Dynamic flow priming programs allow tuning up the cell layers properties for engineered vascular graft

Abstract

Tissue engineered vascular grafts (TEVG) are potentially clear from ethical and epidemiological concerns sources for reconstructive surgery for small diameter blood vessels replacement. Here, we proposed a novel method to create three-layered TEVG on biocompatible glass fiber scaffolds starting from flat sheet state into tubular shape and to train the resulting tissue by our developed bioreactor system. Constructed tubular tissues were matured and trained under 3 types of individual flow programs, and their mechanical and biological properties were analyzed. Training in the bioreactor significantly increased the tissue burst pressure resistance (up to 18 kPa) comparing to untrained tissue. Fluorescent imaging and histological examination of trained vascular tissue revealed that each cell layer has its own individual response to training flow rates. Histological analysis suggested reverse relationship between tissue thickness and shear stress, and the thickness variation profiles were individual between all three types of cell layers. Concluding: a three-layered tissue structure similar to physiological can be assembled by seeding different cell types in succession; the following training of the formed tissue with increasing flow in a bioreactor is effective for promoting cell survival, improving pressure resistance, and cell layer formation of desired properties.

Figure 1

Silicone frame design for a glass fiber sheet set up and cell layer formation. Far right: Actual frames with glass fiber scaffold preconditioned in culture media. A part of the image was created by the rendering function of SOLIDWORKS 2018.

Figure 2

Formation of the tissue layers on a glass fiber sheet at each maturation step during sheet culture period. (A) First layer, white: fibroblasts stained by Calcein-AM, red: dead cells stained by ethidium, (B) Second layers, red fluorescent: RFP-SMCs. Fibroblasts on the background are non-fluorescent, (C) Third layers, red: RFP-SMCs, teal: GFP-HUVECs with automatic white balance. Each picture was taken under inverted fluorescent microscope after completing tissue maturation steps at weekly intervals. (C) Endothelial cell layer (blue) is in front of the smooth muscle cell layer (red), and the cells are close to be evenly distributed on the glass fiber.

Figure 3

Survival of cells in the formed tissues on a GF sheet after maturation in the developed bioreactor. (A) DAPI stained nuclei, (B) Staining with Calcein-AM and Ethidium.

Figure 4

Tensile strength measurement of the glass fiber sheet before and after tissue formation with the developed tensile tester device. **p < 0.01 by two-tailed t-test with an alpha level of 0.05. The error bar relates to the standard deviation of the mean.

Figure 5

Infiltration of fibroblasts into a glass fiber. White arrows: invasive growth areas.

Figure 6

Burst pressure resistance of the tissue trained at different flow rate. *p < 0.05 by two-tailed t-test with an alpha level of 0.05. The error bar represents the standard deviation of the mean.

Figure 7

Matured blood vessels after training in the bioreactor. The tubular shape could be retained without much damage by some experimental procedures. (A) Extraction, (B) Washing with PBS, (C) Cutting/Slicing, (D) Observation.

Figure 8

Cross sections of three-layer blood vessels. (A) 5 ml/min composite of 20 images with different focal planes from the front to the back of the freshly formed tissue. (B) 1 ml/min higher magnification image with wall exposure, white dotted line: defined separation of two layers. Both; red: RFP-SMC, green: GFP-HUVEC, white arrow: flow direction.

Figure 9

(A) Overview of a representative cross section of a 1 ml/min three-layered blood vessel stained by HE. (B) A 5 ml/min three-layered blood vessel. (C) Enlarged view of three layers. Example of thickness calculation of each cell layer.

Figure 10

Trends of layer thickness change for the three types of cells at each flow rate compared with static culture.

Figure 11

Relationship between the total thickness of the three layers in matured tissue (i.e., infiltration depth into the scaffold) and the shear stress.

Figure 12

Tensile strength measurement tester device. (A) The developed tensile holder. (B) Overall appearance of the device assembled with the Load/Displacement Measurement Unit and digital force gauge. A part of the image was created by the rendering function of SOLIDWORKS 2018.

Figure 13

Construction method of three-layer blood vessel using glass fiber as a scaffold. (1) Fibroblast seeding. (2) Smooth muscle cell seeding on top of fibroblast layer. (3) Endothelial cells seeding onto two-layer smooth muscle cells/fibroblast structure. (4) Layer structure conditioning and tissue maturation. (5) Wrapping the flat sheet around the metal rod to form tubular shape. (6) Holding the tube shape with surgical suture. (7) Rod removal/central channel formation. (8) Formed three-layer blood vessel before tissue maturation and training.

Figure 14

The overview of the 4-channel bioreactor for maturation of proto-tissues into the trained vascular tissue by individual training flow programs. Scale bar = 5 cm. A part of the image was created by the rendering function of SOLIDWORKS 2018.

Figure 15

An auxiliary device for aseptic and reproducible tissue wrapping on glass fiber. A part of the image was created by the rendering function of SOLIDWORKS 2018.

Figure 16

Training flow programs.