Physical and Chemical Signals That Promote Vascularization of Capillary-Scale Channels

Abstract

Proper vascularization remains critical to the clinical application of engineered tissues. To engineer microvessels in vitro, we and others have delivered endothelial cells through preformed channels into patterned extracellular matrix-based gels. This approach has been limited by the size of endothelial cells in suspension, and results in plugging of channels below ~30 μm in diameter. Here, we examine physical and chemical signals that can augment direct seeding, with the aim of rapidly vascularizing capillary-scale channels. By studying tapered microchannels in type I collagen gels under various conditions, we establish that stiff scaffolds, forward pressure, and elevated cyclic AMP levels promote endothelial stability and that reverse pressure promotes endothelial migration. We applied these results to uniform 20-μm-diameter channels and optimized the magnitudes of pressure, flow, and shear stress to best support endothelial migration and vascular stability. This vascularization strategy is able to form millimeter-long perfusable capillaries within three days. Our results indicate how to manipulate the physical and chemical environment to promote rapid vascularization of capillary-scale channels within type I collagen gels.

Keywords: collagen; cyclic AMP; endothelial cells; genipin; microvascular tissue engineering; pressure.

FIGURE 1

Methods for the formation and analysis of seeded capillary-scale channels. (A) Gelling and seeding procedure, and corresponding phase-contrast images at each step. (B) Application of forward and reverse pressure, starting one day after seeding (“day 1”). (C) Vascularization metrics for seeded microchannels.

FIGURE 2

Day 4 phase-contrast images of native and genipin-crosslinked collagen gels that were seeded and placed under forward pressure and low-cAMP media culture conditions. (A) 120-μm-diameter channels. (B) 60-μm-diameter channels. (C) 30-μm-diameter channels. (D) 15-μm-diameter channels. Insets show seeded channels on day 1. Seeding was from the left side.

FIGURE 3

Phase-contrast and fluorescence viability images of vessels that were cultured under various conditions, on days 1 and 4 after seeding. Displayed are regions with channel diameters between 10 and 40 μm. Locations of smallest confluent diameters are marked by yellow lines; locations of smallest vascularized diameters are marked by red lines. (A) Forward pressure and low cAMP. (B) Forward pressure and high cAMP. (C) Reverse pressure and low cAMP. (D) Reverse pressure and high cAMP. Arrowheads denote plugs. Seeding was from the left side.

FIGURE 4

Effect of pressure direction and cAMP level on vascularization of tapered microchannels. (A) Smallest confluent diameter (D₁) on days 1 and 4. (B) Smallest vascularized diameter (D₂) on days 1 and 4. (C) Endothelial coverage (f_EC) on days 1 and 4. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

FIGURE 5

Vascularization of 20-μm-diameter channels. (A) Phase-contrast images (day 0, 1, 3, and 5) of seeded 20-μm-diameter channels cultured under high cAMP and low, intermediate, or high reverse pressure. (B) Migration profiles of confluent endothelium over five days for each pressure category: low (0.25-0.5 cm H₂O), intermediate (0.75-1.5 cm H₂O), high (1.75-3 cm H₂O). (C) Retraction distance (δ_EC) of confluent endothelium for each pressure category. *, p < 0.05; **, p < 0.01. (D) Migration rate of confluent endothelium from day 0 to day 2 (v) versus average shear stress on days 1 and 2; fit was to a one-phase decay. (E) Phase-contrast and fluorescence images of a representative vessel cultured under intermediate reverse pressure for five days, then perfused with viability dyes. (F) Phase-contrast and fluorescence images of a representative vessel cultured under intermediate reverse pressure for five days, and then perfused with fluorescent dextran-containing media. Seeding was from the left side.

FIGURE 6

Summary of capillary-scale vascularization in type I collagen channels. (A) Modes of vascularization under different culture conditions. (B) Mechanisms that underlie capillary-scale vascularization.