In this study, 3D endothelial sprout navigation was examined by using a microfluidic platform that enabled the separate and simultaneous tuning of biomaterial parameters and soluble biochemical stimuli. Increasing the collagen density within a blended collagen/fibronectin matrix was observed as resulting in slower sprout initiation, altered sprout pathfinding behavior, and increased VEGF activation and saturation thresholds. The perturbation of a single scaffold variable (collagen density) impacts multiple biomaterial properties including diffusivity, matrix mechanics, fibril morphology, and presentation of cell-binding domains (each of which is briefly discussed next). This tendency to impact multiple material characteristics when making a single change in biomaterial formulation is quite common for both natural and synthetic polymeric biomaterials.4,5
This highly coupled complexity in material properties underscores the need for high-throughput, in vitro
screening tools to help assess the angiogenic potential of various biomaterial formulations. Further, these results support the notion that no single VEGF concentration profile is optimal to support sprouting morphogenesis in all cases, but rather the ideal VEGF concentration profile will be dependent on the choice of biomaterial scaffold for each specific application.
Although increasing collagen density did result in retarded diffusivity of VEGF, all matrix formulations were observed as having similar equilibrium VEGF concentration profiles within 120
min, a time-interval that is negligible compared with the experiment duration of 4 days.19
Since fibronectin has been widely reported as enhancing VEGF activity toward ECs and has the potential to bind VEGF to the matrix,49,50
fibronectin concentration was kept constant across all matrix formulations. To assess potential VEGF binding to the biomaterial, an equilibrium gradient of Texas Red-conjugated dextran (which is not expected to bind to the matrix) was compared with the equilibrium gradient of FITC-conjugated VEGF. The two gradients were identical (), thus demonstrating that VEGF is not appreciably substrate bound in this biomaterial formulation. In addition, since the microfluidic device was constantly perfused with fresh medium and had a cross-sectional height and width (240
mm) less than the diffusion limit of tissue-engineered constructs,51
it is unlikely that any observed differences in EC sprouting were due to changes in diffusivity. This is an important consideration, as recent experiments have reported that matrix-restricted diffusion can affect EC sprouting in some systems.17
In addition to diffusivity effects, changes in collagen density had a profound impact on matrix mechanics. As previously reported, increasing collagen density resulted in stiffer matrices with increased plateau storage moduli.19
Changes in matrix mechanics have been widely reported as altering cell spreading,52
Although many of these studies have been performed on carefully controlled substrates (such as 2D, amorphous polyacrylamide or PDMS gels) that allow mechanics to be altered in a manner which leaves other material properties relatively unchanged,56
as scientists look to apply these insights to more physiologically relevant biomaterials, the ability to tailor single variables will become more complex. For example, variations in collagen density can also result in substantial changes in matrix morphology, with increasing collagen concentration causing greater fiber density and greater fibril clustering into multi-stranded fibers.57,58
Both fiber dimensions and alignment have been reported as affecting cell morphology and function.59,60
In addition to effects on matrix mechanics and fiber morphology, increasing collagen density also results in an increased concentration of collagen cell-binding ligands and a possible alteration in the spatial presentation of those ligands. ECs reportedly interact with collagen cell-binding ligands via α1
These interactions mediate EC sprout formation in collagen matrices by suppressing cyclic AMP and decreasing protein kinase A activation.61
Further, although the overall fibronectin concentration was held constant in all matrix formulations, and, hence, the concentration of fibronectin cell-binding ligands was also constant, the distribution and availability of those ligands may be altered due to the changes in matrix morphology. Numerous reports have detailed the ability of ligand concentration and distribution to control several cellular behaviors including cell spreading,62
Given the inherent complexity of cell–biomaterial interactions in 3D, the development of new experimental platforms that enable direct, real-time visualization of cell-material dynamics offers an ability to perform high-throughput analyses of different biomaterial formulations. In the studies presented here, the dynamic sprout pathfinding processes of initiation, early elongation, and navigational turning were followed over 4 days. Our experiments demonstrated that endothelial sprouts alter their pathfinding sensitivity to VEGF depending on the matrix density. At higher matrix densities, EC sprouts were more sensitive to the local VEGF concentration both during sprout initiation () and sprout elongation (–). In general, steeper VEGF gradients and higher VEGF concentrations were required to induce directionality and proper pathfinding in denser matrices. Steeper VEGF gradients may initiate greater sub-cellular localization of intracellular cascades that lead to subsequent asymmetry in cytoskeletal reorganization and cell polarization.38
Recent studies have also shown that the level of VEGFR2 transcription can be mediated by matrix stiffness14
; therefore, the number of VEGFRs may be altered for various collagen densities, hence regulating VEGF sensitivity in a matrix-dependent manner. This hypothesis is consistent with our observation that the VEGF activation and saturation thresholds are increased in higher density matrices ( and ), thus suggesting that VEGFR2 may be up-regulated during sprout pathfinding within stiffer matrices. Future experiments will quantitatively assess VEGFR2 presentation on tip and stalk cells in various 3D microenvironments.
Finally, we observed and quantified VEGF-induced turning of EC sprouts, a phenomenon that has not been previously described in 3D in vitro
models of angiogenesis to the best of our knowledge (). In lower density matrices, sprouts that had originally misaligned were able to turn and properly reorient parallel to the VEGF gradient; in contrast, this turning phenomenon was only rarely observed in higher density matrices (). These results suggest that only a narrow range of ECM environments may be permissive to allow significant VEGF-induced sprout turning. In our previous studies, we observed statistically significant morphological differences between sprouts formed in 1.2 versus 1.9
mg/mL density matrices.19
Sprouts in lower density matrices were significantly thinner, contained fewer cells per cross-sectional area, were less likely to form a hollow lumen, and displayed faster sprouting speeds.19
This is an indication of the ability of the matrix to control the rate of new sprout formation, potentially due to its control over migration speed of the cells in 3D. It has been previously shown that denser matrices require greater proteolytic activity to induce matrix remodeling, which is a prerequisite for 3D cell migration.67,68
This hypothesis is consistent with our observation that cells on beads embedded in denser matrices (1.9 and 2.7
mg/mL) are restricted to 2D migration on the bead surfaces for the first 48
h of culture; whereas cells in less dense matrices (0.7 and 1.2
mg/mL) have already begun 3D migration into the matrix within 24
h (). If cellular proliferation of stalk cells is maintained at a relatively constant rate regardless of matrix density, then slower growing sprouts will result in a higher number of stalk cells per cross-sectional area. Future experiments will quantitatively assess the action of various proteases during sprout pathfinding and the migration and proliferation rates of tip and stalk cells, respectively.
Once a sprout has formed, the ability to perform a turn in direction may be related to the sprout morphology. Thicker sprouts, which are more likely to form in the higher density matrices,19
require the coordinated motion of a greater number of cells per cross-sectional area compared with thinner sprouts. Further, since thicker sprouts are more likely to have organized to form a stable, hollow lumen,19
it is possible that the mechanical requirements to induce sprout turning may be increased in higher density matrices. These hypotheses are consistent with our observations that sprout turning is more likely to occur in lower density matrices ().