doi:10.1111/j.1365-2591.2010.01700.
Cytotoxicity of chlorhexidine on human
osteoblastic cells is related to intracellular
glutathione levels
T.-H. Lee1,2, C.-C. Hu3, S.-S. Lee4, M.-Y. Chou5 & Y.-C. Chang2,5
1
Institute of Medicine, Chung Shan Medical University, Taichung; 2Department of Dentistry, Chung Shan Medical University
Hospital, Taichung; 3School of Applied Chemistry, Chung Shan Medical University, Taichung; 4School of Public Health, Chung
Shan Medical University, Taichung; and 5School of Dentistry, Chung Shan Medical University, Taichung, Taiwan
Abstract
Lee T-H, Hu C-C, Lee S-S, Chou M-Y, Chang Y-C.
Cytotoxicity of chlorhexidine on human osteoblastic cells is
related to intracellular glutathione levels. International End-
odontic Journal, 43, 430–435, 2010.
Aim To evaluate the mechanisms of cytotoxicity of
chlorhexidine (CHX) in human osteoblastic cells in
vitro.
Methodology Cytotoxicity, cell proliferation and
collagen synthesis assays were performed to elucidate
the toxic effects of CHX on the human osteoblastic cell
line U2OS. To determine whether glutathione (GSH)
levels were important in the cytotoxicity of CHX, cells
were pre-treated with 2-oxothiazolidine-4-carboxylic
acid (OTZ) to boost GSH levels or buthionine sulfoxi-
mine (BSO) to deplete GSH.
Introduction
Chlorhexidine (CHX) is a synthetic cationic bis-guanide
that consists of two symmetric 4-cholorophenyl rings
and two biguanide groups connected by a central
hexamethylene chain. CHX is a bisbiguanide antiseptic
active against gram-positive and gram-negative bacte-
ria, facultative anaerobes and aerobes, moulds, yeasts
and viruses. CHX is a positively charged hydrophobic
and lipophilic molecule that interacts with phospholip-
ids and lipopolysaccharides on the cell membrane of
Correspondence: Prof. Yu-Chao Chang, School of Dentistry,
Chung Shan Medical University, 110, Sec. 1, Chien-Kuo N.
Rd., Taichung, Taiwan (Tel.: 886 4 24718668 ext. 55011;
Fax: 886 4 24759065; e-mail:cyc@csmu.edu.tw).
430 International Endodontic Journal, 43, 430–435, 2010
Results CHX demonstrated a cytotoxic effect to U2OS
cells in a dose-dependent manner (P < 0.05). The 50%
inhibition concentration of CHX was approximately
0.005%. CHX also inhibited cell proliferation and
collagen synthesis (P < 0.05). The addition of OTZ
acted as a protective effect on the CHX-induced cytotox-
icity (P < 0.05). In contrast, the addition of BSO
enhanced the CHX-induced cytotoxicity (P < 0.05).
Conclusions The levels of CHX tested inhibited cell
growth, proliferation and collagen synthesis on U2OS
cells. CHX has significant potential for periapical
toxicity. GSH depletion might be one of the mechanisms
underlying CHX cytotoxicity.
Keywords: chlorhexidine, cytotoxicity, glutathione,
osteoblastic cells.
Received 19 November 2009; accepted 6 January 2010
bacteria and then enters the cell through some type of
active or passive transport mechanism (Hauman &
Love 2003, Kanisavaran 2008). Its efficacy is because
of the interaction of positive charge of the molecule and
negatively charged phosphate groups on the microbial
cell walls (Gomes et al. 2003), thereby altering the
cell’s osmotic equilibrium. This increases the perme-
ability of the cell wall, which allows the CHX molecule
to penetrate into the bacteria.
CHX is a widely used antimicrobial agent for topical
preoperative skin disinfection, skin wounds (including
burns) and general skin cleansing, and as a surgical
hand scrub. The most common oral preparation,
chlorhexidine gluconate, is water soluble and at
physiological pH, readily dissociates and releases the
positively charged chlorhexidine component (Greenstein
ª 2010 International Endodontic Journal

et al. 1986). In endodontics, CHX has been found to be
an effective antimicrobial agent when used as a root
canal irrigant (Delany et al. 1982). Recently,
supplementing the antibacterial activity of calcium
hydroxide-based root canal sealer (Siren et al. 2004)
and root-end filling material mineral trioxide aggregate
(Stowe et al. 2004) with CHX preparations might
therefore be a potential improvement to the overall
therapeutic efficacy of endodontic treatment.
Previous studies have indicated that CHX was
cytotoxic to rat fibroblast cell lines (Giannelli et al.
2008, Faria et al. 2009), human dermal fibroblasts
(Hidalgo & Dominguez 2001), human gingival fibro-
blasts (Pucher & Daniel 1993, Mariotti & Rumpf 1999),
human periodontal ligament cells (Chang et al. 2001a),
human alveolar bone cells (Cabral & Fernandes 2007)
and human osteoblastic cell line (Giannelli et al. 2008).
However, the potential toxicological implications of
CHX still remain to be elucidated.
The sulphydryl group containing tripeptide consti-
tutes a first defence intracellular antioxidant (Meister
1994). It is known that glutathione (GSH) plays a role
in cellular protection from damage produced by free
radicals and electrophiles. When cellular GSH is
depleted, cells become extremely prone to oxidative
damage. It is well known that 2-oxothiazolidine-4-
carboxylic acid (OTZ), a precursor of cysteine, meta-
bolically promotes GSH synthesis and increases intra-
cellular GSH levels by as many as 2–3 times the control
level (Williamsan et al. 1982). However, buthionine
sulfoximine (BSO), a specific inhibitor of r-glutamyl
cysteine synthetase, which inhibits GSH synthesis, is
relatively nontoxic and is quite efficient in decreasing
intracellular GSH levels (Dethmers & Meister 1981).
Osteoblasts are the principal cells responsible for the
periapical tissues. CHX or CHX-containing endodontic
products will come into contact with, or in close
proximity to, the periapical tissues. In this study,
cytotoxicity, cell proliferation and collagen synthesis
assays were used to investigate the effects of CHX on
human osteoblastic cell line U2OS cells. To determine
whether GSH levels were important in the cytotoxicity
of CHX, cells were pre-treated with the GSH precursor,
2-oxothiazolidine-4-carboxylic acid (OTZ), to boost
thiol levels or BSO to deplete GSH.
Materials & methods
All tissue culture biologicals were purchased from
Gibco Laboratories (Grand Island, NY, USA). CHX
was prepared from Hibitane (5% W/V chlorhexidine
ª 2010 International Endodontic Journal
Lee et al. Cytotoxicity of CHX
gluconate) (Zeneca Limited, Macclesfield, UK). Hoechst
33258 (H33258), BSO and OTZ were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). [3H]-thymi-
dine and [3H]-proline were obtained from Amersham
International PLc (Amersham, UK). CHX was directly
dissolved in the culture medium before each experi-
ment. The final concentration of CHX was 0 to
0.00001%.
Cell culture
U2OS cells (American Tissue Type Collection HTB 96),
derived from human osteogenic sarcoma, were cul-
tured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% foetal bovine serum (FBS),
100 lg mL)1 of streptomycin, 100 mg mL)1 of peni-
cillin at 37 C in humidified incubator under ambient
pressure air atmosphere containing 5% CO2 (Ho et al.
2006). Confluent cells were detached with 0.25%
trypsin and 0.05% EDTA for 5 min, and aliquots of
separated cells were subcultured. The cells were sub-
cultured at 1 : 4 splits every third day.
Cytotoxicity assay
H33258 fluorescence assay using a multiwell fluores-
cence scanner was developed for screening cytotoxicity
to cells cultured in 24-well microtiter plates by Skehan
(1995). H33258 fluorescence was measured according
to previous studies (Chang & Chou 2001, Ho & Chang
2007). The cells were plated at an initial density of
2 · 104 cells per well into 24-well culture plates and
allowed to attach for 48 h. Culture medium was
replaced with fresh DMEM, and then cells were exposed
to various concentrations of CHX for 2 h. Finally, the
medium was removed and the plates frozen at )80 C
and stored frozen until ready for further processing.
After thawing plates, 100 lL distilled water was added
to each well and incubated for 1 h at room tempera-
ture. They were refrozen at )80 C for 90 min and
thawed to room temperature. Then 100 lL of TNE
buffer (10 mmol L)1 Tris, 1 mmol L)1 EDTA, 2 mmol
L)1 NaCl, pH 7.4) containing 20 lg mL)1 of H33258
dye was added. After dilution, the final H33258
concentration is 10 lg mL)1. This step is required to
allow the addition of an EDTA-high NaCl buffer that
partly dissociates DNA, allowing better dye access and
therefore brighter fluorescence. After a 90-min incu-
bation period in the dark, the DNA content was
measured using a fluorescent plate reader (CytoFluor
2300, Millipore, Bedford, MA, USA) at an excitation
International Endodontic Journal, 43, 430–435, 2010 431
 Cytotoxicity of CHX Lee et al.

wavelength of 350 nm and an emission wavelength of CHX, then co-incubated for 2 h. Cytotoxicity was
460 nm. judged using H33258 fluorescence assay as described
previously.
Cell proliferation assay
Statistical analysis
[3H]-thymidine incorporation into cellular DNA was
used as a measure of cell proliferation as described Triplicate experiments were performed throughout this
previously (Chang et al. 2001b, Ho et al. 2007). The study. All assays were repeated three times to ensure
cells were plated at an initial density of 5 · 104 cells reproducibility. The significance of the results obtained
per well into 6-well culture plates. After overnight from control and treated groups were statistically
attachment, [3H]-thymidine (0.5 lCi mL)1) was added analysed by paired Student’s t-test.
and cells were exposed to various concentrations of
CHX for 24 h. Finally, the radioactive medium was
Results
discarded and cells were washed three times with 5%
trichloroacetic acid at 4 C. Cells were solubilized with CHX demonstrated a cytotoxic effect on the human
1 mL of 0.1 N NaOH for 15 min at the room temper- osteoblastic cell line U2OS cells (Fig. 1). CHX reduced
ature. Aliquots of the cell lysates were counted in a the fluorescence of H33258 of U2OS cells in a dose-
liquid scintillation counter (Packard model 2100TR; dependent manner (P < 0.05). The 50% inhibition
Packard, Downers Grove, NJ, USA) and expressed as a concentration of CHX was approximately 0.005%.
percentage of control. Cell proliferation using [3H]-thymidine incorporation
into cellular DNA is shown in Fig. 2. CHX at the
concentration higher than 10)5% was found to inhibit
Collagen synthesis
cell proliferation (P < 0.05). The effect of inhibition
Collagen synthesis was measured in 24-well culture was dose dependent (P < 0.05). Elevating the CHX
plates as described previously (Chang et al. 1999). Each concentration up to 0.01% almost completely inhibited
well was incubated with 5 · 104 cells in 1 mL of DNA synthesis (Fig. 2).
DMEM with 10% FBS and incubated overnight before The effect of CHX on collagen synthesis of human
replacement with 1-mL fresh serum-free medium and U2OS cells is shown in Fig. 3. CHX inhibited collagen
various concentrations of CHX. The rate of collagen synthesis at the CHX concentration higher than 10)3%
synthesis was estimated by the incorporation of [3H]-
proline into pepsin-resistant native collagen. After
labelling with tritiated proline for 24 h, the radioactive
collagen produced was assayed by pepsin extraction
and purification of the native collagen by salt precip-
itation. The final precipitates were each dissolved in
0.5 mmol L)1 acetic acid and transferred to scintilla-
tion vial inserts containing 2 mL of a scintillation
cocktail. Radioactivity was measured in a liquid scin-
tillation counter (Packard model 2100TR; Packard,
Downers Grove, NJ, USA) and expressed as a percent-
age of control.

Effects of OTZ and BSO on CHX-induced cytotoxicity

To further elucidate the roles of GSH in CHX-associated
cytotoxicity, 5 mmol L)1 OTZ and 50 lmol L)1 BSO
without cytotoxic concentrations were added to cul-
tures. This protocol was necessary to allow changes in
GSH levels to occur prior to exposure to CHX. U2OS
cells were exposed to OTZ or BSO for 24 h prior to the
addition of the 50% inhibition concentration (IC50) of
Figure 1 Cytotoxicity measured using H33258 fluorescent
dye on U2OS cells after exposure to various concentrations of
chlorhexidine (CHX). Results are expressed as percentage of
fluorescence intensity relative to the untreated control. Data
are shown as mean ± SD. *Denotes significant differences from
control values with P < 0.05.

432 International Endodontic Journal, 43, 430–435, 2010 ª 2010 International Endodontic Journal

Figure 2 Effect of various concentrations of chlorhexidine
(CHX) on cell proliferation of human U2OS cells. Results are
expressed as a percentage of [3H]-thymidine incorporation
relative to untreated control. Data are shown as mean ± SD.
*Denotes significant differences from control values with
P < 0.05.
Figure 3 Effects of chlorhexidine (CHX) on collagen synthesis
of human U2OS cells during 2-h incubation. All values are
means of [3H]-proline incorporation by three independent tests
±SD. *Denotes significant differences from control values with
P < 0.05.
(P < 0.05). At the concentrations of 10)2%, CHX
decreased about 50% collagen synthesis.
To further elucidate the roles of GSH in CHX-induced
cytotoxicity, OTZ was used to increase the intracellular
GSH level and BSO was used to deplete the intracellular
GSH level. The combination effects of 0.005% CHX
with 5 mmol L)1 OTZ or 50 lmol L)1 BSO on U2OS
cells by H33258 fluorescence assay are shown in
Fig. 4. Addition of OTZ extracellularly could protect the
ª 2010 International Endodontic Journal
Lee et al. Cytotoxicity of CHX
Figure 4 Effects of 5 mmol L)1 2-oxothiazolidine-4-carboxylic
acid (OTZ) or 50 lmol L)1 BSO on 0.005% chlorhexidine
(CHX)-induced cytotoxicity to U2OS cells by H33258 fluores-
cence assay. Percentage of H33258 fluorescence after incu-
bation with CHX with or without OTZ and BSO, compared
with that of control was calculated. Each point and bar
represent a mean ± SD. *Denotes significant differences from
CHX alone with P < 0.05.
cells from CHX-induced cytotoxicity (P < 0.05). Addi-
tion of BSO enhanced the cell death on CHX-induced
cytotoxicity (P < 0.05).
Discussion
The human osteoblastic cell line U2OS employed in this
study has been used as a model for osteoblasts, because
these cells express the osteoblast phenotype (Nelissen
et al. 2000). In addition, the selection of such a
permanent cell line has advantages as they are easily
maintained in culture. Furthermore, donor biopsy
variability was eliminated and greater reproducibility
was possible.
CHX is a cationic bisbiguanide with excellent anti-
microbial action. It is active against a wide range of
microorganisms being bacteriostatic at low concentra-
tions and bactericidal at higher concentrations. In this
study, CHX was shown to inhibit cell growth and
proliferation on U2OS cells. CHX exhibited cytotoxic
effects to U2OS cells. Many types of cells have been
applied to evaluate the cytotoxicity of CHX. CHX was
found to exhibit cytotoxicity to rat fibroblast cell lines
(Giannelli et al. 2008, Faria et al. 2009), human
dermal fibroblasts (Hidalgo & Dominguez 2001), hu-
man gingival fibroblasts (Pucher & Daniel 1993,
Mariotti & Rumpf 1999), human periodontal ligament
cells (Chang et al. 2001a), human alveolar bone cells
International Endodontic Journal, 43, 430–435, 2010 433
 Cytotoxicity of CHX Lee et al.
(Cabral & Fernandes 2007) and human osteoblastic cell
line (Giannelli et al. 2008). Data suggest substantial
differences in cytotoxic levels with CHX on different
type of cells. Taken together, CHX is a cytotoxic agent
and the cytotoxicity of CHX is not cell type specific.
In this study, CHX was also demonstrated to inhibit
collagen synthesis in U2OS cells. Previous studies have
shown that CHX inhibited protein synthesis (Pucher &
Daniel 1993, Chang et al. 2001a) and collagen syn-
thesis (Mariotti & Rumpf 1999) in human oral fibro-
blasts. The highly cationic nature of CHX might have a
general inhibitory effect on the cells or the CHX might
affect collagen protein biosynthesis specifically.
Previous study has demonstrated that the cell
growth, proliferation and matrix synthesis play an
important role in wound healing and tissues regener-
ation (MacNeil & Somerman 1993). In this study, CHX
was found to inhibit cell growth, proliferation and
collagen synthesis on U2OS cells. Therefore, these
detrimental effects of CHX might impair the reparative
and regenerative potential of periapical tissues.
GSH is ubiquitous in eukaryotic cells and is implicated
in many cellular functions. It acts as a reducing agent,
and an antioxidant participates in detoxification reac-
tions for reactive oxygen species (ROS) (Shaw & Chou
1986, Deneke & Fanburg 1989). In this study,
pre-treatment of U2OS cells with BSO enhanced the
cytotoxicity of CHX. This is possibly because of irrevers-
ible inhibition of the GSH synthesis by BSO and persistent
consumption by CHX. Furthermore, the cytotoxic effect
of CHX can be prevented by the addition of extracellular
OZT. These findings indicated that GSH depletion might
be one of the mechanisms underlying CHX-induced
cytotoxicity. Yeung et al. (2007) reported that CHX can
induce ROS and act as a pro-oxidant. Recently, CHX was
found to generate ROS in Saos-2 osteoblastic cell line
(Giannelli et al. 2008). Taken together, cytotoxicity of
CHX is related to intracellular GSH levels.
GSH has been documented to have regulatory effects
in cell proliferation activity (Meister & Anderson 1983).
In this study, CHX was found to inhibit cell growth and
proliferation in a dose-dependent manner. Thus, the
depletion of cellular GSH by CHX might partly produce
this growth inhibitory effect.
The cytotoxic effects of CHX on U2OS cells depended
on the exposure dose, frequency and duration. How-
ever, it is difficult, if not impossible, to determine how
much CHX acts on periapical tissue. In this study, it was
found that the lower concentrations of CHX could easily
reach the effective cytotoxic level on U2OS cells during
long-term exposure. It should be emphasized that CHX
434 International Endodontic Journal, 43, 430–435, 2010
has significant potential for periapical toxicity. Care
should be taken to reduce the possibility of periapical
irritation from CHX in clinical treatment. In addition,
the development of agents that induce the cellular
synthesis of GSH level might be useful for protect
periapical tissue against cytotoxicity induced by CHX.
Conclusion
CHX was found to inhibit cell growth, proliferation
and collagen synthesis in U2OS cells. The addition of
OTZ acted as a protective effect on the CHX-induced
cytotoxicity. In contrast, the addition of BSO enhanced
the CHX-induced cytotoxicity. GSH depletion could be
the mechanism CHX-induced cytotoxicity.
References
Cabral CT, Fernandes MH (2007) In vitro comparison of
chlorhexidine and povidone-iodine on the long-term prolif-
eration and functional activity of human alveolar bone cells.
Clinical Oral Investigations 11, 155–64.
Chang YC, Chou MY (2001) Cytotoxicity of fluoride on human
pulp cell cultures in vitro. Oral Surgery Oral Medicine, Oral
Pathology, Oral Radiology and Endodontology 91, 230–4.
Chang YC, Tai KW, Lii CK, Chou LSS, Chou MY (1999)
Cytopathologic effects of arecoline on human gingival
fibroblasts in vitro. Clinical Oral Investigations 3, 25–9.
Chang YC, Huang FM, Tai KW, Chou MY (2001a) The effect of
sodium hypochlorite and chlorhexidine on cultured human
periodontal ligament cells. Oral Surgery Oral Medicine Oral
Pathology, Oral Radiology and Endodontology 92, 446–50.
Chang YC, Lii CK, Tai KW, Chou MY (2001b) Adverse effects
of arecoline and nicotine on human periodontal ligament
fibroblasts in vitro. Journal of Clinical Periodontology 28, 277–
82.
Delany GM, Patterson SS, Miller CH, Newton CW (1982) The
effect of chlorhexidine gluconate irrigation on the root canal
flora of freshly extracted necrotic teeth. Oral Surgery Oral
Medicine & Oral Pathology 53, 518–23.
Deneke SM, Fanburg BL (1989) Regulation of cellular
glutathione. American Journal of Physiology 257, 163–73.
Dethmers JK, Meister A (1981) Glutathione export by human
lymphoid cells: depletion of glutathione by inhibition of its
synthesis decrease export and increases sensitivity to
irradiation. Proceeding of National Academy Science United
State of America 78, 7492–6.
Faria G, Cardoso CRB, Larson RE, Silva JS, Rossi MA (2009)
Chlorhexidine-induced apoptosis or necrosis in L929 fibro-
blasts: a role for endoplasmic reticulum stress. Toxicology and
Applied Pharmacology 234, 256–65.
Giannelli M, Chellini F, Margheri M, Tonelli P, Tani A (2008)
Effect of chlorhexidine digluconate on different cell types: a
ª 2010 International Endodontic Journal

molecular and ultrastructural investigation. Toxicology In
Vitro 22, 308–17.
Gomes BPFA, Souza SFC, Ferraz CCR et al. (2003) Effective-
ness of 2% chlorhexidine gel and calcium hydroxide against
Enterococcus faecalis in bovine root dentine in vitro. Interna-
tional Endodontic Journal 36, 267–75.
Greenstein G, Berman C, Jaffin R (1986) Chlorhexidine: an
adjunct to periodontal therapy. Journal of Periodontology 57,
370–6.
Hauman CHJ, Love RM (2003) Biocompatibility of dental
materials used in contemporary endodontic therapy: a
review. Part 1. Intracanal drugs and substances. Interna-
tional Endodontic Journal 36, 75–85.
Hidalgo E, Dominguez C (2001) Mechanisms underlying
chlorhexidine-induced cytotoxicity. Toxicology In Vitro 15,
271–6.
Ho YC, Chang YC (2007) Effects of a bacterial lipid byproduct
on human pulp fibroblasts in vitro. Journal of Endodontics 33,
437–41.
Ho YC, Huang FM, Chang YC (2006) Mechanisms of
cytotoxicity of eugenol in human osteoblastic cells in vitro.
International Endodontic Journal 39, 389–93.
Ho YC, Huang FM, Chang YC (2007) Cytotoxicity of formal-
dehyde on human osteoblastic cells is related to intracellular
glutathione levels. Journal of Biomedical Materials Research
(Part: B- Applied Biomaterials) 83, 340–4.
Kanisavaran ZM (2008) Chlorhexidine gluconate in endodon-
tics: an update review. International Dental Journal 58, 247–
57.
MacNeil RL, Somerman MJ (1993) Molecular factors regulat-
ing development and regeneration of cementum. Journal of
Periodontal Research 28, 550–9.
Mariotti AJ, Rumpf D (1999) Chlorhexidine-induced changes
to human gingival fibroblasts collagen and non-collagen
protein production. Journal of Periodontology 70, 1443–8.
ª 2010 International Endodontic Journal
Lee et al. Cytotoxicity of CHX
Meister A (1994) Glutathione, ascorbate, and cellular protec-
tion. Cancer Research 54, 1969–75.
Meister A, Anderson M (1983) Glutathione. Annual Reviews of
Biochemistry 52, 711–60.
Nelissen JMDT, Torensma R, Pluyter M et al. (2000) Molecular
analysis of its hematopoiesis supporting osteoblastic cell line
U2-OS. Experimental Hematology 28, 422–32.
Pucher JJ, Daniel JC (1993) The effects of chlorhexidine
digluconate on human fibroblasts in vitro. Journal of
Periodontology 62, 526–32.
Shaw JP, Chou IN (1986) Elevation of in intracellular
glutathione content: association with mitogenic stimulation
of quiescent fibroblasts. Journal of Cell Physiology 129, 193–
8.
Siren EK, Haapasalo MPP, Waltimo TMT, Orstavik D (2004)
In vitro antibacterial effect of calcium hydroxide combined
with chlorhexidine or iodine potassium iodide on Entero-
coccus faecalis. European Journal of Oral Sciences 112, 326–
31.
Skehan P (1995) Assays of cell growth and cytotoxicity. In:
Studizinski GP, ed. Cell growth and apoptosis. A practical
approach. New York: IRL Press, pp. 176–8.
Stowe TJ, Sedgley CM, Stowe B, Fenno JC (2004) The effects of
chlorhexidine gluconate (0.12%) on the antimicrobial
properties of tooth-colored ProRoot MTA. Journal of End-
odontics 30, 429–31.
Williamsan JM, Boettcher B, Meister A (1982) Intracellular
cysteine delivery system that protects against toxicity by
promoting glutathione synthesis. Proceeding of National
Academy Science United State of America 79, 6246–9.
Yeung SY, Huang CS, Chan CP et al. (2007) Antioxidant and
pro-oxidant properties of chlorhexidine and its interaction
with calcium hydroxide solutions. International Endodontic
Journal 40, 837–44.
International Endodontic Journal, 43, 430–435, 2010 435