Abstract:

Objective

To investigate the effects and mechanisms of Schwann cells-derived extracellular vesicles （SCs-EVs） on pulp regeneration.

Methods

Human dental pulp stem cells（hDPSCs） were isolated and cultured in vitro， and treated with SCs-EVs， SCs-EVs combined with Wnt/β-catenin signaling pathway inhibitor IWR-1 （SCs-EVs group， SCs-EVs+IWR-1 group）， and a blank treatment group （Control group） was set up. The cell proliferative activity and cell clonogenesis ability were detected by CCK-8 method and plate cloning method respectively. The formation ability of calcium nodules was detected by alizarin red staining method. The lipid formation capacity in cells was detected by oil red O staining method. The mRNA expression levels of self-renewal marker genes （Oct4， Nanog， Sox2）， osteogenic differentiation marker genes （ALP， Runx2， COL-1），lipogenic differentiation marker genes （ADPN， FABP4， LPL） and neurogenic differentiation marker genes （Gfap， Nestin， Tubb3） were detected by RT-qPCR. The protein expression level of Runx2，ADPN， Nestin and Wnt/β-catenin signaling pathway related proteinswere detected by Western blot.The fluorescence intensity of β-catenin was detected by immunofluorescence. Independent sample t test or corrected t test was used for comparison between two groups， one-way ANOVA was used for comparison between multiple groups， and LSD-t test was used for further pair-to-group comparison（between groups with different SCs-EVs concentrations； SCs-EVs group and Control group；SCs- EVs+IWR-1 group and SCs-EVs grou）.

Results

Compared with the Control group， the cell proliferation activity in SCs-EVs group was enhanced （P ＜ 0.05）， together with an increasing of the number of clones （909.17 ± 52.86 vs 415.38 ± 25.61）， calcium nodule formation ability （0.79 ±0.05 vs 0.24 ± 0.02）， lipid formation ability （0.52 ± 0.03 vs 0.14 ± 0.01）， Oct4， Nanog， Sox2， ALP，Runx2， COL-1， ADPN， FABP4， LPL， Gfap， Nestin， Tubb3 mRNA expression levels， Runx2 （1.05 ±0.16 vs 0.18 ± 0.05）， ADPN （0.91 ± 0.09 vs 0.14 ± 0.04）， Nestin （0.96 ± 0.13 vs 0.15 ± 0.03），β-catenin （1.01 ± 0.12 vs 0.19 ± 0.03） protein expression levels and β-catenin fluorescence intensity（4.27 ± 0.31 vs 1.07 ± 0.12） （all P＜0.05）. Compared with SCs-EVs group， cell proliferation activity in SCs-EVs+IWR-1 group was decreased （P＜0.05）， together with a decreasing of the number of clones （482.55 ± 29.32 vs 909.17 ± 52.86）， calcium nodule formation ability （0.32 ± 0.03 vs 0.79 ±0.05）， lipid formation ability （0.20 ± 0.01 vs 0.52 ± 0.03）， Oct4， Nanog， Sox2， ALP， Runx2， COL- 1，ADPN， FABP4， LPL， Gfap， Nestin， Tubb3 mRNA expression levels， Runx2 （0.21 ± 0.05 vs 1.05 ±0.16）， ADPN （0.37 ± 0.06 vs 0.91 ± 0.09）， Nestin （0.34 ± 0.09 vs 0.96 ± 0.13）， β-catenin （0.31 ±0.05 vs 1.01 ± 0.12） protein expression levels and and β-catenin fluorescence intensity （1.29 ± 0.15 vs 4.27 ± 0.31） （all P ＜ 0.05）.

Conclusion

SCs-EVs promotes pulp regeneration by activating the Wnt/β-catenin signaling pathway to promote the proliferation， self-renewal and pluripotency of hDPSCs.

Key words: Schwann cells, Extracellular vesicles, Pulp regeneration, Dental pulp stem cells, multiplication, Pluripotency