Original Research Comparative evaluation of calcium concentration and ph of the periapical environment after applying calcium hydroxide into root canals - In vitro AVOY KUMAR DASH*# K. S. BANGA **## ABSTRACT Calcium hydroxide as a root canal medicament has been used in endodontics for many years. The use of calcium hydroxide in clinical situations involving necrotic pulp is advantageous as a result of its antiseptic action on the microorganisms present in the main root canal, its ramifications in the dentinal tubules, as well as in the apical cementum. It s biologic and antiseptic effects rely on alkalinity and calcium ion release. Release of calcium and hydroxyl ions into periapical area is dependent upon time and the type of vehicle used. The present in-vitro study was aimed to evaluate and compare calcium concentration and ph of the periapical environment after applying calcium hydroxide into root canals. Fifty single rooted single canal extracted teeth were divided into five groups. After access opening, cleaning and shaping calcium hydroxide was placed inside the canals in following order. Group 1-calcium hydroxide was mixed with distilled water at a powder/water weight ratio of 38%; group 2- calcium hydroxide was mixed with distilled water at 44%; group 3-calcium hydroxide was mixed with distilled water at 50%; group 4-calcium hydroxide powder only was used; and group 5-the control group, in which nothing was applied to the canals. All samples were immersed in distilled water maintained at 37 C. Calcium concentration and ph of the distilled water were measured after 3 days, 7 days, and then at weekly intervals up to 5 weeks, during which time the storage medium was renewed after each measurement. Calcium concentration and the change in ph of the distilled water were statistically quicker and greater in groups 1 to 3 (mixture groups) than group 4 (powder only). The highest calcium concentration (peak Ca 2+ release) was observed after 3 days for the mixture groups, whereas that for the powder only group was found at 7 days. Peak ph change was found after 3 days for the all groups. After peaking, all groups showed a decline of the ph over time. These results suggest that the time required for optimum intracanal activity when using calcium hydroxide mixtures is at least1 week. Key words: Calcium hydroxide, ph and Intracanal medicaments. Introduction The common sources of bacterial infections involving dental pulp are carious exposures, traumatic injuries and periodontal involvement, out of which caries is a major cause. Whatever may be the cause, pulpal infections should be treated by doing a good quality root canal treatment followed by suitable post endodontic restoration. The ultimate goals of endodontic treatment are complete removal of bacteria, their byproducts and pulpal remnants from infected root canals and the complete seal of disinfected root canals. Though single session root canal treatment is popular 1, it is not indicated in chronic nonvital cases in which * Lecturer, ** Professor, Department of Conservative Dentistry and Endodontics, # Chhattisgarh Dental College and Research Institute, G.E Road, Rajnandgaon, Chhattisgarh ## Nair Hospital Dental College, Dr. A. L Nair Road, Mumbai 126
AVOY KUMAR DASH, K. S. BANGA an inter-appointment disinfectant dressing is required 2.There are so many materials developed and used as intracanal medicaments like phenolic preparations, formaldehyde, chlorhexidine, Ledermix paste, Triple antibiotics paste and bioactive glass, but none of them are as popular as Calcium hydroxide. It is the only material, which has been used for almost hundred years. Powder form of calcium hydroxide cannot be used in root canal treatment, as it is intended to reach the narrow and complex root canal architecture. It requires a suitable vehicle to carry it along. Several vehicles have been used for calcium hydroxide, starting from distilled water to camphorated parachlorophenol 3.The main actions of calcium hydroxide come from the ionic dissociation of Ca ++ and OH ions and the action of these ions on vital tissue and bacteria generates the induction of hard tissue deposition and the antibacterial effect respectively 4. Antibacterial activity of calcium hydroxide is also due to its high ph which causes series of changes in the bacterial cell like damage to bacterial cytoplasmic membrane, protein denaturation and damage to DNA which leads to bacterial death 5. Materials and Methods Fifty recently extracted human single rooted teeth with single canal were collected. All the teeth were examined and verified thoroughly to rule out any developmental anomalies or defect. Debris, soft tissues and calculus were cleared from the external root surface mechanically. Further cleaning was done by hydrogen peroxide solution followed by treatment with normal saline and teeth were stored in cool dry place. Access cavity preparation was done in each tooth using high speed airotor handpiece with diamond points. Working length was determined by placing 15-number K file into the canal until it was visible to naked eye at the apex. Then 1 mm short of this length was decided as working length. Cleaning-shaping was done by step back technique using stainless steel hand file (Mani, Inc, Japan). Master apical preparation was done by 30# file. The canals are flared sufficiently to accommodate atleast medium sized plugger to within the 2 mm of working length. Throughout the cleaning and shaping of the canals,5ml of 3% Sodium Hypochlorite (Prime dental) and normal saline were used sequentially for intracanal irrigation(27gauge needle) with final irrigation done with normal saline. After completion of root canal preparation, all the samples were thoroughly washed with distilled water to ensure removal of remains of sodium hypochlorite and normal saline. Then all the canals were dried by using paper points (Densply India). Samples were divided into five groups randomly with 10 teeth each. Following variation of calcium hydroxide powder and distilled water were taken 1. Group 1: Powder-liquid ratio is 38% Group 2: Powder-liquid ratio is 44% Group 3: Powder-liquid ratio is 50% Group 4: Powder only Group 5: Control group For group 1,2 and 3 Paste form of Calcium hydroxide placed inside the canal by micromotor handpiece and lentulospiral. For group 4 powder form of Calcium hydroxide packed with plugger(no- 25) into the canal. Coronal access was sealed with green wax. Samples were placed inside the dark color glass bottle containing 10 ml distilled water 127
COMPARATIVE EVALUATION OF CALCIUM CONCENTRATION AND PH OF THE PERIAPICAL ENVIRONMENT AFTER APPLYING CALCIUM HYDROXIDE INTO ROOT CANALS - IN VITRO at 37 0 C. Teeth in the group V (control group) were left unfilled and coronal access was sealed with wax. Both calcium level and ph changes were determined at the third day, seventh day and then weekly for next five weeks. During the procedure storage media of different groups were renewed after each measurement 1. During the procedure, ph of all sample were measured using digital ph meter (equiptronics). It was standardized by putting it into a glass beaker containing distilled water. The standard ph of distilled water was taken as 6.83. The amount of calcium released or the concentration of calcium in the distilled water of each storage media of sample was measured by using spectro-photometer(sanyo,japan). Procedure Preparation of working reagent Fifty New plastic tests tubes were taken. Pipette-tips and glassware were thoroughly washed with distilled water then dry. Working reagent was prepared by carefully mixing of one volume of reagent 1 with one volume of reagent 2 according to the manufacturer s instructions (Calcium O-CPC Kit). Composition of reagent. 1. o-cresolphthaleine complexone (o-cpc) 50ml 2. Buffer 50ml 3. Standard 5ml Preparation for micro method for discrete analysers (Spectro-PhotoMeter) 1000µL of working reagent was taken and mixed with 20 µl standard provided in the kit and incubated for 3 min at room temperature to test the standard. Similarly, 20 µl of testing sample was taken and mixed with 1000µL of working reagent. First reading was for standard, which is taken as 0.291. Then reading for blank was taken. For all practical purposes it was taken as zero as all other samples were compared with blank. Principle of Spectro-Photo Meter When infra red ray passes through a medium, it gives out a light of particular wave length according to the substance present in that medium. In an acidic medium, calcium binds with o- cresolphthaleine complexone (o-cpc) which produces a purple colour which is absorbed at 570 nm. It is proportional to the concentration of calcium present in that medium. 9 System parameter Reaction End point. Temperature 30-35 o C Wavelength 570nm Standard concentration 10mgs/dl Reaction time 3 min. Procedure for individual sample measurement. In a clean dry plastic test tube 20 µl of testing sample was taken and mixed with 1000µL of working reagent. One ml of this sample put into the plastic tube given in the spectro-photo meter, and inserted into the slot given for the moving tube belt inside the spectro-photo meter. There are eight tube slots. The first slot is for Blank. Remaining seven slots are for testing samples. So at a given point of time seven samples can be tested. Each sample shows a different colour in the spectrophoto meter, according to the amount of calcium present. The reading obtained from the monitor was compared with the blank and noted accordingly. 128
AVOY KUMAR DASH, K. S. BANGA After each measurement the testing sample were discarded. The tubes were thoroughly washed with distilled water and allowed to air dry. Same procedure was followed for other samples also. As the spectro-photo meter is sensitive to remaining solutions, new test tube with all fresh measuring instruments like glass pipette, tip of automated pipette were used. The findings were recorded separately according to the days of measurement i.e 3 rd day, 7 th day, 14 th day, 21 st day, 28 th day and 35 th day. Results: For Calcium:- For group 1; The maximum calcium ion release occurred in to the periapical medium in mg/dl on the third day with mean 7.40 with SD 2.93.On the seventh day the mean was 4.02 which increased to 5.90 on 21 st day and again decreased to 2.26 on the 35 th day. For group 2: The peak calcium release was seen on day 3,with mean 10.41,SD 1.43 which gradually decreases up to 14 th day(mean-6.16,sd- 5.07) and then increases on 21 st day(mean 8.37,SD- 4.72) thereafter decreases further. For group 3: The similar pattern of calcium release as in group 1& 2 but the amount of calcium release is maximum as compare to group 1and 2,ie 12.03 on day 3, which may attributed to high powder- water ratio i.e. 50%. For group 4: It shows maximum calcium release on 7 th day (12.85) not 3 rd day (7.04) as in first three groups. After 1 st week the amount of calcium release decreases up to 35 th day. For group 5: This group is a control group without any calcium hydroxide inside the root canal so it did not show any statistically significant changes. For ph:- For group 1: This group shows ph changes towards alkalinity (mean 11.40) on day 3. Subsequently the ph decreases as days pass and reaches around neutral on thirty fifth days (7.67). For group 2: In this group ph behavior follows the same pattern as group 1 i.e. maximum on 3 rd day and then decrease thereafter reaching around neutral on thirty fifth day. For group 3: This group shows maximum ph on 3 rd day and then decreases up to thirty fifth days. For group 4: This group shows maximum ph changes on 3 rd day but less as compared to the other three mixture groups. This group followed the same pattern as first three groups i.e. peak on 3 rd day and gradually comes down to neutral. For group 5: Though this group did not contain anything for the sake of comparison its ph was still measured similar to other groups. We found that all samples showed ph around 8 which can be considered as neutral. Following results were obtained after statistical analysis: Groups p-value for Ca ++ p-value for ph Group-1 <0.0003 <0.0001 Group-2 <0.0003 <0.0001 Group-3 <0.0001 <0.0001 Group-4 <0.0001 <0.0001 Group-5 0.1417 statistically insignificant Post- hoc analysis by Student- Newman- Keul multiple comparison test 129
COMPARATIVE EVALUATION OF CALCIUM CONCENTRATION AND PH OF THE PERIAPICAL ENVIRONMENT AFTER APPLYING CALCIUM HYDROXIDE INTO ROOT CANALS - IN VITRO For group 1, 2, and 3 the calcium level obtained on day 3 was significantly greater than calcium level obtained on other days. For group 4 the calcium level obtained on day 7 was significantly greater than calcium level obtained on other days. For group 5 similar tests applied and p- value obtained was 0.1417 which is considered non significant. No statistically significant difference among the entire mixture group. Statistically significant difference exists regarding the amount of calcium ion release by mixture group over the powder groups at 3 rd, 14 th and 21 st day. No noticeable changes were seen in control group as far as amount of calcium ion release and ph changes are concerned. Discussion Bacteria in the root canal system initiate and maintain periapical inflammatory lesions. 1 The bacterial micro flora in root canal infections of untreated teeth is dominated by anaerobic bacteria and several different anaerobic species are found 6,7. Root canal treatment aims to eliminate bacteria from the infected root canal and prevent reinfection. Cleaning, shaping and irrigation of canals greatly reduce the number of bacteria 3. It is impossible to obtain complete disinfection in all cases even after thorough cleaning, shaping and irrigation with disinfectant and antiseptics 7. If the canal is not filled or dressed with a disinfectant between two visits, bacteria may multiply rapidly within days to near the original number 7. The amount of calcium released into the testing media was measured using the chelate color former method, Calcium O CPC kit with a calcium measurement reagent. Chelate color former measurement method is very sensitive to react with common detergent, silicate glass tube even tap water washed pipette. Any form of contamination, even mild one, can lead to false results 9. Calcium hydroxide dissolved in distilled water gives a purplish red color by the chelate color former in which calcium is bonded to an orthocleasolphthalain complex 1.Calcium content of the sample was determined by measuring the light absorption of this purplish red color at 570nm. Amount of calcium ion release into the testing medium induces varying degrees of color changes when it reacts with working reagent. Test tube containing the working reagent was placed in the spectrophotometer and taken as blank and the reading is matched with 570nm. That means any color change which is more or less compared to the blank will give reading on the spectrophotometer. So this blank is compared with all the testing samples. The standard for calcium measurement given in the kit was also tested. For better comparison and calculation standard for amount of calcium measurement by spectro-photo meter was taken as 0.291 for all the measurement and calculated as: OD TEST Calcium in mg/dl = 10 OD STD (0.291) The testing medium in each sample bottle was renewed after each measurement and the samples were placed back in the media immediately after measurement. Calcium Hydroxide plays an important role not only as an intracanal medicament but also whole range of conditions in endodontics 11. Calcium ion and Hydroxyl ion derived from calcium hydroxide dressing diffuse through root 130
AVOY KUMAR DASH, K. S. BANGA dentine. Rate of diffusion is faster and reaches higher level cervically than apically 15. Periapical foramen is a major route of calcium ion and Hydroxyl ion diffusion along with dentinal tubules 1. In our study we consider both periapical foramen and dentinal tubules as route for release of calcium ion and Hydroxyl ions. after it decreases slowly. Regarding ph changes, this paste increases the ph in periapical environment which is responsible for its anti bacterial property and periapical healing potential. Though there are several controversies regarding the time period for which calcium hydroxide paste should be left in the canal for optimum result, we found that this medicament should be left inside the canal at least for seven days to show maximum result. As every case is different, clinical signs, symptoms and radiographic features should always be co-related before and during the treatment of nonvital teeth with periapical radiolucency. Further studies are required in this field to verify and ascertain the role of calcium hydroxide in different clinical situation. References: 1. Noriyasu Hosoya, (2001) Calcium Concentration and ph of the Periapical Environment after Applying Calcium Hydroxide into Root Canals in Vitro. Journal of Endodontics; 27 (5), 343-346. 2. Tronstad L (1991) Clinical Endodontics, 1st edn. Stuttgart, Germany: Georg Thieme Verlag, 101. 3. L.R.G. Fava et al (1999) Calcium hydroxide paste Classification and indications: Review of Literature. International Endodontics Journal. 1999; 32, 257-282. Conclusions Calcium hydroxide should be used for treatment of non vital pulp as inter appointment dressing material. Release of calcium and hydroxyl ions into periapical area is dependent upon time and the type of vehicle used. Thinner and aqueous base vehicle when used causes rapid and transient release of calcium and hydroxyl ions, which shows peak around third days and up to seven days. There 4. Estrela C (1994) AnaÂlise quõâmica de pastas de hidroâxido de calico frente aá liberacaäo de ions calcio,de ions hidroxila e formacaäo de carbonato de calcio na presencë a de tecido con i untivo de caäo (Thesis). SaÄo Paulo. 5. J F SEQUERIA et al (1999) Mechanism of antimicrobial activity of calcium hydroxide: A critical review. International Endodontic Journal 1999; 32, 361 369. 6. Bergenholtz G (1974) Micro-organisms from necrotic pulp of traumatized teeth. Odontologisk Revy 25, 347 58. 7. Byström A, Sundqvist G (1981) Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scandinavian Journal of Dental Research 89, 321 8. 8. Byström, Sundqvist G (1985) The antibacterial effect of CMCP, camphorated phenol and calcium hydroxide in the 131
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