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. 2017 Jun 7:12:4323-4333.
doi: 10.2147/IJN.S136281. eCollection 2017.

Involvement of autophagy in tantalum nanoparticle-induced osteoblast proliferation

Chengrong Kang  1   2 , Limin Wei  1 , Bin Song  1 , Liangjiao Chen  3 , Jia Liu  1 , Bin Deng  4 , Xuan Pan  2 , Longquan Shao  1
Affiliations

Involvement of autophagy in tantalum nanoparticle-induced osteoblast proliferation

Chengrong Kang et al. Int J Nanomedicine. .

Abstract

Porous tantalum (Ta) implants are highly corrosion resistant and biocompatible, and they possess significantly better initial stability than that of conventional titanium (Ti) implants. During loading wear, Ta nanoparticles (Ta-NPs) that were deposited on the surface of a porous Ta implant are inevitably released and come into direct contact with peri-implant osteoblasts. The wear debris may influence cell behavior and implant stabilization. However, the interaction of Ta-NPs with osteoblasts has not been clearly investigated. This study aimed to investigate the effect of Ta-NPs on cell proliferation and their underlying mechanism. The Cell Counting Kit-8 (CCK-8) assay was used to measure the cell viability of MC3T3-E1 mouse osteoblasts and showed that Ta-NP treatment could increase cell viability. Then, confocal microscopy, Western blotting, and transmission electron microscopy were used to confirm the autophagy induced by Ta-NPs, and evidence of autophagy induction was observed as positive LC3 puncta, high-LC3-II expression, and autophagic vesicle ultrastructures. The CCK-8 assay revealed that the cell viability was further increased and decreased by the application of an autophagy inducer and inhibitor, respectively. In addition, pre-treatment with autophagy inhibitor 3-methyladenine (3-MA) inhibited the Ta-NP-induced autophagy. These results indicate that the Ta-NPs can promote cell proliferation, that an autophagy inducer can further strengthen this effect and that an autophagy inhibitor can weaken this effect. In conclusion, autophagy was involved in Ta-NP-induced cell proliferation and had a promoting effect.

Keywords: autophagy; osteoblast; proliferation; tantalum nanoparticles.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Characterization of Ta-NPs. TEM (A) and SEM (B) images showing primarily spherical shapes. Magnification ×200,000. (C) DLS measurements. The red, green and blue lines indicate three independent experiments. Abbreviations: DLS, dynamic light scattering; SEM, scanning electron microscopy; Ta-NPs, Ta nanoparticles; TEM, transmission electron microscopy.
Figure 2
Figure 2
Cell viability of MC3T3-E1. The MC3T3-E1 cells were cultured with Ta-NPs at 1, 10, or 20 μg/mL for 24 h; cells cultured with α-MEM alone served as a control. The cell viability of all the Ta-NP-treated groups was higher than that of the control group, especially for the 10 and 20 μg/mL groups (P<0.05). Experiments were repeated three times with at least three wells per condition. Note: *P<0.05 versus control group. Abbreviations: α-MEM, alpha-modified Eagle’s medium; Ta-NPs, Ta nanoparticles.
Figure 3
Figure 3
Cell viability of MC3T3-E1. MC3T3-E1 cells were cultured with 10 and 20 μg/mL Ta-NPs for 24 h, with or without pretreatment with rapamycin (200 nM) or 3-MA (10 mM) for 1 h; cells were also treated with rapamycin or 3-MA alone to identify whether they have an effect on cell viability. Notes: The pretreatment with rapamycin and 3-MA accordingly increased and decreased the cell viability, respectively. Especially, the 20 μg/mL group pretreated with 3-MA had decreased cell viability of nearly 50%. These results showed that rapamycin or 3-MA alone had no obvious influence on cell viability, while autophagy induction further increased cell viability and autophagy inhibition decreased cell viability. Experiments were repeated three times with at least three wells per condition. *P<0.05, ***P<0.001 versus control group; $$P<0.01, $$$P<0.001 versus rapamycin-pretreated group. Abbreviations: 3-MA, 3-methyladenine; Ta-NPs, Ta nanoparticles.
Figure 4
Figure 4
Confocal immunofluorescence images. Notes: The MC3T3-E1 cells were cultured with 20 μg/mL Ta-NPs for 24 h, with or without pretreatment with 3-MA (10 mM) for 1 h; cells treated with α-MEM alone served as a negative control group, and cells treated with 200 nM rapamycin served as a positive control group. The LC3-labeled green puncta (a characteristic of autophagosomes) could be clearly observed in the positive control group and in the 20 μg/mL Ta-NP-treated group but were nearly indistinguishable in the negative control group. When cells were pretreated with 3-MA, the amount of green puncta decreased sharply. Experiments were repeated three times. Scale bar: 40 μm. Magnification ×1,200. Abbreviations: 3-MA, 3-methyladenine; α-MEM, alpha-modified Eagle’s medium; Ta-NPs, Ta nanoparticles.
Figure 5
Figure 5
Expression of autophagy proteins by Western blots. Notes: The MC3T3-E1 cells were cultured with 20 μg/mL Ta-NPs for 24 h, with or without pretreatment with 3-MA (10 mM) for 1 h; cells treated with α-MEM alone served as a negative control group, and cells treated with 200 nM rapamycin served as a positive control group. β-actin was used as the internal control. Compared to the negative control group, 20 μg/mL Ta-NPs treatment increased the LC3-II expression and decreased the p62 expression, while pretreatment with 3-MA counteracted the effect of Ta-NPs. Experiments were repeated three times. “NC” represents the negative control group, and “PC” represents the positive control group. *P<0.05, **P<0.01, ***P<0.001 versus negative control group; $P<0.05, $$P<0.01, $$$P<0.001 versus positive control group; #P<0.05, ##P<0.01 versus Ta-NPs alone treated group. Abbreviations: 3-MA, 3-methyladenine; α-MEM, alpha-Modified Eagle’s Medium; Ta-NPs, Ta nanoparticles.
Figure 6
Figure 6
Ultrastructure of MC3T3-E1 cells imaged by TEM. The MC3T3-E1 cells were cultured with 20 μg/mL Ta-NPs for 24 h, with or without pretreatment with 3-MA (10 mM) for 1 h; cells treated with 200 nM rapamycin served as a positive control group. Notes: In the rapamycin (A) and 20 μg/mL Ta-NP-treated (B and C) cells, the autophagic vesicles (isolated membrane, autophagosomes and autophagolysosomes; arrowheads) could be clearly observed, some even containing organelles such as an endoplasmic reticulum (white triangle). When the cells were pretreated with 3-MA (D), the Ta-NPs could still be taken up by MC3T3-E1 osteoblasts (black triangle), but without autophagic vesicle formation instead of mitochondrial swelling (double arrowheads). Scale bars: 500 nm. Magnification ×40,000. Arrowheads: autophagic vesicles; white triangle: endoplasmic reticulum; black triangle: internalized Ta-NPs; double arrowheads: swollen mitochondria; N: nucleus. Abbreviations: 3-MA, 3-methyladenine; TEM, transmission electron microscopy; Ta-NPs, Ta nanoparticles.
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