Xenogeneic Decellularized Scaffold: A Novel Platform for Ovary Regeneration

Abstract

Women younger than 40 years may face early menopause because of premature ovarian failure (POF). The cause of POF can be idiopathic or iatrogenic, especially the cancer-induced oophorectomy and chemo- or radiation therapy. The current treatments, including hormone replacement therapy (HRT) and cryopreservation techniques, have increased risk of ovarian cancer and may reintroduce malignant cells after autografting. Decellularization technique has been regarded as a novel regenerative medicine strategy for organ replacement, wherein the living cells of an organ are removed, leaving the extracellular matrix (ECM) for cellular seeding. This study aimed to produce a xenogeneic decellularized ovary (D-ovary) scaffold as a platform for ovary regeneration and transplantation. We have developed a novel decellularization protocol for porcine ovary by treatment with physical, chemical, and enzymatic methods. Using hematoxylin and eosin (H&E) staining, DAPI staining, scanning electron microscopy (SEM), and quantitative analysis, this approach proved effective in removing cellular components and preserving ECM. Furthermore, the results of biological safety evaluation demonstrated that the D-ovary tissues were noncytotoxic for rat ovarian cells in vitro and caused only a minimal immunogenic response in vivo. In addition, the D-ovary tissues successfully supported rat granulosa cell penetration ex vivo and showed an improvement in estradiol (E2) hormone secretion.

Keywords: bioengineering in organ transplantation; decellularization; extracellular matrix; premature ovarian failure; regenerative medicine.

FIG. 1.

Flow diagram of study design. Color images available online at www.liebertpub.com/tec

FIG. 2.

Micrograph (A, B), H&E staining (C–F), DAPI staining (G–J), and DNA content (K) of N-ovary and D-ovary samples. (A, B) Scale bar 10 mm; (C–J) Scale bar 100 μm. D-ovary, decellularized ovary; H&E, hematoxylin and eosin; N-ovary, native ovary. Color images available online at www.liebertpub.com/tec

FIG. 3.

Composition of decellularized ECM in D-ovary samples using Alcian Blue and immunohistochemical staining (A–J), and quantitative analysis of glycosaminoglycans and collagen content of ECM (K, L). Scale bar 100 μm. ECM, extracellular matrix. Color images available online at www.liebertpub.com/tec

FIG. 4.

Microarchitecture of decellularized scaffold presented by scanning electron microscopy images. “a”: Ovarian cells in native scaffold; “b” and “c”: follicular cavity was observed both in native and decellularized scaffold; “d”: pore walls of D-ovary became porous because of the lack of intact cells. (A–D) Scale bar 50 μm; (E, F) Scale bar 5 μm.

FIG. 5.

Cytotoxicity of D-ovary samples. The migration and metabolic activities of ovarian cells were measured using the cell migration assay (A–F) and CCK-8 test (G). CCK-8, Cell Counting Kit-8; NS, not significant. Color images available online at www.liebertpub.com/tec

FIG. 6.

H&E and immunohistochemical staining show the immunological response of the host to native and decellularized ECM implanted subcutaneously after 2 and 4 weeks. Scale bar 100 μm. Color images available online at www.liebertpub.com/tec

FIG. 7.

The numerical density of CD68⁺ (pan-macrophage/monocyte marker), CD86⁺ (M1 macrophage marker), and CD3⁺ (T lymphocyte marker) immune cells in D-ovary and N-ovary samples at 2 and 4 weeks. *p < 0.05.

FIG. 8.

Integration of granulosa cells into D-ovary scaffold ex vivo (A–H) and comparison of E2 expression levels of cocultured and isolated ovarian tissues (I). Higher magnification images (E–H) represent the area within the black box in lower magnification images (A–D). Arrows indicate the direction of cell migration. Scale bar 100 μm. E2, estradiol. Color images available online at www.liebertpub.com/tec