Co-cultures of endothelial cells (EC) and mesenchymal stem cells (MSC) in three-dimensional (3D) protein hydrogels can be used to recapitulate aspects of vasculogenesis in vitro. MSC provide paracrine signals that stimulate EC to form vessel-like structures, which mature as the MSC transition to the role of mural cells. In this study, vessel-like network formation was studied using 3D collagen/fibrin (COL/FIB) matrices seeded with embedded EC and MSC and cultured for 7 days. The EC:MSC ratio was varied from 5:1, 3:2, 1:1, 2:3 and 1:5. The matrix composition was varied at COL/FIB compositions of 100/0 (pure COL), 60/40, 50/50, 40/60 and 0/100 (pure FIB). Vasculogenesis was markedly decreased in the highest EC:MSC ratio, relative to the other cell ratios. Network formation increased with increasing fibrin content in composite materials, although the 40/60 COL/FIB and pure fibrin materials exhibited the same degree of vasculogenesis. EC and MSC were co-localized in vessel-like structures after 7 days and total cell number increased by approximately 70%. Mechanical property measurements showed an inverse correlation between matrix stiffness and network formation. The effect of matrix stiffness was further investigated using gels made with varying total protein content and by crosslinking the matrix using the dialdehyde glyoxal. This systematic series of studies demonstrates that matrix composition regulates vasculogenesis in 3D protein hydrogels, and further suggests that this effect may be caused by matrix mechanical properties. These findings have relevance to the study of neovessel formation and the development of strategies to promote vascularization in transplanted tissues.

A major translational challenge in the fields of therapeutic angiogenesis and tissue engineering is the ability to form functional networks of blood vessels. Cell-based strategies to promote neovascularization have been widely explored, and have led to the consensus that co-delivery of endothelial cells (ECs) (or their progenitors) with some sort of a supporting stromal cell type is the most effective approach. However, the choice of stromal cells has varied widely across studies, and their impact on the functional qualities of the capillaries produced has not been examined. In this study, we injected human umbilical vein ECs alone or with normal human lung fibroblasts (NHLFs), human bone marrow-derived mesenchymal stem cells (BMSCs), or human adipose-derived stem cells (AdSCs) in a fibrin matrix into subcutaneous pockets in SCID mice. All conditions yielded new human-derived vessels that inosculated with mouse vasculature and perfused the implant, but there were significant functional differences in the capillary networks, depending heavily on the identity of the co-delivered stromal cells. EC-alone and EC-NHLF implants yielded immature capillary beds characterized by high levels of erythrocyte pooling in the surrounding matrix. EC-BMSC and EC-AdSC implants produced more mature capillaries characterized by less extravascular leakage and the expression of mature pericyte markers. Injection of a fluorescent tracer into the circulation also showed that EC-BMSC and EC-AdSC implants formed vasculature with more tightly regulated permeability. These results suggest that the identity of the stromal cells is key to controlling the functional properties of engineered capillary networks.

Forming functional blood vessel networks in engineered or ischemic tissues is a significant scientific and clinical hurdle. We adapted poly(ethylene glycol) (PEG)-based hydrogels to investigate the role of mechanical properties and proteolytic susceptibility on vascularization. Four arm PEG vinyl sulfone was polymerized by Michael-type addition with cysteine groups on a slowly degraded matrix metalloprotease (MMP) susceptible peptide, GPQG↓IWGQ, or a more rapidly cleaved peptide, VPMS↓MRGG. Co-encapsulation of endothelial cells and supportive fibroblasts within the gels led to vascular morphogenesis in vitro that was robust to changes in crosslinking peptide identity, but was significantly attenuated by increased crosslinking and MMP inhibition. Perfused vasculature formed from transplanted cells in vivo in all gel types; however, in contrast to the in vitro results, vascularization in vivo was not decreased in the more crosslinked gels. Collectively, these findings demonstrate the utility of this platform to support vascularization both in vitro and in vivo.

Fibrin is the primary extracellular constituent of blood clots, and plays an important role as a provisional matrix during wound healing and tissue remodeling. Fibrin-based biomaterials have proven their utility as hemostatic therapies, scaffolds for tissue engineering, vehicles for controlled release, and as platforms for culturing and studying cells in three dimensions. Nevertheless, fibrin presents a complex milieu of signals to embedded cells, many of which are not well understood. Synthetic ECMs provide a blank slate that can ostensibly be populated with specific bioactive cues, including growth factors, growth factor binding motifs, adhesive peptides, and peptide cross-links susceptible to proteases, thereby enabling a degree of customization for specific applications. However, the continued evolution and improvement of synthetic ECMs requires parallel efforts to deconstruct native ECMs and decipher the cues they provide to constituent cells. The objective of this review is to reintroduce fibrin, a protein with a well-characterized structure and biochemistry, and its ability to support angiogenesis specifically. Although fibrin’s structure-function relationships have been studied for decades, opportunities to engineer new and improved synthetic hydrogels can be realized by further exploiting fibrin’s inspiring design.

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