Collagen-polymer guidance of vessel network formation and stabilization by endothelial colony forming cells in vitro

Abstract

Vessel morphogenesis is vital to regenerative medicine strategies. Here, collagen polymers, specified by intermolecular cross-link composition, are used to independently vary microstructure (fibril density, interfibril branching) and physical properties (stiffness) to guide 3D vessel network formation by endothelial colony forming cells (ECFC) in vitro. Increasing stiffness, by modulation of fibril density or interfibril branching, increases vessel diameter, length and branching. Oligomer matrices also induce vessel stabilization via type IV collagen deposition. This work shows that ECFC vessel formation depends on the interplay of collagen microstructure and physical properties and names oligomers and intermolecular cross-links as key design parameters for vascular-inductive matrices.

Keywords: endothelial colony forming cells; microstructure; oligomers; type I collagen; vasculogenesis.

Figure 1. Morphometric analysis of ECFC vessel networks within collagen matrices

ECFC vessel networks were stained with FITC conjugated UEA-1 lectin and imaged using confocal microscopy (26 slices at 2 μm thickness/slice). Images were reconstructed in 3D using Imaris (A), covered with an isosurface (B), masked to create an additional channel (C), and then analyzed using filament tracer. Panel D shows the fit of the filaments in the cone geometry that approximates the morphology of vessel network volume. Scale bar = 20 μm.

Figure 2. ECFC show enhanced in-vitro vessel formation within monomer versus oligomer matrices

ECFC at 5×10⁵ cells/ml were seeded within collagen monomer (A) and oligomer (B) matrices matched at G′ of 200 Pa and cultured for 7 days. Constructs were stained with FITC-conjugated UEA-1 lectin and imaged using confocal microscopy. Panels A and B represent 3D rendered images of z-stacks (21 slices at 5 μm thickness/slice) showing lectin-based detection of 3D ECFC vessel networks. Scale bar = 100 μm.

Figure 3. ECFC vessel network morphology is dependent on oligomer concentration

Schematic representations of 3D ECFC vessel network elongation and branching are shown for oligomer matrices at 0.5-, 1.5-, and 3.0 mg/ml (A). The distributions of vessel diameters (B) were measured for ECFC vessel networks following 7 days of culture within oligomer matrices at 0.5 mg/ml (black), 1.5 mg/ml (gray), and 3.0 mg/ml (white). Cumulative distributions of vessel diameters (C) show statistically significant shifts toward increased vessel diameters with increasing oligomer concentration (n≥123; 0.5 mg/ml vs 1.5 mg/ml, p<0.0001; 1.5 mg/ml vs 3.0 mg/ml, p=0.0446; 0.5 mg/ml vs 3.0 mg/ml, p=<0.0001). Total vessel length (D), average vessel segment length (E), average vessel segment volume (F), and vessel volume percent (G) were measured for all ECFC vessel networks at 0.5 mg/ml (black), 1.5 mg/ml (gray), and 3.0 mg/ml (white). As collagen concentration increased, average segment length and average segment volume increased significantly. Total vessel length decreased progressively over the concentration range. Vessel volume percent showed an increase from 0.5 mg/ml to 1.5 mg/ml, with no further increase at 3 mg/ml (Data represents mean ± SEM; n≥14; asterisk indicates statistically different pairwise comparisons, p<0.05).

Figure 4. Monomer concentration has limited effect on ECFC vessel network morphology

The distributions of vessel diameters (A) were measured for ECFC vessel networks following 7 days of culture within monomer matrices at 0.5 mg/ml (black), 1.5 mg/ml (gray), and 3.0 mg/ml (white). Cumulative distributions of vessel diameters (B) show no statistical difference between any of the concentrations (n≥55; 0.5 mg/ml vs 1.5 mg/ml, p=0.1718; 1.5 mg/ml vs 3.0 mg/ml, p=0.0518; 0.5 mg/ml vs 3.0 mg/ml, p=0.4374). Total vessel length (C), average vessel segment volume (D), and vessel volume percent (E) were measured for all ECFC vessel networks at 0.5 mg/ml (black), 1.5 mg/ml (gray), and 3.0 mg/ml (white). As collagen concentration increased, total vessel length and vessel volume percent decreased with no significance. Average segment volume increased significantly with concentration (Data represents mean ± SEM; n≥14; asterisk indicates statistically different pairwise comparisons, p<0.05).

Figure 5. Increase in oligomer to monomer ratio yields increase in matrix stiffness through increased interfibril branching independent of fibril density

Reconstructed images of fibril microstructure for collagen formulations representing oligomer:monomer ratios of 0:100 (A), 50:50 (B), and 100:0 (C) polymerized at matched concentration (1.5 mg/ml). Matrix stiffness (G′), fibril volume fraction, number of interfibril branch points, and fibril diameter were quantified and compared. As oligomer content increased, the number of interfibril branch points and matrix stiffness increased significantly (p<0.05). No statistical differences were noted for fibril density or diameter values (Data represents mean ± SD; n≥3; letters indicate statistically different groups for each parameter based upon Tukey-Kramer range testing; p<0.05). Scale bar = 10 μm.

Figure 6. ECFC vessel network morphology changes with the addition of oligomers

Schematic representations of 3D ECFC vessel network elongation and branching are shown for 1.5 mg/ml matrices of 0:100, 50:50, and 100:0 oligomer:monomer ratios (A). Vessel diameter distributions (B), total vessel length (C), average vessel segment length (D), and vessel volume percent (E) were measured for ECFC vessel networks following 7 days of culture within 0:100 (white), 50:50 (gray), and 100:0 (black) oligomer:monomer collagen matrices prepared at 1.5 mg/ml. Total vessel length for 50:50 oligomer/monomer is significantly lower than oligomer and monomer. 50:50 oligomer/monomer also shows significantly higher average segment length and volume than the other matrices. Vessel volume percent is highest in oligomer matrices, but there is no difference observed between monomer and 50:50 oligomer/monomer matrices (Data represents mean ± SEM; n≥14; asterisk indicates statistically different groups, p<0.05).

Figure 7. Comparison of ECFC vessel network lumen diameter and morpology for monomer and oligomer matrices prepared at matched stiffness (G’)

Schematic representations of 3D ECFC vessel network elongation and branching are shown for oligomer and monomer matrices matched at 200 Pa (A). Vessel diameter distribution (B), total vessel length (C), average vessel segment volume (D), and vessel volume percent (E) were measured for ECFC vessel networks following 7 days of culture within monomer (black) and oligomer (white) matrices matched at 200 Pa. Total vessel length, average segment volume, and vessel volume fraction were significantly greater for oligomer matrices (Data represents mean ± SEM; n≥13 for C, D, E; asterisks indicates statistically different groups, p <0.05).

Figure 8. Comparison of temporal and spatial patterns of collagen IV deposition by ECFC vessel networks within oligomer and monomer matrices

ECFC at 5×10⁵ cells/ml were seeded in monomer and oligomer matrices matched at G′ of 200 Pa and cultured for 3, 7, and 14 days. Constructs were stained with FITC-conjugated UEA-1 lectin and anti-collagen type IV antibody. Samples were imaged using confocal microscopy (21 slices at 5 μm thickness/slice) to show 3D ECFC vessel networks (green) and basement membrane deposition, collagen type IV (red). (A) ECFC deposition of collagen type IV increased dramatically with time within oligomer matrices. Collagen type IV is secreted from the cells, and Day 3, 7 oligomer cultures show close association of collagen type IV with the basal membrane of the cell/vessel. At Day 14, collagen type IV is associated with the basal membrane of the vessels, as well as in areas distant from the vessel network that could be potential vessel regression sites. ECFC in monomer showed little collagen type IV deposition at all time points. Panel B shows single slice and cross-section views (XY-, XZ-, and YZ planes) of ECFC vessel networks within oligomer. Specimens were stained with anti-collagen type IV antibody. Arrows in the XY plane indicate the lumen areas shown in XZ- and YZ planes. Scale bar = 50 μm.

Figure 9. Comparison of temporal and spatial patterns of laminin deposition by ECFC vessel networks within oligomer and monomer matrices

ECFC at 5×10⁵ cells/ml were seeded in monomer and matrices matched at G′ of 200 Pa and cultured for 3, 7, and 14 days. Constructs were stained with FITC-conjugated UEA-1 lectin and anti-laminin antibody. Samples were imaged using confocal microscopy (21 slices at 5 μm thickness/slice) to show 3D ECFC vessel networks (green) and basement membrane deposition, laminin (red). Laminin deposition is largely cell associated and is deposited to a lesser quantity than collagen type IV. Punctate staining patterns were observed with both monomer and oligomer matrices and remained consistent over time. Scale bar = 50 μm.