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Article by Colin Birkenstock & Katherine Cunningham
From Stretching to Synthesis: How to Cue Macro-vessels to Sprout Capillaries
Source Publication:
The Effect of Mechanical Loading on Sprouting Angiogenesis from Engineered Macro-vessel Model, Small Methods, 2025, Lior Debbi et al., Shulamit Levenberg lab, https://onlinelibrary.wiley.com/doi/abs/10.1002/smtd.202500850
One of the biggest challenges in growing organs or large tissues in the lab is building a working blood vessel system, called vasculature. Cells can’t survive without oxygen and nutrients, which are delivered by blood vessels. If a piece of tissue gets too thick without enough vessels, the cells in the middle begin to die. Solving this problem is no simple task. Our bodies rely on an incredibly dense network of tiny blood vessels called capillaries, some so narrow that red blood cells must squeeze through them single file. In this study, researchers built a larger, engineered “macro” blood vessel and placed it inside a gel that partially mimics the structural features of a native tissue in the body. Then, they gently stretched this system lengthwise (longitudinally) and side-to-side (laterally) to test whether these physical forces could influence the process by which new blood vessels grow, called angiogenesis. Investigating how stretching impacts angiogenesis makes sense as many processes in the body involve stretching and relaxation, such as heartbeats, breathing, and muscle movement. Remarkably, the researchers found that simply changing the direction of stretch was enough to either stimulate or dampen new vessel growth.
What did the researchers do?
To test the effects of external physical stimulation on angiogenesis, the researchers created a new assay to measure and apply a stretching force to the engineered blood vessel. Specifically, they created a new device that contains an engineered blood vessel and a hydrogel meant to provide structure for any new vessels that sprout. Once the vessel model was complete, a machine stretched it at precise intervals. The vessel strain during longitudinal stretching was found to be high and uniform across the vessel, inhibiting angiogenesis. However, when the stretching was lateral, strain varied across the vessel wall, creating areas of the vessel where strain was moderate, stimulating angiogenesis. The difference in angiogenesis across the three groups, including a control group, is apparent in the image below.
To function properly, vessels must be perfusable and well-integrated. Perfusability is what allows for nutrient and oxygen transport. After showing the presence of new vessels in response to stretching, the researchers demonstrated that the vessels were, in fact, perfusable. To do this, the researchers injected fluid into the macro-vessel and showed that the fluid also filled the sprouting capillary-like vessels. If the cells of the new vessels are not well integrated, they leak. This would be a major problem in a clinical context. For example, if the blood vessels in an implanted lab-grown tissue were to leak, it could cause edema, poor perfusion, or more serious bleeding-related complications. Therefore, the researchers also confirmed integration by demonstrating that there was no visible leakage when injecting the fluid. They also showed that VE-cadherin, a protein that holds the cells together, was present throughout the new vessels.
Why is this important?
Currently, when growing tissues in the lab, they are submerged in media which contains nutrients and oxygen. This setup relies on passive diffusion, where the media is in direct, or nearly direct, contact with the cells, to support tissue growth. However, if the tissue grows too large, the core of the tissue dies, as the media can no longer reach and effectively diffuse through the core. This limitation prevents the research of larger tissues in the lab and their implantation in patients. The ability to grow blood vessels is critical for creating ways to reach those core cells and deliver nutrients to them. This paper demonstrates a new strategy by using external physical cues to direct angiogenesis in the macro vessel, unlike existing methods which generally exert force by pushing fluids through the engineered vessel or in between the cells that surround it. Understanding how stretch affects angiogenesis can also help researchers investigate developmental biology. For example, the heart stretches as it pumps, suggesting that fetal heart beating during gestation could provide signaling for further capillary growth.
How did they do this?
The researchers created the model by first 3D printing a negative mold out of a water-soluble material. They then poured polydimethylsiloxane (PDMS) into the negative mold and cured it. Once cured, everything was placed in water to dissolve the negative mold but leave the PDMS mold intact. The researchers then placed a rod through the center of the PDMS and added hydrogel. Once the hydrogel was set, the rod was removed and endothelial cells were added, along with pro-angiogenic factors to create the engineered macro-vessel. Finally, a fixation frame was added to the model to allow for stretching.
The researchers estimated the strain the macro vessel would bear through finite element analysis, a computer simulation that breaks the vessel into tiny pieces, stretches each piece, and then aggregates the degree of the strain felt by each piece. They then tested it in real life by placing fluorescent beads in the hydrogel before stretching the material and tracking the beads under strain. They found the real-life and theoretical results were closely aligned.
To image the constructed vessels, the researchers used both genetic engineering and immunofluorescence. The endothelial cells making up the vessels were genetically engineered ahead of time to express a protein that glows green to be seen under the microscope. However, to highlight the VE-cadherin, the researchers used immunofluorescence, which starts with obtaining an antibody to specifically attach to VE-cadherin. Then, another glowing antibody is specifically attached to the first VE-cadherin antibody. The glow of the second antibody can then be visualized as an indicator of the protein since they occupy the same position.
To see whether the vessels were perfusable, the researchers simply injected a fluorescent dye into the macro-vessel which they could then view through the microscope.
What comes next?
This paper shows a promising new mechanism by which researchers can influence angiogenesis. However, to reach the goal of growing more life-like vascularized tissues in the lab, researchers will have to move beyond creating isolated blood vessels within a simple hydrogel. Researchers are also constructing angiogenesis models where blood vessels are grown alongside the cells of a particular tissue to address this limitation. Further, this experiment only shows that these vessels can be properly perfused for a brief amount of time, whereas scientists will eventually have to demonstrate that the vessels will function for months to years if they want to create tissues that are implanted into humans. Additional research is also needed to identify which angiogenesis-promoting molecular pathways are triggered by the stretching. In the end, this paper provides a great proof of concept for a new tool researchers can use to create new blood vessels, but more scientific work will need to be done to create ever more realistic tissue constructs in vitro.