Perfusion Bioreactor Conditioning of Small-diameter Plant-based Vascular Grafts

Abstract

Background: Vascular grafts are mainly composed of synthetic materials, but are prone to thrombosis and intimal hyperplasia at small diameters. Decellularized plant scaffolds have emerged that provide promising alternatives for tissue engineering. We previously developed robust, endothelialized small-diameter vessels from decellularized leatherleaf viburnum. This is the first study to precondition and analyze plant-based vessels under physiological fluid flow and pressure waveforms. Using decellularized leatherleaf viburnum as tissue-engineered grafts for implantation can have profound impacts on healthcare due to their biocompatibility and cost-effective production.

Methods: A novel perfusion bioreactor was designed, capable of accurately controlling fluid flow rate and pressure waveforms for preconditioning of small-diameter vascular grafts. A closed-loop system controlled pressure waveforms, mimicking physiological values of 50-120 mmHg at a frequency of 8.75 Hz for fluid flow reaching 5 mL/min. Plant-based vascular grafts were recellularized with endothelial and vascular smooth muscle cells and cultured for up to 3 weeks in this bioreactor. Cell density, scaffold structure and mechanics, thrombogenicity, and immunogenicity of grafts were evaluated.

Results: Bioreactor treatment with fluid flow significantly increased luminal endothelial cell density, while pressure waveforms reduced thrombus formation and maintained viable vascular smooth muscle cells within inner layers of grafts compared to static controls. Suture retention of grafts met transplantation standards and white cell viability was suitable for vascular remodeling.

Conclusion: Low thrombogenicity of endothelialized leatherleaf viburnum holds great potential for vascular repair. This study provides insight into benefits of conditioning plant-based materials with hemodynamic forces at higher frequencies that have not previously been investigated.

Keywords: Bioreactor; Plant; Tissue engineering; Vascular graft.

Fig. 1

A Representative photo of culture region in chamber. B Representative photo of the vascular graft. C Side view of graft in chamber in AutoCAD. D Schematic of bioreactor system with its individual components

Fig. 2

A Example of a specimen during a suture retention strength test. B CAD model of custom-made 3D stencil used as suture holder. C Max load of 8–0 and 10–0 sutures looped through plant-based grafts and native vessels. *p < 0.05 and error bars represent standard deviation (N = 3). One-way ANOVA and post hoc Tukey tests were used to compare data between groups with a p value of < 0.05 to determine statistical significance

Fig. 3

Representative 20 × magnification images of endothelial cells seeded on grafts with A 24 h no flow and B 24 h flow (N = 3), and C 3 week no flow and D 3 week flow (N = 3) stained with Hoechst. E Cell count for grafts with seeded endothelial cells under static and flow conditions. *p < 0.05 and error bars represent standard deviation. Cells were counted using 3 images per sample (N = 9). One-way ANOVA and post hoc Tukey tests were used to compare data between groups with a p value of < 0.05 to determine statistical significance

Fig. 4

Representative 10 × magnification images of thrombosis testing on A acellular grafts, B seeded grafts without bioreactor preconditioning and C seeded grafts with bioreactor feedback treatment and their corresponding threshold images D–F. Quantification of thrombosis through G thrombus-free area fraction and H thrombus thickness. *p < 0.05 and error bars represent standard deviation. Unpaired t-tests were used to determine statistical significance

Fig. 5

Representative H&E images of grafts at 40 × magnification with A no endothelial cells (ECs) and vascular smooth muscle cells, B endothelial cells and vascular smooth muscle cells, C endothelial cells and vascular smooth muscle cells with flow, D endothelial cells and vascular smooth muscle cells with flow and pressure. E Distribution of ECM orientation. F Wall thickness, G fiber thickness and H cell wall fiber volume-fraction were measured for 3 images per sample (N = 9). Error bars represent standard deviation

Fig. 6

Representative SEM images of A,E acellular grafts and seeded grafts under B,F static, C,G flow and D,H flow plus pressure treatment and imaged at magnifications of 110 × and 800 × , respectively. Graft I inner diameter, J outer diameter and K pore diameter were measured for 3 images per sample (N = 9). *p < 0.05 and error bars represent standard deviation