title: Endothelialization of chitosan porous conduits via immobilization of a recombinant fibronectin fragment (rhFNIII7-10).
abstract: The present study aimed to develop a pre-endothelialized chitosan (CH) porous hollowed scaffold for application in spinal cord regenerative therapies. CH conduits with different degrees of acetylation (DA; 4% and 15%) were prepared, characterized (microstructure, porosity and water uptake) and functionalized with a recombinant fragment of human fibronectin (rhFNIII(7-10)). Immobilized rhFNIII(7-10) was characterized in terms of amount ((125)I-radiolabelling), exposure of cell-binding domains (immunofluorescence) and ability to mediate endothelial cell (EC) adhesion and cytoskeletal rearrangement. Functionalized conduits revealed a linear increase in immobilized rhFNIII(7-10) with rhFNIII(7-10) concentration, and, for the same concentration, higher amounts of rhFNIII(7-10) on DA 4% compared with DA 15%. Moreover, rhFNIII(7-10) concentrations as low as 5 and 20μg ml(-1) in the coupling reaction were shown to provide DA 4% and 15% scaffolds, respectively, with levels of exposed cell-binding domains exceeding those observed on the control (DA 4% scaffolds incubated in a 20μg ml(-1) human fibronectin solution). These grafting conditions proved to be effective in mediating EC adhesion/cytoskeletal organization on CH with DA 4% and 15%, without affecting the endothelial angiogenic potential. rhFNIII(7-10) grafting to CH could be a strategy of particular interest in tissue engineering applications requiring the use of endothelialized porous matrices with tunable degradation rates.
authors.affiliation: INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. iamaral@ineb.up.pt
authors.forename: I F
authors.initials: IF
authors.lastname: Amaral
authors.forename: I
authors.initials: I
authors.lastname: Neiva
authors.forename: F
authors.initials: F
authors.lastname: Ferreira da Silva
authors.forename: S R
authors.initials: SR
authors.lastname: Sousa
authors.forename: A M
authors.initials: AM
authors.lastname: Piloto
authors.forename: C D F
authors.initials: CD
authors.lastname: Lopes
authors.forename: M A
authors.initials: MA
authors.lastname: Barbosa
authors.forename: C J
authors.initials: CJ
authors.lastname: Kirkpatrick
authors.forename: A P
authors.initials: AP
authors.lastname: Pêgo
mesh_terms: D000327:Adsorption; D048271:Chitosan / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology*; D005353:Fibronectins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D008855:Microscopy, Electron, Scanning; D018919:Neovascularization, Physiologic / Q000187:drug effects; D016062:Porosity; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D017550:Spectroscopy, Fourier Transform Infrared; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D005353:Fibronectins; D005456:Fluorescent Dyes; D055767:Immobilized Proteins; D011994:Recombinant Proteins; D004247:DNA; D048271:Chitosan