Size-dependent Effects of Gold Nanoparticles on Osteogenic Differentiation of Human Periodontal Ligament Progenitor Cells

Abstract

Gold nanoparticles (AuNPs) have been reported to promote osteogenic differentiation of mesenchymal stem cells and osteoblasts, but little is known about their effects on human periodontal ligament progenitor cells (PDLPs). In this study, we evaluated the effects of AuNPs with various diameters (5, 13 and 45 nm) on the osteogenic differentiation of PDLPs and explored the underlying mechanisms. 5 nm AuNPs reduced the alkaline phosphatase activity, mineralized nodule formation and expression of osteogenic genes, while 13 and 45 nm AuNPs increased these osteogenic markers. Compared with 13 nm, 45 nm AuNPs showed more effective in promoting osteogenic differentiation. Meanwhile, autophagy was up-regulated by 13 and 45 nm AuNPs but blocked by 5 nm AuNPs, which corresponded with their effects on osteogenic differentiation and indicated that autophagy might be involved in this process. Furthermore, the osteogenesis induced by 45 nm AuNPs could be reversed by autophagy inhibitors (3-methyladenine and chloroquine). These findings revealed that AuNPs affected the osteogenic differentiation of PDLPs in a size-dependent manner with autophagy as a potential explanation, which suggested AuNPs with defined size could be a promising material for periodontal bone regeneration.

Keywords: autophagy; gold nanoparticles; human periodontal ligament progenitor cells; osteogenic differentiation; size-dependent..

Figure 1

Characterization of AuNPs. (A) photographs (from left to right: 5, 13 and 45 nm), (B) TEM images, (C) size distribution, measured by DLS and (D) UV-vis absorption spectra of AuNPs with different sizes.

Figure 2

Biocompatibility and cellular uptake of AuNPs. PDLPs were treated with AuNPs with the diameters of 5, 13 and 45 nm, respectively. (A) Light microscope images of PDLPs treated with AuNPs (10 μM) for 48 h. (B) Effects of AuNPs on the viability of PDLPs were measured using CCK-8 at 48 h. **p<0.01. (C) TEM images of PDLPs treated with AuNPs (10 μM) for 3 days. (V-VIII) were the high magnification images of the indicated portion in (I-IV), respectively. Arrows indicate internalized AuNPs, the labels “AP” indicate autophagosome, and the labels “AL” indicate autolysosome.

Figure 3

Effects of AuNPs on the ALP activity and mineralization. PDLPs were treated with AuNPs (5, 13 and 45 nm) at concentration of 10 μM. (A) ALP activity levels on day 3 and 7, (B) ALP staining on day 7, and (C) mineralized nodules stained with alizarin red S and von Kossa on day 21. *p<0.05, **p<0.01.

Figure 4

Effects of AuNPs on osteogenic gene expression. PDLPs were treated with AuNPs (5, 13 and 45 nm) at concentration of 10 μM. (A) Real-time PCR analysis of Runx2, ALP, COL1, OPN, and OCN mRNA expression on day 3, 5 and 7. (B) Western blot analysis of Runx2, ALP, COL1 and OPN protein expression on day 7. *p<0.05, **p<0.01.

Figure 5

Effects of AuNPs on autophagy. PDLPs were treated with AuNPs (5, 13 and 45 nm) at concentration of 10 μM. (A) Real-time PCR analysis of LC3 and Beclin1 mRNA expression on day 1 and 3. (B) Western blot analysis of LC3-II and p62 expression on day 3. *p<0.05, **p<0.01.

Figure 6

Effects of autophagy inhibitors on the 45 nm AuNP-induced osteogenic differentiation. PDLPs were treated with 45 nm AuNPs (10 μM) alone, or combine with 3-MA (2 mM) or CQ (5 μM). (A) ALP staining on day 7, alizarin red S and von Kossa staining on day 21. (B) Real-time PCR analysis of Runx2, ALP, COL1, OPN, and OCN mRNA expression on day 5. (C) Western blot analysis of Runx2, ALP, COL1 and OPN protein expression on day 7. P values for the comparison with control were selectively shown. *p<0.05, **p<0.01.