Table 1.
Primer sequences.
Figure 1.
Isolation and identification for human periodontal ligament stem cells.
A: Colonies derived from single cell in the method of limiting dilution was picked up for further culturing of hPDLSCs; B: hPDLSCs were grown in culture medium with a long spindle shape; C: Adipogenic differentiation of hPDLSCs was determined by Oil red O staining after 21 days of adipogenic induction; D: Osteogenic differentiation of hPDLSCs was determined by Alizarin red S staining after 21 days of osteogenic induction; E: Immunophenotype analysis of PDLSCs was determined by Flow cytometry assay.
Figure 2.
Nicotine affects the morphology and suppresses the proliferation of hPDLSCs.
A: Morphology of PDLSC in control group; B: morphology of hPDLSC with stimulation of nicotine in 10−3 mol/L, black arrow indicated the vacuolar degeneration of PDLSCs; C–F: Morphologies of hPDLSC with stimulation of nicotine from 10−4–10−7 mol/L, increased granular substance in PDLSCs was indicated by black arrow; G: Proliferation of hPDLSCs from each group was determined by WST-1 assay. Nicotine with different concentrations suppresses the proliferation of hPDLSC.
Figure 3.
Nicotine deteriorates the osteogenic differentiation capacity of hPDLSCs.
A: hPDLSCs treated by 10−4–10−6 mol/L nicotine were cultured in osteogenic medium and osteogenic differentiation was determined by Alizarin red S staining after 21 days. Representative entire plate views of alizarin red staining in 6-well plates for PDLSCs from each group. B: The quantity result for Alizarin Red S staining; C: The expression of osteogenic related genes Runx2, BSP, ALP and OCN were determined by qRT-PCR after 14 days of culturing with osteogenic supplement. D: The expression of osteogenic related protein Runx2, BSP, ALP and OCN were determined by western blot after 14 days of culturing with osteogenic supplement. Data represent the means ± SD. *p<0.05 (n = 3).
Figure 4.
Nicotine deteriorates the osteogenic differentiation of hPDLSCs through the activation of α7 nAChR.
The expression of α7 nAChR in PDLSCs from each group was detected by (A) qRT-PCR and (B) Western Blot analysis. C, D: hPDLSCs treated with/without nicotine and α-BTX were cultured in osteogenic medium and osteogenic differentiation was determined by Alizarin red S staining after 21 days. E: The expression of osteogenic related genes Runx2, BSP, ALP and OCN were determined by qRT-PCR after 14 days of culturing with osteogenic supplement. F: The expression of osteogenic related protein Runx2, BSP, ALP and OCN were determined by western blot after 14 days of culturing with osteogenic supplement. Data represent the means ± SD. *p<0.05 (n = 3).
Figure 5.
Nicotine deteriorates the osteogenic differentiation of hPDLSCs through α7 nAChR regulating Wnt/β-catenin pathway.
A: The expression of wnt signaling pathway related proteins were determined by western blot, wnt signaling pathway was activated by nicotine in the downstream of α7 nAChR; B, C: hPDLSCs treated with/without nicotine and dkk1 were cultured in osteogenic medium and osteogenic differentiation was determined by Alizarin red S staining after 21 days. D: The expression of osteogenic related genes Runx2, BSP, ALP and OCN were determined by qRT-PCR after 14 days of culturing with osteogenic supplement. E: The expression of osteogenic related proteins Runx2, BSP, ALP and OCN were determined by western blot after 14 days of culturing with osteogenic supplement. Data represent the means ± SD. *p<0.05 (n = 3).