Search

Abstract: The activation of surfaces by covalent attachment of bioactive moieties is an important strategy for improving the performance of biomedical materials. Such techniques have also been used as tools to study cellular responses to particular chemistries of interest. The creation of gradients of covalently bound chemistries is a logical extension of this technique. Gradient surfaces may permit the rapid screening of a large range of concentrations in a single experiment. In addition, the biological response to the gradient itself may provide new information on receptor requirements and cell signaling. The current work describes a rapid and flexible technique for the covalent addition of bioactive peptide gradients to a surface or gel and a simple fluorescence technique for assaying the gradient. In this technique, bioactive peptides with a terminal cysteine are bound via a heterobifunctional coupling agent to primary amine-containing surfaces and gels. A gradient in the coupling agent is created on the surfaces or gels by varying the residence time of the coupling agent across the surface or gel, thereby controlling the extent of reaction. We demonstrate this technique using poly(l-lysine)-coated glass surfaces and fibrin gels. Once the surface or gel has been activated by the addition of the coupling agent gradient, the bioactive peptide is added. Quantitation of the gradient is achieved by measuring the reaction kinetics of the coupling agent with the surface or gel of interest. This can be done either by fluorescently labeling the coupling agent (in the case of surfaces) or by spectrophotometrically detecting the release of pyridine-2-thione, which is produced when the thiol-reactive portion of the coupling agent reacts. By these methods, we can obtain reasonably precise estimates for the peptide gradients without using expensive spectroscopic or radiolabeling techniques. Validation with changes in fibroblast cell migration behavior across a bioactive peptide gradient illustrates preservation of peptide function as well as the usefulness of this technique. [Copyright &y& Elsevier]

Understanding the mechanisms of protein interactions with solid surfaces is critical to predict how proteins affect the performance of materials in biological environments. Low-fouling and ultra-low fouling surfaces are often evaluated in short-term protein adsorption experiments, where 'short-term' is defined as the time required to reach an initial apparent or pseudo-equilibrium, which is usually less than 600 s. However, it has long been recognized that these short-term observations fail to predict protein adsorption behavior in the long-term, characterized by irreversible accumulation of protein on the surface. This important long-term behavior is frequently ignored or attributed to slow changes in surface chemistry over time—such as oxidation—often with little or no experimental evidence. Here, we report experiments measuring protein adsorption on "low-fouling" and "ultralow-fouling" surfaces using single-molecule localization microscopy to directly probe protein adsorption and desorption. The experiments detect protein adsorption for thousands of seconds, enabling direct observation of both short-term (reversible adsorption) and long-term (irreversible adsorption leading to accumulation) protein-surface interactions. By bridging the gap between these two time scales in a single experiment, this work enables us to develop a single mathematical model that predicts behavior in both temporal regimes. The experimental data in combination with the resulting model provide several important insights: (1) short-term measurements of protein adsorption using ensemble-averaging methods may not be sufficient for designing antifouling materials; (2) all investigated surfaces eventually foul when in long-term contact with protein solutions; (3) fouling can occur through surface-induced oligomerization of proteins which may be a distinct step from irreversible adsorption; and (4) surfaces can be designed to reduce oligomerization or the adsorption of oligomers, to prevent or delay fouling. [ABSTRACT FROM AUTHOR]

Poly(ε-caprolactone) (PCL) is a hydrophobic and cytocompatible aliphatic polyester that has been used to produce PCL-based nanofibrous for both wound healing and tissue repair. However, the high hydrophobicity and low water adsorptive have been challenges for developing PCL-based materials for use in tissue engineering field. Here, we report a new polymer (a hydrophilic amino-functionalized tannin (TN)) that is associated with PCL for developing PCL-TN blends at different PCL:TN weight ratios (100:0, 95:5, 85:15 and 78:22). PCL:TN ratio may be tuned to modulate hydrophilicity and cytocompatibility of the nanofibers. The neutralization step and surface wettability played an important role in the attachment of human adipose-derived stem cells (ADSC cells) on PCL-TN membranes. Also, fluorescence images confirmed great proliferation of ADSC cells on the PCL-TN electrospun surfaces. Yet, neutralized PCL-TN nanofibers promoted bactericidal activity against Pseudomonas aeruginosa. These membranes have potential to be used as scaffolds for tissue engineering purposes. [ABSTRACT FROM AUTHOR]