Effect of the Nd:YAG laser on sealer penetration into root canal surfaces: a confocal microscope analysis

Accepted Manuscript Title: Effect of the Nd:YAG laser on sealer penetration into root canal surfaces: a confocal microscope analysis Author: Paloma Montero-Miralles Raquel Castillo-Oyag¨ ue Isabel S´ aez de la Fuente Christopher D. Lynch Gabriel Castillo-Dal´ ı Daniel Torres-Lagares PII: S0300-5712(14)00098-0 DOI: http://dx.doi.org/doi:10.1016/j.jdent.2014.03.017 Reference: JJOD 2273 To appear in: Journal of Dentistry Received date: 11-2-2014 Revised date: 19-3-2014 Accepted date: 30-3-2014 Please cite this article as: Montero-Miralles P, Castillo-Oyag¨ ue R, de la Fuente IS, Lynch CD, Castillo-Dal´ ı G, Torres-Lagares D, Effect of the Nd:YAG laser on sealer penetration into root canal surfaces: a confocal microscope analysis, Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.03.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 22 Accepted Manuscript 1 Title: ‘Effect of the Nd:YAG laser on sealer penetration into root canal surfaces: a confocal microscope analysis’. Short title: Effect of the Nd:YAG laser on sealer penetration. Authors: Paloma Montero-Miralles, DDS;1 Raquel Castillo-Oyagüe, DDS, PhD;2 Isabel Sáez de la Fuente, DDS;1 Christopher D. Lynch, BDS, PhD, MFD, FDS (Rest Dent), FACD, FHEA;3Gabriel Castillo-Dalí, Dr. Biol, PhD;1 Daniel Torres-Lagares DDS, PhD.1 1Faculty of Dentistry, University of Seville (US), C/ Avicena, s/n, 41009, Sevilla, Spain. 2Faculty of Dentistry, Complutense University of Madrid (UCM), Pza. Ramón y Cajal, s/n, 28040, Madrid, Spain. 3School of Dentistry, Cardiff University, CF14 4XY, Cardiff, Wales, U.K. Corresponding author: Raquel Castillo de Oyagüe. Department of Buccofacial Prostheses, Faculty of Dentistry, Complutense University of Madrid (UCM), Pza. Ramón y Cajal, s/n, E-28040, Madrid, Spain. Phone: 0034-607367903. Fax: 0034-913942029. E-mail: [email protected] Key words: Confocal microscope; Nd:YAG laser; endodontic instrumentation; dentinal permeability; endodontic sealer; sealer penetration. Page 2 of 22 Accepted Manuscript 2 ‘Effect of the Nd:YAG laser on sealer penetration into root canal surfaces: a confocal microscope analysis’. Abstract Objectives: The objective of this in vitro study was to evaluate the use of the Neodymium:Yttrium-Aluminium-Garnet (Nd:YAG) laser as part of the root canal treatment on the penetration of sealer into dentinal tubules. Methods: Eighty extracted lower premolars were randomly assigned to two groups (n = 40 each): Control group (CG), subjected to a conventional protocol of endodontic instrumentation and obturation; and Laser group (LG), in which Nd:YAG laser irradiations were combined with conventional preparation and obturation. Endodonted samples were sectioned at 3 and 5 mm from the apex and observed under a confocal scanning microscope (CLSM). The penetration depth into the dentinal tubules and the extension of the intracanal perimeter infiltrated by sealer were measured. The Student-Newman- Keuls test was run for between-group comparisons (α=.05). Results: The depth of sealer penetration into dentinal tubules did not differ among groups. LG samples showed the significantly highest percentage of penetrated perimeter at 3 mm from the root apex. Within each group, the greatest depth of penetration (P=.0001), and the major percentage of penetrated perimeter (P<.001), were recorded at 5 mm. Conclusions: The application of the Nd:YAG laser after instrumentation did not improve the depth of sealer penetration into the dentinal tubules. The laser enlarged the total penetrable perimeter near the apex. Clinical significance: The Nd:YAG laser may be an appropriate complement in root canal treatment, as it enhances the sealer adaptation to the dentinal walls in the proximity of the apex. Page 3 of 22 Accepted Manuscript 3 Introduction The smear layer (i.e., a biofilm of detritus that covers the walls of prepared and unprepared root canals) obstructs the dentinal tubules, reduces the infiltration of irrigant solutions, and interferes with the adhesion of sealer agents.1-5 For ensuring a suitable endodontic obturation, the smear layer, soft-tissue debris, inflammatory irritants, and microorganisms must be totally eradicated.6-9 Although these resist mechanical instrumentation and irrigation, they seem to be more amenable to disruption by pressure waves generated by pulsed lasers.10 Moreover, the filling materials must reach a high level of adaptability to the cleaned root canal walls, which may be achieved thanks to the penetration of sealers into the dentinal tubules.11-13 Conventional protocols of chemomechanical root canal preparation (even in conjunction with chelants, i.e., EDTA), do not completely eliminate the inorganic material at the tubules’ entrance.14 However, laser technology has widened the spectrum of endodontic indications in the last few years by achieving an effective intracanal ablation of the smear layer7,15,16 and melting of dentinal surfaces, so that the underlying tubules would be more impermeable.15,17-20 The Neodymium:Yttrium-Aluminium-Garnet (Nd:YAG) laser system has been the most analyzed because of its important effect on dentinal permeability.8,12,20-26 Goodis et al20 stated that Nd:YAG laser irradiations in combination with manual instrumentation of the root canals could eliminate the smear layer leaving no remaining tissues on them. Dederich et al27 confirmed that the dentinal walls could recrystallize in a glazed non-porous surface devoid of organic tissues with this type of laser, which was supported by Michiels et al.28 Accordingly, several authors12,15,17,21,25 attributed to this laser both an improved cleanliness and a reduced dentinal permeability of the intracanal surfaces caused by dentine fusion that contributed to close and seal the dentinal tubules. Nonetheless, these results may depend upon the different factors that define the energy level, such as power of irradiation, duration of exposure, and color of the dentine.8,12,18,22,27,29 Compared with the scanning electron microscope and other methods used in these studies,8,12,18,21,22,27,29 the analysis with confocal scanning microscope (CLSM) has the advantage of providing detailed information about the Page 4 of 22 Accepted Manuscript 4 presence and distribution of sealers or dental adhesives inside dentinal tubules in the total circumference of the root canal walls at low magnifications (50×-100×) through the use of fluorescent rhodamine-marked sealers.30 Although this technology would seem to be ideal to determine the degree of penetration and adaptation of the filling materials, clinical achievements with a wide variety of sealers, canal preparation, and obturation techniques have not been forthcoming.13 Actually, this is the first investigation in which the effect of the Nd:YAG laser on sealer penetration has been evaluated at different root levels from the foraminal apex using confocal scanning microscopy and related specific software. The aim of this in vitro study was to evaluate the effect of Nd:YAG laser irradiations on the penetration of sealer into dentinal tubules under confocal microscopy. The null hypothesis tested was that neither the use of the Nd:YAG laser after endodontic instrumentation, nor the root level assessed from the apical foramen influenced the depth of sealer penetration into the dentinal tubules and the extension of root canal surfaces infiltrated by sealer. Materials and Methods Preparation of samples Eighty single-rooted human lower premolars with a closed apex that had been extracted because of orthodontic reasons were used in the study. The exclusion criteria were: presence of more than one canal, caries, calculus, open apex, root resorption, and/or root fractures. The teeth were conserved in an injection saline solution of 900 mg NaCl/ 100 ml water (Fresenius Kabi, Barcelona, Spain), and randomly assigned to two groups: Control group (CG) (n = 40), which followed a conventional endodontic instrumentation and obturation protocol; and Laser group (LG) (n = 40), in which Nd:YAG laser irradiations were applied after conventional instrumentation, prior to obturation. The pulp chamber was opened with 330 tungsten-carbide and EndoZ burs (Dentsply- Maillefer, Tulsa, USA) under water cooling. The coronal flaring was prepared using Gates Glidden drills No. #1, #2, and #3 (Dentsply-Maillefer). A K10 file (Dentsply-Maillefer) was Page 5 of 22 Accepted Manuscript 5 introduced through the access cavity to determine the working length, which was established 1 mm short of the apical foramen. The manual glide path was performed with flexofile K15-20 files (Dentsply-Maillefer) and the instrumentation was completed with a rotatory nickeltitanium MTwo system (VDW, Munich, Germany) using 10.04, 15.05, 20.06, 25.06, 30.05, 35.04, and 40.04 files. During the procedure, 15% EDTA gel (Dentaflux, Madrid, Spain) and irrigations with 5 ml of 5.25% sodium hypochlorite (NaOCl) were applied. Root canals were dried by introducing appropriately sized (Ø 40) absorbent paper points (Dentsply-Maillefer) to the working length. The classic 17% EDTA solution was not used for final flush in order to preserve the smear layer.31 An Nd:YAG laser (Deka, Florence, Italy) with a 1064 nm wavelength and a 300 µm optic fiber with a rubber stopper was selected for the study.32,33 The laser parameters programmed were: 1.5 W, 15 Hz and 100 mJ. The laser power emitted at the fiber tip was measured by a wattmeter (Field Master, Coherent Inc., Auburn, CA, USA) before each irradiation to ensure stable and standardized power outputs.34 After the canal was dried, the laser tip was introduced 1 mm short of the working length and then moved in circles inside the canal making contact with the entire walls. Five 5-s cycles with a 20-s break between them were performed. Root canals were filled inserting a size 40, 0.2 tapered gutta-percha master cone (Dentsply-Maillefer) impregnated with resin-based root canal sealer (AH-Plus Jet, Dentsply- Maillefer) mixed with 1% rhodamine B isothiocyanate (Sigma-Aldrich, St. Louis, MO, USA). Lateral condensation was performed using a size A finger spreader (Dentsply-Maillefer) with two size 25, 0.2 tapered gutta-percha points (Dentsply-Maillefer), and size C accessory points (Dentsply-Maillefer). Excess gutta-percha was removed from the coronal cavity up to the level of the cementoenamel junction using a hot instrument (A.S.A. Dental, Massarosa, Italy). Warm vertical compaction of the remaining coronal gutta-percha was performed using a prefitted hand plugger (Machtou, Dentsply-Maillefer). Page 6 of 22 Accepted Manuscript 6 The study was conducted following the ethical principles of medical investigation under the Helsinki Declaration of the World Medical Association (http://www.wma.net), and the Spanish Law 14/2007 of July 3rd for Biomedical Research (http://www.boe.es). The Ethics Committee Approval (Court of Ethics at the University of Seville, US, Spain) was obtained. Confocal microscope examination The root-treated teeth were prepared for confocal microscope analysis. Premolars were cut perpendicularly to the occlusal/apical axis both at the 3 and 5 mm levels from the apex using a 102 × 0.3 mm diamond blade mounted in an IsoMet Low Speed Saw precision cutter (Buehler, Lake Bluff, IL) with running water. The same root levels have been evaluated in related studies on sealer penetration.13,35 Samples were polished with an 8” sanding disk for 5 s to reduce cut irregularities and provide smooth surfaces, allowing for better visibility during confocal microscopic imaging. The specimens were prepared for microscope analysis based on the protocol proposed by Janda36 for natural teeth at room temperature (RT: 23.0±1.0 ºC). This method requires an effective dehydration and drying of the samples for minimizing the appearance of artefacts that may result from preparation. The specimens were embedded in 70%, 80%, 96%, and 100% ethyl alcohol for 24 h each, submerged in 3 consecutive baths of 100% acetone for 24 h, and dried in a vacuum desiccator (vacuum achieved by a glass filter pump)36 at 60 °C for 2 h. Dehydrated samples were examined on an inverted Leica TCS-SPE confocal microscope (Leica, Mannheim, Germany) along the Z-axis with a 60-µm range. The parameters analyzed were: depth of sealer penetration into dentinal tubules (in microns), and penetrated perimeter (both in millimeters and percentages) at 3 and 5 mm from the foraminal apex. The respective absorption and emission wavelengths for rhodamine B were 540 nm and 590 nm. The specimens were first observed using the HC PL Fluotar 10×/0.30 lens (Leica). Visualized layers were selected 10 µm below the sampled surface.13,37 For the analysis of the penetration depth and superficial extension of sealer at each root level (i.e, 3 and 5 mm from the apex), 40 images equally distributed on 5 allocations along the intracanal perimeter of every Page 7 of 22 Accepted Manuscript 7 sample were captured with the HCX PL APO 40×/1.25-0.75 oil lens (Leica), and acquired with the IM50 Image Manager Software, v1.20 (Leica Microsystems, Buffalo Grove, IL, US) (Fig. 1). These 40× pictures were taken by using 40 sections of 1 µm step size each in a format of 1,024 × 1,024 pixels along the Z-axis. Among such images (numbered from 1 to 40), 5 were analyzed per specimen and section from the apex (No.: 1, 8, 16, 32, and 40). On each selected micrograph, the depth of sealer penetration was measured at 0° (N), 45° (NO), 90° (O), 135° (SO), 180° (S), 225° (SE), 270° (E), and 315° (NE). When penetration was observed, the length of the sealer tag from the canal wall along the entire tubule was recorded in microns. The canal wall served as the starting point, and sealer penetration into dentinal tubules (sealer tags) was calculated to a maximum depth of 1,000 µm with the ruler tool of the Leica Application Suite Advanced Fluorescence Lite software (Leica Microsystems) (Fig. 1). The intracanal perimeter of each sample was measured at 3 and at 5 mm from the root apex using the Leica software (Leyca Microsystems). The extension of the intracanal perimeter infiltrated by sealer was registered using the described method and measuring points as for the penetration depth at each root level. Percentages of sealer penetration were also calculated. For statistical analysis, the abovementioned measured points of each variable were averaged per specimen and canal section from the apex. Statistical analysis Descriptive statistics were obtained for each variable. The depth of sealer penetration into dentinal tubules and the penetrated perimeter were reported for each group by both a measure of centrality (mean) and a measure of variability (standard deviation: SD).19,38 The Kolmogorov-Smirnov test confirmed that the data was normally distributed. The Student-Newman-Keuls test was run for between-group comparisons.39,40 Data were processed using the Statistical Package for the Social Sciences (software v.20) (SPSS/PC+, Inc.; Chicago, IL, USA) taking the cut-off level for statistical significance at Page 8 of 22 Accepted Manuscript 8 α = 0.05.5,31,41 The statistical probes utilized in this study adhere to the requirements of Hannigan and Lynch for oral and dental research.38 Results No significant differences were found among CG and LG samples concerning the depth reached by the sealer into the dentinal tubules at 3 and 5 mm from the root apex (Table 1). The extension of the intracanal perimeter penetrated by the sealer (in mm) was significantly higher in LG than in CG specimens regardless of the root level assessed (Table 1). However, given that CG and LG showed significant differences in the total perimeter at 5 mm from the apex (P =.0001), the ‘percentage of penetrated perimeter’ may be a more accurate measurement than the ‘penetrated perimeter in millimetres’. In this regard, while LG showed higher percentages of penetrated perimeter than CG at both root-levels evaluated, significant differences were only recorded at 3 mm from the apex (P = .034) (Table 1). When comparing the sections made at 3 and 5 mm from the apex within each study group (CG and LG), the depth of penetration (P =.0001), the penetrated perimeter (in mm) (P =.0001), and the percentage of penetrated perimeter (P <.001) were significantly greater at 5 mm. Significant differences in the total perimeter were found between both root levels assessed within each study group (P =.0001) (Table 2). Therefore, the percentage of penetrated perimeter may be more representative than the penetrated perimeter in millimetres for these comparisons. Discussion The smear layer acts as a physical barrier that prevents the adhesion and reduces the sealer penetration into dentinal tubules, thus affecting the efficacy of sealers.1-3,5 Its removal may improve the sealer penetration, enhancing the interface between filling material and root canal walls for achieving a proper obturation.6,9 It has been shown that hand preparation with irrigation does not result in complete cleaning of the root canal, mainly when fins and other anatomical irregularities are present, as it usually occurs in the apical third of the root.8 Although the use of lasers in Dentistry have been studied for a number of years,25,39,42 Page 15 of 22 Accepted Manuscript 15 28. Michiels R, Vergauwen TE, Mavridou A, Meire M, De Bruyne M, De Moor RJ. Investigation of coronal leakage of root fillings after smear-layer removal with EDTA or Nd:YAG lasing through capillary-flow porometry. Photomedicine and Laser Surgery 2010; 28: S43-50. 29. Dederich DN, Zakariasen KL, Tulip J. Scanning electron microscopic analysis of canal wall dentin following neodymium-yttrium-aluminum-garnet laser irradiation. Journal of Endodontics 1984; 10: 428-31. 30. D'Alpino PH, Pereira JC, Svizero NR, Rueggeberg FA, Pashley DH. Use of fluorescent compounds in assessing bonded resin-based restorations: a literature review. Journal of Dentistry 2006; 34: 623-34. 31. Hülsmann M, Rümmelin C, Schäfers F. Root canal cleanliness after preparation with different endodontic handpieces and hand instruments: a comparative SEM investigation. Journal of Endodontics 1997; 23: 301-6. 32. Gutknecht N, Behrens VG. Instrumentation of root canal walls with Nd-YAG laser. ZWR 1991; 100: 748-50, 752, 755. 33. Moura-Netto C, Guglielmi Cde A, Mello-Moura AC, Palo RM, Raggio DP, Caldeira CL. Nd:YAG laser irradiation effect on apical intracanal dentin - a microleakage and SEM evaluation. Brazilian Dental Journal 2011; 22: 377-81. 34. 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 in Medical Science 2009; 24: 59-65. 35. Weis MV, Parashos P, Messer HH. Effect of obturation technique on sealer cement thickness and dentinal tubule penetration. International Endodontic Journal 2004; 37: 653-63. 36. Janda R. Preparation of extracted natural human teeth for SEM investigations. Biomaterials 1995; 16: 209-17. 37. Bitter K, Paris S, Martus P, Schartner R, Kielbassa AM. A Confocal Laser Scanning Microscope investigation of different dental adhesives bonded to root canal dentine. Page 16 of 22 Accepted Manuscript 16 International Endodontic Journal 2004; 37: 840-8. 38. Hannigan A, Lynch CD. Statistical methodology in oral and dental research: pitfalls and recommendations. Journal of Dentistry 2013; 41: 385-92. 39. Ariyaratnam MT, Wilson MA, Mackie IC, Blinkhorn AS. A comparison of surface roughness and composite/enamel bond strength of human enamel following the application of the Nd:YAG laser and etching with phosphoric acid. Dental Materials 1997; 13: 51-5. 40. Oyagüe RC, Osorio R, da Silveira BL, Toledano M. Comparison of bond stability between dual-cure resin cements and pretreated glass-infiltrated alumina ceramics. Photomedicine and Laser Surgery 2011; 29: 465-75. 41. Moradi S, Ghoddusi J, Forghani M. Evaluation of dentinal tubule penetration after the use of dentin bonding agent as a root canal sealer. Journal of Endodontics 2009; 35: 1563-6. 42. Castillo-Oyagüe R, Lynch CD, Turrión AS, López-Lozano JF, Torres-Lagares D, Suárez- García MJ. Misfit and microleakage of implant-supported crown copings obtained by laser sintering and casting techniques, luted with glass-ionomer, resin cements and acrylic/urethane-based agents. Journal of Dentistry 2013; 41: 90-6. 43. 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. Journal of Endodontics 2010; 36: 1580-3. 44. Hall MC, Clement DJ, Dove SB, Walker WA 3rd. A comparison of sealer placement techniques in curved canals. Journal of Endodontics 1996; 22: 638-42. 45. Eriksson AR, Albrektsson T. Temperature threshold levels for heat-induced bone tissue injury: a vital microscopic study in the rabbit. Journal of Prosthetic Dentistry 1983; 50: 101- 7. 46. Bahcall J, Howard P, Miserendino L, Walia H. Preliminary investigation of the histological effects of laser endodontic treatment on the periradicular tissues in dogs. Journal of Endodontics 1992; 18: 47-51. Page 17 of 22 Accepted Manuscript 17 47. De Groot SD, Verhaagen B, Versluis M, Wu MK, Wesselink PR, van der Sluis LW. Laseractivated irrigation within root canals: cleaning efficacy and flow visualization. International Endodontic Journal 2009; 42: 1077-83. 48. Todea C, Balabuc C, Sinescu C, Filip L, Kerezsi C, Calniceanu M, et al. Enface optical coherence tomography investigation of apical microleakage after laser-assisted endodontic treatment. Lasers in Medical Science 2010; 25: 629-39. 49. Park DS, Lee HJ, Yoo HM, Oh TS. Effect of Nd:YAG laser irradiation on the apical leakage of obturated root canals: an electrochemical study. International Endodontic Journal 2001; 34: 318-21. 50. Gutknecht N, Kaiser F, Hassan A, Lampert F. Long-term clinical evaluation of endodontically treated teeth by Nd:YAG lasers. Journal of Clinical Laser Medicine and Surgery 1996; 14: 7- 11. 51. AlEisa K, Al-Dwairi ZN, Lynch E, Lynch CD. In vitro evaluation of the effect of different endodontic sealers on retentive strength of fiber posts. Operative Dentistry 2013; 38: 539-44. 52. Nova V, Karygianni L, Altenburger MJ, Wolkewitz M, Kielbassa AM, Wrbas KT. Pull-out bond strength of a fibre-reinforced composite post system luted with self-adhesive resin cements. Journal of Dentistry 2013; 41: 1020-6. Page 18 of 22 Accepted Manuscript 18 Figure legends Figure 1: Confocal image of the inner perimeter of a laser-irradiated sample sectioned at 5 mm from the root apex (HC PL FLUOTAR 10×/ 0.30 oil lens). The depth of sealer penetration into the dentinal tubules and the extension of the intracanal perimeter infiltrated by sealer were recorded using LAS AF Lite software (Leica Microsystems) focussing on the dentinal tubules. (*ROI: region of interest). Figure 2: Confocal image of the sealer penetrating the dentinal tubules of a laser-irradiated sample sectioned at 3 mm from the root apex (HCX PL APO 40×/ 1.25-0.75 oil lens). Page 19 of 22 Accepted Manuscript 19 Tables Table 1: Comparison of the results obtained between control and laser groups (SD: standard deviation; *: P < 0.05). Group Depth of penetration (µm) Total perimeter (mm) Penetrated perimeter (mm) Penetrated perimeter (%) Control (n = 80) 106.81 ± 87.4 1.95 ± 0.2 0.84 ± 0.5 42.57 ± 23.6 Laser (n = 80) 105.13 ± 78.6 2.15 ± 0.4 1.14 ± 0.7 50.90 ± 23.4 P value 0.895 0.0001* 0.001* 0.022* 3 mm from the apex Control (n = 40) 63.11 ± 53.8 1.82 ± 0.2 0.61 ± 0.3 33.29 ± 18.8 Laser (n = 40) 66.73 ± 59.2 1.88 ± 0.2 0.81 ± 0.4 42.86 ± 21.4 P value 0.770 0.121 0.019* 0.034* 5 mm from the apex Control (n = 40) 150.52 ± 93.1 2.01 ± 0.2 1.07 ± 0.5 51.75 ± 24.4 Laser (n = 40) 143.53 ± 77.3 2.42 ± 0.5 1.47 ± 0.7 58.94 ± 22.8 P value 0.71 0.0001* 0. 004* 0.164 Page 20 of 22 Accepted Manuscript 20 Table 2: Comparison of the results obtained at 3 mm and at 5 mm from the root apex (SD: standard deviation; *: P < 0.05). Distance from the apex Depth of penetration (µm) Total perimeter (mm) Penetrated perimeter (mm) Penetrated perimeter (%) 3 mm (n = 80) 65.03 ± 56.4 1.85 ± 0.2 0.71 ± 0.4 38.41 ± 20.6 5 mm (n = 80) 146.82 ± 84.6 2.26 ± 0.4 1.28 ± 0.6 55.55 ± 23.7 P value 0.0001* 0.0001* 0.0001* 0.0001* Control group (CG) 3 mm (n = 40) 63.11 ± 53.8 1.82 ± 0.2 0.61 ± 0.3 33.39 ± 18.8 5 mm (n = 40) 150.52 ± 93.1 2.01 ± 0.2 1.07 ± 0.5 51.75 ± 24.4 P value 0.0001* 0.0001* 0.0001* 0.0001* Laser group (LG) 3 mm (n = 40) 66.73 ± 59.2 1.88 ± 0.2 0.81 ± 0.4 42.86 ± 21.4 5 mm (n = 40) 143.53 ± 77.3 2.42 ± 0.5 1.47 ± 0.7 58.94 ± 22.8 P value 0.0001* 0.0001* 0.0001* 0.001* Page 21 of 22 Accepted Manuscript Figure 1 Page 22 of 22 Accepted Manuscript Figure 2