Novel Midkine-Derived Peptide Promotes Reparative Dentine Formation via DDIT4/mTOR Pathway-Mediated Activation of Autophagy

Aim: Midkine (MK) protein plays a critical regulatory role in tooth development and odontogenic differentiation, and has been shown to significantly promote reparative dentine formation. However, the direct application of recombinant MK protein as a pulp capping material presents clinical challenges and may increase medical costs. In contrast, peptides offer a cost-effective and feasible alternative by overcoming many of the limitations associated with full-length proteins. This study aimed to develop a novel MK-derived peptide for use in vital pulp therapy (VPT).

Methodology: The human MK protein sequence was appropriately cleaved, resulting in the generation of multiple MK-derived peptides. The peptide with the highest mineralisation potential was identified through quantitative polymerase chain reaction (qPCR) and alizarin red S (ARS) staining. Cell proliferation, live/dead cell staining and Apoptosis assays were conducted to evaluate the in vitro biosafety of MK-derived peptide. The optimal concentration of MK-derived peptide for promoting cell migration and odontogenic differentiation was determined using cell migration assays, qPCR, Western blotting, Alkaline Phosphatase staining and ARS staining. RNA Sequencing, bioinformatics analysis, transmission electron microscopy and siRNA were used to explore the potential mechanism by which MK-derived peptide promotes odontogenesis. A rat pulp capping model was established, and in vivo evaluation was conducted through Micro-CT, H&E, Masson and immunohistochemical staining.

Results: Following appropriate cleavage of the human MK protein, a total of 15 peptides were generated, among which MK-12 (KARYNAQCQETI) exhibited the highest mineralisation potential. MK-12 demonstrated favourable in vitro biocompatibility at concentrations below 200 μg/mL, with the optimal effect on promoting cell migration and odontogenic differentiation observed at 100 μg/mL. RNA-sequencing and functional analyses revealed that MK-12 was closely associated with Autophagy and mammalian target of rapamycin (mTOR) signalling pathways. Subsequent experiments demonstrated that MK-12 modulates the mTOR pathway and Autophagy by upregulating the expression of DNA damage-inducible transcript 4 (DDIT4). In vivo studies confirmed that MK-12 effectively promoted the formation of reparative dentine and exhibited favourable biocompatibility.

Conclusion: MK-12, a peptide derived from midkine, was found to promote reparative dentine formation via DDIT4/mTOR pathway-mediated activation of Autophagy, thereby providing a mechanistic basis for its potential clinical application in VPT.

animal model; dental pulp stem cells; dentinogenesis; midkine; peptides; pulp capping material; reparative dentine.