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. 2018 Feb 9:13:877-893.
doi: 10.2147/IJN.S147849. eCollection 2018.

Effects of silica-gentamicin nanohybrids on osteogenic differentiation of human osteoblast-like SaOS-2 cells

Wei He  1 , Dina A Mosselhy  2   3 , Yudong Zheng  1 , Qingling Feng  4 , Xiaoning Li  4 , Xing Yang  4 , Lina Yue  1 , Simo-Pekka Hannula  2
Affiliations

Effects of silica-gentamicin nanohybrids on osteogenic differentiation of human osteoblast-like SaOS-2 cells

Wei He et al. Int J Nanomedicine. .

Abstract

Introduction: In recent years, there has been an increasing interest in silica (SiO2) nanoparticles (NPs) as drug delivery systems. This interest is mainly attributed to the ease of their surface functionalization for drug loading. In orthopedic applications, gentamicin-loaded SiO2 NPs (nanohybrids) are frequently utilized for their prolonged antibacterial effects. Therefore, the possible adverse effects of SiO2-gentamicin nanohybrids on osteogenesis of bone-related cells should be thoroughly investigated to ensure safe applications.

Materials and methods: The effects of SiO2-gentamicin nanohybrids on the cell viability and osteogenic differentiation of human osteoblast-like SaOS-2 cells were investigated, together with native SiO2 NPs and free gentamicin.

Results: The results of Cell Count Kit-8 (CCK-8) assay show that both SiO2-gentamicin nanohybrids and native SiO2 NPs reduce cell viability of SaOS-2 cells in a dose-dependent manner. Regarding osteogenesis, SiO2-gentamicin nanohybrids and native SiO2 NPs at the concentration range of 31.25-125 μg/mL do not influence the osteogenic differentiation capacity of SaOS-2 cells. At a high concentration (250 μg/mL), both materials induce a lower expression of alkaline phosphatase (ALP) but an enhanced mineralization. Free gentamicin at concentrations of 6.26 and 9.65 μg/mL does not significantly influence the cell viability and osteogenic differentiation capacity of SaOS-2 cells.

Conclusions: The results of this study suggest that both SiO2-gentamicin nanohybrids and SiO2 NPs show cytotoxic effects to SaOS-2 cells. Further investigation on the effects of SiO2-gentamicin nanohybrids on the behaviors of stem cells or other regular osteoblasts should be conducted to make a full evaluation of the safety of SiO2-gentamicin nanohybrids in orthopedic applications.

Keywords: ALP activity; SiO2 NPs; cytotoxicity; gentamicin; mineralization.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1
SEM images of the samples. Notes: (A) Native SiO2 NPs. (B) SiO2–gentamicin nanohybrids. Abbreviations: SEM, scanning electron microscope; SiO2, silica; NPs, nanoparticles.
Figure 2
Figure 2
TEM images and size-distribution histograms of the samples. Notes: TEM images of the (A) native SiO2 NPs and (B) SiO2–gentamicin nanohybrids. Size-distribution histograms of the (C) native SiO2 NPs and (D) SiO2–gentamicin nanohybrids generated from images (A) and (B), respectively. Most of the native SiO2 NPs are well dispersed (A). The average size of native SiO2 NPs calculated from the TEM image is 312±26 nm, with a size distribution of 265–405 nm (C). The size of the SiO2–gentamicin nanohybrids increases markedly, compared with the size of the native SiO2 NPs (B). The average size of SiO2–gentamicin nanohybrids is 719±128 nm, and the size distribution ranges from 495 to 965 nm (D). Abbreviations: TEM, transmission electron microscope; SiO2, silica; NPs, nanoparticles.
Figure 3
Figure 3
FTIR spectra and TGA of the samples. Notes: (A) FTIR spectra of the native SiO2 NPs, SiO2–gentamicin nanohybrids, and free gentamicin. (B) TGA of the native SiO2 NPs and SiO2–gentamicin nanohybrids. Abbreviations: FTIR, Fourier-transform infrared; TGA, thermogravimetric analysis; SiO2, silica; SiO2–G, SiO2–gentamicin nanohybrids; NPs, nanoparticles; G, gentamicin.
Figure 4
Figure 4
Cell viability detected by CCK-8 assay. Notes: (A) The cells were incubated in the normal medium for 1, 3, and 5 days. (B) The cells were incubated in the osteogenic induction medium for 7 and 14 days. The values are expressed as mean ± SD of triplicate experiments. *p<0.05 compared with the control group. **p<0.01 compared with the control group. Abbreviations: CCK-8, Cell Count Kit-8; SiO2, silica; SiO2–G, SiO2–gentamicin nanohybrids; G, gentamicin.
Figure 5
Figure 5
Calcein-AM staining for living cells. Notes: The cells were incubated with different concentrations of native SiO2 NPs, SiO2–gentamicin nanohybrids, and free gentamicin in the normal medium for 1, 3, and 5 days. The images are representative of three independent experiments. Abbreviations: SiO2, silica; SiO2–G, SiO2–gentamicin nanohybrids; NPs, nanoparticles; G, gentamicin.
Figure 6
Figure 6
ALP activity of the SaOS-2 cells. Notes: (A) Optical microscopic images and (B) macrograph of ALP staining. (C) ALP activity of SaOS-2 cells after osteogenic induction for 7 days. Values are expressed as mean ± SD of the triplicate experiments. **p<0.01 compared with the control group. Abbreviations: ALP, alkaline phosphatase; SiO2, silica; SiO2–G, SiO2–gentamicin nanohybrids; G, gentamicin.
Figure 7
Figure 7
Collagen secretion of the SaOS-2 cells. Notes: (A) Optical microscopic images and (B) macrograph of Sirius Red staining for the collagen secretion of cells after osteogenic induction for 7 days. (C) The quantitative results of retention of Sirius Red. Data are expressed as mean ± SD (n=3 for each sample). *p<0.05 compared with the control group. Abbreviations: SiO2, silica; SiO2–G, SiO2–gentamicin nanohybrids; G, gentamicin.
Figure 8
Figure 8
Expression of COLI, OPN, and OCN of the SaOS-2 cells. Notes: Immunofluorescent staining for COLI, OPN, and OCN. The cells were incubated with different concentrations of native SiO2 NPs, SiO2–gentamicin nanohybrids, and free gentamicin in the osteogenic induction medium for 7 days (for COLI and OPN) and 14 days (for OCN). The images are representative of three independent experiments. Abbreviations: COLI, type I collagen; OPN, osteopontin; OCN, osteocalcin; SiO2, silica; SiO2–G, SiO2–gentamicin nanohybrids; NPs, nanoparticles; G, gentamicin.
Figure 9
Figure 9
ECM mineralization of the SaOS-2 cells. Notes: (A) Optical microscopic images and (B) macrograph of Alizarin Red S staining for matrix mineralization of cells after osteogenic induction for 14 days. (C) The quantitative results of retention of Alizarin Red S. Data are expressed as mean ± SD (n=3 for each sample). **p<0.01 compared with the control group. Abbreviations: ECM, extracellular matrix; SiO2, silica; SiO2–G, SiO2–gentamicin nanohybrids; G, gentamicin.
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