Bioengineered bilayered grafts for structural and functional posterior lamellar eyelid reconstruction

Eyelid defects involving posterior lamella loss pose significant challenges in reconstructive surgery due to their functional-anatomical complexity. While our previous autologous auricular chondrocyte-derived tissue-engineered cartilage (TEC) grafts successfully maintained normal eyelid morphology, they lacked functional epithelium. This study develops bioengineered bilayered mucosa-cartilage (BMC) grafts through coculture of TEC with oral mucosal explants. The resulting BMC grafts demonstrated a stratified epithelium with barrier integrity and MUC1-producing capacity and a cartilage layer with surgical-grade tensile modulus (1.68 MPa). Upon transplantation into rabbit tarsoconjunctival defects, BMC grafts surpassed both untreated controls and TEC grafts. All grafts demonstrated integration by 2 weeks post-implantation, with transient inflammatory infiltration resolving by 8 weeks. BMC and TEC grafts better preserved eyelid morphology and blinking function than controls throughout the 8-week study. Crucially, BMC-reconstructed eyelids developed continuous stratified epithelia with 5.8-layer MUC1-secreting epithelial cells as early as 2 weeks, progressing to MUC5AC⁺ goblet cell-rich epithelia by 8 weeks post-implantation. In contrast, TEC counterparts formed thinner epithelia with a lower density of goblet cells. These results confirm the structural integrity and secretory functions of BMC grafts, advancing clinical translation of functional eyelid substitutes.