title: Kinetics and isotherm of fibronectin adsorption to three-dimensional porous chitosan scaffolds explored by ¹²⁵I-radiolabelling.
abstract: In this study, (125)I-radiolabelling was explored to follow the kinetics and isotherm of fibronectin (FN) adsorption to porous polymeric scaffolds, as well as to assess the elution and exchangeability of pre-adsorbed FN following incubation in serum-containing culture medium. Chitosan (CH) porous scaffolds with two different degrees of acetylation (DA 4% and 15%) were incubated in FN solutions with concentrations ranging from 5 to 50 µg/mL. The kinetic and isotherm of FN adsorption to CH were successfully followed using (125)I-FN as a tracer molecule. While on DA 4% the levels of adsorbed FN increased linearly with FN solution concentration, on DA 15% a saturation plateau was attained, and FN adsorbed amounts were significantly lower. These findings were supported by immunofluorescent studies that revealed, for the same FN solution concentration, higher levels of exposed cell-binding domains on DA 4% as compared with DA 15%. Following incubation in serum containing medium, DA 4% also revealed higher ability to exchange pre-adsorbed FN by new FN molecules from serum than DA 15%. In accordance, when assessing the efficacy of passively adsorbed FN to promote endothelial cell (EC) adhesion to CH, ECs were found to adhere at higher levels to DA 4% as compared with DA 15%, 5 µg/mL of FN being already efficient in promoting cell adhesion and cytoskeletal organization on CH with DA 4%. Taken together the results show that protein radiolabelling can be used as an effective tool to study protein adsorption to porous polymeric scaffolds, both from single and complex protein solutions.
authors: INEB-Instituto de Engenharia Biomédica; Universidade do Porto; Porto, Portugal.
authors: Isabel F
authors: IF
authors: Amaral
authors: INEB-Instituto de Engenharia Biomédica; Universidade do Porto; Porto, Portugal; REQUIMTE; Instituto Superior de Engenharia do Porto; Instituto Politécnico do Porto; Porto, Portugal.
authors: Susana R
authors: SR
authors: Sousa
authors: INEB-Instituto de Engenharia Biomédica; Universidade do Porto; Porto, Portugal.
authors: Ismael
authors: I
authors: Neiva
authors: IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto; Porto, Portugal.
authors: Lara
authors: L
authors: Marcos-Silva
authors: REPAIR Lab, Institute of Pathology; University Medical Center; Johannes Gutenberg University; Mainz, Germany.
authors: Charles J
authors: CJ
authors: Kirkpatrick
authors: INEB-Instituto de Engenharia Biomédica; Universidade do Porto; Porto, Portugal; Universidade do Porto; Instituto de Ciências Biomédicas Abel Salazar and Faculdade de Engenharia; Porto, Portugal.
authors: Mário A
authors: MA
authors: Barbosa
authors: INEB-Instituto de Engenharia Biomédica; Universidade do Porto; Porto, Portugal; Universidade do Porto; Instituto de Ciências Biomédicas Abel Salazar and Faculdade de Engenharia; Porto, Portugal.
authors: Ana P
authors: AP
authors: Pêgo
mesh_terms: D000327:Adsorption; D001672:Biocompatible Materials / Q000737:chemistry*; D002460:Cell Line; D048271:Chitosan / Q000737:chemistry*; D042783:Endothelial Cells; D005353:Fibronectins / Q000737:chemistry / Q000493:pharmacokinetics*; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D007700:Kinetics; D016062:Porosity; D013499:Surface Properties; D054457:Tissue Scaffolds
chemical_list: D001672:Biocompatible Materials; D005353:Fibronectins; D007457:Iodine Radioisotopes; D048271:Chitosan
keywords: 3-D scaffolds; fibronectin; fluorescence; protein adsorption; protein conformation
references: Biomaterials. 2009 Oct;30(29):5465-75
references: 19615736
references: J Mater Sci Mater Med. 2004 Apr;15(4):387-90
references: 15332604
references: Nat Mater. 2011 Jan;10(1):67-73
references: 21151163
references: Mol Biol Cell. 1999 Mar;10(3):785-98
references: 10069818
references: J Cell Sci. 2002 Jun 15;115(Pt 12):2581-90
references: 12045228
references: Biomaterials. 1997 Apr;18(7):567-75
references: 9105597
references: J Biomed Mater Res A. 2003 Aug 1;66(2):247-59
references: 12888994
references: Biomaterials. 2004 Dec;25(28):5947-54
references: 15183609
references: Biomaterials. 2005 Jun;26(16):3197-206
references: 15603814
references: J Mater Sci Mater Med. 2008 Jan;19(1):343-57
references: 17597372
references: Langmuir. 2004 Oct 26;20(22):9745-54
references: 15491210
references: J Biomed Mater Res A. 2005 Dec 15;75(4):824-31
references: 16138323
references: J Biomed Mater Res A. 2005 Feb 1;72(2):228-36
references: 15625681
references: Lab Invest. 2001 Dec;81(12):1717-27
references: 11742042
references: J Biomater Sci Polym Ed. 2007;18(4):469-85
references: 17540120
references: Biomaterials. 2009 Jan;30(3):307-16
references: 18952279
references: J Biomed Mater Res A. 2009 Jun 15;89(4):1040-8
references: 18478557
references: Acta Biomater. 2009 Jun;5(5):1389-98
references: 19135420
references: Biomaterials. 2002 Dec;23(24):4803-9
references: 12361619
references: J Biomed Mater Res A. 2003 Nov 1;67(2):538-47
references: 14566796
references: J Mater Sci Mater Med. 2011 Apr;22(4):839-44
references: 21431352
references: Biomaterials. 1998 Jul;19(14):1275-85
references: 9720891
references: Biotechnol Bioeng. 2008 Oct 15;101(3):609-21
references: 18461606
references: Microvasc Res. 2002 Nov;64(3):384-97
references: 12453433
references: J Biomed Mater Res A. 2003 Nov 1;67(2):531-7
references: 14566795
references: Biomaterials. 2009 Oct;30(29):5541-51
references: 19595453
references: J Biomed Mater Res A. 2003 Jan 1;64(1):20-37
references: 12483693
references: Colloids Surf B Biointerfaces. 2010 Jun 1;77(2):181-90
references: 20185279