Researchers cannot yet produce large amounts of tissue using tissue engineering approaches such as bioprinting, as there is still no good solution for the creation of a suitable blood vessel network to support sizable tissue sections. However, that hasn't stopped the research community from forging ahead to develop the necessary recipes to produce functional tissue of various types, just in very small amounts. In many cases this artificial tissue isn't exactly the same in structure as the tissue it replaces, but it is nonetheless still capable of carrying out the desired functions. Some organs or crucial parts of organs are small enough to be produced in entirety, however, and hence researchers are now able to carry out demonstrations such the one here, in which artificial mouse ovaries are created, transplanted, and shown to be fully functional. The engineered ovaries produce the desired hormones and are capable of supporting the full process of mammalian reproduction. It is a good example of the quality of tissue being produced these days; once the blood vessel hurdle is overcome, the generation of entire organs will follow shortly thereafter.
Patients undergoing treatment regimens that eradicate their disease, such as cancer, may be left with diminished ovary function. 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 follicles. 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 shape.
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 factor. 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 biomaterial. 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 stiffness. It is believed that this compartmentalization will be critical to providing long-term (multiple decades) function with an implant. 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 requirements for creating a human bioprosthetic ovary, a bioengineered functional tissue replacement. As the first steps towards this goal, here, we investigated porous hydrogel scaffolds with murine follicles seeded throughout the full depth of the scaffold layers to create a murine bioprosthetic ovary. Microporous architectures were achieved through 3D printing partially crosslinked, thermally regulated gelatin. We found that specific scaffold architectures created a 3D feel by providing appropriate depth and multiple contact sites for the ovarian follicle, which resulted in optimal murine follicle survival and differentiation in vitro. The open micropores within the hydrogel scaffold provided sufficient space and nutrient diffusion for follicle survival and maturation in vitro and in vivo, as well as space for vasculature to infiltrate when implanted in vivo without the need for significant scaffold degradation as is required when using hydrogel encapsulation.
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.