Effect of tetrahedral DNA nanostructures on proliferation and osteogenic differentiation of human periodontal ligament stem cells

Abstract

Objective: To explore the effects and underlying biological mechanisms of tetrahedral DNA nanostructures (TDNs) on the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs).

Materials and methods: Real-time cell analysis (RTCA) and CCK8 were used to screen the best concentration of TDN for PDLSCs. Cell proliferation and osteogenic differentiation were assessed after PDLSCs were treated with TDN. Data were analysed using one-way ANOVA.

Results: Tetrahedral DNA nanostructures could play a crucial role in accelerating the proliferation of PDLSCs and had the strongest promotive effect on PDLSCs at a concentration of 250 nmol/L. Simultaneously, the osteogenic differentiation of PDLSCs could be promoted significantly by TDNs and the finding displayed that the Wnt/β-catenin signalling pathway might be the underlying biological mechanisms of TDNs on promoting the osteogenic differentiation of PDLSCs.

Conclusion: Tetrahedral DNA nanostructure treatment facilitated the proliferation of PDLSCs, significantly promoted osteogenic differentiation by regulating the Wnt/β-catenin signalling pathway. Therefore, TDNs could be a novel nanomaterial with great potential for application to PDLSC-based bone tissue engineering.

Keywords: nanomaterials; osteogenic differentiation; periodontal ligament stem cells; tetrahedral DNA nanostructure.

Figure 1

Characterization of TDNs. A, The composition diagram of TDN. B, The result of 8% PAEG. C, The scanning image of TDN by AFM. D, The scanning image of TDN by TEM. Scale bars are 100 nm. E, After treatment with TDNs for 4 h, the ability of TDN to enter PDLSCs without the assistance of vectors was analysed by immunofluorescence staining. (nucleus: blue, cytoskeleton: green, cy5: red). Scale bars are 25 µm. F, After treatment with TDNs for 6 h, the ability of TDN to enter PDLSCs without the assistance of vectors was analysed by immunofluorescence staining. (nucleus: blue, cytoskeleton: green, cy5: red). Scale bars are 25 µm.

Figure 2

Effect of TDNs on proliferation. A, The proliferation of PDLSCs was observed by RTCA. B, Statistical analysis of RTCA results in 12, 24, 36 and 48 h. C, Statistical analysis of CCK8 results. Data are presented as means ±standard deviations (n = 3). ***P < 0.001. D, Flow cytometry was used to explore the role of TDNs on the PDLSCs by detecting cell cycle changes. E, Statistical analysis of cell cycle distribution. Data are presented as means ±standard deviations (n = 3). ***P < 0.001.

Figure 3

Effect of TDNs on osteogenic differentiation in PDLSCs. A, The quantitative analysis results of gene expression obtained by RT‐PCR. Data are presented as means ± standard deviations (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. B, Immunofluorescent micrographs of RUNX 2 protein and OPN protein. (nucleus: blue, RUNX 2 protein and OPN protein: green). Scale bars are 25 µm. C, The results of RUNX 2 and OPN proteins expression measured by WB. D, Statistical analysis of WB. Data are presented as means ± standard deviations (n = 3). ***P < 0.001.

Figure 4

Enhancement of calcium nodule formation and ALP activity. A, Osteogenic differentiation was monitored by ALP staining at day 7. B, Osteogenic differentiation was detected by Alizarin Red staining (bottom) at day 14.

Figure 5

The potential mechanism through which TDN regulated the osteogenic differentiation of PDLSCs. A, Immunofluorescent micrographs of β‐catenin protein, GSK3‐β protein and LEF1 protein. (nucleus: blue, β‐catenin, GSK3‐β protein and LEF1 protein: green). Scale bars are 25 µm. B, WB analysis of the β‐catenin, GSK3‐β and LEF 1 proteins expression. C, Statistical analysis of WB. Data are presented as means ± standard deviations (n = 3). ***P < 0.001.

Figure 6

As novel DNA nanomaterials, TDNs were employed to stimulate PDLSCs to regulate its biological behaviours. The results demonstrated that TDNs could facilitate the proliferation of PDLSCs, and the optimum work concentration was 250 nmol/L. In addition, the findings also demonstrated that TDNs could greatly enhance the osteogenic differentiation of PDLSCs