Engineering primitive multiscale chimeric vasculature by combining human microvessels with explanted murine vessels.

Strategies to separately manufacture arterial-scale tissue engineered vascular grafts and microvascular networks have been well-established, but efforts to bridge these two length scales to create hierarchical vasculature capable of supporting parenchymal cell functions or restoring perfusion to ischemic tissues have been limited. This work aimed to create multiscale vascular constructs by assessing the capability of macroscopic vessels isolated from mice to form functional connections to engineered capillary networks ex vivo. Vessels of venous and arterial origins from both thoracic and femoral locations were isolated from mice, and then evaluated for their abilities to sprout endothelial cells (EC) capable of inosculating with surrounding human cell-derived microvasculature within bulk fibrin hydrogels. Comparing aortae, vena cavae, and femoral vessel bundles, we identified the thoracic aorta as the rodent macrovessel that yielded the greatest degree of sprouting and interconnection to surrounding capillaries. The presence of cells undergoing vascular morphogenesis in the surrounding hydrogel attenuated EC sprouting from the macrovessel compared to sprouting into acellular hydrogels, but ultimately sprouted mouse EC interacted with human cell-derived capillary networks in the bulk, yielding chimeric vessels. We then integrated micromolded mesovessels into the constructs to engineer a primitive 3-scale vascular hierarchy comprising capillaries, mesovessels, and macrovessels. Overall, this study yielded a primitive hierarchical vasculature suitable as proof-of-concept for regenerative medicine applications and as an experimental model to better understand the spontaneous formation of host-graft vessel anastomoses.

Pre-cultured, cell-encapsulating fibrin microbeads for the vascularization of ischemic tissues.

There is a significant clinical need to develop effective vascularization strategies for tissue engineering and the treatment of ischemic pathologies. In patients afflicted with critical limb ischemia, comorbidities may limit common revascularization strategies. Cell-encapsulating modular microbeads possess a variety of advantageous properties, including the ability to support prevascularization in vitro while retaining the ability to be injected in a minimally invasive manner in vivo. Here, fibrin microbeads containing human umbilical vein endothelial cells (HUVEC) and bone marrow-derived mesenchymal stromal cells (MSC) were cultured in suspension for 3 days (D3 PC microbeads) before being implanted within intramuscular pockets in a SCID mouse model of hindlimb ischemia. By 14 days post-surgery, animals treated with D3 PC microbeads showed increased macroscopic reperfusion of ischemic foot pads and improved limb salvage compared to the cellular controls. Delivery of HUVEC and MSC via microbeads led to the formation of extensive microvascular networks throughout the implants. Engineered vessels of human origins showed evidence of inosculation with host vasculature, as indicated by erythrocytes present in hCD31+ vessels. Over time, the total number of human-derived vessels within the implant region decreased as networks remodeled and an increase in mature, pericyte-supported vascular structures was observed. Our findings highlight the potential therapeutic benefit of developing modular, prevascularized microbeads as a minimally invasive therapeutic for treating ischemic tissues.

Manufacturing the multiscale vascular hierarchy: progress toward solving the grand challenge of tissue engineering.

In human vascular anatomy, blood flows from the heart to organs and tissues through a hierarchical vascular tree, comprising large arteries that branch into arterioles and further into capillaries, where gas and nutrient exchange occur. Engineering a complete, integrated vascular hierarchy with vessels large enough to suture, strong enough to withstand hemodynamic forces, and a branching structure to permit immediate perfusion of a fluidic circuit across scales would be transformative for regenerative medicine (RM), enabling the translation of engineered tissues of clinically relevant size, and perhaps whole organs. How close are we to solving this biological plumbing problem? In this review, we highlight advances in engineered vasculature at individual scales and focus on recent strategies to integrate across scales.

Stromal cell identity modulates vascular morphogenesis in a microvasculature-on-a-chip platform.

Supportive stromal cells of mesenchymal origins regulate vascular morphogenesis in developmental, pathological, and regenerative contexts, contributing to vessel formation, maturation, and long-term stability, in part via the secretion of bioactive molecules. In this work, we adapted a microfluidic lab-on-a-chip system that enables the formation and perfusion of microvascular capillary beds with connections to arteriole-scale endothelialized channels to explore how stromal cell (SC) identity influences endothelial cell (EC) morphogenesis. We compared and contrasted lung fibroblasts (LFs), dermal fibroblasts (DFs), and bone marrow-derived mesenchymal stem cells (MSCs) for their abilities to support endothelial morphogenesis and subsequent perfusion of microvascular networks formed in fibrin hydrogels within the microfluidic device. We demonstrated that while all 3 SC types supported EC morphogenesis, LFs in particular resulted in microvascular morphologies with the highest total network length, vessel diameter, and vessel interconnectivity across a range of SC-EC ratio and density conditions. Not only did LFs support robust vascular morphology, but also, they were the only SC type to support functional perfusion of the resultant capillary beds. Lastly, we identified heightened traction stress produced by LFs as a possible mechanism by which LFs enhance endothelial morphogenesis in 3D compared to other SC types examined. This study provides a unique comparison of three different SC types and their role in supporting the formation of microvasculature that could provide insights for the choice of cells for vascular cell-based therapies and the regulation of tissue-specific vasculature.

Design principles for lymphatic drainage of fluid and solutes from collagen scaffolds.

In vivo, tissues are drained of excess fluid and macromolecules by the lymphatic vascular system. How to engineer artificial lymphatics that can provide equivalent drainage in biomaterials remains an open question. This study elucidates design principles for engineered lymphatics, by comparing the rates of removal of fluid and solute through type I collagen gels that contain lymphatic vessels or unseeded channels, or through gels without channels. Surprisingly, no difference was found between the fluid drainage rates for gels that contained vessels or bare channels. Moreover, solute drainage rates were greater in collagen gels that contained lymphatic vessels than in those that had bare channels. The enhancement of solute drainage by lymphatic endothelium was more pronounced in longer scaffolds and with smaller solutes. Whole-scaffold imaging revealed that endothelialization aided in solute drainage by impeding solute reflux into the gel without hindering solute entry into the vessel lumen. These results were reproduced by computational models of drainage with a flow-dependent endothelial hydraulic conductivity. This study shows that endothelialization of bare channels does not impede the drainage of fluid from collagen gels and can increase the drainage of macromolecules by preventing solute transport back into the scaffold. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 106-114, 2018.