Evaluating the potential of an amelogenin-derived peptide in tertiary dentin formation

Abstract

Several novel biomaterials have been developed for dental pulp capping by inducing tertiary dentin formation. The aim of this study was to evaluate the effect of QP5, an amelogenin-based peptide, on the mineralization of dental pulp cells (DPCs) in vitro and in vivo. The cell viability of human DPCs (hDPCs) after treatment with QP5 was determined using the Cell Counting Kit-8 (CCK-8). Migration of hDPCs was assessed using scratch assays, and the pro-mineralization effect was determined using alkaline phosphatase (ALP) staining, alizarin red staining and the expression of mineralization-related genes and proteins. The results showed that QP5 had little effect on the cell viability, and significantly enhanced the migration capability of hDPCs. QP5 promoted the formation of mineralized nodules, and upregulated the activity of ALP, the expression of mRNA and proteins of mineralization-related genes. A pulp capping model in rats was generated to investigate the biological effect of QP5. The results of micro-computed tomography and haematoxylin and eosin staining indicated that the formation of tertiary dentin in QP5-capping groups was more prominent than that in the negative control group. These results indicated the potential of QP5 as a pulp therapy agent.

Keywords: amelogenin-derived peptide; dental pulp cells; pulp capping; tertiary dentin.

Figure 1.

QP5 treatment enhanced the migration ability of hDPCs without cytotoxicity. The effects of QP5 on cytotoxicity and migration of hDPCs. (A) Light microscopy images of scratch wound assays, (B) Quantitative analysis of migration rates at 12 and 24 h. Scratch wound assays revealed that both 10 and 50 μg/mL concentrations of QP5 significantly promoted the motility of hDPCs (*P < 0.05). (C) Effect of QP5 on cell viability determined using the CCK-8 assay, there was no statistically significant difference between the groups (P > 0.05). hDPCs, human dental pulp cells; CCK-8, cell counting kit-8.

Figure 2.

QP5 treatment promoted the mineralization of hDPCs hDPCs were cultured with negative control (Con), 10 μg/mL QP5 (Q10) and 50 μg/mL QP5 (Q50), respectively. (A) ALP staining showed promotion of mineralization with Q10 and Q50 of hDPCs on days 3, 5 and 7 after treatment. ALP activity increased in the Q10 and Q50 group on days 3, 5 and 7, with odontogenic induction. On Day 14, alizarin red staining was performed to indicate the effects of QP5 on the mineralization of hDPCs. (B) On days 7 and 14 after treatment, the expression levels of genes related to odontogenesis (ALP, OCN, DSPP, OPN, DMP1 and OSX) were evaluated using qPCR. (C) The expression levels of the genes related to odontogenesis (DSP, OPN, DMP and OSX) were evaluated using the Western blots on days 7 and 14. (D) The result of the western blot was standardized with GAPDH (*P < 0.05). hDPSs, human dental pulp cells; qPCR, quantitative polymerase chain reaction; ALP, alkaline phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3.

QP5 induced the formation of hard tissues through direct pulp capping in rats. (A) Establishment of the pulp capping model. The teeth were cleaned (a) and their pulps were mechanically exposed (b), the cavity was sealed with GIC, after capping with different materials (c). the ratio (BV/TV) of total dentine volume (BV) and total tooth volume (TV) was determined at 4 and 8 weeks (B) (n = 6, *P < 0.05). (C) Representative μCT images of tertiary dentin formation the pulp was exposed mechanically and divided into three groups: the CH (a–e), QP5 (f–j) and PBS control groups (k–o). Three-dimensional sagittal reconstruction images were presented (a, f, k). Representative sagittal μCT images (b, d, g, i, l, n) and color-coded images of tertiary dentin according to the degree of mineral density (c, e, h, j, m, o) are shown. Areas enclosed in the red rectangle indicate tertiary dentin. CH, calcium hydroxide; P, pulp.

Figure 4.

QP5-treated dental pulp showed a decreased inflammatory response and obvious tertiary dentin formation histological images indicating the pulpal response after pulp capping. The rat molars were divided into three groups: the CH, QP5 and PBS groups, based on the different materials used. The pulp was direct capped used different materials after 4 weeks (A) and 8 weeks (B). Reparative dentin formation was observed adjacent to the existing dentin in the CH and QP5 groups. However, the hard tissue demonstrated an irregular pattern that differed from primary dentin. P, pulp; TD, tertiary dentin; D, dentin; CH, calcium hydroxide; PBS, phosphate-buffered saline.

Figure 5.

QP5 had little effect on lipopolysaccharide-stimulated inflammatory model in hDPCs LPS-stimulated inflammatory hDPCs were cultured with negative control (Con), 10 μg/mL QP5 (Q10) and 50 μg/mL QP5 (Q50), respectively. (A) The CCK-8 assay was used to assess the viability of hDPCs for 12 and 24 h after treatment with different concentrations of E. coli LPS. Values are expressed as means ± SD of three replicates of one representative experiment. (B) The protein levels of IL-6 were determined with the use of ELISA kits (*P < 0.05). (C) The protein levels of TNF-α were determined with the use of ELISA kits. hDPCs, human dental pulp cells; CCK-8, cell counting kit-8; ELISA, enzyme-linked immunosorbent assay.