Macro, Micro, and Everything in Between. Bridging the Gap: A Vision Toward the Creation of Multiscale Vascular Networks

Vascularization is a key issue for the clinical translation of tissue engineering strategies. This has been recognized in the field for almost two decades. Several strategies to solve this issue are proposed but none has decisively tackled the problem. This is in part due to an excessive focus on microvascularization that ignores the need of having macrovessels capable of being surgically connected to the patient's circulation upon implantation. Indeed, a strategy for macrovessel engineering must co‐exist with a strategy for microvessels. And if this is true, all the intermediate scales have to be addressed as well. Therefore, multiscale vascular networks must be the focus of tissue engineering vascularization efforts. In this work, a reflection is made on a possible path forward for researchers and engineers in the field to achieve the ultimate goal of efficient vascularization of engineered tissues and organs.


DOI: 10.1002/adbi.202300291
parts, the newer approaches follow a bottom-up approach that more closely resembles principles of biological development.
An essential condition to ensure the survival of thick and complex engineered tissues is the presence of a functional vascular network.After implantation, this network must anastomose, i.e., link to host circulation, to allow nutrient-and oxygen-rich blood to supply the engineered tissue.Most efforts in TE have focused on the creation of microvasculature within engineered tissues that will spontaneously anastomose with host vessels after implantation.This is now known to be a doomed strategy since, without adequate stimuli such as flow, the engineered microvasculature regresses before anastomosis is complete, leading to implant failure. [1]During transplantation of vascularized thick tissues and organs, the transplanted tissue contains a vascular axis that is surgically anastomosed to the recipient's circulation, ensuring immediate perfusion and tissue survival.This is what must be replicated during the implantation of engineered tissues and organs.Therefore, not only must engineered tissues contain microvessels but a whole multiscale vascular network that ensures seamless continuity from macrovessels right down to capillaries.
One possible approach to achieve this is through the use of the arteriovenous (AV) loop strategy. [2]In the patient's body, an easily accessible vein and artery are connected to create an AV loop (Figure 1).The AV loop and the engineered tissue to be vascularized are isolated inside a chamber and the whole construct is then incubated in vivo until sufficient vessel ingrowth into the engineered tissue is achieved.At this point, the newly vascularized tissue along with the AV loop, which will act as a vascular axis capable of being anastomosed with the local vasculature, are harvested and implanted at the defect site.This technique ingeniously uses the patient's body as a bioreactor to obtain a multiscale vascular network that is completely organic.The same basic concept has been proposed for the vascularization of cell sheetbased TE constructs, [3] where several multilayer grafts are serially implanted on top of each other at an ectopic site, allowing time for vessel ingrowth between implantations to create a fully vascularized thick construct.This construct can then be resected with a vascular axis and implanted at the defect site.However, these strategies require multi-step interventions, which may not be desirable or even possible, depending on the patient.The loop is placed in an isolation chamber together with the scaffold.The chamber is closed and the whole construct implanted in the patient.After sufficient time elapses, the vascularized scaffold is retrieved from the patient together with the vascular axis, i.e., the segmented artery and vein.The latter can be anastomosed with local vessels at the defect site, where the scaffold will be implanted.B) Micro-computed tomography reconstruction of the microvessel network formed in a scaffold vascularized using the AV loop technique, in an animal model.(Adapted with permission [21] Copyright 2016, JoVE).Created with Biorender.com.
But to engineer a functional multiscale vascular network in the lab is a substantial challenge due to the significantly different nature of each vessel type.
Macrovessels are responsible for moving blood over long distances in a circuit around the body starting and ending at the heart.Because of this, they are stronger and more complex than microvessels.Macrovessels range in diameter from 100-150 μm up to several centimeters. [4,5]For patients suffering from peripheral artery disease or those in need of alternative vascular access for hemodialysis, substitutes for larger macrovessels are required.While autologous grafts (i.e., taken from the patient) are, for obvious reasons of compatibility, the gold standard in these cases, their availability is limited, and retrieval causes donor site morbidity and significant discomfort.Vascular grafts made of synthetic materials have been successfully used as alternatives for larger vessels.However, such synthetic materials do not integrate with the host tissue and cause chronic inflammation that significantly increases thrombogenicity, i.e., the probability of generating blood clots.This is a vital issue since thrombotic events are one the main reasons for vascular graft failure, especially for graft diameters smaller than 6 mm. [6]It is in this range that TE macrovessels have been mostly proposed, making use of, for example, biodegradable scaffolding materials to harbor cells that produce robust ECM during a long maturation process, followed by decellularization to yield constructs capable of being used off-the-shelf.Preliminary clinical outcomes are highly encouraging, [7,8] suggesting a viable route to engineer functional vessels at this scale.However, the long and complex biofabrication processes and/or thrombogenicity concerns derived from the use of synthetic materials may complicate widespread clinical adoption.The need for streamlined fabrication methodologies capable of yielding macrovessels displaying anti-thrombotic properties is clear.
By contrast, microvessels span in size from a few micrometers (capillaries) to ≈100 μm (arterioles/venules) and are essential for tissue functioning. [5]Capillaries form capillary beds, dense networks originating from branching arterioles on one end and converging into venules on the other.Capillary beds allow nutrient, waste, and gas exchange between the blood and surrounding tissues.Networks similar to capillary beds can be created in engineered tissues by seeding endothelial cells-the cells that line the interior of blood vessels-and guiding their self-assembly into microvascular networks (Figure 2A) that can work as conduits to transport blood.For the latter, the formation of a hollow space within the tubes of the formed network in a process termed lumenization, [9] is an essential but often overlooked step.If present, adequate stromal cells can be recruited to act as pericytes-cells that wrap around capillaries-potentially stabilizing the network in a native-like manner.While this may seem organic, it requires the addition of extrinsic growth factors that are often associated with tumorigenesis and abnormal vessel formation. [10]But even in the absence of such issues, these microvascular networks tend to regress in the absence of adequate blood flow, which clearly limits their applicability in these conditions.
Although blood vessels have been successfully engineered on either end of the size spectrum, the multiscale nature of organic vasculature has yet to be achieved.In other words, we still need to make all the stuff in between the micro and the macro and assemble them into a functional unit.This unified vascularization strategy is the next major step being pursued by many researchers.
The Levenberg group has put forward strategies where separately produced macro-and microvessels are merged to form a multiscale vascular network.Small macrovessels with diameters of one mm or less are created using custom synthetic scaffolds (temporary 3D matrices) that are lined with endothelial cells.These are then combined with microvascular networks formed in separate scaffolds that will become the main engineered tissue. [11,12]Spontaneous anastomosis occurs between sprouts coming out of the larger vessels, via specially designed openings, and the microvessels in the network.The macrovessels are surgically anastomosed to the host circulation allowing the whole construct to be functionally perfused with blood, demonstrating the effectiveness of the strategy in this context.However, the clinical importance of these works is somewhat limited by the relatively small size of the macrovessels and by the synthetic materials used in their fabrication.But it is nevertheless an effort that may be foundational in creating an integrated solution.
Addressing the specific problem of the synthetic materials used for macrovessels TE, the L'Hereux group has been developing materials based on cell self-assembled extracellular matrix (ECM) to fabricate macrovessels in a scaffold-free manner.In earlier versions, human fibroblasts from target patients were cultured in conditions that maximized ECM production, forming mechanically robust cell sheets.These sheets were then subjected to a maturation process that yielded totally biological tubular structures that were then lined with endothelial cells from the patient.Upon implantation, these vessels engineered with autologous materials (i.e., from the patient) were shown to be largely functional. [13]Interestingly, macrovessels made with allogenic ECM (i.e., from donors other than the patients) were also produced and tested clinically with positive outcomes even in terms of immunogenicity. [14]While the latter results suggest Off-the-shelf macrovessels can be produced by textile methodologies using ECM-derived threads.SVF can be used to create microvascular networks and to endothelize the macrovessels.The latter would be edited to allow endothelial cell sprouting and anastomosis with the microvasculature.The whole construct would then be subjected to dynamic conditioning before or after combination with an engineered tissue.Created with Biorender.com. the viability of this approach for the fabrication of off-the-shelf macrovessels, the long and complex fabrication process, as in other TE strategies, hinders commercial viability.A more recent and relevant development to this approach involves the use of ECM-derived threads to produce woven macrovessels, which are stronger and faster to fabricate in comparison with existing strategies. [15]Future preclinical and clinical experimentation may confirm the significant potential of this method for clinical application.
One of the main takeaways of the described TE strategies is that the production of tissues by self-assembly is still, in many ways, superior to other engineering approaches.[18] The SVF is a cellular fraction that can be isolated from fat and is composed of diverse cell populations, including endothelial and stromal cells, [19] that when seeded and left to grow, spontaneously self-assemble into a lumenized capillary bed-like network (Figure 2B).This process requires neither additional supportive cell populations, nor potentially harmful extrinsic growth factors.Not only does this sim-plify the process of microvasculature production (making clinical translation more likely), but it also allows for an autologous approach by simply harvesting fat tissue from the patient.For example, from the same fat tissue sample, stromal progenitors can be isolated and differentiated to obtain bone, while the rest of the fraction is used for the microvascular network that will feed the new bone.
Combining the different approaches described above could be a potential solution to obtain fully functional multiscale vascular networks (Figure 3).The process begins with off-the-shelf macrovessels produced from woven allogeneic ECM-derived materials that do not induce inflammation nor thrombosis after implantation, maintaining long-term functionality.Stromal vascular fraction cells are isolated from the patient and used to line the macrovessels and produce microvessel networks, in an extrinsic growth factor-free manner, by allowing the self-assembly of cells in an ECM-derived hydrogel.Anastomosis between the macroand microvessels is allowed by specifically designed features in the macrovessels.The key subsequent step would be to provide dynamic stimuli to the system (blood flow), ensuring the maturation and maintenance of the whole network in combination with the tissue or organ to be implanted in the patient.This would require the entire engineered vascular system to be housed in a bioreactor providing the stimulus.Such a setup has been recently proposed by Helms and Zippusch et al. [20] The technological foundations to realize this solution are therefore in place.But their embodiment into a protocol or methodology that can be financially sustainable and clinically viable may not be so easily achieved.The incorporation of the concept of multiscale vascular networks within the broader methodologies to engineer tissues will no doubt be challenging.Yet the promise is there.

Figure 1 .
Figure1.TE scaffold vascularization using an AV loop.A) An AV loop is established by anastomosing an easily accessible artery and vein.The loop is placed in an isolation chamber together with the scaffold.The chamber is closed and the whole construct implanted in the patient.After sufficient time elapses, the vascularized scaffold is retrieved from the patient together with the vascular axis, i.e., the segmented artery and vein.The latter can be anastomosed with local vessels at the defect site, where the scaffold will be implanted.B) Micro-computed tomography reconstruction of the microvessel network formed in a scaffold vascularized using the AV loop technique, in an animal model.(Adapted with permission[21] Copyright 2016, JoVE).Created with Biorender.com.

Figure 2 .
Figure 2. Microvasculature formation in TE scaffolds.A) TE scaffolds can be prevascularized by seeding endothelial cells and culturing them in the presence of extrinsic angiogenic/vasculogenic growth factors.This will cause endothelial cell self-assembly into capillary-like networks.If present, stromal cells can be recruited and act as perivascular cells, stabilizing the network.B) The SVF can be used for the same purpose of prevascularization without adding extrinsic growth factors, in a self-assembly process.Created with Biorender.com.

Figure 3 .
Figure3.Combination of existing cellular self-assembly strategies to obtain multiscale vascular constructs.Off-the-shelf macrovessels can be produced by textile methodologies using ECM-derived threads.SVF can be used to create microvascular networks and to endothelize the macrovessels.The latter would be edited to allow endothelial cell sprouting and anastomosis with the microvasculature.The whole construct would then be subjected to dynamic conditioning before or after combination with an engineered tissue.Created with Biorender.com.