Collagen-Polymer Guidance of Vessel Network Formation and Stabilization by Endothelial Colony Forming Cells In Vitro

Abstract

The dynamic process of vessel formation and remodeling is essential to embryonic development, post-natal tissue homeostasis and function, wound healing, and next-generation therapeutic vascularization and tissue engineering strategies. Here, uncommon collagen polymer building blocks, specified by their intermolecular cross-link composition, were used to tune the fibril microstructure and physical properties of collagen matrices for purposes of guiding three-dimensional (3D) lumenized vessel network formation by endothelial colony forming cells (ECFC) in vitro. A new and comprehensive 3D vessel morphometric analysis approach was also used to quantify vessel network morphology and architecture parameters. Results show that independent variation of collagen concentration (fibril density) and oligomer:monomer ratio (interfibril branching), two independent determinants of matrix stiffness, yield vessel networks with different architectures and persistence. Increases in fibril density decreased the overall number of vessel networks formed, while an increase in interfibril branching led to formation of highly branched vessel networks with increased vessel volume density. In general, increasing matrix stiffness, whether by modulation of fibril density or interfibril branching, contributed to increased lumen expansion and vessel segment elongation. Finally, oligomer, but not monomer, matrices induced maturation and stabilization (>14 days) of ECFC vessel networks, marked by changes in the temporal and spatial deposition of collagen type IV. Collectively, this work highlights that ECFC vessel morphogenesis is not dependent upon matrix stiffness alone, but rather the interplay of collagen fibril microstructure and matrix physical properties. Furthermore, it identifies oligomers and their associated intermolecular cross-links as new and important design parameters for vascular-inductive matrices for use in cell culture, regenerative medicine, and engineered tissue applications.

Microstructural Parameter-Based Modeling for Transport Properties of Collagen Matrices

“…First, fibril microstructures were visualized and quantified from collecting confocal image stacks as reported in the previous studies [24,25]. Geometric information from confocal microscopy and Imaris was exported to a 3D CAD program (PTC Creo parametric 2.0) for reconstruction of a collagen matrix model as shown in Fig.…”

“…3. The resulting geometric information then was exported to ANSYS-ICEM CFD to construct cylindrical fibrils with a diameter size of 400 nm which was measured on a confocal microscopy image [25]. Recently, it was found that confocal microscopy tends to overestimate the size of an object compared to scanning electron microscopy (SEM) [29].…”

“…This is mainly due to the emerging of various novel biomaterials for tissue engineering and to the difficulty of identifying and quantifying their microstructural characteristics. These parameters include fiber/fibril structure, arrangement, and intermolecular crosslinks [24,25].…”

“…For example, even though the matrix porosity is similar, the permeability can be different according to the fiber orientation [26]. Recently, studies showed that the branching point, one of the characteristics that distinguish monomer from oligomer, is another parameter affecting transport and mechanical properties [24,25]. It was observed that even though the porosity was similar by modulation of different fibril diameters and branching points, the resulting values differed with the different numbers of the branching points.…”

“…We employed branching points, as a measure of collagen microstructure [25], since the branching points can be independently controlled while keeping the porosity and fibril diameters constant. As reported previously [24,25], mechanical properties, i.e., storage modulus, could be changed by varying the ratio of monomer to oligomer, and ultimately stem cell differentiation can be guided. In addition, an increase in oligomer content results in a higher number of branching points per total fibril length but it also results in lower segment length.…”