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ORIGINAL RESEARCH
Year : 2015  |  Volume : 19  |  Issue : 3  |  Page : 268-272  

Comparative in vitro SEM study of a novel root canal irrigant-MTAD and conventional root biomodifiers on periodontally involved human teeth


Department of Periodontics, Babu Banarasi Das College of Dental Sciences, Lucknow, Uttar Pradesh, India

Date of Submission22-Dec-2013
Date of Acceptance17-Feb-2015
Date of Web Publication26-Jun-2015

Correspondence Address:
Charu Tandon
Tandon Clinic, Main Market, Barabanki 225 001, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-124X.154192

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   Abstract 

Background: Smear layer removal and collagen fiber exposure may improve regeneration which can be accomplished by use of root biomodifiers. These enhance the degree of connective tissue attachment to denuded roots. The objective of this in vitro study was to evaluate and compare novel root canal irrigant and other root biomodifiers for smear layer removal on periodontally involved human teeth. Materials and Methods: Forty human teeth were collected and stored in saline. After scaling and root planing, two samples were obtained from each tooth. Thus, a total of 80 dentin blocks were randomly divided into four groups: Mixture of tetracycline, acid and detergent (MTAD), tetracycline hydrochloride (TTC HCl), citric acid (CA), and ethylenediaminetetraacetic acid (EDTA). The agents were applied for 3 min by active burnishing. Immediately following treatment, the specimens were rinsed, dehydrated, fixed and prepared for scanning electron microscope and were examined at Χ 3500 magnification. Sampaio's index was evaluated by the previously trained blind examiner using photomicrographs. Groups were compared using analysis of variance followed by Tukey's post-hoc test. Results: Mixture of tetracycline, acid, and detergent is most efficacious in removing the smear layer and showed statistically significant dentinal tubules opening, followed by EDTA, TTC HCl, and CA. Conclusion: Mixture of tetracycline, acid and detergent and conventional root biomodifiers used in the study alters the dentin surface by smear layer removal and exposure of dentinal tubules. Hence, MTAD as a root biomodifier may have a significant role in periodontal regeneration .

Keywords: Citric acid, ethylenediaminetetraacetic acid, MTAD, root biomodifier, scanning electron microscope study, tetracycline hydrochloride


How to cite this article:
Tandon C, Govila V, Pant VA, Meenawat A. Comparative in vitro SEM study of a novel root canal irrigant-MTAD and conventional root biomodifiers on periodontally involved human teeth. J Indian Soc Periodontol 2015;19:268-72

How to cite this URL:
Tandon C, Govila V, Pant VA, Meenawat A. Comparative in vitro SEM study of a novel root canal irrigant-MTAD and conventional root biomodifiers on periodontally involved human teeth. J Indian Soc Periodontol [serial online] 2015 [cited 2020 May 26];19:268-72. Available from: http://www.jisponline.com/text.asp?2015/19/3/268/154192


   Introduction Top


Smear layer is an amorphous, granulated, and irregular layer covering the root surface when observed under scanning electron microscope (SEM). [1] It is constituted of inorganic (calcium, phosphate), and organic material (odontoblastic process, bacteria, and blood cells) and bacterial products (endo and exotoxins) [2] resulting in hypermineralization of root surface [3] which may function as a physical barrier to the growth of a connective tissue attachment to the root surface. [4] Scaling and root planing eliminate the mineralized debris present on periodontally involved root surfaces, but promotes the formation of this thin residous smear layer that might delay the adhesion of new fibroblasts and connective tissue.

Conditioning of root surfaces by topical application of acid solutions had been introduced as a periodontal regenerative procedure. Root biomodifiers have been found to be effective in eliminating adhered organic and inorganic material and retained toxins from the altered root surface, furthermore, they have shown to expose dentin collagen and cementum bound proteins, resulting in a root surface which is conducive in promoting periodontal regeneration. A number of agents have been proposed for the root conditioning including tetracycline hydrochloride (TTC HCl), [5] citric acid (CA), [6] and ethylenediaminetetraacetic acid (EDTA). [7]

The concept of acid demineralization in periodontal therapy was first introduced in the 1800s as a substitute for scaling and calculus removal. CA alters the external characteristics of root surfaces, removes the smear layer after scaling and root planing, demineralizes the treated surface leaving a "mat-like" collagen surface with exposed dentinal tubules, and eliminates bacterial endotoxins from pathologically altered cementum surfaces. [8]

Tetracycline HCl is an effective antibiotic which is absorbed onto the root surface and slowly released in its active form (substantivity). [9] It acts as calcium chelator, resulting in demineralization of the root surface and removal of the smear layer. Furthermore, it is having a potent anti-inflammatory and anti-collagenase activity. [10]

Ethylenediaminetetraacetic acid is a decalcifying agent operating at a neutral pH by chelating the divalent cations. It preserves the vitality of the remaining periodontal cells close to the root surface, and also has the advantage of biocompatibility (pH = 7.0) as compared to other root conditioning agents. [10]

BioPure MTAD ® (Dentsply, Tulsa Dental, USA.) has been used as an antibacterial root canal irrigant. It stands for a mixture of TTC isomer (doxycycline), acid-CA, detergent-tween80 ® . It has the ability to remove the smear layer and also exert a potent antimicrobial activity. [11] In the present study, mixture of tetracycline, acid, and detergent (MTAD) has been used as a novel approach for root biomodification.

On the basis of above facts, the present in vitro study was aimed to evaluate and compare novel root canal irrigant and other root biomodifiers for smear layer removal on periodontally involved human teeth.


   Materials and methods Top


The study sample consisted of 40 freshly extracted single-rooted, periodontally compromised teeth with hopeless prognosis and caries free root surfaces. The teeth with wasting diseases, tooth fracture, endodonticaly treated, and prosthodonticaly restored were excluded from the study. Written informed consent was taken prior to extraction from all the patients and ethical committee clearance from the institution was obtained. Following extraction samples were stored in normal saline.

Preparation of samples

Samples were obtained from the cervical third of the root by making two parallel grooves with a cylindrical bur under copious saline irrigation. The first groove was positioned horizontally at cementoenamel junction and second groove made parallel and 4 mm apical in relation to the first. The diseased root surfaces of all teeth were scaled with an ultrasonic scaler and thoroughly planed with #1-2, 3-4 Gracey curettes (Hu-Freidy) to remove all the diseased cementum. With the help of diamond disc under copious irrigation, the two samples are obtained first by transverse sectioning the root from the grooves and secondly by sectioning the sample longitudinally into two from the middle. The dentin samples of dimension 4 mm × 6 mm were prepared. The labial and lingual surface of each specimen was used for the study.

Total 80 samples were randomly divided into 4 groups:

  • Group A: Biopure MTAD [Figure 1]
  • Group B: Tetracycline HCl (50mg/ml)
  • Group C: Citric acid (pH 1.0)
  • Group D: Ethylene diaminetetraacetic acid (15%).
These agents were freshly prepared every time and were applied by "active burnishing" on the curetted root surfaces for 3 min. The cotton pellets are changed every 30 seconds to ensure consistent solution application. Following treatment, samples were rinsed thoroughly with distilled water.
Figure 1: MTAD

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Preparation of samples for scanning electron microscope study

Following the chemical treatment, all samples were dehydrated in a graded series of ethanol (10-90%) for 30 min each and finally in 100% acetone for 30 min more. The samples were dried under the lamp and then mounted on the aluminum stubs and inserted in SC7640 sputter coater machine for gold/palladium coating on specimens. All the specimens were examined in a Polaron-SEM (Leo-430) at a magnification of ×3500 and photomicrographs were evaluated to ascertain the extent of root biomodification by removal of smear layer, patent dentinal tubules in relation to the total number of dentinal tubules.

Analysis of photomicrographs

The photomicrographs were distributed to three calibrated, trained blind examiners to determine the degree of smear layer removal according to the root surface modification index (Sampaio's index). [8] The scores are as follows:

Score 1

Root surface without smear layer, with dentinal tubules completely opened; no evidence of smear layer in the dentinal tubule gaps.

Score 2

Root surface without smear layer, with dentinal tubules completely opened; evidence of smear layer in the dentinal tubule gaps .

Score 3

Root surface without smear layer, with the dentinal tubules partially opened.

Score 4

Root surface covered with smear layer, with uniform aspect; evidence of dentinal tubule gaps.

Score 5

Root surface covered with smear layer, with uniform aspect; no evidence of dentinal tubule gaps.

Score 6

Root surface covered with smear layer, with irregular aspect and presence of grooves and/or scattered debris.

The number of totally patent smear free dentinal tubules out of the total number of dentinal tubules and percentage patency was calculated. Data were summarized as mean ± standard deviation (SD). The Sampaio's index, patent dentinal tubules and percentage patency of four groups (MTAD, TTC HCl, CA, and EDTA) were compared by analysis of variance (ANOVA) and the significance of mean difference between the groups was done by Tukey's post-hoc test after ascertaining normality by Shapiro-Wilk test and homogeneity of variances by Levene's test. A two-sided (α = 2) P < 0.05 was considered statistically significant. The analyses were performed on STATISCA - Version 6.0 software.


   Results Top


The Sampaio's index scores of Group A, B, C, D ranged from 1-2, 2-4, 4-5, and 1-3 respectively [Figure 2]a-d], with mean ± SD 1.67 ± 0.49, 3.27 ± 0.59, 4.20 ± 0.41 and 2.33 ± 0.62, respectively [Table 1]. Comparing the mean scores of four groups, ANOVA revealed significantly different Sampaio's index scores (F = 64.09, P < 0.001), patent dentinal tubule scores (F = 140.42, P < 0.001) and mean percentage patency scores of four groups (F = 141.70, P < 0.001).
Figure 2: Scanning electron microscope photomicrographs of (a) MTAD, (b) ethylenediaminetetraacetic acid, (c) tetracycline hydrochloride, (d) citric acid

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Table 1: Sampaio's Index, patent dentinal tubules, and percentage patency (mean±SD) of four experimental groups


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Further, Tukey test [Table 2] revealed significantly different and lower mean Sampaio's index, mean patent dentinal tubule and percentage patency scores of MTAD group as compared to TTC HCl (P < 0.001), CA (P < 0.001) and EDTA (P = 0.006) groups. Similarly, the mean Sampaio's index, patent dentinal tubule and percentage patency scores of EDTA group also lowered significantly as compared to both TTC HCl (P < 0.001) and CA (P < 0.001). Further, the mean Sampaio's index scores of TTC HCL group was also found significantly different and lower as compared to CA (P < 0.001). However, like patent dentinal tubules (P = 0.974), the mean percentage patency scores also not differed between TTC HCL and CA (P = 0.427) that is, found to be statistically the same.
Table 2: Significance (P) of mean difference of Sampaio's Index, patent dentinal tubules, percentage patency between four experimental groups


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   Discussion Top


One of the objectives of periodontal therapy is the restoration of the lost periodontium and conversion of the periodontitis-affected root surface into a substrate which is biocompatible for epithelial and connective tissue cell adherence and attachment. Methods to achieve this objective include scaling and root planing, and use of demineralizing agents. Scaling and root planing leads to the formation of smear layer which inhibits the growth of a connective tissue attachment to the root surface. [4],[12] Demineralizing agents have shown to expose the dentinal collagen, widening the orifices of dentinal tubules, remove cementum bound proteins and retained toxins from altered root surface. Thus, the root surface smear layer removal with chemicals was carried out during periodontal therapy to enhance regeneration of the lost periodontal attachment apparatus. [13] Many chemical agents have been proposed as root conditioning agents, some of them are CA, TTC HCl group, EDTA. [4] The other agents used are carbon dioxide laser, [14] neodymium:yttrium, aluminum, garnet laser, [15] and erbium: Yttrium, aluminum, garnet laser [11] and many more.

As previously established in a systematic review by Mariotti (2003) and American Academy of Pediatrics, position paper (2005), TTC HCl, CA, and EDTA remove the smear layer but do not provide clinical benefit to the patient during regenerative procedures. [16],[17] As BioPure MTAD ® (Dentsply Ltd., USA.) is used as an efficacious root canal irrigant for smear layer removal, so in the present study, we have utilized this novel agent as a root biomodifier for smear layer removal on periodontally involved human teeth. Doxycycline is the primary ingredient contributing to its antimicrobial activity. CA removes the inorganic materials and Tween-80 reduces the surface tension and benefits the diffusion of acids into the root canal irregularities and dentinal tubules. Torabinejad et al. (2003) demonstrated that MTAD is an effective solution for the removal of the smear layer; it also did not significantly change the structure of the dentinal tubules. [13] TTC HCl, 50 mg/ml concentration was used according to previous studies in which Ishi et al. (2008), Isik et al. (2000) inferred that TTC HCl concentrations between 50 and 125 mg/ml might alter dentin surfaces by removing smear layer and also maximize tubule openings in a short period of time, if repeated applications were performed. [4],[9] Another agent used was CA pH 1 was also applied for 3 min as recommended by earlier studies. [18],[19],[20] EDTA of 15% concentration, pH of 4.35 was used as given by Lasho et al. (1983) [21] and Sampaio et al. (2003). [22]

The present study photomicrographs were evaluated using the root surface modification index (Sampaio's index) for smear layer removal [8] [Table 2]. The lowest score was achieved in Group A/MTAD which indicates complete smear layer removal and complete opening of dentinal tubules, which was in accordance with the study by Mozayeni et al. (2009) who stated that MTAD revealed the presence of more abundant and larger dentinal tubule opening when compared with other agents. [23] The result of MTAD was statistically significant with TTC HCl, CA, and EDTA. [5],[10],[11] However, to date, no in vitro or in vivo studies have been conducted utilizing MTAD as a root biomodifier.

The patent dentinal tubules and percentage patency intergroup comparison by Z-test showed highly statistically significant difference among the groups. MTAD had higher statistically significant number of patent and percentage patency of dentinal tubules when compared with TTC HCl (P = 0.0001), CA (P < 0.0001) and EDTA (P = 0.0455). This was in agreement with the study done by Torabinejad et al. (2003), and Mozayeni et al. (2009) who inferred that MTAD was more efficacious in removing smear layer and opening dentinal tubules of instrumented root canals because of being a combination agent of doxycycline, CA and a detergent tween-80 which reduces the surface tension and helps in better penetration of these acids into smear layer. Zhang et al. (2003) demonstrated in his study that MTAD is less cytotoxic to the surrounding cells and tissues than EDTA. [24]

Ethylenediaminetetraacetic acid was statistically significant than TTC HCl and CA, because EDTA is better in smear layer removal and exposure of dentinal tubules by making the root surfaces more biocompatible according to Silverio et al. (2007), [1] and Soares et al. (2010) [25] [Table 2]. Blomlöf and Lindskog (1995) also stated that EDTA in a neutral pH was more effective to demineralize dentin than the acidic agents like CA, and selectively remove mineral from a dentin surface, exposing a collagenous matrix. [26] TTC HCl when compared with CA (P = 1.0000) did not had any statistically significant difference which was in accordance with the studies done by Lafferty et al. (1993) who demonstrated comparable results of CA and TTC because of their similar acidic pH (TTC HCl 1.75 pH, CA pH 1). [27]

In summary, our results confirm that the root condition agents/root biomodifiers are effective in removing the smear layer and exposing dentinal tubules. MTAD being most efficacious followed by EDTA, TTC HCl, and CA. Despite the limitations of the study, further longitudinal in vitro and in vivo studies to establish MTAD as a root conditioning agent are warranted.


   Conclusion Top


The study has established that the various root biomodifiers: MTAD, TTC HCl 50 mg/ml, CA pH 1, and EDTA 15% modifies the dentin surface by smear layer removal and exposure of dentinal tubules. Hence, the application of MTAD as a root biomodifier may have a significant role in periodontal regeneration both in vitro and in vivo.


   ACKNOWLEDGeMENT Top


I express my gratitude to Dr.Aarati Kalluri, DDS (USA) for providing MTAD. I am thankful to Dr. Madhav Kumar and Mr.Subodh, for their support in conducting my SEM study at Birbal Sahni Institute of Palaeobotany, Lucknow. I am also grateful to Director, Institute for Data Computing and Training (I.D.C.T.), Lucknow, for providing valuable assistance in data analysis. The authors report no conflicts of interest related to this study.

 
   References Top

1.
Silverio KG, Martinez AE, Rossa C Jr. Effects of basic fibroblast growth factor on density and morphology of fibroblasts grown on root surfaces with or without conditioning with tetracycline or EDTA. J Oral Sci 2007;49:213-20.  Back to cited text no. 1
    
2.
Adriaens PA, De Boever JA, Loesche WJ. Bacterial invasion in root cementum and radicular dentin of periodontally diseased teeth in humans. A reservoir of periodontopathic bacteria. J Periodontol 1988;59:222-30.  Back to cited text no. 2
    
3.
Parashis AO, Tsiklakis K, Tatakis DN. EDTA gel root conditioning: Lack of effect on clinical and radiographic outcomes of intrabony defect treatment with enamel matrix derivative. J Periodontol 2006;77:103-10.  Back to cited text no. 3
    
4.
Isik GA, Tarim B, Hafez AA, Yalcin FS, Onan U, Cox CF. A comparative scanning electron microscopic study on the characteristics of demineralized dentin root surface using different tetracycline HCl concentrations and applications times. J Periodontol 2000;71:219-25.  Back to cited text no. 4
    
5.
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. 5
    
6.
Register AA, Burdick FA. Accelerated reattachment with cementogenesis to dentin, demineralized in situ. II. Defect repair. J Periodontol 1976;47:497-505.  Back to cited text no. 6
    
7.
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. 7
    
8.
Sampaio JE, Theodoro LH, Correa MA, Mendes AJ. A comparative SEM study of smear layer removal by detergents and EDTA on the root surface. Int J Periodontics Restorative Dent 2005;25:157-63.  Back to cited text no. 8
    
9.
Ishi EP, Dantas AA, Batista LH, Onofre MA, Sampaio JE. Smear layer removal and collagen fiber exposure using tetracycline hydrochloride conditioning. J Contemp Dent Pract 2008;9:25-33.  Back to cited text no. 9
    
10.
Cohen ES, editor. Biomechanical root preparation. In: Atlas of Cosmetic and Reconstructive Periodontal Surgery. 3 rd ed.. Hamilton: PMPH-USA, BC Becker Inc.; 2007. p. 271-4.  Back to cited text no. 10
    
11.
Theodoro LH, Zezell DM, Garcia VG, Haypek P, Nagata MJ, Almeida JM, et al. Comparative analysis of root surface smear layer removal by different etching modalities or erbium: Yttrium- aluminium- garnet laser irradiation. A scanning electron microscopy study. Lasers Med Sci 2010;25:485-91.  Back to cited text no. 11
    
12.
Polson AM, Frederick GT, Ladenheim S, Hanes PJ. The production of a root surface smear layer by instrumentation and its removal by citric acid. J Periodontol 1984;55:443-6.  Back to cited text no. 12
    
13.
Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochlorite on the ability of MTAD to remove the smear layer. J Endod 2003;29:233-9.  Back to cited text no. 13
    
14.
Pant V, Dixit J, Agarwal AK, Seth PK, Pant AB. Behavior of human periodontal ligament cells on CO2 laser irradiated dentinal root surfaces: An in vitro study. J Periodontol Res 2004;39:373-9.  Back to cited text no. 14
    
15.
Haghighati F, Arefi V. A comparative study on root surface demineralization using citric acid and tetracycline in vitro: A scanning electron microscopy study. Acta Med Iran 2002;40:247-55.  Back to cited text no. 15
    
16.
Mariotti A. Efficacy of chemical root surface modifiers in the treatment of periodontal disease. A systematic review. Ann Periodontol 2003;8:205-26.  Back to cited text no. 16
    
17.
Wang HL, Greenwell H, Fiorellini J, Giannobile W, Offenbacher S, Salkin L, et al. Periodontal regeneration. J Periodontol 2005;76:1601-22.  Back to cited text no. 17
    
18.
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. 18
    
19.
Hanes PJ, Polson AM, Ladenheim S. Cell and fiber attachment to demineralized dentin from normal root surfaces. J Periodontol 1985;56:752-65.  Back to cited text no. 19
    
20.
Wen CR, Caffesse RG, Morrison EC, Nasjleti CE, Parikh UK. In vitro effects of citric acid application techniques on dentin surfaces. J Periodontol 1992;63:883-9.  Back to cited text no. 20
    
21.
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. 21
    
22.
Sampaio JE, Rached RS, Pilatti GL, Theodoro LH, Batista LH. Effectiveness of EDTA and EDTA-T brushing on the removal of root surface smear layer. Pesqui Odontol Bras 2003;17:319-25.  Back to cited text no. 22
    
23.
Mozayeni MA, Javaheri GH, Poorroosta P, Ashari MA, Javaheri HH. Effect of 17% EDTA and MTAD on intracanal smear layer removal: A scanning electron microscopic study. Aust Endod J 2009;35:13-7.  Back to cited text no. 23
    
24.
Zhang W, Torabinejad M, Li Y. Evaluation of cytotoxicity of MTAD using the MTT-tetrazolium method. J Endod 2003;29:654-7.  Back to cited text no. 24
    
25.
Soares PB, Castro CG, Branco CA, Magalhaes D, Neto AJ, Soares CJ. Mechnical and acid root treatment on periodontally affected human teeth- a scanning electronic microscopy. Braz J Oral Sci 2010;9:128-32.  Back to cited text no. 25
    
26.
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. 26
    
27.
Lafferty TA, Gher ME, Gray JL. Comparative SEM study on the effect of acid etching with tetracycline HCl or citric acid on instrumented periodontally-involved human root surfaces. J Periodontol 1993;64:689-93.  Back to cited text no. 27
    


    Figures

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