Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Summary

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Abstract

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Introduction

In treatment of coronary artery disease (CAD), a patient's own blood vessels are harvested as graft material for bypass surgery. However, oftentimes, ill patients do not have viable vessels to donate to themselves, and in cases where they do, the donor site causes considerable additional harm and has a serious risk for infection.¹ Engineered vascular grafts could fill this need. Scalability is of utmost importance for engineering vessels in order to meet the wide range of patient vessel size requirements. However, present methods for engineering vessels are not easily scalable, and typically require remanufacture of complex molds or polymer....

Protocol

1. Cell Culture Preparation

1. Utilize human aortic smooth muscle cells purchased commercially.
2. Maintain cells in smooth muscle cell growth media composed of 88.6% 231 media, 0.1% each of recombinant human insulin (rH-insulin), recombinant human fibroblast growth factor (rH-FGF), recombinant human epidermal growth factor (rH-FGF), and ascorbic acid; and 5% each of fetal bovine serum (FBS) and L-glutamine; and 1% antibiotic/antimycotic.
   NOTE: Each growth factor, FBS and L-glutamine are purchased as a vascular media growth kit.
3. Change media every 48 hours until the cells are about 90% confluent and ready for tissue seeding.
4. ....

Results

Demonstrated here is fabrication of 3 different engineered vascular graft sizes (Figure 1), showing that the Ring Stacking Method (RSM) is scalable. To prove applicability, the 3 different vessel sizes chosen correlate to actual human vessel size for the left anterior descending artery (small; lumen diameter = 4 mm)¹⁷, descending aorta (intermediate; lumen diameter = 10 mm) and ascending aorta (large; lumen diameter = 20 mm)¹⁸

Discussion

The Ring Stacking Method presents multiple advantages over current vascular tissue engineered construct techniques. The RSM can be adapted to create human vessels of any size by simply customizing the post and outer shell dimensions. Our method allows for development of polymer-free engineered vessels composed solely of human cells and rapidly degrading support material found in the body's natural wound healing process. Polymer grafts are known to cause restenosis in the clinic and could become problematic if contain.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Human Aortic Smooth Muscle Cells	ATCC	PCS-100-012	vascular smooth muscle cells
Medium 231	Gibco (Life Technologies	M-231-500	media specific to vascular smooth muscle cells
Human Aortic Smooth Muscle Cell Growth Kit	ATCC	PSC-100-042	growth factors for maintaining vascular smooth muscle cell viability
Replicator Mini 3D printer	MakerBot	N/A	3D printer
Poly(lactic acid) 3D ink (PLA)	MakerBot	N/A	3D printer filament
Poly(dimethlysiloxane) (PDMS)	Ellworth Adhesives	3097358-1004	polymer for gluing plate parts
Fibrinogen	Hyclone Labratories, Inc.	SH30256.01	fibrin gel component
Thrombin	Sigma Life Sciences	F3879-5G	fibrin gel component
Tranforming Growth Factor-Beta 1	PeproTech	100-21	growth factor for stimulating collagen production
Hemocytometer	Hausser Scientific Co.	3200	for cell counting
Polycarbonate tubing	US Plastics	PCTUB1.750X1.625	material for making tall, ring stacking plates
Polycarbonate sheet	Home Depot	409497	material for making tall, ring stacking plates
Adhesive polymer solvent	SCIGRIP	10799	material for making tall, ring stacking plates
Instron 5940	Instron	N/A	tensile testing machine
U-Stretch	Cell Scale	N/A	tensile testing machine
Smooth Muscle Actin	MA5-11547	Thermo Fisher	antibody
Tropomyosin	MA5-11783	Thermo Fisher	antibody