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. 2022 Sep 28;13(4):165.
doi: 10.3390/jfb13040165.

Construction of Artificial Ovaries with Decellularized Porcine Scaffold and Its Elicited Immune Response after Xenotransplantation in Mice

Tong Wu  1   2   3 , Yue-Yue Gao  1   2   3 , Xia-Nan Tang  1   2   3 , Jin-Jin Zhang  1   2   3 , Shi-Xuan Wang  1   2   3
Affiliations

Construction of Artificial Ovaries with Decellularized Porcine Scaffold and Its Elicited Immune Response after Xenotransplantation in Mice

Tong Wu et al. J Funct Biomater. .

Abstract

Substitution by artificial ovary is a promising approach to restore ovarian function, and a decellularized extracellular matrix can be used as a supporting scaffold. However, biomimetic ovary fabrication and immunogenicity requires more investigation. In this study, we proposed an effective decellularization protocol to prepare ovarian scaffolds, which were characterized by few nuclear substances and which retained the extracellular matrix proteins. The ovarian tissue shape and 3-dimensional structure were well-preserved after decellularization. Electron micrographs demonstrated that the extracellular matrix fibers in the decellularized group had similar porosity and structure to those of native ovaries. Semi-quantification analysis confirmed that the amount of extracellular matrix proteins was reduced, but the collagen fiber length, width, and straightness did not change significantly. Granulosa cells were attached and penetrated into the decellularized scaffold and exhibited high proliferative activity with no visible apoptotic cells on day 15. Follicle growth was compromised on day 7. The implanted artificial ovaries did not restore endocrine function in ovariectomized mice. The grafts were infiltrated with immune cells within 3 days, which damaged the artificial ovary morphology. The findings suggest that immune rejection plays an important role when using artificial ovaries.

Keywords: artificial ovaries; decellularization; immune response; ovarian function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Preparation and evaluation of Dec scaffolds. (A) The decellularization process of porcine ovarian cortical strips. Ovaries displayed comparable shape and homogeneity after decellularization, and the color turned from red to white. (B) H&E staining showed the presence of both basophilic (cell nuclei) and eosinophilic (cell cytoplasm and ECM) materials in native tissues, while cell nuclei were absent in Dec tissues. (C) DAPI staining displayed the presence of cell nuclei in native ovaries, which disappeared after decellularization. (D) DNA quantification demonstrated a significant decrease in DNA content of the Dec scaffolds compared to the native tissues. (E) The dsDNA fragments of Dec tissues were shorter than 200 bp. (F) The appearance of the whole ovarian tissues before and after decellularization. (G) H&E staining showed the absence of cell nuclei in the cortical and medullar regions in decellularized tissues. **** p < 0.0001. Abbreviations: bp, base pair; DAPI, 4′,6-diamidino-2-phenylindole; Dec, decellularized; ECM, extracellular matrix; H&E, hematoxylin-eosin.
Figure 2
Figure 2
The constitution of decellularized ovarian scaffolds. (A) Scanning electron micrographs of native and Dec ovaries. Complex fiber networks with porous structures were visible from both outside and inside of Dec tissues. (B) Masson trichrome staining showed the persistence of collagen fibers (blue) and their comparable distribution. The cellular components (red and claret) were removed. (C) Semiquantitative analysis of collagens using IHC staining. (D) Hydroxyproline assays demonstrated a significant increase in collagen concentration after decellularization. (E) Polarized light micrographs of native and Dec ovaries (left). Collagen fibers were indicated by colorful short lines using FIRE-CT program in MATLAB (right). (F) The width, length and straightness were compared between native and Dec tissues. (G) Alcian blue and PAS staining revealed the loss of GAG and preservation of acid mucopolysaccharide in Dec scaffolds. (H) Semiquantitative analysis of selected ECM proteins. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant. Abbreviations: AMH, anti-Müllerian hormone; BMP, bone morphogenetic protein; COL, collagen; CTGF, connective tissue growth factor; FN, fibronectin; GAG, glycosaminoglycan; IHC, immunohistochemistry; LAMA, laminin A; PAS, periodic acid-Schiff; TGFB, transforming growth factor-beta.
Figure 3
Figure 3
Recellularization of Dec scaffolds with ovarian cells and follicles. (A) Cytoskeleton α-tubulin staining demonstrated the stereoscopic structure was well-preserved on day 7. (B) CYP19A1 indicated the steroid hormone synthesis function of seeding cells on day 7. (C) Ki-67 staining confirmed the presence of proliferating cells on day 15. (D) Representative images of the TUNEL assay showed few apoptotic ovarian cells on day 15. H&E staining of follicles cultured on Dec scaffolds on day 1 (E) and day 7 (F). Abbreviation: CYP19A1, cytochrome P450 family 19 subfamily A member 1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling.
Figure 4
Figure 4
In vivo function of the transplanted artificial ovaries. (A) The condition of vaginal opening in different groups. (B) Representative images of vaginal smear after transplantation indicated the estrus cycles of mice. (C) The dynamic variation of estrus cycles of mice after 4 weeks.
Figure 5
Figure 5
Immune response assessments of transplanted grafts. (A) The macroscopic observation of transplanted scaffolds on day 3 and day 7. (B) H&E staining revealed that inflammatory cells were scattered throughout the grafts and some invaded into the scaffolds on day 3. (C) Individual follicles (arrowhead) could be seen among these inflammatory cells. The total disappearance (D) and partial remnants (E), indicated by asterisks, of scaffolds on day 7. (F) Fluorescence staining for C3 and CD14 were observed in the cells located peripherally on the scaffolds. (G) Cells expressing CD18 and H2AB1 cells could be seen in the central part. Abbreviations: C3, complement C3; CD, cluster of differentiation.
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