The Whitening Effect and Histological Safety of Nonthermal Atmospheric Plasma Inducing Tooth Bleaching

International Journal of Environmental Research and Public Health

Article The Whitening Effect and Histological Safety of Nonthermal Atmospheric Plasma Inducing Tooth Bleaching

Seoul-Hee Nam 1 , Byul Bo Ra Choi 2 and Gyoo-Cheon Kim 2,3,*

1 Department of Dental Hygiene, College of Health Science, Kangwon National University, Samcheok 25949, Korea; [email protected] 2 Feagle Co., Ltd., Yangsan 50614, Korea; [email protected] 3 Department of Oral Anatomy, School of Dentistry, Pusan National University, Yangsan 50612, Korea * Correspondence: [email protected]; Tel.: +82-51-510-8243; Fax: +82-51-510-8241

Abstract: Various light sources have been applied to enhance the bleaching effect. This study was to identify the histological evaluation in oral soft tissues, as well as tooth color change after tooth bleaching by nonthermal atmospheric pressure plasma (NAPP). Nine New Zealand adult female rabbits were randomly divided into three groups (n = 3): group 1 received no treatment; group 2 was

treated with NAPP and 15% carbamide peroxide (CP), which contains 5.4% H2O2, and group 3 was treated with 15% CP without NAPP. Color change (∆E) was measured using the Shade Eye NCC colorimeter. Animals were euthanized one day later to analyze the histological responses occurring in oral soft tissues, including pulp, gingiva, tongue, buccal mucosa, and hard and soft palates. Changes in all samples were analyzed by hematoxylin and eosin staining and Masson’s trichrome. Teeth

treated with plasma showed higher ∆E than that obtained with bleaching agents alone. Overall, the histological characteristics observed no appreciable changes. The combinational treatment of

Citation: Nam, S.-H.; Choi, B.B.R.; plasma had not indicated inﬂammatory responses as well as thermal damages. NAPP did not cause Kim, G.-C. The Whitening Effect and histological damage in oral soft tissues during tooth bleaching. We suggest that NAPP could be a Histological Safety of Nonthermal novel alternative energy source to conventional light sources for tooth bleaching. Atmospheric Plasma Inducing Tooth Bleaching. Int. J. Environ. Res. Public Keywords: nonthermal atmospheric pressure plasma; safety; tooth bleaching; oral soft tissues; Health 2021, 18, 4714. https:// histological damage doi.org/10.3390/ijerph18094714

Academic Editors: YoungJin Jung, Jong-Hoon Kim and Hae Yean Park 1. Introduction Following the initial introduction of tooth bleaching using nonthermal atmospheric Received: 30 March 2021 pressure plasma (NAPP) [1], many articles have reported that NAPP-based bleaching is Accepted: 27 April 2021 Published: 28 April 2021 much more effective than the conventional methods [2–8]. Besides the bleaching efficiency, preserving healthy oral tissues without damage is very important during tooth bleaching

Publisher’s Note: MDPI stays neutral using NAPP. Nam et al. [9] demonstrated that tooth bleaching using NAPP with 15% HP with regard to jurisdictional claims in or CP did not influence the microhardness and the mineral content of dental hard tissues. published maps and institutional afﬁl- In addition, the application of NAPP did not result in any structural–morphological and iations. topographic changes in the enamel [10]. Although hard tissue analysis has been reported after tooth bleaching using NAPP, safety analysis on oral soft tissues has not been made so far. Vital tooth bleaching procedures have been considered one of the most conservative treatments in esthetic dentistry. The most popular bleaching agents are hydrogen peroxide (HP; H O ) and carbamide peroxide (CP; CH N O ), which diffuse through the organic Copyright: © 2021 by the authors. 2 2 6 2 3 Licensee MDPI, Basel, Switzerland. matrix of enamel and dentin [11]. Tooth bleaching results from the redox reaction between This article is an open access article the peroxide-based bleaching agents and the darkened substrate [12]. Currently, tooth distributed under the terms and bleaching with CP is very popular. Higher concentrations of CP contain higher amounts conditions of the Creative Commons of HP, and, for this reason, bleaching results are achieved more quickly than when lower Attribution (CC BY) license (https:// concentrations of CP are used [13]. However, tooth bleaching has been reported to cause creativecommons.org/licenses/by/ several adverse effects [14–16]. The most common adverse effects are tooth hypersensitivity, 4.0/). gingival irritation, and pulpal damage [17,18]. The use of higher concentrations of bleaching

Int. J. Environ. Res. Public Health 2021, 18, 4714. https://doi.org/10.3390/ijerph18094714 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021, 18, 4714 2 of 10

agents can pose a higher risk of tooth and oral soft tissue damage and cause chemical burns when in contact with teeth, gingival tissue, oral mucosa, skin, or eyes [19–21]. Various light sources have been applied to enhance the bleaching effect [16]. They accelerate the decomposition of HP into ·OH radical, which makes bleaching a faster reaction [20]. This theory supports that the production of ·OH radical is hastened by temperature increase [22]. OH radical induces oxidative damage on cells and tissues, so that chronical generation of it could lead to specific diseases [23]. Numerous studies have proved that light-activated bleaching did not produce any perceivable color changes [24]. Moreover, the heat generated by light-activated tooth bleaching is capable of diffusing through enamel and dentin and reaching the pulpal space, potentially leading to irreversible thermal damage of the pulp tissue [25]. Thus, the safety of light-activated bleaching is still a matter of controversy, and more evidence is required to make a precise assertion of its effectiveness and safety. Many efforts have been made to increase patient benefits regarding better bleaching efficacy and safe application with recent bleaching systems [25]. Therefore, in order to be a better and safe tooth bleaching method, an effective and nonthermal energy source is desired. Our previous study has shown that NAPP increased the production of ·OH radicals and yielded a much better bleaching effect than that of conventional light sources [1,3,5,7], while the tooth temperature was maintained near the body temperature [7]. Furthermore, tooth bleaching with NAPP did not affect the microhardness and mineral content of the tooth [7]. The histological investigation of oral tissues after NAPP treatment is essential for its safe utilization. The 15% CP is used for home bleaching and contains a relatively low HP concentration of 5.4%. Llena et al. [26] reported that CP had a less cytotoxic effect on the human dental pulp stem cells than other products. In our previous paper, the bleaching effect of 15% CP itself was insignificant, but the combination treatment of plasma with 15% CP showed similar results to the bleaching effect of 30% HP [1,4]. The procedure of home bleaching using 15% CP takes a long time to achieve a bleaching effect, but the pain complaints of the operator are relatively small. Therefore, NAPP treatment could reduce the time required for the bleaching effect of 15% CP without the injury to the teeth. However, the histological safety of NAPP has not been fully demonstrated in oral soft tissues such as gingiva, tongue, buccal mucosa, and hard and soft palates. If there is an energy source that effectively induces the tooth color change without causing tissue damages, it would be a useful device for tooth bleaching in clinical practice. The aim of this study was to evaluate the histological changes in the oral soft tissues treated with NAPP and 15% CP to prove that NAPP is safe as well as effective.

2. Materials and Methods 2.1. NAPP Device A schematic of the NAPP device was developed (Figure1). The plasma generating module of the inner stainless and outer copper electrode was made of a ceramic body as a dielectric. The outer electrode is grounded, and a sinusoidal high voltage is applied to the inner electrodes by a high voltage source, which can increase the voltage up to 10 kV with a frequency of 15 kHz. Over 2.8 kHz can generate the plasma in an alumina tube when argon was sued as buffer gas at a low rate of 2 standard liters per minute. The temperature of the NAPP ﬂow at 1 cm from the electrode end was maintained around 35 ◦C for 10 min [1,3–7]. The distance of treating subjects from the electrodes was kept at 1 cm. Int. J. Environ. Res. Public Health 2021, 18, x 3 of 10

Int. J. Environ. Res. Public Health 2021, 18, x 3 of 10 Int. J. Environ. Res. Public Health 2021, 18, 4714 3 of 10

94 Figure 1. Illustration of the methodology used in the study. Schematic drawing of the NAPP de- 95 vice. 96 94 FigureFigure 1. 1. IllustrationIllustration of of the the methodology methodology used used in in the the study. study. Schematic Schematic drawing of the NAPP device.de- 95 2.2.vice. Tooth Bleaching Procedure 97 96 2.2. Tooth Bleaching Procedure Nine New Zealand adult female rabbits, older than six months and ranging in weight 98 2.2. ToothNine Bleaching New Zealand Procedure adult female rabbits, older than six months and ranging in weight 97 from 2.5 to 3.5 kg, were used in this study. Animals were housed individually in separate 99 from 2.5 to 3.5 kg, were used in this study. Animals were housed individually in separate rabbit cagesNine at New the Zealandambient adulttemperature female rabbits,of 20 °C older and athan 12/12 six- hmonths light/dark and cycle.ranging Animals in weight 100 98 rabbit cages at the ambient temperature of 20 ◦C and a 12/12-h light/dark cycle. Animals hadfrom free 2.5access to 3.5 to drinkingkg, were waterused in and this standard study. Animals laboratory were pellets. housed At theindividually time of the in study, separate 101 99 had free access to drinking water and standard laboratory pellets. At the time of the study, thererabbit were cages no clinical at the ambientsigns of diseasetemperature in any of animal. 20 °C and The a protocol 12/12-h waslight/dark reviewed cycle. and Animals ap- 102 100 there were no clinical signs of disease in any animal. The protocol was reviewed and provedhad freeby the access Pusan to drinking National water University and standard Institutional laboratory Animal pellets. Care Atand the Use time Committee of the study, 103 101 approved by the Pusan National University Institutional Animal Care and Use Committee (PNUthere-IACUC), were no Pusan, clinical Korea, signs in of 2012 disease (approval in any animal.2012-0086). The The protocol overall was experimental reviewed and de- ap- 104 102 (PNU-IACUC), Pusan, Korea, in 2012 (approval 2012-0086). The overall experimental signproved of this bystudy the isPusan depicted National in Figure University 2. All rabbits Institutional were subjected Animal Care to general and Use anesthesia, Committee 105 103 design of this study is depicted in Figure2. All rabbits were subjected to general anesthesia, performed(PNU-IACUC), with an Pusan,intramuscular Korea, in 5:1 2012 mixture (approval of ketamine 2012-0086). HCl The(50 mg/kg,overall Ketalasrexperimental®, Yu- de- 106 104 performed with an intramuscular 5:1 mixture of ketamine HCl (50 mg/kg, Ketalasr®, sign of this study is depicted in Figure 2. All ®rabbits were subjected to general anesthesia, 105 han,Yuhan, Korea) Korea) and xylazine and xylazine (10 mg/kg, (10 mg/kg, Rumpun Rumpun, Bayer,®, Bayer, Korea), Korea), and operated and operated on under on under 107 performed with an intramuscular 5:1 mixture of ketamine HCl (50 mg/kg, Ketalasr®, Yu- 106 sterilesterile conditions conditions prior prior to the to bleaching the bleaching procedure. procedure. The Thenine nine animals animals were were then then randomly randomly 108 han, Korea) and xylazine (10 mg/kg, Rumpun®, Bayer, Korea), and operated on under 107 divideddivided into intothree three groups groups of three of three animals animals each, each, according according to the to treatment. the treatment. Group Group 1, the 1, the 109 sterile conditions prior to the bleaching procedure. The nine animals were then randomly 108 controlcontrol group, group, was wasnot treated. not treated. Group Group 2 was 2 wastreated treated with with NAPP NAPP in combination in combination with with 15% 15% 110 divided into three groups of three animals each, according to the treatment. Group 1, the 109 CP (KoolCP (Kool white white 15%, 15%, Pac- Pac-dentdent International, International, USA). Walnut, A uniform CA, USA).1 mm A-thick uniform layer1 of mm-thick 15% 111 control group, was not treated. Group 2 was treated with NAPP in combination with 15% 110 CP waslayer applied of 15% over CP wasthe labial applied surface over of the the labial upper surface incisor of teeth. the upperDuring incisor NAPP teeth.exposure, During 112 CP (Kool white 15%, Pac-dent International, USA). A uniform 1 mm-thick layer of 15% 111 the NAPPNAPP tip exposure, was positioned the NAPP approximately tip was positioned 1 cm approximatelyaway from the 1enamel cm away surface. from theGroup enamel 113 CP was applied over the labial surface of the upper incisor teeth. During NAPP exposure, 112 3 wassurface. treated Group with 15% 3 was CP treated without with NAPP 15% and CP withoutleft undisturbed. NAPP and After left undisturbed.the 30-min bleach- After the 114 the NAPP tip was positioned approximately 1 cm away from the enamel surface. Group 113 ing treatment,30-min bleaching the gel treatment,was gently the wiped gel waswith gently sterile wiped gauze. with sterile gauze. 115 3 was treated with 15% CP without NAPP and left undisturbed. After the 30-min bleach- 114 ing treatment, the gel was gently wiped with sterile gauze. 115

116 FigureFigure 2. Flow 2. Flow chart chart of the of experimental the experimental design design for the for in the vivo in vivostudy. study. 117 116 Figure 2. Flow chart of the experimental design for the in vivo study. 117

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Int. J. Environ. Res. Public Health 2021,2.3.18, 4714 Color Changes 4 of 10 118 The outcome of color change (ΔE) was determined by the colorimeter (Shade Eye 119 NCC, Shofu Inc., Kyoto, Japan). The color values were based on the lab color system, CIE 120 2.3.(Commission Color Changes Internationale de L’Eclairage), a widely used tooth color evaluation [27]. 121 The valueThe outcome differences of color of L*, change a*, and ( ∆bE* )in was each determined group were by measured the colorimeter using Adobe (Shade Pho- Eye 122 NCC,toshop Shofu CS2 (Adobe Inc., Kyoto, Systems, Japan). San The Jose, color CA, values USA). were The basedoverall on Δ theE for lab the color total system, difference CIE 123 (Commissionin the values Internationaleof L*, a*, and deb* were L’Eclairage), measured a widely and calculated used tooth according color evaluation to the following [27]. The 124 valueformula: differences of L*, a*, and b* in each group were measured using Adobe Photoshop 125 CS2 (Adobe Systems, San Jose, CA, USA). The overall ∆E for the total difference in the 222 values of L*, a*, and b* were measured=++ELb ( and*) calculated( a**) according( ) to the following formula:(1) q ∆E = (∆L∗)2 + (∆a∗)2 + (∆b∗)2 (1) 2.4. Histological Evaluation 126 2.4. HistologicalTissue biopsies Evaluation were performed one day after bleaching, as it is the optimal time to 127 evaluateTissue any biopsies reaction. were Rabbits performed were euthanized, one day after and bleaching, teeth, gingiva, as it is thetongue, optimal buccal time mu- to 128 evaluatecosa, and any hard reaction. and soft Rabbits palates were were euthanized, harvested andfor histological teeth, gingiva, analysis. tongue, All buccal biopsied mucosa, tis- 129 andsues hardwere andplaced soft in palates different were labeled harvested vials containing for histological 4% paraformaldehyde analysis. All biopsied fixative tissues solu- 130 weretion at placed room intemperature different labeled for at least vials 48 containing h. Teeth were 4% paraformaldehyde decalcified in neutral fixative 10% ethylene- solution 131 atdiaminetetraacetic room temperature acid for (EDTA) at least 48 for h. four Teeth weeks, were anddecalcified the EDTA in neutral solution 10% was ethylenedi- changed 132 aminetetraaceticevery day to accelerate acid (EDTA) demineralization. for four weeks, The and fixed the tissues EDTA solutionwere embedded was changed in paraffin every 133 dayunder to acceleratevacuum. Serial demineralization. sections of 4 The μm fixed thickness tissues were were cut embedded using a rotary in paraffin microtome, under 134 vacuum.mounted Serialon glass sections slides, of and 4 µ msubjected thickness to werehematoxylin cut using and a rotary eosin microtome,(H&E) and mountedMasson’s 135 ontrichrome glass slides, (MST) and staining. subjected H&E to staining hematoxylin was performed and eosin to (H&E) evaluate and epidermal Masson’s trichromeor dermal 136 (MST)changes staining. and morphological H&E staining changes was performed of tissue; MST, to evaluate the three epidermal-color staining or dermal in histology, changes 137 andwas morphologicalused to detect collagen changes offibers. tissue; The MST, stained the three-colortissues were staining examined in histology, microscop wasically, used in 138 toa blinded detect collagen manner, fibers. using The a light stained microscope tissues were (CKX41, examined Olympus, microscopically, Tokyo, Japan) in a adapted blinded 139 manner,with a digital using camera a light microscope (Pixel link PL (CKX41,-B686 Olympus,CU, Canada), Tokyo, at a Japan) magnification adapted withof ×100, a digital ×200, 140 cameraand ×400. (Pixel Images link were PL-B686 loaded CU, into Canada), a computer at a magnification and processed of with×100, Image×200,-Pro and Plus×400. 5.1 141 Imagessoftware were (Media loaded cybernetics into a computer Inc., Washington, and processed DC, USA). with Image-Pro Plus 5.1 software 142 (Media cybernetics Inc., Washington, DC, USA). 3. Results 143 3. Results 3.1. Change in Tooth Color 144 3.1. Change in Tooth Color The mean ΔE values using colorimeter were shown in Figure 3. The mean ΔE± stand- 145 The mean ∆E values using colorimeter were shown in Figure3. The mean ∆E± standard ard deviations (S.D) were 12.36 ± 0.61 and 7.61 ± 0.32 after 30 min treatment with plasma 146 deviations (S.D) were 12.36 ± 0.61 and 7.61 ± 0.32 after 30 min treatment with plasma and withoutand without plasma, plasma, respectively. respectively. After After 24 h, 24 the h, the mean mean values values of ∆ofE Δ±E SD± SD were were 5.60 5.60± ± 0.17,0.17, 147 andand 4.234.23 ±± 0.080.08 after after 1 1 day day of of treatment treatment with with plasma plasma and without plasma, respectively. AnAn 148 improvementimprovement ofof toothtooth colorcolor waswas observedobserved atat thethe applicationapplication ofof NAPP.NAPP. 149

150

FigureFigure 3.3. ChangeChange inin ∆ΔEEvalues values afterafter tooth tooth bleaching bleaching using using plasma plasma using using Shade Shade Eye Eye NCC NCC colorimeter. colorime- 151 ter. 152 3.2. Response of Pulp Tissue After H&E and MST staining, samples were examined under a light microscope (Figure4). H&E staining revealed that the three groups of pulp tissues had normal characteristics in all respects, presenting normal cellular structure and conﬁguration. Pulp Int. J. Environ. Res. Public Health 2021, 18, x 5 of 10

3.2. Response of Pulp Tissue 153 Int. J. Environ. Res. Public Health 2021, 18, 4714 5 of 10 After H&E and MST staining, samples were examined under a light microscope (Fig- 154 ure 4). H&E staining revealed that the three groups of pulp tissues had normal character- 155 istics in all respects, presenting normal cellular structure and configuration. Pulp tissues 156 tissues from groups 2 and 3 were indistinguishable from those of the control, group 1 from groups 2 and 3 were indistinguishable from those of the control, group 1 (Figure 4a). 157 (Figure4a). None of the groups showed cellular displacement of odontoblast nuclei into None of the groups showed cellular displacement of odontoblast nuclei into dentin tu- 158 dentin tubules. The odontoblasts were arranged beneath the dentin in the form of one bules. The odontoblasts were arranged beneath the dentin in the form of one layer with 159 layer with normal size and shape. In addition, none of the pulp tissues presented cellular normal size and shape. In addition, none of the pulp tissues presented cellular hyperac- 160 hyperactivity, collagen disorganization, or intense vascularization, which is evidence of tivity, collagen disorganization, or intense vascularization, which is evidence of thermal 161 thermal damage and inﬂammation in MST (Figure4b). damage and inflammation in MST (Figure 4b). 162

163

Figure 4. 4. HistologicalHistological response response of of pulp pulp tissue tissue to to bleaching bleaching treatment treatment in in each each group; group; (a ()a Repre-) Representative 164 sentative H&E image and (b) MST image. Magnification ×200. 165 H&E image and (b) MST image. Magniﬁcation ×200.

3.3. Effects Effects on on Oral Oral Soft Soft Tissues Tissues 166 TissuesTissues of of gingiva, gingiva, tongue, tongue, buccal buccal mucosa, mucosa, and andhard hardand soft and palates soft palates were stained were stainedby 167 H&Eby H&E after after tooth tooth bleaching bleaching using usingNAPP NAPP (Figures (Figures 5a–9a).5 a–The9a). cornified The cornified layer was layer not wassep- not 168 aratedseparated from from the underlying the underlying basal layer, basal and layer, the and configuration the configuration of both epithelial of both epithelial and con- and 169 nectiveconnective tissue tissue cell layers cell layers was intact was intactin H&E in staining. H&E staining. A columnar A columnar shape of shapebasal cells of basal with cells 170 wellwith-defined well-defined cell membrane cell membrane,, followed followed by the cuboidal by the and cuboidal squamous and shape squamous of the shapestratum of the 171 spinosum is observed in all three groups. Additionally, the structures of epithelial and 172 stratum spinosum is observed in all three groups. Additionally, the structures of epithelial dermal layers in the gingiva, tongue, buccal mucosa, and in hard and soft palates in 173 and dermal layers in the gingiva, tongue, buccal mucosa, and in hard and soft palates in groups 2 and 3 were consistent with those of the control group, by examination after MST 174 groups 2 and 3 were consistent with those of the control group, by examination after MST (Figures 5b–9b). Densely packed collagen fibers were not different from the normal state, 175 (Figures5b–9b). Densely packed collagen fibers were not different from the normal state, and fibroblasts, which were numerous and clear in all samples, were observed scattered 176 and fibroblasts, which were numerous and clear in all samples, were observed scattered throughout the connective tissue. Blood vessels were lined with endothelial cells, and red 177 throughout the connective tissue. Blood vessels were lined with endothelial cells, and blood corpuscles were observed in the vascular lumen. All oral soft tissues in each exper- 178 red blood corpuscles were observed in the vascular lumen. All oral soft tissues in each imental group exhibited histologically normal characteristics without disorganization of 179 Int. J. Environ. Res. Public Health 2021, 18, x 6 of 10 collagenexperimental fibers, group changes exhibited of cell morphology, histologically thermal normal damage, characteristics or inflammatory without disorganization response. 180 of collagen fibers, changes of cell morphology, thermal damage, or inflammatory response.

181 FigureFigure 5. 5. RepresentativeRepresentative histological histological image image of gingival of gingival tissue tissue of each of each group group with with(a) H&E (a) H&Eand (b and) 182 MST(b) MST staining. staining. Magnification Magniﬁcation ×200.× 200. 183

184 Figure 6. Representative histological image of tongue tissue of each group with (a) H&E and (b) 185 MST staining. Magnification ×200. 186

187 Figure 7. Representative histological image of buccal mucosal tissue of each group with (a) H&E 188 and (b) MST staining. Magnification ×200. 189

Int. J. Environ. Res. Public Health 2021, 18, x 6 of 10 Int. J. Environ. Res. Public Health 2021, 18, x 6 of 10

181 Int. J. Environ. Res. Public Health 2021, 18, 4714 6 of 10 181 Figure 5. Representative histological image of gingival tissue of each group with (a) H&E and (b) 182 MSTFigure staining. 5. Representative Magnification histological ×200. image of gingival tissue of each group with (a) H&E and (b) 183182 MST staining. Magnification ×200. 183

184 184 FigureFigure 6. 6. RepresentativeRepresentative histological imageimage ofof tonguetongue tissue tissue of of each each group group with with (a ()a H&E) H&E and and (b ()b MST) 185 Figure 6. Representative histological image of tongue tissue of each group with (a) H&E and (b) 185 MSTstaining. staining. Magniﬁcation Magnification×200. ×200. 186 MST staining. Magnification ×200. 186

187 187 Figure 7. Representative histological image of buccal mucosal tissue of each group with (a) H&E 188 Int. J. Environ. Res. Public Health 2021, FigureFigure18, x 7. 7. RepresentativeRepresentative histological imageimage ofof buccalbuccal mucosalmucosal tissue tissue of of each each group group with with (a ()a H&E) H&E7 of and 10 189188 and (b) MST staining. Magnification ×200. and(b) MST(b) MST staining. staining. Magniﬁcation Magnification×200. ×200. 189

190 FigureFigure 8. 8. RepresentativeRepresentative histological histological image of hard palate tissue of each groupgroup withwith ((aa)) H&EH&E andand ( b) 191 (MSTb) MST staining. staining. Magniﬁcation Magnification×200. ×200. 192

193 Figure 9. Representative histological image of soft palate tissue of each group with (a) H&E and 194 (b) MST staining. Magnification ×200. 195

4. Discussion 196 In tooth bleaching treatment, preserving healthy pulp and oral soft tissues are the 197 most important issues. A novel tooth bleaching technique using NAPP has been shown 198 superior in efficacy, but its safety has not been verified. Therefore, if the combination treat- 199 ment of NAPP and 15% CP is shown to cause no histological damage, NAPP is a safe tool 200 for tooth bleaching. 201 In clinical tooth bleaching, a high concentration of HP or CP has been recommended 202 to be used with a high-intensity light source. However, the possible adverse effects result- 203 ing from tooth bleaching have prompted scientific deliberations in dentistry [28]. The high 204 peroxide penetration into the pulp may result in significant damage to tissue health [29]. 205 Within 5 to 15 min, peroxide penetrates the pulp, where it irritates nerves, consequently 206 producing reversible pulpitis [30], which dramatically inhibits pulpal enzyme activity [31] 207 and causes cytotoxic effects to odontoblast-like cells [32]. A number of reports have 208 demonstrated the deleterious effects of bleaching agents on oral soft tissues [33]. Attia- 209 Zouair et al. [34] revealed that the high concentration of HP in bleaching agents might 210 adversely affect not only the gingival epithelium but also the subepithelial tissue. A form 211 of tissue ulceration or blister may be noted in the gingival tissues at the cervical area, or 212 on the lips or other mucosa, as a distinct white change in the tissue with associated un- 213 derlying erythema [35]. Consequently, severe dentinal pain is often felt by patients after 214 bleaching [16]. Therefore, the use of bleaching agents with low concentrations of HP is 215

Int. J. Environ. Res. Public Health 2021, 18, x 7 of 10

190 Int. J. Environ. Res. Public Health 2021Figure, 18, 4714 8. Representative histological image of hard palate tissue of each group with (a) H&E and7 of 10 191 (b) MST staining. Magnification ×200. 192

193 FigureFigure 9. 9. RepresentativeRepresentative histological histological image image of of soft soft palate palate tissue tissue of of each each group group with with ( (a)) H&E H&E and and (b) 194 (MSTb) MST staining. staining. Magniﬁcation Magnification×200. ×200. 195

4.4. Discussion Discussion 196 InIn tooth tooth bleaching bleaching treatment, treatment, preserving preserving healthy healthy pulp pulp and and oral oral soft soft tissues tissues are are the the 197 mostmost important important issue issues.s. A A novel novel tooth tooth bleaching bleaching technique technique using using NAPP NAPP has has been been shown shown 198 superiorsuperior in in efficacy, efficacy, but but its its safety safety has has not not been been verified. verified. Therefore, Therefore, if the ifcombination the combination treat- 199 menttreatment of NAPP of NAPP and 15% and CP 15% is CPshown is shown to cause to cause no histological no histological damage, damage, NAPP NAPP is a safe is a tool safe 200 fortool tooth for tooth bleaching. bleaching. 201 InIn clinical clinical toothtooth bleaching,bleaching, a a high high concentration concentration of HPof HP or CPor CP has has been been recommended recommended to be 202 used with a high-intensity light source. However, the possible adverse effects resulting from to be used with a high-intensity light source. However, the possible adverse effects result- 203 tooth bleaching have prompted scientific deliberations in dentistry [28]. The high peroxide ing from tooth bleaching have prompted scientific deliberations in dentistry [28]. The high 204 penetration into the pulp may result in significant damage to tissue health [29]. Within 5 peroxide penetration into the pulp may result in significant damage to tissue health [29]. 205 to 15 min, peroxide penetrates the pulp, where it irritates nerves, consequently producing Within 5 to 15 min, peroxide penetrates the pulp, where it irritates nerves, consequently 206 reversible pulpitis [30], which dramatically inhibits pulpal enzyme activity [31] and causes producing reversible pulpitis [30], which dramatically inhibits pulpal enzyme activity [31] 207 cytotoxic effects to odontoblast-like cells [32]. A number of reports have demonstrated the and causes cytotoxic effects to odontoblast-like cells [32]. A number of reports have 208 deleterious effects of bleaching agents on oral soft tissues [33]. Attia-Zouair et al. [34] revealed demonstrated the deleterious effects of bleaching agents on oral soft tissues [33]. Attia- 209 that the high concentration of HP in bleaching agents might adversely affect not only the Zouair et al. [34] revealed that the high concentration of HP in bleaching agents might 210 gingival epithelium but also the subepithelial tissue. A form of tissue ulceration or blister may adversely affect not only the gingival epithelium but also the subepithelial tissue. A form 211 be noted in the gingival tissues at the cervical area, or on the lips or other mucosa, as a distinct of tissue ulceration or blister may be noted in the gingival tissues at the cervical area, or 212 white change in the tissue with associated underlying erythema [35]. Consequently, severe on the lips or other mucosa, as a distinct white change in the tissue with associated un- 213 dentinal pain is often felt by patients after bleaching [16]. Therefore, the use of bleaching agents derlying erythema [35]. Consequently, severe dentinal pain is often felt by patients after 214 with low concentrations of HP is preferred in order to minimize the side effects caused by bleachingperoxide penetration.[16]. Therefore, The conventionalthe use of bleaching light sources agents may with affect low cell concentrations membrane proteins of HP and is 215 trigger an autocatalytic reaction, which may result in mutagenesis, carcinogenesis, irreversible cell membrane damage, reduced cell proliferation, and cell death [32]. The high temperature of light-activated bleaching constitutes a serious threat to the vitality of the pulp and oral soft tissues. The healthy pulp of vital teeth exhibits dentinal fluid flow produced by intrapulpal pressure, cytoplasmatic prolongations of odontoblasts, and other intratubular compo- nents [36], which may prevent the diffusion of chemicals through the dentinal tubules [37]. Heat from a light source causes expansion of the liquid in the dentinal tubules and affects the vessels of the pulp [38]. Bowles and Ugwuneri [39] have also shown that the increase of the temperature in the pulp may cause inactivation of several pulp enzymes that are important to the maintenance of physiological cellular functions. The histological studies associated with heat have reported the disappearance of the odontoblastic layer under the treated area and a dense inflammatory infiltrate in the pulps of dogs [40]. Zach and Cohen [41] have shown that pulpal temperature increases of 5.5 ◦C, 11.1 ◦C, and 16.6 ◦C caused 15%, 60%, and 100% irreversible pulp damage, respectively, in Macaca rhesus. If the temperature transmitted towards the cervical region leads to an increase Int. J. Environ. Res. Public Health 2021, 18, 4714 8 of 10

exceeding 10 ◦C, it may cause periodontal injury [42]. Thus, light sources reached a limit temperature for pulp (5.5 ◦C) and periodontal tissue (10 ◦C). To preserve the healthy tissues, low thermal conductivity is important to avoid possible thermal damage [43]. Therefore, evaluation of the safety of light-activated bleaching has received considerable attention. In our previous studies, the temperature of the tooth treated with NAPP stabilized near 37 ◦C, similar to human body temperature, and this state was continuously maintained throughout the tooth bleaching process [1,3,5.7]. The NAPP would be employed with a low and safe temperature. Additionally, the immune system genes of rabbits are apparently more similar to those of the human immune system than rodent genes [44]. For these reasons, rabbits were used as the animal model. In this study, H&E and MST staining were conducted to determine whether NAPP is safe to pulp tissue and oral soft tissues of rabbits after tooth bleaching using NAPP and 15% CP. In all pulp tissues after tooth bleaching with NAPP, the odontoblasts and endothelial cells were intact, with columnar shape and normal size. No inflammatory response or thermal damage was observed in pulp tissues. Besides pulpal tissues, all analyzed oral soft tissues showed the characteristics of normal tissues with a multi-layer epithelium consisting of keratinocytes, well-organized collagen fibers, and active fibroblasts. No significant difference was found in any of the epithelial and dermal layers after tooth bleaching with NAPP. These results demonstrate that combined tooth bleaching of NAPP and 15% CP does not damage oral tissues, as NAPP does not generate high temperature and quickly dissolves toxic HP to nontoxic ·OH. Because tissue safety in this study was investigated in a short period, it is necessary to investigate it over a long period in the future.

5. Conclusions NAPP has a greater capability for effective tooth bleaching than a low concentration of HP alone. This study provides evidence for the histological safety of the combined action of NAPP and low concentration of CP (15%) in tooth bleaching. This treatment does not induce thermal damage and inﬂammatory response in pulp and oral soft tissues. The treatment with NAPP will bring further developments in tooth bleaching procedures. NAPP is as it maximizes the bleaching effect and minimizes the potential risks of tissue damage.

Author Contributions: Conceptualization, S.-H.N.; data curation, B.B.R.C.; methodology, S.-H.N.; resources, S.-H.N.; supervision, G.-C.K.; validation, B.B.R.C. and G.-C.K.; writing the original draft, S.-H.N. and B.B.R.C.; writing review and editing, G.-C.K. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020R1C1C1005306). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Pusan National University Institutional Animal Care and Use Committee (PNU-IACUC) (Approval no. PNU-2012-0086). Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions. Conﬂicts of Interest: B.B.R.C. and G.-C.K. are an employee of Feagle Co., Ltd. The authors declare no conﬂict of interest.

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