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.

Matrix density regulates adipocyte phenotype.

Alterations of the extracellular matrix contribute to adipose tissue dysfunction in metabolic disease. We studied the role of matrix density in regulating human adipocyte phenotype in a tunable hydrogel culture system. Lipid accumulation was maximal in intermediate hydrogel density of 5 weight %, relative to 3% and 10%. Adipogenesis and lipid and oxidative metabolic gene pathways were enriched in adipocytes in 5% relative to 3% hydrogels, while fibrotic gene pathways were enriched in 3% hydrogels. These data demonstrate that the intermediate density matrix promotes a more adipogenic, less fibrotic adipocyte phenotype geared towards increased lipid and aerobic metabolism. These observations contribute to a growing literature describing the role of matrix density in regulating adipose tissue function.

Crossover of surface waves and capillary-viscous-elastic transition in soft biomaterials detected by resonant acoustic rheometry.

Viscoelastic properties of hydrogels are important for their application in science and industry. However, rheological assessment of soft hydrogel biomaterials is challenging due to their complex, rapid, and often time-dependent behaviors. Resonant acoustic rheometry (RAR) is a newly developed technique capable of inducing and measuring resonant surface waves in samples in a non-contact fashion. By applying RAR at high temporal resolution during thrombin-induced fibrin gelation and ultraviolet-initiated polyethylene glycol (PEG) polymerization, we observed distinct changes in both frequency and amplitude of the resonant surface waves as the materials changed over time. RAR detected a series of capillary-elastic, capillary-viscous, and visco-elastic transitions that are uniquely manifested as crossover of different types of surface waves in the temporally evolving materials. These results reveal the dynamic interplay of surface tension, viscosity, and elasticity that is controlled by the kinetics of polymerization and crosslinking during hydrogel formation. RAR overcomes many limitations of conventional rheological approaches by offering a new way to comprehensively and longitudinally characterize soft materials during dynamic processes.

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.

A combination of matrix stiffness and degradability dictate microvascular network assembly and remodeling in cell-laden poly(ethylene glycol) hydrogels.

The formation of functional capillary blood vessels that can sustain the metabolic demands of transplanted parenchymal cells remains one of the biggest challenges to the clinical realization of engineered tissues for regenerative medicine. As such, there remains a need to better understand the fundamental influences of the microenvironment on vascularization. Poly(ethylene glycol) (PEG) hydrogels have been widely adopted to interrogate the influence of matrix physicochemical properties on cellular phenotypes and morphogenetic programs, including the formation of microvascular networks, in part due to the ease with which their properties can be controlled. In this study, we co-encapsulated endothelial cells and fibroblasts in PEG-norbornene (PEGNB) hydrogels in which stiffness and degradability were tuned to assess their independent and synergistic effects on vessel network formation and cell-mediated matrix remodeling longitudinally. Specifically, we achieved a range of stiffnesses and differing rates of degradation by varying the crosslinking ratio of norbornenes to thiols and incorporating either one (sVPMS) or two (dVPMS) cleavage sites within the matrix metalloproteinase- (MMP-) sensitive crosslinker, respectively. In less degradable sVPMS gels, decreasing the crosslinking ratio (thereby decreasing the initial stiffness) supported enhanced vascularization. When degradability was increased in dVPMS gels, all crosslinking ratios supported robust vascularization regardless of initial mechanical properties. The vascularization in both conditions was coincident with the deposition of extracellular matrix proteins and cell-mediated stiffening, which was greater in dVPMS conditions after a week of culture. Collectively, these results indicate that enhanced cell-mediated remodeling of a PEG hydrogel, achieved either by reduced crosslinking or increased degradability, leads to more rapid vessel formation and higher degrees of cell-mediated stiffening.

Coupling Osteogenesis and Vasculogenesis in Engineered Orthopedic Tissues.

Inadequate vascularization of engineered tissue constructs is a main challenge in developing a clinically impactful therapy for large, complex, and recalcitrant bone defects. It is well established that bone and blood vessels form concomitantly during development, as well as during repair after injury. Endothelial cells (ECs) and mesenchymal stromal cells (MSCs) are known to be key players in orthopedic tissue regeneration and vascularization, and these cell types have been used widely in tissue engineering strategies to create vascularized bone. Coculture studies have demonstrated that there is crosstalk between ECs and MSCs that can lead to synergistic effects on tissue regeneration. At the same time, the complexity in fabricating, culturing, and characterizing engineered tissue constructs containing multiple cell types presents a challenge in creating multifunctional tissues. In particular, the timing, spatial distribution, and cell phenotypes that are most conducive to promoting concurrent bone and vessel formation are not well understood. This review describes the processes of bone and vascular development, and how these have been harnessed in tissue engineering strategies to create vascularized bone. There is an emphasis on interactions between ECs and MSCs, and the culture systems that can be used to understand and control these interactions within a single engineered construct. Developmental engineering strategies to mimic endochondral ossification are discussed as a means of generating vascularized orthopedic tissues. The field of tissue engineering has made impressive progress in creating tissue replacements. However, the development of larger, more complex, and multifunctional engineered orthopedic tissues will require a better understanding of how osteogenesis and vasculogenesis are coupled in tissue regeneration. Impact statement Vascularization of large engineered tissue volumes remains a challenge in developing new and more biologically functional bone grafts. A better understanding of how blood vessels develop during bone formation and regeneration is needed. This knowledge can then be applied to develop new strategies for promoting both osteogenesis and vasculogenesis during the creation of engineered orthopedic tissues. This article summarizes the processes of bone and blood vessel development, with a focus on how endothelial cells and mesenchymal stromal cells interact to form vascularized bone both during development and growth, as well as tissue healing. It is meant as a resource for tissue engineers who are interested in creating vascularized tissue, and in particular to those developing cell-based therapies for large, complex, and recalcitrant bone defects.

Injectable pre-cultured tissue modules catalyze the formation of extensive functional microvasculature in vivo.

Revascularization of ischemic tissues is a major barrier to restoring tissue function in many pathologies. Delivery of pro-angiogenic factors has shown some benefit, but it is difficult to recapitulate the complex set of factors required to form stable vasculature. Cell-based therapies and pre-vascularized tissues have shown promise, but the former require time for vascular assembly in situ while the latter require invasive surgery to implant vascularized scaffolds. Here, we developed cell-laden fibrin microbeads that can be pre-cultured to form primitive vascular networks within the modular structures. These microbeads can be delivered in a minimally invasive manner and form functional microvasculature in vivo. Microbeads containing endothelial cells and stromal fibroblasts were pre-cultured for 3 days in vitro and then injected within a fibrin matrix into subcutaneous pockets on the dorsal flanks of SCID mice. Vessels deployed from these pre-cultured microbeads formed functional connections to host vasculature within 3 days and exhibited extensive, mature vessel coverage after 7 days in vivo. Cellular microbeads showed vascularization potential comparable to bulk cellular hydrogels in this pilot study. Furthermore, our findings highlight some potentially advantageous characteristics of pre-cultured microbeads, such as volume preservation and vascular network distribution, which may be beneficial for treating ischemic diseases.