| The generation of functional vascular networks by endothelial colony-forming cells (ECFCs) has the potential to improve treatment for vascular diseases and to facilitate successful transplantation of tissue-engineered organs. ECFCs are recruited from a bone marrow niche to the site of vascularization, where cues from the extracellular matrix (ECM) instigate vascular morphogenesis. Although this process has been elucidated using natural matrices, little is known about vascular morphogenesis of ECFCs in synthetic matrices where properties can be tuned towards both the basic understanding of tubulogenesis in modular environments as well as a clinically relevant alternative to natural materials for regenerative medicine.;In this work, we utilized tunable hyaluronic acid (HA) hydrogels to investigate ECFCs morphogenesis. First, we develop a 2D tube-formation assay using HA-gelatin hydrogel, where matrix stiffness can be modulated. We found that VEGF and matrix stiffness co-regulate ECFCs tubulogenesis. High level of VEGF is required to initiate vascular morphogenesis and to activate membrane type -1 matrix metalloproteinase (MT1-MMP), MMP-1, and MMP-2, to enable ECFCs migration. With decreased substrate elasticity, we observed decreased MMPs expression and increased cellular elongation, with intracellular vacuole extension and coalescence to open lumen compartments. Then, we utilized HA-gelatin hydrogel to explore 3D vascular morphogenesis. We demonstrate that ECFCs express CD44 and CD168, which are specific receptors for HA, and produce hyaluronidase (Hyal) in response to vascular endothelial growth factor (VEGF). We found that decreasing matrix viscoelasticity, which corresponds to a loose ultrastructure, significantly increases ECFC vascular tube length and area, and that the effect of local delivery of VEGF within the hydrogel depends on the makeup of the synthetic environment.;To further control the signaling pathways of ECFCs vascular morphogenesis, we developed arylated HA (AHA) hydrogel, where RGD binding peptide and MMP-sensitive-peptide can be incorporated within this modular culture system. We showed that vacuole and lumen formation are RGD dose-dependent and are recognized by the ECFCs through integrin alpha5beta1 and alphaVbeta 3 subunits. Integration of MMP-sensitive-peptide enabled ECFCs to sprout, branch, and form complex vascular networks. Along the culture period, increased expression of hyaluronidase isoforms Hyal-1, -2, as well as MMPs by the vascular networks resulted in decreased matrix stiffness, release of the encapsulated growth factors, demonstrating the modularity of the synthetic environment.;We tested the functionality of the engineered vascular networks using a subcutaneous implantation model and a burn wound model. In both cases, by two weeks post implantation, 85% of the AHA hydrogels were degraded and replaced by macrophages and tissue ingrowth. Blood vessels in various sizes were found at the periphery and center of the hydrogels. Most microvasculatures at the periphery of the gels were of murine origin. Two types of microvessels at the center of the hydrogels were found: about 60% of the blood vessels contain both human ECFCs and host cells, while the remaining vessels contain only human ECFCs. Perfusion with blood cells was detected in both types of microvasculature, demonstrating that the implanted ECFCs participate in the angiogenesis of the host vasculature and form functional human vascular networks that anastomose with the host vasculature to form functional vessels (vasculogenesis). In the burn wound model, we observed a more rapid integration of the engineered vasculature with the host vessels, which started at day 5, then regressed at day 11, and remained at the end of the healing process (day 14). Overall, we demonstrated that the engineered vascular network can be integrated with the host vasculature in a subcutaneous implantation model and may be useful for the healing process in a burn wound model.;Finally, we exploited the modifiability and physical property of the AHA hydrogels to provide spatial control over vasculogenesis and angiogenesis. We utilized UV light to photopattern the AHA hydrogels to create distinct regions, where vasculogenesis and angiogenesis are either "on" or "off." Using this technique, we were also able to promote or inhibit ex ovo chorioallantoic (CAM) vessels invasion into the AHA hydrogels. Collectively, we showed that the signaling pathways of ECFCs vascular morphogenesis can be precisely and spatially regulated in a synthetic matrix, resulting in a robust and functional microvasculature useful for the study of stem cell vascular biology and towards a range of vascular disorders and approaches in tissue regeneration. |
