Cell-Secreted Alp-Activated Amelogenin Hydrogel Facilitates Dentin-Pulp Regeneration

By exploiting elevated alkaline phosphatase (ALP) activity in odontogenic-differentiated human dental pulp cells (HDPCs), this study develops an ALP-responsive amelogenin hydrogel to enhance odontogenic differentiation, proposing a new dentine-pulp regeneration strategy. HDPCs from patients (14-20 years) were ethically approved. Amelogenin peptide (KWYQNMIR) with Nap-GFFpY self-assembling motif was synthesized. Material characterization included nanofiber microstructure (TEM), secondary structure (CD), critical assembly concentration (microplate), time-dependent viscoelasticity (rheometer), and dissolution stability. Cell-derived ALP-triggered hydrogelation was quantified via fluorescence/microplate analysis. Cytocompatibility (CCK-8), migration (scratch assay), and odontogenic differentiation (Alizarin Red mineralization, qPCR) were systematically evaluated. Both D- and L-configuration hydrogels formed nanofibrous structures, with D-gel showing a higher critical micelle concentration (63 μM, α-helix-dominated loose fibers) compared to L-gel (8 μM, β-sheet-structured ordered nanofibers). ALP-triggered gelation produced elastic solids within 15 minutes, and D-gel retained 34% mass after 30 days.Cell-derived ALP-triggered hydrogel self-assembly was demonstrated by sodium fluorescein staining and microplate reader analysis, with both hydrogels exhibiting no cytotoxicity and enhanced odontogenic differentiation, and the D-gel significantly enhancing HDPC migration. This study firstly reports HDPC-secreted ALP drives peptide self-assembly into enzyme-responsive hydrogels, elucidating molecular configuration's critical role in hydrogel performance. Specifically, the D-type hydrogel exhibited exceptional biocompatibility and stability, offering valuable insights for the design of advanced biomaterials.