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Self-complementary synthetic peptides, composed by 8 and 16 residues, were analyzed by CD, NMR and small angle neutron scattering (SANS) techniques in order to investigate the relevance of charge and hydrophobic interactions in determining their self-assembling properties. All the sequences are potentially able to form fibrils and membranes as they share, with the prototype EAK16, a strictly alternating arrangement of polar and nonpolar residues. We find that 16-mer peptides show higher self-assembling propensities than the 8-mer analogs and that the aggregation processes are favored by salts and neutral pH. Peptide hydrophobic character appears as the most relevant factor in determining self-assembling. Solution conformational analysis, diffusion and SANS measurements all together show that the sequences with a higher self-assemble propensity are distributed, in mild conditions, between light and heavy forms. For some of the systems, the light form is mostly constituted by monomers in a random conformation, while the heavy one is constituted by beta-aggregates. In our study we also verified that sequences designed to adopt extended conformation, when dissolved in alcohol-water mixtures, can easily fold in helix structures. In that media, the prototype of the series appears distributed between helical monomers and beta-aggregates. It is worth noticing that the structural conversion from helical monomer to beta-aggregates, mimics beta-amyloid peptide aggregation mechanisms.

The two dipeptides AE (Lalanine–Lglutamic acid) and AK (Lalanine–Llysine), that constitute the “building blocks” of the 16-unit self-complementary amphiphilic oligopeptide EAK16, have been investigated by XPS (X-ray photoelectron spectroscopy) and NEXAFS (near-edge X-ray absorption fine structure) spectroscopy. Thin films of both dipeptides on TiO2, a distinguished biocompatible surface, were prepared by incubation from aqueous solutions. Thick films of dipeptides on inert Au substrates were also studied for comparison. The chemical structure and composition were investigated by XPS spectroscopy; furthermore, molecular orientation of dipeptides on TiO2 was checked by angular dependent NEXAFS measurements at both C–K and N–K edges. In order to yield some insight on adsorption geometry and molecular orientation MD (molecular dynamic) simulations were also carried out. The performed molecular and electronic characterization of AE and AK provides an excellent model for the interpretation of more complex peptide spectra.

Infectious HIV-1 requires gp160 cleavage by furin at the REKR511 downward arrow motif (site1) into the gp120/gp41 complex, whereas the KAKR503 (site2) sequence remains uncleaved. We synthesized 41mer and 51mer peptides, comprising site1 and site2, to study their conformation and in vitro furin processing. We found that, while the previously reported 19mer and 13mer analogues represent excellent in vitro furin substrates, the present extended sequences require heparin for optimal processing. Our data support the hypothesis of a direct binding of heparin with site1 and site2, allowing selective exposure/accessibility of the REKR sequence, which is only then optimally cleaved by furin.

Investigations on the relationships between the properties of biomaterial surfaces and cell adhesion/proliferation processes have recently gained increasing interest. To describe the behaviour of cells adhering and proliferating over different types of (and/or differently treated) substrates, some mathematical models have been also suggested in literature; these models consider both the dependence of cell adhesion/proliferation over time, and the influence of substrate morphology in allowing (or even hampering) cell attachment. Major developments in the biochemical functionalization of the materials used for the production of endosseous devices have been achieved; the ability of the so-called "biomimetic" surfaces to promote cell adhesion, thus favoring the osseointegration process, is already well acknowledged. The aim of this study was to formulate a mathematical model for osteoblast adhesion, mediated by an adhesion peptide (sequence 351-359 mapped on the Human Vitronectin Protein) covalently grafted to a titanium-based surface. To assure a highly homogenous orientation of the peptide to cells, the "specific functionalization" strategy was properly designed. Enzymatic detachment assays allowed comparing osteoblast behaviour over three differently treated titanium substrates (i.e., oxidized, silanized, and peptide-grafted), thus determining how and how much the bioactive peptide can improve the strength of cell adhesion. The results confirmed the capacity of the peptide to increase cell adhesion and adhesion strength; moreover, the role of the peptide was described by a mathematical equation characterizing cells behaviour.

We present a spectroscopic study of the structure of two peptides deposited on Au and TiO2: – PeptA, (EAK16-RGD) bringing the adhesive RGD sequence linked to EAK16; – PeptB, having RGD linked to a “scrambled” sequence of the EAK16 peptide. Previously reported NEXAFS investigations on thin films of the self-assembling peptide EAK16 deposited on Au and TiO2, revealed molecular order and orientation for both substrates. IR spectra show a β-sheet conformation for PeptA and a random structure for PeptB. Angular-dependent NEXAFS measurements reveal an ordered structure with preferential molecular orientation only for PeptA. XPS analysis indicates that PeptA is adsorbed on TiO2 in a larger amount than PeptB.

A properly engineered biomaterial for dental/orthopaedic applications must induce specific responses from the osteoblasts at the implant site. A most desirable response is an efficient adhesion, as it represents the first phase in the cell/material interaction and the quality of this phase will influence the cell's capacity to organize into a new functional tissue. The four osteoblast-adhesive peptides discussed in this paper are mapped on the 339–364 sequence (339MAPRPSLAKKQRFRHRNRKGYRSQRG364) located in the primary heparin-binding site of human vitronectin (HVP). Adsorbed on a polystyrene scaffold, these peptides display different adhesive activities towards osteoblasts. In this paper we report on the structural analysis in solution of the peptides through NMR and computational techniques. We find that the peptides with the highest adhesive activities display a hydrophobic patch opposite to the charged surface candidate to interact with heparin. These findings suggest that the peptides might adsorb on the polystyrene support in a favourable orientation for their activity. Furthermore, molecular models obtained for the four peptides in solution were used in rigid docking simulations with a heparin model. Assuming that the peptide solution conformations are not very different from the polystyrene-adsorbed structures, the simulations reveal that peptide adhesive activity is also affected by the number of ionic interactions and spacing between charged residues.

A fundamental goal in the field of implantology is the design of innovative devices suitable foil promoting, implant-to-tissue integration. This result can be achieved by means of surface modifications aimed at optimizing tissue regeneration. In the framework of oral and orthopedic implantology, surface modifications concern both the optimization of titanium/titanium alloy surface roughness and the attachment of biochemical factors able to guide cellular adhesion and/or growth. This article focuses on the covalent attachment of two different adhesive peptides to rough titanium disks. The capability of biomimetic surfaces to increase osteoblast adhesion and the specificity of their biological activity due to the presence of cell adhesion signal-motif have also been investigated. In addition, surface analyses by profilometry, X-ray photoelectron spectroscopy, and time of flight-secondary ion mass spectrometry have been carried out to investigate the effects and modifications induced by grafting procedures.

Bioactive molecules have been proposed to promote beneficial interactions at bone-implant interfaces for enhancing integration. The main objective of this study was to develop novel methods to functionalize oxidized titanium surfaces by the covalent immobilization of bioactive peptides, through selective reaction involving single functional groups. In the first protocol, an aminoalkylsilane was covalently linked to the Ti oxide layer, followed by covalent binding of glutaric anhydride to the free NH(2) groups. The carboxylic group Of glutaric anhydride was used to condense the free N-terminal group of the side-chain protected peptide sequence. Finally, the Surface was treated with trifluoroacetic acid to deprotect side-chain groups. In the second protocol, the peptide was directly anchored to the Ti oxide surface via UV activation of an arylazide peptide analogue. X-ray photoelectron spectroscopy analyses confirmed that modifications induced onto surface composition were in agreement with the reactions performed. The peptide density of each biomimetic Surface was determined on the basis of radiolabeling and XPS derived reaction yields. The in vitro cellular response of the biomimetic surfaces was evaluated using a primary human osteoblast cell model. Cell adhesion, proliferation, differentiation, and mineralization were examined at initial-, short-, and long-time periods. In was shown that the biomimetic surface obtained through photoprobe-marked analogue that combines an easily-performed modification provides a favorable surface for an enhanced cellular response.

Self-complementary synthetic peptides, composed by 8 and 16 residues, were analyzed by CD, NMR and small angle neutron scattering (SANS) techniques in order to investigate the relevance of charge and hydrophobic interactions in determining their self-assembling properties. All the sequences are potentially able to form fibrils and membranes as they share, with the prototype EAK16, a strictly alternating arrangement of polar and nonpolar residues. We find that 16-mer peptides show higher self-assembling propensities than the 8-mer analogs and that the aggregation processes are favored by salts and neutral pH. Peptide hydrophobic character appears as the most relevant factor in determining self-assembling. Solution conformational analysis, diffusion and SANS measurements all together show that the sequences with a higher self-assemble propensity are distributed, in mild conditions, between light and heavy forms. For some of the systems, the light form is mostly constituted by monomers in a random conformation, while the heavy one is constituted by beta-aggregates. In our study we also verified that sequences designed to adopt extended conformation, when dissolved in alcohol-water mixtures, can easily fold in helix structures. In that media, the prototype of the series appears distributed between helical monomers and beta-aggregates. It is worth noticing that the structural conversion from helical monomer to beta-aggregates, mimics beta-amyloid peptide aggregation mechanisms.

Peptides of alternating charge and hydrophobic amino acids have a tendency to adopt unusually stable β-sheet structures that can form insoluble macroscopic aggregates under physiological conditions. In this study, analogues of a well-known self-assembling peptide, characterized by the same polar/nonpolar periodicity but with different residues, were designed to study the relationship between sequence, conformation in solution and film-forming capacity in saline solution. Peptide conformation, evaluated by circular dichroism, correlated with film forming capacity observed by inverted optical microscopy after addition of saline solution and subsequent drying. We found that polar/nonpolar periodicity of several analogues is not criterion enough to induce β-sheet and thus film formation and that conformations different from β-sheet also allow self-assemblage. Furthermore, addition of the short adhesive sequence RGD to a known self-assembling sequence was shown to not prevent the self-assembling process. This finding might prove useful for the design of biomimetic scaffolds. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 906–915, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Peptides consisting of an alternation of hydrophobic and hydrophilic (positively and negatively charged) amino acids can generate extended ordered structures by self-assembling (SA) from aqueous solutions. In this paper we present XPS, near-edge X-ray absorption fine structure (NEXAFS) and Fourier transform infrared (FTIR) investigations on a series of SA peptides with the aim of determining the effect of side-chain length on molecular arrangement and orientation. Peptides were immobilised on the surface of titanium, a well-known biocompatible material, or deposited as thick films on inert gold surfaces. FTIR analysis yields information on the backbone conformation. XPS spectroscopy was used to investigate the peptide adsorption on the TiO 2 surface. The orientation of the peptide chains was investigated by angular-dependent NEXAFS.

Biomaterials to be used for the production of endosseous devices in dental, orthopedic and maxillo-facial applications, might be designed to support, guide and enhance osteoblast adhesion. Cell recruitment onto biomaterial surface is a fundamental step within the complex process responsible for implant osseointegration; this process involves several proteins from the extra cellular matrix (ECM), cytoskeleton and cell membrane. A new strategy to improve endosseous implant integration is based on preparing biomimetic surfaces able to present adhesive factors to cells. Osteoblast adhesion takes place by at least two different mechanisms: the most investigated one implies the interaction with RGD sequences via cell-membrane integrin receptors; a further mechanism concerns the interaction between cell-membrane heparan sulfate proteoglycans and heparin-binding sites of ECM proteins. In the present study two different biomimetic surfaces were obtained by covalently grafting two adhesive peptides on oxidized titanium substrates after silanization: an RGD-containing peptide and a peptide mapped on human vitronectin. The two sequences are known to act via different adhesive mechanisms. The amount of human osteoblasts adhered onto peptide-enriched or not enriched titanium oxidized surfaces and the strength of cell binding were estimated, thus comparing the capacity of the bioactive substrates in promoting cell adhesion.

The covalent attachment of an adhesive peptide, reproducing the 351–359 sequence of human vitronectin, to oxidized titanium surfaces was investigated by XPS spectroscopy. The peptide enhances osteoblast adhesion to titanium, the most used biomaterial for implants and prostheses. Core level spectra of the TiO2 surface and of the biomimetic surface were investigated. Novel selective covalent immobilization of (351–359) HVP was carried out by treatment of the TiO2 surface with (3-aminopropyl) triethoxysilane, glutaric anhydride and a side chain protected peptide sequence presenting only a free terminal amino group, followed by side chain deprotection. An alternative strategy for covalent attachment consists in photoactivation of physisorbed (351–359) HVP directly on the TiO2 surface; samples were incubated with HVP solution and subsequently irradiated with UV light. A comparison with the results previously obtained for non-selective HVP immobilization will be discussed.

A wide range of biochemical signals promoting cell functions (adhesion, migration, proliferation, and differentiation) and thereby improving the osseointegration process are currently investigated. Unfortunately, their application for the production of bioactive implantable devices is often hampered by their insolubility; instability; and limited availability of a large amount of inexpensive, high-purity samples. An attractive alternative is the use of short peptides carrying the minimum active sequence of the natural factors. Synthetic peptides mapped on fibronectin and vitronectin have been demonstrated to enhance cell adhesion both to polystyrene and acellular bone matrix; in particular, a nonapeptide sequence from human vitronectin works via an osteoblast-specific adhesion mechanism. In this study, we incorporated these peptides into a sol-gel silica dressing applied to coat sand-blasted and acid-attacked titanium samples; measured the kinetic of peptide release; and used titanium disks, coated with a peptide-enriched film, as substrates to determine the peptide concentration that maximizes cell adhesion in vitro. We also evaluated in vivo the capacity of the vitronectin-derived peptide to improve osteogenic activity: histologic analysis revealed markedly improved osteogenic activity around peptide-enriched samples. This article also discusses the role of surface characteristics and the importance of bioactive peptides.

EAK16 is a 16 amino acid peptide consisting of an alternation of polar and non-polar pending groups and of positively and negatively charged residues, that makes this material capable of self-assembling, producing an extended ordered structure. Thin films of EAK16 were prepared on TiO2, and An surfaces and investigated by surface-sensitive techniques such as XPS (X-ray photoelectron spectroscopy), NEXAFS (near edge X-ray absorption fine structure) and IR spectroscopies. XPS analysis allowed to check the chemical structure of the samples and to determine the film thickness. IR experiments yielded evidence of the beta-sheet conformation of the peptide backbone. Polarization dependent NEXAFS measurements allowed estimating the angle between the axis of the peptide backbone and the sample surface. (c) 2005 Elsevier B.V. All rights reserved.

The quality of the early cell/material interactions is responsible for the long-term functional properties of any implanted device. Accordingly, "next generation" dental/orthopedic biomaterials should be able to promote osteoblast adhesion thus improving the integration process between surgically placed implants and biological tissues. Recent studies have identified a wide range of biochemical signals that can be exploited to promote adhesion, migration, proliferation and differentiation of cells. The clinical use of natural factors to promote osteoblast adhesion is complicated because those are often insoluble and unstable macromolecules and, in addition, it is difficult to obtain them in high quantities, with good purity grade and at low cost. A valid alternative could be the use of short peptides carrying the minimum active sequence of the natural macromolecular factor. This paper describes the properties of two classes of peptides, promoting different adhesion mechanisms, to enhance rat bone marrow osteoblast adhesion both to polystyrene and to acellular bone matrix.

Next generation dental/orthopedic biomaterials must be designed to enhance and support osteoblast adhesion. The osteoblasts use different ways to adhere, that is, integrin- and proteoglycan-mediated mechanisms. The present study reports on the synthesis and osteoblast-adhesive properties of peptides carrying RGD motifs and of sequences mapped on human vitronectin. Our data suggest that osteoblast adhesion on polystyrene plates modified with a linear peptide, in which the GRGDSP sequence is repeated four times, was significantly higher when compared to the adhesion obtained using branched peptides, interestingly containing the same motif. Osteoblast adhesion assays on acellular bone matrix using this active peptide gave very promising results. We also demonstrated that a novel peptide, carrying the X-B-B-B-X-B-B-X motif (where B is a basic amino acid and X is a nonbasic residue), promotes proteoglycan-mediated osteoblast adhesion more efficiently with respect to the KRSR sequence that was recently proposed as heparan-sulfate binding peptide.