Effects of the association between a calcium hydroxide


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Effects of the association between a calcium hydroxide

  1. 1. Basic Research—BiologyEffects of the Association between a Calcium HydroxidePaste and 0.4% Chlorhexidine on the Development of theOsteogenic Phenotype In VitroRaquel 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†AbstractThe present study aimed to evaluate whether the as-sociation between a calcium hydroxide paste (Calenpaste) 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 dentinaleters 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 withsialoprotein immunolocalization, and mineralized nod- chronic periapical lesions, calcium hydroxide (Ca(OH)2) has been regarded as the bestule 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 dissociationbetween 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 thedevelopment 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) andviability, 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 slowerCa(OH)2 and CHX to promote a desirable synergistic liberation of hydroxyl ions, maintaining its action for a longer period, decreasing itsantibacterial effect during endodontic treatment in vivo solubility, and increasing its root dentin penetrability (8). In addition, it presents pHmight 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). ConcerningAlkaline phosphatase, bone sialoprotein, calcium hy- the improvement of antibacterial activity, chlorhexidine gluconate (CHX) has emergeddroxide, cell culture, cell viability, chlorhexidine, as an effective antimicrobial medication in endodontic therapy (12, 13), exhibiting amineralization, 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 dissolutionSocial Dentistry, †Division of Oral Histology, and ‡Department (18). When used as a root canal treatment, CHX has been shown to be more effectiveof Oral and Maxillofacial Surgery and Periodontology, School than Ca(OH)2 in eliminating Enterococcus faecalis infection inside dentinal tubulesof Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão (11). In this context, different protocols of CHX therapy have been proposed to bePreto, SP, Brazil. effective in reducing bacteria in the root canal system, with varying concentrations Address requests for reprints to Dr Raquel Assed Bezerrada Silva, Departamento de Clínica Infantil, Odontologia Pre- (0.12%–2%) and exposure times, depending on the application, whether irrigation orventiva 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 of0099-2399/$0 - see front matter Copyright © 2008 American Association of Endodontists. such strategy is supported by several in vitro studies, showing that there were nodoi: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. 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 processeddigestion of calvarial bone from newborn (2– 4 days) Wistar rats, as with Adobe Photoshop software (version 7.0; Adobe Systems Inc, Sanpreviously 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 CellRochester, NY) in 24-well polystyrene plates (Falcon, Franklin Lakes, Viability/Proliferation AssayNJ) 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 activityCarlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), of intact mitochondria. Therefore, this conversion only occurs in living7 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 wasevery 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 HCltrast 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 wasHydroxide– 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 fromS.S. White. The Calen CHX paste was prepared by mixing the Calen Alkaline Phosphatase Activity and Total Protein Contentpaste 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 commercialCalen CHX pastes from day 1 on, whereas control cultures were only kit (Labtest Diagnóstica, MG, Brazil). Briefly, 50 L of thymolphthaleinexposed to the osteogenic medium. The concentration chosen was monophosphate was mixed with 0.5 mL of 0.3 mol/L diethanolaminebased 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 thenCalen CHX concentrations (50 or 100 g/mL) induced significant added to 50 L of the lysates obtained from each well for 10 minutes atcytotoxic 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 measuredCell Morphology and Localization of Noncollagenous Bone at 590 nm, and ALP activity was calculated from a standard curve byMatrix 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 forature (RT) by using 4% paraformaldehyde in 0.1 mol/L sodium phos- total protein content. Some cultures were also stained with Fast red atphate buffer (PB), pH 7.2. For dual staining with alizarin red (AR; day 7, as described elsewhere (25), for in situ histochemical detectionSigma) 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 oflow). After washing in PB, cultures were processed for immunofluores- the method of Lowry et al. (26). Briefly, proteins were extracted fromcence labeling (22). Briefly, they were permeabilized with 0.5% Triton each well with 0.1% sodium lauryl sulphate (Sigma) for 30 minutes andX-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 extractin PB for 30 minutes. Primary monoclonal antibodies to BSP (WVID1- was diluted in Folin and Ciocalteau’s phenol reagent (Sigma) for 309C5, 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 noduleProbes) was used in some cases as a marker of the actin cytoskeleton. formation, cultures at day 14 were washed in Hanks’ balanced saltReplacement of the primary monoclonal antibody with PB was used as solution (Sigma) and fixed in 70% ethanol for 60 minutes at 4°C. Thecontrol. All antibody incubations were performed in a humidified envi- samples were washed in phosphate-buffered saline and dH2O and thenronment 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 profuselyples were washed in PB (3 5 minutes). Before mounting for micro- washed in dH2O, they were also processed for triple labeling with BSPscope 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 cameradihydrochloride (DAPI; Molecular Probes) for 5 minutes. Thermanox (Canon EOS Digital Rebel Camera, 6.3 Megapixel CMOS sensor, with acoverslips 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). Theround glass coverslips (Fisher Scientific, Suwanee, GA) mounted with percentage of the substrate area occupied by AR-stained nodules wasProlong antifade (Molecular Probes). The samples were then examined determined by analyzing the macroscopic images by using Image Toolunder 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. 3. Basic Research—BiologyStatistical 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 polygonalWallis test for independent samples (level of significance, 5%) by means shape (Fig. 1A–C). Cell-cell contacts and mitotic figures were alsoof 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 regiontrol, 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 microscopicallyof the experimental group. (Fig. 1J–L) and macroscopically (Fig. 1M–O). Cells associated withFigure 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, greenfluorescence) and DNA stain (DAPI, blue fluorescence) revealed that the cells were adherent and spread in all groups, exhibiting mostly a polygonal shape. At day7, areas of initial cell multilayering in confluent cultures showed ALP activity (Fast red–stained; D–F, transmitted light; D–F insets, epifluorescence) and BSPimmunolabeling (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 barrepresents 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. 4. Basic Research—BiologyTABLE 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 NSData 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 osteogeniccell viability, ALP activity, total protein content, and AR-stained areas cells to 25 g/mL of either Calen or Calen CHX from day 1 onamong the experimental groups. Data are summarized in Table 1. MTT throughout the culture interval. In the in vivo situation because of thevalues increased for cultures at day 7, remaining similar/constant at day capacity of Calen to diffuse throughout the periapical tissues (28), the10 (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 complexitythe 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 systemcould promote a synergistic antibacterial effect (13), ultimately leading (29). Under standard osteogenic conditions, rat calvaria– derived cellsto benefits to the endodontic treatment, including promotion of the generate woven bone-like nodules in areas of cell multilayering duringrepair processes of periapical tissues. Thus, it would be important to the second week of primary cultures, which result from the clonalverify 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 theassociation between Calen paste and 0.4% CHX did not affect the devel- osteoblastic phenotype was detected in all experimental groups by usingopment 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 foractivity, and the total amount of bone-like nodule formation amongcontrol, Calen, or Calen CHX groups. In a previous study with mousefibroblasts, Sirén et al. (27) also showed no changes in cytotoxic effectsof the calcium hydroxide– 0.5% CHX mixture compared with those ofthe pure calcium hydroxide. The cytotoxic effects of exposure to chemical compounds dependon several factors, including concentration and time and duration ofFigure 2. Viability/proliferation analysis (MTT assay) of control, Calen , and Figure 3. Quantitative analysis of AR-stained areas for control, Calen , andCalen 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 the570 – 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. 5. Basic Research—Biologythe 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 canalmultilayered areas and also during the matrix mineralization phase (day dressings on the regeneration of inflamed periapical tissue. Acta Odontol Scand14), associated with the AR-stained bone-like nodules. The total area of 2005;63:143–52.calcified matrix for the Calen or Calen CHX groups was similar to the 16. Yesilsoy C, Whitaker E, Cleveland D, Phillips E, Trope M. Antimicrobial and toxic 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 ofshowing no significant changes among the groups in terms of cell via- some irrigating solutions on bacterial endotoxin in dogs. Braz Dent J 2004;bility. 15:109 –14. 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 pulpCalen paste at a concentration of 25 g/mL does not affect the progres- tissue by aqueous solution of chlorhexidine digluconate and chlorhexidine diglu-sion of osteogenic cell cultures, allowing the formation of mineralized conate gel. Int Endod J 2004;37:38 – 41. 19. Podbielski A, Spahr A, Haller B. Additive antimicrobial activity of calcium hydroxidenodules in vitro. Although higher concentrations of the Calen CHX and chlorhexidine on common endodontic bacterial pathogens. J Endod 2003;29: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 manualdesirable synergistic antibacterial effect during endodontic treatment in instrumentation, with or without a calcium hydroxide/1% chlorhexidine intracanalvivo might not significantly affect osteoblastic cell biology, especially in dressing, on the healing of experimentally induced chronic periapical lesions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:628 –36.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 antibacterialoutward 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.animal models after the placement of Calen CHX paste. 22. De Oliveira PT, Zalzal SF, Beloti MM, Rosa AL, Nanci A. Enhancement of in vitro osteogenesis on titanium by chemically produced nanotopography. J Biomed Mater Res A 2007;80:554 – 64. Acknowledgments 23. Silva RAB, Leonardo MR, Silva LAB, Faccioli LH, Medeiros AI. Effect of a calcium The authors thank Roger Rodrigo Fernandes and graduate stu- hydroxide-based paste associated to chlorhexidine on raw 264.7 macrophage celldent Lucas Novaes Teixeira (University of São Paulo at Ribeirão line culture. Oral Surg Oral Med Oral Pathol Oral Radiol Endod (doi: 10.1016/Preto) for technical assistance. The mouse monoclonal antibody j.tripleo.2008.06.027).anti-rat BSP (WVID1-9C5), developed by Michael Solursh and 24. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55– 63.Ahnders Franzen, was obtained from the Developmental Studies 25. Majors AK, Boehm CA, Nitto H, Midura RJ, Muschler GF. Characterization of humanHybridoma Bank developed under the auspices of the National In- bone marrow stromal cells with respect to osteoblastic differentiation. J Orthop Resstitute of Child Health and Human Development and maintained by 1997;15:546 –57.the Department of Biological Sciences of the University of Iowa. 26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. References 27. Sirén EK, Haapasalo MP, Waltimo TM, Ørstavik D. In vitro antibacterial effect of calcium hydroxide combined with chlorhexidine or iodine potassium iodide on 1. Manzur A, González AM, Pozos A, Silva-Herzog D, Friedman S. Bacterial quantifica- Enterococcus faecalis. Eur J Oral Sci 2004;112:326 –31. tion in teeth with apical periodontitis related to instrumentation and different intra- 28. Leonardo MR, da Silva LA, Leonardo Rde T, Utrilla LS, Assed S. Histological evaluation canal medications: a randomized clinical trial. J Endod 2007;33:114 – 8. of therapy using a calcium hydroxide dressing for teeth with incompletely formed 2. Leonardo MR, Hernandez ME, Silva LA, Tanomaru-Filho M. Effect of a calcium apices and periapical lesions. J Endod 1993;19:348 –52. hydroxide-based root canal dressing on periapical repair in dogs: a histological 29. Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation in- study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:680 –5. terrelationships during progressive development of the osteoblast phenotype. En- 3. Murray PE, García Godoy C, García Godoy F. How is the biocompatibilty of dental docr Rev 1993;14:424 – 42. biomaterials evaluated? Med Oral Pathol Oral Cir Bucal 2007;12:e258 – 66. 30. Nanci A, Zalzal S, Gotoh Y, McKee MD. Ultrastructural characterization and immu- 4. Allard U, Stromberg U, Stromberg T. Endodontic treatment of experimentally in- duced apical periodontitis in dogs. Endod Dent Traumatol 1987;3:240 – 4. nolocalization of osteopontin in rat calvarial osteoblast primary cultures. Microsc 5. Hasselgren G, Olsson B, Cvek M. Effects of calcium hydroxide and sodium hypochlo- Res Tech 1996;33:214 –31. rite on the dissolution of necrotic porcine muscle tissue. J Endod 1988;14:125–7. 31. Bellows CG, Aubin JE, Heersche JN, Antosz ME. Mineralized bone nodules formed in 6. Safavi KE, Nichols FC. Alteration of biological properties of bacterial lipopolysaccha- vitro from enzymatically released rat calvaria cell populations. Calcif Tissue Int ride by calcium hydroxide treatment. J Endod 1994;20:127–9. 1986;38:143–54. 7. Silva L, Nelson-Filho P, Leonardo MR, Rossi MA, Pansani CA. Effect of calcium hy- 32. Bellows CG, Aubin JE. Determination of numbers of osteoprogenitors present in droxide on bacterial endotoxin in vivo. J Endod 2002;28:94 – 8. isolated fetal rat calvaria cells in vitro. Dev Biol 1989;133:8 –13. 8. Leonardo MR. Endodoncia: tratamiento de conductos radiculares. 1st ed. São Paulo: 33. Bonucci E. Main suggested calcification mechanisms: cells. In: Biological calcifica- Artes Médicas, 2005. tion: normal and pathological processes in the early stages. Berlin-Heidelberg: 9. Leonardo MR, da Silva LA, Leonardo Rde T, Utrilla LS, Assed S. Histological evaluation Springer, 2007:491–506. of therapy using a calcium hydroxide dressing for teeth with incompletely formed 34. Tye CE, Rattray KR, Warner KJ, et al. Delineation of the hydroxyapatite-nucleating apices and periapical lesions. J Endod 1993;19:348 –52. domains of bone sialoprotein. J Biol Chem 2003;278:7949 –55.10. Nelson Filho P, Silva LAB, Leonardo MR, Utrilla LS, Figueiredo F. Conective tissue 35. Tye CE, Hunter GK, Goldberg HA. Identification of the type I collagen-binding domain responses to calcium hydroxide based root canal medicaments. Int Endod J of bone sialoprotein and characterization of the mechanism of interaction. J Biol 1999;32:303–11. Chem 2005;280:13487–92.JOE — Volume 34, Number 12, December 2008 Effects of Ca(OH)2 Paste and CHX on Development of Osteogenic Phenotype 1489