Mechanisms of tubulogenesis and endothelial phenotype expression by MSCs.

Stem cell-based therapies are a promising new avenue for treating ischemic disease and chronic wounds. Mesenchymal stem cells (MSCs) have a proven ability to augment the neovascularization processes necessary for wound healing and are widely popular as an autologous source of progenitor cells. Our lab has previously reported on PEGylated fibrin as a unique hydrogel that promotes spontaneous tubulogenesis of encapsulated MSCs without exogenous factors. However, the mechanisms underlying this process have remained unknown. To better understand the therapeutic value of PEGylated fibrin delivery of MSCs, we sought to clarify the relationship between biomaterial properties and cell behavior. Here we find that fibrin PEGylation does not dramatically alter the macroscopic mechanical properties of the fibrin-based matrix (less than 10% difference). It does, however, dramatically reduce the rate of diffusion through the gel matrix. PEGylated fibrin enhances the tubulogenic growth of encapsulated MSCs demonstrating fluid-filled lumens by interconnected MSCs. Image analysis gave a value of 4320 ± 1770 µm total network length versus 618 ± 443 µm for unmodified fibrin. PEGylation promotes the endothelial phenotype of encapsulated MSCs--compared to unmodified fibrin--as evidenced by higher levels of endothelial markers (von Willebrand factor, 2.2-fold; vascular endothelial cadherin, 1.8-fold) and vascular endothelial growth factor (VEGF, up to 1.8-fold). Prospective analysis of underlying molecular pathways demonstrated that this endothelial-like MSC behavior is sensitively modulated by hypoxic stress, but not VEGF supplementation as evidenced by a significant increase in VEGF and MMP-2 secretion per cell under hypoxia. Further gain-of-function studies under hypoxic stress demonstrated that hypoxia culture of MSCs in unmodified fibrin could increase both vWF and VE-cadherin levels to values that were not significantly different than cells cultured in PEGylated fibrin. This result corroborated our hypothesis that the diffusion-limited environment of PEGylated fibrin is augmenting endothelial differentiation cues provided by unmodified fibrin. However, MSC networks lack platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, which indicates incomplete differentiation towards an endothelial cell type. Collectively, the data here supports a revised understanding of MSC-derived neovascularization that contextualizes their behavior and utility as a hybrid endothelial-stromal cell type, with mixed characteristics of both populations.

Fibrin-based 3D matrices induce angiogenic behavior of adipose-derived stem cells.

Engineered three-dimensional biomaterials are known to affect the regenerative capacity of stem cells. The extent to which these materials can modify cellular activities is still poorly understood, particularly for adipose-derived stem cells (ASCs). This study evaluates PEGylated fibrin (P-fibrin) gels as an ASC-carrying scaffold for encouraging local angiogenesis by comparing with two commonly used hydrogels (i.e., collagen and fibrin) in the tissue-engineering field. Human ASCs in P-fibrin were compared to cultures in collagen and fibrin under basic growth media without any additional soluble factors. ASCs proliferated similarly in all gel scaffolds but showed significantly elongated morphologies in the P-fibrin gels relative to other gels. P-fibrin elicited higher von Willebrand factor expression in ASCs than either collagen or fibrin while cells in collagen expressed more smooth muscle alpha actin than in other gels. VEGF was secreted more at 7 days in fibrin and P-fibrin than in collagen and several other angiogenic and immunomodulatory cytokines were similarly enhanced. Fibrin-based matrices appear to activate angiogenic signaling in ASCs while P-fibrin matrices are uniquely able to also drive a vessel-like ASC phenotype. Collectively, these results suggest that P-fibrin promotes the angiogenic potential of ASC-based therapeutic applications.

Three-dimensional image quantification as a new morphometry method for tissue engineering.

Morphological analysis is an essential step in verifying the success of a tissue engineering strategy where the presence of a desired cellular phenotype must be determined. While morphometry has transitioned from observational grading to computational quantification, established quantitative methods eliminate information by relying on two-dimensional (2D) analysis to describe three-dimensional (3D) niches. In this study, we demonstrate the validity and utility of 3D morphological quantification using two common angiogenesis assays in our fibrin-based in vitro model: (1) the microcarrier bead assay with human mesenchymal stem cells and (2) the rat aortic ring outgrowth assay. The quantification method is based on collecting and segmenting fluorescent confocal z-stacks into 3D models with 3D Slicer, an open-source magnetic resonance imaging/computed tomography analysis program. Data from 3D models are then processed into biologically relevant metrics in MATLAB for statistical analysis. Metrics include descriptive parameters such as vascular network length, volume, number of network segments, and degree of network branching. Our results indicate that 2D measures are significantly different than their 3D counterparts unless the vascular network exhibits anisotropic growth along the plane of imaging. Additionally, the statistical outcomes of 3D morphological quantification agreed with our initial qualitative observations among different test groups. This novel quantification approach generates more spatially accurate and objective measures, representing an important step toward improving the reliability of morphological comparisons.