Silk-ELR co-recombinamer covered stents obtained by electrospinning

Abstract

In the field of tissue engineering the choice of materials is of great importance given the possibility to use biocompatible polymers produced by means of biotechnology. A large number of synthetic and natural materials have been used to this purpose and processed into scaffolds using Electrospinning technique. Among materials that could be used for the fabrication of scaffold and degradable membranes, natural polymers such as collagen, elastin or fibroin offer the possibility to design structures strictly similar to the extracellular matrix (ECM). Biotechnology and genetic engineering made possible the advent of a new class of biopolymers called protein-based polymers. One example is represented by the silk-elastin-proteins that combine the elasticity and resilience of elastin with the high tensile strength of silk-fibroin and display engineered bioactive sequences. In this work, we use electrospinning technique to produce a fibrous scaffold made of the co-recombinamer Silk-ELR. Obtained fibres have been characterized from the morphological point of view. Homogeneity and morphology have been explored using Scanning Electron Microscopy. A thorough study regarding the influence of Voltage, flow rate and distance have been carried out to determine the appropriate parameters to obtain the fibrous mats without defects and with a good distribution of diameters. Cytocompatibility has also been in vitro tested. For the first time we use the co-recombinamer Silk-ELR for the fabrication of a 2.5 angioplasty balloon coating. This structure could be useful as a coated scaffold for the regeneration of intima layer of vessels.

Keywords: elastin-like-recombinamers; electrospinning; silk; tissue engineering.

Figure 1

Co-recombinamer Silk-ELR primary structure (A). The highlighted blue part is the SILK portion, while the red one is the RGD bioactive sequence. Scheme of Silk-ELR co-recombinamer electrospinning and related fibre mats obtained (B)

Figure 2

Co-recombinamer Silk-ELR fibres obtained by changing voltage values from 13 to 20 kV (A, C and E) and related distribution diameter histograms (B, D and F). Tip-to-collector distance and flow rate were kept respectively at 15 cm and 0.3 ml/h. Histogram of fibre size as a function of changing tip-to-collector distance (G). P-values < 0.001

Figure 3

Co-recombinamer Silk-ELR fibres obtained by varying feed rate from 0.2 to 0.5 ml/h (A and C) and related distribution diameter histograms (B and D). Voltage and distance were kept respectively at 20 kV and 15 cm Histogram of fibre size as a function of changing flow rate (E). Silk-ELR fibres obtained increasing P-values < 0.001

Figure 4

Co-recombinamer Silk-ELR fibres obtained by varying tip-to-collector distance from 13 to 17 cm (A, C and E) and related distribution diameter histograms (B, D and F). Voltage and feed rates were kept respectively at 20 kV and 0.2 ml/h. Histogram of fibre size as a function of changing tip-to-collector distance (G). P-values < 0.001

Figure 5

Confocal Microscopy images of HUVECs on gelatin coated dishes (A, B and C) and the respective histogram with adhesion of cells as a function of seeding time (G). HUVECs on Silk-ELRs co-recombinamer scaffolds (D, E and F) and the related histogram with adhesion of cells as a function of seeding time (H). Scale bar 20 µm. P–values < 0.001

Figure 6

Images of angioplasty catheters provided by Conic Vascular and used for the fabrication of Silk-ELR matrices. Balloon with its coverage (A) and without the coverage (B). Fibre matrix obtained by electrospinning Silk-ELR solutions for 2 h (C)

Figure 7

Scanning Electron Microscopy angioplasty catheters structure (A and B). Picture A shows the internal part of the balloon without the Silk-ELR fibre matrix. Picture B shows the structure of the balloons with the adhered Silk-ELR matrix around it. Scanning Electron Microscopy was used for the evaluation of fibre morphology (C). Picture D shows Scanning Electron Microscopy measurements of layer thickness