ABSTRACT
The present study assessed the supportive roles of the decellularized human ovarian tissue in homing of mouse fetal ovarian cells into the scaffold as well as the formation of the follicular-like structure. The human ovarian cortical tissues were decellularized by three freeze-thaw cycles and then, treated with Triton X-100 for 15 h and 0.5% sodium dodecyl sulfate for 72 h. After isolation and preparation of mouse fetal ovarian cells (19 dpc) they were seeded into the decellularized scaffolds and cultured for 7 days, then using a light microscope, laser confocal scanning microscope, and scanning electron microscope these scaffolds were studied. Analysis of gene expression related to oocyte and follicular cells such as Ddx4, Nobox, Gdf9, and Connexin37 was assessed by real-time RT-PCR and the DDX4 and GDF9 proteins were detected by immunohistochemistry. The result showed that the human ovarian tissue was decellularized properly and the tissue elements and integrity were well preserved. After 7 days of in vitro culture, the fetal ovarian cells attached and penetrated into different sites and depths of the scaffold. The formed organoid within the scaffold showed large round, small polyhedral, and elongated spindle cells similar to the follicle structure. The molecular analysis and immunohistochemistry were confirmed an increase in the expression of genes and proteins related to oocyte and follicular cells in these reconstructed structures. In conclusion, the recellularization of human ovarian scaffolds by mouse fetal ovarian cells could support the follicular-like structure formation and it provides an in vitro model for follicle reconstitution and offers an alternative approach for clinical usage.
ABSTRACT
Decellularized tissue has been used as a natural extracellular matrix (ECM) or bioactive biomaterial for tissue engineering. The present study aims to compare and analyze different decellularization protocols for mouse liver fragments and cell seeding and attachment in the created scaffold using human endometrial mesenchymal cells (hEMCs).After collecting and dissecting the mouse liver into small fragments, they were decellularized by Triton X-100 and six concentrations of sodium dodecyl sulfate (SDS; 0.025, 0.05, 0.1, 0.25, 0.5, and 1%) at different exposure times. The morphology and DNA content of decellularized tissues were studied, and the group with better morphology and lower DNA content was selected for additional assessments. Masson's tri-chrome and periodic acid Schiff staining were performed to evaluate ECM materials. Raman confocal spectroscopy analysis was used to quantify the amount of collagen type I, III and IV, glycosaminoglycans and elastin. Scanning electron microscopy and MTT assay were applied to assess the ultrastructure and porosity and cytotoxicity of decellularized scaffolds, respectively. In the final step, hEMCs were seeded on the decellularized scaffold and cultured for one week, and finally the cell attachment and homing were studied morphologically.The treated group with 0.1% SDS for 24 h showed a well preserved ECM morphology similar to native control and showing the minimum level of DNA. Raman spectroscopy results demonstrated that the amount of collagen type I and IV was not significantly changed in this group compared to the control, but a significant reduction in collagen III and elastin protein levels was seen (P < 0.001). The micrographs showed a porous ECM in decellularized sample similar to the native control with the range of 2.25 µm to 7.86 µm. After cell seeding, the infiltration and migration of cells in different areas of the scaffold were seen. In conclusion, this combined protocol for mouse liver decellularization is effective and its recellularization with hEMCs could be suitable for clinical applications in the future.
ABSTRACT
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.
