3D-bioprinted active gingival hydrogel for periodontal soft tissue regeneration via TGF-β/ Smad and Wnt/β-Catenin-mediated extracellular matrix remodeling

Soft tissue management is essential in periodontal, orthodontic, and implant therapies, yet autologous grafts remain limited by donor-site morbidity, inconsistent tissue quality, and restricted availability. To address these challenges, we developed an active gingival hydrogel (AGH) composed of gelatin methacrylate (GelMA), chondroitin sulfate methacrylate (ChsMA), and gingival fibroblasts, which was fabricated into cell-laden hydrogels using extrusion-based 3D bioprinting. The AGH exhibited excellent rheological performance, print fidelity, and interconnected porous microstructures that supported nutrient diffusion and cell migration. Gingival fibroblasts cocultured with AGH showed robust adhesion, proliferation, and collagen matrix deposition, accompanied by significant upregulation of fibronectin (FN) and type I collagen (COL1). Mechanistic studies revealed that these effects were mediated through activation of the Wnt/β-catenin and TGF-β/Smad signaling pathways, which synergistically regulate extracellular matrix remodeling and epithelial keratinization. In vivo experiments demonstrated that AGH implantation significantly enhanced gingival thickness, collagen density, and neovascularization while reducing inflammatory infiltration, as verified by MRI, histological, and immunohistochemical analyses. Furthermore, co-culture with gingival epithelial cells promoted upregulation of KRT10 and KRT14, indicating improved epithelial differentiation. Collectively, this study establishes a 3D bioprinted active gingival hydrogel as a biomimetic and functional substitute for autologous grafts, offering a promising strategy for periodontal and peri-implant soft tissue regeneration.