Research Article. University Journal of Dental Sciences. University J Dent Scie 2017; No. 3, Vol. 1

Transcription

1 COMPARATIVE EVALUATION OF CALCIUM ION, HYDROXYL ION RELEASE AND PH LEVELS BETWEEN NANO CALCIUM HYDROXIDE AND OTHER CALCIUM HYDROXIDE BASED INTRACANAL MEDICAMENTS : AN IN VITRO STUDY Sonali, Garg Amit Kumar, Paul Rohit, Hans Manoj, Nagpal Ajay Department of Conservative Dentistry and Endodontics K. D. Dental College and Hospital, Mathura ABSTRACT : Aims And Objectives: To compare calcium ion, hydroxyl ion release and ph levels between nano calcium hydroxide and other calcium hydroxide based intracanal medicaments in vitro. Materials And Methods: Calcium hydroxide based substances were divided into six groups (n=5): Group A (Calcium hydroxide powder with distilled water), Group B (Nano calcium hydroxide powder with distilled water), Group C (Vitapex), Group D (RC Cal), Group E (Dentocal) & Group F (Calcium hydroxide points). For determining the particle morphology of different calcium hydroxide products, TEM technique was used. 30 polyethylene cylindrical tubes were taken, one end of which was sealed with temporary material and filled with the materials of each group. Tubes were then immersed in separate calibrated beakers, each with 10 ml of distilled water. For each group, 5 samples were analyzed after 0, 10, and 20 minutes; and after 1, 2, 24 and 48 hours, 1 week and 1 month. The ph value was measured by calibrated ph meter. Ion liberation was measured by an ICP-atomic emission spectrometry. Statistical analysis was done by ANOVA and Tukey's post hoc tests. Results: Group B had the lowest particle size. According to calcium ion and hydroxyl ion liberation, RC Cal was better and according to ph value, Dentocal was better than other groups. Conclusion: Aqueous based preparations of calcium hydroxide should be chosen over points or oil-based calcium hydroxide preparations. University Journal of Dental Sciences Research Article Keywords : Calcium hydroxide, ph, Calcium ions, Hydroxyl ions Source of support : Nil Conflict of interest: None INTRODUCTION: Calcium hydroxide, widely used in endodontics is a strong alkaline substance having ph nearly It dissociates into calcium and hydroxyl ions in aqueous medium. Antimicrobial activity, inhibition of tooth resorption and induction of repair by hard tissue formation are its biological properties. (1-3) That's why it has been recommended for use in several clinical situations. (4) Release of hydroxyl ions as highly oxidant free radicals show extreme reactivity with several biomolecules. (5) The microorganisms penetrate into infected dentin tubules from 50 to 100 ìm depth. (6) The application of calcium hydroxide into instrumented and irrigated root canals eliminates microorganisms effectively is known. (7) Due to buffering capacity of hydroxyapatite selective permeability of the hydroxide ions in the dentin tubules is there. (8) Because of a high local ph particles inserted into the open dentin tubules may act as a direct source of dissociated calcium hydroxide which dissolve continuously in aqueous form, enhancing antimicrobial effectiveness. (9) Nano-particles are microscopic particles of less than 100 nm which are different in properties such as active surface area, chemical and biological reac- tivity. (10) They are getting popular in dentistry and medicine as antibacterial agents. The higher surface to volume ratio and charge density results in their greater interaction with the environment and thus causes a higher antibacterial activity. (11) So this study was done to find out the effect of nano-particles & other calcium hydroxide based intracanal medicaments on the release of calcium & hydroxyl ions and on ph levels. University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 50

2 University J Dent Scie 2017; No. 3, Vol. 1 MATERIALS AND METHOD : This study was carried out in the Department of Conservative Dentistry and Endodontics, K. D. Dental College and Hospital, Mathura, Uttar Pradesh, in the Department of University Sophisticated Instrument Facility, Aligarh Muslim University, Aligarh, Uttar Pradesh and at the Atmy Analytical Labs Private Limited, DLF Industrial Area, Faridabad, Haryana. Calcium hydroxide based substances were divided into six groups (n=5): Group A: Calcium hydroxide powder with distilled water (Sigma-Aldrich, St.Louis, Missouri, United States) Group B: Nano calcium hydroxide powder with distilled water (Pankaj Enterprises, New Delhi, India) Group C: Vitapex (J. Morita, Tokyo, Japan) Group D: RC Cal (Prime Dental Products, Mumbai, India) Group E: Dentocal (Anabond Stedman Pharma Research, Chennai, India) Group F: Calcium hydroxide points (Coltene whaledent, Mahwah, NJ, USA) For determining the particle size and shape of different calcium hydroxide products TEM (transmission electron microscopy) (JEOL, JEM-2100Plus, USA) technique was used. Measurements were performed on the samples dried under vacuum. Thirty polyethylene cylindrical tubes were taken, one end of which was sealed with 1 mm layer of temporary material (Orafil-GTM, Prevest DenPro, Jammu, India) and filled with the respective materials of each group (n=5). An endodontic file was used to introduce carefully calcium hydroxide pastes into the tubes through the opening, avoiding bubble formation. RC Cal, Dentocal and Vitapex were placed according to manufacturer's instructions. Calcium hydroxide points (#40) were introduced until tubes were completely filled. Tubes were then immersed immediately in separate calibrated beakers, each with 10 ml of distilled water with neutral ph which was used as extraction solution. The extraction solution was maintained at room temperature and without agitation. For each group, 5 samples were analyzed. For each group, samples were analyzed after 0, 10, and 20 minutes; and after 1, 2, 24 and 48 hours, 1 week and 1 month. The ph value was measured with a calibrated ph meter (Eutech Instruments, Ayer Rajah Crescent, Singapore). Calcium ion liberation was measured by an inductively coupled plasma-atomic emission spectrometry (ICP-AES) at same time intervals used for ph readings. ICP spectrometer (Perkin Elmer USA Model Optima 3300RL) was used at a wavelength of nm specific for calcium quantification. From the calculation of calcium ions liberated, it was possible to determine the amount of hydroxyl ions liberated. The molecular weight of 2 mol hydroxyl ion is 34, and molecular weight of 1 mol calcium ion is 40.08, and molecular weight of complete molecule is The percent of the two in the total weight was calculated to be 45.89% and 54.11%, respectively. Therefore in 1 mol of calcium hydroxide, there was 45.89% hydroxyl ions and 54.11% calcium ions, the quantity of hydroxyl ions was calculated as they were directly proportional. The antimicrobial action of calcium hydroxide depends on the concentration of hydroxyl ions in the solution. Then it was also analyzed that whether particle morphology do have any effect on the ph, calcium ion release and hydroxyl ion release. Statistical analysis was carried out using one way analysis of variance (ANOVA) and Tukey's honestly significant difference (HSD) post hoc tests with PASW statistics version 18 to compare the statistical difference. Significance level was set at P-value less than RESULTS: Figures: TEM images of A) Calcium hydroxide (SigmaAldrich) powder showing irregular particle morphology. B) Calcium hydroxide (nano) powder showing circular particle morphology. C) Vitapex showing circular particle morphology. D) RC Cal showing circular particle morphology. E) Dentocal showing irregular particle University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 51

3 morphology. F) Calcium hydroxide points showing irregular particle morphology. Mean particle size (nm) of different calcium hydroxide intracanal medicaments in TEM represented the largest particle size of Group C followed by Group D, E, A and F respectively. While, Group B having the lowest particle size as revealed by One-Way ANOVA. Table 1. Mean ph of Different Calcium Hydroxide Intracanal Medicaments. ph than other groups. At 0 min & 10 min, Group A had least ph value than all other groups. Group B had least ph value at 20 min, 1 hr, 2 hrs & 1 week. Table 2. ICP AES Mean Calcium Ion Release (ppm) of Different Calcium Hydroxide Intracanal Medicaments. F=ANOVA Test N=Sample Size Table 1 represents that at 0 min, 10 min & 20 min, Group C reached the higher ph value than all other groups. At 1 hr, 2 hrs, 24 hrs, 1 week & 1 month, Group E reached the highest ph than other groups. At 48 hrs, Group D reached the highest F=ANOVA test N=Sample Size Table 2 represents that calcium release was highest in Group D followed by Group E at all time intervals. Group A had least calcium release at 0, 20 min, 1 hr, 2 hrs, 1 week & 1 month but higher than Group F at 10 min, 24 hrs & 48 hrs. University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 52

4 Table 3. Mean Hydroxyl Ion Release (ppm) of Different Calcium Hydroxide Intracanal Medicaments. F=ANOVA Test N=Sample Size Table 3 represents that hydroxyl ion release was highest in Group D followed by Group E at all time intervals. Group A had least hydroxyl ion release at 0, 20 min, 1 hr, 2 hrs, 1 week & 1 month but higher than Group F at 10 min, 24 hrs & 48 hrs. DISCUSSION : Calcium hydroxide aqueous paste, widely used as interim antimicrobial dressing in root canal treatment dissociates at body temperature into calcium ions (Ca2+) and hydroxide ions (OH- ), leaving mostly particles undissolved. (12) The antibacterial action is by the control of bacterial enzymatic activity. (13) The release of hydroxyl ions raises the ph of the medium inactivating the essential enzyme system of bacteria. The mineralizing effect can be explained by the activation of alkaline phosphatase enzyme due to its elevated ph. (13) The best ph for the activation of this enzyme ranges from 8.6 to (14) The permeability of dentin is due to tubule anatomy, density, diameter, and length as well as size and charge, studied by various investigators. (15, 16) Mjor et al. reported that the tubules were irregular in direction and density at apical root dentin having a diameter of 2 to 5 ìm. (15) Dentin is a substrate, whereas calcium hydroxide is a material and the size of the dentin tubules correlates with the size of the calcium hydroxide particles. The geometry of the small particles allow calcium hydroxide to enter the open dentinal tubules. The TEM images in our study revealed that morphology of different particles of Group A, E & F was irregular whereas that of Group B, C & D was circular. Also it was found that width of the particles of nano calcium hydroxide was less than 10 nm and had the least value among all the groups. These results were in agreement with the study performed by Yasaei M et al. (17) Taglieri G et al. (18) studied calcium hydroxide nanoparticles in aqueous suspensions structurally and morphologically with X-Ray diffraction (XRD) and transmission electron microscopy (TEM), respectively and found that they were crystalline, regularly shaped, hexagonally plated with dimensions 30 nm to 300 nm or less. In an another study under TEM, (19) he found the presence of linear chains constituting very small particles of diameters less than 10 nm. Presence of very small particles was because of the higher amounts of surfactant. It was found that undissolved fine particles may cause antimicrobial action inside dentin tubules. For effective therapeutic action of calcium hydroxide preparations, time seems to be a vital factor. Zmener et al. tested the ph changes over a period of 30 days using a mixture of calcium hydroxide and distilled water and two commercial calcium hydroxide products in a simulated periapical environment and found that there was a rapid increase in the ph at l hour and 24 hours, followed by continuous but more gradual increase from 15 to 30 days. (20) In our study all the University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 53

5 groups reached at maximum ph at one week which gradually decreased with time as observed after one month. Many substances have been added to the calcium hydroxide powder to improve its properties such as the antibacterial action, radiopacity, flow, and consistency. The ideal vehicle should allow a gradual and slow release of calcium and hydroxide ions. The vehicles suggested can be classified as aqueous and oily. Safavi et al. studied the effect of mixing vehicle on dissociation of calcium hydroxide in solution. They found that the use of non-aqueous mixing vehicles may impede the effectiveness of calcium hydroxide as a root canal dressing. (21) Similarly to our study Vitapex having oily vehicle presented less effectiveness in both ph and release of calcium and hydroxyl ions than aqueous vehicles. Carvalho CN et al. (22) evaluated ph and release of calcium, sodium and phosphate ions from different medications in human dentin and measured ions by (ICP/AES) at 10 min, 24 h, 7, 14, 21 and 30 days. It was found that CH had the highest level of ph & calcium ions release at 30 days because of different vehicles used and the concentration of chlorhexidine which may affect its ability to release ions. (23) Similarly in our study also mixing of vehicles had an effect on release of ions and ph. But in our study release of ions rapidly increased after 2 hrs, reached maximum value after 1 week and gradually decreased till 1 month. Pawinska M et al. (24) evaluated in vitro release of hydroxyl ions from several calcium hydroxide preparations nonsetting and setting canal sealers and points. It was found that nonsetting preparations have a significantly higher capability of hydroxyl ions release. The ph values of samples of all materials correlated positively with time. Almost all materials reached a maximum on the 8th day of the experiment same as in our study. Eppendorf tubes were preferred over natural teeth because the difference in size of apical foramina and anatomic variations like cul-de-sacs fins and lateral canals etc. could have lead to various dentinal tubules opening onto the root surface and thus variable results could have achieved. (25) RC Cal and Dentocal were used in the study along with calcium hydroxide distilled water as they are highly alkaline, non toxic and water-based radiopaque calcium hydroxides pastes with barium sulphate in ready to use paste form which could be easily cleaned and removed from canal whenever required. They have better chemical stability and non drying nature. They are both antibacterial and bacteriostatic in nature. Barium sulphate in these pastes is added in order to increase the radioopacity. Estrela et al. described liberation of calcium and hydroxide ions faster and more significant when calcium hydroxide distilled water paste was used. (26) The results of present study favour more liberation of calcium and hydroxyl ion from the RC Cal and Dentocal pastes as compared to calcium hydroxide with distilled water paste. This difference may be caused by the different methodologies used as well as addition of barium sulphate in these pastes. According to Sahrawat KL et al., the ICP-AES method provided a better precision than the colorimetric method for the determination of hot water extractable B in the soil samples. (27) ICP-AES method describes multi-elemental determinations by using sequential or simultaneous optical systems and axial or radial viewing of the plasma. The instrument measures characteristic emission spectra by optical spectrometry. Samples are nebulized and the resulting aerosol is transported to the plasma torch. Element-specific emission spectra are produced by a radio-frequency inductively coupled plasma. The spectra are dispersed by a grating spectrometer, and the intensities of the emission lines are monitored by photosensitive devices. (28) So we used the ICP-AES method in the present study. A gutta-percha point is newly introduced for calcium hydroxide delivery with fewer residues, composing 52% Ca(OH)2, 42% gutta percha, sodium chloride, surfactant and coloring agents. They are the hygienic time release preparations releasing Ca(OH)2 from a gutta percha matrix. They are firm yet flexible for easy introduction into root canal. Pure calcium hydroxide is homogenously distributed throughout a gutta percha matrix. Sodium chloride and surfactant improve the solubility of calcium hydroxide and mobility of ions. They are ISO standard and have light brown color to avoid confusion with gutta percha points. A drop of sterile water may be used together with the point for initial release of ions. However after insertion into root canal, sufficient fluid flows into space between point and canal wall from dentinal tubules and apical area to activate the Ca(OH)2 even without additional water. Due to low solubility of calcium hydroxide only small amounts are released at one time. The surrounding fluid is quickly saturated. However, as further moisture flows into the canal calcium hydroxide is continuously released maintaining high ph. Economides et al. evaluated the release of hydroxyl ions and found that calcium hydroxide containing gutta-percha points showed a significantly lower alkalinizing potential than the University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 54

6 non-setting preparation and calcium hydroxide mixed with distilled water. (29) The gutta-percha matrix probably binds the hydroxyl ions and blocks their release at the site of application. In our study also the ph was not significantly increased by gutta-percha point. Economides et al. and Azabal-Arroyo et al. reported maximum ph values of 9.5 and 10.9, respectively. (30) Calt et al. showed that calcium hydroxide gutta-percha points did not induce any changes in ph and calcium ion levels. (31) Larsen and Horsted - Bindslev concluded that hydroxyl ion liberation from the gutta-percha points is limited compared to that from calcium hydroxide pastes. These results are comparable to those of our study. Lohbauer et al. evaluated calcium ion release and ph characteristics of calcium hydroxide plus points (new formula, greater dissociation and even more effective) and found that calcium hydroxide plus points had a greater release of calcium ions compared to conventional calcium hydroxide points. (32) This result is due to greater dissociative ability of calcium hydroxide plus points having similar composition as compared to conventional calcium hydroxide points. The diffusion of hydroxyl ions through dentin from different calcium hydroxide medicaments was determined by Sevimay et al. (33) They found that non-setting calcium hydroxide based materials have an effective release of hydroxyl ions compared with calcium hydroxide plus points. The results of present study are comparable to those observed in previous studies. Vitapex (Yellow soft paste with iodoform odor) contains calcium hydroxide with iodoform in silicon oil with excellent accessibility. It is composed of 40.4% iodoform, 22.4% silicone oil, 6.9 % inert substances and calcium hydroxide. Iodoform provides bacteriostatic property and increased radioopacity. Silicone oil act as lubricant and ensures complete coating of canal walls. Due to inert substances it never hardens and gets solubilized as well as calcium hydroxide remains active in root canal. Also it remains chemically stable. It was observed that oil paste containing calcium hydroxide was largely lacking in both ion release and antimicrobial properties. Larsen and Bindslev observed that the aqueous suspension exhibited the highest ph and calcium ion liberation. (34) The low solubility and poor ability to diffuse make it difficult for oil paste containing calcium hydroxide compounds to reach maximum ph levels in a short period of time. The results of our in vitro study show that they should be used for a minimum of 7 days to achieve maximum therapeutic effectiveness. On the other hand, nano calcium hydroxide showed least ph which was in contrast with the fact that smaller particle size leads to better penetration into dentinal tubules and rise in ph. The present study showed that particle size did not have any effect on the ph, Ca++ ion release & OH- ion release. Also further investigations should be performed. Hence, from this study it can be formulated that Non-setting premixed calcium hydroxide pastes are better in antimicrobial activity as compared to calcium hydroxide powder and calcium hydroxide points. REFERENCES 1. Bystrom A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTA in 60 cases of endodontic therapy. Int Endod J 1985;18: Tronstad L. Root resorption etiology, terminology and clinical manifestations. Endod Dent Traumatol 1988;4: Foreman PC, Barnes F. A review of calcium hydroxide. Int Endod J 1990;23: Heithersay GS. Calcium hydroxide in the treatment of pulpless teeth with associated pathology. J Br Endod Soc 1975;8: Freeman BA, Crapo JD. Biology of disease: Free radicals and tissue injury. Lab Investig 1982;47: Safavi KE, Spangberg LS, Langeland K. Root canal dentinal tubule disinfection. J Endod 1990;16: Bystrom A, Claesson R, Sundqvist G. The a n t i b a c t e r i a l e f f e c t o f c a m p h o r a t e d paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatol 1985;1: Wang JD, Hume WR. Diffusion of hydrogen ion and hydroxyl ion from various sources through dentine. Int Endod J 1988;21: Renedo MJ, Fernandez J, Garea A, et al. Influence of particle size and structural properties of sorbents prepared from fly-ash and Ca(OH)2 on the SO2 removal ability. Chem Eng Comm 2000;182: Allaker RP, Memarzadeh K. Nanoparticles and the control of oral infections. Int J Antimicrob Agents 2014;43(2): Wu D, Fan W, Kishen A, et al. Evaluation of the antibacterial efficacy of silver nanoparticles against University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 55

7 Enterococcus faecalis biofilm. J Endod 2014;40(2): Lange's handbook of chemistry. New York: McGraw-Hill; Estrela C, Blitzkow G, Baumann L, et al. Mechanism of action of calcium and hydroxyl ions of calcium hydroxide on tissue and bacteria. Braz Dent J 1995;6: Tronstad L, Andreassen JO, Haselgreen G, et al. ph changes in dental tissues after root canal filling with calcium hydroxide. J Endod. 1981;7: Mjor IA, Smith MR, Ferrari M, et al. The structure of dentine in the apical region of human teeth. Int Endod J 2001;34: Pashley DH. Dentin: a dynamic substrate a review. Scanning Microsc 1989;3: Yasaei M, Ghaffari M, Zamanian A, et al. Characterization of calcium hyd- roxide cements modified by addition of nanohydroxyapatite. ICNS4, Kish Island, I.R. Iran; Taglieri G, Mondelli C, Daniele V, et al. Synthesis and x-ray diffraction ana- lyses of calcium hydroxide nanoparticles in aqueous suspension. Adv Mater Phy Chem 2013; Taglieri G, Mondelli C, Daniele V, et al. Synthesis, textural and structural properties of calcium hydroxide nanoparticles in hydro-alcoholic suspension. Adv Mater Phy Chem 2014;4: Zmener O, Pameijer CH, Banegas G. An in vitro study of the ph of three calcium hydroxide dressing materials. Dent Traumatol 2007;23: Safavi K, Nakayama TA. Influence of mixing vehicle on dissociation of calcium hydroxide in solution. J Endod 2000;26: Carvalho CN, Freire LG, Lima De Carvalho AP, et al. Ions release and ph of calcium hydroxide, chlorhexidine and bioactive glass based endodontic medicaments. Braz Dent J 2016;27(3): Pacios MG, de la Casa ML, de Bulacio MA, et al. Influence of different vehicles on the ph of calcium hydroxide pastes. J Oral Sci 2004;46: Pawinska M, Skrzydlewska E. Release of hydroxyl ions from calcium hydroxide preparations used in endodontic treatment. Rocz Akad Med Bialymst 2003; Eldeniz AU, Erdemir A. Evaluation of ph and calcium ion release of acroseal sealer in comparison with apexit and sealapex sealers. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103: SM, Qualtrough AJE, Worthington HV. An in vitro comparison of ph changes in root dentin following canal dressing with calcium hydroxide points and a conventional calciun hydroxide paste. Int endod J 2002;35: Estrela C. Chemical analysis of the liberation of calcium and hydroxyl ions from calcium hydroxide pastes in connective tissue in the dog. Braz Dent J 1996;7: Sahrawat KL, Shirisha K, Murthy KVS, et al. Comparative evaluation of inductively coupled plasma atomic emission spectroscopy and colorimetric methods for determining hot-waterextractable boron in soils. Int Cr Res Inst Sem Ar Trop 2012;43(6): Economides N, Koulaouzidou EA, Beltes P, et al. In vitro release of hyd- roxyl ions from calcium hydroxide gutta-percha points. J Endod 1999;23: Azabal-Arroyo M, Menasalvas-Ruiz G, Martín- Alonso J, et al. Loss of hyd- roxyl ions from guttapercha points with calcium hydroxide in their composition: An in vivo study. J Endod 2002;28: Calt S, Serper A. Time dependant effects of EDTA on dentin structures. J Endod 2002;28: Lohbauer U, Gambarini G, Ebert J, et al. Calcium release and ph characteristics of calcium hydroxide plus points. Int Endod J 2005;38: Sevimay S, Kalayci A, Yilmaz S. In vitro diffusion of hydroxyl ions through root dentine from various calcium hydroxide medicaments. J Oral rehabil 2003;30: Larsen MJ, Horsted-Bindslev P. A laboratory study evaluating the release of hydroxyl ions from various calcium hydroxide products in narrow canal-like tubes. Int Endod J 2000;33: CORRESPONDING AUTHOR: Dr. Amit Kumar Garg Department of Conservative Dentistry and Endodontics K.D. Dental College and Hospital, Mathura amit_kgdu@rediff.com University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 56