Potential Application of Human β-Defensin 4 in Dental Pulp Repair

Abstract

When pulp tissue is damaged by caries or trauma, vital pulp therapy (VPT) can help preserve the pulp tissue for long-term retention of teeth. However, the choice of pulp capping agent used in VPT is important for the successful preservation of the pulp tissue. Here we investigated the expression and biological function of human β-defensin 4 (HBD4) in dental pulp stem cells (DPSC) and explored its potential as a pulp capping agent. We examined the expression of HBD4 in DPSC in vitro using qPCR and immunofluorescence staining. We also looked at the effect of HBD4 on inflammatory factors in lipopolysaccharide (LPS)-stimulated DPSC, and its effects on mineralizing cell phenotype differentiation, via qPCR and western blot. Finally, we examined the ability of HBD4 to promote the restoration of the pulp-dentin complex in vivo, using male Wistar rats with reversible pulpitis. We found HBD4 was highly expressed in DPSC stimulated by TNF-α and IL-1α. HBD4 down-regulated the expression of inflammatory mediators (i.e., IL-1α, IL-1β, IL-6, TNF-α) in LPS-stimulated DPSC, and suppressed MAPK activity and the NF-κB pathway. HBD4 also enhanced the differentiation of DPSC into osteoblasts or odontoblasts, potentially by modulating the Notch pathway. Furthermore, HBD4 controlled the degree of pulp inflammation in a rat model of reversible pulpitis and induced the formation of restorative dentin. Together our findings indicate HBD4 may be a useful pulp capping agent for use in VPT.

Keywords: anti-inflammatory; dental pulp stem cells; human β-defensin 4; stem cell differentiation; vital pulp therapy.

FIGURE 1

Characterization of dental pulp stem cells (DPSC). (A) Expression of DPSC surface markers. DPSC could be induced to form mineralized nodules (B), lipid droplets (C), and chondrocytes (D).

FIGURE 2

Expression of human beta defensin (HBD) in dental pulp stem cells (DPSC) and the biological activity of HBD4. (A) Expression of HBD1-4 in DPSC determined by qPCR and agarose gel electrophoresis. (B) Relative expression of HBD1-4 in CT and TNF-α + IL-1α stimulation group. (C) The expression of HBD4 in DPSC detected by immunofluorescence (fluorescence microscope, 1000 ×). Blue fluorescence indicates the nucleus stained by DAPI, and green fluorescence indicates HBD4 expression. (D) Effect of HBD4 on cell viability in DSPC. DPSC were treated with different concentrations of HBD4 (0, 5, 10, 20, and 50 μg/mL) for 1, 2, and 3 days before measuring cell viability using the CCK-8 assay. (E) Representative images of migrated cells in control, 20 μg/mL HBD4, 40 μg/mL HBD4, and 10% FBS groups in transwell assay. (F) Relative comparison of migrated cell numbers among the four groups described in (E). All experiments were performed three times. ns, no significance; ^∗p < 0.05; ^∗∗p < 0.01; ^****P < 0.0001 (CT, control; 3D, 3 days; 4D, 4 days).

FIGURE 3

Anti-inflammatory effects of HBD4 in dental pulp stem cells (DPSC). (A) Effects of HBD4 on lipopolysaccharide (LPS)-induced expression of inflammatory mediators in DPSC. Statistical comparisons are between LPS alone vs. LPS with HBD4 at the same time point in each group. (B) The effect of HBD4 on the distribution of p65 in DPSC detected by immunofluorescence (fluorescence microscope, 1000 ×). Blue fluorescence indicates the nucleus stained by DAPI, and green fluorescence marks p65 expression. (C) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cellular proteins. (D) Quantification of the band density observed in (C) determined using Image J software. All experiments were performed three times. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance (CT, control; 6H, 6 h; 12H, 12 h).

FIGURE 4

Effects of HBD4 on mineralizing cell phenotype differentiation in dental pulp stem cells (DPSC). DPSC were treated with HBD4 (10 μg/mL) and lipopolysaccharide (LPS, 1 μg/mL). Alkaline phosphatase (ALP) staining at 7 days (A,B) and alizarin red staining (ARS) staining at 21 days (C,D) of DPSC after HBD4 and LPS treatment. Effects of HBD4 on osteoblast/odontoblast gene (E) and osteoblast/odontoblast protein (F,G) expressions of DPSC in control and inflammatory microenvironments at 21 days. Effects of HBD4 on (H,I) HES1 expression detected by western blot. All experiments were performed three times. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance (CT, control).

FIGURE 5

Effects of HBD4 in a rat model of reversible pulpitis. (A) HE staining, (B) Masson staining, and (C) DSPP/VEGF immunohistochemical staining of the maxillary first molar of Wistar rats at week 8 after the application of different pulp capping materials (optical microscope, 1000x). CT, control group; GS, gelatin sponge group; GS + HBD4, gelatin sponge and HBD4 group; MTA, mineral trioxide aggregate group.