|Year : 2014 | Volume
| Issue : 1 | Page : 24-28
An in vitro scanning electron microscopic study comparing MTAD (intracanal irrigant) and various root biomodifiers on periodontally involved human teeth
Charu Tandon, Vivek Govila, Vandana A Pant, Ajita Meenawat
Department of Periodontics, Babu Banarasi Das College of Dental Sciences, Lucknow, Uttar Pradesh, India
|Date of Web Publication||18-Aug-2014|
W/O. Dr. Manas Tandon, Tandon Clinic, Main Market, Barabanki - 225 001, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Smear layer removal and collagen fiber exposure may improve regeneration outcome, 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 scanning electron microscopic study was to comparatively evaluate mixture of tetracycline (TTC) and acid and detergent (MTAD) 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 planning, two samples were obtained from each tooth. A total of 80 dentin blocks were randomly divided into four groups: MTAD, TTC hydrochloride (TTC HCl), citric acid (CA), and normal saline. 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 was examined at Χ3500 magnification. Previously trained blind examiners evaluated photomicrographs using Sampaio's index (2005). Statistical analysis was performed. Results: MTAD is most efficacious in removing smear layer and showed statistically significant dentinal tubules opening, followed by TTC HCl and CA. Conclusion: MTAD 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, ethylene diaminetetraacetic acid, mixture of tetracycline and acid and detergent, root biomodifier, scanning electron microscope, tetracycline hydrochloride
|How to cite this article:|
Tandon C, Govila V, Pant VA, Meenawat A. An in vitro scanning electron microscopic study comparing MTAD (intracanal irrigant) and various root biomodifiers on periodontally involved human teeth. J Int Clin Dent Res Organ 2014;6:24-8
|How to cite this URL:|
Tandon C, Govila V, Pant VA, Meenawat A. An in vitro scanning electron microscopic study comparing MTAD (intracanal irrigant) and various root biomodifiers on periodontally involved human teeth. J Int Clin Dent Res Organ [serial online] 2014 [cited 2021 Jun 23];6:24-8. Available from: https://www.jicdro.org/text.asp?2014/6/1/24/139089
| Introduction|| |
Scaling and root planning eliminates the mineralized debris but leads to the formation of a thin residous smear layer that delays the adhesion of new fibroblasts and connective tissue on periodontally involved root surfaces. Smear layer is an amorphous, granulated, and irregular layer covering the root surface when observed under scanning electron microscope (SEM).  It constitute of inorganic (calcium, phosphate), organic material (odontoblastic process, bacteria, and blood cells) and bacterial products (endo and exotoxins)  resulting in hypermineralization of root surface,  which may function as a physical barrier to the growth of a connective tissue attachment to the rootsurface. 
Conditioning of root surfaces by topical application of acid solutions had been introduced as a periodontal regenerative procedure. Root biomodifiers are efficient 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),  citric acid (CA),  ethylene diaminetetraacetic acid (EDTA). ,
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 planning, demineralizes the treated surface leaving a "mat-like" collagen surface with exposed dentinal tubules, and eliminates bacterial endotoxins from pathologically altered cementum surfaces. ,
Tetracycline hydrochloride is an effective antibiotic which is absorbed onto the root surface and slowly released in its active form (substantivity).  It act as calcium chelator, resulting in demineralization of the root surface and removal of the smear layer. Furthermore, it is having a potent antiinflammatory and anticollagenase activity. 
BioPure MTAD® is a mixture of TTC, acid and detergent (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-Tween-80® . It has the ability to remove the smear layer and also exert a potent antimicrobial activity.  This is the first study, which aim to use MTAD as a root biomodifier. The aim of the present in vitro SEM study was to comparatively evaluate the efficacy of MTAD (novel intracanal irrigant) and traditional root biomodifiers for smear layer removal and dentinal tubule opening on periodontally involved human teeth.
| Materials and methods|| |
Forty freshly extracted single rooted, periodontally compromised teeth with hopeless prognosis and caries free root surfaces were included in the study. The teeth with wasting diseases, tooth fracture, endodontically treated, and prosthodontically restored were excluded from the study. Written informed consent was obtained prior to extraction from all the patients and ethical committee clearance from the institution was obtained. Following extraction samples was stored in normal saline (NS).
Samples were obtained from the cervical third of 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 ultrasonic scaler and thoroughly planed with number 1-2, 3-4 Gracey curettes (Hu-Freidy) to remove all the altered cementum. With the help of diamond disc under copious irrigation, the two samples are obtained firstly 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 [Figure 1]. The labial and lingual surface of each specimen was used for the study. A total of 80 samples were randomly divided into four groups:
|Figure 1: Sample preparation: (a) Root planning, (b) tooth sectioning, (c) dentine blocks|
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Group I: Biopure MTAD™ (pH 1.28) [Figure 2]a
Group II: TTC HCl (50 mg/ml) (pH 1.75) [Figure 2]b
Group III: CA (pH 1.0) [Figure 2]c
Group IV: NS (control group).
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 s to ensure consistent solution application. Following treatment, samples were rinsed thoroughly with distilled water.
Preparation of samples for scanning electron microscope study
The SEM analysis was done in Birbal Sahani Institute of Palaeobotany, Lucknow. 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 specimen 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).  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 numbers of totally patent smear free dentinal tubules out of the total number of dentinal tubules and percentage (%) patency was calculated. The obtained data were tabulated and statistically analyzed by nonparametric Kruskal-Wallis analysis of variance (ANOVA) and Z-test.
| Results|| |
The Sampaio's index scores of Groups I, II, III, IV ranged from 1-2, 2-4, 4-5 and 4-5, respectively [Figure 3]a-d, with mean (±standard deviation) 1.67 ± 0.49, 3.27 ± 0.59, 4.20 ± 0.41 and 4.53 ± 0.52, respectively [Table 1]. Group I has the lowest score signifying that it was more efficacious in removing the smear layer and exposing the dentinal tubules than other groups. The number of patent dentinal tubules and percentage (%) patency of Groups I, II, and III showed statistically significant results [Table 2].
|Table 2: Comparison (Z value) and signifi cance (P value) of three experimental groups|
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|Figure 3: Scanning electron microscope photomicrographs of (a) MTAD, (b) TTC hydrochloride, (c) CA, (d) normal saline|
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Intergroup comparison with Kruskal-Wallis ANOVA revealed statistically significant difference in scores of Sampio's index, number of patent dentinal tubules and % patency of dentinal tubules [Table 2]. Group I results were highly statistically significant than Groups II and III, IV. The result were statistically insignificant was between II and III.
| Discussion|| |
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, is one of the objectives of periodontal therapy. Methods to achieve this objective include scaling and root planning, and use of demineralizing agents. Scaling and root planning leads to formation of smear layer which inhibits the growth of a connective tissue attachment to the root surface. , 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.  Many chemical agents have been proposed as root conditioning agents, some of them are CA, TTC HCl group, EDTA.  The other methods used are carbon dioxide laser,  neodymium:yittrium, aluminum, garnet (YAG) laser,  and erbium:YAG laser  and many more.
As BioPure MTAD® is used as an efficacious intra 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.  TTC HCl, 50 mg/ml concentration, pH 1.75 was used according to previous studies in which Ishi et al. (2008), Isik et al. (2000) inferred that TTC-HCl concentrations between 50 mg/ml 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. , Another agent used was CA pH 1 was also applied for 3 min as recommended by earlier studies. ,, Whereas NS was used as control.
Root Surface Modification Index (Sampaio's Index) was used for evaluating the photomicrographs for smear layer removal  [Table 2]. The lowest score was achieved in Group I/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.  The result of group I was statistically significant when compared with group II, III, IV. ,, However until date, no in vitro or in vivo studies have been conducted utilizing MTAD as a root biomodifier and comparing these four agents.
The patent dentinal tubules and % patency intergroup comparison by Z-test showed highly statistically significant difference among the groups. MTAD had higher statistically significant number of patent and % patency of dentinal tubules when compared with TTC HCl (P = 0.0001), CA (P < 0.0001) and NS (P < 0.0001).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. 
Tetracycline hydrochloride 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),  Bouchard et al. (1997),  Babay (2000)  who demonstrated comparable results of CA and TTC because of their similar acidic pH (TTC HCl 1.75 pH, CA pH 1) and causes efficient removal of smear layer by demineralization of the organic component. The comparable results can be attributed because of the use of TTC capsules containing significant amount of filler and other substances, which leads to its incomplete dissolution and lead to deposition of TTC crystals on the dentine surface as seen in photomicrographs. , Statistically insignificant results were seen with NS as it removes only the superficial portion of the smear layer due to its application by active burnishing. It leaves the dentinal tubules occluded with debris as observed by Madison and Hokett (1997). 
In summary, our results confirm that the root conditioning agents/root biomodifiers are effective in removing smear layer and exposing dentinal tubules. MTAD being most efficacious followed by TTC HCl and CA. Despite of the limitations of the study, further longitudinal in vitro and in vivo studies to establish MTAD as a root conditioning agent are warranted.
| Conclusion|| |
The overall result of this study have established that root conditioning agents that are MTAD, TTC HCl 50 mg/ml, and CA pH 1 alters 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 wound healing and future new attachment both in vitro and in vivo.
| Acknowledgment|| |
I express my gratitude to Dr. Aarati Kalluri 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 Paleaobotany, Lucknow. I am also grateful to Director, Institute for Data Computing and Training, Lucknow, for providing valuable assistance in data analysis. The authors report no conflicts of interest related to this study.
| References|| |
|1.||Silvério 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. |
|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. |
|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. |
|4.||Isik AG, Tarim B, Hafez AA, Yalçin 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 application times. J Periodontol 2000;71:219-25. |
|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. |
|6.||Register AA, Burdick FA. Accelerated reattachment with cementogenesis to dentin, demineralized in situ. II. Defect repair. J Periodontol 1976;47:497-505. |
|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. |
|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. |
|9.||Cohen ES. Biomechanical root preparation. In: Cohen E editor. Atlas of Cosmetic and Reconstructive Periodontal Susrgery. 3 rd ed. Hamilton: PMPH-USA, BC Becker Inc.; 2007. p. 271-4. |
|10.||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. |
|11.||Theodoro LH, Zezell DM, Garcia VG, Haypek P, Nagata MJ, de Almeida JM, et al. Comparative analysis of root surface smear layer removal by different etching modalities or erbium:yttrium-aluminum-garnet laser irradiation. A scanning electron microscopy study. Lasers Med Sci 2010;25:485-91. |
|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. |
|13.||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. |
|14.||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. |
|15.||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. |
|16.||Garrett JS, Crigger M, Egelberg J. Effects of citric acid on diseased root surfaces. J Periodontal Res 1978;13:155-63. |
|17.||Hanes PJ, Polson AM, Ladenheim S. Cell and fiber attachment to demineralized dentin from normal root surfaces. J Periodontol 1985;56:752-65. |
|18.||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. |
|19.||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. |
|20.||Zhang W, Torabinejad M, Li Y. Evaluation of cytotoxicity of MTAD using the MTT-tetrazolium method. J Endod 2003;29:654-7. |
|21.||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. |
|22.||Bouchard P, Nilveus R, Etienne D. Clinical evaluation of tetracycline HCl conditioning in the treatment of gingival recessions. A comparative study. J Periodontol 1997;68:262-9. |
|23.||Babay N. Comparative SEM study on the effect of root conditioning with EDTA or tetracycline Hcl on periodontally involved root surfaces. Indian J Dent Res 2000;11:53-7. |
|24.||Misra V, Mehrotra KK, Dixit J, Maitra SC. Effect of a carbon dioxide laser on periodontally involved root surfaces. J Periodontol 1999;70:1046-52. |
|25.||Madison JG 3 rd , 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. |
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]