Stimulated Human Umbilical Cord Mesenchymal Stem Cells Enhance the Osteogenesis and Cranial Bone Regeneration through IL-32 Mediated P38 Signaling Pathway.

OBJECTIVE: Our previous study found that it could significantly increase the expression of IL32 after stimulating the human umbilical cord mesenchymal stem cells (S-HuMSCs). However, its role on the osteogenesis and cranial bone regeneration is still largely unknown. Here, we investigated the possible mechanism of this effect. Material and Methods . A series of experiments, including single-cell sequencing, flow cytometry, quantitative real-time polymerase chain reaction, and western blotting, were carried out to evaluate the characteristic and adipogenic-osteogenic differentiation potential of IL-32 overexpression HuMSCs (IL-32high HuMSCs) through mediating the P38 signaling pathway. Moreover, a rat skull bone defect model was established and treated by directly injecting the IL-32high HuMSCs to conduct its role on the cranial bone regeneration.

RESULTS: In total, it found that compared to HuMSCs, IL32 was significantly increased and promoted the osteogenic differentiation (lower expressions of PPAR γ , Adiponectin, and C/EBP α , and increased expressions of RUNX2, ALP, BMP2, OPN, SP7, OCN, and DLX5) in the S-HuMSCs ( P < 0.05). Meanwhile, the enhanced osteogenic differentiation of HuMSCs was recovered by IL-32 overexpression (IL-32high HuMSCs) through activating the P38 signaling pathway, like as the S-HuMSCs ( P < 0.05). However, the osteogenic differentiation potential of IL-32high HuMSCs was significantly reversed by the P38 signaling pathway inhibitor SB203580 ( P < 0.05). Additionally, the HuMSCs, S-HuMSCs, and IL-32high HuMSCs all presented adipogenic-osteogenic differentiation potential, with higher levels of CD73, CD90, and CD105, and lower CD14, CD34, and CD45 ( P > 0.05). Furthermore, these findings were confirmed by the rat skull bone defect model, in which the cranial bone regeneration was more pronounced in the IL-32high HuMSCs treated group compared to those in the HuMSCs group, with higher expressions of RUNX2, ALP, BMP2, and DLX5 ( P < 0.05).

CONCLUSION: We have confirmed that S-HuMSCs can enhance the osteogenesis and cranial bone regeneration through promoting IL-32-mediated P38 signaling pathway, which is proved that IL-32 may be a therapeutic target, or a biomarker for the treatment of cranial bone injuries.