Investigating the Effects of Conditioned Media from Stem Cells of Human Exfoliated Deciduous Teeth on Dental Pulp Stem Cells

Abstract

Pulp regeneration has recently attracted interest in modern dentistry. However, the success ratio of pulp regeneration is low due to the compromising potential of stem cells, such as their survival, migration, and odontoblastic differentiation. Stem cells from human exfoliated deciduous teeth (SHED) have been considered a promising tool for regenerative therapy due to their ability to secrete multiple factors that are essential for tissue regeneration, which is achieved by minimally invasive procedures with fewer ethical or legal concerns than those of other procedures. The aim of this study is to investigate the potency of SHED-derived conditioned media (SHED CM) on dental pulp stem cells (DPSCs), a major type of mesenchymal stem cells for dental pulp regeneration. Our results show the promotive efficiency of SHED CM on the proliferation, survival rate, and migration of DPSCs in a dose-dependent manner. Upregulation of odontoblast/osteogenic-related marker genes, such as ALP, DSPP, DMP1, OCN, and RUNX2, and enhanced mineral deposition of impaired DPSCs are also observed in the presence of SHED CM. The analysis of SHED CM found that a variety of cytokines and growth factors have positive effects on cell proliferation, migration, anti-apoptosis, and odontoblast/osteogenic differentiation. These findings suggest that SHED CM could provide some benefits to DPSCs in pulp regeneration.

Keywords: conditioned medium; cytokines; dental pulp stem cell; human exfoliated deciduous teeth; odontoblast/osteogenic differentiation.

Figure 1

Graphic abstract. SHED were collected from the pulp tissue of primary teeth, and DPSC were collected from the pulp tissue of permanent teeth. The cytokines/growth factors contained in SHED-cultured conditioned media promoted DPSC proliferation, survival, migration, and cell differentiation in pulp regenerative therapy.

Figure 2

Effect of stem cells isolated from human pulp of exfoliated deciduous tooth-derived conditioned media (CM) on dental pulp stem cells (DPSCs) regarding proliferation and migration. (a) Presentative live and dead images of SHED 24 h after treatment with 100% CM versus 50% CM and SFM. Scale bar represents 200 µm. (b) The effect of SHED CM after treatment for 24 h on the proliferation of SHED was quantified by CCK-8 assay. n = 6. (c) Schematic of migration assays. A total of 4 × 10⁴ cells in 100 µL were seeded in the upper compartment, while the lower compartment contained 350 µL 100% CM, 50% CM or SFM. (d) Representative images of migrated SHED in the lower compartment of membrane after 6 h of treatment. (e) Cells counted in the lower compartment in 6 random fields. All data are represented as the mean ± standard deviation (SD). The statistical significance was calculated using one-way analysis of variance (ANOVA) to compare groups. Represents ** p < 0.01, *** p < 0.001, **** p < 0.0001, scale bar = 200 µm.

Figure 3

Treatment with SHED CM improved the viability of DPSC after H₂O₂ exposure. DPSCs were cultured with 200 µM H₂O₂ for 12 h before shifting to 100% CM, 50% CM and SFM for another 12 h. (a) Representative live and dead images of DPSCs confirmed the antioxidative stress effect of SHED-CM. (b) Cell viability was determined by CCK-8 analysis. All data are represented as the mean ± SD. The statistical significance was calculated using one-way analysis of variance (ANOVA) to compare groups. Represents ** p < 0.01, *** p < 0.001, ns = not significant, scale bar = 200 µm.

Figure 4

Cytokine profiling array. (a) The process of cytokine analysis is briefly described. SHED at the 5th passage achieved 70%–80% confluence and was replenished with serum-free αMEM. After 48 h, the conditioned medium (CM) was collected. Control conditioned media was collected from serum-free αMEM (SFM) under the same conditions as conditioned medium. All CM and SFM were stored at −80 °C to maintain the biological properties before cytokine antibody analysis. The Fullmoonbio Cytokine Profiling antibody array features 310 antibodies for profiling cytokines, and related biomarkers were used. The data were analyzed with the ExDEGA GraphicPlus and DAVID tools based on the Gene Ontology (GO) database. (b) A clustering heatmap based on the 50 top cytokines showed a significant difference between SFM and 100% CM. (c) Using the 50 top cytokine lists, the DAVID v6.8 analysis tool with the Gene Ontology Term Biological Process database and ExDEGA Graphic Plus were used to build a functional annotation chart. The graph shows that SHED CM containing growth factors and cytokines has a positive effect on cell proliferation, migration, survival, and osteogenic differentiation. Pathways/biological activities related to upregulation of proliferation, migration, odontoblast/osteogenic differentiation, and multiple biological functions (proliferation, migration, downregulation of cell apoptosis, differentiation including downregulation of cell apoptosis) are marked in different colored boxes (green, blue, orange, and red, respectively).

Figure 4

Cytokine profiling array. (a) The process of cytokine analysis is briefly described. SHED at the 5th passage achieved 70%–80% confluence and was replenished with serum-free αMEM. After 48 h, the conditioned medium (CM) was collected. Control conditioned media was collected from serum-free αMEM (SFM) under the same conditions as conditioned medium. All CM and SFM were stored at −80 °C to maintain the biological properties before cytokine antibody analysis. The Fullmoonbio Cytokine Profiling antibody array features 310 antibodies for profiling cytokines, and related biomarkers were used. The data were analyzed with the ExDEGA GraphicPlus and DAVID tools based on the Gene Ontology (GO) database. (b) A clustering heatmap based on the 50 top cytokines showed a significant difference between SFM and 100% CM. (c) Using the 50 top cytokine lists, the DAVID v6.8 analysis tool with the Gene Ontology Term Biological Process database and ExDEGA Graphic Plus were used to build a functional annotation chart. The graph shows that SHED CM containing growth factors and cytokines has a positive effect on cell proliferation, migration, survival, and osteogenic differentiation. Pathways/biological activities related to upregulation of proliferation, migration, odontoblast/osteogenic differentiation, and multiple biological functions (proliferation, migration, downregulation of cell apoptosis, differentiation including downregulation of cell apoptosis) are marked in different colored boxes (green, blue, orange, and red, respectively).

Figure 5

SHED CM enhanced DPSC osteogenic differentiation after exposure to 25 μM H₂O₂ for 24 h. (a) Relative gene expression, early marker at Day 3 and late marker at Day 7. (b) Alkaline phosphatase (ALP) and Alizarin red staining of DPSCs cultured in osteogenic differentiation media (OM) and 50% CM or SFM at Days 3, 7, 21 and 28. DPSCs cultured in growth media (αMEM, 10% FBS, 1% PS) were used as a negative control. At day 7, the upregulation of ALP activity expressed darker blue in SHED CM treatment. The red color of ARS showed stronger in SHED CM group due to the increase in the amount of mineral deposition, compared to SFM at day 21 and 28. All data are represented as the mean ± SD. The statistical significance was calculated using one-way analysis of variance (ANOVA) to compare groups. Represents * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = nonsignificant, scale bar = 200 µm.

Figure 6

Schematic diagram of the effect of SHED CM on DPSC culture and odontoblast differentiation. SHED CM increased cell proliferation and progenitor cell recruitment, protected cells from oxidative stress, and enhanced cell differentiation.