Engineered blood vessel networks connect to host vasculature via wrapping-and-tapping anastomosis

Abstract

Rapid blood perfusion is critical for postimplantation survival of thick, prevascularized bioartificial tissues. Yet the mechanism by which implanted vascular networks inosculate, or anastomose, with the host vasculature has been unknown, making it difficult to develop optimized strategies for facilitating perfusion. Here we show that implanted vascular networks anastomose with host vessels through a previously unidentified process of "wrapping and tapping" between the engrafted endothelial cells (ECs) and the host vasculature. At the host-implant interface, implanted ECs first wrap around nearby host vessels and then cause basement membrane and pericyte reorganization and localized displacement of the underlying host endothelium. In this way, the implanted ECs replace segments of host vessels to divert blood flow to the developing implanted vascular network. IntroductionA major obstacle in tissue engineering is poor postimplantation graft survival because of insufficient blood perfusion. A potential solution is to populate the engineered tissue with endothelial cells (ECs) or endothelial progenitor cells (EPCs), which can quickly organize into interconnected networks, undergo lumenogenesis, and anastomose with the host vasculature to redirect blood flow into the graft. 1 Human umbilical vein endothelial cells (HUVECs), [2][3][4] ECs derived from human embryonic stem cells, 5 and human adult and cord blood EPCs 6,7 are all capable of generating such patent vascular networks in vivo. This approach has proven effective in improving the quality of engineered skeletal muscle 8 and bone 9 tissues.Limited studies suggest that, during embryonic vasculogenesis and sprouting angiogenesis, anastomosis is accomplished via connection of extended cellular processes followed by lumen propagation through intracellular and intercellular vacuole fusion, 10,11 with macrophages playing an accessory role. 12 However, it is not known whether this is the only mechanism for connecting vessels. Without a basic understanding of the cellular mechanisms of anastomosis, it is difficult to develop strategies for accelerating this critical step for perfusing engrafted tissues.To investigate the process of anastomosis, we used a previously established model in which HUVECs and mouse mesenchymal precursor cells are embedded in collagen-fibronectin gels and placed in cranial window preparations of severe combined immunodeficient mice 2 (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). In this system, anastomosis between host vessels and implanted EC networks occurs as early as 2 weeks after implantation, and the engineered vessels remain stable and functional for one year in vivo. 2 Similar results can be achieved when the mouse mesenchymal precursor cells are replaced with human mesenchymal stem cells 13 or human lung fibroblasts. 14,15 Tracking fluorescently labeled implanted ECs and host ECs simultaneously in live animals, we found that tip cell connections and va...

Integration and Regression of Implanted Engineered Human Vascular Networks During Deep Wound Healing

“…Once the macrophages allowed host vessels to grow into the hydrogels and the wounds reached the proliferation period (day 5), localization of the human and mouse vessels was observed around the wound area. Since burn wounds stimulate a rapid angiogenic response, the integration kinetics observed here agreed with a different wound healing model where human vessels began to integrate with the host vessels at day 3 when a Matrigel plug was used [12] and at day 7 when collagen and fibrin gels were used [42]. In the middle of the proliferation period (day 7), mouse host vessels grew into the wound area, integrating with the human vascular networks to support the regenerating tissue.…”

“…Tissue engineering requires an understanding of vascularized tissue construct kinetics as they integrate with their host vasculature [3,12,42]. This study showed that engineered human vascular networks using ECFCs in HA hydrogels can rapidly integrate with host vasculature during the healing process in a third-degree burn model.…”

Generation of functionally competent and durable engineered blood vessels from human induced pluripotent stem cells

“…Whole-mount gel staining at 6 mo postimplantation showed an infiltration of the gel by the host CD31 + murine ECs, which anastomosed with the engineered vessels at the host-implant interface (Fig. 3A), as recently described, via stabilization or a "wrapping and tapping" mechanism (23). The engineered vessels were associated with both implanted 10T1/2 supporting cells and desmin-positive host-derived cells (Fig.…”

The Hematopoietic Chemokine CXCL12 Promotes Integration of Human Endothelial Colony Forming Cell–Derived Cells into Immature Vessel Networks

“…For sprouting angiogenesis, the extracellular matrix surrounding the vasculature is degraded and mural cells detach from capillaries and microvessels (<100 mm in diameter) allowing the endothelial tip cells to become invasive and to form filopodia and lamellipodia in response to guidance cues, while stalk cells that lie behind the tip cells increase in number, extend the vessels, and form extracellular matrix, junctions, and lumens [19][20][21][22]. Once the tip cells anastomose or inosculate with other tip cells [23], vessel maturation takes place and this involves mural cell recruitment, extracellular matrix deposition, and the commencement of blood flow. A key feature of vasculogenesis and angiogenesis is central vascular lumen formation, the complexity of which has recently been reviewed [24][25][26][27].…”