Implantable arterial grafts from human fibroblasts and fibrin using a multi-graft pulsed flow-stretch bioreactor with noninvasive strength monitoring

Abstract

Tissue-engineered arteries based on entrapment of human dermal fibroblasts in fibrin gel yield completely biological vascular grafts that possess circumferential alignment characteristic of native arteries and essential to their mechanical properties. A bioreactor was developed to condition six grafts in the same culture medium while being subjected to similar cyclic distension and transmural flow resulting from pulsed flow distributed among the graft lumens via a manifold. The lumenal pressure and circumferential stretch were noninvasively monitored and used to calculate stiffness in the range of 80-120 mmHg and then to successfully predict graft burst strength. The length of the graft was incrementally shortened during bioreactor culture to maintain circumferential alignment and achieve mechanical anisotropy comparable to native arteries. After 7-9 weeks of bioreactor culture, the fibrin-based grafts were extensively remodeled by the fibroblasts into circumferentially-aligned tubes of collagen and other extracellular matrix with burst pressures in the range of 1400-1600 mmHg and compliance comparable to native arteries. The tissue suture retention force was also suitable for implantation in the rat model and, with poly(lactic acid) sewing rings entrapped at both ends of the graft, also in the ovine model. The strength achieved with a biological scaffold in such a short duration is unprecedented for an engineered artery.

Figure 1

Pulsed flow-stretch bioreactor. a. 2 mm grafts on glass mandrel during static culture of 2 weeks, b. 2 mm grafts mounted in the bioreactor. c. Schematic of bioreactor with syringe mounted on reciprocating pump causing pulsed flow into the upper manifold, with medium flowing transmural through the tissue and through the lumens collecting on the ablumenal side of the grafts and reinjected into the upper manifold (images and schematic are not to the same scale).

Figure 2

Noninvasive monitoring of graft burst strength. a. Average pressure-strain curve of the grafts of different maturation (harvested at 2 wk and 7 wk), exhibiting different burst pressures, b. Average pressure-strain curve with data for 0–15% strain, showing no difference in pressure-strain behavior, c. Correlation of stiffness between 80-120mmHg with burst pressure; solid line is linear regression of the data points (R² = 0.97) with 95% confidence intervals shown by dotted lines, d. Predicted and measured burst pressure for grafts after 1 and 3 week of bioreactor culture. Stiffness was calculated as defined in Eqn. 2.

Figure 3

Comparison of grafts cultured under pulsed and constant flow. a. Thickness, b. Collagen/Cell, and c. Burst pressure of grafts conditioned with pulsed flow (stretch amplitude of 7%, average flow of 3.75 mL/min/graft) versus constant flow (3.33 ml/min/graft). Significant difference between groups is shown by paired symbols (*).

Figure 4

Comparison of constant length mounting and axial shortening. a. Fiber alignment maps, b. Modulus, c. Anisotropy index (the ratio of modulus in circumferential direction to axial direction) with comparison to ovine femoral artery (Native), and d. UTS with or without incremental shortening of the grafts. Significant difference between circumferential and axial graft properties is shown by paired symbols (* or #).

Figure 5

Properties of 2 mm and 4 mm grafts. a. Thickness, b. Collagen concentration, c. Burst pressure, and d. Compliance of 2 mm and 4 mm grafts in comparison to the ovine femoral artery. The grafts were cultured in the bioreactor for 5 and 7 weeks for 2 mm and 4 mm grafts. Compliance was defined and calculated as Eqn. 3. Significant difference between the 2 mm and 4 mm grafts is shown by paired (*) symbol, while (#) indicates a significant difference of the native artery from both the 2 mm and 4 mm grafts.

Figure 6

Histology of 2 mm and 4 mm engineered vascular grafts. Lillie's trichrome and picrosirius red stained sections of grafts and a sheep femoral artery. The green color indicates collagen, with residual fibrin (and/or other proteins) in red visible in the 2 mm graft. Muscle fibers in native tissue also stain red. The cells were evenly distributed through the thickness of the grafts. Picrosirius red stain under cross-polarized light shows red bands in the circumferential direction. The red intensity was comparable between samples.