An amelogenin-based peptide hydrogel promoted the odontogenic differentiation of human dental pulp cells

Abstract

Amelogenin can induce odontogenic differentiation of human dental pulp cells (HDPCs), which has great potential and advantages in dentine-pulp complex regeneration. However, the unstability of amelogenin limits its further application. This study constructed amelogenin self-assembling peptide hydrogels (L-gel or D-gel) by heating-cooling technique, investigated the effects of these hydrogels on the odontogenic differentiation of HDPCs and explored the underneath mechanism. The critical aggregation concentration, conformation, morphology, mechanical property and biological stability of the hydrogels were characterized, respectively. The effects of the hydrogels on the odontogenic differentiation of HDPCs were evaluated via alkaline phosphatase activity measurement, quantitative reverse transcription polymerase chain reaction, western blot, Alizarin red staining and scanning electron microscope. The mechanism was explored via signaling pathway experiments. Results showed that both the L-gel and D-gel stimulated the odontogenic differentiation of HDPCs on both Day 7 and Day 14, while the D-gel showed the highest enhancement effects. Meanwhile, the D-gel promoted calcium accumulation and mineralized matrix deposition on Day 21. The D-gel activated MAPK-ERK1/2 pathways in HDPCs and induced the odontogenic differentiation via ERK1/2 and transforming growth factor/smad pathways. Overall, our study demonstrated that the amelogenin peptide hydrogel stimulated the odontogenic differentiation and enhanced mineralization, which held big potential in the dentine-pulp complex regeneration.

Keywords: amelogenin; human dental pulp cells; odontogenic differentiation; peptide hydrogel.

Graphical abstract

Figure 1.

Chemical structures of (A) AMG-P8 (KWYQNMIR); (B) Comp.1 (Nap-GFFYGKWYQNMIR) and (C) Comp.2 (Nap-G^DF^DF^DYGKWYQNMIR). TEM and optical images of (D) L-gel and (E) D-gel. Dynamic frequency and strain sweep measurements of L-gel and D-gel (F, G). (H) Circular dichroism spectra of L-gel and D-gel. Determination of CAC values of L-gel (I) and D-gel (J). (K) Compound remaining of the AMG-P8, L-gel and D-gel for an initial concentration of 1.0 mM incubated with 0.1 mg/ml of proteinase K for 24 h.

Figure 2.

Effects of AMG-P8 and supramolecular hydrogels on the odontogenic differentiation of HDPCs. (A, B) The relative mRNA expression of DSPP, DMP-1, RUNX-2, COL-1 on Day 7 and Day 14 (n = 3). (C, D) The protein expression of DSPP, DMP-1 and ALP on Day 7 and Day 14 (n = 3). (E, F) The quantitative protein expression analysis. (G, H) The protein expression and quantitative analysis of DSPP, DMP-1 and ALP in the D-gel and EMPs groups on Day 14. (I) ALP activity of all groups. The data were presented as means ± SD. *P < 0.05, **P < 0.01.

Figure 3.

(A) Effects of AMG-P8 and supramolecular hydrogels on the mineralization of HDPCs. Alizarin red staining on Day 21 (n = 3). Scale bar = 100 μm. (B) The quantitative analysis of alizarin red staining. (C–F) The microscopic morphology of mineralized nodules on Day 21. Scale bar = 50 μm. (G) HDPCs in D-gel group. Scale bar = 20 μm. (H) HDPCs in EMPs group. Scale bar = 4 μm. (I) Calcium–phosphorus (Ca/P) ratio of all groups (n = 3). The data were presented as means ± SD. *P < 0.05, **P < 0.01.

Figure 4.

D-Gel activated ERK 1/2 pathways in HDPCs. (A) Western blot analysis of p-ERK1/2 and ERK1/2 from HDPCs pretreated with specific inhibitors U0126 for 1 h followed by stimulation with D-gel for 1 h (n = 3). (B) The quantitative protein analysis of p-ERK1/2. (C, D) Representative images of ERK1/2 and p-ERK1/2 immunofluorescence staining of HDPCs after being treated with D-gel for 1 h, nuclei were counter stained with DAPI. Scale bar = 20 μm (n = 3). (E, F) The protein expression and quantitative result of AP-1 family (Jun B, p-c Jun, c Fos and p-c Fos) after being treated with D-gel for 1 h (n = 3). The data were presented as means ± SD. *P < 0.05, **P < 0.01.

Figure 5.

D-Gel enhanced HDPCs odontogenic differentiation through MAPK-ERK1/2 and TGF-β/smad signaling pathway. (A, B) The protein expression of DSPP and ALP of HDPCs treated with D-gel and U0126 for 7 and 14 days (n = 3). (C, D) The quantitative protein analysis of DSPP and ALP. (E) Alizarin red staining of HDPCs treated with D-gel and U0126 for 21 days (n = 3). (F) The quantitative analysis of alizarin red staining. (G, H) The protein expression of smad2/3 and p-smad2/3 after being treated with D-gel and U0126 for 7 and 14 days (n = 3). (I, J) The quantitative protein analysis of p-smad2/3. The data were presented as means ± SD. *P < 0.05, **P < 0.01.