)I radiolabelling at maximum to be 3.1-3.4 x 10(-2)mg/mg collagen when reacted with the bulk and 88.9-95.6 microg/cm(2) when attached to the surface. The amount of YIGSR could be tuned by varying the amount of peptide reacted with the dendrimer or the amount of modified dendrimer used in the crosslinking reaction. It was found that YIGSR incorporation into the bulk and YIGSR modification of surface promoted the adhesion and proliferation of human corneal epithelial cells as well as neurite extension from dorsal root ganglia.     

Corneal epithelial cell growth over tethered-protein/peptide surface-modified hydrogels

In this study, we investigated the corneal epithelial cell growth rate and adhesion to novel hydrogels with (1) extracellular matrix proteins [fibronectin, laminin, substance P, and insulin-like growth factor-1 (IGF-1)] and (2) peptide sequences [RGD and fibronectin adhesion-promoting peptide (FAP)] tethered to their surface on poly(ethylene glycol) (PEG) chains. The growth rate to confluence of primary rabbit cornea epithelial cells was compared for plain polymethacrylic acid-co-hydroxyethyl methacrylate (PHEMA/MAA) hydrogels, PHEMA/MAA hydrogels coated with extracellular matrix proteins or peptides, and PHEMA/MAA hydrogels with tethered extracellular matrix proteins or peptides on the surface. The development of focal adhesions by the epithelial cells grown on the surfaces was determined by F-actin staining. Little to no epithelial cell growth occurred on the plain hydrogel surfaces throughout the 15-day culture period. Of the coated hydrogels, only the fibronectin-coated surfaces showed a significant increase in cell growth compared to plain hydrogels (p < 0.009). However, even these surfaces reached a maximum of only 20% confluence. Laminin, fibronectin adhesion-promoting peptide (FAP), and fibronectin/laminin (1:1) tether-modified hydrogels all achieved 100% confluence by the end of the culture period, although the rates at which confluence was reached differed. F-actin staining showed that focal adhesions were formed for the laminin, FAP, and fibronectin/laminin tether-modified surfaces. The results support the hypothesis that tethering certain extracellular matrix proteins and/or peptides to the hydrogel surface enhances epithelial cell growth and adhesion, compared with that seen for protein-coated or plain hydrogel surfaces.     

The effects of cell-matrix interactions on encapsulated beta-cell function within hydrogels functionalized with matrix-derived adhesive peptides

The influence of matrix-derived adhesive peptide sequences on encapsulated beta-cell survival and glucose-stimulated insulin release was explored by covalently incorporating synthetic peptide sequences within a model encapsulation environment. Photopolymerized poly(ethylene glycol) (PEG) hydrogels were functionalized via the addition of acrylate-PEG-peptide conjugates to the polymer precursor solution prior to beta-cell photoencapsulation. Individual MIN6 beta-cells were encapsulated in the presence of the laminin-derived recognition sequences, IKLLI, IKVAV, LRE, PDSGR, RGD, and YIGSR, and the collagen type I sequence, DGEA. In the absence of cell-cell and cell-matrix contacts, encapsulated MIN6 beta-cell survival diminishes within one week; however, in PEG hydrogel derivatives including the laminin sequences IKLLI and IKVAV, encapsulated beta-cells exhibit preserved viability, reduced apoptosis, and increased insulin secretion. Interactions with the laminin sequences LRE, PDSGR, RGD, and YIGSR contribute to improved viability, but insulin release from these samples was not statistically greater than that from controls. MIN6 beta-cells were also encapsulated with various concentrations of IKLLI and IKVAV (0.05-5.0mm), individually, and the peptide combinations IKLLI-IKVAV, IKVAV-YIGSR, and PDSGR-YIGSR to explore synergistic effects. The presented results give evidence that synthetic peptide epitopes may be useful in the design of an islet encapsulation environment that promotes cell survival and function via targeted cell-matrix interactions.     

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.     

Surface chemical immobilization of bioactive peptides on synthetic polymers for cardiac tissue engineering

The aim of this work was the development of new synthetic polymeric systems, functionalized by surface chemical modification with bioactive peptides, for myocardial tissue engineering. Polycaprolactone and a poly(ester-ether-ester) block copolymer synthesized in our lab, polycaprolactone–poly(ethylene oxide)–polycaprolactone (PCL–PEO–PCL), were used as the substrates to be modified. Two pentapeptides, H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) from fibronectin and H-Tyr-Ile-Gly-Ser-Arg-OH (YIGSR) from laminin, were used for the functionalization. Polymeric membranes were obtained by casting from solutions and then functionalized by means of alkaline hydrolysis and subsequent coupling of the bioactive molecules through 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride/N-hydroxysuccinimide chemistry. The hydrolysis conditions, in terms of hydrolysis time, temperature, and sodium hydroxide concentration, were optimized for the two materials. The occurrence of the coupling reaction was demonstrated by infrared spectroscopy, as the presence on the functionalized materials of the absorption peaks typical of the two peptides. The peptide surface density was determined by chromatographic analysis and the distribution was studied by infrared chemical imaging. The results showed a nearly homogeneous peptide distribution, with a density above the minimum value necessary to promote cell adhesion. Preliminary in vitro cell culture studies demonstrated that the introduction of the bioactive molecules had a positive effect on improving C2C12 myoblasts growth on the synthetic materials.     

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.     

Adhesion of alpha5beta1 receptors to biomimetic substrates constructed from peptide amphiphiles

Biomimetic membrane surfaces functionalized with fragments of the extracellular matrix protein, fibronectin, are constructed from mixtures of peptide and polyethylene glycol (PEG) amphiphiles. Peptides from the primary binding loop, GRGDSP, were used in conjunction with the synergy site peptide, PHSRN, in the III(9-10) sites of human fibronectin. These peptides were attached to dialkyl lipid tails to form peptide amphiphiles. PEG amphiphiles were mixed in the layer to minimize non-specific adhesion in the background. GRGDSP and PEG amphiphiles or GRGDSP, PHSRN, and PEG amphiphiles were mixed in various ratios and deposited on solid substrates from the air-water interface using Langmuir-Blodgett techniques. In this method, peptide composition, density, and presentation could be controlled accurately. The effectiveness of these substrates to mimic native fibronectin is evaluated by their ability to generate adhesive forces when they are in contact with purified activated alpha5beta1 integrin receptors that are immobilized on an opposing surface. Adhesion is measured using a contact mechanical approach (JKR experiment). The effects of membrane composition, density, temperature, and peptide conformation on adhesion to activated integrins in this simulated cell adhesion setup were determined. Addition of the synergy site, PHSRN, was found to increase adhesion of alpha5beta1, to biomimetic substrates markedly. Increased peptide mobility (due to increased experimental temperature) increased integrin adhesion markedly at low peptide concentrations. A balance between peptide density and steric accessibility of the receptor binding face to alpha5beta1 integrin was required for highest adhesion.     

Endothelial cell migration on surfaces modified with immobilized adhesive peptides

Endothelial cell (EC) migration has been studied on aminophase surfaces with covalently bound RGDS and YIGSRG cell adhesion peptides. The fluorescent marker dansyl chloride was used to quantify the spatial distribution of the peptides on the modified surfaces. Peptides appeared to be distributed in uniformly dispersed large clusters separated by areas of lower peptide concentrations. We employed digital time-lapse video microscopy and image analysis to monitor EC migration on the modified surfaces and to reconstruct the cell trajectories. The persistent random walk model was then applied to analyze the cell displacement data and compute the mean root square speed, the persistence time, and the random motility coefficient of EC. We also calculated the time-averaged speed of cell locomotion. No differences in the speed of cell locomotion on the various substrates were noted. Immobilization of the cell adhesion peptides (RGDS and YIGSRG), however, significantly increased the persistence of cell movement and, thus, the random motility coefficient. These results suggest that immobilization of cell adhesion peptides on the surface of implantable biomaterials may lead to enhanced endothelization rates.     

Incorporation of cell-adhesion peptides into collagen scaffolds promotes corneal epithelial stratification

The objective of this study was to examine the effects of cell-adhesion peptides incorporated into collagen scaffolds on corneal epithelial cell stratification. Peptides (YIGSR, YIGSRIKVAV, IKVAVYIGSR and negative control YISGR) were first chemically attached to dendrimers. The peptide-modified dendrimers were then used as collagen cross-linkers. This permitted the incorporation of the peptides into the bulk structure of the collagen gels. The amount of peptide incorporated into the collagen gels was determined by 125I radiolabelling to be between 0.064 and 6.4 microg/mg collagen for YIGSR, and between 0.1187 to 11.87 microg/mg collagen for YIGSRIKVAV and IKVAVYIGSR. Corneal epithelial cell monolayers were grown on the surface of the collagen scaffolds and then exposed to conditions that promoted stratification as a stratified epithelial layer is desired in a tissue-engineered cornea. It was found that all of the incorporated peptides promoted stratification of the cells with the exception of the negative control YISGR. A synergistic effect of the combined sequences from laminin was observed, with the orientation of the peptide sequences having a great impact on the ability of the materials to promote cell stratification.     

Corneal epithelial adhesion strength to tethered-protein/peptide modified hydrogel surfaces

In this study, we investigated the suitability of microjet impingement for use on hydrogel materials to determine the cellular adhesion strength of corneal epithelial cells grown on novel hydrogels with extracellular matrix proteins (laminin and/or fibronectin) or a peptide sequence (fibronectin adhesion promoting peptide, FAP) tethered to their surface with poly(ethylene glycol) chains. The deformation of the hydrogel surface in response to the force of the microjet was analyzed both visually and mathematically. After the results of these experiments and calculations determined that no deformation occurred and that the pressure required for indentation (1.25 x 10(6) Pa) was three factors of 10 greater than the maximum pressure of the microjet, the relative mean adhesion strength of primary rabbit corneal epithelial cells grown on the novel poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogels was determined and compared with that of the same type of cells grown on control glass surfaces. Only confluent cell layers were tested. Cells grown on control glass surfaces adhered with a mean relative adhesion strength of 488 +/- 28 dynes/cm2. Under identical conditions, cells grown on laminin- and FAP-tethered hydrogel surfaces were unable to be removed, indicating an adhesion strength greater than 516 dynes/cm2. Cells grown on fibronectin- and fibronectin/laminin (1:1)-tethered surfaces showed significantly lower relative adhesion strengths (201 +/- 50 and 189 +/- 11 dynes/cm2, respectively), compared with laminin- and FAP-tethered surfaces (p = 0.001). Our results demonstrate that the microjet impingement method of cell adhesion analysis is applicable to hydrogel substrates. Additionally, analysis of our test surfaces indicates that fibronectin tethered to this hydrogel in the quantity and by the method used here does not induce stable ligand/receptor bonding to the epithelial cell membrane to the same degree as does laminin or FAP.     

Bio-functionalized thermoresponsive interfaces facilitating cell adhesion and proliferation

Bio-functionalized thermoresponsive culture interfaces co-immobilized with cell adhesive peptide, RGDS, and cell growth factor, insulin (INS), are investigated to promote initial cell adhesion and cell growth for further cell sheet engineering applications. These bio-functionalized interfaces were prepared by electron beam-induced copolymerization of N-isopropylacrylamide (IPAAm) with its carboxyl-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), and grafting onto tissue culture polystyrene dishes, followed by immobilization of RGDS and/or INS to CIPAAm carboxyls. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on the RGDS-INS co-immobilized thermoresponsive interfaces. Immobilized RGDS facilitated initial EC adhesion on the surfaces and INS modification was demonstrated to induce EC proliferation, respectively. More pronounced EC growth was indicated by co-immobilization of appropriate amount of RGDS and INS. This may be due to synergistic effect of direct co-stimulation of adhered ECs by surface-immobilized RGDS and INS molecules. ECs grown on the RGDS-INS co-immobilized thermoresponsive interfaces can also be recovered spontaneously as viable tissue monolayers by solely reducing culture temperature. RGDS-INS co-immobilized thermoresponsive interfaces strongly supported initial EC adhesion and growth than unmodified thermoresponsive surfaces even under serum-free culture. Addition of soluble growth factors to serum-free culture medium effectively induced EC proliferation to confluency. Co-immobilization of cell adhesion peptides and growth factors on thermoresponsive surfaces should be effective for rapid preparation of intact cell sheets and their utilization to regenerative medicine.     

Cell adhesion peptide modification of gold-coated polyurethanes for vascular endothelial cell adhesion

Gold-coated polyurethanes were chemisorbed with three cell-adhesion peptides having an N-terminal cysteine: cys-arg-gly-asp (CRGD), cys-arg-glu-asp-val (CREDV), and the cyclic peptide cys-cys-arg-arg-gly-asp-try-leu-cys (CCRRGDWLC). The peptides were selected based on their presumed preferential interactions with the cell-surface integrins on vascular endothelial cells. The ability of the surfaces to support the preferential adhesion of human vascular endothelial cells was studied by comparing in vitro adhesion results for these cells with those from mouse 3T3 fibroblasts. Surface modification with the peptides was confirmed by water-contact angles and XPS. Surface morphology was determined by AFM and SEM. In vitro cell-culture studies in conjunction with plasma-protein adsorption and immunoblotting were performed on the various modified surfaces. The data suggest that peptide-modified surfaces have significant potential for supporting cell adhesion. Little or no cell adhesion was noted on gold- or cysteine-modified control surfaces. Human vascular endothelial cells showed the greatest adhesion to the CCRRGDWLC-modified surfaces, and the 3T3 fibroblasts adhered best to the CREDV-modified surfaces. Protein adsorption studies suggest that the preferential adsorption of the cell-adhesive proteins fibronectin and vitronectin is not likely mediating the differences noted. It is concluded that the cell-adhesive peptide-modified gold-coated polymers have significant potential for further development both as model substrates for fundamental studies and for use in biomaterials applications.     

Adhesion of human T-lymphoid cells to fibronectin is mediated by two different fibronectin domains.

Lymphocyte adhesion to components of extracellular matrices (i.e. fibronectin) is important for their proper localization in tissues and inflammatory sites. We have studied the attachment of the human cell line HUT-78 (mature T lymphocytes) to fibronectin and to several tryptic fragments of fibronectin. HUT-78 cells effectively adhered to surfaces coated with two Hep II domain-containing fragments of 38,000 and 58,000 MW derived from the A and B chains of fibronectin, respectively. Cells also bound to an 80,000 MW fragment containing the RGDS sequence of fibronectin. Cell adhesion to the 38,000 MW fragment was completely inhibited (100%) by cell preincubation with the soluble 38,000 MW fragment; it was partially inhibited (30-37%) by preincubation with the 58,000 MW fragment or with a synthetic peptide CS-1, comprising the first 25 amino acid residues of the alternatively spliced connecting segment (IIICS), which is present in the A chain of fibronectin and in the 38,000 MW fragment. Cell preincubation with RGDS-containing synthetic peptides or with the 80,000 MW fragment, did not affect attachment to 38,000 MW-coated surfaces. Moreover, preincubation of HUT-78 cells with 38,000 MW fragment had no effect on cell adhesion to 80,000 MW-coated wells, while preincubation with 80,000 MW fragment completely inhibited cell attachment to these surfaces. These results strongly suggest the involvement of two different cell surface receptors which recognize the Hep II/IIICS site and the RGDS site independently. Preincubation with either 38,000 or 80,000 MW fragments prevented cell attachment to fibronectin, indicating that adhesion to the intact molecule requires interaction with both regions. Therefore T-lymphocyte adherence to fibronectin-containing matrices may be regulated by the co-expression of both receptors at the cell surface.     

Study on the potential of RGD- and PHSRN-modified alginates as artificial extracellular matrices for engineering bone

Alginate is a polysaccharide that can be crosslinked by divalent cations, such as calcium ions, to form a gel. Chemical modification is typically used to improve its cell adhesive properties for tissue engineering applications. In this study, alginates were modified with peptides containing RGD (arginine–glycine–aspartic acid) or PHSRN (proline–histidine–serine–arginine–asparagine) sequences from fibronectin to study possible additive and synergistic effects on adherent cells. Alginates modified with each peptide were mixed at different ratios to form gels containing various concentrations and spacing between the RGD and PHSRN sequences. When normal human osteoblasts (NHOsts) were cultured on or in the gels, the ratio of RGD to PHSRN was found to influence cell behaviors, especially differentiation. NHOsts cultured on gels composed of RGD- and PHSRN-modified alginates showed enhanced differentiation when the gels contained >33 % RGD-alginate, suggesting the relative distribution of the peptides and the presentation to cells are important parameters in this regulation. NHOsts cultured in gels containing both RGD- and PHSRN-alginates also demonstrated a similar enhancement tendency of calcium deposition that was dependent on the peptide ratio in the gel. However, calcium deposition was greater when cells were cultured in the gels, as compared to on the gels. These results suggest that modifying this biomaterial to more closely mimic the chemistry of natural cell adhesive proteins, (e.g., fibronectin) may be useful in developing scaffolds for bone tissue engineering and provide three-dimensional cell culture systems which more closely mimic the environment of the human body.     

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.     

Immunofluorescence and flow cytometry analysis of fibronectin and laminin binding to Sporothrix schenckii yeast cells and conidia

The adherence of Sporothrix schenckii yeast cells to several extracellular matrix (ECM) components has already been demonstrated, but the mechanisms of these interactions remained to be defined. In indirect immunofluorescence assays with polyclonal antibodies directed towards the ECM proteins, both hyphae and yeast cells of S. schenckii exhibited the ability to bind laminin and fibronectin. Flow cytometry confirmed the binding of these proteins, and revealed a significant greater binding capability for the yeast cells than for the conidia. Fibronectin and laminin binding was dose-dependent and specific. In addition, competition experiments with synthetic peptides mimicking the adhesive sequences of these proteins, or with cell wall fractions and carbohydrates constitutive of their sugar chains, were performed in order to specify the peptide or carbohydrate motifs involved in the recognition process. A 50% reduction was noticed in fibronectin binding in the presence of the synthetic peptide RGD, and a 38% reduction in laminin binding with the peptide YIGSR. Some carbohydrate-containing fractions of the yeast cell wall also inhibited the binding of fibronectin, but had no significant effect on laminin binding. Together, these results suggest the presence at the yeast surface of distinct receptors for laminin and fibronectin.     

Fibronectin modulates macrophage adhesion and FBGC formation: the role of RGD, PHSRN, and PRRARV domains.

To probe the role of human plasma fibronectin in modulating human blood-derived macrophage adhesion and fusion to form multinucleated foreign-body giant cells (FBGC), a series of biomimetic oligopeptides based on the functional structure of fibronectin was designed and synthesized. Peptides incorporated the RGD and PHSRN integrin-binding sequences from FIII-10 and FIII-9 modules, respectively, and the PRRARV sequence from the C-terminal heparin-binding domain, either alone or in combination. Peptides were immobilized onto a polyethyleneglycol-based polymer substrate. The following conclusions were reached. Fibronectin modulated macrophage adhesion and the extent (i.e., size) of FBGC formation on control surfaces in the presence of serum proteins. Macrophages adhered to all substrates with relatively subtle differences between adhesion mediated by RGD, PHSRN, PRRARV, or combinations thereof. beta1-integrin subunit was essential in macrophage adhesion to peptide-grafted networks in a receptor-peptide specific manner, whereas beta3-integrin subunit was less important. Macrophage adhesion to PRRARV was mediated primarily by the direct interaction with integrins. RGD or PHSRN alone did not provide an adequate substrate for macrophage fusion to form FBGCs. However, the PHSRN synergistic site and the RGD site in a single oligopeptide provided a substrate for FBGC formation that was statistically comparable to that on the positive control material in the presence of serum proteins. This response was highly dependent upon the relative orientation between RGD and PHSRN. PRRARV did not support FBGC formation. These results demonstrate the importance of fibronectin and, specifically, the synergy between RGD and PHSRN domains, in supporting macrophage fusion to form FBGCs.     

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.