PEDIATRIC DENTAL JOURNAL 18(1): 57–63, 2008 57

Case Report Evaluation of transitional changes of sub-gingival bacterial species in twins with gingival fibromatosis

Eriko Miyamoto, Kazuhiko Nakano, Ryota Nomura, Kazuyo Fujita, Rena Okawa and Takashi Ooshima

Department of Pediatric , Osaka University Graduate School of Dentistry 1-8 Yamada-oka, Suita, Osaka 565-0871, JAPAN

Abstract Gingival fibromatosis is a condition of uncommon gingival over- Key words growth, with hereditary causes regarded to be associated with the most common Gingival fibromatosis, form. The condition is generally non-inflammatory, though secondary gingival Inflammation, inflammation occurs in some cases due to formation of anaerobic spaces Polymerase chain reaction, between gingiva and teeth. We present a case of identical twin brothers aged 11 Subgingival plaque, years 9 months who came to our clinic with complaints of gingival esthetic Twins problems. They were both diagnosed with hereditary gingival fibromatosis and were performed. At 14 years 11 months of age, they returned with gingival swelling, though the inflammation and corresponding conditions were significantly different from those seen at the first visit, as the older twin showed severe gingival inflammation, while the younger had moderate inflam- mation. A microbiological analysis was carried out using a PCR technique, which specifically identified , , and Tannerella forsythensis in plaque samples taken from sites of severe inflammation in both patients. The numbers of periodontitis-related species decreased as gingival conditions improved with treatment, including removal of and tooth brushing instruction, and microbiological findings were correlated with clinical conditions. Our findings show that monitoring of periodontitis-related bacterial species is beneficial for evaluating the effects of periodontal treatment with a scientific basis.

Introduction between the gingiva and teeth caused by gingival overgrowth, which provides a favorable environment Gingival fibromatosis is a condition of uncommon for the colonization of anaerobic periodontitis-related gingival overgrowth associated with increased levels bacterial species to display their virulence. of mature collagen, with hereditary causes regarded is commonly seen in children and to be associated with the most common genetic form, adolescents, while periodontitis rarely occurs in in which autosomal-dominant inheritance is reported teenage individuals5). Both conditions are considered to be the most frequently identified1,2). Gingival over- to be caused by virulent periodontal bacterial species growth caused by gingival fibromatosis is typically and several candidates have been analyzed. We non-inflammatory, with surgical removal generally surveyed past articles regarding the detection of carried out to solve esthetic problems in severe periodontal bacteria by PCR and selected 10 species, cases3,4). Another possible clinical complication in Porphyromonas gingivalis, Tannerella forsythensis, cases of gingival fibromatosis is the onset of gingival , Prevotella nigrescens, Campy- inflammation due to formation of anaerobic spaces lobacter rectus, Eikenella corrodens, Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, Received on June 12, 2007 ochracea, Capnocytophaga sputi- Accepted on February 19, 2008 gena, and Treponema denticola, based on results

57 58 Miyamoto, E., Nakano, K., Nomura, R. et al.

Fig. 1 Oral photographs of the twin brothers with gingival fibromatosis Shown are images from the first visit at 11Y9M of age (A and B, older and younger, respectively) as well as at 14Y1M (C and D, older and younger, respectively).

Fig. 2 Hematoxylin-eosin staining of excised gingival tissue (A) Older twin, (B) Younger twin

showing that their distribution in periodontitis as tooth brushing instruction. We also compared patients was significantly different from that in clinical and microbiological findings for the patients, healthy subjects6,7). In addition, the correlation of who both had gingival inflammation complicated specific bacterial species and periodontitis has been with gingival fibromatosis. analyzed, with the red complex species, P. gingi- valis, T. denticola, and T. forsythensis, known to be Case Report major causative agents of periodontitis and highly associated with its severity8,9). Identical twin brothers were referred to the Pedo- In the present study, those 10 periodontal dontics Clinic of Osaka University Dental Hospital bacterial species were monitored in identical twin with complaints of esthetic problems due to gingival brothers during treatment for gingival inflammation overgrowth at the age of 11 years 9 months (11Y9M) using conventional methods, such as mechanical (Fig. 1A and B). Intraoral examinations of both removal of and calculus, as well patients found non-inflammatory fibrous swelling, IDENTICAL TWINS WITH GINGIVAL FIBROMATOSIS 59

As a result, the numbers of teeth associated with gingival inflammation were reduced with the repeated periodontal treatments, and at 14Y8M the gingival conditions were improved in both patients and found to be stable at 14Y10M. Clinical parameters, including probing depth, (BOP), pus discharge, plaque index (PI)10), and gingival index (GI)11), were examined at each visit. Periodontal pocket depth (PD) was measured to the nearest millimeter at 6 points around the circumference of each tooth (mesio-, mid-, and disto-buccal; and disto-, mid-, and mesio-lingual) from the to the deepest probing point, using a round-ended probe (ם) tip 0.4 mm in diameter. BOP was scored as for no (מ) for immediate bleeding on probing, or for (ם) bleeding, while pus discharge was scored as .for no discharge (מ) spontaneous pus discharge or Microbiological analyses were performed using a method described previously6,7), following approval of the study protocol was approved by Ethics Committee of Osaka University Dental Hospital. Fig. 3 Orthopantomographic radiographs taken at 14Y1M Briefly, subgingival plaque samples were collected (A) Older twin, (B) Younger twin with sterile Gracey curettes from 6 representative teeth (maxillary right central incisor, maxillary right central canine, maxillary right first molar, mandibular which covered more than half of the tooth crowns left central incisor, mandibular left primary or of all maxillary and mandibular teeth. No systemic permanent canine and mandibular left first molar) diseases were present, though their mother also and suspended in sterile saline, then centrifuged to had a similar gingival overgrowth condition. We pellet the bacterial cells. Next, bacterial genomic diagnosed the brothers with hereditary gingival DNA was extracted from each pellet using a DNA fibromatosis and gingivectomies were performed isolation kit (Puregene, Gentra Systems, Minneapolis, under local anesthesia at their next visit. Histopatho- MN, USA). Ten periodontitis-associated bacterial logical examinations of excised gingiva specimens species, P. gingivalis, T. denticola, C. ochracea, C. showed the typical appearance of gingival fibro- sputigena, P. intermedia, P. nigrescens, A. actino- matosis (Fig. 2). After the esthetic problems were mycetemcomitans, T. forsythia, C. rectus, and E. cor- solved, the patients did not return to our clinic until rodens, were then analyzed by PCR using species- the age of 14Y1M, when they complained of gingival specific sets of primers. swelling with bleeding (Fig. 1C and D). Intraoral The correlations between clinical parameters examinations showed that the swelling was inflam- (PD, BOP, PI, and GI) and total number of the 10 matory, which was significantly different from that species were also analyzed utilizing 30 subgingival of the first visit. At that time, the older twin still plaque samples from each patient. All statistical had mixed dentition with 7 primary teeth identified, analyses were performed using computer software whereas only permanent teeth were identified in the (Prism 4.0, GraphPad Software, Inc., San Diego, younger twin. Further, orthopantomography revealed CA, USA). Correlations between the total number of that the dental age of the older twin was significantly the 10 bacterial species and PD, PI, and GI were younger than that of the younger twin (Fig. 3). analyzed by regression analysis, while those between We decided to perform conventional periodontal BOP and total number of the 10 bacterial species treatment for both patients, including mechanical and the parameters PD, PI, and GI were analyzed removal of dental plaque and calculus, as well as by a Mann-Whitney t-test. In addition, the presence tooth brushing instruction, at 2- to 3-month intervals. of a specific bacterial species in association with 60 Miyamoto, E., Nakano, K., Nomura, R. et al.

Fig. 4 Transitional evaluations of clinical parameters and detection of 10 periodontitis-related bacterial species from 6 representative teeth in the older (A) and 6 in the younger (B) brothers from the age of 14Y1M to 14Y10M a Locations are indicated as follows: UR1, maxillary right central incisor; UR3, maxillary right canine; UR6, maxillary right first molar; LL1, mandibular left central incisor; LLC, mandibular left primary canine; LL3, mandibular left canine; LL6, mandibular left first molar b Clinical parameters are indicated as follows: PD, periodontal pocket depth; BOP, bleeding on probing; Pus, pus discharge; PI, plaque index; GI, gingival index c The tested periodontal bacteria species are as follows: Pg, Porphyromonas gingivalis; Td, Treponema denticola; Co, Capnocytophaga ochracea; Cs, Capnocytophaga sputigena; Pi, Prevotella intermedia; Pn, Prevotella nigrescens; Aa, Aggregatibacter actinomycetem- comitans; Tf, Tannerella forsythensis; Cr, Campylobacter rectus; Ec, Eikenella corrodens Closed and open boxes indicate detected and not detected, respectively.

PD, PI, and GI was analyzed using a Mann-Whitney visit (11Y9M), when the patients showed non- t-test. Fisher’s exact probability test was used for inflammatory gingival swelling, revealed that only 2 analysis of the presence of specific species and BOP. of the tested species, T. denticola and P. nigrescens, were present in the older brother, while none were Results detected in the younger. In contrast, greater numbers of species were identified at 14Y1M, when both Microbiological examinations conducted at the first patients demonstrated gingival swelling derived from IDENTICAL TWINS WITH GINGIVAL FIBROMATOSIS 61

Fig. 5 Analysis of clinical parameters in locations where plaque samples were collected Three clinical parameters, periodontal pocket depth, plaque index, and gingival index, were compared sites (A, B and C), and between (51סand -negative (N (9סbetween bleeding on probing (BOP)-positive (N sites (D, E and F). A Mann-Whitney (48סand -negative (N (12סred complex species (RC)-positive (N t-test was performed for the analysis (*: PϽ0.05, **: PϽ0.01, ***: PϽ0.001).

inflammation rather than fibromatosis (Fig. 4). The respectively). In addition, the values for PD, PI, and average number of the 10 tested periodontitis-related GI were significantly greater in sites positive for bacterial species at 6 representative tooth sites was BOP as compared to those negative (Fig. 5A, B, and 3.8 in the older brother and 3.2 in the younger. C). As for the effects of the presence of red complex Conventional treatments for gingival inflammation species (P. gingivalis, T. denticola, and T. forsythensis), successfully led to a reduction in inflammation and PD and GI were significantly greater for teeth with the average numbers of bacterial species also tended at least 1 red complex species detected as compared to decrease as their gingival conditions improved. to those without any detected (Fig. 5D, E, and F), At 14Y10M, the average number of the 10 tested and the detection frequency of red complex species periodontitis-related bacterial species at 6 represen- was also significantly higher in positive BOP sites tative tooth sites were 1.3 and 0.8, in the older and than negative sites (PϽ0.001). younger brother, respectively. The clinical parameters PD, PI, and GI were Discussion positively correlated with the total number of species Correlations between the presence of specific bacterial ,0.0082סand P 0.34סat the corresponding sites (r species and periodontal conditions have been analyzed ,0.0006סand P 0.43סand r ,0.0003סand P 0.45סr 62 Miyamoto, E., Nakano, K., Nomura, R. et al. in several studies8,9,12). However, such analyses that patients were also positive for P. gingivalis, which is use clinical data tend to be influenced by individual considered to be the most important infectious agent differences, except for bacterial presence, thus causing several types of periodontal disease14). researchers prefer to collect data from as many P. gingivalis is classified into 6 genotypes (type I subjects as possible. In the present study, we to V and Ib) based on the fimA gene encoding analyzed identical twins who showed quite similar fimbrillin, a subunit of fimbriae, and types II and IV gingival swelling at the first visit (age 11Y9M), are frequently identified in periodontitis patients, indicating that the background conditions were very while subjects with a healthy gingival condition similar. Thereafter, the major difference between the have been found to carry the type I genotype at a subjects was the degree of gingival inflammation higher rate15,16). In the present patients, P. gingivalis identified at the age of 14Y1M. We considered that was positive in 6 samples from the older brother and the results of our comparison of bacterial species in 2 from the younger, all of which were classified as present in the twins indicated specific species type IV (data not shown), indicating that the twin associated with inflammation. brothers possessed disease-associated strains and This is the first known study to analyze the that the P. gingivalis organisms may have been the distribution of bacterial species using a molecular same clones. approach in patients with gingival fibromatosis. At Clinical parameters revealed gingival inflamma- the first visit, no inflammatory signs were detected tion and detection of bacterial species detection in and only a few species detected. In contrast, when subgingival locations in the present patients showed inflammatory conditions were investigated 2 years that PD, PI, and GI results were positively correlated 4 months following surgery in both with the total number of species at corresponding patients (age 14Y1M), the average number of the 10 sites. In addition, PD was shown to be greater with tested periodontitis-related bacterial species were 3.8 teeth with at least 1 red complex species detected and 3.2 in the older and younger brother, respectively. as compared to those without any detected. These Fortunately, conventional treatment for gingival results indicate that evaluation of these 10 bacterial inflammation, including scaling and tooth brushing species using a molecular approach can reveal instruction, successfully led to a reduction in the current clinical condition as well as provide inflammation and the average number of the 10 important information for estimation of prognosis. tested species also tended to decrease as the gingival conditions improved. At 14Y10M, the periodontal Acknowledgments conditions were significantly improved, and the average numbers of bacteria decreased to 1.3 and This study was supported by the 21st Century 0.8, respectively, indicating that the presence of COE program entitled “Origination of Frontier periodontitis-related bacterial species and clinical BioDentistry” at Osaka University Graduate School conditions may be correlated. of Dentistry supported by the Ministry of Education, The red complex species (P. gingivalis, T. denti- Culture, Sports, Science and Technology of Japan. cola, and T. forsythensis) are known to be highly associated with periodontitis severity8,9), which was References confirmed by the present findings. In another analysis of 107 Japanese subjects at 15 years of 1) Hart, T.C., Pallos, D., Bozzo, L., Almeida, O.P., age, T. forsythensis and C. rectus were proposed as Marazita, M.L., O’Connel, J.R. and Cortelli, J.R.: potential risk markers for the onset of periodontitis12). 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