Effect of Pressure Conditions in Uterine Decellularization Using Hydrostatic Pressure on Structural Protein Preservation.

Uterine regeneration using decellularization scaffolds provides a novel treatment for uterine factor infertility. Decellularized scaffolds require maximal removal of cellular components and minimal damage to the extracellular matrix (ECM). Among many decellularization methods, the hydrostatic pressure (HP) method stands out due to its low cytotoxicity and superior ECM preservation compared to the traditional detergent methods. Conventionally, 980 MPa was utilized in HP decellularization, including the first successful implementation of uterine decellularization previously reported by our team. However, structural protein denaturation caused by exceeding pressure led to a limited regeneration outcome in our previous research. This factor urged the study on the effects of pressure conditions in HP methods on decellularized scaffolds. The authors, therefore, fabricated a decellularized uterine scaffold at varying pressure conditions and evaluated the scaffold qualities from the perspective of cell removal and ECM preservation. The results show that by using lower decellularization pressure conditions of 250 MPa, uterine tissue can be decellularized with more preserved structural protein and mechanical properties, which is considered to be promising for decellularized uterine scaffold fabrication applications.

Internal radial perfusion bioreactor promotes decellularization and recellularization of rat uterine tissue.

The advances in infertility treatment technologies such as in vitro fertilization (IVF) help many infertile women to be able to get pregnant. However, these infertility treatments cannot be applied to women who are suffering from absolute uterine factor. Fabrication of functional scaffold in tissue engineering approach is believed to play an important role for uterine regeneration and uterus replacement for treating absolute uterine factor infertility. In this research, we developed an internal radial perfusion bioreactor to promote decellularization and recellularization for fabrication of functional engineered uterine tissue. As a result, the DNA contents of the decellularized uterine tissue with high hydrostatic pressure followed by 7 days internal perfusion washing decreased by 90% compared to native tissue. Collagen and proteoglycan contents in the pressurized uterine tissue with the internal perfusion bioreactor, static (control) and shaking treatment with high hydrostatic pressure showed no significant change compared to the native tissue. The newly developed perfusion bioreactor also enabled to recellularize in the decellularized tissue with statistically significant increase of DNA by 614% compared to non-seeded cell groups. Vimentin and 4',6-diamidino-2-phenylindole (DAPI) was homogeneously expressed in the seeded endometrial stromal cells in the recellularized tissue fabricated using the bioreactor. With the developed internal radial perfusion bioreactor, we are the first group to successfully recellularized uterine tissue in all layers including epithelium, endometrium and myometrium. These results showed that the internal perfusion bioreactor has potential to be utilized for fabrication of functional engineered tissue to promote tissue regeneration.