1. Basic Research—Biology
Effects of the Association between a Calcium Hydroxide
Paste and 0.4% Chlorhexidine on the Development of the
Osteogenic Phenotype In Vitro
Raquel Assed Bezerra da Silva, DDS, PhD,* Mário Roberto Leonardo, DDS, PhD,*
Léa Assed Bezerra da Silva, DDS, PhD,* Larissa Moreira Spinola de Castro, DDS,†
Adalberto Luiz Rosa, DDS, PhD,‡ and Paulo Tambasco de Oliveira, DDS, PhD†
Abstract
The present study aimed to evaluate whether the as-
sociation between a calcium hydroxide paste (Calen
paste) and 0.4% chlorhexidine (CHX) affects the devel-
T he objective of an intracanal dressing is to reduce the number of microorganisms to
levels lower than those achieved by mechanical instrumentation, ultimately prevent-
ing reinfection of the root canal lumen. This medication should have a broad antibac-
opment of the osteogenic phenotype in vitro. With rat terial spectrum, not be cytotoxic, have a sufficient time of action to eliminate bacteria,
calvarial osteogenic cell cultures, the following param- and possess physicochemical properties that permit its diffusion through the dentinal
eters were assayed: cell morphology and viability, al- tubules and lateral ramifications of the root canal system.
kaline phosphatase activity, total protein content, bone Among several intracanal dressings used for teeth with no pulp vitality and with
sialoprotein immunolocalization, and mineralized nod- chronic periapical lesions, calcium hydroxide (Ca(OH)2) has been regarded as the best
ule formation. Comparisons were carried out by using option, and it has been widely recommended and used because of its proven antibac-
the nonparametric Kruskal-Wallis test (level of signifi- terial properties (1), periapical tissue healing stimulation (2), biocompatibility (3),
cance, 5%). The results showed that the association antiexudate activity (4), and necrotic tissues dissolution propriety (5). The dissociation
between Calen paste and 0.4% CHX did not affect the of Ca(OH)2 into calcium and hydroxyl ions is responsible for the alkalinization of the
development of the osteogenic phenotype. No signifi- cavity, which is not conducive to bacterial development and proliferation. In addition,
cant changes were observed in terms of cell shape, cell Ca(OH)2 mediates the neutralization of bacterial lipopolysaccharide (LPS) (6, 7) and
viability, alkaline phosphatase activity, and the total stimulates periapical hard-tissue healing.
amount of bone-like nodule formation among control, The Calen paste (S.S. White, Rio de Janeiro, RJ, Brazil) is a Ca(OH)2-based med-
Calen, or Calen CHX groups. The strategy to combine ication associated with a viscous vehicle (polyethylene glycol 400) that permits slower
Ca(OH)2 and CHX to promote a desirable synergistic liberation of hydroxyl ions, maintaining its action for a longer period, decreasing its
antibacterial effect during endodontic treatment in vivo solubility, and increasing its root dentin penetrability (8). In addition, it presents pH
might not significantly affect osteoblastic cell biology. around 12.4 (9), high antibacterial activity (8), and biocompatibility (9, 10).
(J Endod 2008;34:1485–1489) Several substances have been associated with Ca(OH)2 to improve its radiopacity,
viscosity, flowing, spectrum of antibacterial activity, ionic dissociation, and other phys-
Key Words icochemical properties, ultimately favoring its clinical applications (11). Concerning
Alkaline phosphatase, bone sialoprotein, calcium hy- the improvement of antibacterial activity, chlorhexidine gluconate (CHX) has emerged
droxide, cell culture, cell viability, chlorhexidine, as an effective antimicrobial medication in endodontic therapy (12, 13), exhibiting a
mineralization, osteoblast broad spectrum of activity against gram-positive and gram-negative bacteria (14),
capacity to adsorb onto dental tissues and mucous membrane with prolonged gradual
release at therapeutic levels (12), biocompatibility (15), and low toxicity (16). How-
From the *Department of Pediatric Clinic, Preventive and
ever, CHX itself fails to inactivate bacterial LPS (17) and to promote tissue dissolution
Social Dentistry, †Division of Oral Histology, and ‡Department (18). When used as a root canal treatment, CHX has been shown to be more effective
of Oral and Maxillofacial Surgery and Periodontology, School than Ca(OH)2 in eliminating Enterococcus faecalis infection inside dentinal tubules
of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão (11). In this context, different protocols of CHX therapy have been proposed to be
Preto, SP, Brazil. effective in reducing bacteria in the root canal system, with varying concentrations
Address requests for reprints to Dr Raquel Assed Bezerra
da Silva, Departamento de Clínica Infantil, Odontologia Pre- (0.12%–2%) and exposure times, depending on the application, whether irrigation or
ventiva e Social, Faculdade de Odontologia de Ribeirão Preto– intracanal medication.
USP, Av. do Café, s/n Monte Alegre, 14040-904, Ribeirão The association of Ca(OH)2 and CHX could therefore result in a desirable syner-
Preto, SP, Brasil. E-mail address: raquel@forp.usp.br. gistic antibacterial effect (14) with benefits to the endodontic treatment. The viability of
0099-2399/$0 - see front matter
Copyright © 2008 American Association of Endodontists.
such strategy is supported by several in vitro studies, showing that there were no
doi:10.1016/j.joen.2008.08.031 adverse effects on either solubility or activity of both medications (11, 19). Although
experimental studies have already been undertaken that aim to evaluate the effects of
Ca(OH)2 and CHX (hereafter referred to as Calen CHX paste) on different aspects of
the endodontic therapy (11, 13, 20, 21), none of these assessed the impact of such paste
on osteoblastic cell biology, including morphology, differentiation, and function. Thus,
the present study aimed to evaluate whether the association between Calen paste and
0.4% CHX affects the development of the osteogenic phenotype in vitro.
JOE — Volume 34, Number 12, December 2008 Effects of Ca(OH)2 Paste and CHX on Development of Osteogenic Phenotype 1485

2. Basic Research—Biology
Materials and Methods with N Plan (X10/0.25, X20/0.40) and HCX PL Fluotar (X40/0.75) ob-
Cell Isolation and Primary Culture of Osteogenic Cells jectives, outfitted with a Leica DC 300F digital camera, 1.3 megapixel
Osteogenic cells were isolated by sequential trypsin/collagenase charge-coupled device. The acquired digital images were processed
digestion of calvarial bone from newborn (2– 4 days) Wistar rats, as with Adobe Photoshop software (version 7.0; Adobe Systems Inc, San
previously described (22). All animal procedures were in accordance Jose, CA).
with guidelines of the Animal Research Ethics Committee of the Univer-
sity of São Paulo. Cells were plated on Thermanox coverslips (Nunc,
Methyl-Thiazol-Diphenyl-Tetrazolium Cell
Rochester, NY) in 24-well polystyrene plates (Falcon, Franklin Lakes, Viability/Proliferation Assay
NJ) at a cell density of 20,000 cells/well. The plated cells were grown for The methyl-thiazol-diphenyl-tetrazolium (MTT) assay for cell via-
periods up to 14 days by using an osteogenic medium composed of bility/proliferation is based on the reductive cleavage of yellow tetrazo-
Gibco -Minimum Essential Medium with L-glutamine (Invitrogen, lium salt to a purple formazan compound by the dehydrogenase activity
Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), of intact mitochondria. Therefore, this conversion only occurs in living
7 mmol/L -glycerophosphate (Sigma, St Louis, MO), 5 g/mL ascor- cells. At days 3, 7, and 10, cells were incubated with 100 L of 3-[4,5-
bic acid (Sigma), and 50 g/mL gentamicin (Invitrogen) at 37°C in a dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; 5 mg/
humidified atmosphere with 5% CO2. The culture medium was changed mL) in phosphate-buffered saline at 37°C for 4 hours. The medium was
every 3 days. The progression of cultures was examined by phase con- then aspirated from the well, and 1 mL of acid isopropanol (0.04 N HCl
trast microscopy. in isopropanol) was added to each well. The plates were then agitated
on a plate shaker for 5 minutes, and 100 L of this solution was trans-
ferred to a 96-well format by using opaque-walled transparent-bot-
Exposure of Osteogenic Cell Cultures to Calcium
tomed plates (Fisher Scientific, Pittsburgh, PA). The optical density was
Hydroxide– based Pastes read at 570 – 650 nm on the plate reader ( Quant; Biotek, Winooski,
The Calen paste (2.5 g Ca(OH)2, 0.5 g zinc oxide p.a., 0.05 g VT), and data were expressed as absorbance (24).
colophony, 1.75 mL polyethylene glycol 400) (20) was purchased from
S.S. White. The Calen CHX paste was prepared by mixing the Calen Alkaline Phosphatase Activity and Total Protein Content
paste with 0.4% chlorhexidine digluconate 20% (w/v) aqueous solution Alkaline phosphatase (ALP) activity was assayed in the same lysates
(Farmoderm, Ribeirão Preto, SP, Brasil). Treated cultures were ex- used for determining total protein content as the release of thymol-
posed to the osteogenic medium supplemented with 25 g/mL Calen or phthalein from thymolphthalein monophosphate by using a commercial
Calen CHX pastes from day 1 on, whereas control cultures were only kit (Labtest Diagnóstica, MG, Brazil). Briefly, 50 L of thymolphthalein
exposed to the osteogenic medium. The concentration chosen was monophosphate was mixed with 0.5 mL of 0.3 mol/L diethanolamine
based on a recent study of our group, which showed that higher Calen or buffer, pH 10.1, and left for 2 minutes at 37°C. The solution was then
Calen CHX concentrations (50 or 100 g/mL) induced significant added to 50 L of the lysates obtained from each well for 10 minutes at
cytotoxic effects in the macrophage cell line RAW264.7 (23). 37°C. For color development, 2 mL of 0.09 mol/L Na2CO3 and 0.25
mol/L NaOH were added. After 30 minutes, absorbance was measured
Cell Morphology and Localization of Noncollagenous Bone at 590 nm, and ALP activity was calculated from a standard curve by
Matrix Proteins using thymolphthalein to give a range from 0.012– 0.4 mol thymol-
At days 3 and 7, cells were fixed for 10 minutes at room temper- phthalein/h/mL. Data were expressed as ALP activity normalized for
ature (RT) by using 4% paraformaldehyde in 0.1 mol/L sodium phos- total protein content. Some cultures were also stained with Fast red at
phate buffer (PB), pH 7.2. For dual staining with alizarin red (AR; day 7, as described elsewhere (25), for in situ histochemical detection
Sigma) and immunolabeling with bone sialoprotein (BSP) at day 14, of ALP activity during the onset of differentiation phase of the cultures.
cells were fixed in 70% ethanol for 60 minutes at 4°C (described be- Total protein content was determined by using a modification of
low). After washing in PB, cultures were processed for immunofluores- the method of Lowry et al. (26). Briefly, proteins were extracted from
cence labeling (22). Briefly, they were permeabilized with 0.5% Triton each well with 0.1% sodium lauryl sulphate (Sigma) for 30 minutes and
X-100 in PB for 10 minutes followed by blocking with 5% skimmed milk mixed 1:1 with Lowry solution (Sigma) for 20 minutes at RT. The extract
in PB for 30 minutes. Primary monoclonal antibodies to BSP (WVID1- was diluted in Folin and Ciocalteau’s phenol reagent (Sigma) for 30
9C5, 1:200; Developmental Studies Hybridoma Bank-DSHB, Iowa City, minutes at RT. Absorbance was measured at 680 nm by using a spec-
IA) and osteopontin (MPIIIB10-1, 1:800; DSHB) were used followed by trophotometer (Cecil CE3021, Cambridge, UK). The total protein con-
Alexa Fluor 488 (green fluorescence)– conjugated or Alexa Fluor 594 tent was calculated from a standard curve and expressed as g/mL.
(red fluorescence)– conjugated goat anti-mouse secondary antibody
(1:200; Molecular Probes, Invitrogen, Eugene, OR). Alexa Fluor 488 Mineralized Bone-like Nodule Formation
(green fluorescence)– conjugated phalloidin (1:200; Molecular For the histochemical detection of mineralized bone-like nodule
Probes) was used in some cases as a marker of the actin cytoskeleton. formation, cultures at day 14 were washed in Hanks’ balanced salt
Replacement of the primary monoclonal antibody with PB was used as solution (Sigma) and fixed in 70% ethanol for 60 minutes at 4°C. The
control. All antibody incubations were performed in a humidified envi- samples were washed in phosphate-buffered saline and dH2O and then
ronment for 60 minutes at RT. Between each incubation step, the sam- stained with 2% AR, pH 4.2, for 15 minutes at RT. After being profusely
ples were washed in PB (3 5 minutes). Before mounting for micro- washed in dH2O, they were also processed for triple labeling with BSP
scope observation, samples were briefly washed with dH2O, and cell and DAPI. AR-stained cultures were imaged by epifluorescence micros-
nuclei were stained with 300 nmol/L 4=,6-diamidino-2-phenylindole, copy and also photographed with a high-resolution digital camera
dihydrochloride (DAPI; Molecular Probes) for 5 minutes. Thermanox (Canon EOS Digital Rebel Camera, 6.3 Megapixel CMOS sensor, with a
coverslips were placed face up on glass slides and covered with 12-mm Canon EF 100 mm f/2.8 macro lens; Canon, Lake Success, NY). The
round glass coverslips (Fisher Scientific, Suwanee, GA) mounted with percentage of the substrate area occupied by AR-stained nodules was
Prolong antifade (Molecular Probes). The samples were then examined determined by analyzing the macroscopic images by using Image Tool
under epifluorescence by using a Leica DMLB light microscope (Leica), software (University of Texas Health Science Center, San Antonio, TX).
1486 Silva et al. JOE — Volume 34, Number 12, December 2008

3. Basic Research—Biology
Statistical Analysis Epifluorescence revealed that at day 3, cells were adherent and
Comparisons were carried out with the nonparametric Kruskal- well-spread on Thermanox, exhibiting predominantly a polygonal
Wallis test for independent samples (level of significance, 5%) by means shape (Fig. 1A–C). Cell-cell contacts and mitotic figures were also
of GraphPad Prism 4 software (GraphPad Software Inc, San Diego, CA). clearly observed. At day 7 at the end of the proliferative phase, cultures
were at confluence, showing focal areas of initial cell multilayering.
Results Such areas were stained with Fast red (Fig. 1D–F), with cells positively
The development of the osteogenic phenotype was similar for con- immunolabeled with anti-BSP antibody mostly in the perinuclear region
trol, Calen, and Calen CHX groups. The common aspects of the and in some dots throughout the cytoplasm (Fig. 1G–I). At day 14,
progression of the cultures are therefore described below, irrespective AR-stained nodular areas were clearly noticed both microscopically
of the experimental group. (Fig. 1J–L) and macroscopically (Fig. 1M–O). Cells associated with
Figure 1. Calvaria-derived osteogenic cell cultures grown on Thermanox coverslips at days 3 (A–C), 7 (D–I), and 14 (J–O). (A, D, G, J, M) Control cultures. (B, E,
H, K, N) Calen-treated cultures. (C, F, I, L, O) Calen CHX–treated cultures. At day 3 (A–C), epifluorescence of actin cytoskeleton labeling (phalloidin labeling, green
fluorescence) and DNA stain (DAPI, blue fluorescence) revealed that the cells were adherent and spread in all groups, exhibiting mostly a polygonal shape. At day
7, areas of initial cell multilayering in confluent cultures showed ALP activity (Fast red–stained; D–F, transmitted light; D–F insets, epifluorescence) and BSP
immunolabeling (G–I, red fluorescence). At day 14, all groups exhibited areas of mineralized bone-like nodule formation, which were AR-stained (J–L, epifluores-
cence; M–O, macroscopic observation) and also immunoreactive for BSP and osteopontin (OPN) (green fluorescence in J–L and J inset, respectively). The scale bar
represents 100 m for A–C and G–L; 200 m for D–F and J inset; 300 m for D–F insets; and 2.3 mm for M–O.
JOE — Volume 34, Number 12, December 2008 Effects of Ca(OH)2 Paste and CHX on Development of Osteogenic Phenotype 1487

4. Basic Research—Biology
TABLE 1. Quantitative Analysis of Cell Viability, Protein Content, ALP Activity, and AR-stained Areas in Osteogenic Cell Cultures Grown on Thermanox in Groups I
(Control), II (Calen), and III (Calen CHX)
Time Points Group I Group II Group III Kruskal-Wallis
Parameters
(Days) (Control) (Calen) (Calen CHX) Test
Cell viability (OD) 3 0.21 0.02 0.22 0.02 0.20 0.01 NS
7 0.64 0.05 0.64 0.05 0.68 0.05 NS
10 0.50 0.12 0.58 0.11 0.54 0.17 NS
Protein content ( g/mL) 7 386.74 9.92 385.50 7.7 388.12 6.62 NS
10 97.31 10.31 98.75 4.97 93.01 4.57 NS
ALP activity ( mol thymolphthalein/h/mg) 7 0.45 0.12 0.63 0.16 0.46 0.13 NS
10 31.55 4.31 20.44 8.98 21.15 5.58 NS
AR-stained areas (%) 14 7.2 1.2 8.17 1.3 11.1 3.2 NS
Data represent mean values standard deviation (n).
NS, not significant (P .05).
these calcified areas were labeled with BSP and OPN antibodies exposure. Recently, Silva et al. (23) demonstrated that Calen or Calen
(Fig. 1J–L). CHX concentrations of 50 or 100 g/mL were highly cytotoxic for mac-
Quantitative analysis revealed no significant differences in terms of rophagic cells in vitro. Thus, we have opted to expose the osteogenic
cell viability, ALP activity, total protein content, and AR-stained areas cells to 25 g/mL of either Calen or Calen CHX from day 1 on
among the experimental groups. Data are summarized in Table 1. MTT throughout the culture interval. In the in vivo situation because of the
values increased for cultures at day 7, remaining similar/constant at day capacity of Calen to diffuse throughout the periapical tissues (28), the
10 (Fig. 2). Although at day 7 ALP activity was minimal for all groups, at highest cytotoxic concentrations of the paste might prevent the deposi-
day 10 cultures exhibited peak levels. At day 14, the mean proportion of tion of mineralized matrix for closing the apical foramen. In this con-
AR-stained areas indicated that the experimental conditions supported text, the present results represent only in part the range and complexity
the development of the osteogenic phenotype. In addition, a tendency of cell and tissue response that takes place in vivo.
for the Calen CHX group to support enhanced bone-like nodule The rat calvarial cell culture model was chosen to assess the de-
formation was noticed (Fig. 3). velopment of the osteogenic phenotype when culture medium was sup-
plemented with Calen or Calen CHX. Isolation procedures, culture
Discussion conditions, the temporal sequence of osteoblastic differentiation, and
The rationale for the association of Ca(OH)2 and CHX is that it expression of matrix proteins have been well-defined for this system
could promote a synergistic antibacterial effect (13), ultimately leading (29). Under standard osteogenic conditions, rat calvaria– derived cells
to benefits to the endodontic treatment, including promotion of the generate woven bone-like nodules in areas of cell multilayering during
repair processes of periapical tissues. Thus, it would be important to the second week of primary cultures, which result from the clonal
verify the effects of such mixture on cells associated with production of expansion of osteoprogenitors and their entry into the osteoblast differ-
mineralized matrix. The results of the present study showed that the entiation sequence (29 –31). In the present study, the acquisition of the
association between Calen paste and 0.4% CHX did not affect the devel- osteoblastic phenotype was detected in all experimental groups by using
opment of the osteogenic phenotype in rat calvarial cell cultures. In- ALP activity, BSP immunolabeling, and AR staining for calcium deposits.
deed, no significant changes were observed in terms of cell viability, ALP It has been demonstrated that ALP is crucial for the initiation, but not for
activity, and the total amount of bone-like nodule formation among
control, Calen, or Calen CHX groups. In a previous study with mouse
fibroblasts, Sirén et al. (27) also showed no changes in cytotoxic effects
of the calcium hydroxide– 0.5% CHX mixture compared with those of
the pure calcium hydroxide.
The cytotoxic effects of exposure to chemical compounds depend
on several factors, including concentration and time and duration of
Figure 2. Viability/proliferation analysis (MTT assay) of control, Calen , and Figure 3. Quantitative analysis of AR-stained areas for control, Calen , and
Calen CHX–treated calvaria-derived osteogenic cell cultures grown on Ther- Calen CHX–treated calvaria-derived osteogenic cell cultures grown on Ther-
manox coverslips for 3, 7, and 10 days. No statistically significant differences manox coverslips for 14 days. No statistically significant differences were ob-
were observed among the experimental groups. The optical density was read at served among the experimental groups, although there was a tendency for the
570 – 650 nm, and data were expressed as absorbance. Calen CHX group to support enhanced bone-like nodule formation.
1488 Silva et al. JOE — Volume 34, Number 12, December 2008

5. Basic Research—Biology
the progression/maintenance, of the matrix mineralization process 11. Basrani B, Tjaderhane L, Santos M, et al. Efficacy of chlorhexidine and calcium
(32). Although the role of ALP is still not fully understood, it has been hydroxide containing medicaments against Enterococcus faecalis in vitro. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod 2003;96:618 –24.
proposed that such enzyme generates the Pi needed for hydroxyapatite 12. Rosenthal S, Spångberg L, Safavi K. Chlorhexidine substantivity in root canal dentin.
crystallization and might also hydrolyze pyrophosphate, a mineraliza- Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:488 –92.
tion inhibitor, to facilitate mineral precipitation and growth (33, 34). 13. Zerella JA, Fouad AF, Spångberg LS. Effectiveness of a calcium hydroxide and chlo-
Concerning BSP, this multifunctional, matricellular protein has been rhexidine digluconate mixture as disinfectant during retreatment of failed endodon-
considered an early marker of differentiating osteoblasts, which plays a tic cases. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:756 – 61.
crucial role in the hydroxyapatite nucleation on the collagen matrix 14. Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza-Filho FJ. In vitro
evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite.
(35). Indeed, in the present study, BSP immunolabeling was detected at Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:79 – 84.
the end of the proliferative phase (day 7) in the majority of cells in 15. Dammaschke T, Schneider U, Stratmann U, Yoo JM, Schäfer E. Effect of root canal
multilayered areas and also during the matrix mineralization phase (day dressings on the regeneration of inflamed periapical tissue. Acta Odontol Scand
14), associated with the AR-stained bone-like nodules. The total area of 2005;63:143–52.
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effects of established and potential root canal irrigants. J Endod 1995;21:513–5.
control, a finding that is supported by the results of the MTT assay, 17. Silva LA, Leonardo MR, Assed S, Tanomaru Filho M. Histological study of the effect of
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In conclusion, we have shown that the addition of 0.4% CHX to the 18. Okino LA, Siqueira EL, Santos M, Bombana AC, Figueiredo JA. Dissolution of pulp
Calen paste at a concentration of 25 g/mL does not affect the progres- tissue by aqueous solution of chlorhexidine digluconate and chlorhexidine diglu-
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paste have been demonstrated to be cytotoxic at least to macrophagic 340 –5.
cells (24), the strategy to combine Ca(OH)2 and CHX to promote a 20. De Rossi A, Silva LA, Leonardo MR, Rocha LB, Rossi MA. Effect of rotary or manual
desirable synergistic antibacterial effect during endodontic treatment in instrumentation, with or without a calcium hydroxide/1% chlorhexidine intracanal
vivo might not significantly affect osteoblastic cell biology, especially in dressing, on the healing of experimentally induced chronic periapical lesions. Oral
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bone sites permeated with higher dilutions of the paste as it diffuses 21. Soares JA, Leonardo MR, Tanomaru Filho M, Silva LA, Ito IY. Residual antibacterial
outward into the periapical tissues. Further studies are needed to verify activity of chlorhexidine digluconate and camphorated p-monochlorophenol in cal-
whether the findings presented here correlate with bone repair events in cium hydroxide-based root canal dressings. Braz Dent J 2007;18:8 –15.
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Acknowledgments 23. Silva RAB, Leonardo MR, Silva LAB, Faccioli LH, Medeiros AI. Effect of a calcium
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