In vitro evaluation of periapical lesion-derived stem cells for dental pulp tissue engineering

Abstract

Dental pulp tissue engineering is a promising alternative treatment for pulpitis and periapical periodontitis, and dental pulp stem cells (DPSCs) are considered to be the gold standard for dental seed cell research. Periapical lesions harbor mesenchymal stem cells with the capacity for self-renewal and multilineage differentiation. However, it remains unknown whether these periapical lesion-derived stem cells (PLDSCs) are suitable for dental pulp tissue engineering. To investigate this possibility, PLDSCs and DPSCs were isolated using the tissue outgrowth method and cultured under identical conditions. We then performed in vitro experiments to investigate their biological characteristics. Our results indicate that PLDSCs proliferate actively in vitro and exhibit similar morphology, immunophenotype and multilineage differentiation ability as DPSCs. Simultaneously, PLDSCs exhibit stronger migrative ability and express more vascular endothelial growth factor and glial cell line-derived neurotrophic factor than DPSCs, and PLDSC-derived conditioned medium was more effective in tube formation assay. The mRNA expression levels of immunomodulatory genes HLA-G, IDO and ICAM-1 were also higher in PLDSCs. However, regarding osteo/odontogenic differentiation, PLDSCs showed weaker alkaline phosphatase staining and lower calcified nodule formation compared to DPSCs, as well as lower expression of ALP, RUNX2 and DSPP, as confirmed by a quantitative RT-PCR. The osteo/odontogenic protein expression levels of DSPP, RUNX2, DMP1 and SP7 were also higher in DPSCs. The present study demonstrates that PLDSCs demonstrate potential use as seed cells for dental pulp regeneration, especially for achieving enhanced neurovascularization.

Keywords: dental pulp regeneration; immunomodulatory; odontogenesis; osteogenesis; periapical lesion-derived stem cells; pro-angiogenesis.

Fig. 1

Identification of mesenchymal stem cell properties of DPSCs and PLDSCs. (A) Images obtained from phase‐contrast microscopy showing PLDSCs with a spindle‐like shape and adhesive properties similar to those of DPSCs. Scale bar = 500 μm. (B) Oil red O and Alcian blue staining showing that both DPSCs and PLDSCs can achieve adipogenic and chondrogenic differentiation. Scale bar = 100 μm (upper); 50 μm (below). (C) Immunophenotype of different cell surface markers.

Fig. 2

Comparison of the proliferative and migrative abilities of cells. (A) Growth curve showing the relative attenuance value from the CCK‐8 assays at different time points (n = 3, Student’s t‐test). (B) Results of the colony‐forming unit assay; there was no statistical difference between DPSCs and PLDSCs on day 14 (n = 3, Student’s t‐test). (C) Results of the transwell assay; after 24 h of culture, PLDSCs exhibited stronger migration ability (n = 3, Student’s t‐test). Scale bar = 100 μm. **P < 0.01. Data are shown as the mean ± SD.

Fig. 3

Comparison of the osteo/odontogenic capacities of the cells. (A) ALP staining after osteo/odontogenic induction for 3 and 7 days. Scale bar = 500 μm. (B) Alizarin red staining showing the presence of calcified nodules after 7, 14, 21 and 28 days of osteo/odontogenic induction. Scale bar = 500 μm. (C) Gene expression patterns of ALP, RUNX2 and DSPP after osteogenic induction for 3 and 7 days; the expression level was normalized to that of ACTB (n = 3, Student’s t‐test). *P < 0.05, **P < 0.01. (D) Western blotting detection (upper) and imagej analysis (below) of DSPP, DMP1, RUNX2 and SP7 after osteogenic induction for 7, 14 and 21 days. Data are shown as the mean ± SD.

Fig. 4

Comparison of pro‐angiogenesis properties of DPSCs and PLDSCs. (A) qRT‐PCR was used to compare the expression levels of VEGF; the expression levels were normalized to those of ACTB (n = 3, Student’s t‐test). **P < 0.01. (B) Western blotting detection of VEGF expression. (C) Immunofluorescence staining showing the protein expression of VEGF in the cytoplasm of DPSCs and PLDSCs. Scale bar = 50 μm. (D) Representative images for each group via inverted phase‐contrast microscopy. Scale bar = 100 μm. (E) Analysis of tube formation parameters (number of junctions, nodes, segments and meshes; total segment length; total mesh area) using imagej (n = 5, one‐way analysis of variance). *P < 0.05 with serum‐free, ^& P < 0.05 with DPSCs‐CM. DPSCs‐CM, dental pulp stem cells‐conditioned medium; PLDSCs‐CM, periapical lesion‐derived stem cells‐conditioned medium. Data are shown as the mean ± SD.

Fig. 5

Comparison of neurotrophic ability of DPSCs and PLDSCs. (A) qRT‐PCR was used to compare the expression level of GDNF between PLDSCs and DPSCs; the expression level was normalized to that of ACTB (n = 3, Student’s t‐test). (B) Western blotting results of GDNF expression. (C) Immunofluorescence staining for GDNF expression in DPSCs and PLDSCs. Scale bar = 50 μm. **P < 0.01. Data are shown as the mean ± SD.

Fig. 6

Comparison of the immunomodulatory abilities of the cells. Expression of immunomodulatory genes HLA‐G, IDO, HGF and ICAM‐1 were evaluated via qRT‐PCR; ACTB was used for normalization (n = 3, Student’s t‐test). **P < 0.01. Data are shown as the mean ± SD.