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1 Eff ects of Er,Cr:YSGG Laser Treatment on Human Gingival Fibroblast Attachment, Viability and Morphology of Root Surface: An In Vitro Study Reza Amid, DDS, MSc; Elahe Azizi, DDS; Maryam Torshabi, DDS, PhD; Mohammad Reza Talebi Ardakani, DDS, MSc; Sajjad Ashnagar, DDS; and Seyed Masoud Mojahedi, DDS, MSc ABSTRACT Rehabilitation of periodontal support is the main goal of therapies for periodontitis. Hand instrumentation with curettes, piezoelectric ultrasonic scalers and lasers, such as Er,Cr:YSGG, are used for this purpose. This study was designed to evaluate human gingival fibroblast viability attachment to root surface after modification with the mentioned therapeutic alternatives. Lasers showed significantly lower cell viability after 72 hours compared to hand instrumentation and ultrasound, probably due to more irregular root surfaces after treatment. AUTHORS Reza Amid, DDS, MSc, is a periodontologist who earned his fellowship in laser dentistry in He is an associate professor in the department of periodontics at the Shahid Beheshti University of Medical Sciences, School of Dentistry in Tehran, Iran. Elahe Azizi, DDS, is an independent researcher at the Shahid Beheshti University of Medical Sciences, School of Dentistry in Tehran, Iran Maryam Torshabi, DDS, PhD, holds a doctorate in pharmaceutical biotechnology and specializes in tissue engineering and gene therapy. She is involved in several research projects at the Shahid Beheshti University of Medical Sciences, School of Dentistry in Tehran, Iran. Mohammad Reza Talebi Ardakani, DDS, MSc, is an assistant professor in the department of periodontics at the Shahid Beheshti University of Medical Sciences, School of Dentistry in Tehran, Iran. Sajjad Ashnagar, DDS, is a periodontics resident in the periodontics and oral medicine department at the University of Michigan, School of Dentistry in Ann Arbor, Mich. Seyed Masoud Mojahedi, DDS, MSc, is a dental laser surgeon and an academic co-worker in the Aachen Center for Laser Dentistry at RWTH Aachen University in Aachen, Germany. It has been decades since the fi rst use of ruby laser on hard tissue of a tooth was reported. 1 Since then, outstanding efforts have been made by studying the effects of various lasers in the dental fi eld. Among others, carbon dioxide (CO 2 ), Nd:YAG, Er:YAG, diode and Er,Cr:YSGG lasers clearly exemplify the deep focus of both researchers and clinicians on the utilization of lasers in dentistry. 2 The erbium, chromiumdoped:yttrium, scandium, gallium, garnet hydrokinetic laser system (Er,Cr:YSGG laser) is highly absorbed by water because of its wavelength (2.78 μm). Thus, it is presumed to MAY

2 benefi t medicine and dentistry. This laser can ablate enamel and dentin of the tooth. Concerning specifi c laser energy interaction with water, it provides delicate, clean cuts in hard tissues, 3-5 leading to noticeable removal of caries and preparation of cavities in operative dentistry. 6 Many studies have confi rmed the safety of this laser. The Food and Drug Administration (FDA) has cleared this laser for soft tissue usages, including incision and coagulation, and hard tissue processes like osseous cutting and contouring. 6 There is no thermal damage, including melting and carbonization, upon using Er,Cr:YSGG on mandibular bone or root surface. 7,8 Periodontology is the other domain for Er,Cr:YSGG laser use. Periodontal support must be rehabilitated during periodontal therapy for a patient experiencing periodontitis. All periodontal tissues shall regenerate, including new bone, cementum and fresh periodontal ligaments (PDL). Diseased root surfaces must be decontaminated and smoothed in order to favor the attachment of the critical cells of the periodontal tissue. 9 Several approaches are indicated for this purpose. Hand instrumentation, ultrasonic scalers and lasers such as Er:YAG and Er,Cr:YSGG are implemented. 10 Decontamination and detoxifi cation of the root surface and dentinal tubules are guaranteed using Er,Cr:YSGG laser. At this wavelength, the laser can remove lipopolysaccharides (bacterial endotoxins), smear layer and residual cementum. 11,12 On the contrary, hand instrumentation of root surfaces with curettes leaves a smear layer obliterating dentinal tubules, which contain microbiota and their products. 13 Surface modifi cation is mandatory for the formation of oriented PDL and cell attachment. Roughened root 292 MAY 2016 surface due to subgingival calculus hampers fi broblast migration and attachments, resulting in attachment loss. This attachment is crucial for differentiation and synthesis of extracellular matrix proteins needed for organization of functional attachment tissues. 9 This laser not only removes calculus effi ciently from root surfaces, 7 but also leaves a smooth surface similar to other therapies. Mineral content of the root surface would be more similar to an intact tooth using this laser, while an increase in Laser protocol and confi guration can signifi cantly aff ect the clinical outcomes, as excess energies might result in roughened surfaces with crater-like unevenness. calcium and decrease in phosphate was observed in other treatments. 7 Regardless of the above-mentioned studies on laser utilization as an adjunctive approach in the treatment of periodontitis, several questions remain. Worthy of notice is the fact that laser protocol and confi guration can signifi cantly affect the clinical outcomes, as excess energies might result in roughened surfaces with crater-like unevenness. This endangers fi broblast attachment and viability. Therefore, we aimed to evaluate in vitro viability and attachment confi guration of human gingival fi broblasts on root surfaces treated with hand instruments, ultrasonic scalers and Er,Cr:YSGG laser using MTT assay and scanning electron microscope (SEM) observation. Materials and Methods General Design This comparative cell culture study was carried out according to the Declaration of Helsinki of Fifty single-rooted premolars extracted because of periodontal disease were included in the study. None of the teeth had any cracks, fractures, fillings or endodontic treatment. Informed consent was taken from the patients at the periodontology department of the Shahid Beheshti University of Medical Sciences prior to teeth collection. Patients who had periodontal debridement during the last six months were excluded from the study. Immediately following extraction, any remnant of soft tissue or blood was eliminated using light scrubbing and rinsing with sterile saline solution. Specimens were kept in normal saline at room temperature (20 degrees Celsius) until further operations were done. Specimens were randomly assigned to the following five groups (n=10): Control group: untreated but rinsed with normal saline. Hand instrumentation group: removed both calculus and diseased cementum with Gracey curettes 7 and 8 for 10 times until a smooth surface was reached. Er,Cr:YSGG group I (wavelength = 2.79 μm): hand instrumentation followed by laser irradiation with configuration of 120 mj/pulse and 10 Hz. Er,Cr:YSGG group II (wavelength = 2.79 μm): hand instrumentation followed by laser irradiation with configuration of 160 mj/pulse and 15 Hz. Ultrasonic group: ultrasonic scaler (Piezosurgery, Mectron, Carasco, Italy) used for 60 seconds until all visible calculus was removed.

3 Preparation of Root Specimens Using a diamond disk with water spray perpendicular to the root surface, a coronal section was carried out 1 mm below the cementoenamel junction (CEJ) and an apical section 4 mm from the root apex. Prior to cell culture, root specimens were sterilized using 70% alcohol, antibiotic and antimycotic agents and UV light (class II biologic hood). Laser Treatment An Er,Cr:YSGG dental laser (Biolase Technology, Irvine, Calif.) with a wavelength of 2.79 μm was used in this project. Irradiation was carried out apicocoronally with an angle of 20 to 25 degrees. An optic fiber with a round section of 0.5 mm x 1.65 mm with a chisel-shaped glass fiber tip was utilized for irradiation. The distance of the laser tip to root surface was 1.5 mm. Irradiation was continued until all calculus was removed, with a range of 45 to 60 seconds. Air water spray was supplied during irradiation. Cell Culture Human gingival fibroblasts (HGF.1-PI, NCBI: C-165) were obtained (Cell Bank of Pasteur Institute, Tehran, Iran). They were cultured in DMEM media (Dulbecco s Modified Eagle Medium, Gibco, Waltham, Mass.), containing 10% fetal bovine serum (FBS) (fetal bovine serum, Gibco) and 1% antibiotic and antimycotic agent (Penicillin-Streptomycin Amphotericin B, Gibco) and incubated in a humidified atmosphere of 5% CO 2 and 95% O at 37 degrees Celsius (Memmert incubator, Schwabach, Germany). After sterilization, the samples were rinsed with Dulbecco s phosphatebuffered saline (PBS) and placed into cell culture plates (SPL, Seoul, Korea). There were three plates, one for MTT assay 24 hours after culture, one for MTT assay 72 hours after culture and one for Cell viability (percent of control) 120% 100% 80% 60% 40% 20% 0% FIGURE 1. Cell viability of samples using MTT assay after 24 and 72 hours. 120: laser group I, 160: laser group II, P: Piezo ultrasonic, H: hand instruments. SEM observation 24 hours after culture. Fibroblasts were placed on root slices with a density of 20,000 cells in 400 μl cell culture medium. The samples were then incubated in a CO 2 condition at 37 degrees Celsius for up to three days. MTT Assay 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) assay was carried out for the evaluation of human gingival fibroblast survival on days one and three. The MTT assay (Chemie GmbH, Sigma-Aldrich, St. Louis) was performed for evaluation of cell viability and proliferation. It was carried out 24 and 72 hours after human gingival fibroblasts were placed on root slices. After the designated incubation period, culture media of each plate was changed with medium containing 10% MTT (5 mg/ml) and incubated for three hours. After that time, each well was washed with PBS. Dimethyl sulfoxide (DMSO) was then added to each well in order to solubilize formazan crystals. The volume of 100 μl of content of each well was transferred to a 96 well plate of an ELISA reader for measurement. Optic density was measured in 570 nm (MTT wavelength) and 620 nm (reference wavelength) by an automatic microplate reader. Control P H 24 h 72 h % Viability (Mean ± SE, n+10) Control P H 24 h 100 ± ± ± ± ± h 100 ± ± ± ± ± 10.6 Scanning Electron Microscope (SEM) Observation and Cell Counting After a 24-hour incubation period, the samples were rinsed gently with PBS and fixed with 2.5% glutaraldehyde for 24 hours in refrigerator temperature (4 degrees Celsius). After rinsing with distilled water (three times, each for 10 minutes), 1% osmium solution was added to each well. Then the samples were incubated for two hours at room temperature in darkness. Afterward, rinsing with distilled water was performed again, the same as before. Specimens were dehydrated in increasing concentration of ethanol (30%, 50%, 70%, 80%, 90% and 100%). At the end, specimens were placed under a biologic hood for 48 hours until complete dehydration occurred. Root samples were then gold sputter coated three times and examined using an SEM (KYKY-EM3200 Digital Scanning Electron Microscope, Madell Technology, Ontario, Calif.). Statistical Analysis Viability and proliferation data were reported as mean ± standard deviation (SD). Viability is reported as a percentage of viability compared to control. The proliferation rate is reported as average absorbance at 570 nm. One-way analysis MAY

4 FIGURE 2. SEM picture of fi broblast cultures on control root slice with no surface treatment after 24 hours. FIGURE 3. SEM picture of fi broblast cultures on root slice with hand instrumentation after 24 hours. FIGURE 4. SEM picture of fi broblast cultures on root slice with ultrasonic scaler after 24 hours. of variance (ANOVA) followed by Tukey s test was performed using Prism version 6 (GraphPad Prism, GraphPad Software, La Jolla, Calif.) for statistical analysis. A significance level of 0.05 was designated. According to ISO (2005), any material leading to 70 percent of control group viability is determined cytotoxic. Results Cell viability was similar at 24 hours among all groups, but was significantly lower in laser groups I and II at 72 hours. Proliferation was similar at 24 hours among all groups, but was significantly slowed after 72 hours in lased groups compared to other groups. SEM observation revealed higher irregularity on the root surface. Abnormal and apoptotic fibroblasts were also more prevalent in the lased groups. Viability of Human Gingival Fibroblasts The viability of human gingival fibroblasts 24 hours after experimental procedures were reported as follows: laser I-120mj (86 percent), laser II-160mj (85.6 percent), ultrasonic scaler (90.9 percent) and hand instrument (86.3 percent) (FIGURE 1). These data had no statistically significant difference with the control group (100 percent) (p<0.05). FIGURES 2 6 show SEM images of fibroblast cells of all control and experimental groups after 24 hour. There was no significant difference between the viability of the cells treated with an ultrasonic scaler and hand 294 MAY 2016 instrumentation after 72 hours compared to the control group. Nonetheless, specimens treated with both laser I and II settings showed significantly lower cell viability after 72 hours compared to the control group (p<0.05).this decrease in cell viability was 25.2 percent in laser I and 26.9 percent in laser II. Cell viability of the lased groups was also statistically different from the same data of the hand instrumentation and ultrasonic scaler groups. This difference shows the effect that time has on the cell viability of laser-treated cells. Proliferation of Human Gingival Fibroblasts As illustrated in FIGURE 7, statistically higher proliferation was observed among all specimens as time passed. Proliferation is defi ned as the increase in the number of cells attached to the root slices. Increment in proliferation means that neither of the treatment approaches could hamper or halt cell division and proliferation. It should be underlined that the rate of increase in cell proliferation in the hand instrumentation and ultrasonic scaler groups was similar to the same data in the control group. However, the increase in cell proliferation in both I and II lased groups was lower than the control group (p<0.05). This means that the laser could not stop cell proliferation, but could signifi cantly slow down the rate of the proliferation. Root Surface Observation and Cell Morphology SEM analysis was done for assessment of morphology and attachment of human gingival fi broblasts on the root surfaces at 24 hours. The SEM observation was conducted using the KYKY-EM3200 digital scanning electron microscope. When comparing images taken from root surfaces, the lased specimens showed the highest irregularity. Hand instrumentation and ultrasonic scaler root surfaces showed more regular patterns than the laser groups. When observing fi broblasts on root surfaces, cells in the control group showed a healthier profi le and morphology. Other experimental groups showed abnormal and apoptotic cells, which were more highlighted in the lased groups. Discussion Several tools and protocols are indicated for nonsurgical debridement of bacterial remnant and diseased cementum from root surfaces of patients with periodontitis. Common approaches involve hand instrumentation and ultrasonic scaler. However, a growing trend in the use of lasers in dentistry has led clinicians to benefi t from lasers such as diode, Er:YAG and Er,Cr:YSGG for this purpose. 14,15 This is while some researchers have reported that laser protocols do not provide any clinical advantage in nonsurgical

5 FIGURE 5. SEM picture of fi broblast cultures on root slice with laser (group I, 120mj) after 24 hours. Cell proliferation (average absorbance at 570 nm) FIGURE 7. Cell proliferation of samples using MTT assay after 24 and 72 hours. 120: laser group I, 160: laser group II, P: Piezo ultrasonic, H: hand instrument. periodontal therapies. 16 Among other types of dental lasers, Er,Cr:YSGG was used in this study. Different types of lasers such as Er:YAG, CO2 and Er,Cr:YSGG have been exploited for calculus removal and root planing of periodontally involved teeth. 11,17-19 According to the results of our study, Er,Cr:YSGG laser irradiation resulted in reduced cell attachment, density and count after a 72-hour incubation compared to control samples. Moreover, lased groups I and II, using 120 mj and 160 mj per pulse, respectively, signifi cantly reduced the proliferation rate of fi broblasts. On the other hand, Hakki et al., 9 Control P H FIGURE 6. SEM picture of fi broblast cultures on root slice with laser (group II, 160mj) after 24 hours. 24 h 72 h Absorbance (Mean ± SE, n+10) Control P H 24 h.080 ± ± ± ± ± h.440 ± ± ± ± ±.042 in a project studying attachment of PDL fi broblasts on root surfaces using Er,Cr:YSGG (150 mj/pulse, 10 Hz) and hand instrumentation reported improved cell density in the lased group compared to hand instrumentation samples. In addition, short-pulsed lasers provided more convenient root surfaces for PDL attachment and orientation. This contradiction can be justifi ed by the fact that our study used human gingival fi broblasts instead of PDL fi broblasts. This is more reasonable, because the aim of this study design was to evaluate attachment of new connective tissue to the root surface in nonsurgical periodontal procedures. Results of this study are in accordance with the Tsurumaki et al. 18 investigation, which assessed morphology and attachment of blood components to root surfaces using hand instrumentation, ultrasonic scaler and Er,Cr:YSGG laser irradiation. It was found that hand instrumentation produced a smooth surface with smear layer and grooves with obliterated dentinal tubules. Using laser alone or in conjunction with other approaches led to a rough and irregular surface. SEM observation confi rmed different degrees of surface modifi cation on root surface after laser irradiation. It seems this irregular pattern interferes with fi broblast attachment to the fresh root surface. The degree of roughness and depth of craters depends on output energy, output power, pulse length, shape of laser tip and angle of laser irradiation. Kimura et al. 8 investigated the effect of Er,Cr:YSGG laser irradiation on root surfaces. They found that irradiation with this laser is responsible for irregularity, rough surface and craters with sharp edges. However, this rough surface was free of debris and no sign of carbonization or melting was observed. Other reports, such as the Rotundo et al. 16 study, also state the lack of adjunctive benefi t of Er:YAG in nonsurgical periodontal therapies. They used an Er:YAG laser in combination with hand instrumentation for treatment of patients with periodontitis. After three and six months of follow-up, it showed that laser treatment had no additional clinical merit than hand instrumentation alone. They followed Consolidated Standards of Reporting Trials (CONSORT) guidelines for their clinical trial, so many of the vague points found in some clinical studies were absent. In a project studying the effect of Er:YAG laser and ultrasonic instrumentation on patients with MAY

6 periodontal disease, Sculean et al. 20 also confi rmed similar clinical results for both therapies. Because the 2.94 μm wavelength of the Er:YAG laser is very close to that of the Er,Cr:YSGG, its effect on root surfaces can be relevant. 8 It is worth noting that according to the Ting et al. 6 study, root surface roughness made by Er,Cr:YSGG laser irradiation depends on laser output power. It was revealed that as the output power increased from 1W to 1.5W, signifi cant roughness was made upon irradiation. This was also refl ected in the depth of the craters made by the laser. This study used 1.2W and 1.6W setting for irradiation. As in vivo positive clinical outcomes using Er,Cr:YSGG for treatment of periodontitis were obtained at 1W, 10 this might be concluded that excessive energy made the surface inconvenient for human gingival fi broblasts to survive and attach. However, higher powers might still be suitable for PDL fi broblast attachment. 9,12 But according to our knowledge, no in vitro study confi rmed human gingival fi broblast attachment at a power higher than 1W. To put the issue into perspective, Er,Cr:YSGG yielded no advantage in fibroblast attachment and survival, a key factor for periodontal regeneration after therapies, compared to other conventional treatments. It is suggested to adjust the laser settings from 0.5W to 1W for the same study design in the future. Conclusion Concerning the results of this study, the Er,Cr:YSGG laser did not reveal any satisfactory outcomes in terms of fibroblast attachment and survival at output power of 1.2 and 1.6 watts. Further studies with larger sample size are needed to confirm the validity of these results. 296 MAY 2016 REFERENCES 1. Goldman L, Hornby P, Meyer R, Goldman B. Impact of the laser on dental caries. Nature 1964 Jul 25;203: Pick R, Powell G. Laser in dentistry. 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J Clin Periodontol 2010;37(6): Miyazaki A, Yamaguchi T, Nishikata J, Okuda K, Suda S, Orima K, et al. Effects of Nd:YAG and CO 2 laser treatment and ultrasonic scaling on periodontal pockets of chronic periodontitis patients. J Periodontol 2003;74(2): Tsurumaki JdN, Souto BHM, Oliveira GJPLd, Sampaio JEC, Marcantonio Júnior E, Marcantonio RAC. Effect of instrumentation using curettes, piezoelectric ultrasonic scaler and Er,Cr:YSGG laser on the morphology and adhesion of blood components on root surfaces: A SEM study. Braz Dent J 2011;22(3): Liu CM, Shyu YC, Pei SC, Lan WH, Hou LT. In vitro effect of laser irradiation on cementum-bound endotoxin isolated from periodontally diseased roots. J Periodontol 2002;73(11): Sculean A, Schwarz F, Berakdar M, Romanos GE, Arweiler NB, Becker J. Periodontal treatment with an Er:YAG laser compared to ultrasonic instrumentation: A pilot study. J Periodontol 2004;75(7): THE CORRESPONDING AUTHOR, Sajjad Ashnagar, DDS, can be reached at sajjadashna@yahoo.com.