A Sweet Solution for Regenerative Medicine

Using Sugar to Create 3D Vascular Networks

Lollipops that dissolve at an ideal pace. Intricate sugar lattices that garnish restaurant desserts. The candy layer that coats M&M'S and Skittles. Borrowing concepts from such unlikely realms as candy manufacturing and an open source hacker-inventor community, Christopher S. Chen, Professor of Bioengineering, leads a scientific collaboration that recently advanced the science of tissue engineering. In a lab often suffused with the scent of fresh cotton candy, his postdoctoral fellow Jordan Miller worked with collaborators at MIT to adapt confectionery-making techniques to create precision-designed vasculature using molds made of sugar. Their breakthrough keeps liver cells alive within engineered 3D tissues that can be created rapidly and inexpensively, and was published in July in Nature Materials.

Candy-makers & Hackers

"This innovation fills an important scientific need: we can now readily design and create 3D tissues to study human biology," says Chen, M.D., Ph.D., and Skirkanich Professor of Innovation. "This powerful model for vascular biology mimics the environment of human tissues and will help us understand how things get plumbed and vascularized, what controls how blood vessels grow or don't grow, and what happens to tissues when they get re-vascularized or have too few or too many blood vessels."

Adapting lost wax casting techniques used by sculptors and jewelers for thousands of years, the research team uses an open source printer to cast molds made of a laboratory-grade sugar formulation that dissolves in a gel pre-polymer moments after that gel solidifies. The result: a precise architecture of blood vessels between 150 to 750 microns in diameter that supply nutrients and remove waste for living cells in a gel. The vasculature forms its own capillary sprouts and so far has nurtured liver cells, fibroblasts and myocyte progenitors within 3D tissues.

Over the past decade, scientists have refined techniques for creating thin tissues such as engineered skin, cornea, bladder and trachea. But their efforts to create thicker tissues such as in the liver, heart, kidneys or muscles were stymied because cells in the core of thicker structures would starve without blood vessels that would feed them. Bio-printing layer-by-layer didn't work due to seams and structural weaknesses in the vasculature, and also because primary cells such as liver cells are too fragile to survive that process. Prior molding techniques also used organic solvents that killed living cells.

"This discovery is the next step in liver tissue engineering—how to go from thinner tissues fed by diffusion to a thicker tissue that will require networks of blood flow within it," says Sangeeta Bhatia, M.D., Ph.D., and Wilson Professor at the Massachusetts Institute of Technology, and Chen's collaborator. "All tissues thicker than a millimeter need vascular networks, the piping that brings nutrients close enough to the cells. Chris and his team came up with a way to make this piping. This is a powerful research tool for a broad spectrum of tissue types and diseases."

Odd Combination

Inspiration for this breakthrough came from two improbable topics: dead bodies and desserts. Shortly after coming to Penn, Miller saw the Franklin Institute's Body Worlds exhibit featuring plasticized casts of human organs and bodies. He realized this reverse-mold technique could work better than bio-printing to create engineered vasculature. Later, at a restaurant, Miller had an epiphany. A sugar lattice garnished his dessert. "It looked like vasculature. I immediately realized sugar would be the best material for the mold because it's nontoxic for cells," says Miller, who began creating sugar lattices at home, using cookbooks and a candy thermometer.

As he pondered ways to precision-print sugar, Miller drew on his interests outside the scientific community. He came across the RepRap printer, an inexpensive device often used by hobbyists to print figurines or replacement parts for appliances or toys. He began working with a group of inventors and artists at Hive 76, a Philadelphia-area hacker space, to learn how to build a RepRap. He tweaked the device, replacing a plastic frosting extruder with a glass syringe head wrapped with toaster wire that keeps the sugar solution molten until printed.

It took two years to optimize those sugars. First, they tested recipes from a 19th century German candy-making textbook that strengthened sugar with a starch additive. But that made the sugar too cloudy, disrupting photo-polymerization. Another additive, glycerol, weakened the sugar. Ultimately, a blend of sucrose and glucose plus dextran (a sugar polymer similar to starch used in blood perfusion in human patients to stabilize blood pressure) kept the sugars clear and strong.

Next, they needed to protect the printed sugar filaments so the mold would dissolve at the right moment. Studying lollipops, M&M'S and Skittles, they decided to coat the sugar template with a layer of corn-derived polymer that would allow the sugar to dissolve moments after the gel cross-links. "That thin coating gives us time and control over everything," says Miller. "That allows the sugar to flow out of the gel instead of through the gel, which protects living cells from sugar at a concentration that's toxic."

Future Challenges

The next goals involve designing thicker and more complex vascularized tissues. "Creating capillaries is not a problem," says Chen. "We've been able to print vessels that are the size of what would feed the stem of a maple leaf and all the little vessels inside that leaf. Now we need to be able to print vessels that would feed a branch with 100 leaves on it."

"Too many or not enough blood vessels are central to the ischemic diseases that are a major aspect of mortality and morbidity," says Chen. "The question of how a tissue gets the right amount of vascularity or not is critical, for instance, in cardiac ischemia, peripheral artery disease, chronic wound healing for individuals with diabetes, and revascularization of the brain after stroke."

Bhatia, whose postdoc Kelly Stevens worked closely with Miller, says, "Together, we have created two important building blocks: we have learned how to support cells in artificial environments and how to make tissues thicker. Until now the field of microcirculation has been largely observational. With these tools, we'll be able to recreate the architecture of vasculature in thicker tissues. This is an important step for regenerative medicine as we advance the translational science of creating replacement parts for aging, injured and diseased tissues."

View the article in Penn Engineering magazine "A Sweet Solution for Regenerative Medicine" by Jessica Stein Diamond.

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