Purpose To develop a new protocol for whole-ovary decellularization for the production of a 3D bioscaffold suitable for in vitro/ ex vivo studies and for the reconstruction of a bioengineered ovary. Methods Porcine ovaries were subjected to the decellularization process (DECELL; n = 20) that involved a freeze-thaw cycle, followed by sequential incubations in 0.5% SDS for 3 h, 1% Triton X-100 for 9 h, and 2% deoxycholate for 12 h. Untreated ovaries were used as a control (CTR; n = 6). Both groups were analyzed to evaluate cell and DNA removal as well as ECM preservation. DECELL bioscaffolds were assessed for cytotoxicity and cell homing ability. Results DECELL ovaries maintained shape and homogeneity without any deformation, while their color turned from red to white. Histological staining and DNA quantification confirmed a decrease of 98.11% in DNA content, compared with the native tissue (CTR). Histochemical assessments demonstrated the preservation of intact ECM microarchitecture after the decellularization process. This was also confirmed by quantitative analysis of collagen, elastin, and GAG contents. DECELL bioscaffold showed no cytotoxic effects in co-culture and, when re-seeded with homologous fibroblasts, encouraged a rapid cell adhesion and migration, with repopulating cells increasing in number and aggregating in cluster-like structures, consistent with its ability to sustain cell adherence, proliferation, and differentiation. Conclusion The protocol described allows for the generation of a 3D bioscaffold that may constitute a suitable model for ex vivo culture of ovarian cells and follicles, as well as a promising tool for the reconstruction of a bioengineered ovary.

Ovarian failure is the most common cause of infertility and affects about 1% of young women. One innovative strategy to restore ovarian function may be represented by the development of a bioprosthetic ovary, obtained through the combination of tissue engineering and regenerative medicine. We here describe the two main steps required for bioengineering the ovary and for its ex vivo functional reassembling. The first step aims at producing a 3D bioscaffold, which mimics the natural ovarian milieu in vitro. This is obtained with a whole organ decellularization technique that allows the maintenance of microarchitecture and biological signals of the original tissue. The second step involves the use of magnetic activated cell sorting (MACS) to isolate purified female germline stem cells (FGSCs). These cells are able to differentiate in ovarian adult mature cells, when subjected to specific stimuli, and can be used them to repopulate ovarian decellularized bioscaffolds. The combination of the two techniques represents a powerful tool for in vitro recreation of a bioengineered ovary that may constitute a promising solution for hormone and fertility function restoring. In addition, the procedures here described allow for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies.

Our results show that nab-paclitaxel penetrates into the abdominal wall better than CRE-paclitaxel, in terms of effective penetration and peritoneal tissue concentration.

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Different bioengineering strategies can be presently adopted and have been shown to have great potential in the treatment of female infertility and ovarian dysfunction deriving from chemotherapy, congenital malformations, massive adhesions as well as aging and lifestyle. One option is transplantation of fresh or cryopreserved organs/fragments into the patient. A further possibility uses tissue engineering approaches that involve a combination of cells, biomaterials and factors that stimulate local ability to regenerate/ repair the reproductive organ. Organ transplant has shown promising results in large animal models. However, the source of the organ needs to be identified and the immunogenic effects of allografts remain still to be solved before the technology may enter the clinical practice. Decellularization/ repopulation of ovary with autologous cells or follicles could represent an interesting, still very experimental alternative. Here we summarize the recent advancements in the bioengineering strategies applied to the ovary, we present the principles for these systems and discuss the advantages of these emerging opportunities to preserve or improve female fertility.