Effect of decellularization protocols on the biomechanical properties of porcine ovarian extracellular matrix

Decellularized extracellular matrix (d-ECM) serves as an ideal scaffold for constructing artificial ovaries, a promising approach to fertility preservation for patients experiencing premature ovarian failure. The biomechanical properties of d-ECM are crucial for the development and maturation of follicles. However, there is no standardized or comprehensive framework for evaluating the various decellularization methods proposed in the literature. In this study, we developed a novel decellularization protocol for porcine ovaries using liquid nitrogen and hypertonic saline methods, comparing its effectiveness against conventional chemical and enzymatic techniques through histological analysis, quantitative assessments and biomechanical testing. Histological analyses demonstrated that our d-ECM protocols effectively removed cellular and nuclear materials (at least 95% reduction) while preserving the structural integrity of elastin and collagen fibers (maximum 15% reduction). Furthermore, tensile testing results indicated that the novel decellularization methods using liquid nitrogen and hypertonic saline retained mechanical properties most similar to those of the fresh group. Our findings expand the evaluation of decellularization techniques by incorporating the biomechanical properties of d-ECM. Additionally, we provide valuable insights for enhancing decellularization methods and identifying optimal scaffolds for artificial ovaries.