Using self-assembled monolayers to model the extracellular matrix

The extracellular matrix is an insoluble aggregate of large proteins and glycosoaminoglycans that comprises the microenvironment of cells in tissue. The matrix displays a host of ligands that interact with cell-surface receptors to mediate the attachment and spreading of cells and regulate signaling processes. Studies of cell–matrix interactions and downstream signaling processes commonly employ substrates having an adsorbed layer of protein and are challenged by the difficulty in controlling the structure and activity of the immobilized protein. Significant effort has been directed towards the development of model substrates that present adhesion ligands in defined densities, orientations and environments. Among these approaches, self-assembled monolayers of alkanethiolates on gold offer a high level of control over the molecular structure of the surface and are well-suited to studies of cell adhesion. This review describes the design and use of monolayers for applications in cell biology, including the use of monolayers to evaluate the roles of peptide and protein ligands in cell–matrix interactions, the development of methods to pattern ligands on monolayers and applications to cell biology, the development of dynamic monolayers that can switch the activities of ligands presented to an adherent cell, and the rewiring of interactions between a cell and its substrate. These examples illustrate the flexibility inherent to monolayers for applications in cell biology.     

Modulation of marrow stromal osteoblast adhesion on biomimetic oligo[poly(ethylene glycol) fumarate] hydrogels modified with Arg-Gly-Asp peptides and a poly(ethyleneglycol) spacer

Novel oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels functionalized with cell adhesion peptides were prepared, and the effects of incorporated peptide density and macromolecular structure of hydrogels on attachment and morphology of marrow stromal cells (MSCs) were evaluated. Poly(ethylene glycol) (PEG; number average molecular weight of 930, 2860, and 6090) was used to synthesize OPF. A model peptide, Gly-Arg-Gly-Asp (GRGD), was incorporated into OPF hydrogels after being coupled to acrylated PEG of molecular weight 3400. The increase of incorporated peptide concentration enhanced MSC attachment to OPF hydrogels of PEG of molecular weight of 930 and 2860. However, the number of attached MSCs to OPF hydrogels of PEG (molecular weight 6090) remained constant regardless of the peptide density. The length of PEG in OPF also influenced cell attachment. When 1 micromole peptide/g hydrogel was incorporated into the OPF hydrogels, the degree of cell attachment at 12 h relative to the initial seeding density was 93.9 +/- 5.9%, 64.7 +/- 8.2%, and 9.3 +/- 6.6% for OPF hydrogels prepared with PEG of molecular weights of 930, 2860, and 6090, respectively. However, the crosslinking density of hydrogels did not significantly affect cell attachment. The interaction was sequence specific, in that MSC attachment to GRGD-modified hydrogels was competitively inhibited when cells were incubated in the presence of 0.5 mM soluble GRGD before cell seeding. These results suggest that we can modulate MSC attachment to OPF hydrogels by altering the peptide density and the molecular structure of OPF hydrogels.     

RGD-Functionalized polymer brushes as substrates for the integrin specific adhesion of human umbilical vein endothelial cells

This report demonstrates the feasibility of surface-initiated atom transfer radical polymerization to prepare thin polymer layers ("brushes") that can be functionalized with short peptide ligands and which may be of use as coatings to promote endothelialization of blood-contacting biomaterials. The brushes are composed of poly(2-hydroxyethyl methacrylate) (PHEMA) or poly(poly(ethylene glycol) methacrylate) (PPEGMA), which do not only suppress non-specific adhesion of proteins and cells but also contain hydroxyl groups that can be used to introduce small peptide ligands. A protocol has been developed that allows functionalization of the brushes with RGD containing peptide ligands resulting in surface concentrations ranging from approximately 0.5-12 pmol/cm(2). At peptide surface concentrations >1-5.3 pmol/cm(2), human umbilical vascular endothelial cells (HUVECs) were found to adhere and spread rapidly. A difference in size and morphology of focal adhesions between HUVECs immobilized on PHEMA and PPEGMA brushes was observed. It is proposed that this is due to the increased ethylene glycol spacer length and hydrophilicity of the PPEGMA brushes, which may lead to increased ligand mobility and reduced ligand-integrin affinity. HUVECs immobilized on the polymer brushes were also found to be able to retain homeostasis when exposed to shear stresses that simulated arterial blood flow.     

Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering

Poly(ethylene glycol) (PEG) hydrogels were investigated as encapsulation matrices for osteoblasts to assess their applicability in promoting bone tissue engineering. Non-adhesive hydrogels were modified with adhesive Arg-Gly-Asp (RGD) peptide sequences to facilitate the adhesion, spreading, and, consequently, cytoskeletal organization of rat calvarial osteoblasts. When attached to hydrogel surfaces, the density and area of osteoblasts attached were dramatically different between modified and unmodified hydrogels. A concentration dependence of RGD groups was observed, with increased osteoblast attachment and spreading with higher RGD concentrations, and cytoskeleton organization was seen with only the highest peptide density. A majority of the osteoblasts survived the photoencapsulation process when gels were formed with 10% macromer, but a decrease in osteoblast viability of approximately 25% and 38% was seen after 1 day of in vitro culture when the macromer concentration was increased to 20 and 30wt%, respectively. There was no statistical difference in cell viability when peptides were added to the network. Finally, mineral deposits were seen in all hydrogels after 4 weeks of in vitro culture, but a significant increase in mineralization was observed upon introduction of adhesive peptides throughout the network.     

Co-assembling peptides as defined matrices for endothelial cells

Self-assembling peptides and peptide derivatives bearing cell-binding ligands are increasingly being investigated as defined cell culture matrices and as scaffolds for regenerative medicine. In order to systematically refine such scaffolds to elicit specific desired cell behaviors, ligand display should ideally be achieved without inadvertently altering other physicochemical properties such as viscoelasticity. Moreover, for in vivo applications, self-assembled biomaterials must exhibit low immunogenicity. In the present study, multi-peptide co-assembling hydrogels based on the β-sheet fibrillizing peptide Q11 (QQKFQFQFEQQ) were designed such that they presented RGDS or IKVAV ligands on their fibril surfaces. In co-assemblies of the ligand-bearing peptides with Q11, ligand incorporation levels capable of influencing the attachment, spreading, morphology, and growth of human umbilical vein endothelial cells (HUVECs) did not significantly alter the materials' fibrillization, β-turn secondary structure, or stiffness. RGDS-Q11 specifically increased HUVEC attachment, spreading, and growth when co-assembled into Q11 gels, whereas IKVAV-Q11 exerted a more subtle influence on attachment and morphology. Additionally, Q11 and RGDS-Q11 were minimally immunogenic in mice, making Q11-based biomaterials attractive candidates for further investigation as defined, modular extracellular matrices for applications in vitro and in vivo.     

Polymer latexes for cell-resistant and cell-interactive surfaces

Novel polymer latexes were prepared that can be applied in several ways for the control and study of cell behavior on surfaces. Acrylic latexes with glass transitions ranging from -30 to 100 degrees C were synthesized by dispersion polymerization in a water and alcohol solution using an amphiphilic comb copolymer as a stabilizing agent. The comb had a poly(methyl methacrylate) backbone and hydrophilic poly(ethylene glycol) (PEG) side chains, which served to stabilize the dispersion and create a robust hydrophilic coating on the final latex particles. The end groups of the comb stabilizer can be selectively functionalized to obtain latex particles with a controlled density of ligands tethered to their surfaces. Latexes were prepared with adhesion peptides (RGD) linked to the surface of the acrylic beads to induce attachment and spreading of cells. Coalesced films obtained from the RGD-bearing latex particles promoted attachment of WT NR6 fibroblasts, while films from unmodified latex particles were resistant to these cells. Additionally, RGD-linked beads were embedded in cell-resistant comb polymer films to create cell-interactive surfaces with discrete clustered-ligand domains. Cell attachment and morphology were seen to vary with the surface density of the RGD-bearing latex beads.     

Attachment, proliferation, and migration of marrow stromal osteoblasts cultured on biomimetic hydrogels modified with an osteopontin-derived peptide

We prepared oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels modified with a rat osteopontin-derived peptide (ODP), Asp-Val-Asp-Val-Pro-Asp-Gly-Arg-Gly-Asp-Ser-Leu-Ala-Try-Gly (DVDVPDGRGDSLAYG), as well as Gly-Arg-Gly-Asp-Ser (GRGDS) and investigated the modulation of marrow stromal osteoblast function on the peptide-modified hydrogels. Osteoblast attachment was competitively inhibited by a soluble peptide suggesting that the interaction of osteoblasts with the hydrogel was ligand specific. The proliferation index of osteoblasts relative to the initial seeding density was similar on the hydrogels modified with ODP (1.18+/-0.13) and GRGDS (1.27+/-0.12). However, fibroblasts proliferated faster on GRGDS-modified hydrogels than on ODP-modified hydrogels as evidenced by the proliferation indices of 4.89+/-0.03 and 2.42+/-0.16, respectively. A megacolony migration assay conducted for 3 days with a seeding density of 53,000 cells/cm(2) showed that osteoblasts migrated to a longer distance on ODP-modified hydrogels (0.23+/-0.06 mm/day) than on hydrogels modified with GRGDS (0.15+/-0.02 mm/day). In addition, osteoblasts migrated faster than fibroblasts seeded at the same density on ODP-modified hydrogels (0.15+/-0.11 mm/day). The migration of osteoblasts on the peptide-modified hydrogels was dependent on the peptide concentration of the hydrogels resulting in an increased migration distance with increasing the peptide concentration for the concentrations tested. These results show that OPF-based biomimetic hydrogels hold promise for modulating cell proliferation and migration for specific applications by altering the specific ligand and its concentration in the hydrogels.     

Biomimetic modification of titanium dental implant model surfaces using the RGDSP-peptide sequence: a cell morphology study

Surface topography and (bio)chemistry are key factors in determining cell response to an implant. We investigated cell adhesion and spreading patterns of epithelial cells, fibroblasts and osteoblasts on biomimetically modified, smooth and rough titanium surfaces. The RGD bioactive peptide sequence was immobilized via a non-fouling poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) molecular assembly system, which allowed exploitation of specific cell-peptide interactions even in the presence of serum. As control surfaces, bare titanium and bio-inactive surfaces (scrambled RDG and unfunctionalized PLL-g-PEG) were used. Our findings demonstrated that surface topography and chemistry directly influenced the attachment and morphology of all cell types tested. In general, an increase in cell number and more spread cells were observed on bioactive substrates (containing RGD) compared to bio-inactive surfaces. More fibroblasts were present on smooth than on rough topographies, whereas for osteoblasts the opposite tendency was observed. Epithelial cell attachment did not follow any regular pattern. Footprint areas for all cell types were significantly reduced on rough compared to smooth surfaces. Osteoblast attachment and footprint areas increased with increasing RGD-peptide surface density. However, no synergy (interaction) between RGD-peptide surface density and surface topography was observed for osteoblasts neither in terms of attachment nor footprint area.     

Osteogenic differentiation of rat bone marrow stromal cells cultured on Arg-Gly-Asp modified hydrogels without dexamethasone and beta-glycerol phosphate

In this study, we investigated the effect of signaling peptides incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels on in vitro differentiation and mineralization of marrow stromal cells (MSCs) cultured in media without soluble osteogenic supplements (dexamethasone and beta-glycerol phosphate). When MSCs were cultured for 16 days on OPF hydrogels modified with Arg-Gly-Asp (RGD) containing peptides, the normalized cell number was dependent on the peptide concentration between days 0 and 5 and reached comparable values at day 10 regardless of the concentration. The alkaline phosphatase (ALP) activity of MSCs on the peptide-modified OPF hydrogels was also concentration-dependent: ALP activity showed peaks on day 10 or day 13 on OPF hydrogels modified with 2.0 and 1.0 micromol peptide/g, which were significantly greater than those on the OPF hydrogels modified with 0.1 micromol peptides/g or no peptide. A characteristic marker of osteoblastic differentiation, osteopontin (OPN), was detected for all the test groups. However, OPN secretion between days 0 and 10 was significantly higher on the peptide modified hydrogels compared to that on tissue culture-treated polystyrene. Taken together, the results indicate that the presence of signaling peptide allows for a favorable microenvironment for MSCs to differentiate into osteoblasts and produce mineralized matrix, although the soluble factors may further enhance calcium deposition. These findings further support the usefulness of OPF hydrogels as scaffolds for guided bone regeneration, and represent an initial step in exploring the complex relationship between soluble and insoluble factors in osteogenic differentiation on biodegradable materials.     

Surface polyethylene glycol enhances substrate-mediated gene delivery by nonspecifically immobilized complexes

Substrate-mediated gene delivery describes the immobilization of gene therapy vectors to a biomaterial, which enhances gene transfer by exposing adhered cells to elevated DNA concentrations within the local microenvironment. Surface chemistry has been shown to affect transfection by nonspecifically immobilized complexes using self-assembled monolayers (SAMs) of alkanethiols on gold. In this report, SAMs were again used to provide a controlled surface to investigate whether the presence of oligo(ethylene glycol) (EG) groups in a SAM could affect complex morphology and enhance transfection. EG groups were included at percentages that did not affect cell adhesion. Nonspecific complex immobilization to SAMs containing combinations of EG- and carboxylic acid-terminated alkanethiols resulted in substantially greater transfection than surfaces containing no EG groups or SAMs composed of EG groups combined with other functional groups. Enhancement in transfection levels could not be attributed to complex binding densities or release profiles. Atomic force microscopy imaging of immobilized complexes revealed that EG groups within SAMs affected complex size and appearance and could indicate the ability of these surfaces to preserve complex morphology upon binding. The ability to control the morphology of the immobilized complexes and influence transfection levels through surface chemistry could be translated to scaffolds for gene delivery in tissue engineering and diagnostic applications.     

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

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

Mechanisms underlying the attachment and spreading of human osteoblasts: From transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces

The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24 h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration.     

NCO-sP(EO-stat-PO) surface coatings preserve biochemical properties of RGD peptides

We have previously reported that star shaped poly(ethylene oxide-stat-propylene oxide) macromers with 80% EO content and isocyanate functional groups at the distal ends [NCO-sP(EO-stat-PO)] can be used to generate coatings that are non-adhesive but easily functionalized for specific cell adhesion. In the present study, we investigated whether the NCO-sP(EO-stat-PO) surfaces maintain peptide configuration-specific cell-surface interactions or if differences between dissimilar binding molecules are concealed by the coating. To this end, we have covalently immobilized both linear-RGD peptides (gRGDsc) and cyclic-RGD (RGDfK) peptides in such coatings. Subsequently, SaOS-2 or human multipotent mesenchymal stromal cells (MSC) were seeded on these substrates. Cell adhesion, spreading and survival was observed for up to 30 days. The time span for cell adherence was not different on linear and cyclic RGD peptides, but was shorter in comparison to the unmodified glass surface. MSC proliferation on cyclic RGDfK modified coatings was 4 times higher than on films functionalized by linear gRGDsc sequences, underlining that the NCO-sP(EO-stat-PO) film preserves the configuration-specific biochemical peptide properties. Under basal conditions, MSC expressed osteogenic marker genes after 14 days on cyclic RGD peptides, but not on linear RGD peptides or the unmodified glass surfaces. Our results indicate specific effects of these adhesion peptides on MSC biology and show that this coating system is useful for selective testing of cellular interactions with adhesive ligands.     

Micropatterned surfaces with controlled ligand tethering

Microcontact printing (micro-CP) is a facile, cost-effective, and versatile soft-lithography technique to create two-dimensional patterns of domains with distinct functionalities that provides a robust platform to generate micropatterned biotechnological arrays and cell culture substrates. Current micro-CP approaches rely on nonspecific immobilization of biological ligands, either by direct printing or adsorption from solution, onto micropatterned domains surrounded by a nonfouling background. This technique is limited by insufficient control over ligand density. We present a modified micro-CP protocol involving stamping mixed ratios of carboxyl- and tri(ethylene glycol)-terminated alkanethiols that provides for precise covalent tethering of single or multiple ligands to prescribed micropatterns via standard peptide chemistry. Processing parameters were optimized to identify conditions that control relevant endpoint pattern characteristics. This technique provides a facile method to generate micropatterned arrays with tailorable and controlled presentation of biological ligands for biotechnological applications and analyses of cell-material interactions.     

Integrin interactions with immobilized peptides in polyethylene glycol diacrylate hydrogels

This study employs tissue-engineering technologies to evaluate neutrophil interactions with extracellular matrix (ECM)-mimetic peptides. We have used a polyethylene glycol (PEG) diacrylate derivative to form a hydrogel as a biologically inert surface. Covalent attachment of bioactive moieties to the hydrogel makes it bioactive. The goal is to define the mechanisms by which these moieties influence the interactions of neutrophils with this bioactive hydrogel, and thus understand the likely effects of similar ligands in the ECM. The current experiments analyze the interactions of isolated human neutrophils with PEG hydrogels modified with Arg-Gly-Asp-Ser (RGDS), a known ligand for some beta(1) and beta(3) integrins, and Thr-Met-Lys-Ile-Ile-Pro-Phe-Asn-Arg-Leu-Thr-Ile-Gly-Gly (TMKIIPFNRLTIGG), a ligand for Mac-1, a beta(2) integrin. Our results demonstrate that neutrophils, independent of chemotactic stimulation, show little ability to adhere to unmodified PEG hydrogels. However, cell adhesion and spreading are robust on peptide-modified hydrogels. Incorporating distinct bioactive peptides, either alone or in combination, has enabled recognition of differential functions of alpha(v)beta(3), beta(1), and beta(2) integrins on neutrophil adhesion and spreading. Combined interactions result in activity that differs markedly from that seen with either integrin independently engaged. This model allows investigation of specific ligand-induced leukocyte functions and the development of engineered matrices with defined bioactive properties.     

Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.     

The effect of ligand type and density on osteoblast adhesion, proliferation, and matrix mineralization

Polystyrene surfaces grafted with a nonfouling interfacial interpenetrating polymer network (IPN) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] were modified with several peptide ligands adapted from bone sialoprotein (BSP). IPNs were modified with both single ligands and ligand blends to study the correlation between a simple metric, ligand-receptor adhesion strength, and the extent of matrix mineralization for osteoblast like cells (rat calvarial osteoblasts). The ligands studied included RGD cell-binding [CGGNGEPRGDTYRAY (l-RGD), CGGEPRGDTYRA (s2-RGD), CGPRGDTYG (lc-RGD), cyclic(CGPRGDTYG) (c-RGD), and CGGPRGDT (s-RGD)], heparin binding (CGGFHRRIKA), and collagen binding (CGGDGEAG) peptides, with the appropriate controls. Adhesion strength scaled with ligand density (1-20 pmol/cm(2)) and was dependent on ligand type with the following trend: l-RGD > s2-RGD approximately c-RGD > s-RGD approximately lc-RGD > FHRRIKA approximately DGEA. Independent of ligand density, % matrix mineralization varied with ligand type resulting in the following trend: lc-RGD > s2-RGD > l-RGD approximately c-RGD > s-RGD > FHRRIKA. The Tyr (Y) residue immediately following the RGD cell-binding domain proved to be critical for stable cell proliferation and mineralization, since removal of this residue resulted in erratic cell attachment and mineralization behavior. The minimum BSP sequence necessary for strong adhesion and extensive mineralization was CGGEPRGDTYRA; the minimal sequence suitable for extensive mineralization but lacking strong adhesion was CGPRGDTYG. The cyclic peptide (c-RGD) had much greater adhesion strength compared to its linear counterpart (lc-RGD). The calculated characteristic adhesion strength (F(70)) obtained using a centrifuge adhesion assay proved to be a poor metric for predicting % mineralized area; however, in general, surfaces possessing a F(70) > 100g promoted extensive matrix mineralization. Percent mineralization and number of mineralized nodules scaled with number of cells seeded suggesting a critical dependence on the initial number of osteoprogenitors in culture. This study demonstrates matrix mineralization dependence on ligand type, ligand density, and adhesion strength. The high-throughput character of these surfaces allowed efficient investigation of multiple ligands at multiple densities providing an excellent tool for studying ligand-receptor interactions under normal cell culture conditions with serum present.     

Adhesion Behavior of Human Adipo-Stromal Cells on Self-Assembled Monolayers with Different Surface Densities or Gradients of RGD Peptide

The objective of this study is to investigate the adhesion of human adipo-stromal cells on self-assembled monolayers (SAM) with different surface densities and gradients of Arg-Gly-Asp (RGD) peptide. The different densities and gradients of carboxyl groups on the SAM surface were prepared by a SAM exchange technique, and then RGD was chemically immobilized to allow the RGD surface density on the SAM to change over the range of 2.3–8.9 ng/cm² (3.0–12 pmol/cm²). The spreading area and survival percent of cells increased as the density of RGD immobilized on the SAM became high, whereas no dependence of the RGD density on the number of cells adhered was observed. The survival percent of cells tended to increase with an increase in the RGD immobilization for the carboxyl group-gradient SAM. This finding suggests the possibility that the cell adhesion can be regulated by controlling the RGD density or gradient of cell substrate.     

Extracellular matrix-mimetic poly(ethylene glycol) hydrogels engineered to regulate smooth muscle cell proliferation in 3-D

The goal of this project is to engineer a defined, synthetic poly(ethylene glycol) (PEG) hydrogel as a model system to investigate smooth muscle cell (SMC) proliferation in three-dimensions (3-D). To mimic the properties of extracellular matrix, both cell-adhesive peptide (GRGDSP) and matrix metalloproteinase (MMP) sensitive peptide (VPMSMRGG or GPQGIAGQ) were incorporated into the PEG macromer chain. Copolymerization of the biomimetic macromers results in the formation of bioactive hydrogels with the dual properties of cell adhesion and proteolytic degradation. Using these biomimetic scaffolds, the authors studied the effect of scaffold properties, including RGD concentration, MMP sensitivity, and network crosslinking density, as well as heparin as an exogenous factor on 3-D SMC proliferation. The results indicated that the incorporation of cell-adhesive ligand significantly enhanced SMC spreading and proliferation, with cell-adhesive ligand concentration mediating 3-D SMC proliferation in a biphasic manner. The faster degrading hydrogels promoted SMC proliferation and spreading. In addition, 3-D SMC proliferation was inhibited by increasing network crosslinking density and exogenous heparin treatment. These constructs are a good model system for studying the effect of hydrogel properties on SMC functions and show promise as a tissue engineering platform for vascular in vivo applications.     

Thermolysin-catalyzed peptide synthesis in aqueous polymer two-phase systems

A proteolytic enzyme, thermolysin, was covalently immobilized to dextran which has a small number of carboxyl groups. Enzymatic synthesis of aspartame derivative (Boc-Asp-Phe-OMe) using the dextran-conjugated thermolysin was carried out in an aqueous polymer two-phase system, where poly(ethylene glycol) (PEG) and dextran dissolved in water make a phase separation. The yield of the peptide catalyzed by the dextran-conjugated thermolysin was higher than that using a native enzyme in the aqueous polymer two-phase system partly due to the effect of the microenvironment around the modified enzyme and partly due to the “mass-law” effect (the preferential dissolution of the peptide product into the PEG phase). The aqueous polymer two-phase system would be very useful to carry out enzymatic organic syntheses in high yields.