The inhibitory effect of an RGD-human chitin-binding domain fusion protein on the adhesion of fibroblasts to reacetylated chitosan films

Biomaterials used for tissue engineering applications must provide a structural support for the tissue development and also actively interact with cells, promoting adhesion, proliferation, and differentiation. To achieve this goal, adhesion molecules may be used, such as the tripeptide Arg-Gly-Asp (RGD). A method based on the use of a carbohydrate-binding module, with affinity for chitin, was tested as an alternative approach to the chemical grafting of bioactive peptides. This approach would simultaneously allow the production of recombinant peptides (alternatively to peptide synthesis) and provide a simple way for the specific and strong adsorption of the peptides to the biomaterial. A fusion recombinant protein, containing the RGD sequence fused to a human chitin-binding module (ChBM), was expressed in E. coli. The adhesion of fibroblasts to reacetylated chitosan (RC) films was the model system selected to analyze the properties of the obtained proteins. Thus, the evaluation of cell attachment and proliferation on polystyrene surfaces and reacetylated chitosan films, coated with the recombinant proteins, was performed using mouse embryo fibroblasts 3T3. The results show that the recombinant proteins affect negatively fibroblasts anchorage to the materials surface, inhibiting its adhesion and proliferation. We also conclude that this negative effect is fundamentally due to the human chitin-binding domain.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/27359/2006, POCTI/BIO/45356/200

Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor

A novel high-throughput label-free resonant waveguide grating (RWG) imager biosensor, the Epic® BenchTop (BT), was utilized to determine the dependence of cell spreading kinetics on the average surface density (vRGD) of integrin ligand RGD-motifs. vRGD was tuned over four orders of magnitude by co-adsorbing the biologically inactive PLL-g-PEG and the RGD-functionalized PLL-g-PEG-RGD synthetic copolymers from their mixed solutions onto the sensor surface. Using highly adherent human cervical tumor (HeLa) cells as a model system, cell adhesion kinetic data of unprecedented quality were obtained. Spreading kinetics were fitted with the logistic equation to obtain the spreading rate constant (r) and the maximum biosensor response (Δλmax), which is assumed to be directly proportional to the maximum spread contact area (Amax). r was found to be independent of the surface density of integrin ligands. In contrast, Δλmax increased with increasing RGD surface density until saturation at high densities. Interpreting the latter behavior with a simple kinetic mass action model, a 2D dissociation constant of 1753 ± 243 μm−2 (corresponding to a 3D dissociation constant of ~30 μM) was obtained for the binding between RGD-specific integrins embedded in the cell membrane and PLL-g-PEG-RGD. All of these results were obtained completely noninvasively without using any labels

Adhesive ligand tether length affects the size and length of focal adhesions and influences cell spreading and attachment.

Cells are known to respond to physical cues from their microenvironment such as matrix rigidity. Discrete adhesive ligands within flexible strands of fibronectin connect cell surface integrins to the broader extracellular matrix and are thought to mediate mechanosensing through the cytoskeleton-integrin-ECM linkage. We set out to determine if adhesive ligand tether length is another physical cue that cells can sense. Substrates were covalently modified with adhesive arginylglycylaspartic acid (RGD) ligands coupled with short (9.5 nm), medium (38.2 nm) and long (318 nm) length inert polyethylene glycol tethers. The size and length of focal adhesions of human foreskin fibroblasts gradually decreased from short to long tethers. Furthermore, we found cell adhesion varies in a linker length dependent manner with a remarkable 75% reduction in the density of cells on the surface and a 50% reduction in cell area between the shortest and longest linkers. We also report the interplay between RGD ligand concentration and tether length in determining cellular spread area. Our findings show that without varying substrate rigidity or ligand density, tether length alone can modulate cellular behaviour.This work was supported by the European Research Council to ADRH (grant agreement 282051). We wish to thank all CMBL members for help with this project