Effect of an Experimental Direct Pulp-capping Material on the Properties and Osteogenic Differentiation of Human Dental Pulp Stem Cells

Abstract

Effective pulp-capping materials must have antibacterial properties and induce dentin bridge formation; however, many current materials do not satisfy clinical requirements. Accordingly, the effects of an experiment pulp-capping material (Exp) composed of an antibacterial resin monomer (MAE-DB) and Portland cement (PC) on the viability, adhesion, migration, and differentiation of human dental pulp stem cells (hDPSCs) were examined. Based on a Cell Counting Kit-8 assay, hDPSCs exposed to Exp extracts showed limited viability at 24 and 48 h, but displayed comparable viability to the control at 72 h. hDPSC treatment with Exp extracts enhanced cellular adhesion and migration according to in vitro scratch wound healing and Transwell migration assays. Exp significantly upregulated the expression of osteogenesis-related genes. The hDPSCs cultured with Exp exhibited higher ALP activity and calcium deposition in vitro compared with the control group. The novel material showed comparable cytocompatibility to control cells and promoted the adhesion, migration, and osteogenic differentiation of hDPSCs, indicating excellent biocompatibility. This new direct pulp-capping material containing MAE-DB and PC shows promise as a potential alternative to conventional materials for direct pulp capping.

Figure 1. Flow cytometry of mesenchymal-related antigens in human dental pulp stem cells (hDPSCs).

Representative diagrams are given for the negative control, CD146, CD90, CD45, CD34, and CD29 expression.

Figure 2

Results of cell viability assays after hDPSCs were exposed to various material extracts at 24 h (A), 48 h (B), and 72 h (C). The viability of cells under treatments with different material extracts was investigated using the Cell Counting Kit-8. Data are shown as means ± standard deviation of three independent experiments. *P < 0.05 represents a significant change compared with the control.

Figure 3. The effect of material extracts on adhesion ability in hDPSCs.

Cells were incubated with various material extracts for 1 h. Adherent cells were fixed and stained. The coloured solution was quantified at 595 nm on a microplate reader. (A) Representative diagrams for stained cells. (B) Quantification of the adherent cells. Data are presented as means ± standard deviation and measurements were performed in triplicate, with results summarised as the mean for each experiment. *P < 0.05 represents a significant change compared with the control.

Figure 4. Effects of material extracts on migration in hDPSCs.

(A) Wound-healing assay. Cells were incubated with different material extracts for 24 h. Microphotographs of the scratches were obtained at 24-h post-wounding. (B) Cell migration assays were performed using a two-chamber Transwell system. Cells were treated with different material extracts for 24 h, and the migrated cells were fixed and stained. Representative photos of migrated hDPSCs were observed under a phase-contrast microscope. (C) Quantification of migrated cells. Data are presented as means ± standard deviation of three independent experiments. *P < 0.05 represents a significant change compared with the control.

Figure 5. Alizarin Red staining and quantification.

hDPSCs were co-cultured with different material extracts supplemented with serum-free α-MEM for 2 weeks. Normal culture medium containing α-MEM supplemented with 10% foetal bovine serum was used as blank control medium (Control/N). Cells cultured with osteogenic differentiation medium were used as positive controls (Control/M). (A) Mineralised nodules formed by differentiated cells after incubation were stained with Alizarin Red S. (B) Quantitative measurements of Alizarin Red S staining of hDPSCs. The results are expressed as means ± standard deviation and *P < 0.05 represents a significant difference compared with the control/N group.

Figure 6. Alkaline phosphatase (ALP) staining and quantification.

Cells were co-cultured with different material extracts supplemented with serum-free α-MEM. Normal culture medium containing α-MEM supplemented with 10% FBS was used as blank control medium (Control/N). Cells cultured with osteogenic differentiation medium were used as positive controls (Control/M). ALP staining after 9 days of cultivation was performed using an ALP Staining Kit (A). ALP activity was evaluated at 3 days, 5 days, 7 days, and 9 days using an ALP Kit (B). The results are expressed as means ± standard deviation and *P < 0.05 represents a significant difference compared with the control/N group.

Figure 7. The effects of material extracts on the expression of ALP, BMP-1, ON, DSPP, and OCN in hDPSCs.

Cells were co-cultured with different material extracts supplemented with serum-free α-MEM for 2 weeks. Normal culture medium containing α-MEM supplemented with 10% FBS was used as blank control medium (Control/N). Cells cultured with osteogenic differentiation medium were used as positive controls (Control/M). ALP, BMP-1, ON, DSPP, and OCN mRNA expression levels in hDPSCs were determined by qRT-PCR. The results are expressed as means ± standard deviation and *P < 0.05 represents a significant difference compared with the control/N group.