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Case Report Histologic Analysis of Clinically Healthy Human Gingiva in Patients with Altered Passive Eruption

Andrea Pilloni 1, Lorenzo Marini 1,*, Blerina Zeza 2, Amedeo Ferlosio 3 and Rustam Aghazada 1

1 Section of Periodontics, Department of Oral and Maxillofacial Sciences, “Sapienza” University of Rome, 00161 Rome, Italy; [email protected] (A.P.); [email protected] (R.A.) 2 Division of , Department of Dentistry, Albanian University, 1001 Tirana, Albania; [email protected] 3 Anatomic Pathology, Department of Biomedicine and Prevention, Tor Vergata University of Rome, 00133 Rome, Italy; [email protected] * Correspondence: [email protected]; Tel.:+39-0649-918152; Fax: +39-0644-230811

Abstract: The purpose of this study was to histologically examine the clinically healthy gingiva of patients with altered passive eruption (APE). Five patients with type 1 APE were enrolled. They underwent scaling and polishing and received instructions. After 6 months of supervised plaque control and uninterrupted gingival clinical health (Gingival Index (GI) = 0 and no (BoP)), upper anterior teeth were surgically treated. During the surgical procedure, the excised was collected to be histologically examined. In four out of five patients, signs of inflammation including spongiosis and neutrophil exocytosis could be found in the of the . Ulceration with exposure of the lamina propria and inflammatory granulation   tissue were evident in the most severe cases. Normal density and orientation of collagen fibers could be seen within the superficial and the deep portions of connective tissue, with an increase in size and Citation: Pilloni, A.; Marini, L.; Zeza, number of the deep collagen fibers and a reduced laxity of the superficial ones. In conclusion, the B.; Ferlosio, A.; Aghazada, R. Histologic Analysis of Clinically clinically healthy gingiva of APE patients showed features compatible with persistent inflammation, Healthy Human Gingiva in Patients possibly due to recurrent traumatisms caused by an incisally placed gingival margin. with Altered Passive Eruption. Dent. J. 2021, 9, 29. https://doi.org/ Keywords: anatomy; gingiva; health; histology; inflammation; ; eruption 10.3390/dj9030029

Academic Editors: Jiiang-Huei Jeng and Claude Jaquiéry 1. Introduction Altered passive eruption (APE) is a highly prevalent condition, occurring in 12.1% Received: 6 January 2021 and 35.8% of the population depending on the diagnostic criteria [1,2]. It is defined as Accepted: 2 March 2021 a situation in which “the gingival margin in the adult is located incisal to the cervical Published: 6 March 2021 convexity of the and removed from the cemento–enamel junction of the tooth” [3]. APE was classified by Coslet et al. [4] into a type 1 or 2, depending on the location of the Publisher’s Note: MDPI stays neutral in relation to the alveolar bone crest. While in the type 2 the distance with regard to jurisdictional claims in between the gingival margin and the mucogingival line is within the normally accepted published maps and institutional affil- width (3.0–4.2 and 2.5–2.6 mm in the maxilla and in the mandible, respectively) and the iations. mucogingival line is at the level of the cemento–enamel junction (CEJ), in the type 1 the band of attached gingiva is wider and the mucogingival line is apical to the alveolar ridge. Each type was further divided into a subgroup A or B, based on the location of the alveolar bone crest with respect to the CEJ. In the subgroup A the distance between the bone crest Copyright: © 2021 by the authors. and the CEJ is normal (1–2 mm), whereas in the subgroup B the bone crest is at the level of Licensee MDPI, Basel, Switzerland. or coronal to the CEJ. This article is an open access article Even though the gingiva of the patients with APE is usually healthy in the absence distributed under the terms and of plaque, several studies have related APE to periodontal health, assuming that APE is conditions of the Creative Commons a possible risk factor for . Coslet et al. [4] theorized that APE type Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 2A (because the gingiva is unsupported by connective tissue fibers) and APE subtype 4.0/). B (because of the absence of collagen bundles of the gingival apparatus) are susceptible

Dent. J. 2021, 9, 29. https://doi.org/10.3390/dj9030029 https://www.mdpi.com/journal/dentistry Dent. J. 2021, 9, 29 2 of 9

to periodontal breakdown. Prichard [5] hypothesized that the higher risk of develop- ing periodontal disease in APE patients is due to an incisally placed gingival margin, which causes constant and repeated trauma and subsequent chronic inflammation of a bulbous marginal gingiva. Weinberg and Eskow [6] suggested that excessive keratinized mucosa may cause pseudopockets and interfere with adequate oral hygiene, determin- ing the consequential plaque accumulation and inflammatory response. Volchanky and Cleaton-Jones [1] reported an association between APE and acute necrotizing ulcerative . The authors argued that the association with this infectious disease could be due to the anaerobic environment predisposed by the presence of a deep gingival sulcus. Recently, Aghazada et al. [7] used a 21-day experimental gingivitis model to evaluate the inflammatory response of patients with APE compared to patients with normal gingival anatomy. In the presence of comparable amounts of plaque deposits, APE patients were observed to exhibit early development and delayed resolution of inflammation. The gingival tissue structure is composed of a stratified epithelial tissue overlying a densely collagenous lamina propria. The epithelium facing the oral cavity constitutes the oral epithelium while the portion facing the tooth represents the , which continues with the . The lamina propria comprises the supra-alveolar fiber apparatus, blood and lymphatic vessels and nerves. In the last decades, an attempt was made to distinguish between strictly normal and clinically healthy gingiva [8]. While strictly normal gingiva was considered an artifact that could be present under experimental circumstances, clinically healthy gingiva could exist in the presence of a physiological microbial and antigenic challenge. The clinically healthy gingiva shows a defensive ar- chitecture with the following characteristics: (i) a sulcular and a junctional epithelium with enlarged intercellular spaces, in which a considerable number of neutrophiles and crevicular fluid are present; (ii) a small area of macrophages, lymphocytes and plasma cells in the context of the lamina propria and a subepithelial plexus of venules [9]. On average, buccal gingiva is composed of 27% oral epithelium, 4% junctional epithelium and 69% connective tissue [10]. In the context of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions, a great effort was made to review the clinical and histological determinants of periodontal health. In fact, defining periodon- tal health was considered of fundamental importance to have a common reference point for evaluating periodontal disease and defining significant treatment outcomes [11]. Based on previous studies, it was stated that, in humans, a condition of pristine or clinically healthy gingiva with normal anatomy, even after a 6-month period of supervised oral hygiene practices, always histologically presents a slight inflammatory cell infiltrate [12,13]. It was reiterated that this is due to polymorphonuclear leukocyte physiological surveillance rather than a pathological process [11]. To the best of our knowledge, no study histologically described a clinically healthy gingiva in patients with APE. However, this information could be relevant since it could elucidate if in patients with this condition there are histological findings that could jus- tify the above-mentioned assumptions of a higher susceptibility for periodontal disease. Therefore, the aim of this study was to histologically examine the gingival margin excised during the surgical treatment of patients with APE.

2. Materials and Methods 2.1. Study Design For this histological human study, five patients requiring surgical treatment for altered passive eruption of the teeth comprised in the second sextant were enrolled. In the context of the surgical procedures, the gingival collar excised after the submarginal and sulcular incisions was collected to be histologically examined (Figure1). The present investigation was conducted at the Section of Periodontics of the Depart- ment of Oral and Maxillofacial Sciences, “Sapienza” University of Rome, Rome, Italy from June 2017 to December 2017. Dent.Dent. J. J.20212021, 9,, 9x, FOR 29 PEER REVIEW 3 of3 of9 9

A

B

C

D

FigureFigure 1. 1.StudyStudy design. design. (a) ( APatients) Patients with with type type 1 altered 1 altered passi passiveve eruption eruption (APE) (APE) were were selected. selected. (b) (B) AfterAfter 6 months 6 months of ofsupervised supervised plaque plaque control control and and uninterrupted uninterrupted gingival gingival clinical clinical health, health, patients patients werewere surgically surgically treated. treated. Submarginal Submarginal an andd sulcular sulcular incisions incisions were were performed. performed. (c (C) The) The gingival gingival col- collar larcomprising comprising the the gingival gingival margin margin was was removed removed and and fixed fixed in in 10% 10% neutral neutral buffered buffered formalin. formalin. ( D(d)) The Theexcised excised tissue tissue was was histologically histologically analyzed. analyzed.

2.2.The Ethical present Consideration investigation was conducted at the Section of Periodontics of the Depart- ment ofThe Oral study and protocolMaxillofacial was approvedSciences, “Sapienza” by the Ethical University Committee of Rome, of “Sapienza” Rome, Italy University from Juneof Rome 2017 to on December 11 November 2017. 2015 (Prot. 2448/15). All patients signed a written informed consent document prior to participation. The investigation was performed in accordance 2.2.with Ethical the DeclarationConsideration of Helsinki of 1975, as revised in 2013 [14]. The study protocol was approved by the Ethical Committee of “Sapienza” University 2.3. Participants of Rome on 11 November 2015 (Prot. 2448/15). All patients signed a written informed con- sent documentPatients prior with typeto participation. 1 and subtype The A/Binvestigation altered passive was performed eruption in affecting accordance the upperwith theanterior Declaration teeth of requiring Helsinki surgical of 1975, treatmentas revised forin 2013 esthetic [14]. concerns were selected for this study, according the following exclusion criteria: (1) <18 years; (2) presence of systemic 2.3.diseases; Participants (3) cigarette consumption; (4) presence of any edentulism in the anterior upper arch; (5) presence of attachment loss; (6) presence of probing depth (PD) > 3 mm; (7) Patients with type 1 and subtype A/B altered passive eruption affecting the upper intake of drugs with a known effect on the gingival inflammatory response to oral biofilm anterior teeth requiring surgical treatment for esthetic concerns were selected for this (e.g., phenytoin, calcium channel blockers and cyclosporine); (8) history of periodontal study, according the following exclusion criteria: (1) <18 years; (2) presence of systemic surgical treatment; (9) pregnant or lactating; (10) the CEJ was not measurable with a diseases; (3) cigarette consumption; (4) presence of any edentulism in the anterior upper ; (11) presence of gingival overgrowth/hyperplasia, or inflammation; arch;and (5) (12) presence presence of ofattachment anatomical loss; alterations (6) presence of the of toothprobing crown depth (e.g., (PD) restorations, > 3 mm; (7) attrition, intake ofor drugs traumatic with a injury). known effect on the gingival inflammatory response to oral biofilm (e.g., phenytoin,Diagnosis calcium of APEchannel was blockers performed and clinically cyclosporine); and confirmed (8) history radiographically. of periodontal surgical Prelimi- treatment;narily, patients (9) pregnant with teeth or lactating; with short (10) clinical the CEJ crowns was andnot excessivemeasurable gingival with a display periodontal (EGD) probe;were examined.(11) presence Then, of gingival a differential overgrowth diagnosis/hyperplasia, to distinguish or APEinflammation; from other and conditions (12) pres- that encepresent of anatomical comparable alterations clinical features of the tooth was accomplishedcrown (e.g., restorations, (e.g., hypermobile attrition, upper or traumatic , short injury).upper lip, teeth attrition, excessive upper jaw growth and dentoalveolar upper extrusion).

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For the clinical investigation, a periodontal probe was employed. If the distance from the gingival margin to the CEJ was > 2 mm, the diagnosis of APE was made. [2,7]. For the radiographic analysis, a long-cone, parallel technique, periapical radiograph was used together with a gutta-percha cone to detect the gingival margin. The magnification of the image was adjusted by matching the real length of the gutta-percha cone with its length on the radiograph. The true length of the anatomical crown was estimated by measuring the distance between the CEJ and the gingival margin. The diagnosis of APE was confirmed if the length of the clinical crown (gingival margin to incisal edge) was significantly different (≥3 mm) to the length of the radiographic crown (CEJ to incisal edge) [15]. To discriminate between APE type 1 and 2, the attached gingiva was examined. Type 1 APE was diagnosed when the band of keratinized gingiva (from mucogingival junction to the gingival margin) was wider than a normally recognized mean width of 3.0–4.2 mm in the upper arch [16,17]. To distinguish between APE subtype A and B, “bone sounding”, under anesthesia, was used. A diagnosis of APE subtype A was made when the transgingival probing revealed a subgingivally positioned CEJ. Furthermore, the diagnosis of APE subtype A was certified when two distinct lines were identified in the radiographs (the most apical for the bone crest and the other for the CEJ) [15].

2.4. Pre-Surgical Procedures Prior to the surgical treatment, patients underwent professional scaling and polishing to achieve gingival health and standardize baseline conditions. A medium (Elmex Inter X, GABA International AG, Muenchenstein, Switzerland), standard toothpaste (Elmex, GABA International AG) and unwaxed dental floss (Elmex, GABA International AG) were provided. Polishing and oral hygiene instructions were repeated until patients were clinically healthy, as characterized by the absence of Bleeding on Probing (BoP) [18] measured on 6 sites and a Gingival Index (GI) [19] score of 0. Both of these assessments were made at the level of the upper anterior teeth (canine to canine). Patients were scheduled to have follow-up visits at 6 months and were carefully supervised for plaque control and optimal gingival health. After 6 months of uninterrupted clinical health, they were surgically treated.

2.5. Surgical Procedures As suggested by Garber and Salama [20], in cases of altered passive eruption type 1A, a simple to expose the hidden anatomy of the crown was performed; in the cases of type 1B, an apically repositioned full-thickness flap with osseous resective surgery was made. In all of the cases, a first submarginal incision was made on the buccal surface of each papilla, leaving the interproximal tissue totally intact. Then, a sulcular incision was executed. The remaining gingival collar was excised with a . The removed tissue was immediately fixed in 10% neutral buffered formalin and the surgical treatment was continued and finally completed.

2.6. Histological Analysis After being fixed in 10% neutral buffered formalin for 24 h, the samples were oriented through the identification of the oral epithelium and perpendicularly sectioned along the longitudinal diameter by 2 mm cuts. Each slide comprised the lining epithelium and the underlining lamina propria. All samples were embedded in paraffin wax and serial sections of 4 µm were cut at different levels and subsequently stained from each block with haematoxylin and eosin. The slices were examined under light microscopy with Nikon Eclipse E1200 and pictures taken by means of a Nikon camera system. Microscopically, the following have been evaluated: (i) the integrity of epithelium or the presence of erosion/ulceration; (ii) the presence and characteristic of inflammatory infil- tration (i.e., acute with neutrophils or chronic with lymphocytes and plasma cells); (iii) the orientation of collagen fibers and characteristic of capillary vessels have been considered. Dent. J. 2021, 9, x FOR PEER REVIEW 5 of 9

haematoxylin and eosin. The slices were examined under light microscopy with Nikon Eclipse E1200 and pictures taken by means of a Nikon camera system. Microscopically, the following have been evaluated: (i) the integrity of epithelium or the presence of erosion/ulceration; (ii) the presence and characteristic of inflammatory in- filtration (i.e., acute with neutrophils or chronic with lymphocytes and plasma cells); (iii) the orientation of collagen fibers and characteristic of capillary vessels have been consid- ered.

Dent. J. 2021, 9, 29 5 of 9 3. Results Five patients were treated, and the gingival collar excised during each surgical pro- 3.cedure Results was histologically examined. The study population consisted of four females and oneFive male, patients aged were 19 treated,to 27 years and the with gingival a mean collar age excised at baseline during each of surgical22.75 years proce- ± 3.03. Two pa- duretients was received histologically a diagnosis examined. of The type study 1 subgroup population consistedA APE, ofwhereas four females three and received one a diagnosis male,of type aged 1 19subgroup to 27 years B. with a mean age at baseline of 22.75 years ± 3.03. Two patients receivedAt a diagnosisthe 6-month of type follow-up 1 subgroup visits A APE, and whereas the day three of received the surgery, a diagnosis all ofpatients type exhibited GI 1 subgroup B. = 0 Atand the no 6-month BoP measured follow-up visits on andsix sites the day at of the the level surgery, of allthe patients upper exhibited anterior GI teeth. = 0 and noFour BoP measured out of five on six patients sites at theshowed level of histological the upper anterior features teeth. comparable to gingivitis with differentFour out levels of five of patients severity. showed histological features comparable to gingivitis with different levels of severity.

3.1.3.1. Epithelial Epithelial Tissue Tissue TheThe sulcular sulcular epithelium epithelium exhibited exhibited characteristic characteristic signs of inflammation signs of inflammation comprising comprising neutrophilneutrophil exocytosis exocytosis and spongiosis and spongiosis (Figure2 a,b).(Figure 2a,b).

FigureFigure 2. 2.Light Light microscopy microscopy of 4 µ ofm 4 sections µm sections of the epithelial of the epithelial and connective and connective tissues of a patient tissues of a patient withwith APE APE and and stained stained with with haematoxylin haematoxylin and eosin. and (eosin.a) Histological (a) Histological section corresponding section corresponding to to the thegingival gingival margin margin at aa magnificationmagnification of 4of× .(4×.b )(b Enlargement) Enlargement of the of rectangular the rectangu arealar in Aarea at a in A at a magnifi- magnificationcation of 10×. of 10 In× .the In the sulcular sulcular epit epitheliumhelium (circled (circled area), area), note thenote typical the ty signspical of inflammation,signs of inflammation, such suchas spongiosis as spongiosis and and neutrophil neutrophil exocytosis. OE OE = = oral oral epithelium; epithelium; SE =SE sulcular = sulcular epithelium; epithelium; SCT = SCTsuperficial = superficial layer layer of of connective connective tissue;tissue; DCT DCT = deep = deep layer layer of connective of connective tissue. tissue. Ulceration with exposure of the underlying lamina propria and proliferation of capil- laries, distinctiveUlceration signs with of anexposure inflammatory of the granulation underlyin tissue,g lamina were evidentpropria in and the most proliferation of ca- severepillaries, cases distinctive (Figure3). signs of an inflammatory granulation tissue, were evident in the most severe cases (Figure 3).

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Figure 3. Light microscopy of 4 µm sections of the sulcular epithelial tissue of a patient with APE and stained with haematoxylin and eosin. In the circled area, it is possible to observe an extensive ulceration of the epithelium, a sign of severe inflammation at a magnification of 10×.

The gingival epithelium exhibited areas of reactive hyperplasia with increased ex- FigureFigurepression 3. Light3. Lightof microscopythe microscopy rete pegs of 4 µ mand of sections 4 acanthosis,µm ofsections the sulcular associatedof the epithelial sulcular with tissue epithelial ofvarying a patient tissue degrees with APE of a of patient intensity with of APE a andandchronic stained stained lympho-plasmacellular with with haematoxylin haematoxylin and eosin. and Ininflammatory theeosin. circled In area,the circled itinfi is possibleltrate area, (from to it observe is possibleperivascular an extensive to observe to diffuse an extensive infil- ulceration of the epithelium, a sign of severe inflammation at a magnification of 10×. ulcerationtrates) (Figure of the 4). epithelium, a sign of severe inflammation at a magnification of 10×.

The gingival epithelium exhibited areas of reactive hyperplasia with increased ex- pression of the rete pegs and acanthosis, associated with varying degrees of intensity of a chronic lympho-plasmacellular inflammatory infiltrate (from perivascular to diffuse infil- trates) (Figure 4).

FigureFigure 4. 4.Light Light microscopy microscopy of 4 µm of sections 4 µm ofsections the sulcular of the epithelial sulcular tissue epithelial of a patient tissue with APE of anda patient with APE stainedand stained with haematoxylin with haematoxylin and eosin. Inan thed eosin. circled area,In the an circled obvious area, chronic an inflammatory obvious chronic plasma inflammatory cellplasma infiltrate cell is infiltrate present, at is a magnificationpresent, at a of magnification 10×. of 10×. 3.2. Connective Tissue 3.2. TheConnective collagen fibersTissue maintained a normal orientation and a regular difference in density according with their position in the connective tissue. In particular, in the deep layer of FigureThe 4. Lightcollagen microscopy fibers maintained of 4 µm sections a normal of the orie sulcularntation epithelial and a regular tissue of difference a patient with in den- APE thesity sub-epithelial according connectivewith their tissue, position collagen in the fibers conne werective denser tissue. and more In particular, parallel to the in the deep layer gingivaland stained epithelium. with haematoxylin The less numerous and and eosin. thinner In the fibers circled of the area, superficial an obvious layer showed chronic inflammatory aplasmaof “sunburst” the sub-epithelial cell patterninfiltrate with is connectivepresent, respect to at the atissue, magnification root surface collag anden of thefibers10×. alveolar were bone denser but withand amore parallel to perpendicularthe gingival fashion epithelium. related to theThe gingival less numerous lining epithelium and (Figurethinner2a). Infibers the superficial of the superficial layer layer3.2.showed ofConnective the a connective “sunburst” Tissue tissue pattern there were with elongated respect capillaries, to the whileroot insurface the deep and layer the there alveolar bone but werewith arterioles a perpendicular with orientation fashion parallel related to the to collagen the gingival fibers. Particular lining aspectsepithelium could be(Figure 2a). In the foundThe in the collagen increased fibers number maintained and size of the a deepnormal collagen orie fibers,ntation due and to a substantiala regular difference in den- sclerotizationsitysuperficial according (Figurelayer with of5), andthetheir theconnective position lower laxity intissue the of the connethere superficial ctivewere ones.tissue. elongated Moreover, In particular, capillaries, in the in while the deep in the layer scleroticofdeep the layer areas,sub-epithelial there a reduced were number connective arterioles of vessels with tissue, were orientatio present.collagenn parallel fibers wereto the denser collagen and fibers. more Particular parallel to aspects could be found in the increased number and size of the deep collagen fibers, due the gingival epithelium. The less numerous and thinner fibers of the superficial layer to a substantial sclerotization (Figure 5), and the lower laxity of the superficial ones. More- showed a “sunburst” pattern with respect to the root surface and the alveolar bone but over, in the sclerotic areas, a reduced number of vessels were present. with a perpendicular fashion related to the gingival lining epithelium (Figure 2a). In the superficial layer of the connective tissue there were elongated capillaries, while in the deep layer there were arterioles with orientation parallel to the collagen fibers. Particular aspects could be found in the increased number and size of the deep collagen fibers, due to a substantial sclerotization (Figure 5), and the lower laxity of the superficial ones. More- over, in the sclerotic areas, a reduced number of vessels were present.

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FigureFigure 5. 5.Light Light microscopy microscopy of 4 µm of sections 4 µm showingsections the showing substantial the sclerotization substantial of thesclerotization deep of the deep collagencollagen fibers, fibers, at a magnification at a magnification of 10×. * = of sclerotic 10×. areas.* = sclerotic areas. 4. Discussion 4. DiscussionPeriodontal health could be thought of as a stable periodontium that functions com- fortablyPeriodontal in a person with health psychological could be and thought social well-being of as a stable about theirperiodontium mouth [21]. that functions com- Biological, environmental, systemic, economic, social and psychological factors could all havefortably an influence in a person on an individual’s with psychological periodontal health and [21 social]. well-being about their mouth [21]. Bio- logical,APE wasenvironmental, suggested to be systemic, an anatomical economic, condition thatsocial could and negatively psychological affect pe- factors could all have riodontalan influence health [on1,4– an6]. However,individual’s only one periodontal clinical study health was carried [21]. out to clarify the hypothesis of a higher predisposition for periodontal diseases in patients with this condi- tion. AghazadaAPE was et al.suggested [7] conducted to abe study an anatomical using an experimental condition gingivitis that modelcould and negatively affect perio- comparingdontal health patients [1,4–6]. with APE However, to patients withoutonly one APE. clinical The first study group ofwas patients carried was out to clarify the hy- morepothesis prone toof developa higher gingival predisposition inflammation whenfor periodontal plaque was allowed diseases to accumulate in patients on with this condition. their teeth than the second. In addition, once instructed to resume oral hygiene procedures, theAghazada resolution ofet theal. inflammation[7] conducted took a longer study in theusing APE an group experimental than in patients gingivitis with model and com- normalparing gingival patients anatomy. with APE to patients without APE. The first group of patients was more proneOur to investigation develop isgingival the first histological inflammation study performed when plaque to identify was specific allowed features to accumulate on their of the clinically healthy gingival margin of the patients with APE, aiming to provide fur- therteeth information than the to second. understand In addition, this little-known once clinical instructed entity. to In theresume literature, oral there hygiene procedures, the areresolution few studies of on the histologic inflammation analysis of took human longer gingiva in withthe APE normal group anatomy. than Brecx in patients with normal etgingival al. [12] evaluated anatomy. the cellular composition of developing infiltrated connective tissue in volunteers enrolled in a 21-day experimental gingivitis trial. As the clinical parameter for inflammationOur investigation (GI) increased, is the the first infiltrated histological connective study tissue showedperformed a substantial to identify specific features increaseof the inclinically lymphocytes healthy (from 17.0% gingival to 29.9%) margin associated of withthe apatients reduction inwith the numericalAPE, aiming to provide fur- densityther information of fibroblasts (from to understand 48.1% to 34.9%). this No little-know significant differencesn clinical in entity. the numerical In the literature, there are density of polymorphonuclear leukocytes was observed (20.8% to 22.6%). In a second study,few studies the gingival on tissue histologic of five dental analysis hygienists of human with clinical gingiva indices forwith plaque normal and GI anatomy. Brecx et al. close[12] toevaluated zero was analyzed the cellular at 0, 1, composition 4, and 6 months. of During developing this long-standing infiltrated optimal connective tissue in vol- oralunteers hygiene enrolled regime, the in volumetric a 21-day density experimental of infiltrated connectivegingivitis tissue trial. progressively As the clinical parameter for decreased. In particular, the numerical density of lymphocytes within the infiltrate de- creasedinflammation considerably (GI) (from increased, 18.4% to 5.6%), the whereas infiltrated the numerical connective density tissue of fibroblasts showed a substantial in- increasedcrease in (from lymphocytes 57.7% to 71.0%). (from As in 17.0% the previous to 29.9%) investigation, associated the numerical with densitya reduction of in the numerical polymorphonucleardensity of fibroblasts leukocytes (from was rather48.1% stable to 34.9%). (from 20.6% No to significant 17.7%) [13]. Both differences these in the numerical studies indicated that a small inflammatory cell infiltrate is always present in clinically healthydensity gingiva of polymorphonuclear with normal anatomy. leukocytes was observed (20.8% to 22.6%). In a second study,In the the present gingival study, thetissue increase of five in the dental number hygienists and size of the with deep clinical collagen fibers,indices for plaque and GI dueclose to ato considerable zero was sclerotization, analyzed at and 0, the1, 4, lower and laxity 6 months. of the superficial During fibers this didlong-standing not optimal oral seem to substantially differ from the studies by Brecx et al. [12,13], probably due to the prolongedhygiene statusregime, of clinical the volumetric health. However, density as an alternativeof infiltrated explanation, connective there could tissue progressively de- creased. In particular, the numerical density of lymphocytes within the infiltrate de- creased considerably (from 18.4% to 5.6%), whereas the numerical density of fibroblasts increased (from 57.7% to 71.0%). As in the previous investigation, the numerical density of polymorphonuclear leukocytes was rather stable (from 20.6% to 17.7%) [13]. Both these studies indicated that a small inflammatory cell infiltrate is always present in clinically healthy gingiva with normal anatomy. In the present study, the increase in the number and size of the deep collagen fibers, due to a considerable sclerotization, and the lower laxity of the superficial fibers did not seem to substantially differ from the studies by Brecx et al. [12,13], probably due to the prolonged status of clinical health. However, as an alternative explanation, there could be an increase in collagen synthesis or abnormal fibroblast function in patients with APE. On the other hand, in four out of five cases, the histological analysis of the epithelium of pa-

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be an increase in collagen synthesis or abnormal fibroblast function in patients with APE. On the other hand, in four out of five cases, the histological analysis of the epithelium of patients with APE showed features compatible with gingivitis which contrast with the condition of physiological immune surveillance observed in the above-mentioned investigations [12,13]. This exacerbated inflammatory response at the epithelial level could be justified, in the absence of plaque deposits and pseudopockets, by an incisally placed gingival margin which reduces the defensive capacity of the periodontium against recurrent traumatism, as proposed by several studies [22–24]. The results of this analysis could represent a possible explanation for the assumed predisposition of patients with APE for periodontal pathologies. Even in the absence of microbial biofilm-induced inflammation, the APE condition may create a “circle” that would maintain a gingival irritation over time with subsequent difficulties in maintaining domiciliary oral hygiene and further plaque accumulation. In this study, the status of clinical health was ensured by the absence of BoP and GI = 0 from baseline until the surgical treatment of patients. Moreover, patients did not have probing depths > 3 mm or attachment loss. These criteria agree with the definitions of periodontal health proposed by the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions [11]. A time lapse of 6 months occurred from the moment that patients were clinically healthy and the surgical treatment was selected, since it was observed that a persistent excellent oral hygiene condition is essential for any histological decrease in the inflammatory infiltrate [13]. Treatment of APE is basically surgical and consists of a crown-lengthening procedure. Two different approaches were described by Gaber and Salama [20] depending on the APE subtype and subgroup. In the type 1 subgroup A, the preferred option is the gingivectomy. In the type 2, the elective treatment is an apically repositioned full-thickness flap with, in the subgroup A, osseous resective surgery. Literature regarding the treatment of APE is scarce and merely discusses the outcomes of surgical procedures aimed at improving aesthetics. In this regard, Cairo et al. [25] showed that a carefully pre-operative planning and periodontal plastic surgery with or without osseous resective surgery can achieve predictable results. To date, no studies evaluated the potential beneficial effect of the treatment of APE on periodontal health. However, the findings of the present histological analysis are in agreement with the outcomes of our previous clinical study [7] and suggest that surgical restoration of normal gingival anatomy should be considered to reduce the increased tendency toward plaque-induced inflammation in patients with this condition. The analysis was not able to address any specific cause for the histological findings and was not able to detect any differences among patients based on age, gender, and APE subgroups. In fact, all patients showed similar histological characteristics. This could be probably due to the main limitations of the present study: (i) the enrollment of only five subjects for this investigation, even though the same number of patients was selected in a comparable research [13]; (ii) patients with type 2 APE were excluded from this study, because the surgical treatment of these patients would not allow for submarginal incisions and the removal of a gingival collar for the histological examination of the gingival margin, lacking an adequate width of keratinized gingiva; (iii) the absence of a control site or group with gingiva with normal anatomy. For these reasons, case-control studies with a larger sample size are needed to confirm the results of the present study. Furthermore, the investigation of the biomolecular features of healthy, inflamed, wounded and unwounded human gingiva in APE patients is encouraged, to clarify if the healing process is somehow impaired in this specific population, leading, for example, to keloids or scar formation.

5. Conclusions In their limits, the results of this first histologic study of the gingiva of APE patients show signs of inflammation even after an extended time interval of uninterrupted peri- odontal health. Dent. J. 2021, 9, 29 9 of 9

Author Contributions: Conceptualization, A.P. and R.A.; methodology, L.M., B.Z. and R.A.; investi- gation, A.F. and R.A.; data curation, A.F. and R.A.; writing—original draft preparation, L.M., B.Z. and R.A..; writing—review and editing, A.P. and A.F.; visualization, L.M.; supervision, A.P.; project administration, R.A. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Sapienza, University of Rome (protocol code 2448/2015). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. 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 and ethical restrictions. Conflicts of Interest: The authors declare no conflict of interest.

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