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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 8  |  Issue : 1  |  Page : 57-61

Fibrin clot adhesion to instrumented conditioned root surfaces by MTAD and normal saline: A scanning electron microscopy study


1 Dental Hospital and Implant Centre, Mehatpur, Himachal Pradesh, India
2 Department of Orthodontics, Himachal Institute of Dental Sciences, Paonta Sahib, Himachal Pradesh, India
3 Department of Pedodontics, Surendra Dental College, Sriganganagar, Rajasthan, India

Date of Submission21-Oct-2019
Date of Decision14-Feb-2020
Date of Acceptance16-Feb-2020
Date of Web Publication20-Jun-2020

Correspondence Address:
Dr. Gaurav Didhra
Dental Hospital and Implant Centre, Opp. UCO Bank Main Bazar, Mehatpur - 174 315, Himachal Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/amhs.amhs_149_19

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  Abstract 


Background and Aim: Smear layer removal and formation of a stable bond between the blood clot and root surface are desirable for periodontal healing, which can be achieved by root biomodifiers. The objective of this in-vitro study was to evaluate the root surface changes in terms of fibrin clot adhesion subsequent to the application of MTAD (mixture of doxycycline (a tetracycline isomer), citric acid, and polysorbate-80 (a detergent) and normal saline. Materials and Methods: Twenty human single-rooted teeth were collected and grouped into Saline (Group I) and MTAD Biopure (Group II) treatment groups containing 10 teeth in each group. After root conditioning, fresh human blood was applied to each root specimen and was allowed to clot, and subsequently, samples were dehydrated in a graded series of ethanol and visualized under scanning electron microscope. Results: The highest blood clot attachment score was observed with roots treated with MTAD and the least results were observed with control group saline. Conclusion: Specimens treated with MTAD best supported the fibrin clot attachment. Saline was least effective of all the samples, showing the presence of the smear layer.

Keywords: Fibrin clot, MTAD, periodontal regeneration, root biomodification


How to cite this article:
Didhra G, Upadhyay S, Sharma A, Sambyal H. Fibrin clot adhesion to instrumented conditioned root surfaces by MTAD and normal saline: A scanning electron microscopy study. Arch Med Health Sci 2020;8:57-61

How to cite this URL:
Didhra G, Upadhyay S, Sharma A, Sambyal H. Fibrin clot adhesion to instrumented conditioned root surfaces by MTAD and normal saline: A scanning electron microscopy study. Arch Med Health Sci [serial online] 2020 [cited 2020 Oct 30];8:57-61. Available from: https://www.amhsjournal.org/text.asp?2020/8/1/57/287349




  Introduction Top


Regeneration of the periodontal ligament and bone destroyed by infectious processes is the ultimate goal of periodontal surgery. It has been demonstrated that the formation and maintenance of a stable bond between the blood clot and the root surface are critical for optimal periodontal healing, preventing the down growth of gingival epithelial cells and promoting new connective tissue attachment. However, the root surfaces of periodontally involved teeth are heavily contaminated by bacteria and their endotoxins, and this contamination may hinder the outcome of periodontal regenerative procedures by preventing new connective tissue attachment. Therefore, to enhance the periodontal regenerative procedure, root conditioning is the method to restore the biocompatibility of the root surface to promote the migration, attachment, proliferation, and synthesis of connective tissue macromolecules by connective tissue cells.[1]

Root biomodifiers have shown to remove inorganic and organic materials and toxins from the root surfaces; furthermore, they have shown to expose dentin collagen and cementum bound proteins, resulting in a root surface which is conducive in promoting periodontal regeneration. Many chemical agents have been proposed for the purpose of root conditioning, including citric acid, tetracycline hydrochloride group, and ethylenediaminetetraacetic acid. The other agents used are carbon dioxide laser, neodymium: Yttrium–aluminum–garnet laser, erbium: yttrium–aluminum–garnet laser, and many more.[2]

Biopure MTAD™ (Dentsply Tulsa Dental, Tulsa, OK, USA) is a material originally developed for use during endodontic treatment to remove the smear layer from the root canal walls. The use of MTAD has, so far, been confined to endodontics to aid bacterial removal from dentine tubules within the canal and enhancing better attachment of the sealant to the canal walls.[3] Many of the unique properties of MTAD which contribute to its status as an endodontic irrigant may be transferable to the conditioning process in periodontal therapy.[4]

In this study, an effort has been made to compare the effect of normal saline and MTAD on adhesion of fibrin clot to periodontally diseased root surfaces.


  Materials and Methods Top


Twenty human single-rooted teeth were extracted due to severe periodontitis and were collected as study specimens from the Department of Oral and Maxillofacial Surgery of Himachal Institute of Dental Sciences, Paonta Sahib. Inclusion criteria consisted of teeth with Grade III mobility or hopeless prognosis, bleeding on gentle probing, and proximal bone loss on radiographic examination. Patients with a history of systemic disease, oral prophylaxis in the previous 6 months, carious teeth along with periapical infection, and any history of acute pain and swelling necessitating tooth removal were not included in the study.

Following extraction, the teeth were washed with distilled water and stored in normal saline 0.9% at room temperature till further study.

Preparation of samples

Two parallel grooves were made on the proximal surface of the roots of each tooth using diamond disk bur with micromotor handpiece under copious irrigation, the first groove is prepared at the cementoenamel junction and another at 3 mm apical to it.

The area between two grooves is debrided and planned with apico-cervical stroke movements till the surface becomes smooth and shiny, using Gracey curette No. 5–6 (HU-Friedy, USA). After root planing, the area between the two grooves was sectioned to obtain dentinal blocks of adequate size. In such a way, 20 dentinal blocks are obtained and stored in normal saline until use.

Chemical treatment of the dentinal blocks – All 20 specimens are divided into two groups:

  • Group 1: Ten root specimens are treated with normal saline by cotton pellets saturated with it and changed after every 20 s for a period of 3 min
  • Group 2: Ten root specimens are conditioned with Biopure MTAD™ by placing cotton pellets saturated with it and changed every 20 s for 3 min.


Root conditioning was done by burnishing soaked cotton pellets with light pressure. Then, the specimens were washed with 10 ml saline solution. Venous blood was collected from a healthy volunteer and a drop of blood was placed on each chemically treated root surface. The drop of blood was allowed to clot for 20 min at 37°C in a humidifier chamber. Then, the specimens were subjected to 5-min rinses in phosphate-buffered saline (PBS) thrice times; this was done to test the adhesion of clot.[5] After that, each block of specimen was fixed in 2.5% glutaraldehyde for 30 min. Subsequently, the blocks were subjected thrice in PBS for 5 min; after that, each block was subjected for dehydration in graded series of ethanol (10%–90%) for 30 min each and the final dehydration carried out in 100% acetone for 30 min more.[6] The samples were dried overnight in a dehydration jar, mounted on metallic stubs with adhesive tape, and sputter coated with gold. Finally, the specimens were observed using a scanning electron microscopy (SEM) unit. The surfaces of the roots were scanned and representative photomicrographs were obtained on the computer screen at ×5000 magnification.

Analysis of photomicrographs

Scoring of the samples was done according to the criteria given by Theodoro et al. 2006.[7]

  • Score 0: Absence of fibrin network and blood cells
  • Score 1: Scarce fibrin network with no blood cells
  • Score 2: Moderate fibrin network and moderate quantity of blood cells
  • Score 3: Dense fibrin network and trapped blood cells.


Statistical analysis

Comparison of the median of different groups was done at 25th, 50th, and 75th percentile. Intergroup analysis was done by applying Mann–Whitney U-test. Significance is assessed at a 5% level of significance.


  Results Top


The photomicrographs of the samples treated with saline showed the presence of a heavy smear layer throughout the entire sample. There was no evidence of fibrin network and blood cells in all the samples. All the samples belonged to score 0. The median value of saline at maximum and minimum interval is 0 [Table 1] and [Table 2], [Figure 1] and [Figure 2].
Table 1: Scoring of samples using criteria of Theodoro LH et al. 2006

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Table 2: Comparison of median of groups

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Figure 1: Frequencies of scores within groups

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Figure 2: Photomicrograph of dentine samples treated with saline (magnification ×5000)

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The photomicrographs of the samples treated with MTAD showed a maximum number of samples with dense fibrin network and trapped cells, i.e., score 3. The median values of MTAD at the minimum and maximum intervals are 1 and 3, respectively [Table 1] and [Table 2], [Figure 1] and [Figure 3].
Figure 3: Photomicrograph of dentine samples treated with MTAD (magnification ×5000)

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The mean rank on intergroup comparison between Group 1 and Group 2 mean rank was 5.50 and 15.50, respectively, on applying Mann–Whitney U-test, when both the experimental groups were compared, P value was found to be statistically significant (P < 0.001), showing that Group 2 is better than Group 1 in terms of fibrin clot attachment [Table 3].
Table 3: Inter group comparison between normal saline (Group 1) and MTAD (Group 2)

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  Discussion Top


The prime objective of all the periodontal therapy is to convert a periodontitis-affected root surface into a substrate which is biologically acceptable for epithelial and connective tissue adherence.[8] However, the periodontitis-affected root surfaces are hypermineralized and contaminated with cytotoxic and other biologically active substances, such surfaces are not biocompatible with the adjacent periodontal cells that play an important role in periodontal wound healing,[9] and it is not possible to decontaminate the periodontitis-affected root surface by mechanical mean alone.[10]

With age systemic and local changes occur inside the oral cavity that may lead to alterations at microscopic level causing hindrance in fibrin clot attachment hence the teeth that were easy to remove and appeared clinically normal without any root surface alterations were selected.

Root biomodification is a periodontal regenerative technique which has received much attention. It has been shown to expose collagen fibrils and creates a zone of demineralized matrix of 3–20 μm thick. The tooth collagen exposed by this root demineralization pretreatment procedure is thought to augment periodontal wound healing, thereby enhancing periodontal regeneration. A variety of agents have been used in conjunction with root demineralization new attachment procedures such as hydrochloric acid, ethylenediaminetetraacetic acid, phosphoric acid, tetracycline, stannous fluoride, and citric acid.[11]

Considering the above facts, an effort has been made in this study to determine the surface characteristics of the diseased root surface by conditioning with normal saline and MTAD BIOPURE™ under the scanning electron microscope.

Biopure™ MTAD (Dentsply Tulsa Dental, Tulsa, Oklahoma, USA) is a mixture of doxycycline (a tetracycline isomer), citric acid, and polysorbate-80 (a detergent). It has been reported to remove the smear layer effectively, eliminate microbes that are resistant to conventional endodontic irrigants and dressings, and provide sustained antimicrobial activity.

In the present study, 20 single-rooted teeth affected by periodontitis with grade III mobility were extracted from the patients with no history of systemic disease. Multirooted teeth during extraction were marred by extraction forceps causing alteration in the quality of samples, so we preferred to go with single rooted as they were easily studied under SEM without any error in sample preparation. Teeth affected by caries were not included in this study, as it could have adversely affected the root surface topography. Minimal instrumentation during extraction was considered to avoid chipping of the root structure, and teeth with the immediate past history of scaling and root planing were excluded, as they may alter the root surface. Teeth with attrition, abrasion, and erosion were not included in the study, as they have shown to produce secondary changes in tooth structure like alteration in mineral composition and formation of sclerotic dentine.

Only proximal root surfaces were preferred because facial and lingual surfaces were marred by the extraction forceps during tooth extraction.[11] The teeth in this study were root planed until the roots felt hard, velvety smooth, and glass-like to the touch of an explorer and until no rough spots or deposits could be detected. After root planing, the samples are stored in saline to avoid dehydration of the specimens.[12]

In the present study, active burnishing of the samples with cotton pellets saturated with the respective conditioner is done and changed after every 20 s for a period of 3 min. This active burnishing was found to increase the size of dentinal tubules and expose a large amount of collagen matrix.[4] After root conditioning, the samples were washed with 10 ml of saline solution to rid the specimens off any remaining/pooled conditioning agent on the root surface.[13]

On visualizing the samples under the scanning electron microscope, the photomicrographs of the samples treated with saline showed the presence of a heavy smear layer throughout the entire sample. All the samples belonged to score 0. When the instrumented conditioned root surface of samples treated with saline was rinsed in PBS following 20-min incubation with blood, the blood clot washed away from all the samples along with SEM evaluation showing smear layer. This is in accordance with the study of Baker et al. 2000.[14]

The photomicrographs of the samples treated with MTAD showed a maximum number of samples with dense fibrin network and trapped cells, i.e., score 3 (seen in six samples).

Doxycycline (a tetracycline isomer) is the primary ingredient of MTAD contributing to its antimicrobial activity. Citric acid has been shown to remove the smear layer in combination with doxycycline and allowed the penetration of doxycycline into the root canal irregularities and dentinal tubules to show its antimicrobial activity, both of them have an influential hold as root conditioner in our esteemed periodontal literature. The third component, i.e., TWEEN ® 80, reduces the surface tension and enhances the penetrating ability and diffusion of acids into the root canal wall and irregularities.[15] Moreover, its low pH (1.28) causes more demineralization of matrix and exposes collagen fibers enhancing more fibrin clot attachment, so clinically, when we will use this product as a root conditioner, it will give better results in terms of attachment gain as compared to other conditioner individually.

Studies from the past had shown that individual components of MTAD have been used previously as periodontal conditioners. Boyko et al. showed that the citric acid demineralization of the root surfaces resulted in enhanced healing of periodontal defects by improving the adhesiveness of root surface as a substrate to which cells can adhere.[16] Polson et al. had shown that citric acid application to the root planned surface removed smear layer and exposed collagenous matrix that appeared to provide a more hospitable environment for connective tissue attachment.[17] Baker et al. showed better efficiency of citric acid demineralization of the root surface to promote fibrin clot adhesion as compared to ethylenediaminetetraacetic acid and protein constructs.[5] Wikesjö et al. showed that tetracycline HCL removed the smear layer and exposed dentinal tubules and can act as an improved substrate for connective tissue components vital to periodontal healing.[18] Minocha and Rahul, found increased attachment of fibrin clot when a combination of citric acid and tetracycline is used as compared to when citric acid alone was used.[12]

On the basis of statistical analysis when both the groups, i.e., Group 1 and Group 2, were compared, the value of P value was found to be statistically significant (P < 0.001), showing that Group 2 is better than Group 1 in terms of fibrin clot attachment. This is in accordance with the study done by Zia et al. (2014),[13] Gupta et al. (2015),[19] and Houshmand et al. (2011).[4]

When both the experimental groups were compared, i.e., Group 1 and Group 2, the value of P value was found to be significant at the level P < 0.001. The above results show that normal saline is not able to remove smear layer and does not show fibrin clot attachment and is less efficacious than of MTAD BIOPURE™. The better results for MTAD can be attributed due to its low pH (1.28) and the presence of detergent (TWEEN ® 80 ) enhancing its penetration and thus better removal of the smear layer and subsequently better attachment of fibrin clot. This is in accordance with the study done by Zia et al. (2014)[13] and Tandon et al. (2015).[2]

Hence, the overall statistics shows that the Group II (MTAD) is better than Group I (Normal saline), indicating superior efficacy of MTAD as a better root surface modifier as compared to normal saline.


  Conclusion Top


Within the limits of this study, it was concluded that MTAD is an effective root conditioning agent and can be used in removing the smear layer. It may be have its own demerits also, but this can be conclusive only when we get enough evidence clinically to explain the efficiency of this material in periodontics.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Rompen EH, Goffinet GH, Nusgens B. Human periodontal ligament fibroblast behaviour on chemically conditioned dentine: An in-vitro study. J Periodontol 1999;70:1144-52.  Back to cited text no. 1
    
2.
Tandon C, Govila V, Pant VA, Meenawat A. Comparative in vitro SEM study of a novel root canal irrigantMTAD and conventional root biomodifiers on periodontally involved human teeth. J Indian SocPeriodontol 2015;19:268-72.  Back to cited text no. 2
    
3.
Ghandi M, Houshmand B, Nekoofar MH, Tabor RK, Yadeghari Z, Dummer PM. The effect of MTAD, an endodontic irrigant, on fibroblast attachment to periodontally affected root surfaces: A SEM analysis. J Indian Soc Periodontol 2013;17:188-92.  Back to cited text no. 3
[PUBMED]  [Full text]  
4.
Houshmand B, Ghandi M, Nekoofar M, Gholamii GA, Tabor RK, Dummer PM. SEM analysis of MTAD efficacy for smear layer removal from periodontally affected root surfaces. J Dent (Tehran) 2011;8:157-64.  Back to cited text no. 4
    
5.
Baker DL, Pavlow SA, Wikesjo UM. Fibrin clot adhesion to dentin conditioned with protein constructs: An in vitro proof-of-principle study. J Clin Periodontol 2005;32:561-6.  Back to cited text no. 5
    
6.
Tandon C, Govilla V, Pant AV, Meenawat A. An in vitro scanning electron microscopic study comparing MTAD (intracanalirrigant) and various root biomodifiers on periodontally involved human teeth. J Int Clin Dent Res Org 2014;6:24-8.  Back to cited text no. 6
    
7.
Theodoro LH, Sampaio JE, Haypek P, Bachmann L, Zezell DM, Garcia VG. Effect of Er:YAG and diode lasers on the adhesion of blood components and on the morphology of irradiated root surfaces. J Periodontal Res 2006;41:381-90.  Back to cited text no. 7
    
8.
Hanes PJ, O'Brien NJ, Garnick JJ. A morphological comparison of radicular dentine following root planning and treatment with citric acid or tetracycline HCL. J Clin Periodontol 1991;18:660-8.  Back to cited text no. 8
    
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Blomlof JP, Blomlof LB, Lindskog SF. Smear removal and collagen exposure after non-surgical root planning followed by etching with an EDTA gel preparation. J Periodontol 1996;67:841-5.  Back to cited text no. 9
    
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Jones WA, O'Leary TJ. The effectiveness of in-vivo root planning in removing bacterial endotoxin from the roots of periodontally involved teeth. J Periodontol 1978;49:337-42.  Back to cited text no. 10
    
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Sterrett JD, Bankey T, Murphy HJ. Dentin demineralization. The effects of citric acid concentration and application time. J Clin Periodontol 1993;20:366-70.  Back to cited text no. 11
    
12.
Minocha T, Rahul A. Comparison of fibrin clot adhesion to dentine conditioned with citric acid, tetracycline, and ethylene diamine tetra acetic acid: An in vitro scanning electron microscopic study. J Indian Soc Periodontol 2012;16:333-41.  Back to cited text no. 12
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13.
Zia A, Andrabi SM, Bey A, Kumar A, Fatima Z. Endodontic irrigant as a root conditioning agent: An in vitro scanning electron microscopic study evaluating the ability of MTAD to remove smear layer from periodontally affected root surfaces. Singapore Dent J 2014;35:47-52.  Back to cited text no. 13
    
14.
Baker PJ, Rotch HA, Trombelli L, Wikesjö UM. An in vitro screening model to evaluate root conditioning protocols for periodontal regenerative procedures. J Periodontol 2000;71:1139-43.  Back to cited text no. 14
    
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Torabinejad M, Shabahang S, Aprecio RM, Kettering JD. The antimicrobial effect of MTAD: An in vitro investigation. J Endod 2003;29:400-3.  Back to cited text no. 15
    
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Boyko GA, Brunette DM, Melcher AH. Cell attachment to demineralized root surfaces in vitro. J Periodontal Res 1980;15:297-303.  Back to cited text no. 16
    
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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. 17
    
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Wikesjö UM, Baker PJ, Christersson LA, Genco RJ, Lyall RM, Hic S, et al. A biochemical approach to periodontal regeneration: Tetracycline treatment conditions dentin surfaces. J Periodontal Res 1986;21:322-9.  Back to cited text no. 18
    
19.
Gupta PK, Mahajan UP, Gupta K, Sheela NV. Comparative evaluation of a new endodontic irrigant – Mixture of a tetracycline isomer, an acid, and a detergent to remove the intracanal smear layer: A scanning electron microscopic study. J Int Oral Health 2015;7:1-6.  Back to cited text no. 19
    


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    Tables

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