In silico and in vitro characterization of GH12, an innovative peptide for dental pulp regeneration

To characterize the mechanism of action of GH12 and to assess its cytotoxicity on dental pulp mesenchymal stem cells (DP-MSCs).

GH12 mechanism of action was characterized using in silico predictions and lipid bilayer liposome assays. GH12 cytotoxicity was assessed in 2D culture using DP-MSCs through Live/Dead assay and lysosome staining after 2, 4, and 6 h of incubation. Finally, GH12 cytotoxicity was assessed in a fibrin-based 3D hydrogel after 48 h.

In silico predictions suggested a trimeric or tetrameric pore-forming mechanism for GH12, and its capacity to destabilize membranes was further validated by liposome permeabilization assays. In 2D cultures, GH12 significantly decreased DP-MSCs viability and increased lysosomal staining at 50 µg/mL after 2, 4, and 6 h (p < 0.05). In the 3D model, GH12 did not exhibit a significant effect on DP-MSCs viability (92 % at 50 µg/mL after 48 h; p = 0.18).

In silico predictions suggested that GH12 may adopt trimeric or tetrameric assemblies consistent with a pore-forming mechanism. While cytotoxicity and cellular stress were observed in 2D cultures of DP-MSCs, no toxicity was detected in the fibrin-based 3D model.

These findings support the potential integration of the promising antimicrobial peptide GH12 into advanced regenerative endodontic models, paving the way for further preclinical investigations and its eventual translation into clinical applications.