Helping tissue grafts build a blood supply: Less is more

blood vessels in vivo

For a tissue graft to survive in the body — whether it’s a surgical graft or bioengineered tissue — it needs to be nourished by blood vessels, and these vessels must connect with the recipient’s circulation. While scientists know how to generate blood vessels for engineered tissue, efforts to get them to connect with the recipient’s vessels have mostly failed.

“Surgeons will tell you that when putting tissue in a new location in the body, the small blood vessels don’t connect at the new site,” says Juan Melero-Martin, PhD, a researcher in Cardiac Surgery in Boston Children’s Hospital. “If you want to engineer a tissue replacement, you’d better understand how the vessels get connected, because if the vessels go, the graft goes.”

Melero-Martin and colleagues have uncovered several strategies to help these connections form, as they describe online today in Nature Biomedical Engineering. The strategies could help improve the success of such procedures as heart patching, bone grafting, fat transplants and islet transplantation.

Less is more

Tissue engineers have a choice: They can faithfully build a 3D replica of the tissue in question, complete with blood vessels. Or they can provide something less fully baked: a mix of cells that includes the cells that make up blood vessels, leaving them to form vessels on their own once implanted.

Melero-Martin, first author Ruei-Zeng Lin, PhD, and colleagues compared the two approaches, putting human tissue grafts into mice. When they pre-assembled the tissue, incorporating lab-grown vessels, the grafts were actually less able to connect with the animals’ own vessels. When they implanted tissue with the raw cells, which had not yet matured into blood vessels, the grafts were much better perfused.

“We then tried to understand why un-connected cells do better,” says Melero-Martin.

Neutrophils needed

Through further experiments they learned the following:

  • Once implanted, the more “primitive” unassembled grafts engaged with cells from the animals’ innate immune system — specifically, non-inflammatory neutrophils called N2s. When mice were depleted of N2s, vascular connections no longer formed. The pre-assembled grafts didn’t engage with neutrophils.
  • The unassembled grafts secreted factors that stimulate vessels to connect. When the team injected the fluid surrounding these grafts into the mice, it drew neutrophils to the injection site. Fluid from the pre-assembled grafts produced no such response, but when fluid from the unassembled grafts was added to the pre-assembled tissue, vessels successfully hooked up with the mouse vessels.
  • The unassembled grafts had high levels of three cytokines (chemical signals): IL-6, CXCL1 and CXCL8. As the grafts were allowed to mature, these cytokines disappeared.
  • The unassembled grafts had less Notch signaling, which is known to increase as blood vessels mature and suppress vessel growth. By adding Notch-inhibiting drugs to pre-assembled grafts, the researchers got them to connect with the mouse vessels.
These grafts are both made up of unassembled human vascular cells, suspended in a hydrogel and implanted in mice just under the skin. Before receiving the graft, the mouse at left underwent irradiation, which depeleted it of circulating neutrophils. At day 7, its graft shows no perfusion of blood vessels. The graft at right, in a mouse that still had neutrophils, shows robust perfusion.

Improving graft blood perfusion

These insights could make an immediate impact in surgical grafting, or to improve the engraftment of bioengineered tissues, says Melero-Martin. “The good news is you can activate these grafts by adding factors that stimulate host factors, or by inhibiting Notch.”

Lin and Melero-Martin

Notch inhibitors are already available and in clinical trials for other indications, he adds.

But when a highly constructed tissue isn’t necessary, he still favors implanting cells in their “free” state. “We think that when grafts are too pre-assembled, they are not triggering a repair response; they’re not calling for help,” Melero-Martin says. “And we think non-inflammatory neutrophils are at the core of that repair response.”

Sitaram Emani, MD, a cardiac surgeon at Boston Children’s who was a co-author on the paper, sees implications for his practice.

“We are constantly trying to reduce the rate of reoperation in children who need tissue patches for reconstruction of heart vessels and valves,” says Emani. “This technology is a major leap forward in our understanding and design of tissue-engineered patches.”

In this “Behind the Paper” blog post on Nature‘s site, Lin describes how the discoveries grew out of what was initially a failed hypothesis. The study was supported by the National Institutes of Health (grants R00EB009096, R01AR069038, R01HL128452 and R21AI123883).