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.

The endocrine system dynamically controls tissue differentiation and homeostasis, but has not been studied using dynamic tissue culture paradigms. Here we show that a microfluidic system supports murine ovarian follicles to produce the human 28-day menstrual cycle hormone profile, which controls human female reproductive tract and peripheral tissue dynamics in single, dual and multiple unit microfluidic platforms (Solo-MFP, Duet-MFP and Quintet-MPF, respectively). These systems simulate the in vivo female reproductive tract and the endocrine loops between organ modules for the ovary, fallopian tube, uterus, cervix and liver, with a sustained circulating flow between all tissues. The reproductive tract tissues and peripheral organs integrated into a microfluidic platform, termed EVATAR, represents a powerful new in vitro tool that allows organ–organ integration of hormonal signalling as a phenocopy of menstrual cycle and pregnancy-like endocrine loops and has great potential to be used in drug discovery and toxicology studies.

Clinical interventions to preserve fertility and restore hormone levels in female patients with therapy-induced ovarian failure are insufficient, particularly for pediatric cancer patients. Laproscopic isolation of cortical ovarian tissue followed by cryopreservation with subsequent autotransplantation has temporarily restored fertility in at least 27 women who survived cancer, and aided in pubertal transition for one pediatric patient. However, reintroducing cancer cells through ovarian transplantation has been a major concern. Decellularization is a process of removing cellular material, while maintaining the organ skeleton of extracellular matrices (ECM). The ECM that remains could be stripped of cancer cells and reseeded with healthy ovarian cells. We tested whether a decellularized ovarian scaffold could be created, recellularized and transplanted to initiate puberty in mice. Bovine and human ovaries were decellularized, and the ovarian skeleton microstructures were characterized. Primary ovarian cells seeded onto decellularized scaffolds produced estradiol in vitro. Moreover, the recellularized grafts initiated puberty in mice that had been ovariectomized, providing data that could be used to drive future human transplants and have broader implications on the bioengineering of other organs with endocrine function.

No abstract

Women exposed to diethylstilbestrol (DES) in utero frequently develop vaginal adenosis, from which clear cell adenocarcinoma can arise. Despite decades of extensive investigation, the molecular pathogenesis of DES-associated vaginal adenosis remains elusive. Here we report that DES induces vaginal adenosis by inhibiting the BMP4/Activin A-regulated vaginal cell fate decision through a downregulation of RUNX1. BMP4 and Activin A produced by vaginal mesenchyme synergistically activated the expression of ΔNp63, thus deciding vaginal epithelial cell fate in the Müllerian duct epithelial cells (MDECs) via direct binding of SMADs on the highly conserved 5′sequence of ΔNp63. Therefore, mice in which Smad4 was deleted in MDECs failed to express ΔNp63 in vaginal epithelium and developed adenosis. This SMAD-dependent ΔNp63 activation required RUNX1, a binding partner of SMADs. Conditional deletion of Runx1 in the MDECs induced adenosis in the cranial portion of vagina, which mimicked the effect of developmental DES-exposure. Furthermore, neonatal DES exposure downregulated RUNX1 in the fornix of the vagina, where DES-associated adenosis is frequently found. This observation strongly suggests that the downregulation of RUNX1 is the cause of vaginal adenosis. However, once cell fate was determined, the BMP/Activin-SMAD/RUNX1 signaling pathway became dispensable for the maintenance of ΔNp63 expression in vaginal epithelium. Instead, the activity of the ΔNp63 locus in vaginal epithelium was maintained by a ΔNp63-dependent mechanism. This is the first demonstration of a molecular mechanism through which developmental chemical exposure causes precancerous lesions by altering cell fate.