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ORIGINAL ARTICLE
Year : 2014  |  Volume : 8  |  Issue : 1  |  Page : 8-13

Effectiveness of Er: YAG (PIPS) and Nd: YAG activation on final irrigants for smear layer removal - SEM observation


Department of Conservative Dentistry and Endodontics, M. A Rangoonwala Dental College and Research Centre, Pune, Maharashtra, India

Date of Web Publication9-Jun-2014

Correspondence Address:
Sucheta Sathe
Department of Conservative Dentistry and Endodontics, Ma Rangoonwala College of Dental Sciences and Research Centre, Azam Campus, Camp, Pune
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0976-2868.134110

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  Abstract 

Aim and Objectives: To determine the effectiveness of laser on final irrigants and observe smear layer removal from coronal, middle, and apical third region of root canal. Materials and Methods: Thirty single-rooted premolars were selected for the study. Access opening was done followed by which cleaning and shaping was completed till F3 using rotary protaper (Dentsply). The samples were randomly divided into three groups (n = 10), Group I - Hand activation using 15 no. K file, Group II - neodymium-doped yttrium aluminum garnet (Nd: YAG)-activated, and Group III - erbium-doped yttrium aluminum garnet (Er: YAG)-activated; which were further divided into two subgroups (n = 5) depending upon the final rinse used, subgroup A - 5.25% sodium hypochlorite (NaOCl) and subgroup B - 17% ethylenediaminetetraacetic acid (EDTA). The samples were then sectioned and taken for scanning electron microscope (SEM) observation. Results: Within the limitations of the study, photon-induced photoacoustic streaming, that is, Er: YAG PIPS showed maximum smear layer removal in coronal, middle and apical third region on 17% EDTA activation.

Keywords: Diode laser, final irrigant, neodymium-doped yttrium aluminum garnet laser, photon-induced photoacoustic streaming, smear layer, scanning electron microscope


How to cite this article:
Sathe S, Hegde V, Jain PA, Ghunawat D. Effectiveness of Er: YAG (PIPS) and Nd: YAG activation on final irrigants for smear layer removal - SEM observation. J Dent Lasers 2014;8:8-13

How to cite this URL:
Sathe S, Hegde V, Jain PA, Ghunawat D. Effectiveness of Er: YAG (PIPS) and Nd: YAG activation on final irrigants for smear layer removal - SEM observation. J Dent Lasers [serial online] 2014 [cited 2017 May 26];8:8-13. Available from: http://www.jdentlasers.org/text.asp?2014/8/1/8/134110


  Introduction Top


The complete obliteration of root canal space with an inert filling material and creation of a fluid tight seal are the goals for successful endodontic therapy. [1] In order to create a fluid tight seal, it is imperative that the endodontic filling material closely adapts or bonds to the tooth structure. This however is impaired by the presence of smear layer, which invariably forms after endodontic instrumentation. [2],[3] The smear layer contains organic material, odontoblastic processes, bacteria, and blood cells.

Various materials and techniques have been reported with wide variations in their efficacy regarding removal of the intracanal smear layer. [2],[4] The most widely used chemical for the purpose is ethylenediaminetetraacetic acid (EDTA) in different formulations. [5] They have been reported to consistently produce canals with patent dentinal tubules. [6] However, it has been found to be less efficient in narrow portions of the canal, [7] requires a long application time for optimum results, [8] and can seriously damage the dentin, if used in excess. [9]

Clinically, endodontic procedures use both mechanical instrumentation and chemical irrigants in the attempt to three-dimensionally debride, clean, and decontaminate the endodontic system. [10],[11]

Some of these irrigation techniques include manual irrigation with needles and cannulas, and the use of machine-assisted agitation systems and sonic and ultrasonic energy sources. [12] Irrigation with 5.25% sodium hypochlorite (NaOCl) alone is unable to remove debris and the smear layer. [13] Other irrigants such as 2% chlorhexidine gluconate, 17% EDTA, and 10% citric acid have been used to help remove debris.

Even after doing this meticulously, still fall short of successfully removing all of the infective microorganisms and debris. This is because of the complex root canal anatomy and the inability of common irrigants to penetrate into the lateral canals and the apical ramifications. It seems, therefore, appropriate to search for new materials, techniques, and technologies that can improve the cleaning and decontamination of these anatomical areas. [14]

The effectiveness of lasers in dentistry continues to be an area of discussion. The use of lasers for nonsurgical endodontic treatment of the root canal system has been reported since the early 1970s. [15],[16] Laser treatment can be a valuable tool for the removal of the dentinal smear layer, [17],[18],[19],[20],[21] as a debridement device during endodontic treatment. The erbium-doped yttrium aluminium garnet (Er: YAG) laser (wavelength 2,940 nm) is approved by the Food and Drug Administration (FDA) in 1997 for cleaning, shaping, and enlarging the root canal. [22] Other lasers like different wavelengths of diode and neodymium-doped YAG (Nd: YAG) have also been used for the same purpose.


  Materials and Methods Top


Sample Preparation

Thirty single-rooted extracted human teeth were used in the study. The teeth with fracture, cracks, or any other defects were excluded. Subsequently, they were scaled with ultrasonics for removal of calculus or any soft tissue debris, washed with distilled water, and they were then stored in normal saline which was changed daily till required number of teeth were collected. Standard endodontic access cavity preparations were performed and then a 15# K file (Mani K File) was inserted into the canal until the tip was just visible at the apical foramen to check for the patency. Chemomechanical preparation was done till F3 using rotary protapers (Dentsply, Maillefer) for all the samples. Irrigation of all the samples during preparation was accomplished using a combination of 17% aqueous EDTA, 5 ml of 5.25% NaOCl, and saline. Samples were then divided randomly divided into three groups depending upon method of activation.

Group I :Hand activation using K file

Group II - Nd: YAG activation

Group III - Er: YAG activation using photon-induced photoacoustic streaming (PIPS) tip.

These groups were further divided into two subgroups depending upon the final irrigant used.

Subgroup A - 5.25% NaOCl

Subgroup B - 17% EDTA.

Activation of irrigant for Group I was done mechanically by agitating 15# K file in the canal when it was filled with the final irrigant solution. For Group II, Nd: YAG laser with a wavelength of 1,064 nm (Fidelis; Fotona) was used for laser irradiation, the setting used were 1.5W at very short pulse mode of 15 Hz, three cycles of 5 s. Flexible laser fiber of 200 μm was used with constant circular motion from apical to coronal direction and kept 1 mm short of apex.

An Er: YAG laser with a wavelength of 2,940 nm (Fidelis; Fotona) was used to irradiate the root canals for Group III. A newly designed 12 mm long, 400 μm quartz tip was used. The tip was tapered and had 3 mm of the polyamide sheath stripped back from its end. The laser operating parameters used for all the samples (using the free-running emission mode) were as follows: 40 mJ per pulse, 20 Hz, at very short pulse mode. The coaxial water spray feature of the hand piece was set to 'off', while air settings were kept as 2. The tip was placed into the coronal access opening of chamber only, and was kept stationary and not advanced into the orifice of the canal. During the laser irradiation cycles, the root canals were continuously irrigated with final irrigant to maintain hydration and levels using a hand syringe with a 27 gauge needle positioned above the laser tip in the coronal aspect of the access opening, accordingly to the above protocol.

After preparation, root canal walls were dried using paper points. Longitudinal grooves were made on the distal and mesial root surfaces, preserving the inner shelf of the dentin surrounding the canal. Roots were then sectioned with the help of chisel and mallet. Samples were then subjected to scanning electron microscope (SEM) for smear layer removal.


  Results Top


Control group specimens (Group I) consistently exhibited a thick smear layer with NaOCl (subgroup A) [Figure 1], while comparatively less smear layer was observed with EDTA (subgroup B) [Figure 2]. SEM examination demonstrated that when NaOCl irrigation was applied, a noticeable smear layer and occluded dentinal tubules remained on the treated surface. Debris, defined as dentin chips and pulp remnants loosely attached to the internal surface of the root canals, was present in specimens' subgroup A (Group I). On the other hand, in specimens of EDTA in most of the specimens open dentinal tubules were observed in the coronal and the middle third, while in the apical third of all specimens occluded tubules were observed.
Figure 1: NaOCl (hand activation)

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Figure 2: EDTA (hand activation)

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Group II specimens treated with the Nd: YAG laser showed improved cleaning compared to Group I specimens for both the subgroups. The root canal surfaces exhibited open tubules, scattered residual debris, and a thinner smear layer in subgroup B [Figure 3] compared to the subgroup A [Figure 4] specimens. While within subgroup B of Groups I and II comparatively more dentinal tubules were open and less smear layer was observed in Group II.

Group III specimens treated with the Er: YAG laser showed the most effective removal of the smear layer from the root canal walls compared to Group II specimens and Group I (control) specimens. SEM images at higher magnifications (from ×1000 to ×2000) showed exposed and intact collagen fibers and open dentinal tubules even in the apical third of subgroup B [Figure 5], while open dentinal tubules along with scattered dentinal chips were observed in subgroup A [Figure 6]. None of the SEM micrographs indicated signs of dentin melting from excessive heat.
Figure 3: EDTA (Nd: YAG)

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Figure 4: NaOCl (Nd:YAG)

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Figure 5: EDTA (Er: YAG)

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Figure 6: NaOCl (Er:YAG)

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  Discussion Top


Current instrumentation techniques using rotary instruments and chemical irrigation still fall short of successfully removing the smear layer from inside the root canal system. This was confirmed by the results seen in the control group (Group I) where the conventional technique was employed.

To overcome this, different laser activation was carried out. The concept of laser-activated irrigation is based on cavitation. Because of the high absorption of water by the mid-infrared wavelength of lasers, the cavitation process generates vapor-containing bubbles, which explode and implode in a liquid environment. [23] This subsequently initiates pressure waves/shock waves by inducing the shear force on dentinal wall. In a water-filled root canal, the shock waves could potentially detach the smear layer and disrupt bacterial biofilms. [23],[24] To efficiently activate irrigant and generate shock waves in the root canal, lasers with wavelength from 940 to 2,940 nm have been used. [24],[25],[26],[27],[28],[29],[30],[31]

To remove both organic and inorganic debris of the smear layer, the alternate use of NaOCl and EDTA had been recommended. [32] However, alternating NaOCl with EDTA decreases the antibacterial and tissue-dissolving activity of NaOCl per se. [33] Furthermore, prolonged use of EDTA can cause dentinal erosion of the root canal by decalcifying the peritubular dentin. [8] This could be accelerated with following use of NaOCl by dissolving the already exposed organic matrix. [21] Therefore, instead of alternating regimen, the sole effect of laser-activated irrigation on smear layer removal in the presence of either NaOCl or EDTA was a matter of interest in this study.

The results of this study indicate that NaOCl subgroups did not remove smear layer from the middle and apical third of the canal wall. This is in agreement with studies done by earlier researchers. [22],[25] EDTA is efficient in removing the smear layer which is evident in this study for all three groups. Effects of EDTA were limited to coronal and middle third in Groups I and II, while it was effective even in apical third for Group III (Er: YAG-PIPS). Ciucchi et al., stated that, there was definite decline in the efficiency of irrigating solutions along the apical part of the canals. [34] This can probably be explained to the fact that dentin in apical third is much more sclerosed and the number of dentinal tubules present there is less. [35] A 1,064-nm Nd: YAG laser-activated irrigation with either NaOCl or EDTA was much better than using NaOCl irrigation alone and as effective as EDTA final flush for smear layer removal.

The Er: YAG laser used in this investigation was equipped with a novel 400 μm diameter radial and stripped tip. Using subablative parameters (average power 0.8 W, 40 mJ at 20 Hz) proved to be more effective than other two techniques at removing the smear layer. This finding could be attributed to the photomechanical effect seen when light energy is pulsed in liquid. [36],[37],[38] When activated in a limited volume of fluid, the high absorption of the Er: YAG wavelength in water, combined with the high peak power derived from the short pulse duration that was used for 5 s (three cycles), resulted in a photomechanical phenomenon. A profound "shockwave-like" effect is observed when radial and stripped tips are submerged in a liquid-filled root canal. As a result of the very small volume, this effect may remove the smear layer and residual tissue tags and potentially decrease the bacterial load within the tubules and lateral canals. [39],[40],[41] By using lower subablative energy (40 mJ) and restricting the placement of the tip to within the coronal portion of the tooth only, the undesired effects of the thermal energy, previously described in the literature, was avoided. [42],[43],[44] In the current study the smear layer and debris were not removed by thermal vaporization, but probably by photomechanical streaming of the liquids, which were laser activated in the coronal part of the tooth. The authors describe this light energy phenomenon as PIPS. The effect of irradiation with the Er: YAG laser equipped with a tip of novel design at subablative power settings (0.8 W, 40 mJ) is synergistically enhanced by the presence of EDTA; this leads to significantly better debridement of the root canal contributing to an improvement in treatment efficacy.

With conventional treatment protocols (without a laser), an irrigation syringe is more effective when the tip is placed closer to the working length. With this new laser system, the laser tip is not placed within the canal itself, but is rather confined to the coronal chamber above the orifice. It is suggested that this allows easy access for the photomechanical effects to occur within the root canal, which may assist in cleaning canals of various shapes. A standard ISO size #30 file preparation is needed to allow traditional laser tips (200-320 μm) to reach close to the apex. [39],[40],[41] Using the radial and stripped design with PIPS, the apex can be reached without the need to negotiate the tip close to the apex. Irrigation with chelating agents following the current conventional instrumentation procedure requires more time to initiate a satisfactory debridement (EDTA placed passively into the prepared root canal). [42],[43] The PIPS technique resulted in pronounced smear layer removal when used together with EDTA and at the settings outlined.


  Conclusion Top


The Er: YAG laser used in this study showed significantly better smear layer removal than traditional syringe irrigation. At the energy levels and with the operating parameters used, no thermal effects or damage to the dentin surface was observed. In this study, the Er: YAG laser with the current settings produced a photomechanical effect demonstrating its potential as an improved alternative method for debriding the root canal system in a minimally invasive manner.

 
  References Top

1.Ingle JI, Beveridge EE. Endodontics, 2 nd ed. Philadelphia: Lea and Biger; 1976. p. 216.  Back to cited text no. 1
    
2.Baumgartner JC, Brown CM, Mader CL, Peters DD, Shulman JD. A scanning electron microscopic evaluationof root canal debridement using saline, NaOCl and citric acid. J Endod 1984;10:525-31.  Back to cited text no. 2
[PUBMED]    
3.McComb D, Smith DC. A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endod 1975;1:238-42.  Back to cited text no. 3
[PUBMED]    
4.Cunningham WT, Martin H. A scanning electron microscope evaluation of root canal debridement with the endosonic ultrasonic synergistic system. Oral Surg Oral Med Oral Pathol 1982;53:527-31.  Back to cited text no. 4
[PUBMED]    
5.O′Connell MS, Morgan LA, Beeler WJ, Baumgartner JC. A comparative study of smear layer removal using different salts of EDTA. J Endod 2000;26:739-43.  Back to cited text no. 5
    
6.Goldman LB, Goldman M, Kronman JH, Lin PS. The efficacy of several irrigating solutions for endodontics: A SEM study. Oral Surg Oral Med Oral Pathol 1981;52:197-204.  Back to cited text no. 6
[PUBMED]    
7.Patterson SS. In vivo and in vitro studies of the effects of disodium salt of ethylene diamine tetra acetate on human dentin and its endodontic implications. Oral Surg Oral Med Oral Pathol 1963;16:83-103.  Back to cited text no. 7
[PUBMED]    
8.Goldberg F, Spielberg C. The effects of EDTAC and the variation of its working time analyzed with scanning electron microscopy. Oral Surg Oral Med Oral Pathol 1982;53:74-7.  Back to cited text no. 8
[PUBMED]    
9.Calt S, Serper A. Time dependent effects of EDTA on dentin structures. J Endod 2002;28:17-9.  Back to cited text no. 9
    
10.Peters OA. Current challenges and concepts in the preparation of root canal systems: A review. J Endod 2004;30:559-67.  Back to cited text no. 10
[PUBMED]    
11.Hülsmann M, Peters OA, Dummer PM. Mechanical preparation of root canals: Shaping goals, techniques and means. Endod Top 2005;10:30-76.  Back to cited text no. 11
    
12.Gu LS, Kim JR, Ling J, Choi KK, Pashley DH, Tay FR. Review of contemporary irrigant agitation techniques and devices. J Endod 2009;35:791-804.  Back to cited text no. 12
    
13.Baumgartner JC, Cuenin PR. Efficacy of several concentrations of sodium hypochlorite for root canal irrigation. J Endod 1992;18:605-12.  Back to cited text no. 13
    
14.Olivi G, DiVito E. REVIEW Photoacoustic Endodontics using PIPS™: Experimental background and clinical protocol. J Laser Health Acad 2012:22-5.  Back to cited text no. 14
    
15.Weichman JA, Johnson FM, Nitta LK. Laser use in endodontics. II. Oral Surg Oral Med Oral Pathol 1972;34:828-30.  Back to cited text no. 15
[PUBMED]    
16.Weichman JA, Johnson FM. Laser use in endodontics. A preliminary investigation. Oral Surg Oral Med Oral Pathol 1971;31:416-20.  Back to cited text no. 16
[PUBMED]    
17.Sulewski JG. Making the most of the 16 th Annual Conference and Exhibition: A practical orientation for attendees. Academy of Laser Dentistry 16 th Annual Conference and Exhibition, 22-26 April 2009, Las Vegas, Nevada.  Back to cited text no. 17
    
18.Tomita Y, Shima A. Mechanisms of impulsive pressure generation and damage pit formation by bubble collapse. J Fluid Mech 1986;169:535-64.  Back to cited text no. 18
    
19.Hmud R, Kahler WA, George R, Walsh LJ. Cavitational effects in aqueous endodontic irrigants generated by near-infrared lasers. J Endod 2010;36:275-8.  Back to cited text no. 19
    
20.Blanken J, Verdaasdonk R. Cavitation as a working mechanism of the Er, Cr: YSGG laser in endodontics: A visualization study. J Oral Laser Appl 2007;7:97-106.  Back to cited text no. 20
    
21.Blanken J, De Moor RJ, Meire M, Verdaasdonk R. Laser induced explosive vapor and cavitation resulting in effective irrigation of the root canal: Part 1 - a visualization study. Lasers Surg Med 2009;41:514-9.  Back to cited text no. 21
    
22.De Moor RJ, Blanken J, Meire M, Verdaasdonk R. Laser induced explosive vapor and cavitation resulting in effective irrigation of the root canal: Part 2 - evaluation of the efficacy. Lasers Surg Med 2009;41:520-3.  Back to cited text no. 22
    
23.George R, Meyers IA, Walsh LJ. Laser activation of endodontic irrigants with improved conical laser fiber tips for removing smear layer in the apical third of the root canal. J Endod 2008;34:1524-7.  Back to cited text no. 23
    
24.De Moor RJ, Meire M, Goharkhay K, Moritz A, Vanobbergen J. Efficacy of ultrasonic versus laser-activated irrigation to remove artificially placed dentin debris plugs. J Endod 2010;36:1580-3.  Back to cited text no. 24
    
25.de Groot SD, Verhaagen B, Versluis M, Wu MK, Wesselink PR, van der Sluis LW. Laser-activated irrigation within root canals: Cleaning efficacy and flow visualization. Int Endod J 2009;42:1077-83.  Back to cited text no. 25
    
26.Levy G, Rizoiu I, Friedman S, Lam H. Pressure waves in root canals induced by Nd: YAG laser. J Endod 1996;22:81-4.  Back to cited text no. 26
    
27.Levy G. Cleaning and shaping the root canal with a Nd: YAG laser beam: A comparative study. J Endod 1992;18:123-7.  Back to cited text no. 27
[PUBMED]    
28.Yamada RS, Armas A, Goldman M, Lin PS. A scanning electron microscopic comparison of a high volume final flush with several irrigating solutions: Part 3. J Endod 1983;9:137-42.  Back to cited text no. 28
[PUBMED]    
29.Grawehr M, Sener B, Waltimo T, Zehnder M. Interaction of ethylene diamine tetra acetic acid with sodium hypochlorite in aqueous solutions. Int Endod J 2003;36:411-7.  Back to cited text no. 29
    
30.Calt S, Serper A. Smear layer removal by EGTA. J Endod 2000;26:459-61.  Back to cited text no. 30
    
31.Niu W, Yoshioka T, Kobayashi C, Suda H. A scanning electron microscopic study of dentinal erosion by final irrigation with EDTA and NaOCl solutions. Int Endod J 2002;35:934-9.  Back to cited text no. 31
    
32.Mader CL, Baumgartner JC, Peters DD. Scanning electron microscopic investigation of the smear layer on root canal walls. J Endod 1984;10:477-83.  Back to cited text no. 32
[PUBMED]    
33.Ciucchi B, Khettabi M, Holz J. The effectiveness of different endodontic irrigation procedures on the removal of the smear layer: A SEM study. Int Endod J 1989;22:21-8.  Back to cited text no. 33
[PUBMED]    
34.Schoop U, Goharkhay K, Klimscha J, Zagler M, Wernisch J, Georgopoulos A, et al. The use of the erbium, chromium: yttrium-scandium-gallium-garnet laser in endodontic treatment: The results of an in vitro study. J Am Dent Assoc 2007;138:949-55.  Back to cited text no. 34
    
35.Schoop U, Barylyak A, Goharkhay K, Beer F, Wernisch J, Georgopoulos A, et al. The impact of an erbium, chromium: yttrium-scandium-gallium-garnet laser with radial-firing tips on endodontic treatment. Lasers Med Sci 2009;24:59-65.  Back to cited text no. 35
    
36.Gordon W, Atabakhsh VA, Meza F, Doms A, Nissan R, Rizoiu I, et al. The antimicrobial efficacy of the erbium, chromium: yttrium-scandium-gallium-garnet laser with radial emitting tips on root canal dentin walls infected with Enterococcus faecalis. J Am Dent Assoc 2007;138:992-1002.  Back to cited text no. 36
    
37.Matsuoka E, Kimura Y, Matsumoto K. Studies on the removal of debris near the apical seats by Er: YAG laser and assessment with a fiberscope. J Clin Laser Med Surg 1998;16:255-61.  Back to cited text no. 37
    
38.Takeda FH, Harashima T, Eto JN, Kimura Y, Matsumoto K. Effect of Er: YAG laser treatment on the root canal walls of human teeth: An SEM study. Endod Dent Traumatol 1998;14:270-3.  Back to cited text no. 38
    
39.Takeda FH, Harashima T, Kimura Y, Matsumoto K. Efficacy of Er: YAG laser irradiation in removing debris and smear layer on root canal walls. J Endod 1998;24:548-51.  Back to cited text no. 39
    
40.Takeda FH, Harashima T, Kimura Y, Matsumoto K. A comparative study of the removal of smear layer by three endodontic irrigants and two types of laser. Int Endod J 1999;32:32-9.  Back to cited text no. 40
    
41.Pecora JD, Brugnera-Junior A, Cussioli AL, Zanin F, Silva R. Evaluation of dentin root canal permeability after instrumentation and Er: YAG laser application. Lasers Surg Med 2000;26:277-81.  Back to cited text no. 41
    
42.Khedmat S, Shokouhinejad N. Comparison of the efficacy of three chelating agents in smear layer removal. J Endod 2008;34:599-602.  Back to cited text no. 42
    
43.Hulsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment: Mode of action and indications for their use. Int Endod J 2003;36:810-30.  Back to cited text no. 43
    
44.Pecora JD, Brugnera-Junior A, Cussioli AL, Zanin F, Silva R. Evaluation of dentin root canal permeability after instrumentation and Er: YAG laser application. Lasers Surg Med 2000;26:277-81.  Back to cited text no. 44
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]


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