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

Efeitos citotóxicos do peróxido de hidrogénio em células periodontais

r e s u m o

Palavras-chave: Objectivos: Avaliar in vitro a citotoxicidade do Peróxido de Hidrogénio (H O ) em células pe-
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Citotoxicidade riodontais através do efeito de soluções H O na viabilidade e morfologia de culturas de fi-
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Fibroblasto broblastos gengivais e osteoblastos humanos imortalizados, em diferentes concentrações e
Periodonto tempos de exposição.
Peróxido de hidrogénio Métodos: Foram usadas linhagens imortalizadas de fibroblastos gengivais e osteoblastos
Osteoblasto fetais humanos, as quais foram cultivadas, separadamente, em placas de 96 poços, que ao
atingir a confluência, foram expostas a concentrações de H O de 0,05 µg/ml a 10 µg/ml (16
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concentrações diferentes), durante 1 h, 24 h ou 72 h, em ensaios triplicados (n=24), sendo
utilizado exclusivamente meio de cultura como controlo. A viabilidade celular foi avaliada
por métodos fluorométricos previamente descritos através da conversão da resazurina e a
morfologia celular por microscopia ótica invertida com contraste de fase. Os dados foram
analisados estatisticamente através do teste ANOVA one-way com post-hoc de Tukey e
Dunnet’s e do coeficiente de correlação de Pearson (r) conforme apropriado (α=0,05).
Resultados: O H O induziu uma redução da viabilidade, superior a 50% tanto nos fibroblas-
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tos como nos osteoblastos, visível em todas as concentrações testadas após 1h de exposição
e superior a 70% a 24h e 72h (p<0,05). Foi detetada uma correlação significativa e negativa
aos tempos 1h e 72h para osteoblastos (r=-0,471) e fibroblastos (r=-0,12), respetivamente. A
análise das micrografias obtidas apresentou destacamento celular e menor densidade ce-
lular em concordância com estes resultados.
Conclusão: A exposição ao H O resultou em alterações celulares e efeitos citotóxicos de
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moderados a severos em fibroblastos gengivais e osteoblastos. (Rev Port Estomatol Med Dent
Cir Maxilofac. 2019;60(3):111-117)
© 2019 Sociedade Portuguesa de Estomatologia e Medicina Dentária.
Published by SPEMD. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

whitening procedures, H O is able to diffuse from the pulp
Introduction 2 2 (11,12)
chamber to the root surface. This observation is support-
Hydrogen peroxide (H O ) is a well-described reactive oxygen ed by the documented risk of external root resorption associ-
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species (ROS) commonly used in Dentistry procedures. Name- ated with non-vital whitening procedures, in which the diffu-
ly, it is used in periodontal and peri-implantitis treatments in- sion of H O is considered to be the most probable etiological
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cluding implant surface decontamination, as well as in tooth factor. 13,14 While the potential toxicity of H O in external
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whitening products, mouth rinses and toothpastes. 2-6 H O bleaching techniques has been previously assessed in pulp
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concentrations depend on the type of treatment: 30 µg/ml for cells, little is known on the risks presented to periodontal cells
peri-implantitis treatments and 30 µg/ml to 380 µg/ml for tooth by external and internal bleaching procedures, which may re-
bleaching. However, it has been well-described that H O has a lease up to 0.34 to 4.4 µg/ml of H O into the periodontal space
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broad spectrum of biological effects that raise several concerns after 24 h of exposure. 1,15,16
regarding its safety, even at low concentrations. Among those These considerations suggest a potential risk to periodon-
biological effects is the overproduction of ROS, which results in tal cells, especially fibroblasts and osteoblasts. Human gingival
DNA, lipid and protein oxidation that may eventually lead to fibroblasts (HGF) play an important role in the homeostasis of
cell necrosis or apoptosis mechanisms in oral cells. 7,8 periodontal tissues since fibroblasts have an intrinsic ability
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During oral care procedures, periodontal cells are poten- to differentiate into other cells. Osteoblasts, on the other
tially exposed to H O from endogenous and exogenous sourc- hand, are the primary cells responsible for the formation of
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es. In periodontitis and peri-implantitis treatments, H O is bone, by synthesizing the components of the extracellular ma-
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conventionally used as a decontaminating agent. A validated trix and regulating its mineralization. While the current lit-
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protocol for peri-implantitis is based on a 30-µg/ml solution erature has focused mostly on the effects of H O on the den-
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of H O applied on the implant surface. 3,9,10 During these treat- tal pulp after bleaching procedures, the effects of H O on HGFs
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ments, connective tissue and bone in the peri-implant space and osteoblasts may also be assessed using odontoblasts and
are in contact with H O , thus raising questions on its toxicity dental pulp cells as models.
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and impact on tissue healing. Previous studies using HGFs have shown that a high con-
H O is also the main active product in bleaching treat- centration of H O (15% v/v) can delay cell division and induce
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ments. It has been demonstrated in vitro that, during internal alterations in cell morphology. On the other hand, in studies