Luminal endothelialization of small caliber silk tubular graft for vascular constructs engineering

Abstract

The constantly increasing incidence of coronary artery disease worldwide makes necessary to set advanced therapies and tools such as tissue engineered vessel grafts (TEVGs) to surpass the autologous grafts [(i.e., mammary and internal thoracic arteries, saphenous vein (SV)] currently employed in coronary artery and vascular surgery. To this aim, in vitro cellularization of artificial tubular scaffolds still holds a good potential to overcome the unresolved problem of vessel conduits availability and the issues resulting from thrombosis, intima hyperplasia and matrix remodeling, occurring in autologous grafts especially with small caliber (<6 mm). The employment of silk-based tubular scaffolds has been proposed as a promising approach to engineer small caliber cellularized vascular constructs. The advantage of the silk material is the excellent manufacturability and the easiness of fiber deposition, mechanical properties, low immunogenicity and the extremely high in vivo biocompatibility. In the present work, we propose a method to optimize coverage of the luminal surface of silk electrospun tubular scaffold with endothelial cells. Our strategy is based on seeding endothelial cells (ECs) on the luminal surface of the scaffolds using a low-speed rolling. We show that this procedure allows the formation of a nearly complete EC monolayer suitable for flow-dependent studies and vascular maturation, as a step toward derivation of complete vascular constructs for transplantation and disease modeling.

Keywords: bioreactor for vascular tissue engineering endothelialization of silk tubular scaffolds; cardiovascular tissue engineering; high seeding efficiency; in vitro dynamic cell seeding and culture; silk fibroin scaffold; tissue engineered vascular graft.

FIGURE 1

Illustration of bioreactors and scaffolds derivation. (A) Overall hydraulic scheme of the Minibreath bioreactor. It is shown the compartment used for scaffold housing and the inlet point where cells were injected to proceed with seeding. (B) The picture on the left and the scheme on the right show, respectively, an illustration of the rotating seeding phase and the configuration of the bioreactor. P indicates the peristaltic pump and R the medium reservoir. During the seeding phase, cell suspension was injected inside the luminal compartment of the scaffold after which, the system was allowed to rotate the scaffold for a period of 3 days. (C) After 3 days of seeding phase, the scaffold was perfused with a physiological venous flow (0.5 mL/min) provided by a closed loop hydraulic circuit (scheme on the right), consisting of tubing (1), a reservoir (2) and a peristaltic pump (3). (D) Description of the tools and the procedure employed to optimize the seeding of the silk scaffolds. Panels 1 and 2 show, respectively, the molds used to fabricate the PDMS holders used to house the circular silk patches in cell culture, while panel 3 shows one of the tubular scaffolds still in its sterile packaging. Panels 4–7 contain a sequence of pictures illustrating how we derived circular patchs from a tubular scaffold and placed them in a multiwell culture plate after housing into the PDMS mold. Static cell seeding and culture experiments were performed by gently pipetting ECs suspension over pre-wetted scaffold.

FIGURE 2

Optimization of ECs seeding onto circular silk scaffold patches. (A) Increasing amounts (75 × 10³–900 × 10³) of cells were seeded onto the circular silk scaffold patches produced as in Figure 1D. The pictures on the upper side of the panel show the MTT staining of both sides of the scaffold halves, where the increasing purple color indicates an increased coverage of the seeding surface (side a). The lack of staining on sides b indicates that there was no migration of the cells on the opposite scaffold side. The micrographs on the bottom part of the panel show the appearance of the endothelial cell (EC) layer as observed in transversal section of the scaffold. It is evident the formation of an EC monolayer at 300 × 10³ ECs concentration. (B) SEM micrographs of the sides a of the silk scaffold circular patches cellularized with the increasing amounts of ECs. Also in this case, 300 × 10³ was the optimal cell concentration, favoring the formation of a monolayer made of firmLy adhering cells.

FIGURE 3

Efficiency of the rotation-dependent ECs seeding and firm adhesion of ECs under a slow flow. (A) The picture on the top-left of the panel shows a nearly complete coverage by ECs of the luminal scaffold surface, as detected by MTT assay. The micrographs on the center and the right indicate, respectively, the structure of the EC layer in transversal section and the spreading of the cells onto the silk scaffold. Application of a constant flow (0.5 mL/min for 3 days), did not produce substantial modifications in the ECs coverage, neither at a macroscopic level (MTT staining and transversal sectioning), nor at a microscopic observation. This witnesses a good compatibility of the scaffold for firm adhesion of ECs resistant to application of a steady flow. Quantification of the area covered by ECs onto SEM micrograph showed that the cell coverage did not decrease in presence of a flow (B), and that cells exposed to flow clearly synthesized a basal lamina, to reinforce their own firm adhesion (C).