Osteoblast population migration characteristics on substrates modified with immobilized adhesive peptides

The process of cell migration is inextricably linked with the process of cell adhesion and, therefore, with cell/substrate adhesiveness. The present study adapted an under-agarose cell migration assay to quantitatively examine population migration characteristics of osteoblasts, on substrates modified with adhesive peptides, in the absence and presence of growth factors. Short-term, that is, 48 h osteoblast migration distances on substrates modified with adhesive Arg-Gly-Asp-Ser peptides were significantly (P < 0.05) less than migration distances on substrates modified with non-adhesive Arg-Asp-Gly-Ser peptides, demonstrating that osteoblast population haptokinesis was significantly decreased on substrates modified with adhesive peptides. Random motility coefficients calculated in the present study for osteoblast populations were an order of magnitude lower than a published random motility coefficient for leukocytes, proving quantitatively that, compared to leukocytes, osteoblasts migrate via haptokinesis more slowly. The 48 and 72 h osteoblast population migration differentials in the presence of an initial mass of 60 ng of basic Fibroblast Growth Factor, on substrates modified with Arg-Gly-Asp-Ser or with Arg-Asp-Gly-Ser, were larger than all other chemotactic differentials on these substrates. Quantitative investigations (such as the present study) of cell population migration characteristics on model biomaterial surfaces will become increasingly necessary as the discipline of cell/tissue engineering matures.     

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.     

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.     

Cell adhesion peptides alter smooth muscle cell adhesion, proliferation, migration, and matrix protein synthesis on modified surfaces and in polymer scaffolds

The effects of cell adhesion peptides (RGDS, KQAGDV, VAPG) on vascular smooth muscle cells grown on modified surfaces and in tissue-engineering scaffolds were examined. Cells were more strongly adhered to surfaces modified with adhesive ligands than to control surfaces (no ligand or a nonadhesive ligand). Cell migration was higher on surfaces with 0.2 nmol/cm(2) of adhesive ligand than on control surfaces, but it was lower on surfaces with 2.0 nmol/cm(2) of adhesive ligand than it was on control surfaces. Further, cell proliferation was lower on adhesive surfaces than it was on control surfaces, and it decreased as the ligand density increased. Similarly, in the peptide-grafted hydrogel scaffolds, cell proliferation was lower in scaffolds containing the adhesive peptides than it was in control scaffolds. After 7 days of culture, more collagen per cell was produced in control scaffolds than in scaffolds containing adhesive peptides. In addition, collagen production decreased in the scaffolds as the ligand concentration increased. While modification of a surface or scaffold material with adhesive ligands initially increases cell attachment, it may be necessary to optimize cell adhesion simultaneously with proliferation, migration, and matrix production.     

PEG-variant biomaterials as selectively adhesive protein templates: model surfaces for controlled cell adhesion and migration

Our study focused on the role of poly(ethylene glycol) (PEG) in actively regulating the biological responsiveness of protein-adsorbed biomaterials. To this end, we designed PEG-variant biomaterials from a family of tyrosine/PEG-derived polycarbonates to present surfaces ranging from low to intermediate levels of PEG concentration, below the PEG level requisite for complete abolition of protein adsorption. We analyzed the effect of PEG concentration on the amount, conformation and bioactivity of an adsorbed model protein, fibronectin, and on the attachment, adhesion strength and motility of L929 fibroblasts. Our results demonstrate that low levels of PEG can regulate not only the extent but also the conformation and specific bioactivity of adsorbed fibronectin. As the PEG concentration was increased from 0 to 6 mol%, the amount of adsorbed fibronectin decreased linearly yet the fibronectin conformation was altered such that the overall bioactivity of adsorbed fibronectin was uncompromised. We report that the degree of cell attachment varied with PEG concentration in a manner similar to the dependence of fibronectin bioactivity on PEG. In contrast, the nature of cell adhesion strength dependence on PEG paralleled the pattern observed for fibronectin surface concentration. Our studies also indicated that the rate of cell migration was inversely correlated with PEG concentration over a narrow range of PEG concentration. Overall, these results highlight the striking ability of PEG-variant biomaterials to systematically regulate the behavior of adsorbed cell adhesion proteins and, consequently, effect cell functions.     

Binding and orientation of fibronectin on polystyrene surfaces using immobilized bacterial adhesin-related peptides

Fibronectin (FN) is known to bind to bacteria via high affinity receptors on bacterial surfaces known as adhesins. The binding of bacteria to FN is thought to have a key role in foreign device associated infections. For example, previous studies have indicated that Staphylococcus aureus adhesins bind to the 29 kDa NH(3) terminus end of FN, and thereby promote bacteria adherence to surfaces. Recently, the peptide sequences within the S. aureus adhesin molecule that are responsible for FN binding have been identified. Based on these observations, we hypothesize that functional FN can be bound and specifically oriented on polystyrene surfaces using bacterial adhesin-related (BRP-A) peptide. We further hypothesize that monoclonal antibodies that react with specific epitopes on the FN can be used to quantify both FN binding and orientation on these surfaces. Based on this hypothesis, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces using BRP-A peptide. To test this hypothesis, the binding and orientation of the FN to immobilized BRP-A was quantified using (125)I-FN, and monoclonal antibodies. (125)I-FN was used to quantitate FN binding to peptide-coated polystyrene surfaces. The orientation of bound FN was demonstrated by the use of monoclonal antibodies, which are reactive with the amine (N) or carboxyl (C) termini of the FN. The results of our studies demonstrated that when the BRP-A peptide was used to bind FN to surfaces that: 1. functional FN was bound to the peptide; 2. anti-C terminus antibodies bound to the peptide FN; and 3. only limited binding of anti-N terminus antibodies to peptide-bound FN occurred. We believe that the data that indicate an enhanced binding of anti-C antibodies reactive to anti-N antibodies are a result of the FN binding in an oriented manner with the N termini of FN bound tightly to the BRP-A on the polystyrene surface.     

Endothelial cell adhesion to vascular prosthetic surfaces

Improving the long term patency of small diameter prosthetic grafts remains an important but elusive objective in vascular surgery. In pursuit of a non-thrombogenic surface, we have cultured human-adult endothelial cells, examined their adhesive properties and their ability to colonize the inner surface of a Dacron graft. To examine cell adhesion, endothelial cells were labelled with 111In-oxine and inoculated onto prosthetic wells previously prepared with either cold insoluble globulin (CIG), 1% gelatin, alginate or left untreated as a control. At 100 min, mean percentage adhesion to CIG and gelatin precoated wells was 86.0 +/- 9.9% (+/- SD) and 81.6 +/- 2.9% respectively, whereas alginate at 57.5 +/- 7.5% and the control well at 48.3 +/- 9.9% showed significantly less adhesion. Further experiments examined the adhesion of Indiumoxine labelled endothelial cells to Dacron graft (2 cm X 6 mm ID) placed in an in vitro arterial circuit. An immediate loss of approximately 20% of the cells occurred within the first 30 s, whereafter, a stable population were adherent to the graft material. By 102 min, 73.4 +/- 1.8% of cells remained attached when exposed to tissue culture medium, but only 64.1 +/- 6.9% after exposure to blood. Cultured human adult endothelial cells adhere most effectively to prosthetic surfaces precoated with CIG or gelatin, and remain attached following exposure to shear forces.     

Three-dimensional nanofibrillar surfaces covalently modified with tenascin-C-derived peptides enhance neuronal growth in vitro

Current methods to promote growth of cultured neurons use two-dimensional (2D) glass or polystyrene surfaces coated with a charged molecule (e.g. poly-L-lysine (PLL)) or an isolated extracellular matrix (ECM) protein (e.g. laminin-1). However, these 2D surfaces represent a poor topological approximation of the three-dimensional (3D) architecture of the assembled ECM that regulates neuronal growth in vivo. Here we report on the development of a new 3D synthetic nanofibrillar surface for the culture of neurons. This nanofibrillar surface is composed of polyamide nanofibers whose organization mimics the porosity and geometry of the ECM. Neuronal adhesion and neurite outgrowth from cerebellar granule, cerebral cortical, hippocampal, motor, and dorsal root ganglion neurons were similar on nanofibers and PLL-coated glass coverslips; however, neurite generation was increased. Moreover, covalent modification of the nanofibers with neuroactive peptides derived from human tenascin-C significantly enhanced the ability of the nanofibers to facilitate neuronal attachment, neurite generation, and neurite extension in vitro. Hence the 3D nanofibrillar surface provides a physically and chemically stabile cell culture surface for neurons and, potentially, an exciting new opportunity for the development of peptide-modified matrices for use in strategies designed to encourage axonal regrowth following central nervous system injury.     

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.     

Endothelial cell adhesion and proliferation to PEGylated polymers with covalently linked RGD peptides

A nonfouling peptide grafted polymer was synthesized that can promote endothelial cell (EC) binding. The polymer was composed of hexyl methacrylate, methyl methacrylate, poly(ethylene glycol) methacrylate, and CGRGDS peptide. The peptide was incorporated into the polymer system either by a chain transfer reaction or by coupling to an acrylate-PEG-N-hydroxysuccinimide (NHS) comonomer. The introduction of PEG chains minimizes protein adsorption. Human umbilical vein ECs and endothelial colony forming cells were cultured on these surfaces in short term and long-term studies. A difference in number and morphology of ECs was observed depending on the method of peptide incorporation. Both cell types adhered better to polymer films containing NHS coupled RGD peptide after 2 h even in the presence of albumin but significant cell detachment occurred after 4 days. Polymer solutions were electrospun into fibrous scaffolds. Both nonfouling and peptide binding characteristics were retained after processing.     

Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration

Basic fibroblast growth factor (bFGF) was immobilized to hydrogel scaffolds with retention of mitogenic and chemotactic activity. The bFGF was functionalized in order to incorporate it covalently within polyethylene glycol (PEG) hydrogel scaffolds by reaction with acryloyl-PEG-NHS. Hydrogels were formed by exposing aqueous solutions of PEG diacrylate, acryloyl-PEG-RGDS, and acryloyl-PEG-bFGF to long-wavelength ultraviolet light in the presence of a photoinitiator. These bFGF-modified hydrogels with RGD adhesion sites were evaluated for their effect on vascular smooth muscle cell (SMC) behavior, increasing SMC proliferation by approximately 41% and migration by approximately 15%. A covalently immobilized bFGF gradient was formed using a gradient maker to pour the hydrogel precursor solutions and then photopolymerizing to lock in the concentration gradient. Silver staining was used to detect the bFGF gradient, which increased linearly along the hydrogel's length. Cells were observed to align on hydrogels modified with a bFGF gradient in the direction of increasing tethered bFGF concentration as early as 24 h after seeding. SMCs also migrated differentially, up the concentration gradient, on bFGF-gradient hydrogels compared to control hydrogels with and without a constant bFGF concentration. These hydrogel scaffolds may be useful for studying protein gradient effects on cell behavior and for directing cell migration in tissue-engineering applications.     

Covalently immobilized RGD gradient on PEG hydrogel scaffold influences cell migration parameters

Understanding the influence of a controlled spatial distribution of biological cues on cell activities can be useful to design “cell instructive” materials, able to control and guide the formation of engineered tissues in vivo and in vitro. To this purpose, biochemical and mechanical properties of the resulting biomaterial must be carefully designed and controlled. In this work, the effect of covalently immobilized RGD peptide gradients on poly(ethylene glycol) diacrylate hydrogels on cell behaviour was studied. We set up a mechanical device generating gradients based on a fluidic chamber. Cell response to RGD gradients with different slope (0.7, 1 and 2 mM cm^?1) was qualitatively and quantitatively assessed by evaluating cell adhesion and, in particular, cell migration, compared to cells seeded on hydrogels with uniform distribution of RGD peptides. To evaluate the influence of RGD gradient and to exclude any concentration effect on cell response, all analyses were carried out in a specific region of the gradients which displayed the same average concentration of RGD (1.5 mM). Results suggest that cells recognize the RGD gradient and adhere onto it assuming a stretched shape. Moreover, cells tend to migrate in the direction of the gradient, as their speed is higher than that of cells migrating on hydrogels with a uniform distribution of RGD and increases by increasing RGD gradient steepness. This increment is due to an augmentation of bias speed component of the mean squared speed, that is, the drift of the cell population migrating on the anisotropic surface provided by the RGD gradient.     

Binding and orientation of fibronectin to silanated glass surfaces using immobilized bacterial adhesin-related peptides

Previously, we have demonstrated the suitability of bacterial adhesin-related peptides, directly immobilized on polystyrene surfaces, to bind and orient fibronectin (FN). For these studies a method to bind the large protein FN in a desired orientation on a solid substratum was developed which utilizes a bacterial adhesin-related peptide (designated BRP-A), which is known to bind specifically to the NH3-terminus end of FN. Glass substrata was first coated with an amine-terminated silane, followed by streptavidin (SA), which was used as an intermediate tether to bind the biotinylated bacterial adhesin-related peptide. The BRP-A peptide, used for these studies was synthesized with a terminal biotin to assure irreversible coupling of the BRP-A to the streptavidin. The biotinylated BRP-A was next immobilized on the SA-silanated glass surfaces. 125I-FN was used to quantify the amount of FN binding to the (BRP-A):SA-silanated glass surface. Monoclonal antibodies, which react with specific epitopes at either the NH3- or -COOH-termini of FN, were used to quantify the binding and orientation of FN. The results of these studies indicated: (1) FN bound to the BRP-A:SA-silanated glass surface; and (2) the bound FN was oriented such that NH2-terminal region of FN was bound towards the glass surface and the COOH-terminus was oriented away from the glass surface. These studies demonstrate that small peptides can be used to specifically bind and orient large proteins such as FN on the surfaces.     

Modular peptides promote human mesenchymal stem cell differentiation on biomaterial surfaces

Molecular design strategies in biomedical applications often involve creating modular “fusion” proteins, in which distinct domains within a single molecule can perform multiple functions. We have synthesized a new class of modular peptides that include a biologically active sequence derived from the growth factor BMP-2 and a series of hydroxyapatite-binding sequences inspired by the N-terminal α-helix of osteocalcin. These modular peptides can bind in a sequence-dependent manner to the surface of “bone-like” hydroxyapatite coatings, which are nucleated and grown on a biodegradable polymer surface via a biomimetic process. The BMP-2-derived sequence of the modular peptides is biologically active, as measured by its ability to promote osteogenic differentiation of human mesenchymal stem cells. Our study indicates that the modular peptides described here are multifunctional, and the characteristics of this approach suggest that it can potentially be applied to a range of biomaterials for regenerative medicine applications.     

Endothelial cell differentiation into capillary structures by copolymer surfaces with phenylboronic acid groups

A ternary copolymer composed of m-acrylamidophenylboronic acid, N,N-dimethylaminopropylmethacrylamide and N-isopropylacrylamide was synthesized. Long-term culture of bovine aortic endothelial cells on this copolymer substrate demonstrated adhesion and proliferation of the cells. After 26 days in culture, endothelial cells spontaneously developed into capillary networks. The interactions between phenylboronic acid groups in copolymer and glycoconjugates on endothelial cell plasma membranes are proposed to regulate the induction of tissue formation, since phenylboronic acid groups are known to specifically form reversible complexes with cis-diol compounds such as glucose. This copolymer is a novel material capable of mediating specific signals analogous to extracellular matrix to promote proliferation of endothelial cells, inducing capillary structures and prompt angiogenesis.     

Endothelial cell growth on silicon modified hydrogenated amorphous carbon thin films

As part of a method development for peel testing, an interlaboratory comparison among Food and Drug Administration-Center for Drug Evaluation and Research, Food and Drug Administration-Center for Devices and Radiological Health and Southwest Research Institute was conducted using medical tapes. The aim was to determine which readily available substrate [stainless steel (SS), high density polyethylene (HDPE) or Vitro-Skin(R)] would best distinguish among various medical tapes. Five medical tapes (3M 1523, 3M 1525L, 3M 1776, Mepiform(R) and Mediderm(R) 3505) were evaluated on four different substrates (SS, HDPE, Vitro-Skin, and human cadaver skin) using the following peel parameters: approximately 3 min dwell time, 90 degrees peel angle, and 300 mm/min peel rate. No substrate mimics cadaver skin for all five tapes. SS had the best ability to distinguish among the medical tapes. Overall, for quality control purposes (yielding good discrimination and precision), SS would be the optimal substrate.     

Directing cell migration using micropatterned and dynamically adhesive polymer brushes

Micropatterning techniques, such as photolithography and microcontact printing, provide robust tools for controlling the adhesive interactions between cells and their extracellular environment. However, the ability to modify these interactions in real time and examine dynamic cellular responses remains a significant challenge. Here we describe a novel strategy to create dynamically adhesive, micropatterned substrates, which afford precise control of cell adhesion and migration over both space and time. Specific functionalization of micropatterned poly(ethylene glycol methacrylate) (POEGMA) brushes with synthetic peptides, containing the integrin-binding arginine–glycine–aspartic acid (RGD) motif, was achieved using thiol–yne coupling reactions. RGD activation of POEGMA brushes promoted fibroblast adhesion, spreading and migration into previously non-adhesive areas, and migration speed could be tuned by adjusting the surface ligand density. We propose that this technique is a robust strategy for creating dynamically adhesive biomaterial surfaces and a useful assay for studying cell migration.     

Endothelial cell migration and morphogenesis on silk fibroin scaffolds containing hydroxyapatite electret

The purpose of this study was to evaluate the effects of composite wound dressing films made of silk fibroin (SF) containing hydroxyapatite (HA) or polarized HA (pHA) powders on endothelial cell (EC) behaviors that have important roles in the wound-healing process. XRD revealed the SF films to be semicrystalline, with a broad peak centered at about 20.7° which is characteristic of β-sheets embedded within an amorphous matrix. The SF composite films with 0.6 (w/v)% in concentration of HA powder (HA/SF) or pHA powder (pHA/SF) contained HA crystals of amorphous and silk II crystalline structures. SEM observation showed that there were differences in SF morphology between HA/SF and pHA/SF. The pHA/SF exhibited a furry texture around the pHA crystals, most likely due to the stored charged and zeta potentials. The HA/SF and pHA/SF films enhanced EC migration compared with that on the SF film. The number of migrated cells on the HA/SF and pHA/SF was ~1.5 times larger than that on the SF. The quantitative analysis of the endothelial morphogenesis indicated that the pHA/SF film enhanced the formation of capillary-like structures compared with SF and HA/SF. Thus, pHA/SF may potentially stimulate and contribute to the enhancement of angiogenesis in the wound-healing process.     

Endothelial cell labeling with indium-111-oxine as a marker of cell attachment to bioprosthetic surfaces

Canine vascular endothelium labeled with indium-111-oxine was used as a marker of cell attachment to vascular prosthetic surfaces with complex textures. Primarily cultured and freshly harvested endothelial cells both took up the label rapidly. An average of 72% of a 32 micro Ci labeling dose was taken up by 1.5 X 10(6) cells in 10 min in serum-free medium. Over 95% of freshly labeled cells were viable by trypan blue tests and only 5% of the label was released after 1 h incubations at 37 degrees C. Labeled and unlabeled cells had similar rates of attachment to plastic dishes. Scanning electron microscopic studies showed that labeled cells retained their ability to spread on tissue culture dishes even at low (1%) serum levels. Labeled endothelial cells seeded onto Dacron or expanded polytetrafluoroethylene vascular prostheses by methods used in current surgical models could be identified by autoradiography of microscopic sections of the prostheses, and the efficiency of cell attachment to the prosthesis could be measured by gamma counting. Indium-111 labeling affords a simple and rapid way to measure initial cell attachment to, and distribution on, vascular prosthetic materials. The method could also allow measurement of early cell loss from a flow surface in vivo by using external gamma imaging.