Introduction
“Periodontal regeneration is defined histologically as regeneration of the tooth-supporting tissues, alveolar bone, periodontal ligament, and cementum over a previously diseased root surface.”[ 1 , 2 ] Mechanical instrumentation of the root surface removes bacterial deposits and calculus that leaves a layer called the smear layer which contains residual calculus, bacteria, and bacterial products.[ 3–6 ] The root conditioning agents eliminate bacteria and their endotoxins.[ 7 , 8 ] The tetracyclines are a group of bacteriostatic antimicrobials, useful against a broad spectrum of microorganisms.[ 9 , 10 ] Ethylene-diamine-tetra-acetic acid (EDTA) is a chelating agent that exhibits effects of demineralization by chelating divalent cations at a neutral pH. Erbium, chromium-doped yttrium, scandium, gallium and garnet (Er, Cr: YSGG) laser are used for the purpose of pocket debridement, wound healing, and other surgical procedures. This laser is used on hard tissue.[ 11 ]
There is no literature available as yet on the effectiveness of Er, Cr: YSGG laser on blood component adhesion (BCA) and fibrin network formation on dentin.
The aim of the study was to compare the fibrin network formation and smear layer removal (SLR) among Er, Cr: YSGG laser, ethylene-diamine-tetra-acetic acid, and tetracycline groups on periodontally affected root surfaces in vitro .
Subjects and Methods
Collection and preparation of the specimens
Twenty freshly extracted single-rooted teeth were collected from patients attending Amrita School of Dentistry who were affected by the diseases in the periodontal tissues. The teeth extracted were free of fillings, caries, and restorations. The teeth extracted were straight away rinsed in distilled water. The teeth were stored in 0.9% saline, until the start of the study. On the proximal surfaces of the root of each extracted tooth, two parallel grooves were made with the help of a diamond-tapered fissure bur attached to an airotor handpiece under profuse irrigation. The first groove was made at the cementoenamel junction and another groove was made 3 mm apical to the first groove. Mechanical instrumentation was done in the area between the two grooves with the use of Gracey curettes No5/6 GDC. After instrumentation, each tooth was sectioned with microtome to achieve four dentin sections of about 4 mm × 3 mm × 1 mm which were prepared from each tooth, and one section was allotted to each group. The sections were then stored in bottles with phosphate-buffered saline.
Experimental procedures
A total of 40 dentin sections had been prepared from 20 extracted teeth. The following treatments were performed. (a) Treated with saline for 3 min, (b) root conditioned with 24% EDTA gel (pH 7.2) with cotton pellets saturated with the agent and changed at an interval of 20 s for 3 min, (c) exposed to a laser with a wavelength of 2.78 mm, power of 0.75W, pulse duration of 140 ms, repetition rate of 20 Hz, and using 33% water and 43% air. Each sample was irradiated for about 5–7 s. (d) Root conditioned with 10 mg/ml tetracycline hydrochloride (HCl) solution (pH 1.3) with cotton pellets saturated with this agent that was then changed at an interval of 20 s for 3 min. The root sections were washed with 10 ml saline solution. From a healthy male volunteer, venous blood was collected and placed on each root surface that was treated. The drop of blood was permitted to clot for 20 min on five specimens from each group. The remaining specimens were subjected to 5 min rinses in phosphate-buffered saline three times.
Preparation for scanning electron microscopy study
Sections of dentinal surfaces were placed for 30 min in 2.5% glutaraldehyde. Afterward, these sections were introduced three times in phosphate-buffered saline. The sections were then kept for dehydration by placing them in graded ethanol (70%, 90%, and 95%) for 5 min. The samples were allowed to be dried overnight and were kept on metallic stubs with adhesive tape, and sputter coating was done with gold. Using a scanning electron microscopy (SEM) unit, the specimens were observed. The sections were scanned and their photomicrographs were achieved on a monitor at × 500, ×1000, ×2000, ×2500, and × 5000. Scoring for adhesion of the components of blood (BCA) and removal of smear layer (SLR) was executed with the photomicrographs taken. A trained, single-blinded operator had performed the scoring.
“BCA scoring (Polson, 2004):
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.”
“SLR scoring (Theodoro, 2004):
Score 1 – No smear layer and open dentinal tubules
Score 2 – No smear layer and partially open dentinal tubules
Score 3 – No smear layer and obliterated dentinal tubules
Score 4 – Moderate smear layer and open dentinal tubules
Score 5 – Moderate smear layer and partially open dentinal tubules
Score 6 – Heavy smear layer and open dentinal tubules
Score 7 – Heavy smear layer and partially open dentinal tubules.”
Results
In the control group (Group A), five samples showed a moderate smear layer with partially open dentinal tubules. Four samples showed scarce fibrin network and blood components on the root surface. One sample showed an absence of fibrin network. In the EDTA group (Group B), three samples showed no smear layer but dentinal tubules were obliterated. Two samples showed no smear layer but dentinal tubules were partially open. Three samples showed dense fibrin network and trapped blood cells and two samples showed moderate fibrin and blood components. In the Er, Cr: YSGG laser group (Group C), four samples showed the absence of smear layer and partially open dentinal tubules. One sample showed an absence of smear layer with obliterated dentinal tubules. Four samples showed dense fibrin networks and trapped blood cells. In the tetracycline group (Group D), three samples showed no smear layer with open dentinal tubules and three samples showed no smear layer with obliterated dentinal tubules. Three samples showed few blood cells and two samples showed dense fibrin network and blood cells. There was a statistically significant difference between tetracycline, Er, Cr: YSGG laser, and EDTA with the control group in terms of the SLR scoring and the BCA scoring. Graph 1 and Tables 1 and 3 show the comparison of SLR, among the groups with the median interquartile range SLR of 5 in scaling and root planing (SRP) category, tetracycline HCl category was 1, EDTA category was 3, and Er, Cr: YSGG laser was 2 and comparison shows statistically significant difference with a P = 0.005. Graph 2 and Tables 2 and 4 shows the comparison of BCA, among groups with median BCA of 1 in SRP category, tetracycline category was 3, EDTA category was 3, and Er, Cr: YSGG laser category was 3 and comparison shows statistically significant difference with a P = 0.004, Figures 1 and 2 are SEM image showing the effect of SRP on dentin (moderate smear layer with partially open dentinal tubules and scarce fibrin network with blood cells, respectively). Figures 3 and 4 show the effects of EDTA on dentin (absence of smear layer with obliterated dentinal tubules and dense fibrin network with trapped blood cells, respectively). Figures 5 and 6 show the effect of tetracycline HCl on dentin (absence of smear layer with open dentinal tubules and dense fibrin network with blood cells respectively). Figures 7 and 8 show the effect of E, Cr: YSGG laser on dentin (absence of smear layer with obliterated tubules and abundant presence of fibrin network with blood cells, respectively).
Graph 1: Comparison of the mean SLR scores among groups. SLR: Smear layer removal
Table 1: Comparison of the smear layer removal score among treatment groups with control group (scaling and root planing)
Graph 2: Comparison of the mean BCA scores among groups. BCA: Blood component adhesion
Table 2: Comparison of the blood component adhesion among treatment groups with the control group (scaling and root planing)
Table 3: Comparison of the smear layer removal score among groups
Table 4: Comparison of the blood component adhesion score among groups
Figure 1: Effect of scaling and root planing on removal of smear layer on dentin
Figure 2: Effect of scaling and root planing on blood component adhesion on dentin
Figure 3: Effect of 24% EDTA gel on removal of smear layer on dentin
Figure 4: Effect of 24% EDTA gel on blood component adhesion on dentin
Figure 5: Effect of 10mg/ml tetracycline hydrochloride on removal of smear layer on dentin
Figure 6: Effect of 10mg/ml tetracycline hydrochloride on blood component adhesion on dentin
Figure 7: Effect of Er,Cr:YSGG laser on removal of smear layer on dentin
Figure 8: Effect of Er,Cr:YSGG laser on blood component adhesion on dentin
Statistical analysis
The statistical analysis was performed using IBM SPSS version 20.0 software. Biostatistics Department, Amrita Institute of Medical Science and Research, (Kochi, Kerala). Categorical variables are expressed using frequency and percentage. Numerical variables are presented using mean and standard deviation. In the case of mean comparison between more than two groups, the analysis of variance test was used and for nonnormal variables, Kruskal–Wallis test was used. A P < 0.05 is considered to be statistically significant.
Discussion
The current study was focused on comparing the root surface conditioning techniques, the fibrin network formation, and SLR by SRP, Er, Cr: YSGG laser, EDTA, and tetracycline and thereby assessing parameters such as SLR score and BCA score. In this study, the samples were equally distributed into four groups: SRP, tetracycline, EDTA, and Er, Cr: YSGG laser at a percentage of 25% with 10 samples in each group. In the present study, there was significant difference among groups Er, Cr: YSGG laser – SRP, EDTA – SRP, and tetracycline HCl – SRP according to SLR scoring and BCA scoring, that showed effective removal of smear layer in all areas when compared to the SRP group [Tables 1 and 2 ]. The added advantage of the Er, Cr: YSGG laser is in the removal of smear layer, due to its capability to ablate mineralized tissue and produce dentinal tubules exposition. Furthermore, in this study, we were able to appreciate a dense fibrin network formation with blood cell adhesion on the root surface when compared with SRP and other treatment groups which is of immense significance in bringing about periodontal regeneration, unlike other studies, where there is SLR alone. According to a study conducted by Rocha et al . and Coldiron et al . who observed that after instrumentation there was a presence of a heavy smear layer, fewer fibrin network formation, or appearance of blood cells.[ 12 , 13 ] According to Matsuoka et al ., they stated that Er, Cr: YSGG laser utilized with water was successful in debris and SLR when compared to the control group (SRP).[ 14 ] The mechanism of the removal of smear layer by the Er, Cr: YSGG laser is based on the expansion and implosion of vapor lock and secondary cavitation effect.[ 15 ] In the current study, there was no significant difference between the EDTA, tetracycline, and Er, Cr: YSGG laser according to SLR scoring and BCA scoring when compared to the SRP group [Tables 3 and 4 ]. Although there was complete elimination of the smear layer in the samples exposed to Er, Cr: YSGG laser, the dentinal tubules were partially open. This indicates that with the application of Er, Cr: YSGG laser an additional desensitizing effect was achieved which results in reduced dentin hypersensitivity, and at the same time, exposes the underlying collagen fibers for periodontal regeneration to take place without damaging the surrounding tissues. There was also appreciable dense fibrin network and BCA onto the root surface. The EDTA group showed a complete absence of smear layer but obliterated dentinal tubules when compared to the tetracycline HCl group that showed wide open dentinal tubules and the Er, Cr: YSGG laser group that showed partially open dentinal tubules. Dense fibrin network formation and adhesion of blood cell components were seen in the EDTA group. Being a calcium chelating agent, this might be due to increased amounts of remaining calcium ions that could have had attracted EDTA molecules resulting in harder complete removal of contaminants. Tetracycline HCl showed a complete absence of smear layer and open dentinal tubules when compared to the EDTA group which showed obliterated dentinal tubules and Er, Cr: YSGG laser that showed partially open dentinal tubules. Similar blood cell component adhesion results were seen in comparison to EDTA and Er, Cr: YSGG laser. A unique property of tetracycline HCl is its high substantivity, where there is sustained release of the tetracycline from the root surface which represents antibacterial property. EDTA was found to be more conductive to gingival fibroblast attachment during in vitro studies.[ 16 ] A study by Nanda et al . stated that EDTA and tetracycline HCL were successful in eliminating the smear layer. However, EDTA was found to be a better root conditioning agent as it helped in enlarging the diameter of dentinal tubules more when compared to tetracycline HCl.[ 17 ] Er, Cr: YSGG laser conditioning was found to be effective in the stem cells attachment within periodontal ligament after periodontal therapies such as free gingival grafts and connective tissue grafts.[ 18 ] According to Montero-Miralles, studied the effect of 17% EDTA gel and Er, Cr: YSGG laser (BIOLASE) with 2.780 nm wavelength and Radial firing tips RFT-2 with 275 microns diameter in the removal of smear layer after instrumentation, they found greater cleaning capacity than EDTA.[ 19 ] According to a study by Al Habdan et al . assessed the outcome of Er, Cr: YSGG laser on the occlusion of dentinal tubules with other agents (such as desensitizing toothpaste, desensitizing varnish, and desensitizing paste) used on exposed dentin surface at varying intervals of time with the help of SEM and they observed that the Er, Cr: YSGG laser (Waterlase) caused the opening of dentinal tubules to various degrees over different periods.[ 20 ] Gholami et al . evaluated the outcome of Er, Cr: YSGG laser, Nd: YAG, and 810 nm diode lasers on the occlusion of dentinal tubules and they concluded that the lasers have the capability to melt peritubular dentin, can partially or completely cause occlusion of dentinal tubules, and therefore, reduces hypersensitivity.[ 21 ]
The rationale of root biomodification agents is to create biocompatible root surface, with the eradication of bacterial products, exposure of dentinal collagen for the attraction to the periodontal fibroblasts, and enable new attachment of connective tissue.
It can be concluded that the treatment agents used in this study can establish attachment of new connective tissue in the regeneration of surrounding tissues. In this study, Er, Cr: YSGG laser-treated samples presented with an added advantage to the removal of smear layer, formation of fibrin network, and adhesion of BCA along with desensitizing effect that can result in better reduction in dentin hypersensitivity.
Limitations
The consideration of a small sample size is the limitation of the present study. A sample size larger would have been effective with respect to the parameters investigated in this study. Further research may help rectify these limitations.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
1. Saini R, Marawar PP, Shete S, Saini S. Periodontitis, a true infection. J Glob Infect Dis 2009;1:149–50.
2. American Academy of Periodontology. Glossary of Periodontal Terms Chicago American Academy of Periodontology 2001.
3. Aukhil I. Biology of wound healing. Periodontol 2000 2000;22:44–50.
4. Wikesjö UM, Nilvéus RE, Selvig KA. Significance of early healing events on periodontal repair:A review. J Periodontol 1992;63:158–65.
5. 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.
6. Hanes P, Polson A, Frederick T. Citric acid treatment of periodontitis-affected cementum. A scanning electron microscopic study. J Clin Periodontol 1991;18:567–75.
7. Blomlöf L, Jonsson B, Blomlöf J, Lindskog S. A clinical study of root surface conditioning with an EDTA gel. II. Surgical periodontal treatment. Int J Periodontics Restorative Dent 2000;20:566–73.
8. Mariotti A. Efficacy of chemical root surface modifiers in the treatment of periodontal disease. A systematic review. Ann Periodontol 2003;8:205–26.
9. Ciancio SG. Antibiotics in periodontal therapy Newman MG, Kornman K. Antibiotics/Antibacterial use in Dental Practice Chicago Quintessence 1990.
10. Hanes PJ, O'Brien NJ, Garnick JJ. A morphological comparison of radicular dentin following root planing and treatment with citric acid or tetracycline HCl. J Clin Periodontol 1991;18:660–8.
11. Harashima T, Kinoshita J, Kimura Y, Brugnera A, Zanin F, Pecora JD, et al. Morphological comparative study on ablation of dental hard tissues at cavity preparation by Er:YAG and Er, Cr:YSGG lasers. Photomed Laser Surg 2005;23:52–5.
12. Rocha FR, Zandim-Barcelos DL, Rossa C, Sampaio JE. The smear layer created by scaling and root planing is physiologically eliminated in a biphasic process. Braz Oral Res 2015;29:1–7.
13. Coldiron NB, Yukna RA, Weir J, Caudill RF. A quantitative study of cementum removal with hand curettes. J Periodontol 1990;61:293–9.
14. Matsuoka E, Jayawardena JA, Matsumoto K. Morphological study of the Er, Cr:YSGG laser for root canal preparation in mandibular incisors with curved root canals. Photomed Laser Surg 2005;23:480–4.
15. Blanken JM, Verdaasdonk RM, Meire M. Cavitation as a working mechanism of the Er, Cr:YSGG laser in endodontics:A visualization study. J Oral Laser Appl 2007;7:97–106.
16. Babay N. The effect of EDTA on the attachment and growth of cultured human gingival fibroblasts in periodontitis-affected root surface. J Contemp Dent Pract 2001;2:13–23.
17. Nanda T, Jain S, Kaur H, Kapoor D, Nanda S, Jain R. Root conditioning in periodontology –Revisited. J Nat Sci Biol Med 2014;5:356–8.
18. Fekrazad R, Lotfi G, Harandi M, Ayremlou S, Kalhori KA. Evaluation of fibroblast attachment in root conditioning with Er, Cr:YSGG laser versus EDTA:A SEM study. Microsc Res Tech 2015;78:317–22.
19. Montero-Miralles P, Torres-Lagares D, Segura-Egea JJ, Serrera-Figallo MÁ, Gutierrez-Perez JL, Castillo-Dali G. Comparative study of debris and smear layer removal with EDTA and Er, Cr:YSGG laser. J Clin Exp Dent 2018;10:e598–602.
20. Al Habdan AH, Amal AA, Anoud M, Lamia M, Reem AS. The effectiveness of Er. Cr. YSGG laser in sustained dentinal tubules occlusion using scanning electron microscopy. J Dent Health Oral Disord Ther 2017;7:368–73.
21. Gholami GA, Fekrazad R, Esmaiel-Nejad A, Kalhori KA. An evaluation of the occluding effects of Er;Cr:YSGG, Nd:YAG, CO
2 and diode lasers on dentinal tubules:A scanning electron microscope
in vitro study. Photomed Laser Surg 2011;29:115–21.
