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116 rev port estomatol med dent cir maxilofac. 2019;60(3):111-117

moderately cytotoxic at 50-30% cell viability; and severely cy- gest that, within this range of concentrations, there is a
totoxic at <30% cell viability (ISO 10993-5:2009). dose-dependent response in the viability of HGFs at 72 h of
The results of our study showed, for the first time, that, in exposure (small effect), thus rejecting the third and fourth
both the osteoblast and gingival fibroblast cell lines tested, null hypotheses.
exposure to a 0.05-µg/ml solution of H O (10-fold lower than An important limitation of our study is that only one cell
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the reportedly safe concentration of 0.68 µg/ml) elicited a line was used for each cell type. Thus, further studies using
cell-viability decrease of up to 50% (P<0.05), thus rejecting the different cell lines and primary osteoblasts or fibroblasts
first two null hypotheses. This effect was more pronounced should be undertaken to confirm these findings. IC50 was not
with longer incubation times. According to some authors, ex- calculated because the scope of this study was to evaluate the
posure to H O concentrations lower than 0.68 µg/ml is con- effects of clinically relevant concentrations on cell viability.
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sidered safe regardless of the cell type, resulting in limited However, further studies should characterize the toxicological
effects in many animal cells. A significant impact on the cell profile of H O exposure in these cell types in order to fully
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viability would only be expected in exposures to concentra- elucidate on the safe threshold concentration of this agent, as
tions over 1.7 µg/ml. As stated before, our results contradict well as its impacts on cell function and differentiation. Finally,
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this assumption, suggesting that H O toxicity for periodontal this was an in vitro study, with the inherent limitations that
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cells might be underestimated. This divergence could be ex- turn direct extrapolation to in vivo impossible, namely the ab-
plained by the use of distinct and more-resistant cell lines in sence of antioxidant mechanisms, or the rate of H O degra-
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these previous studies, or by other differences in experimental dation.
design, including broader concentration ranges or shorter ex- Within the limits of this study, the results suggest that
posure times. H O exposure in the range of concentrations used in peri-im-
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Different toxicity effects were observed in the two cell plantitis debridement and in internal bleaching procedures,
types used in this assay. After the exposure of HGF to the potentially reaching the periodontal space, may severely de-
lowest-concentration H O solution (0.05 µg/ml), mean via- crease periodontal cell viability. These findings raise new ques-
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bility decreases of 50% – slightly cytotoxic – (1 h) and 80% – tions regarding H O safety for those applications. Whether
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severely cytotoxic (24 and 72 h) – were observed. In previous this cytotoxicity happens in vivo and to what extent H O ex-
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studies with human fibroblast cell lines, concentrations of posure impacts cell function is yet to determine. These obser-
H O ranging between 1.7 and 42.5 µg/ml resulted in a prolif- vations need to be integrated into more complex models to
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eration decrease, morphology alterations and cell death. (29–34) clarify the role of endogenous and exogenous antioxidant
However, these studies were based on shorter exposure times systems in reverting these alterations.
(90 seconds), while, in our study, the shortest exposure was
1 h, and most were performed in types of fibroblasts other
that gingival fibroblasts. Our results presented a similar de- Conclusions
crease in cell viability at a 100-fold lower concentration of
H O for the HGF cell line (0.05 µg/ml), thus suggesting that The results of this study suggest that direct exposure to H O
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the tolerance threshold for gingival fibroblasts may be lower within clinically relevant concentrations resulted in moder-
than previously reported, considering exposure times com- ate to severe cytotoxic effects in periodontal cells – osteo-
prised between 1 h and 72 h. 1,19 However, this effect might be blasts and gingival fibroblasts in vitro. A small negative corre-
specifically related to the cell line used in this study, which lation between H O concentration and cell viability was
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may be more sensitive to H O exposure than other cell lines observed in osteoblasts but not in fibroblasts.
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and primary cells. Further studies are necessary to determine the exact safe-
Higher resistance to H O after 1 h of exposure, with ty threshold of the direct application of H O on these cells, as
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non-cytotoxic to slightly cytotoxic effects (25-50% viability well as its impact on cell differentiation and function.
decrease) but similar severely cytotoxic effects in longer ex-
posures, was observed in hFOBs. A previous study with hFOB
cells reported a safe threshold of a 24-h exposure to 3.4 µg/ml Ethical disclosures
35
H O . Our results suggest that this limit is potentially below
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a concentration of 0.05 µg/ml of H O in osteoblasts, parti- Protection of human and animal subjects. The authors
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cularly after 24 h of exposure. This difference may result from declare that no experiments were performed on humans or
the use of a different method for viability assessment, as the animals for this study.

authors of the previous study used formazan reduction. Confidentiality of data. The authors declare that no patient
While these methods are generally considered equivalent,
some reports state different performances of resazurin and data appear in this article.

formazan methods, with resazurin generally causing higher Right to privacy and informed consent. The authors declare
sensitivity. 36,37 that no patient data appear in this article.
A small negative overall correlation between H O con-
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centration and cell viability was detected in osteoblasts, spe-
cifically at 1 h of exposure (moderate effect). It can be there- Conflict of interest
fore speculated that osteoblasts may have a dose-dependent
response to short-term H O exposures. The results also sug- The authors have no conflicts of interest to declare.
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