Figure 1.
Morphology (A–D) and cytotoxicity (E) of ZnO-T structures synthesized at different dates.
Ages of ZnO-T used for cell treatment were: A = 16–20 weeks, B = 13–17 weeks, C = 8–12 weeks and D = 3–7 weeks. Passage number of normal human dermal fibroblasts (NHDF): P7–P11. Seeded cell number: 50000 cells/cm2; ZnO-T concentration: 0.05–10 mg/ml; time prior treatment: 48 h; duration of treatment: 24 h. [Each symbol represents the mean ± SE of n = 3–5 independent experiments with fourfold determinations. CTRL is short abbreviation for control].
Table 1.
Impact of age of ZnO nano-microtetrapods on cell toxicity.
Figure 2.
Cytotoxic effect of ZnO-T with different surface charges.
Cytotoxicity of untreated or either pre-treated ZnO-T with O2 or UV-light. Cell viability of normal human dermal fibroblasts (NHDF) was determined by the MTT assay. Passage number of fibroblasts: P8–P20. Seeded cell number: 50000 cells/cm2 ZnO-T concentration: 0.05–10 mg/ml; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of at least n = 3–5 independent experiments with fourfold determinations.
Table 2.
Impact of surface charge on cell toxicity.
Figure 3.
Effect of ZnO-T morphology on cytotoxicity: Phase contrast microscopy (A–D) and scanning electron microscopy (SEM) (E–H).
A, E: ZnO-T “thick”; B, F: ZnO-T “fine”; C, G: ZnO-T reference “crushed”; D, H: ZnO-T “reference”, (I–K) Viability (MTT-assay) of treated human dermal fibroblasts (NHDF). Passage number of NHDF: P10–P14. Seeded cell number: 50000 cells/cm2; ZnO-T concentration: 0.05–10 mg/ml; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3–5 independent experiments with fourfold determinations.
Figure 4.
Percentage increase or decrease of cytotoxicity depending on size/shape of ZnO-T compared to the reference probe (ZnO-T reference) at each concentration [% viable cells of probe/% viable cells of reference * 100].
Each value shown is the mean of three independent experiments (± SE. Statistical significance determined by one way analysis of variance: thick vs. crushed 2 mg/ml (** = p<0.01); thick vs. fine 5 mg/ml (* = p<0.05); thick vs. crushed 5 mg/ml (*** = p<0.001) (post hoc test: Bonferroni).
Table 3.
Impact of morphology on cell toxicity.
Figure 5.
Effect of ZnO-T on cell viability determined by the MTT-assay depending on passage number of normal human dermal fibroblasts (NHDF).
Seeded cell number: 50000 cells/cm2; ZnO-T concentration: 0.2–5 mg/ml; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3 independent experiments. Statistical significance determined by one way analysis of variance: P7–P12 vs. P14–P19 2 mg/ml (** = p<0.01) and 5 mg/ml (* = p<0.05).
Table 4.
Impact of passage number on cell toxicity.
Figure 6.
Effect of cell density on ZnO-T cytotoxicity.
Cell viability of normal human dermal fibroblasts (NHDF) was determined by the MTT assay. Passage number: P11–P15. Seeded cell numbers: 25000 cells/cm2, 50000 ells/cm2 and 100000 cells/cm2; ZnO-T concentration: 0.05–5 mg/ml; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3 independent experiments with fourfold determinations. Statistical significance determined by one way analysis of variance: 25000 vs. 50000 cells/cm2 2 mg/ml (*** = p<0.001) and 5 mg/ml (*** = p<0.001); 100,000 vs. 50000 cells/cm2 and 5 mg/ml (*** = p<0.001).
Table 5.
Impact of seeding density on cell toxicity.
Figure 7.
Phase contrast images of normal human dermal fibroblasts (NHDF) depending on passage numbers and seeding cell density after 24 h treatment with ZnO-T and ZnCl2.
A, D, G and J: untreated NHDF; B, E, H and K: 0.5 mg/ml ZnO-T; C, F, I and L: 30 µg/ml ZnCl2. Passage numbers: P11–P15 (A–C and G–I) vs. P22–P26 (D–F and J–L). Seeded cell number: 25000 (A–F) vs. 100000 cells/cm2 (G–L); time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3 independent experiments with fourfold determinations. Additionally experiments were done with three different frozen stocks of NHDF to verify the result.
Figure 8.
Effect of passage number and seeding cell density of NHDF on ZnO-T and ZnCl2 cytotoxicity.
A and D: cell protein concentrations of controls determined by the Lowry-assay. Passage numbers: P11–P15 vs. P22–P26. Seeded cell numbers: 25000 (A–C) vs. 100000 cells/cm2 (D–F); ZnO-T concentration: 0.05–5 mg/ml; ZnCl2 concentration: 25–45 µg/ml; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3 independent experiments with fourfold determinations. Additionally experiments were done with three different frozen stocks of NHDF to verify the result. Statistical significance determined by one way analysis of variance: cell density: 100000 cells/cm2, ZnO-T: P11–P15 vs. P22–P26 1 mg/ml (** = p<0.01), 2 mg/ml (*** = p<0.001) and 5 mg/ml (*** = p<0.001). Cell density: 100000 cells/cm2, ZnCl2: P11–P15 vs. P22–P26 35 µg/ml (* = p<0.05).
Table 6.
Impact of seeding density + fibroblast passage number on cell toxicity.
Figure 9.
Comparing the cytotoxic effect of ZnCl2, spherical ZnO NP and ZnO-T on normal human dermal fibroblasts (NHDF) determined by the MTT-assay.
Zn content is calculated in [mM] according to the used probe concentration. Passage number: P6–P9. Seeded cell number: 50000 cells/cm2; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3 independent experiments with fourfold determinations. Statistical significance determined by one way analysis of variance: EC50 values are highly significant (p<0,001) different (Table 7). The typical SEM images of spherical ZnO NP (average diameter is ∼ 150 nm) and ZnO are shown as inset images which were used for investigation in present case.
Table 7.
EC50 values for ZnCl2, spherical ZnO NP and ZnO-T.
Figure 10.
Role of direct cell contact for ZnO-T toxicity shown by phase contrast microscopy and changes in cell viability (MTT-Assay).
Column diagram shows the cytotoxicological effects of different probes on normal human dermal fibroblasts (NHDF). CTRL (Image: A) = cell culture medium as control; CTRL (S) (Image: B) = supernatant of cell culture medium 24 h pre-incubated on NHDF as control; ZnO-T = ZnO-T in dispersion directly on NHDF (10 mg/ml or 20 mg/ml (Images: not shown due to overlapping of ZnO tetrapods layer)); ZnO-T (S) – disp. + cells = supernatant of ZnO-T in culture medium pre-incubated on NHDF for 24 h (10 mg/ml (Image: C) or 20 mg/ml (Image: D)); ZnO-T (S) – disp. = supernatant of ZnO-T in culture medium pre-incubated for 24 h without NHDF (10 mg/ml (Image: G) or 20 mg/ml (Image H); ZnO-T in Transwells = ZnO-T in dispersion (10 mg/ml (Image: E) or 20 mg/ml (Image: F) into transwells to prevent cell contact. Each value shown is the mean of three independent experiments (± SE). Passage number: P12–P14. Seeded cell number: 50000 cells/cm2; time prior treatment: 48 h; duration of treatment: 24 h. Each column represent the mean ± SE of n = 3 independent experiments. Statistical significance determined by one way analysis of variance: ZnO-T vs. C/D, G/H and E/F (* = p<0.05, ns = not significant).