Endocrine Abstracts

Patients with gonadotoxicity due to disease treatment, or who face primary gonadal insufficiency as a result of genetic causes, have limited options for long-term endocrine and fertility support. Current fertility options, such as egg or embryo banking, exclude women with hormone-responsive cancers and pre-pubertal children. This highlights the urgency of addressing this currently unmet need with innovative engineering solutions. Our objective is to create an ovary bioprosthesis that can restore function. We have implanted a bioinspired, scalable 3D-printed scaffold in an ovariectomized mouse that models human disease.

To better understand the natural ovarian environment, we decellularized, or removed all of the cells, from bovine ovaries or human ovary slices and examined the remaining organ skeleton. Through scanning electron microscopy, we identified defined regions where quiescent primordial follicles reside in the cortex and where large growing follicles reside in the medulla. The extracellular matrix of the cortex contained stiffer laminin fibers and the medulla contained more flexible fibronectin fibers, as both regions contained mostly collagen. This natural ovary skeleton composition, architecture and mechanical properties inspired our design for an ovarian implant. We developed a new method for printing gelatin, a collagen-derived biomaterial, into scaffolds for the purpose of creating a self-supporting ovary bioprosthesis skeleton. Using this method, we created scaffolds with bioinspired pore structures and a stiffness after cross-linking (compressive elastic modulus ~30 kPa) similar to that of the ovary (elastic modulus 5–20 kPa). We tested several scaffold designs to optimize ovarian follicle survival and function. Our criteria were to maintain high mechanical properties of the implant for desirable surgical handling while creating space from scaffold porosity to enable follicle expansion, ovulation and release of an egg, vascularization and passage of follicle paracrine signals. Our intersecting 30° and 60° advancing angle designs, that allow for multiple strut contacts, supported folliculogenesis at a significantly higher rate than scaffolds created with a 90° advancing angle containing through-pores (30°, 78.6% ±3.6 survival; 60°, 75.9% ±4.0 survival; 90°, 48.5% ±8.3, P=0.01). The 30° and 60° scaffolds supported two or more follicle contacts at a higher rate than the 90° scaffolds. These additional contacts were essential for sustained follicle health over the 8-day culture (74.3% ±6.5 survival with two or more strut contacts versus 33.2% ±11.3 with one strut contact, P=0.01). Three-dimensional confocal analysis confirmed that the follicles were supported within the manufactured follicle niche structures of the 30° and 60° scaffolds and interacted with the struts. Follicular somatic cells robustly expressed vinculin, a focal adhesion protein, along the pore struts and spread those struts. The length of spreading was significantly less with more contacts, which provided support for the follicle’s spheroid structure as the basement membrane was maintained (average contact length when contacting one strut 198.7 μm ±42.8; two struts 104.8 μm ±14.0; three struts 59.5 μm ±5.4; P<0.005). Follicles within the intersecting scaffold designs stained positive for 3β hydroxysteroid dehydrogenase, released estradiol over an 8-day in vitro culture, and upon exposure to human chorionic gonadotropin, ovulated and released MII eggs through the open porosity without mechanical or enzymatic manipulation of the follicular cells or scaffold. The implantable ovary bioprosthesis was constructed by down-selecting to a 60° 3D printed scaffold and seeded with green fluorescent protein (GFP)-positive quiescent primordial and growing primary and secondary murine follicles. The bioprostheses were implanted into the native organ site in ovariectomized GFP-negative mice. These follicle-seeded bioactive scaffolds supported vascular infiltration without the addition of angiogenic factors, restored the estrous cycle (as observed through vaginal cytology) and produced live offspring. These healthy offspring were supported by the lactating implant recipient mother until weaning.

These data underscore the importance of the bioactive scaffold architecture in supporting folliculogenesis and demonstrate a functional ovary bioprosthesis. This research expands beyond the current state of tissue engineering toward developing a common scheme for complex soft tissue replacement through bioinspired-manufacturing of bioactive scaffolds.