Abstract
BackgroundHuman dental pulp stem cell (DPSC)-mediated regenerative endodontics is a promising therapy for damaged teeth; however, the hypoxic environment in root canals can affect tissue regeneration. In this study, we investigate the characteristics and possible regulatory mechanisms of DPSC function under hypoxic conditions.MethodsHuman DPSCs were cultured under normoxia (20% O2) and hypoxia (3% O2). DPSC proliferation and osteo/odontogenic differentiation potential were assessed by Cell Counting Kit-8 (CCK8) assay, carboxyfluorescein succinimidyl ester (CFSE) assay, alkaline phosphatase (ALP) activity, Alizarin red staining, real-time RT-PCR assays, and western blot analysis. Microarray and bioinformatic analyses were performed to investigate the differences in the mRNA, lncRNA, and miRNA expression profiles of DPSCs.ResultsDPSCs exhibited a more powerful proliferation ability and lower osteo/odontogenic differentiation potential in hypoxic conditions. A total of 60 mRNAs (25 upregulated and 35 downregulated), 47 lncRNAs (20 upregulated and 27 downregulated), and 14 miRNAs (7 upregulated and 7 downregulated) in DPSCs were differentially expressed in the hypoxia group compared with the normoxia group. Bioinformatic analysis identified that 7 mRNAs (GRPR, ERO1L, ANPEP, EPHX1, PGD, ANGPT1, and NQO1) and 5 lncRNAs (AF085958, AX750575, uc002czn.2, RP3-413H6.2, and six-twelve leukemia (STL)) may be associated with DPSCs during hypoxia according to CNC network analysis, while 28 mRNAs (including GYS1, PRKACB, and NQO1) and 13 miRNAs (including hsa-miR-3916 and hsa-miR-192-5p) may be involved according to miRNA target gene network analysis. The depletion of one candidate lncRNA, STL, inhibited the osteo/odontogenic differentiation potentials of DPSCs.ConclusionsOur results revealed that hypoxia could enhance the proliferation ability and impair the osteo/odontogenic differentiation potential of DPSCs in vitro. Furthermore, our results identified candidate coding and noncoding RNAs that could be potential targets for improving DPSC function in regenerative endodontics and lead to a better understanding of the mechanisms of hypoxia’s effects on DPSCs.
Highlights
Human dental pulp stem cell (DPSC)-mediated regenerative endodontics is a promising therapy for damaged teeth; the hypoxic environment in root canals can affect tissue regeneration
Hypoxia enhanced the proliferation ability and inhibited the osteo/odontogenic differentiation potential of DPSCs To investigate the effects of hypoxia on DPSCs, DPSCs were cultured in both normoxic and hypoxic conditions
The results showed that hypoxia resulted in a significant decrease in the alkaline phosphatase (ALP) activity of DPSCs (Fig. 2a)
Summary
Human dental pulp stem cell (DPSC)-mediated regenerative endodontics is a promising therapy for damaged teeth; the hypoxic environment in root canals can affect tissue regeneration. Pulpitis and periapical periodontitis are the most prevalent oral diseases, and root canal treatment is the current primary treatment. Regenerative endodontics (RE) is a clinical procedure that aims to regenerate the dentin-pulp complex (DPC) inside root canals [3]. RE is a more ideal treatment for teeth that are damaged from pulpitis and periapical periodontitis, which is garnering increased attention [4,5,6]. A previous study reported that pulp-like tissues were formed by transplanting 3D DPSC constructs into human root canals after subcutaneous implantation into immunodeficient mice [7]. To improve the outcomes of RE, a key issue is to improve DPSC function in dental clinic conditions; it is necessary to explore the mechanisms controlling the directed differentiation of DPSCs in these conditions
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