3rd meeting of young researchers at UP 1 - IJUP - Universidade do ...

Surface modification of chitosan porous scaffolds with recombinant
fragments of fibronectin to promote endothelial cell adhesion
I. R. Neiva 1 , I. F. Amaral 1 , F. Ferreira da Silva 2 , S. R. Sousa 1,3 , A. M. Piloto 1 ,
M. A. Barbosa 1 and A. P. Pêgo 1
1 INEB – Instituto de Engenharia Biomédica, Divisão de Biomateriais, Universidade do Porto, Portugal;
2 IBMC – Instituto de Biologia Celular e Molecular, Unidade de Produção e Purificação de Proteínas,
Universidade do Porto, Portugal; 3 ISEP – Instituto Superior de Engenharia do Porto, Departamento de
Engenharia Química, Porto, Portugal
Neural stem cell (NCS) therapies are among the most promising strategies for the treatment of
spinal cord injuries. The transplantation of NCSs within a pre-endothelialised porous scaffold
is expected to promote NCS survival as well as neuronal differentiation. Previous work showed
that porous chitosan (Ch) scaffolds colonization by endothelial cells (ECs) could be achieved
by physiadsorbing fibronectin (FN) to Ch [1]. In this study, rhFNIII7-10, a recombinant
fragment of FN including the central cell-binding domain, was investigated regarding its ability
to promote EC adhesion to Ch scaffolds.
For this purpose, rhFNIII7-10 was produced and purified. Ch tubular porous scaffolds with two
degrees of acetylation (DAs), namely 4 and 15%, were prepared by thermally induced phase
separation (TIPS). Scaffolds were incubated in FN or rhFNIII7-10 solutions, and the amount of
adsorbed protein, as well as the exposure of cell-binding domains quantified using 125 I
radiolabelling and immunofluorescence staining, respectively. The ability of Ch scaffolds
physiadsorbed with rhFNIII7-10 to promote EC adhesion was investigated using a human cell
line of microvasculature ECs (HPMEC ST1.6R). Cell adhesion was assessed at 24h of cell
culture using a resazurin-based assay, while cell cytoskeleton organization was analysed by
confocal laser scanning microscopy (CLSM) after F-actin/DNA staining.
For the range of concentrations tested, the radioiodination assay showed an increase of
adsorbed protein as a function of rhFNIII7-10 concentration. Upon physiadsorption,
immunofluorescence studies revealed for FN and rhFNIII7-10 similar exposure of cell-binding
domains. Physiadsorption of rhFNIII7-10 to Ch scaffolds with DA 4% promoted EC adhesion to
Ch and cytoskeleton organization, no statistical significant differences being found in terms of
cell numbers as compared to FN, except for the highest concentration tested (80 µg/mL). In
contrast, rhFNIII7-10 was unable to promote EC adhesion to DA 15%, except for the highest
concentration evaluated.
Present results suggest that physiadsorbed rhFNIII7-10 can be used as a strategy to promote EC
adhesion to Ch scaffolds, its effectiveness being dependent on the DA. Presently, the covalent
grafting rhFNIII7-10 to Ch scaffolds is being explored.
[1] Amaral, I.F., Unger, R.E., Fuchs, S., Mendonça, A.M., Sousa, S.R., Barbosa, M.A., Pêgo, A.P.,
Kirkpatrick, C,. (2009), Fibronectin-mediated endothelialisation of chitosan porous matrices,
Biomaterials, 30 (29), 5465-5475
Work supported by FCT (FEDER funding, PTDC/SAU-BEB/65328/2006)
3 rd meeting of young researchers at UP 63