Department of Obstetrics and Gynecology, The Second Hospital of Hebei Medical University, 215 Heping West Road, Shijiazhuang, 050000, Hebei, China.
Stem Cell Res Ther. 2022 Jul 26;13(1):347. doi: 10.1186/s13287-022-03035-3.
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, various issues have greatly restricted its clinical application. Transplantation of adipose-derived stem cells (ADSCs) has promising roles in restoring ovarian function of rats with POI, but insufficient retention has greatly hampered their efficiency. Here, we designed a 3D-bioprinted engineering ovary composed of drug-free IVA and 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.
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 calculate follicle number and assess proliferation and apoptosis of granulosa cells (GCs). 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.
CM-Dil-labeled ADSCs were distributed in the interstitium of ovaries and had significantly higher retention in the 3D-bioprinted engineering ovary group. Several regions of the co-staining for CM-Dil and CD31 were in the area of vascular endothelial cells. Meanwhile, the follicle counts, GCs proliferation, 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.
Our results suggested that 3D-bioprinted engineering ovary had great potential for restoring impaired ovarian function of rats with POI, which could compensate for the disadvantages of drug-free IVA to some extent.
新兴的无药物体外激活（IVA）技术使卵巢早衰（POI）患者能够恢复卵巢功能并孕育自己的遗传后代。然而，各种问题极大地限制了其临床应用。脂肪来源干细胞（ADSCs）的移植在恢复 POI 大鼠的卵巢功能方面具有广阔的前景，但由于保留率不足，极大地阻碍了其效率。在这里，我们设计了一种由无药物 IVA 和 ADSCs 组成的 3D 生物打印工程卵巢，这可能会延长 ADSCs 的保留时间，并构建早期血管微环境，从而在一定程度上弥补无药物 IVA 的不足，改善 POI 大鼠受损的卵巢功能。
环磷酰胺腹腔注射后，将 POI 模型大鼠随机分为 5 组：（1）POI 组；（2）卵巢碎片组；（3）3D 支架联合卵巢碎片组；（4）卵巢碎片联合 ADSCs 组；（5）3D 支架与 ADSCs 联合卵巢碎片作为 3D 生物打印工程卵巢组。正常大鼠被鉴定为对照组。观察 CM-Dil 标记的 ADSCs 的定位和与 CD31 的共定位，以检查分化的分布和潜在机制。进行组织形态学和免疫组织化学分析，计算卵泡数，并评估颗粒细胞（GCs）的增殖和凋亡。免疫荧光染色用于评估血管生成。测量激素水平以评估内分泌轴的恢复情况。进行 Western blot 分析和 RT-PCR 以探讨潜在机制。
CM-Dil 标记的 ADSCs 分布在卵巢间质中，在 3D 生物打印工程卵巢组中保留率显著更高。CM-Dil 和 CD31 的共染色区域有几个位于血管内皮细胞区域。同时，3D 生物打印工程卵巢组的卵泡计数、GCs 增殖、新生血管形成和激素水平均显著改善，优于其他组。此外，通过调节 PI3K/AKT 通路，改善了卵巢功能并促进了血管生成。
我们的结果表明，3D 生物打印工程卵巢具有恢复 POI 大鼠受损卵巢功能的巨大潜力，在一定程度上可以弥补无药物 IVA 的不足。
