Collagen matrix physical properties modulate endothelial colony forming cell-derived vessels in vivo

Abstract

Developing tissue engineering approaches to generate functional vascular networks is important for improving treatments of peripheral and cardiovascular disease. Endothelial colony forming cells (ECFCs) are an endothelial progenitor cell (EPC) population defined by high proliferative potential and an ability to vascularize collagen-based matrices in vivo. Little is known regarding how physical properties of the local cell microenvironment guide vessel formation following EPC transplantation. In vitro evidence suggests that collagen matrix stiffness may modulate EPC vessel formation. The present study determined the ability of 3D collagen matrix physical properties, varied by changing collagen concentration, to influence ECFC vasculogenesis in vivo. Human umbilical cord blood ECFCs were cultured within matrices for 18 h in vitro and then fixed for in vitro analysis or implanted subcutaneously into the flank of immunodeficient mice for 14 days. We report that increasing collagen concentration significantly decreased ECFC derived vessels per area (density), but significantly increased vessel sizes (total cross sectional area). These results demonstrate that the physical properties of collagen matrices influence ECFC vasculogenesis in vivo and that by modulating these properties, one can guide vascularization.

Fig. 1

3D collagen matrix physical properties varied with collagen concentration. Fibril density increased linearly with collagen concentration as shown in 3D CRM images of 0.5 (A) and 2.5 (B) mg/ml matrices (scale bar = 10 μm). Shear storage modulus (G′ lines = linear regression trend lines), increased with increasing collagen concentration (p<0.05 between concentrations, n=3–5 for mechanical analyses) and with the addition of ECFCs (2 × 10⁶cells/ml, gray lines, p<0.05 at each concentration except 3.5 mg/ml). Matrix fluid-like behavior, indicated by δ (D), was significantly affected by collagen concentration (* denotes p<0.05 within cell groups) and ECFC addition (p<0.05 at each concentration except 1.5 mg/ml). Matrix E_c (E) similarly increased with collagen concentration (* denotes p<0.05 within cell groups), but did not change significantly with ECFC addition (p>0.05 at all concentrations).

Fig. 2

Confocal analysis of ECFC morphology within 3D collagen matrices with varied physical properties after 18 hours of in vitro culture and immediately prior to subcutaneous implantation. Cellularized matrices were labeled with DAPI (red), and UEA-1 Lectin (green) for visualization of nuclei and ECFCs, respectively. ECFC showed cord like networks but no apparent lumen formation in any collagen matrix formulation at this time point. While low concentration matrices (A, 0.5 mg/ml) appear to have a lower cell density compared to higher concentration matrices (B, 3.5 mg/ml, Scale bar A,B = 100 μm) the cell number is not different between the two conditions.

Fig. 3

Histochemical analysis of explanted ECFC matrices. Matrices, removed after 14 days, remodeled to a different extent dependent on collagen concentration. Representative sections (of n≥6 implants in different mice) are shown for 0.5 (B,C), 1.5 (C,H), 2.5 (D,I), and 3.5 (E,J) mg/ml matrices (scale bars A–E = 1 mm, F–J = 250 μm). Lower concentration matrices contracted and degraded to a greater degree than higher concentration matrices (A–D). All matrices, except no cell controls (0.5 mg/ml A,F), were able to direct ECFCs to form functional hCD31⁺ blood vessels which contained RBCs.

Fig. 4

Quantification of RBC-containing vessel density within explanted ECFC matrices. Average total vessels per area (A) and hCD31⁺ vessels per area (B) decreased with increasing collagen concentration (* denotes p<0.05 between groups). Interestingly, the percentage of hCD31⁺ vessels (out or the total ECFC and chimeric vessels) increased with increasing concentration (C).

Fig. 5

Analysis of RBC-containing vessels areas within explanted ECFC matrices. Light micrographs of explant histological sections show representative hCD31⁺ vessels from multiple matrices with different areas between 51 and 100 μm² (A), between 501 and 1000 μm² (B), between 1001 and 2000 μm² (C), and greater than 4000 μm² (D, scale bar A–D = 100 μm, A–D arrows –RBCs, asterisks -hCD31+ vessels). The distribution of hCD31⁺ vessel areas (E), average hCD31⁺ vessel area (F), and total hCD31⁺ vascular area (G) were measured for each collagen concentration (*denotes p<0.05 between groups). Vessel morphology was significantly altered by matrix collagen concentration, with increasing concentration shifting towards increased average areas and total vessel areas.