Stress urinary incontinence (SUI) is a common disease in middle-aged and elderly women, severely affecting patients' quality of life. Urinary suspension surgery is the most effective treatment for SUI, but complications related to implanted materials limit its clinical application. The use of autologous stem cells cultured in vitro to prepare pelvic ligament suspensions holds great promise in the treatment of stress urinary incontinence, and the stress environment during the cultivation process plays an important role in the mechanical properties of ligament tissue and the cultivation period. This study aims to select the optimal scaffold material for in vitro cultivation of pelvic ligaments, investigate the effects of stress environment on the mechanical properties and cultivation period of in vitro cultivated ligaments, and provide support for the refined in vitro cultivation of pelvic ligaments, with the aim of significantly improving their mechanical properties and shortening the cultivation period.

Surface structure and macroscopic mechanical analysis of commonly used scaffold materials Silk, PCL, and PLA for in vitro cultivation of ligaments were conducted. Fibroblast activity, proliferation, and adhesion on different scaffold materials were determined at different time points. Meanwhile, the Young's modulus of cells and the mRNA expression of extracellular matrix-related genes were measured to select the most suitable scaffold material for pelvic ligament cultivation. The selected scaffold materials were applied to in vitro cultivation of pelvic ligaments to construct cell-scaffold complexes, and fibroblast viability, proliferation, and Young's modulus under different stress stimuli were measured. Transcriptomics and PPI network analysis revealed the collagen assembly mechanism mediated by stress stimuli, and the mRNA expression levels of genes related to the TGF-β signaling pathway and downstream extracellular matrix target genes were validated. The effects of stress stimuli on mechanical properties and cultivation period were evaluated.

Among Silk, PCL, and PLA scaffolds, the diameter and pore size of Silk scaffold were larger than those of PCL and PLA scaffolds, which were 11.894 ± 0.138μm and 110.667 ± 43.036μm, respectively. Silk scaffold exhibited higher Young's modulus and ultimate stress compared to PCL and PLA (P < 0.05). Fibroblast proliferation activity was highest on Silk scaffold, and cells grew in elongated spindle shapes on different scaffold materials, but cell alignment on Silk scaffold was more regular, with higher levels of COL1 and COL3 in the extracellular matrix. Fibroblasts grown on Silk scaffold also had higher Young's modulus compared to other scaffolds (P < 0.05). These results suggest that Silk scaffold is more suitable for constructing in vitro cultivation systems for pelvic ligaments. When applying Silk scaffold to construct cell-ligament complexes and applying different magnitudes of stress, cell proliferation activity increased with increasing stress within a certain range, with the highest proliferation activity observed at 60g of stress, resulting in a 40% increase in cultivation efficiency. Transcriptomics analysis and PPI network under 60g stress stimulation revealed significant effects on the TGF-β signaling pathway and ECM expression. Under stress stimulation, gene expression of TGF-β and ECM components COL1, COL3, and ELN significantly increased (P < 0.05), while ELN, MMP2, and TIMP showed a decreasing trend (P < 0.05). Particularly, COL1, COL3, and ELN as the main components of ligament ECM increased by 5.11-fold, 4.48-fold, and 2.36-fold, respectively, under stress conditions.

This study demonstrates that: (1) A comparison of the biological and mechanical properties between natural polymer Silk scaffolds and synthetic polymers PCL and PLA scaffolds reveals that the natural polymer Silk scaffold has larger fiber diameter and pore size, which can provide mechanical support for stress loading. The orderly arrangement of scaffold fibers induces more regular cell alignment, promotes cell proliferation on the scaffold, increases cell stiffness, and enhances the expression of matrix genes, indicating that the natural polymer Silk scaffold is the optimal material for pelvic ligament tissue engineering. (2) Applying stress stimulation to pelvic ligament cells cultured in vitro can improve cell proliferation efficiency to a certain extent by promoting the expression levels of the TGF-β1 signaling pathway and downstream COL1, COL3, and ELN genes, thereby enhancing the quality of the in vitro ligaments.

This study analyzed the effects of different scaffold materials on in vitro cultivation of ligaments, selected the most suitable scaffold material, constructed an in vitro cultivation model of pelvic ligaments under stress stimulation, determined the effects of different stress stimuli on in vitro cultivated ligaments, obtained the optimal stimulating stress, achieved precise control of in vitro cultivation of pelvic ligaments under mechanical stimulation, thereby improving cultivation efficiency, shortening cultivation time, and obtaining pelvic ligaments with mechanical properties suitable for human use. This lays a theoretical foundation for its early application in clinical practice.