title: The inhibitory effect of an RGD-human chitin-binding domain fusion protein on the adhesion of fibroblasts to reacetylated chitosan films.
abstract: 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.
authors.affiliation: IBB, Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors.forename: Vera
authors.initials: V
authors.lastname: Carvalho
authors.forename: Lucília
authors.initials: L
authors.lastname: Domingues
authors.forename: Miguel
authors.initials: M
authors.lastname: Gama
mesh_terms: D016475:3T3 Cells; D000107:Acetylation / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002686:Chitin / Q000737:chemistry / Q000494:pharmacology*; D048271:Chitosan / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects*; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology*; D011137:Polystyrenes; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D009842:Oligopeptides; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins; D002686:Chitin; C047981:arginyl-glycyl-aspartic acid; D048271:Chitosan