Evaluation of decellularized ovarian scaffolds by triton, ammonium hydroxide and SDS

Background: Decellularization is a novel technique in regenerative medicine. Recently decellularized ovary introduced as a scaffold in the field of human fertility preservation. So the aim of the present study was producing a decellularized ovarian scaffold for fertility preservation approaches.Objective: The aim of the present study was the evaluation ovarian cortex decellularization by Triton, Ammonium hydroxide and SDS.Materials and Methods: In the present study 2 mm pieces of bovine and human ovarian cortex were prepared. In the SDS group, ovarian cortex was decellularized with 0.1% SDS for 24 hr, in the SDS-Triton-ammonium group these pieces were decellularized with 0.5% SDS for 2 hr, 1% Triton and 0.1% Ammonium hydroxide for 22 hr and in SDS-Triton group pieces were decellularized with 0.5% SDS for 3 hr and 1% Triton for 9 h. Human ovarian cortex was decellularized in three steps. First, it was treated with 0.1% SDS for 24 hr then according to freezing and thawing, it was treated with 0.2% SDS for 10 hours. After decellularization, all of the scaffolds were washed with deionized water for 24 hr. The intact ovarian cortex was used as a control group. H&E and DAPI staining were applied to prove decellularization. Elastin and Masson s trichrome staining was carried out to evaluate the presence of elastin and collagen respectively in decellularized tissue. Furthermore, MTT test was done to assess the in vitro scaffold’s cytocompatibility.Results: According to the results of H&E staining, the bovine ovarian cortex was decellularized in all groups. No residual nuclei were observed by DAPI staining. Preservation of the ECM was evaluated by Masson Trichrome and Gomori s aldehyde-fuchsin staining. Elastic and collagen fibers were kept after the decellularization process in all groups. OD values of eluted formazan of MTT test showed that fibroblasts on the SDS-Triton-Amonium decellularized scaffolds were more viable than other groups. Human ovarian cortex was decellularized completely with mentioned protocol. Furthermore elastic and collagen fibers were kept in decellularized human ovarian cortex too. There was no significant difference in the Proliferation rate of the fibroblast cells in the human decellularized scaffold and two-dimensional conventional culture system.Conclusion: In conclusion, human ovarian cortex was decellularized with the combination of different SDS solution and the bovine ovarian cortex was decellularized by SDS, SDS-Triton and SDS-Triton-Ammonium. Structure of ECM was not damaged in any groups. However, MTT test results of SDS-Triton-Ammonium were better than other groups.

Keywords:

Fertility preservation , Ovarian cortex