Cytotoxicity of a novel nano-silver particle endodontic irrigant

Figures & data

Figure 1 Transmission electron microscopy image of silver nanoparticles.

Notes: The average size of these nanoparticles is about 25.2±6.5 nm. Courtesy of Prof WT Wong; unpublished data.

Figure 2 Illustration of the assembly.

Note: A plastic ring was placed on the agar (0.5 mm thick) overlaying the cell culture which formed a sample well for the test material.

Figure 3 The cytotoxic zone.

Notes: (A) A montage view of live PDL cells (fluorescent green) distributed across the diameter of the well after incubation with 3% sodium hypochlorite for 24 hours. (B) High-power view showing the boxed area of (A). The transition of cell lysis zone to cell detachment, and then the unaffected zone could be observed. (C) Being affected by the sample solution, the PDL cell was detached from the base plate. The morphology of the cell changed from spindle to oval.
Abbreviation: PDL, periodontal ligament.

Figure 4 After reducing the diaphragm size of the light source of the phase contrast microscope, at 10× magnification, the field of view covered around 15 PDL cells of about 200 μm diameter.

Abbreviation: PDL, periodontal ligament.

Figure 5 The traveling stage of the microscope was traveling along the diameter of the culture well.

Table 1 Classification of cytotoxic score

Cytotoxic score	Cytotoxic range
0	Cytotoxic range =0
1	Cytotoxic range ≤0.6 cm
2	Cytotoxic range ≤1.1 cm
3	Cytotoxic range ≤1.6 cm
4	Cytotoxic range >1.6 cm
5	Whole culture well was affected

Notes: For each sample well, a cytotoxic score was assigned according to the cytotoxic range; (0.6 cm was the internal diameter of the sample well that contained the test material).

Table 2 Interpretation of cytotoxic score

Cytotoxic score	Interpretation
0–0.5	Non-cytotoxic
0.6–1.9	Mildly cytotoxic
2.0–3.9	Moderately cytotoxic
4.0–5.0	Markedly cytotoxic

Note: The cytotoxic score was classified into four toxicity classes.

Figure 6 Dose-response relationship between the novel nano-silver irrigant to both NIH 3T3 and hPDLSCs.

Notes: (A) Relative cell viability (48 hours) vs dilution of the stock nano-silver solution on NIH 3T3 cells. (B) Relative cell viability (48 hours) vs dilution of the stock nano-silver solution on hPDLSCs.
Abbreviations: hPDLSCs, human periodontal ligament stem cells; vs, versus.

Figure 7 A significant linear relationship of survivability of hPDLSCs and Mouse fibroblast NIH3T3 in different dilutions of nano-silver solution.

Abbreviation: hPDLSCs, human periodontal ligament stem cells.

Figure 8 Survival curves (generated by the regression model) of hPDLSCs and Mouse fibroblast NIH3T3 (directly cultured for 48 hours with different dilutions of nano-silver).

Abbreviations: hPDLSCs, human periodontal ligament stem cells; PDL, periodontal ligament.

Table 3 Comparison of cytotoxic score of various irrigants on hPDLSCs and Mouse fibroblast NIH3T3

	Human PDL cells				Mouse fibroblast
	Mean	SD	95% CI for Mean		Mean	SD	95% CI for Mean
			Lower bound	Upper bound			Lower bound	Upper bound
Control	0.2500	0.50000	0	1.0456	0.0000	0.00000	0.0000	0.0000
Nano-silver	0.1250	0.25000	0	0.5228	0.2500	0.50000	0	1.0456
3% Hypo	2.5000*,**	0.57735	1.5813	3.4187	3.7500*,**	0.50000	2,9544	4,5456

Notes: The mean cytotoxic score of hPDLSCs of the 3% sodium hypochlorite group was significantly higher than control *(P<0.05) and nano-silver group **(P<0.05). The same comparison was performed on mouse fibroblasts NIH 3T3. The mean cytotoxic score of NIH 3T3 of the 3% hypo group was significantly higher than control *(P<0.05) and nano-silver group **(P<0.05). The cytotoxic scores of both cell types in various test solutions were compared by crosstab analysis resulting in a 100% Kappa score.

Abbreviations: hPDLSCs, human periodontal ligament stem cells; PDL, periodontal ligament; hypo, hypochlorite.