I’m standing in the laboratory of Monica Laronda, on Northwestern’s Chicago campus when the assistant professor of pediatrics walks down the hallway, smiling and holding a jar that she brought from her office. Inside is a cow ovary, which she takes out and hands to me. It’s not a functioning organ, but one that has had the cells removed from it. What remains is the extracellular matrix, the part that provides structure.
Laronda is director of pediatric fertility and hormone preservation and restoration research at Ann and Robert H. Lurie Children’s Hospital of Chicago and assistant professor of pediatrics at Northwestern University Feinberg School of Medicine. She aims to create a bioprosthetic ovary that could be used as an alternative to hormone replacement therapy in children who have decreased hormone function and fertility, a common side effect for kids being treated for cancer. The research is on the forefront of two interdisciplinary fields: oncofertility and 3D biomaterial printing, both areas where Northwestern scientists have made trailblazing contributions.
The ovary Laronda handed me is white and fairly small. The texture seems familiar. “It feels like …”
“A packing peanut,” she says.
She’s deconstructing cow ovaries to learn how to build replacements that function normally. Laronda explains that the follicle is the part of the ovary that contains the oocyte, the cell that is capable of developing into an egg, as well as the cells that create hormones.
Laronda is trying to create a niche for ovarian follicles that mimics the one in a natural ovary. Egg cells have complex requirements. They must remain non-growing until the body is ready to recruit them. Then they need to transform into an egg, eventually expanding up to 600 times their size.
Laronda’s research could one day help girls who have had pediatric cancer and a decrease in fertility as a result of treatment. Chemotherapy and radiation therapies can damage the reserve of egg cells within a female’s body. Before starting her own lab, Laronda worked with Teresa Woodruff, now dean of The Graduate School, who pioneered the interdisciplinary field of oncofertility. Woodruff is an internationally recognized expert in ovarian biology. “She is the one who voiced the fact that some oncologists had observed that their patients were less likely to get pregnant, or else had some reduction in ovarian hormones,” Laronda says. “They discovered that treatment did actually affect patient fertility.”
In 1975, only 50 percent of children diagnosed with cancer before the age of 20 survived more than five years. Now, that number is up to 84 percent, according to National Cancer Institute statistics. That means that there is now a focus shift toward maintaining quality of life after treatment is completed.
While pediatric endocrinologist can provide synthetic hormones to pediatric cancer survivors who have lost the ability to go through puberty, lifelong, daily hormone pills (the same pills used as contraceptives) only restore the hormonal endocrine effects on bone or muscle. The reproductive tissue no longer functions and so fertility would be lost.
“If you imagine a person who is very, very young undergoing this treatment, then it requires decades of synthetic hormones to maintain normal endocrine health and function,” says Laronda, who is also a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University. “It would be better if we could actually restore that normal endocrine and fertility functions.”
Engineering an Ovary Replacement
In many instances, adult women with cancer can freeze their eggs or bank embryos. Children don’t have that option – they have yet to go through puberty and do not make eggs. However, doctors can freeze the tissue where the potential egg cells are. Humans have the highest amount of potential egg cells at birth, the number of which decreases until menopause.
Laronda says one consideration is that the greatest cancer survival rates are associated with certain blood cancers, like leukemia. In these cases, ovary tissue has been shown to contain some cancerous cells and could be dangerous to implant later. One way to get around the issue is to ensure the transplantation of only healthy, cancer-free ovarian follicles. Doing so would require a replacement for the tissue environment that holds and nurtures follicles, allowing them to grow.
To recreate this tissue, Woodruff and Laronda turned to 3D printing.
3D printing has existed for decades as a method of creating product prototypes. It wasn’t until recently, however, that 3D printing gained traction as a method for creating scaffolds — structures that cells can grow on — for tissue engineering.
Soon after Ramille Shah, materials science and engineering and surgery, bought her first 3D printer in 2010, she started an effort to create more biomaterial inks, materials that are meant to interact with cells and are able to be 3D printed. Biomaterial inks can contain particles that provide physical and functional cues, sending specific signals to cells, or even contain cells within the ink.
Woodruff was already trying to create scaffolds for an artificial ovary when she learned about 3D printing for manufacturing. “It really struck me as a next-generation idea, that we might be able to do something biological,” Woodruff says. “I was elated to begin this kind of work because to me it represented a whole new generation of biology that we couldn’t study otherwise.”
She heard about Shah’s efforts from a mutual collaborator and reached out to her. Woodruff, Laronda, Shah, and a graduate student Alexandra Rutz sat down to brainstorm a plan of attack.
Shah, who is a member of the Simpson Querrey Institute for BioNanotechnology, and Rutz worked on material development and settled on a gelatin-based ink. Shah’s team also printed a porous scaffold that the follicles could be seeded onto later. The follicle being printed in the ink can limit the growth of the follicle, the travel of nutrients to the cells, and ovulation.
Once the design for printing the scaffold was complete, the researchers tested it in a small-animal model.
Steps to Human Trials
In January 2016, Laronda and lab manager Kelly Even went downstairs to weigh their mice. They had removed the ovaries from each mouse in a surgical site just a millimeter in length. Next, they implanted a scaffold that had been seeded with ovarian follicles that contained a florescent green protein. The female mice with bioprosthetic ovaries were mated with male mice that had sired previous litters. Then the researchers waited.
About a month later, they found a pup mixed in with the expectant mothers. Laronda and Even immediately directed a blue light on the cage and the pup glowed lime green. The experiment was a success. Even made a birth announcement for the pup and sent it to everyone in the lab.
The researchers published their work in Nature Communications, and Laronda went from being a postdoc in Woodruff’s lab to earning her own faculty position, where she could begin to focus on continuing the research in larger animal models.
“It’s phenomenal to have exceptional students who train with you who then transition to a faculty position,” Woodruff says, “To have someone local that I can talk with and work with and see how she develops is an unparalleled treat.”
“We performed the procedure successfully in mice and that was a really great step, but mice are not humans. We’re still adjusting the 3D printing to eventually test this in humans,” Laronda says. “We have a 3D printer in a good manufacturing facility which allows us to produce clinical-grade products. The thought is that once this is ready, we could quickly move it into human trials.”
Laronda can visualize the process and is already doing parallel research on areas where she foresees problems might arise. “I’m trying to predict what our bioprosthetic ovary 2.0 will need to look like.”
The researchers are looking to engineer biomaterial ink for this next version, knowing that what worked for the simpler mouse follicles might not work in larger animals.
Luckily, 3D printing is an ideal tool to create and test environments and biological responses to them. And an artificial organ doesn’t necessarily require all of the same features present in the real organ, which is nearly impossible to replicate. Instead, the goal is to create the simplest artificial template possible along with providing the right signals that can allow cells to remodel the artificial environment into a more natural architecture that functions appropriately.
“My hope for the future is that we completely eliminate the field of oncofertility,” says Woodruff. “We do that by having more targeted chemotherapy or smart biologics that treat the cancer and don’t have off-target effects on ovarian follicles. In the meantime, we hope to protect and extend reproductive health through the work being done in Monica’s lab.”
Brittany Callan earned her master of science degree in journalism from Northwestern in June. She is currently pursuing a career in science writing.