Silver nanoparticles promote osteogenic differentiation of human urine-derived stem cells at noncytotoxic concentrations

Hui Qin,^1,* Chen Zhu,^2,* Zhiquan An,¹ Yao Jiang,¹ Yaochao Zhao,¹ Jiaxin Wang,¹ Xin Liu,¹ Bing Hui,¹ Xianlong Zhang,¹ Yang Wang<sup>1

1</sup>Department of Orthopedics, Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, ²Department of Orthopaedic Surgery, Provincial Hospital Affiliated to Anhui Medical University, HeFei, People's Republic of China

*These authors contributed equally to this work

Abstract: In tissue engineering, urine-derived stem cells are ideal seed cells and silver nanoparticles (AgNPs) are perfect antimicrobial agents. Due to a distinct lack of information on the effects of AgNPs on urine-derived stem cells, a study was conducted to evaluate the effects of silver ions and AgNPs upon the cytotoxicity and osteogenic differentiation of urine-derived stem cells. Initially, AgNPs or AgNO₃ were exposed to urine-derived stem cells for 24 hours. Cytotoxicity was measured using the Cell Counting kit-8 (CCK-8) test. The effects of AgNPs or AgNO₃ at the maximum safety concentration determined by the CCK-8 test on osteogenic differentiation of urine-derived stem cells were assessed by alkaline phosphatase activity, Alizarin Red S staining, and the quantitative reverse transcription polymerase chain reaction. Lastly, the effects of AgNPs or AgNO₃ on "urine-derived stem cell actin cytoskeleton organization" and RhoA activity were assessed by rhodamine-phalloidin staining and Western blotting. Concentration-dependent toxicity was observed starting at an AgNO₃ concentration of 2 µg/mL and at an AgNP concentration of 4 µg/mL. At these concentrations, AgNPs were observed to promote osteogenic differentiation of urine-derived stem cells, induce actin polymerization and increase cytoskeletal tension, and activate RhoA; AgNO₃ had no such effects. In conclusion, AgNPs can promote osteogenic differentiation of urine-derived stem cells at a suitable concentration, independently of silver ions, and are suitable for incorporation into tissue-engineered scaffolds that utilize urine-derived stem cells as seed cells.

Keywords: silver nanoparticles, osteogenic differentiation, stem cell, cytotoxicity

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