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ORIGINAL ARTICLE
Year : 2015  |  Volume : 19  |  Issue : 5  |  Page : 520-524  

An in vitro scanning electron microscope study to evaluate the efficacy of various root conditioning agents


1 Department of Periodontics, Institute of Dental Sciences, Barelly, India
2 Department of Orthodontics, 21, Corps Dental Unit, Bhopal, Madhya Pradesh, India
3 Department of Orthodontics, Institute of Dental Sciences, Barelly, India
4 Department of Periodontics, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow, Uttar Pradesh, India

Date of Web Publication13-Oct-2015

Correspondence Address:
Rajkumar Maurya
21, Corps Dental Unit, Bhopal, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-124X.167168

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   Abstract 

Aim: The purpose of present study was to investigate the scanning electron microscopic alterations produced on scaled and root planed dentin surfaces after application of different etching modalities namely citric acid, tetracycline hydrochloride and ethylenediaminetetraacetic acid (EDTA). Materials and Methods: Eighty specimens of teeth were collected and categorized into four groups. In group 1, control group specimens were treated with normal saline for 3 min, group 2 specimens were treated with citric acid, group 3 specimens were treated with tetracycline hydrochloride for 3 min and group 4 specimens were treated with 15% EDTA. Results: Opening of dentinal tubules was seen in all specimens except for control group that was treated with normal saline. The total number of tubules seen was highest in citric acid group as compared to tetracycline hydrochloride and EDTA Group. The total number of patent tubule opening was highest in citric acid as compared to EDTA and tetracycline. The diameter of patent dentinal tubules in citric acid group was more than EDTA and tetracycline. Conclusion: The study showed that all the three agents were effective in removing the smear layer however number of patent and wider diameter dentinal tubules was seen in vitro best in citric acid than EDTA and then in tetracycline hydrochloride. Hence, citric acid group is more efficient than EDTA and tetracycline HCl group in removing the smear layer and in opening of number of patent dentinal tubules.

Keywords: Citric acid, ethylenediaminetetraacetic acid, root conditioning agents, tetracyline HCl


How to cite this article:
Garg J, Maurya R, Gupta A, Tandon P, Gupta KK, Srivastava A. An in vitro scanning electron microscope study to evaluate the efficacy of various root conditioning agents. J Indian Soc Periodontol 2015;19:520-4

How to cite this URL:
Garg J, Maurya R, Gupta A, Tandon P, Gupta KK, Srivastava A. An in vitro scanning electron microscope study to evaluate the efficacy of various root conditioning agents. J Indian Soc Periodontol [serial online] 2015 [cited 2020 Mar 28];19:520-4. Available from: http://www.jisponline.com/text.asp?2015/19/5/520/167168




   Introduction Top


The major goal of periodontal therapy is the re-establishment of connective tissue attachment to the diseased root surfaces of the teeth. The periodontitis affected root surfaces are hypermineralized and contaminated with cytotoxic and other biologically active substances.[1],[2],[3],[4],[5] Such surfaces are not biocompatible with adjacent periodontal cells, the proliferation of which is pivotal for periodontal wound healing.[6] It is not possible to decontaminate a periodontitis affected root surface completely by mechanical means alone.[7] The instrumented surface will inevitably be covered by a smear layer following root planing.[8]

Conditioning of root surfaces by the topical application of acid solutions has been introduced as a periodontal regenerative procedure to dissolve the smear layer and to aid in detoxification of any root surface contaminant remaining after root planing.[8] There are number of agents for conditioning of root surfaces that have been proposed for demineralization purposes. These include citric acid, phosphoric acid, tetracycline, doxycycline, minocydine, fibronectin, ethylenediaminetetraacetic acid (EDTA), sodium deoxycholate and Cohn's fraction IV.[9] Citric acid has been shown to alter the surface characteristics of treated root surfaces by removing smear layer.[8] It causes demineralization of root surfaces and removes bacterial endotoxins from the pathologically altered cementum surfaces. The effects of citric acid application have been reviewed by Fialkoff and Fry that its use would accelerate cementogenesis by widening dentinal tubules.[8],[10] Etching at neutral pH with agents such as EDTA has been shown to be quite effective in exposing collagen fibers on dentine surfaces. EDTA etching also appeared to promote early cell colonization by providing a more biocompatible surface for cell tissue attachment.[11] Tetracyclines, which are well known as effective agents in control of periodontal pathogens, have been shown to produce a dentine surface that can enhance periodontal regeneration.[12]In vitro study of Terranova et al. investigating the effect of tetracycline on dentine, have suggested its potential usefulness in the regenerative procedure.[12]

The investigations have shown that the application of these chemical agents results in the exposure of fibrils of the dentinal collagen matrix with inter-digitations of new and old collagen fibrils in soft tissues-root interface. In the present scanning electron microscope (SEM) study, an attempt has been made to compare the surface alterations on planed root surfaces following application of citric acid, EDTA and tetracycline HCl.


   Materials and Methods Top


Selection of teeth and preparation of specimens: Eighty human single-rooted teeth affected with severe periodontitis that were free from caries, cervical-restorations, erosion were extracted and used in the present study. Care was taken, during extraction, not to instrument the root surface to be studied. Following extraction the teeth were washed with distilled water and stored in normal saline. After extraction, the border between healthy and diseased root surfaces was marked with a bur. The diseased root surfaces of all teeth were scaled with ultrasonic scaler and thoroughly planed with Gracey curettes (No. 1/2, 3/4, 5/6). After scaling and root planing, crown and healthy portion of root along with 2 or 3 mm of apical portion of the root was removed with a water cooled high-speed bur. The dentine specimens of dimension 5 mm × 5 mm were prepared for the study. The labial surface of each specimen was used for the study.

Preparation of chemical agents



  1. Tetracycline HCl-500 mg of tetracycline HCl capsule was dissolved in 5 ml of sterile H2O that is, distilled H2O under continuous stirring for 10 min that gave tetracycline solution at pH of 1.3 as checked using pH meter [10]
  2. Citric acid-15 g of anhydrous citric acid crystals were dissolved in 30 ml of distilled H2O at room temperature till the solution became saturated to obtain citric acid at pH of 1.2 as checked using pH meter [13]
  3. Ethylenediaminetetraacetic acid-25 ml of distilled H2O with 2.31 ml of 5 Normality NaOH and then adding 4.25 g of the disodium salt of EDTA to prepare 15% EDTA solution. This solution had a pH of 7.5 as checked using a pH meter.[9]
  4. All 80 roots divided into 4 groups
  5. Group I: The root specimens were treated with normal saline for 3 min and used as control group
  6. Group II: The root specimens were treated with tetracycline HCl solution by placing the cotton pellets saturated solution that was changed every 20 s for the total time of 3 min
  7. Group III: The root specimens were treated with citric acid solution by placing the cotton pellets saturated solution when were changed every 20 s for total time of 3 min
  8. Group IV: The root specimens were treated with 15% EDTA solution by placing the cotton pellets saturated solution that was changed every 20 s for the total time of 3 min.


The solution was applied to specimen surfaces by light pressure burnishing.[13] The specimens were divided into four groups. After etching, the teeth were immediately immersed in approximately twenty ml of distilled water for 20 s and gently swirled to stop the chemical reaction. All specimens were dehydrated in a graded series of ethanol using l00% acetone as the final step. The teeth were dried for half an hour and were mounted on the stubs with an adhesive tape with the labial side of the toots facing the beam of the SEM and in such a way that the roots were placed in the center of the stub. The specimens were sputter coated with gold-palladium as to ensure a proper conducting surface to the nonconducting specimen.

The surface of the roots was scanned and observed on the computer screen fitted with the SEM at 3000×. The representative areas were photographed. The roots were examined with respect to presence or absence of smear layer, total number of open dentinal tubules and diameter of open dentinal tubules.

Statistical analysis

All statistical analyses were carried out using SPSS (Version 11.0, SPSS, Chicago, IL, USA). Analysis was done using the arithmetic mean and the standard deviation for the mean was used as measure of deviation. Student's unpaired t-test was done to compare between groups.


   Results Top


The opening of dentinal tubules was not evident in Saline group [Figure 1]. The total number of dentinal tubules opening was highest for citric acid group (601) [Figure 2] followed by EDTA group (464) [Figure 3] and then tetracycline HCl group (354) [Figure 4]. The least number of tubules opening was seen in Tetracycline Hydrochloride group. On statistically comparing the difference between citric acid and tetracycline hydrochloride group of the total dentinal tubules the difference was significant. The difference was also significant between EDTA and tetracycline hydrochloride, citric acid and EDTA [Table 1].
Figure 1: The opening of dentinal tubules in Group I in Saline group

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Figure 2: The opening of dentinal tubules in Group II in citric acid group

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Figure 3: The opening of dentinal tubules in Group III in ethylenediaminetetraacetic acid Group

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Figure 4: The opening of dentinal tubules in Group IV in Tetracycline HCl group

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Table 1: Comparison of mean of total tubules in three experimental groups

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The percentage of patent dentinal tubules was seen maximum in citric acid (452) followed by EDTA (252) and tetracycline HCl (168) [Table 2]. On comparing the mean of patent tubules in three experimental groups significant difference was found between the groups [Table 3].
Table 2: Comparison of percentage of patent tubules in three experimental groups

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Table 3: Comparison of mean of patent tubules in three experimental groups

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Same observations were also observed for mean diameter of dentinal tubule orifice in the different groups. The mean diameter of patent dentinal tubules orifice was greater in citric acid (0.608 µm) as compared to EDTA group (0.571 µm) followed by tetracycline hydrochloride (0.468 µm) group which represented the minimum opening of the dentinal tubules [Table 4].
Table 4: Comparison of mean diameter of dentinal tubule orifice (μm) in different experimental groups

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   Discussion Top


The objective of the present study was to compare the efficacy of different chemical agents for opening of dentinal tubules on root planed surfaces.

The obtained specimens were categorized into 4 groups (one control and three experimental) consisting of 20 specimens in each group. The teeth used in this study were sectioned, scaled, and root planed and conditioned as soon as possible after extraction Normal saline solution was used for temporary storage of teeth. Scaling was carried out by EMS ultrasonic scaler and root planing was carried out by 1/2, 3/4 and 5/6 Gracey Curettes to obtain a smooth hard glass like surface. The method of application of root conditioners has varied among clinicians. In the present study, the light pressure burnishing technique was preferred over passive application. The solutions were applied for a total time of 3 min. The control group specimens were treated with normal saline. Counting of dentinal tubules orifice in saline group was not possible as smear layer covered the root surface. At × 3000 control group specimens were characterized by an irregular amorphous surface that served to correspond to smear layer [Figure 1]. Depressions that seemed to correspond to dentinal tubules orifice were rare. These findings are in agreement with Lasho et al.[9] Citric acid conditioning of roots to produce new connective tissue attachment has been used since many years.[14] Some of these studies have reported reestablishment of connective tissue attachment on roots following citric acid conditioning. However due to its acid pH, it impairs the vitality of surrounding periodontal tissues Blomlof and Lindskog (1995).[15] Hence proper isolation of the soft tissue and bone is required before condition with citric acid, it is one of its limitations. EDTA has been used in endodontics, both to enlarge the orifice of dentinal tubules and increase their permeability.[15] EDTA exerts its action by chelating divalent cations at neutral PH. It has no deleterious effect on the surrounding periodontal tissues (Brannstrom et al. 1980,[16] Blomlof and Lindskog 1995).[15] EDTA etching has also been reported to promote early cell colonization and tissue colonization by providing more biocompatible surface for cell and tissue attachment. The tetracyclines have been shown to produce a dentine surface that can potentially further enhance periodontal regeneration.In vitro study of Terranova et al.[12] investigating the effect of tetracycline on dentine have suggested its potential usefulness. Tetracycline studies demonstrate multiple beneficial properties like enhanced attachment and growth of gingival fibroblast, good anti-collagenase activity, high substantivity and inhibition of parathyroid hormone that causes bone resorption. All the three experimental groups showed difference in mean number of dentinal tubules opening.

Total number of dentinal tubules in tetracycline HCl group was 354, in EDTA group it was 464, and in the citric acid group it was 601. The number of tubules opened by the citric acid group was higher than the other 2 groups.[17] Mean number of tubule opening in the citric acid group was 30.05 greater than EDTA group which is 23.2 and tetracycline HCl 17.7 The difference between tetracycline group and EDTA group for total number of dentinal tubules is statistically significant [Table 1]. This can be due to the substantivity of tetracycline hydrochloride on dentine surface as compared to EDTA. The proportion of patent tubules to the total number of dentinal tubules was 75.2% in citric acid group, compared to EDTA 54.3% and tetracycline hydrochloride group 47.5% [Table 2]. The lesser number of patent dentinal tubules in tetracycline HCl group may be attributed to the fact that tetracycline have a high substantivity on dentine surface. As the experimental agent was lightly burnished, the tetracycline powder remained on the test specimen and was seen, occluding the dentinal tubules. Partially occluded tubules were also counted as nonpatent or occluded. Same observations were also made by Madison and Hokett (1997)[18] with the use of tetracycline powder in their experimental study. The comparison of citric acid group showed higher number of patent dentinal tubules when compared to tetracycline HCl and EDTA group and results were statistically significant for comparison between tetracycline HCl group and citric acid group [17] and between EDTA and tetracycline HCl. The results of total number patent dentinal tubules in three experimental groups were statistically significant between tetracycline HCl, EDTA and citric acid group [Table 3].

The comparison of mean diameter of the three experimental groups showed that the citric acid group had higher diameter (0.608 mm) as compared to the EDTA and tetracycline HCl group. EDTA group showed higher value of mean diameter opening when compared to tetracycline hydrochloride group. The mean diameter of EDTA is (0.571 mm). The mean diameter of tetracycline HCl is 0.468 mm [Table 4]. The tetracycline hydrochloride group showed the least diameter of dentinal tubule. It can be partially because of occupancy of tubules by substativity of tetracycline HCl and partially because of chelator effect of EDTA. In the present study, it was established that root conditioning in all the three experimental groups helped in the removal of smear layer and exposure of dentinal tubules. The agents not only remove debris but their demineralizing action also removes peritubular dentine, resulting in a wider orifice of the dentinal tubules (Goldburg and Ambranovich 1977).[18] The clean surfaces with wider tubular openings appear to offer a more favorable environment for close adhesion of fibroblasts or a true organic attachment with new cementum formation (Lasho DJ et al., 1983).[9] Hence, their application as root conditioner may have a significant role in periodontal wound healing and future new attachment in vivo.


   Conclusion Top


The present study was carried out on dentin slabs prepared from periodontally diseased human teeth after application of the three root conditioning agent's namely citric acid, tetracycline hydrochloride, ethylenediaminetetraaceticacid under SEM. From the study, it was concluded that all the three agents namely citric acid, tetracycline hydrochloride, ethylenediaminetetraacetic acid were effective in removing smear layer. Opening of dentinal tubules was seen in all specimens except for control group that was treated with normal saline. The total number of tubules seen was highest in citric acid group as compared to tetracycline hydrochloride and EDTA Group. The total number of patent tubule opening was highest in citric acid as compared to EDTA and tetracycline. The diameter of patent dentinal tubules in citric acid group was more than EDTA and tetracycline.

Hence, citric acid group is more efficient than EDTA and tetracycline HCl group in removing the smear layer and in opening of number of patent dentinal tubules.

 
   References Top

1.
Sculean A, Berakdar M, Willershausen B, Arweiler NB, Becker J, Schwarz F. Effect of EDTA root conditioning on the healing of intrabony defects treated with an enamel matrix protein derivative. J Periodontol 2006;77:1167-72.  Back to cited text no. 1
    
2.
Selvig KA, Zander HA. Chemical analysis and microradiography of cementum and dentin from periodontally diseased human teeth. J Periodontol 1962;33:303-10.  Back to cited text no. 2
    
3.
Hatfield CG, Baumhammers A. Cytotoxic effects of periodontally involved surfaces of human teeth. Arch Oral Biol 1971;16:465-8.  Back to cited text no. 3
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4.
Aleo JJ, De Renzis FA, Farber PA.In vitro attachment of human gingival fibroblasts to root surfaces. J Periodontol 1975;46:639-45.  Back to cited text no. 4
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5.
Aleo JJ, De Renzis FA, Farber PA, Varboncoeur AP. The presence and biologic activity of cementum-bound endotoxin. J Periodontol 1974;45:672-5.  Back to cited text no. 5
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Polson AM, Hanes PJ. Cell and fiber attachment to demineralized dentin. A comparison between normal and periodontitis-affected root surfaces. J Clin Periodontol 1987;14:357-65.  Back to cited text no. 6
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Jones WA, O'Leary TJ. The effectiveness of in vivo root planing in removing bacterial endotoxin from the roots of periodontally involved teeth. J Periodontol 1978;49:337-42.  Back to cited text no. 7
    
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Pitaru S, Gray A, Aubin JE, Melcher AH. The influence of the morphological and chemical nature of dental surfaces on the migration, attachment, and orientation of human gingival fibroblasts in vitro. J Periodontal Res 1984;19:408-18.  Back to cited text no. 8
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9.
Lasho DJ, O'Leary TJ, Kafrawy AH. A scanning electron microscope study of the effects of various agents on instrumented periodontally involved root surfaces. J Periodontol 1983;54:210-20.  Back to cited text no. 9
    
10.
Labahn R, Fahrenbach WH, Clark SM, Lie T, Adams DF. Root dentin morphology after different modes of citric acid and tetracycline hydrochloride conditioning. J Periodontol 1992;63:303-9.  Back to cited text no. 10
    
11.
Blomlöf J, Jansson L, Blomlöf L, Lindskog S. Root surface etching at neutral pH promotes periodontal healing. J Clin Periodontol 1996;23:50-5.  Back to cited text no. 11
    
12.
Terranova VP, Franzetti LC, Hic S, DiFlorio RM, Lyall RM, Wikesjö UM, et al. A biochemical approach to periodontal regeneration: Tetracycline treatment of dentin promotes fibroblast adhesion and growth. J Periodontal Res 1986;21:330-7.  Back to cited text no. 12
    
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Garrett JS, Crigger M, Egelberg J. Effects of citric acid on diseased root surfaces. J Periodontal Res 1978;13:155-63.  Back to cited text no. 13
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14.
Blomlöf J, Lindskog S. Root surface texture and early cell and tissue colonization after different etching modalities. Eur J Oral Sci 1995;103:17-24.  Back to cited text no. 14
    
15.
Brännström M, Nordenvall KJ, Glantz PO. The effect of EDTA-containing surface-active solutions on the morphology of prepared dentin: An in vivo study. J Dent Res 1980;59:1127-31.  Back to cited text no. 15
    
16.
Pant V, Dixit J, Agrawal AK, Seth PK, Pant AB. Behavior of human periodontal ligament cells on CO2 laser irradiated dentinal root surfaces: An in vitro study. J Periodontal Res 2004;39:373-9.  Back to cited text no. 16
    
17.
Madison JG 3rd, Hokett SD. The effects of different tetracyclines on the dentin root surface of instrumented, periodontally involved human teeth: A comparative scanning electron microscope study. J Periodontol 1997;68:739-45.  Back to cited text no. 17
    
18.
Goldberg F, Abramovich A. Analysis of the effect of EDTAC on the dentinal walls of the root canal. J Endod 1977;3:101-5.  Back to cited text no. 18
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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