Abstract: Objective: To investigate the effect of DDIT3 overexpression on proliferation and osteogenic/odontoblastic differentiation of HDPCs. Methods: DDIT3 overexpression lentiviral construct was confirmed by sequencing. Generation of lentiviral vectors was accomplished using a three-plasmid transfection procedure. Cells were allocated into three groups: HDPCs-WT group, HDPCs-GFP group, and HDPCs-DDIT3-overexpression group. Expression of DDIT3 was quantified by qRT-PCR and Western blot. Cell proliferation and osteogenic/odontoblastic differentiation were detected by MTT, qRT-PCR, ALP staining, Alizarin red staining, and von Kossa staining. Results: A lentiviral vector system was used to efficiently overexpress DDIT3 in primary HDPCs to levels >90%. It was found that DDIT3 mRNA was overexpressed over 38-fold. MTT assay revealed DDIT3 overexpression reduced HDPC proliferation by 72 and 96 hours compared to control groups. DDIT3 overexpression led to no significant difference in ALP staining area after 7 days culture in odontoblastic medium. However, DDIT3 overexpression enhanced calcium deposition by day 14, as examined by alizarin red and von Kossa staining. qRT-PCR results revealed that DDIT3 overexpression did not affect ALP and RUNX2 mRNA levels, however, it significantly increased OSX, DSPP, DMP1, and OCN mRNA levels by day 14. Western blot analysis revealed that DSPP protein levels were higher in the HDPCs-DDIT3-overexpression group. Conclusion: Lipid metabolism related gene DDIT3 may correlate with HDPCs late osteogenic/odontoblastic differentiation.

Key words: DDIT3, HDPCs, Osteogenesis and adipogenesis