猪子宫脱细胞基质水凝胶及其微阵列对猪子宫内膜基质细胞行为的调控

Porcine uterine decellularized extracellular matrix hydrogels and engineered microarrays regulate porcine endometrial stromal cell behavior

摘要:

猪子宫脱细胞外基质(decellularized extracellular matrix, DECM)水凝胶是子宫内膜组织工程的理想候选材料，但其脱细胞效率与细胞外基质(extracellular matrix, ECM)活性保留常受限。为探究DECM水凝胶的生化和力学特性对子宫内膜基质细胞(porcine endometrial stromal cells, pESCs)的调控机制，改善传统工艺中脱细胞效率与ECM活性的矛盾，本研究优化了猪子宫DECM水凝胶制备工艺：采用冻融联合SDS/Triton X-100脱细胞法，经正交实验确定最佳胃蛋白酶消化参数(72 h, 4 ℃, 10 mg/mL)，显著提升了脱细胞效率(P<0.001)并完整保留了胶原、糖胺聚糖、生长因子及多孔结构(经蛋白组学与扫描电镜结果证实)。鉴于纯DECM力学性能不足，开发了0.5% DECM+5%甲基丙烯酰化明胶(methacryloyl gelatin, GelMA)复合水凝胶，其弹性模量接近天然子宫内膜(P<0.001)，且降解稳定性与溶胀比更优。细胞研究表明，高DECM含量通过整合素α5β1-FAK通路促进pESCs迁移与聚集体自组装。针对传统培养的局限，构建了半球形/圆柱形DECM-GelMA复合微阵列，利用接触引导效应成功诱导细胞定向生长。本研究实现了DECM高效制备与生物活性协同优化，阐明了其通过整合素信号及细胞骨架重构调控细胞行为的机制，为仿生子宫内膜支架开发提供了新策略。

Abstract:

The porcine uterine decellularized extracellular matrix (DECM) hydrogel represents a promising candidate for endometrial tissue engineering. However, conventional preparation methods face challenges in balancing decellularization efficiency and bioactive extracellular matrix (ECM) preservation. To investigate the regulatory mechanisms of the biochemical and biomechanical properties of the DECM hydrogel on porcine endometrial stromal cells (pESCs), while addressing the inherent conflict between decellularization efficiency and preserved ECM bioactivity in conventional processing techniques. In this study, we established an optimized protocol: combining freeze-thaw cycles with SDS/Triton X-100 decellularization, followed by orthogonal experimental determination of optimal pepsin digestion parameters (72 h, 4 ℃, 10 mg/mL). The refined protocol enhanced decellularization efficiency (P<0.001) while maintaining intact collagen architecture, glycosaminoglycans, growth factors, and porous microstructure, as validated through proteomic analysis and scanning electron microscopy. To address the inherent mechanical limitations of pure DECM hydrogels, we developed a composite hydrogel system, which was composed of 0.5% DECM and 5% methacrylated gelatin (GelMA). This composite hydrogel system demonstrated elastic modulus comparable to native endometrium (P<0.001), with improved degradation stability and optimized swelling ratio compared with single-component systems. Cellular investigations revealed that higher DECM content promoted porcine endometrial stromal cell migration and spheroid self-organization through activating the integrin α5β1-FAK signaling pathway. To overcome limitations in conventional 2D culture, we engineered DECM-GelMA composite microarrays with hemispherical/cylindrical configurations, successfully inducing directional cellular alignment via contact guidance effects. The present study achieved concurrent optimization of DECM preparation efficiency and bioactivity retention, while elucidating its regulatory mechanisms through integrin-mediated signaling and cytoskeletal reorganization. These findings provide new biomimetic strategies for designing functional endometrial scaffolds with improved histocompatibility and mechanobiological responsiveness.