How did the team come together to start working on this project?
MML: Our motivation for creating an engineered ovary starts with the need to restore hormone function and the option of fertility in young girls or women who have insufficient ovarian function, and this can most notably, occur in children who undergo chemotherapy or radiation to eradicate their cancer. Girls who have not undergone puberty, are not able to stimulate and preserve eggs prior to treatment, like some adult women may choose to do. There is also a chance that some girls do not undergo normal puberty, due to the effects of their treatment on their ovaries. Ovarian tissue slices that contain the ovarian reserve cells have been transplanted back into patients by groups such as Donnez & Dolmans in Brussels, Andersen in Copenhagen and Silber in St. Louis. The risk with using this tissue is that the tissue can contain cancer cells, especially from patients with metastatic disease. We want to create a method for restoring their ovarian function, in a way that could be safe and include their own cells. We sought to create a scaffold that would provide the necessary support of ovarian cell aggregates or follicles and be handled during surgery. Teresa Woodruff, my mentor and a senior author on this work, began the Oncofertility Consortium. Her work has brought a variety of scientists, clinicians, ethicists, engineers and patients together to learn more about these issues of treatment-causing infertility. To address the goal of developing a safe ovarian transplant we sought an expert in materials science and 3D printing to helps us address these fundamental tissue-engineering feats.
RNS: Over the past several years, my lab has been developing new biomaterials that are compatible with room temperature extrusion-based 3D printing to create optimal biomimetic microenvironments for regenerating different tissues and organ structures. Teresa Woodruff, whose lab is right next door to mine, had found out about our work in this area and was very interested to see if we can use our 3D printable biomaterials and constructs to create an artificial ovary microenvironment or “niche” that can support follicle survival and control function (i.e. vary the niche to be able to influence follicle maturation) for the reasons MML described above. We met for our first brainstorming meeting about 3.5 yrs ago and the rest is history!
How did you start on this line of work, and what inspired the research on 3D printed ovaries?
MML: Our motivation for creating an artificial ovary starts with the need to restore hormone function and the option of fertility in young girls or women who have insufficient ovarian function. For example, this can occur in children who undergo chemotherapy or radiation treatments to irradiate their cancer. Teresa Woodruff, my mentor and a senior author on this work, began the Oncofertility Consortium to bring awareness to and address these issues of treatment-causing infertility. Girls who have not undergone puberty, are not able to stimulate and reserve eggs prior to treatment, like some adult women may choose to do. There is also a chance that some girls do not undergo normal puberty, due to the effects of their treatment on their ovaries. Ovarian tissue slices that contain the bank of immature cells have been transplanted back into patients by groups such as Donnez & Dolmans in Brussels, Andersen in Copenhagen and Silber in St. Louis. The risk with using this tissue is that we and others have shown that the tissue can contain cancer cells, especially from patients with metastatic disease. We want to create a method for restoring their ovarian function, in a way that could be safe and standardized with their own cells. We sought to create a scaffold that would provide the necessary support of ovarian cell aggregates and be handled during surgery.
Why was this area of research so important for you?
MML: I have found reproductive biology fascinating throughout my career, and I especially find the way that cells behave in different environments interesting. We know that the sex hormone producing cells of the ovary support the oocyte, or potential egg cell, and we needed to find a way to enable the ovarian follicles to do what they do – which is differentiate, mature and grow until the oocyte is ready to break away and ovulate as an egg that can be fertilized. Not only is this fascinating to me on a scientific level, but it is important to recognize the importance of the ovary as an organ. In some patients, natural or induced ovarian insufficiencies result in the inability to undergo puberty or cause menopause at a young age. Because sex hormones play a critical role in brain, muscle, bone, cardiovascular health, especially during puberty, I am most excited about learning more about these effects on our patients and the possibility of restoring these ovarian hormones for whole-body health.
RNS: One of my primary motivations for being involved in biomedical research is to be able to engineer biomaterials and implants that can enhance patient quality of life. Engineering an artificial ovary bioprosthesis with the potential to help female cancer survivors live healthy and normal lives by preserving their hormone production and fertility would be a significant accomplishment. Furthermore, in this work we are identifying specific parameters that significantly affect the survival and behavior of follicles when cultured within biomaterial scaffolds, such as the pore architecture. One of the major reasons why I purchased the 3D printer in my lab is to be able to study the role of pore architecture on cell survival and behavior – so these findings are very exciting since they validate that pore architecture is important.
Why is the gelatin you are using so crucial to the printing process?
RNS: To be able to engineer an artificial environment that supports the survival and function of cells or cell aggregates, the material needs to be non-toxic to cells, promote cell adhesion and interaction, be tunable with regard to material properties, and be able to be remodeled by cells so they can create their own natural matrix. Furthermore, to be clinically translated, the material needs to be scalable and low cost. Gelatin is a natural material derived from collagen that has all these properties and is already being used in clinically approved devices. Furthermore, we have been able to use gelatin as a 3D printable hydrogel ink that results in structures with well-defined pore architectures by simply controlling the printing temperature – this has enabled our experiments to study how pore architecture can affect the biological response.
You’re using a 3D printer to create scaffolding made out of biological material to support hormone producing cells and immature egg cells, called oocytes. What kind of 3D printer do you use, and how did you come up with the material that it prints?
RNS and ALR: We use an extrusion-based 3D printer, the Bioplotter, manufactured by Envisiontec. We chose gelatin as our material for this work as well as other tissues we are exploring in the Shah Lab because it is derived from collagen, which is the most abundant protein in tissues and organs. Collagen is responsible for giving tissues and organs structure, and therefore, it makes sense to use such a material as a scaffold. Cells can respond to gelatin in that they can adhere and remodel the material, allowing cells to replace the scaffold with their own synthesized tissue. Furthermore, gelatin is relatively cheap and already has several FDA-approved uses, which can facilitate the translation of our 3D printable gelatin devices for clinical use.
Once the scaffolding is implanted in the body, does normal ovarian function return quickly?
MML: How quickly normal ovarian function resumes depends a lot on the state of the patient or patient model, and the type of follicles that the ovarian bioprosthesis contains. In some cases, where the ovarian tissue is transplanted, the patient is primed with hormones to trigger normal cyclicity and this could be done with our transplant as well.
What are some of the biggest challenges of printing body parts?
RNS: Specific tissues and organs all are different with regard to mechanical properties, biological properties, composition, and function, and a lot is still not known with regard to what material and structural factors can lead to the optimal environment for regenerating specific complex tissue or organ structures. Furthermore, cell source is a big challenge especially for more complex organ structures where multiple cell types are needed and in large quantities to be able to regenerate relevant-sized human tissues.
What are the steps that need to take place to take this from being viable in mice to viable in humans?
MML: This was a great accomplishment in mice, but we are now expanding this research into pigs as part of the Fertility and Hormone Preservation and Restoration (FHPR) Program at the Stanley Manne Research Institute at Lurie Children’s Hospital. Piglets have commonly been used as a model for pediatric surgery and transplants due to the similarities in anatomy, physiology and immunology between pigs and humans. The pig female reproductive tract is more similar to human than that of a mouse; however, there are still major differences. In addition to this, my lab is working with human induced pluripotent stem cells to develop patient-specific hormone producing cells of the ovary. We are also working alongside the physicians in our FHPR program to develop best practices for monitoring ovarian function in our pediatric patients and preserving and restoring ovarian function in a safe way.
RNS: To get to human use, we also need to produce and 3D print the bioprosthesis using clinical grade materials and in a regulated manufacturing environment (i.e. one that is qualified for Good Manufacturing Practices (GMP)). We are currently pursuing an effort called, “3D Biofabrication for Restorative Integrative Devices in a GMP Environment” or “3D BRIDGE” at NU in order to create 3D printed devices for different tissue targets including the ovary bioprosthesis.
Being able to print and embed ovaries opens a myriad of possibilities for women’s health, from restoring fertility in women cancer survivors to providing a solution for trans women. Who do you believe this innovation will have the most impact on, and what opportunity you most excited about?
MML: I think that a successful ovarian bioprosthesis would benefit a wide array of patients, including those with disorders of sex development and others in the sex and gender minority groups. I think it would specifically benefit the significant number of pediatric cancer patients, as approximately 85% of them will survive their cancer. There is an increased risk for these patients to display hormone insufficiencies and difficulties getting pregnant. In some cases the hormone insufficiencies result in the inability to undergo puberty or cause menopause at a young age. Because sex hormones play a critical role in brain, muscle, bone, cardiovascular health, especially during puberty, I am most excited about learning more about these effects on our patients and the possibility of restoring these ovarian hormones for whole-body health.
Are there other topics in women’s health that you believe could be impacted by new tech, such as 3D printing?
MML: There are many women who suffer from uterine, fallopian tube and cervical diseases that could benefit from a restorative implant. But women are also more affected by kidney disease, for example, and could benefit from a kidney transplant.
RNS: The Shah TEAM lab is currently developing 3D printable structures for a variety of other organ and tissue targets such as liver, kidney, cardiac, and breast tissue, which can be used for disease modeling and toxicity screening (when made on smaller scales), and hopefully one day as future artificial functional implants (either partial or whole organ structures).
Is there anything else you would like to add?
MML: I think that a successful ovarian bioprosthesis would benefit a wide array of patients, including those with disorders of sex development and others in sex and gender minority groups. Research into women’s health has been under-represented and now, as of January 2016, consideration of sex as a biological variable is required to be reported in NIH-funded research. I am honored to share that our institution - Northwestern University, Teresa Woodruff and the Women’s Health Research Institute have had a hand in creating this change – and because of this increased inclusiveness in research reporting, I think we will begin to learn more and more about sex differences in disease. This will play a role in how we protect and restore ovarian (and testicular) function in our patients.
RNS: This work is the first demonstration of a 3D printed artificial environment that has led to the functional recovery of an organ structure. There are many more exciting things to come as we develop new biomaterials and strategies using 3D printing to further refine the ovary bioprosthesis as well as other structures for different tissue and organ targets.