Biomimetic 3D-printed bilayered scaffold with stem cell delivery for rabbit vaginal regeneration

Traditional biological scaffolds for vaginal reconstruction often fail to restore the physiological bilayered structure of vaginal tissue, leading to post-operative complications such as contraction, stenosis, and fibrosis. To address this, we developed a biomimetic bilayered vaginal scaffold using decellularized porcine vaginal extracellular matrix (vECM), gelatin methacryloyl (GelMA), and silk fibroin (SF) hydrogels. The optimized bioink (vECM/GelMA/SF) was characterized via SEM, rheology, FTIR, swelling tests, and H&E staining, confirming its morphology, stability, and biocompatibility. Rabbit bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured, then directionally induced to differentiate into epithelial cells and smooth muscle cells. The differentiated cells were spatially loaded onto the inner and outer layers of the biomimetic scaffold for vaginal reconstruction. Following transplantation into rabbit vaginal defects, the scaffold mediated structural and functional restoration of vaginal tissue. Histological analyses at 12 weeks post-implantation confirmed: regeneration of mature stratified epithelium, organization of muscular bundles, and absence of complications including contraction, stenosis, or fibrosis. The uniqueness of this study lies in the first application of directionally differentiated BMSCs for vaginal reconstruction was synergistically combined with a bilayered 3D-printed architecture mimicking native vaginal stratification, enabling stem cell-driven regeneration of both epithelial and muscular tissues. This integrated approach achieved functional restoration of vaginal defects through precise spatial organization of differentiated cells within biomimetic layers.