Emerging additive manufacturing techniques enable investigation of the effects of pore geometry on cell behavior and function. Here, we 3D print microporous hydrogel scaffolds to test how varying pore geometry, accomplished by manipulating the advancing angle between printed layers, affects the survival of ovarian follicles. 30° and 60° scaffolds provide corners that surround follicles on multiple sides while 90° scaffolds have an open porosity that limits follicle–scaffold interaction. As the amount of scaffold interaction increases, follicle spreading is limited and survival increases. Follicle-seeded scaffolds become highly vascularized and ovarian function is fully restored when implanted in surgically sterilized mice. Moreover, pups are born through natural mating and thrive through maternal lactation. These findings present an in vivo functional ovarian implant designed with 3D printing, and indicate that scaffold pore architecture is a critical variable in additively manufactured scaffold design for functional tissue engineering.
Introduction
Patients undergoing treatment regimens that eradicate their disease, such as cancer, may be left with diminished ovary function, including the inability to undergo puberty, early menopause and infertility1,2,3. Current assisted reproductive technologies and hormone restoration procedures, including in vitro fertilization and ovarian transplants, do not provide long-term solutions and leave paediatric patients with metastatic disease without options4,5,6,7,8. Therefore, the oncofertility field is tasked to develop a whole organ replacement that restores long-term hormone function and fertility for all patients. In past work, we and others have sought to create an engineered ovary with biomaterials and isolated follicles9,10,11,12,13,14. Ovarian follicles are spherical, multicellular aggregates that include a centralized oocyte (female gamete) and surrounding support cells, granulosa and theca, that produce hormones in response to stimulation from the pituitary. The spheroid shape of a follicle is critical to its survival in that the support cells must maintain contact with the oocyte until it has matured and is ready for ovulation. Consequently, a three-dimensional (3D) material environment is critical to maintaining these cell–cell interactions and follicle shape15,16,17.
Thus far, there have been several reports of live births from biomaterial implants in mice, and all have used isolated follicles or whole ovarian tissue encapsulated in a plasma clot or similar fibrin hydrogel bead containing growth factor components or purified vascular endothelial growth factor18,19,20,21. These results are very encouraging and have validated both the model procedure and the need for graft vascularization for complete restorative organ function of isolated follicles in a biomaterial18,19. However, hydrogel encapsulation of follicles poses several challenges, especially with respect to the size of anticipated transplants. Specifically, when translating this work to a large animal or human, the implant must house a significantly larger population of follicles and therefore must be considerably larger than those used in mice. At these scales, diffusion limits may become a concern. Future strategies must permit channels within the hydrogels (to facilitate host vasculature infiltration) or including pre-embedded vasculature to sustain follicle viability and circulate follicular hormones. Moreover, the ovary is a heterogeneous organ that compartmentalizes different follicle pools (quiescent and growing) into the cortex and medulla regions that have varying stiffness5,22. It is believed that this compartmentalization will be critical to providing long-term (multiple decades) function with an implant23,24. Therefore, a biomaterial strategy that can produce a mimetic construct of spatially varying material properties may be required for optimal implant function and longevity.
3D printing can be used to address all of these future implant requirements25,26,27,28,29 for creating a human bioprosthetic ovary, a bioengineered functional tissue replacement. As the first steps towards this goal, here, we investigated tortuous 3D printed porous hydrogel scaffolds with murine follicles seeded throughout the full depth of the scaffold layers to create a murine bioprosthetic ovary. We hypothesized that these scaffolds would provide 3D support to follicles; yet, allow for immediate vascularization and ovulation through the designed porosity. 3D printing also provides unprecedented control over scaffold architectural features. It is unclear, however, how manipulation of pore design may influence cell behavior. Only a few studies to date have investigated the influence of pore architecture on cell–matrix interactions and function30,31,32,33. Notably, in another study—and the only pore geometry investigation in soft tissue—Freed and co-workers33 discovered that a scaffold with a honey-comb structure provided anisotropic mechanical properties that led to improved directional electrical excitation and cell alignment in a cardiac cells in vitro. Here, we sought to optimize pore geometry to support murine follicle survival and function in our bioprosthetic ovary. Our promising results validate exploration in future preclinical large animal models.
