Drug-free in vitro activation combined 3D-bioprinted adipose-derived stem cells restore ovarian function of premature ovarian insufficiency

Abstract

Background Emerging drug-free in vitro activation (IVA) technique enables patients with premature ovarian insufficiency (POI) to restore ovarian function and conceive their own genetic offspring. However, many issues have greatly restricted its clinical application. Mesenchymal stem cells (MSCs) could significantly stimulate neovascularization and increase blood perfusion to reduce the loss of primordial follicles, and effectively improve the quality and number of oocytes in aged mice. Here, we designed a 3D-bioprinted engineering ovary composed of drug-free IVA and adipose-derived stem cells (ADSCs), which may prolong the retention of ADSCs and construct an early vascular microenvironment, thus compensating for the disadvantages of drug-free IVA to some extent and ameliorating impaired ovarian function in the POI rats. Methods After intraperitoneal injection of cyclophosphamide, the POI model rats were randomized into 5 groups: (1)POI group; (2)ovarian fragments group; (3)3D scaffold combined with ovarian fragments group; (4)ovarian fragments combined with ADSCs group; (5)3D scaffold with ADSCs combined with ovarian fragments as 3D-bioprinted engineering ovary group. Normal rats were identified as the control group. The localization of CM-Dil labeled ADSCs and co-localization with CD31 were observed to examine the distribution and underlying mechanism of differentiation. Histomorphological and immunohistochemical analyses were performed to enumerate follicle number and assess granulosa cells (GCs) proliferation and apoptosis. Immunofluorescence staining was used to evaluate angiogenesis. Hormone levels were measured to evaluate the restoration of endocrine axis. Western blot analysis and RT-PCR were conducted to explore the potential mechanism. Results CM-Dil-labeled ADSCs were distributed in the interstitium of ovaries and had significantly higher retention in the 3D-bioprinted engineering ovary group. Some regions of the CM-Dil (+) staining co-expressed with CD31 staining were in the area of vascular endothelial cells. Meanwhile, the follicle counts, GCs proliferation and apoptosis, neoangiogenesis and hormone levels were significantly improved in the 3D-bioprinted engineering ovary group, as compared with other groups. Furthermore, the ovarian function was ameliorated and angiogenesis was promoted through regulating the PI3K/AKT pathway. Conclusion Our results suggested that 3D-bioprinted engineering ovary had great potential for restoring impaired ovarian function of POI, which could compensate for the disadvantages of drug-free IVA to some extent.