Chirality effects at each amino acid position on tripeptide self-assembly into hydrogel biomaterials.

Hydrogels formed by ultrashort peptides are emerging as cost-effective materials for cell culture. However, L-peptides are labile to proteases, while their D-isomers are thought to not support cell growth as well. In contrast, the self-assembly behaviour and biological performance of heterochiral peptides (i.e., made of both d and l amino acids) are largely unknown. In this study, we evaluate the effects of amino acid chirality on tripeptide self-assembly and hydrogelation at physiological pH, and cytocompatibility in fibroblast cell culture. A series of uncapped hydrophobic tripeptides with all combinations of d, l amino acids was prepared, tested for self-assembly under physiological conditions, and analysed by circular dichroism, FT-IR, cryo-TEM, AFM, and Thioflavin T fluorescence imaging. Amino acid chirality has a profound effect on the peptides' supramolecular behaviour. Only selected isomers form hydrogels, and of amyloid structure, as confirmed by rheology and XRD. Importantly, they are able to maintain the viability and proliferation of fibroblasts in vitro. This study identifies two heterochiral gels that perform well in cell culture and will assist in the design of innovative and cost-effective peptide gel biomaterials.

Biological responses of human osteoblasts and osteoclasts to flame-sprayed coatings of hydroxyapatite and fluorapatite blends.

The aim of this study was to determine how the activities of human osteoblastic cells and osteoclasts respond to substrates of thermal-sprayed mechanical blends of hydroxyapatite and fluorapatite with a view of determining an optimal blend ratio for osseointegration. Human osteoblastic cells and osteoclasts were grown on titanium alloy discs coated with blends of hydroxyapatite and fluorapatite, with concentrations ranging from 0 to 100% fluorapatite. Human osteoblastic cells attached in greater numbers and proliferated at a greater rate on blends containing 40% fluorapatite. Human osteoblastic cells grown on blends containing 40% fluorapatite for 7 days also expressed the highest levels of mRNA for several proteins involved with regulating bone metabolism (osteoprotegerin and receptor activator nuclear factor kappa B ligand), and bone formation (osteopontin, osteonectin and bone sialoprotein 1). Osteoclasts resorbed the dentine but poorly resorbed the hydroxyapatite-fluorapatite blends, particularly at high levels of fluorapatite. This in vitro study demonstrates that thermal-sprayed hydroxyapatitecoatings containing 40% fluorapatite may promote optimal bone growth and improve osseointegration of implants.

Surface-MALDI mass spectrometry in biomaterials research.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used for over a decade for the determination of purity and accurate molecular masses of macromolecular analytes, such as proteins, in solution. In the last few years the technique has been adapted to become a new surface analysis method with unique capabilities that complement established biomaterial surface analysis methods such as XPS and ToF-SSIMS. These new MALDI variant methods, which we shall collectively summarize as Surface-MALDI-MS, are capable of desorbing adsorbed macromolecules from biomaterial surfaces and detecting their molecular ions with high mass resolution and at levels much below monolayer coverage. Thus, Surface-MALDI-MS offers unique means of addressing biomaterial surface analysis needs, such as identification of the proteins and lipids that adsorb from multicomponent biological solutions in vitro and in vivo, the study of interactions between biomaterial surfaces and biomolecules, and identification of surface-enriched additives and contaminants. Surface-MALDI-MS is rapid, experimentally convenient, overcomes limitations in mass resolution and sensitivity of established biochemical techniques such as SDS-PAGE, and can in some circumstances be used for the quantitative analysis of adsorbed protein amounts. At this early stage of development, however, limitations exist: in some cases proteins are not detectable, which appears to be related to tight surface binding. This review summarizes ways in which Surface-MALDI-MS methods have been applied to the study of a range of issues in biomaterials surfaces research.

XPS and surface-MALDI-MS characterisation of worn HEMA-based contact lenses.

XPS and MALDI-MS were used to analyse initial adsorption events in the fouling of HEMA-based contact lenses. All of the lenses tested accumulated tear film deposits within 10 min of wear. XPS indicated the presence of mainly proteinaceous deposits, with indications of some contributions by mucins or lipids on some lenses and the nature of the deposit being influenced by the lens chemistry. MALDI-MS detected the presence of surface-adsorbed species with molecular weights < 15 kDa. While lysozyme could be identified by comparison of MALDI-MS signals with known protein mass and assignments are suggested for some other signals, several other species, with MWs less than that of lysozyme, could not be identified as no ocular proteins with corresponding MWs had been reported in previous biochemical tear film analyses. These species, and others, were also detected in MALDI-MS analysis of reflex tear film, suggesting that the adsorbed unidentified species were not simply products of surface-induced dissociation of adsorbing higher-MW proteins. This short-term wear study detected rapid interface conversion and demonstrated the utility and surface sensitivity of XPS and MALDI-MS in characterising contact lens deposits at the initial stages when sub-monolayer adsorbed amounts are present on lenses.

A review of the development of a synthetic corneal onlay for refractive correction.

A synthetic corneal onlay, or implantable contact lens, could obviate the need for spectacles or conventional contact lenses in patients who seek convenient, reversible correction of refractive error. Several research groups have attempted to develop such a product in the past but much of the data from these studies remains unpublished due to commercial interests. This article reviews relevant papers and patents in the corneal implant field and discusses our efforts to develop a synthetic corneal onlay using a perfluoropolyether-based polymer.

The architecture of a collagen coating on a synthetic polymer influences epithelial adhesion.

The current study sought to identify a collagen coating methodology for application to polymer surfaces that would provide for the development of adhesive structures responsible for the sustained adhesion of corneal epithelial tissue. We compared an uncoated microporous polycarbonate surface and equivalent surfaces coated with either covalently immobilized collagen I or chemically crosslinked collagen I gel in a corneal explant outgrowth assay over 21 days. Electron microscopy was used to examine the formation of hemidesmosomes, basal lamina, and anchoring fibrils at the tissue-polymer interface. The crosslinked collagen gel preparation supported the overlying epithelial tissue across the pore openings and allowed for the formation of identifiable basal lamina, hemidesmosomes, and anchoring fibrils between the epithelial tissue and the polymer surface. Hemidesmosomal plaque, but no basal lamina or anchoring fibril formation, occurred on the uncoated surface or on that coated with covalently immobilized collagen I. We propose that the collagen matrix provided by the crosslinked collagen gel was reorganized by the epithelial tissue and that this, combined with the secretion of ECM molecules, served to limit the diffusion of basement membrane components, which permitted an increase in the local concentration of these molecules, which favored the assembly of epithelial adhesive structures.

Peptoid-containing collagen mimetics with cell binding activity.

Collagen mimetic peptides containing the peptoid residue Nleu (Goodman Bhumralkar, Jefferson, Kwak, Locardi. Biopolymers 1998;47:127-142) were tested for interactions with epithelial cells and fibroblasts. Molecules containing the sequence Gly-Pro-Nleu with a minimum of nine repeats showed cell binding activity. The activity of these molecules appeared to be conformationally sensitive, with the triple-helical form being preferred. When immobilized on a surface, the (Gly-Pro-Nleu)(10)-Gly-Pro-NH(2) sequence stimulated the attachment and growth of corneal epithelial cells and fibroblasts and the migration of epithelial tissue. The peptide sequence KDGEA inhibited cell attachment to the (Gly-Pro-Nleu)(10)-Gly-Pro-NH(2) sequence, suggesting that cell binding to this collagen mimetic involves the alpha2beta1 heterodimer integrin receptor. Interestingly, peptides containing the sequence (GlyNleu-Pro-)(10)-NH(2) did not have cell binding activity. The discovery that triple-helical peptides containing the Gly-Pro-Nleu sequences interact with cells opens up new opportunities in the design of collagen mimetic biomaterials.

Effect of porosity and surface hydrophilicity on migration of epithelial tissue over synthetic polymer.

The relative effects of porosity and surface chemistry on the migration of epithelial tissue over the surface of a polymer were determined in vitro. These studies compared nonporous polymers with those having 0.1-microm diameter track-etched pores and were conducted on polycarbonate and polyester. Epithelial tissue migration over the polymer surface was stimulated by the presence of these pores. The surface chemistries of the polymers were modified by deposition of various polymer films using radio frequency gas deposition, giving a range of surfaces that varied in air:water sessile contact angle (SCA) of between 26 and 100 degrees. Tissue migration on the nonporous surfaces was affected by the surface chemistry, being generally linear as a function of the SCA and higher on hydrophilic than on hydrophobic surfaces but reduced if the hydrophilic surface had a mobile chemistry. The effects of the 0.1-microm diameter pores and the surface hydrophilicity were additive with the maximal level of epithelial tissue migration occurring on a porous, hydrophilic polymer surface.

Increased resistance to ionizing and ultraviolet radiation in Escherichia coli JM83 is associated with a chromosomal rearrangement.

Cells cope with radiation damage through several mechanisms: (1) increased DNA repair activity, (2) scavenging and inactivation of radiation-induced radical molecules, and (3) entry into a G0-like quiescent state. We have investigated a chromosomal rearrangement to elucidate further the molecular and genetic mechanisms underlying these phenomena. A mutant of Escherichia coli JM83 (phi 80dlacZ delta M15) was isolated that demonstrated significantly increased resistance to both ionizing and ultraviolet radiation. Surviving fractions of mutant and wild-type cells were measured following exposure to standardized doses of radiation. Increased radioresistance was directly related to a chromosomal alteration near the bacteriophage phi 80 attachment site (attB), as initially detected by the LacZ- phenotype of the isolate. Southern hybridization of chromosomal DNA from the mutant and wild-type E. coli JM83 strains indicated that a deletion had occurred. We propose that the deletion near the attB locus produces the radioresistant phenotype of the E. coli JM83 LacZ- mutant, perhaps through the alteration or inactivation of a gene or its controlling element(s).

Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis.

A ca. 10-kilobase (kb) HindIII fragment of plasmid DNA from Bacillus thuringiensis subsp. israelensis was cloned into plasmid pUC9 and transformed into Escherichia coli. Extracts of the recombinant strain contained a 27-kilodalton (kDa) peptide that reacted with antibodies to a 27-kDa peptide isolated from crystals produced by B. thuringiensis subsp. israelensis. Extracts of the recombinant strain were hemolytic and toxic to Aedes aegypti larvae. Full expression of the 27-kDa peptide required the presence of a ca. 0.8-kb region of DNA located 4 kb upstream from the structural gene; the 0.8-kb region could be present in cis or trans relative to the gene and apparently acted post-transcriptionally. Analysis of maxicells showed that the 10-kb insert also coded for peptides of 67, 20, and 16 kDa; data obtained with different subclones suggest that the 20-kDa peptide is encoded in the 0.8-kb DNA region.