Abdominal Aortic Aneurysm (AAA) is caused by a weakening of the aortic wall due to the degradation of the elastic fibers, which brings to inflammation and to the apoptosis of smooth muscle cells of the media layer (Figure 1). EVAR (Figure 2) is a less invasive than sirurgical open repair procedure because the graft is placed into the patient thanks to a catheter. In both cases the solution is a mechanical support inside the vessel affected by AAA. These two methods try only to repair aortic wall, a tissue engineering approach is strongly necessary to reach healing by regeneration. Therefore a biodegradable scaffold has been designed to be implanted by open repair in patient.

Background

Two different concentrations of PCL have been tested: 10% and 12% solubilized in DiChlorumMethane (DCM), DiMethylFormamide (DMF) and Methanol in aqueous solution in the ratio of 5:3:2.The final goal is to obtain a tube with a lenght of 10-20 cm and a diameter in the range of 1.2-2.4 cm. The first characterizations have been done on layers: two different films have been generated by electrospinning (Figure 3).Electrospinning settings. 10% PCL: 0,8 ml/hrs; 27 kV. 12% PCL: 1,2 ml/hrs; 22 kV. During elctrospinning the effective evaporation of the solvent has been checked at the optical microscope.The obtained films has been characterized at the Scanning Electron Microscope (SEM).

Methods

ResultsOptical microscope. The 10% PCL has some bubbles within the fibers, this mean that the organic solvent is not at all evaporated.The 12% concentration is made of good fibers without bubbles. For this reason the final tubular scaffold has been built with the 12% PCL (Figure 4E).

SCAFFOLD FOR AORTIC WALL TISSUE BY ELECTROSPINNING

Eloina Corradi, Sabrina Eccheli, Stefania Masella, Solmaz Khaghani.University of Trento, Master in Cellular and Molecular Biotechnology

Laboratory tutor: Winston ElliottCourse: Tissue engineering and regenerative medicine, Professor Antonella Motta

porosity (μm)

diameter (μm)

10%12%

21,97 2,896,97 1,62

Figure 2. The two main approaches to treat AAA are the surgical open repair and the EndoVascularAneurysmRepair (EVAR). The aorta gain strenght thanks to a metallic or synthetic stent graft.http://www.musc.edu/radiology/interventional/index.htm

F i g u r e 3 . E l e c t r o s p i n n i n g machinery during the scaffold bu i l d i ng . A t ube has been generated and the films analysed have been cut from it. To visualize better the scaffold, rhodamine has been added to the solution, that has been elctrospun.

SEM. In the table on the right the mean values of porosity and diameters of the two samples.

In the 10% sample the fibers are more regular and homogenous also in term of diameter and are more oriented comparing to the 12% sample. In the 12% sample the fibers are not regular, not cylindrical and are randomly distributed.

10% PCL0,8ml/hrs

27kV

12% PCL1,2 ml/hrs

22 kV

1 KX

5 KX

Figure 4. SEM images of PCL layers (A,B,C,D).A: 10% PCL at 1KX magnification. B: 12% PCL at 1KX. C: 10% PCL at 5 KX. D: 12% PCL at 5 KX. E: Tubular scaffold generated with the 12% PCL. The red color is given by rhodamine.

ConclusionsThe 12% PCL solution fibers show less bubbles inside the net, this means that organic solvent has evaporated completely. This is an important feature if the scaffold has to be implanted into a patient because the solvent, trapped into fibers, will be released during the degradation of the scaffold causing toxicity. For this reason the 12% PCL seems to be more attractive than the 10% PCL for our purpose. However the mean porosity (6,97 μm) and diameter (1,62 μm) of fibers obtained allow the cell adhesion and migration. If the endothelial cells adhere and migrate on the graft, stenosis is likely to occur. To improve the future PCL scaffold, heparin and silk fibroin could be added in order to avoid the thrombus formation and to increase the hemocompatibility.The initial idea for this work was to build a scaffold (Figure 5) made of a net of silk fibroin/heparin/VEGF and PLGA/Calcium phosphate to give strength and provide a gradual release of the growth factor. VEGF promotes the endothelial cells to release bFGF and TGFβ, which stimulate the regeneration of the smooth muscle cells in the media layer. Since PLGA is hydrolyzed too quickly in physiological conditions, attention was focused on PCL: this polymer has a lower degradation rate and gives more strength to the final scaffold. Apart from the replacement of PLGA/Calcium phosphate with PCL, the initial designed scaffold remains. Further analysis are needed to evaluate the mechanical and chemical properties of the scaffold, which is going to interact with a specific pathological environment.

Figure 5. Ideal scaffold. A: Silk fibroin and heparin. B: Silk fibroin, heparin, PLGA, Calcium phosphate and VEGF.

Figure 1. The three layers of the aortic wall: Tunica Intima, media, externa (adventitia).Copyright 2004 Pearson Education, Inc. publishing as Benjamin Cummings.

AORTA TISSUE

! contact with blood! blood pressure control! Windkessel effect

The first characterization has been performed with PolyCaproLactone (PCL), a synthetic polymer with a long degradation time, that is already used for long term implantable device.

A B

C D

E

University of Trento Master of Science in Cellular and Molecular Biotechnology Gene Therapy 08/05/2014 Mattia Bolzan Fabio Marsoner Michele Olivieri

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University of Trento Master of Science in Cellular and Molecular Biotechnology Gene Therapy 08/05/2014 Mattia Bolzan Fabio Marsoner Michele Olivieri

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AA

B

Reference: Farokhi et al., Bio-hybrid silk fibroin/calcium phosphate/PLGA nanocomposite scaffold to control the delivery of vascular endothelial growth factor, Materials Science & Engineering. C, Materials for Biological Applications 2014 February 1, 35: 401-10

lunedì 30 giugno 2014