Development of a Semi-Automated, Bulk Seeding Device for Large Animal Model Implantation of Tissue Engineered Vascular Grafts

Abstract

Vascular tissue engineering is a field of regenerative medicine that restores tissue function to defective sections of the vascular network by bypass or replacement with a tubular, engineered graft. The tissue engineered vascular graft (TEVG) is comprised of a biodegradable scaffold, often combined with cells to prevent acute thrombosis and initiate scaffold remodeling. Cells are most effectively incorporated into scaffolds using bulk seeding techniques. While our group has been successful in uniform, rapid, bulk cell seeding of scaffolds for TEVG testing in small animals using our well-validated rotational vacuum technology, this approach was not directly translatable to large scaffolds, such as those required for large animal testing or human implants. The objective of this study was to develop and validate a semi-automated cell seeding device that allows for uniform, rapid, bulk seeding of large scaffolds for the fabrication of TEVGs appropriately sized for testing in large animals and eventual translation to humans. Validation of our device revealed successful seeding of cells throughout the length of our tubular scaffolds with homogenous longitudinal and circumferential cell distribution. To demonstrate the utility of this device, we implanted a cell seeded scaffold as a carotid interposition graft in a sheep model for 10 weeks. Graft remodeling was demonstrated upon explant analysis using histological staining and mechanical characterization. We conclude from this work that our semi-automated, rotational vacuum seeding device can successfully seed porous tubular scaffolds suitable for implantation in large animals and provides a platform that can be readily adapted for eventual human use.

Keywords: bulk seeding; carotid implantation; mesenchyaml stem cells; sheep model; vascular tissue engineering.

FIGURE 1

Overview of translating-rotating seeding device. (A) Schematic of the linear sliding stage system used to translate the Stylet/Diffuser component within the rotating scaffold lumen during seeding under vacuum. (B) Image of the Stylet/Diffuser system emerging from the proximal mounting tee. (C) Image of a mounted scaffold with the Diffuser infusing cell suspension within the scaffold lumen. (D) Schematic and (E) image of the novel cell seeding device developed in this study to bulk seed human-sized tubular scaffolds. Scale bars depict 1 cm.

FIGURE 2

In vitro analysis of seeding device performance. (A) Schematic of the sectioning technique used to quantitatively assess the distribution of cells seeded within scaffolds using the novel seeding device. (B) Separation of each section for histological staining and metabolic activity assessment. Cell metabolic activity assay sections were further divided into quadrants to estimate circumferential cell distribution within the scaffold. (C) Scaffold seeding efficiency for each seeding configuration examined in this study. (D) Longitudinal distribution of cells across seven longitudinal sections for each seeding configuration. The dashed line indicates ideal cell distribution. (E) Circumferential distribution of cells across four quadrants for each seeding configuration. (F) Combined average of all three seeding configurations for longitudinal, and (G) circumferential distribution. (H) Cumulative difference between longitudinal cell distribution and the ideal distribution for each seeding configuration. (I) H&E staining of the seeded scaffold (seeded at a Diffuser displacement speed of 2.5 mm/s) to visualize the distribution of cells within the scaffold pores. Scale bars depict 1 mm.

FIGURE 3

Large animal implantation and explant analysis of a large seeded construct. (A,B) Macroscopic image of the explanted TEVG (A, intact and B, longitudinally sectioned) after 10 weeks as a carotid interposition graft in a sheep model. Scale bars depict 5 mm. (C) Brightfield images of sections L1 to L7. (D) H&E staining of sections L1 to L7 demonstrating cellular distribution in the graft and neo-intima formation. (E) VVG staining of sections L1 to L7 demonstrating elastin distribution in the graft neo-tissue. (F) Auto-fluorescence of the PEUU material remaining in the graft. Scale bars depict 100 μm. (G) Failure strength, (H) modulus in the low strain region and (I) modulus in the high strain region for the unseeded scaffold, the explanted TEVG and the native carotid arterial tissue. ^∗∗ indicates statistical significance at p < 0.01 and ^**** indicates statistical significance at p < 0.0001.