Biomaterials for Improving Ovarian Tissue Transplantation

Cryopreservation of ovarian tissue and its subsequent transplantation represent a big hope to preserve fertility in young women who have defeated cancer. Ovary revascularization is a crucial factor impacting the outcome of the engraftment. Limited oxygenation may have severe consequences on the ovarian reserve, with a significant loss of follicles. New frontiers in reproductive technology aim to reduce the ischemic/hypoxic window following auto-transplantation procedures. Biomaterials supplemented with ovarian-derived endothelial cells could be the solution to enhance vascular regeneration in the transplanted tissue.

In this study, we propose a combined Advanced Therapeutic Medicinal Product (ATMP) obtained from the association of cryopreserved ovarian tissue with a 3D dermal substitute — a biocompatible and bioactive scaffold employed in regenerative medicine — pre-seeded with vascular system cells previously isolated from the same ovarian tissue. This pre-seeding, known as inosculation, is a bioengineering approach aimed at enhancing revascularization by promoting the formation of novel vascular networks within the scaffold prior to implantation. The goal of the research is to demonstrate that a such graft can boost the growth of new vessels (Fig. 1), potentially improving the ovary survival and functionality.

Figure 1: Scheme summarizing the experimental pipeline adopted for sample preparation and subsequent graft revascularization analysis. Adapted from graphical abstract from Spazzapan et al., Bioactive Materials (2025), under a Creative Common license.

To evaluate the effectiveness of this approach, several techniques were employed including synchrotron radiation-based X-ray phase-contrast microtomography (SR PC-microCT). As a volumetric imaging technique, SR PC-microCT enables three-dimensional visualization of the inner anatomical structures of the proposed ATMP at high spatial and contrast resolution, with the additional advantage of being non-destructive. Scans were carried out at the SYRMEP Imaging beamline of Elettra. The findings obtained by the X-ray images were complemented by histology and immunohistochemical analyses, adhesion and proliferation assays, gene expression and immunofluorescence. 

A bovine collagen-based scaffold, Integra®, was selected among various dermal substitute materials tested and was used as a support for ovarian transplantation in subsequent in vivo experiments on mouse models. Histology clearly demonstrates the presence of endothelial cells within the Integra^® matrix, exhibiting a tendency to form vascular structures. Red-blood cells can be also observed inside the developing vessels (Fig. 2a). Similarly, Fig. 2b shows a virtual slice obtained by X-ray PC microCT of a sample region at the interface between the ovarian tissue and the Integra^® support. In agreement with the histological data, the X-ray image shows a massive accumulation of dense structures within the scaffold, which may be attributed to a high concentration of endothelial cells. Notably, SR PC microCT enables the cells distribution within the scanned blocks to be tracked, revealing a migration of the endothelial cells from the matrix into the tissue with a preferential side of accumulation. Supplementary videos are available on the full paper website (please follow the link at bottom of this page). 

Figure 2: (a) Histological section showing the ovarian tissue grafted onto the scaffold below the mouse skin. Red dots indicate the endothelial cells proliferation. (b) SR PC microCT image showing a similar portion of sample where dense structures are visible within the matrix, potentially blood cells. (c) SR PC microCT image of a sample stained with iodine. The arrows point a massive cell accumulation, especially at the interface between the ovarian tissue and Integra^®. (d) Close-up at the contact area between tissue and scaffold where the yellow circle highlights the channel established by the endothelial cells migrating from the scaffold to the tissue. Adapted from Spazzapan et al., Bioactive Materials (2025), under a Creative Common license.

Additionally, SR PC microCT was employed to image grafted ovarian tissue after iodine staining, a contrast agent known to enhance cells visualization in X-ray imaging. Fig. 2 c-d display a SR PC microCT cross-section, which simultaneously highlights the ovarian vascular network, the dermal scaffold and the accumulation of endothelial cells at the border between the two structures — suggesting that Integra^® may indeed promote cell proliferation.

In conclusion, the morphological data, together with multi-photon microscopy and gene expression, suggest that enhanced vascularization is linked to the use of the Integra^® biomaterial. These results may pave the way for successful ovarian transplantation, with significant implications for fertility preservation technologies.

This research was conducted by the following research team:

Mariagiulia Spazzapan¹, Silvia Pegoraro², Roman Vuerich^1,3, Gabriella Zito², Andrea Balduit², Elena Longo⁴, Lorella Pascolo², Miriam Toffoli⁵, Giorgia Meshini⁵, Alessandro Mangogna², Gloria Ros⁶, Francesca Buonomo², Federico Romano², Letizia Lombardelli⁷, Giovanni Papa⁵, Marie-Pierre Piccinni⁷, Serena Zacchigna^3,5, Chiara Agostinis², Roberta Bulla¹, Giuseppe Ricci^2,5

¹ Department of Life Sciences, University of Trieste, Trieste, Italy
² Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
³ Cardiovascular Biology Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
⁴ Elettra - Sincrotrone Trieste S.C.p.A., Trieste, Italy
⁵ Department of Medical, Surgical and Health Science, University of Trieste, Trieste, Italy
⁶ Central RNA Laboratory, Istituto Italiano di Tecnologia (IIT), Genova, Italy
⁷ Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

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