|Year : 2012 | Volume
| Issue : 3 | Page : 145-150
Comparative evaluation of fibrin clot adhesion to root modified with citric acid, tetracycline hydrochloride, minocycline, and ethylenediaminetetraacetic acid: An in vitro study
Ashutosh Nirola, Madhu Gupta, Priyanka Batra, Kanika Singla, Ruby Singla
Department of Periodontology and Oral Implantology, Luxmi Bai Institute of Dental Sciences and Hospital, Patiala, Punjab, India
|Date of Submission||18-Sep-2012|
|Date of Acceptance||24-Jan-2013|
|Date of Web Publication||29-Apr-2013|
Department of Periodontology and Oral Implantology, Luxmi Bai Institute of Dental Sciences and Hospital, Patiala, Punjab
Source of Support: None, Conflict of Interest: None
Background: The objective of periodontal therapy is to convert periodontitis affected root surface into a surface which is biologically compatible for connective tissue adherence and attachment. The present study was to compare the efficacy of citric acid, ethylenediaminetetraacetic acid (EDTA), tetracycline hydrochloride (TTC-HCl), and minocycline as root conditioning agents in terms of fibrin clot adhesion to the dentinal surfaces under Scanning Electron Microscope (SEM).
Materials and Methods: A total of 40 specimens were obtained from fresh extracted human teeth which were divided into four groups comprising 10 specimens in each group. The root conditioning agents used were: citric acid, EDTA, TTC-HCl, and minocycline. The root surfaces were planed and specimen blocks (7 × 5 mm) were obtained. They were subjected to various conditioning agents and then exposed to fresh blood which was allowed to clot. These specimens were then rinsed and subjected to SEM analysis.
Results: The results showed that citric acid conditioned root specimens presented with the best fibrin clot adhesion than any other group.
Conclusion: It was concluded that root conditioning agents have a significant role in periodontal wound healing and future new attachment.
Keywords: Blood, fibrin, root conditioning, smear layer
|How to cite this article:|
Nirola A, Gupta M, Batra P, Singla K, Singla R. Comparative evaluation of fibrin clot adhesion to root modified with citric acid, tetracycline hydrochloride, minocycline, and ethylenediaminetetraacetic acid: An in vitro study. Indian J Oral Sci 2012;3:145-50
|How to cite this URL:|
Nirola A, Gupta M, Batra P, Singla K, Singla R. Comparative evaluation of fibrin clot adhesion to root modified with citric acid, tetracycline hydrochloride, minocycline, and ethylenediaminetetraacetic acid: An in vitro study. Indian J Oral Sci [serial online] 2012 [cited 2019 Nov 20];3:145-50. Available from: http://www.indjos.com/text.asp?2012/3/3/145/111176
| Introduction|| |
Periodontal disease, although multifactorial, has been shown to have bacteria in the form of plaque as its primary etiologic agent. Bacteria initiate disease in many ways, one of which is by production of endotoxins.  Endotoxins' adsorption has been found on diseased root surface  and it adversely affects cell attachment to the substrate.  Periodontitis-affected root surfaces are hypermineralized and contaminated with cytotoxic and other biologically active substances. ,
One of the objectives of periodontal therapy is to convert periodontitis-affected root surface into a surface which is biologically compatible for connective tissue adherence and attachment.  The traditional treatment of such pathologically altered root surfaces has relied on mechanical removal of plaque, calculus, root-bound toxins, and contaminated cementum. But such mechanical debridement and planing of teeth generate a smear layer. This smear layer acts as a barrier between the periodontal tissues and the root surface, inhibiting the formation of new connective tissue attachment to the root surface, and also serves as a reservoir for the microbial growth. Root conditioning by topical application of acidic solutions has been demonstrated to remove not only this smear layer but also any remaining root surface contaminants. Different etching and chelating agents such as citric acid, phosphoric acid, tetracycline hydrochloride (TTC-HCl), ethylenediaminetetraacetic acid (EDTA), fibronectin, doxycycline hydrochloride, sodium deoxycholate, minocycline, etc., have been used to remove the smear layer and endotoxins from the root surface. Surface demineralization of dentin by these agents exposes the collagen matrix, thereby providing a substrate that supports the chemotaxis, migration, and attachment of cells involved in wound healing. In vitro studies have shown enhanced fibrin clot adhesion to cleansed root surface.  This fibrin network promotes a temporary protection of matrix through which cells can migrate during the healing process. It consists of platelets embedded in a mesh of cross-linked fibrin fibers derived from thrombin cleavage of fibrinogen, smaller amount of extracellular molecules such as plasma fibronectin, cytokines, and growth factors which will determine the wound healing process. 
| Materials and Methods|| |
Preparation of specimen
A total of 40 samples were prepared. All tooth cuts were made with a double-sided diamond disk in a slow-speed handpiece under copious water irrigation. To obtain an experimental surface, the crown of each tooth at the level of cemento-enamel junction (CEJ) and apical third of each root was removed and the remaining root was sectioned longitudinally through the root canal to produce a 7×5 mm tooth section. All samples were rinsed and stored in phosphate-buffered saline (PBS), pH 7.4, at 4°C until use. The dentin root surface was then instrumented by using a sharp Gracey 1-2 periodontal curette with six to eight strokes per area to achieve a smooth, glass-like surface.
Preparation of chemical agents
Citric acid solution
The saturated solution was made by slowly adding anhydrous citric acid to 10 ml of distilled water using a magnetic stirrer to mix the solution. The pH of solution was 1, as checked using pH meter.
Ethylenediaminetetraacetic acid solution
15% EDTA solution was prepared by combining 25 ml of distilled water with 2.31 ml of 5 N NaOH, and then adding 4.25 g of the disodium salt of EDTA. The solution had a pH of 7.5. 
Tetracycline hydrochloride solution
Five hundred milligrams of TTC-HCl powder was dissolved in 5 ml of distilled water, which gave TTC-HCl solution at a pH of 1.8, as checked using pH meter. 
Minocycline (100 mg) was prepared by mixing a standard 1 ml of sterile water with the content of one capsule. This solution had a pH of 3.2.
Dentin specimens were prepared by placing the cotton pellets saturated with test solutions for 5 min. Then, these were subjected to three 5-min rinses in PBS and allowed to air-dry for 30 min. Blood applied on groups after conditioning was allowed to clot for 20 min in humidifier. Then, they were subjected to three 5-min rinses in PBS. Immediately after rinsing, fixation was done at room temperature.
Scanning electron microscope analysis preparation
After the dehydration process, the specimens were air-dried. Dried samples were mounted on scanning electron microscope (SEM) stubs. Specimens were then sputter coated with gold in a fine coat (JEOL 10N sputter JFC 1100) sputtering device. The mounted specimens were evaluated using model JEOL 6100 (SEM).
- All specimens were prepared by fixation and dehydration at room temperature.
- Blocks were fixed in 2.5% gluteraldehyde for 30 min.
- Blocks were subsequently subjected to three 5-min rinses in PBS.
Scanning electron microscope examination
Scanning photomicrographs were taken from the center of the test area at ×3000 magnification. Photographs of the samples were obtained by developing the saved photomicrographs from the SEM.
The micrographs were taken and evaluated for fibrin clot adhesion to specimen estimated by the scores given by Lafferty TA, et al. 1993 as seen in [Table 1] and [Graph 1[Additional file 1]].
The data so obtained were compiled and subjected to statistical analysis.
- Score 0: Absence of fibrin network and blood cells
- Score 1: Scarce fibrin network and/or blood cells
- Score 2: Moderate fibrin network and moderate quantity of blood cells
- Score 3: Dense fibrin network and trapped blood cells
Statistical analysis was done with standard computer software. The non-parametric Kruskal-Wallis test (P < 0.05) was used to compare the rank of the evaluated groups. This procedure was followed by non-parametric Mann-Whitney U test when the Kruskal-Wallis test suggested a significant difference between the groups (P < 0.05). The Mann-Whitney U test (P < 0.05) was used to determine the differences between individual groups.
| Results|| |
SEM analysis of fibrin clot adhesion to experimental groups
In group A1, i.e. citric acid treated samples, there was dense fibrin network and entrapped blood cells (score 3) in eight samples, while two samples showed moderate fibrin and blood cell attachment (score 2).
In group B1, i.e. EDTA treated samples, eight samples showed scarce fibrin and blood cell attachment (score 1), while only two samples showed moderate fibrin and blood cell attachment (score 2).
In group C1, i.e. TTC-HCl treated samples, seven samples showed moderate fibrin and blood cell attachment (score 2), while only three samples showed scarce fibrin and blood cell attachment (score 1).
In group D1, i.e. minocycline treated samples, nine samples showed scarce fibrin or blood cell attachment (score 1), while one sample showed moderate fibrin and blood cell attachment (score 2).
Comparison between the mean ranks of the groups (Mann-Whitney Test) showed a significant difference between the following groups: groups A1 and B1 (P = 0.0001), groups A1 and C1 (P = 0.0001), groups A1 and D1 (P = 0.0001), groups B1 and C1 (P = 0.028), and groups C1 and D1 (P = 0.008) as seen in [Table 2], [Graph 2[Additional file 2]].
Comparison between experimental group B1 and experimental group D1 showed that fibrin clot adhesion was insignificant between groups B1 and D1 (P = 0.542).
| Discussion|| |
Root surface conditioning by topical application of acidic solutions has been demonstrated to remove the smear layer and also any remaining root surface contaminants. It uncovers and widens the orifice of dentinal tubules with unmasking of the intra- and peri-tubular dentin collagen matrix  due to demineralization. This is indispensable for adhesion of fibrin clot. The clot adhesion appears vitally dependent on the formation of a resilient unit between the clot (fibrin network) and the collagen fibers exposed at the root surface. The present study was designed to evaluate the extent of the fibrin clot adhesion on cleansed root conditioned surfaces. 
In the present study, maxillary and mandibular anterior teeth with grade III mobility indicated for extraction due to chronic periodontitis were used.
A total of 40 specimens were obtained from the roots of the extracted maxillary and mandibular anterior teeth, which were categorized into four groups (citric acid, EDTA, TTC-HCl, and minocycline) consisting of equally divided specimens in each group.
The teeth used in this study were sectioned near the CEJ to obtain the experimental surface because the coronal part of the root contains less cementum as compared to its apical part,  making it easy to remove the cementum and obtain a glass-like dentin surface for root conditioning. Instrumentation prior to application of root conditioning agents was done to remove the hypermineralized surface layer present on the periodontitis-affected roots.
In the present study, passive application was preferred over burnishing technique as the latter may itself form smear layer which partially or completely obliterates the dentinal tubule opening.
The obtained specimens were categorized into four groups consisting of 10 specimens in each group. PBS was used for temporary storage of the teeth. The solution used during this short holding time should not have affected the final surface characteristics. These findings are consistent with the findings of Lafferty. 
Group A1 (citric acid) exhibited vast three-dimensional array of dense interconnected fibrin strands enmeshing the trapped blood cells (observed in eight samples) to moderate fibrin network and blood cells observed in two samples [Figure 1].
|Figure 1: Showing citric acid treated dentin specimen after blood application (A1)|
Click here to view
The results obtained in this study are in accordance with those obtained by Baker et al. in 2000  and Baker et al. in 2005  in their in vitro studies.
In contrast, animal studies by Nyman et al. that utilized citric acid for root conditioning have failed to accomplish new attachment. Speculative explanation for these inconsistent findings have included variations in animal models, inconsistent flap adaptation, inadequate demineralization of periodontitis-affected root surface, ,, and repopulation of the root surface with inappropriate cell types.
Group B1 (EDTA) exhibited the presence of scarce fibrin network and attachment of blood cells (in eight samples) to moderate fibrin network and blood cells (in two samples). It was observed that most samples in group B1 inhibited blood element adsorption and adhesion to the root surface. This is because of calcium chelating property of EDTA that inhibits or retard coagulation events in accordance with findings by Delazari, et al.  , Sculean A, et al.  [Figure 2]
|Figure 2: Showing EDTA treated dentin specimen after blood application (B1)|
Click here to view
In contrast, studies conducted by Blomlof and Lindskog  have shown that etching at neutral pH with EDTA is equally, if not more efficient, compared to agents operating at low pH in exposing collagen fibres on dentin surfaces.
Group C1 (TTC-HCl) exhibited the presence of moderate fibrin network and moderate attachment of blood cells (in seven samples) to scarce fibrin network and blood cells (in three samples). These results were obtained because TTC-HCl was successful in removing the smear layer and exposed the opening of dentinal tubules with collagen matrix. , [Figure 3]
|Figure 3: Showing tetracycline HCl treated dentin specimen after blood application (C1)|
Click here to view
In the present study, TTC-HCl fared better than the EDTA group as regards to the fibrin network and blood cell attachment. It can be due to greater demineralization potential of tetracycline than EDTA. Several in vivo studies have demonstrated that TTC-HCl-demineralized dentin surfaces showed greater number of attached cells, greater connective tissue attachment,  reattachment, and new cementum formation.
In contrast, Delazari et al. found in their study that fibrin network formation in situ was not improved by application of TTC-HCl.
Saturated citric acid successfully removes the smear layer and provides greater depth of demineralization as compared to TTC-HCl, as well as increases the wettability of dentin resulting in enhanced attachment of the fibrin clot imposed onto the root surfaces, as continuous adhesion of fibrin clot appears to be dependent on the wettability of the substrate. It is observed that although citric acid is an anticoagulant, it has shown no adverse effects on early fibrin polymerization.
Group D1 (minocycline) exhibited the presence of scarce fibrin network and attachment of blood cells (in nine samples) to moderate fibrin network and blood cells (in one sample). This difference was probably due to the higher pH (3.2) and lower concentration of minocycline, therefore a higher concentration of minocycline may be required to achieve comparable results. [Figure 4]
|Figure 4: Showing minocycline treated dentin specimen after blood application (D1)|
Click here to view
Comparison of mean ranks of the groups (Mann-Whitney Test) showed a significant difference between the following groups: groups A1 and B1 ( P = 0.0001), groups A1 and C1 (P = 0.0001), groups A1 and D1 (P = 0.0001), groups B1 and C1 (P = 0.028), and groups C1 and D1 (P = 0.008). These observations are consistent with the findings of Baker, et al. 
Comparison between experimental group B1 (EDTA) and experimental group D1 (minocycine) showed that fibrin clot adhesion was insignificant between these groups (P = 0.542). These observations are in consistent with the findings of Baker et al. 
Surface demineralization of dentin by these chemical agents exposes the collagen matrix, which is valuable in enhancing periodontal healing, since a collagen rich surface is more conducive to cell colonization than a hydroxyapatite-rich surface. This fibrin network promotes a temporary protection of matrix through which cells can migrate during the healing process. It consists of platelets embedded in a mesh of cross-linked fibrin fibers derived from thrombin cleavage of fibrinogen, smaller amount of extracellular molecules such as plasma fibronectin, cytokines, and growth factors which will determine the wound healing process.
In the present study, it was observed that root conditioning with citric acid resulted in best fibrin clot adhesion than any other experimental group. Thus, root conditioning agents have a significant role in periodontal wound healing and future new attachment. However, results of the present study are limited to physical findings of the root surface changes and do not present in vivo differences that may result from the physiologic effects of these root conditioning agents.
Differences between the results of the present study and those of other studies may be related to the disease status of the dentin specimen utilized, the concentration, time and mode of application of the demineralizing agent, or a combination of these variables. Hence, additional studies including microbial studies, both in vitro and in vivo, of these variables are needed.
| Conclusion|| |
Within the limits of the study, it can be concluded that root conditioning may have a significant role in periodontal wound healing. It creates biologically acceptable root surfaces which are necessary for regeneration and new attachment in vivo. In vitro, citric acid,  EDTA, TTC-HCl,  and minocycline agents resulted in the fibrin clot attachment to the dentin surface.
| References|| |
|1.||Trombelli L, Scabbia A, Zangari F, Griselli A, Calura G. Effect of Tetracycline HCl on Periodontally affected human root surfaces. J Periodontol 1995;66:685-91. |
|2.||Baker DL, Pavlow SS, Wikesjo UM. Fibrin clot adhesion to dentin conditioned with protein constructs: An in vitro study. J Clin Periodontol 2005;32:561-6. |
|3.||Nyman S, Lindhe J, Karring T. Healing following surgical treatment and root demineralization in monkeys with periodontal disease. J Clin Periodontol 1981;8:249-58. |
|4.||Delazari FM, Gerlach RF, Joly JC, Lima AF. Scanning electron microscopy study of the effect of tetracycline HCl on smear layer removal and fibrin network formation. Braz Dent J 1999;10:81-7. |
|5.||Blomlof J, Lindskog S: Periodontal tissue-vitality after different etching modalities. J Clin Periodontol 1995;22:464-8. |
|6.||Jones WA, O'Leary TJ. The effectiveness of in vivo root planning in removing bacterial endotoxin from the roots of Periodontolly involved teeth. J Periodontol 1978;49:337-42. |
|7.||Baker PJ, Rotch HA, Trombelli L, Wikesjo UM. An in vitro screening model to evaluate root conditioning protocols for Periodontol regenerative procedures. J Periodontol 2000;71:1139-43. |
|8.||Borghetti A, Mattout P, Mattout C. How much root planning is necessary to remove the cementum from the root surface? Int J Periodont Restorat Dent 1987;4:23-9. |
|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. |
|10.||Lafferty TA, Gher ME, Gray JL. Comparative SEM study on the effect of acid etching with tetracycline HCl or citric acid on instrumented Periodontolly involved human root surfaces. J Periodontol 1993;64:689-93. |
|11.||Lowenberg B, Thibault J, Lawrence C, Sodek J. The influence of chemically-induced modifications of root surfaces on cell migration, attachment, and orientation. J Dent Res 1986;65:1010-5. |
|12.||Hanes PJ, Polson AM. Cell and fiber attachment to demineralized cementum from normal root surfaces. J Periodontol 1989;60:188-98. |
|13.||Wikesjo UM, Claffey N, Nilveus R, Egelberg J. Periodontal repair in dogs: Effect of root surface treatment with stannous fluoride orcitric acid on root resorption. J Periodontol 1991;62:180-4. |
|14.||Polson AM, Proye MP. Effect of root surface alterations on periodontal healing II: Citric acid treatment of the denuded root. J Clin Periodontol 1982;9:441-54. |
|15.||Sculean A, Berakdar M, Willershausen B, Arweiler NB, 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. |
|16.||Babay N. The effect of tetracycline HCl on the smear layer formed by various root planning modalities: A scanning electron microscope study. Saudi Dent J 2000;12:156-60. |
|17.||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. |
|18.||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. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]