RESUMEN
The present study makes assessments by analyzing the efficacy of combined decellularization protocol for human ovarian fragments. Tissues were decellularized by freeze-thaw cycles, and treated with Triton X-100 and four concentrations (0.1, 0.5, 1 and 1.5%) of sodium dodecyl sulfate (SDS) at two exposure times. The morphology and DNA content of decellularized tissues were analyzed, and the group with better morphology and lower DNA content was selected for further assessments. The Acridine orange, Masson's trichrome, Alcian blue, and Periodic Acid-Schiff staining were used for extracellular matrix (ECM) evaluation. The amount of collagen types I and IV, glycosaminoglycans (GAGs), and elastin was quantified by Raman spectroscopy. The fine structure of the scaffold by scanning electron microscopy was studied. The endometrial mesenchymal cells were seeded onto decellularized scaffold by centrifugal method and cultured for 7 days. After 72 h the treated group with 0.5% SDS showed well-preserved ECM morphology with the minimum level of DNA (2.23% ± 0.08). Raman spectroscopy analysis confirmed that, the amount of ECM components was not significantly decreased in the decellularized group (P < 0.001) in comparison with native control. The electron micrographs demonstrated that the porosity and structure of ECM fibers in the decellularized group was similar to native ovary. The endometrial mesenchymal cells were attached and penetrated into the decellularized scaffold. In conclusion this combined protocol was an effective method to decellularize human ovarian tissue with high preservation of ECM contents, and human endometrial mesenchymal cells which successfully interacted with this created scaffold.
