Human β-defensin 3-combined gold nanoparticles for enhancement of osteogenic differentiation of human periodontal ligament cells in inflammatory microenvironments

Abstract

Objective: It is a great challenge to absorb and conduct biophysicochemical interactions at the nano-bio interface. Peptides are emerging as versatile materials whose function can be programmed to perform specific tasks. Peptides combined nanoparticles might be utilized as a new approach of treatment. Human β-defensin 3 (hBD3), possesses both antimicrobial and proregeneration properties. Gold nanoparticles (AuNPs) have shown promising applications in the field of tissue engineering. However, the coordinating effects of AuNPs and hBD3 on human periodontal ligament cells (hPDLCs) remain unknown. In this study, we systematically investigated whether AuNPs and hBD3 would be able to coordinate and enhance the osteogenic differentiation of hPDLCs in inflammatory microenvironments, and the underlying mechanisms was explored.

Methods: hPDLCs were stimulated with E. coli-LPS, hBD3 and AuNPs. Alkaline phosphatase (ALP) and alizarin red S staining were used to observe the effects of hBD3 and AuNPs on the osteogenic differentiation of hPDLCs. Real-time PCR and western blot were performed to evaluate the osteogenic differentiation and Wnt/β-catenin signaling pathway related gene and protein expression.

Results: In the inflammatory microenvironments stimulated by E. coli-LPS, we found that AuNPs and hBD3 increased the proliferation of hPDLCs slightly. In addition, hBD3-combined AuNPs could significantly enhance ALP activities and mineral deposition in vitro. Meanwhile, we observed that the osteogenic differentiation-related gene and protein expressions of ALP, collagenase-I (COL-1) and runt-related transcription factor 2 (Runx-2) were remarkably upregulated in the presence of hBD3 and AuNPs. Moreover, hBD3-combined AuNPs strongly activated the Wnt/β-catenin signaling pathway and upregulated the gene and protein expression of β-catenin and cyclin D1. Furthermore, hBD3-combined AuNPs induced osteogenesis, which could be reversed by the Wnt/β-catenin signaling pathway inhibitor (ICG-001).

Conclusion: The present study demonstrated that hBD3 combined AuNPs could significantly promote the osteogenic differentiation of hPDLCs in inflammatory microenvironments via activating the Wnt/β-catenin signaling pathway.

Keywords: Wnt/β-catenin signaling; gold nanoparticles; hBD3; inflammatory microenvironments; osteogenesis; periodontal ligament cells.

Figure 1

Effects of hBD3 on the cell viability and osteogenic differentiation of hPDLCs in inflammatory microenvironments. Notes: hPDLCs were treated with hBD3 (5 μg/mL) and E. coli-LPS (1 μg/mL). (A) The cell viability of hPDLCs was analyzed with a CCK-8 assay on day 7. (B) ALP staining and (C) ALP activity of hPDLCs after hBD3 treatment. (D) ALP, COL-1, and Runx-2 mRNA expressions on day 7 analyzed by real-time PCR. ^#P<0.05 and ^##P<0.01 compared with the control group; *P<0.05, **P<0.01, and ****P<0.0001. Abbreviations: ALP, alkaline phosphatase; CCK-8, cell counting kit-8; COL-1, collagenase-I; E. coli, Escherichia coli; hBD3, human β-defensin 3; hPDLCs, human periodontal ligament cells; LPS, lipopolysaccharides; PCR, polymerase chain reaction; Runx-2, runt-related transcription factor 2.

Figure 2

Effects of hBD3-combined AuNPs on cell viability of hPDLCs and cellular uptake of AuNPs in inflammatory microenvironments. Notes: hPDLCs were treated with hBD3 (5 μg/mL), AuNPs (45 nm, 10 μM), and E. coli-LPS (1 μg/mL). (A) The biocompatibility of hPDLCs measured by CCK-8 on day 7. (B) TEM images of uptake of AuNPs on day 7. Arrows indicate internalized AuNPs. ^#P<0.05 and ^##P<0.01 compared with the control group. Abbreviations: AuNPs, gold nanoparticles; CCK-8, cell counting kit-8; E. coli, Escherichia coli; hBD3, human β-defensin 3; hPDLCs, human periodontal ligament cells; LPS, lipopolysaccharides; TEM, transmission electron microscopy.

Figure 3

Effects of hBD3-combined AuNPs on the ALP activity and calcium deposition of hPDLCs in inflammatory microenvironments. Notes: hPDLCs were treated with hBD3 (5 μg/mL), AuNPs (45 nm, 10 μM), and E. coli-LPS (1 μg/mL). (A) ALP staining on day 7 and calcium deposition staining with ARS on day 21. (B) ALP activity levels on day 7 and (C) calcium deposition quantification on day 21. ^#P<0.05, ^##P<0.01, ^###P<0.001, and ^####P<0.0001 compared with the group control; *P<0.05 and ****P<0.0001. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S; AuNPs, gold nanoparticles; E. coli, Escherichia coli; hBD3, human β-defensin 3; hPDLCs, human periodontal ligament cells; LPS, lipopolysaccharides.

Figure 4

Effects of hBD3-combined AuNPs on osteogenic gene and protein expressions of hPDLCs in inflammatory microenvironments. Notes: hPDLCs were treated with hBD3 (5 μg/mL), AuNPs (45 nm, 10 μM), and E. coli-LPS (1 μg/mL). (A) The mRNA expression levels of ALP, COL-1, and Runx-2 analyzed by real-time PCR on day 7. (B) The protein expression levels of ALP, COL-1, and Runx-2 analyzed by Western blot on day 7. ^#P<0.05, ^##P<0.01, ^###P<0.001, and ^####P<0.0001 compared with the control group; *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Abbreviations: ALP, alkaline phosphatase; AuNPs, gold nanoparticles; COL-1, collagenase-I; E. coli, Escherichia coli; hBD3, human β-defensin 3; hPDLCs, human periodontal ligament cells; LPS, lipopolysaccharides; PCR, polymerase chain reaction; Runx-2, runt-related transcription factor 2.

Figure 5

Effects of Wnt/β-catenin signaling pathway on osteogenic differentiation of hPDLCs induced by hBD3-combined AuNPs in inflammatory microenvironments. Notes: hPDLCs were treated with hBD3 (5 μg/mL), AuNPs (45 nm, 10 μM), E. coli-LPS (1 μg/mL) and ICG-001 (10 μM). (A) The Wnt/β-catenin pathway target gene cyclin D1 mRNA expression on day 7 analyzed by real-time PCR. (B) β-Catenin and cyclin D1 protein expressions on day 7 analyzed by Western blot. (C) ICG-001 blocked the mRNA expression of the target gene cyclin D1 and (D) the protein expressions of β-catenin and cyclin on day 7. ^#P<0.05, ^##P<0.01, ^###P<0.001, and ^####P<0.0001 compared with the control group; *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Abbreviations: AuNPs, gold nanoparticles; E. coli, Escherichia coli; hBD3, human β-defensin 3; hPDLCs, human periodontal ligament cells; LPS, lipopolysaccharides; PCR, polymerase chain reaction.

Figure 6

Effects of ICG-001 on osteogenic differentiation of hPDLCs induced by hBD3-combined AuNPs in inflammatory microenvironments. Notes: hPDLCs were treated with hBD3 (5 μg/mL), AuNPs (45 nm, 10 μM), E. coli-LPS (1 μg/mL) and ICG-001 (10 μM). (A) ALP staining on day 7 and mineralized nodules staining with ARS on day 21 and (B) ALP activity levels on day 7 and mineralized nodules activity levels on day 21. (C) ALP, COL-1, and Runx-2 mRNA expressions on day 7 analyzed by real-time PCR and (D) ALP, COL-1, and Runx-2 protein expressions on day 7 analyzed by Western blot. ^#P<0.05, ^###P<0.001, and ^####P<0.0001 compared with the control group; *P<0.05, ***P<0.001, and ****P<0.0001. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S; AuNPs, gold nanoparticles; COL-1, collagenase-I; E. coli, Escherichia coli; hBD3, human β-defensin 3; hPDLCs, human periodontal ligament cells; PCR, polymerase chain reaction; Runx-2, runt-related transcription factor 2.