A Combined Method for Bilayered Vascular Graft Fabrication

INTRODUCTION Different approaches and biomaterials have been applied to solve thrombosis and intimal hyperplasia associated with failure of small diameter grafts. Electrospinning is a technique able to produce vascular graft with fibers in the nanometer range that resembles the extracellular matrix of native tissues, thus supporting endothelial cell attachment, alignment, proliferation and maintenance of their in vivo function [1, 2]. Moreover spray, phase-inversion technique was successfully employed to fabricate microporous polyurethane vascular graft able to support wall thickness cell colonization in 3D structure [3-4]. With the aim to take the advantage of both techniques we produced a bilayered polyurethane vascular graft combining electrospinning and spray technique. EXPERIMENTAL METHODS A 12% (w/v) solution of Poly(ether urethane) Estane® 5714 (hereinafter PU) was prepared by dissolving the PU in a solvent mixture of tetrahydrofurane (THF), N,N-dimethylformamide (DMF) and deionized water (58/40/2, v/v/v) for electrospinning. The electrospun inner-layer of the composite graft (ID 5 mm, length 200 mm) was produced using a custom-made electrospinning apparatus equipped with a rotating cylindrical collector [5]. Electrospinning was performed using needle-to-collector distance of 22 cm, solution flow rate of 0.5 mL/h and voltage of 26kV. Electrospun fibers were deposited on a grounded stainless steel rod rotating at 125 rpm to produce a tubular mat. The process was prolonged for 2 h to obtain a 100 ?m thick electrospun network. The external layer was obtained using spray, phase-inversion method as previously described [3]. A 0.2% (w/v) PU solution was prepared by dissolving PU in THF:1,4-dioxane (DX) (1:1, v/v) added with 17% (v/v) of distilled water. The PU solution and distilled water were sprayed on the electrospun scaffold rotating at 88 rpm at a flow rate of 2 mL/min for 50 min. After fabrication, grafts were maintained for 24 h in distilled water. For ultrastructural evaluation SEM micrographs were acquired and analyzed with ImageJ to quantify the thickness of electrospun and sprayed layers and the diameters of PU nanofibers. A peel tests, were performed according to modified ASTM method D 903-93 with dynamometric tension/compression machine (Z1.0, Zwick/Roell Germany) equipped with a 100 N load to evaluated adhesion strengths between electrospun and sprayed layers. After the peeling test the grafts were analyzed by SEM to evaluated morphological structure. RESULTS AND DISCUSSION Bilayered graft after fabrication appeared porous and without any gross defects. SEM images of the grafts evidenced different morphologies (Fig. 1A): 1) a nanofibrous inner layer with 101±6 ?m of thicknesses; 2) a microporous external layer with 130±13 ?m of thicknesses and a pore size measuring 90±55 nm (Fig. 1C); 3) an intermediate layer with a structure containing a mixing of electrospun and sprayed structures. No significant difference of electrospun layer thickness and fiber diameter was observed before and after spray deposition (Fig. 1B). The peel test between the electrospun and sprayed layers showed a 27±1.7 g/mm2 adhesion force. The SEM analysis of morphological structure of electrospun surface after peel test revealed a firm adhesion of sprayed layer onto the electrospun surface of graft indicated by the presence a thin layer of the microporous structure attached to the underlining electrospun layer. CONCLUSION This study demonstrated the possibility to combine two different consolidated techniques to manufacture a bilayered polyurethane vascular graft with a homogeneous microporous layer firmly attached on electrospun layer with the aim to supply a provisional cell template for in situ tissue regeneration.