The hDPSC-CM-derived peptide P1DC promotes osteogenic differentiation by regulating the p38/MAPK pathway

Abstract

Human dental pulp stem cells (hDPSCs) are dental-derived mesenchymal stem cells with robust multipotent differentiation potential, indicating that they are promising for bone tissue engineering (BTE). We previously screened a secreted peptide, P1DC, derived from differentiated hDPSC-CM, that markedly promoted the proliferation of hDPSCs. However, its effect on osteogenesis and bone regeneration is unknown. HDPSCs were isolated from dental pulp tissue. The effects of P1DC on the osteogenic activity of hDPSCs were evaluated by quantitative real-time polymerase chain reaction (qRT‒PCR), western blotting, alkaline phosphatase (ALP) staining, and Alizarin Red S (ARS) staining. Additionally, the role of P1DC in bone formation in vivo was validated in a rat calvarial defect model. Micro-CT, hematoxylin-eosin, Masson’s trichrome staining and immunohistochemistry were used to verify the effects of P1DC on bone regeneration. Moreover, RNA sequencing (RNA-Seq) analysis was performed to analyze the underlying mechanisms of P1DC-induced osteogenesis. In hDPSCs, 2.5 µM P1DC significantly upregulated the mRNA and protein levels of osteogenic markers (Runx2, ALP, and OSX), increased ALP activity, and accelerated matrix mineralization, with all differences statistically significant ( p < 0.05). Furthermore, micro-CT analyses in vivo rat calvarial defect models revealed that 2.5 μM P1DC markedly augmented bone regeneration capacity ( p < 0.05). Hematoxylin‒eosin and Masson’s trichrome staining also verified substantially more mature bone matrix deposition within P1DC-treated group, with significantly higher new bone area fraction ( p < 0.05). Mechanistically, RNA-sequencing profiling indicated activation of the MAPK signaling pathway following P1DC treatment. Further Western blot validation revealed markedly elevated phosphorylation levels of p38 in P1DC-treated hDPSCs ( p < 0.05), and the osteogenic differentiation of P1DC + p38 inhibitor group was statistically inferior to that of the P1DC single-treatment group ( p < 0.05). Collectively, our results indicated that P1DC effectively increased osteogenic differentiation in hDPSCs in vitro and facilitated bone regeneration in vivo, at least partially via the p38/MAPK signaling pathway, suggesting new avenues for enhancing bone regeneration strategies.