gingival overgrowth induced by cyclosporine-A - Oral Surgery, Oral ...

Vol. 120 No. 3 September 2015

Impact of three different mouthwashes on the incidence of gingival overgrowth induced by cyclosporine-A: a randomized controlled experimental animal study Nesma Sultan Mohamed, BDS,a Rehab Rizk Abas El-Zehery, BDS, MSc, PhD,b Mohamed I. Mourad, BDS, MSc, PhD,c and Mohammed El-Awady Grawish, BDS, MSc, PhDd Objective. This study aimed to evaluate the effects of three different mouthwashes on the incidence of cyclosporineAeinduced gingival overgrowth. Study Design. One hundred albino rats were divided into five equal groups. Group I rats received no treatment. Group II rats were administered cyclosporine-A. Group III, IV, and V rats were handled as group II and concomitantly treated with chlorhexidine gluconate, cetylpyridinium chloride, and essential oil mouthwashes, respectively. Ten rats from each group were euthanized after the first week of treatment, and the remaining rats were euthanized after the second week of treatment. The obtained specimens were stained with hematoxylin and eosin, Masson’s trichrome, and tumor growth factor beta (TGF-b), matrix metalloproteinase 1 (MMP-1), and interleukin 6 (IL-6) immunohistochemical stains. Results. The histologic findings at the end of the first and second weeks revealed marked increases in gingival overgrowth for groups II and IV in comparison with groups III and V. Increased epithelial thickness and collagen accumulation were prominent in groups II and IV compared with the other groups. Groups II and IV revealed the highest immunoreactivities for TGF-b and for IL-6. Groups I, III, and V revealed the highest level of MMP-1 expression. Conclusions. Essential oils and chlorhexidine gluconate mouthwashes significantly reduced the incidence of gingival overgrowth compared with cetylpyridinium chloride. (Oral Surg Oral Med Oral Pathol Oral Radiol 2015;120:346-356)

Gingival overgrowth (GO) has many risk factors. Among these risk factors are hereditary risk factors, metabolic imbalances, and certain medications (phenytoin, an antiepileptic drug; nifedipine, a calcium channel blocker; and cyclosporine-A [CsA], an immunosuppressive drug).1 The potent immunosuppressive drug CsA is used to prevent graft rejection. The prevalence of GO ranges from 20% to 35% for CsAtreated patients. Certain risk factors directly correlate with and have an effect on the degree of GO. These factors include the level of plaque control, gender (males are three times more sensitive), daily drug dose, or inverse correlations, such as age.2 No effective and predictable method of managing this condition exists. Treatment may involve optional drug therapy, such as replacing cyclosporine with tacrolimus as an alternative immunosuppressant.3 Tooth brushing and interdental cleaning are the mechanical aids of treatment, and chemical plaque control agents have proven to be ideal adjuncts to mechanical

plaque control. Among these chemical plaque control agents is chlorhexidine (CHX), which is considered the gold standard because of its property of substantivity in preventing plaque formation. Mouthwashes composed of essential oils (EOs) have demonstrated greater reductions in supragingival plaque compared with cetylpyridinium chloride (CPC).4 To the best of our knowledge, no published papers have compared the potent antibacterial activity of EO mouthwashes with that of CHX. The antimicrobial actions of EOs are intimately associated with their major property, namely, hydrophobicity, which produces an increase in bacterial membrane permeability and the consequent loss of their primary cellular elements.5 In addition, CPC was shown to inhibit bacterial coaggregation, thus interfering with plaque maturation, and to inhibit insoluble glucan synthesis.6 The null hypothesis of the present study was that the effects of the three different mouthwashes on CsA-induced GO would not differ.

a

Demonstrator of Oral Biology, Faculty of Dentistry, Mansoura University, Egypt. b Assistant Professor of Oral Biology, Faculty of Dentistry, Mansoura University, Egypt. c Assistant Professor of Oral Pathology, Faculty of Dentistry, Mansoura University, Egypt. d Professor of Oral Biology, Faculty of Dentistry, Mansoura University, Egypt. Received for publication Feb 5, 2015; returned for revision Apr 28, 2015; accepted for publication May 10, 2015. Ó 2015 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2015.05.005

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Statement of Clinical Relevance In experiments performed in rats, essential oil and chlorhexidine gluconate mouthwashes significantly reduced the incidence of cyclosporine A-induced gingival overgrowth. The best results were for essential oil mouthwash. Cetylpyridinium chloride had no significant effect on drug-induced gingival overgrowth.

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MATERIALS AND METHODS Animals One hundred male, white albino rats weighing 150 to 200 g were selected. All experimental procedures were performed under the approved protocol of the ethical committee of the Faculty of Dentistry, University of Mansoura, Egypt. The rats were fed a commercial diet and water. The rats were divided randomly into five equal groups as follows: The negative control group (I) rats received no treatment. The positive control group (II) rats were administered CsA (30 mg/kg) daily for 2 weeks. The rats in groups III, IV, and V were administered the same dose of CsA as group II and concomitantly treated with CHX/G, CPC, and EO mouthwashes, respectively. The three mouthwashes were administered twice daily for the same period as CsA administration. Medicine supplementation A soft gelatinous capsule of CsA (100 mg, Sandimmune, Novartis Pharmaceuticals Corporation, Hanover, Germany) was dissolved in 16.5 cm3 corn oil. The dissolved drug was administered by gastric syringe; each rat received 30 mg/kg daily. CHX/G (Listermix, Sigma Pharmaceutical Industries, Egypt), CPC (Ezafluor, Cairo Pharmaceutical and Chemical Industries, Egypt) and EO (Listerine, Johnson & Johnson, Italy) mouthwashes were used topically. The method of mouthwash application was in accordance with that described by Gau et al. in 2013.7 The rats were handled properly by two operators; one operator controlled rat movement, and the other applied a small cotton pellet soaked in the mouthwash solution on the rat’s mandibular gingiva posteriorly for 30 seconds. The ingredients of the three different mouthwashes used in the study are illustrated in Table I. Biopsy collection Ten rats from each group were euthanized at the end of the first week of treatment, whereas the remaining rats were euthanized after the second week of treatment. First, the rats were anesthetized with a ketamine and valium mix (75 mg/mL ketamine, Parke Davis, Sydney, Australia, and 5 mg/mL valium, Roche Products Ltd., Selwyn Garden City, UK). Then, the rats were euthanized with an overdose of halothane. Scissors were used to cut the angles of the mouth backward between the upper and lower jaws, and the muscles were completely removed. Then, the lower jaw was dislocated by pulling it down, and biopsy specimens were collected by the following two methods: First, a spoon excavator was used to reflect the marginal gingiva, and then it was continually resected from the right side of the mandible opposite the molar region by using a

ORIGINAL ARTICLE Mohamed et al. 347

Table I. The three mouth washes used in the study and their ingredients Product name

Ingredients

CHX (Listermix)

Chlorhexidine gluconate, thymol, eucalyptol, menthol, clove oil Cetylpyridinium chloride, amine fluoride Aqua, ethanol denatured, sorbitol, poloxamer 407, benzoic acid, sodium saccharin, eucalyptol, methyl salicylate, aroma, thymol, menthol, sodium benzoate

CPC (Ezafluor) EO (Listerine)

CHX, chlorhexidine; CPC, cetylpyridinium chloride; EO, essential oils.

scalpel. Second, mandibular specimens from the left side of the mandible, including molar teeth with surrounding bone and gingival tissue, were cut in a buccolingual direction by using a scalpel. Routine tissue processing and staining The soft tissue specimens were fixed immediately in 10% formaldehyde in phosphate-buffered saline (PBS) for 24 hours. Specimens containing teeth were decalcified in 4% EDTA solution (Hexis Cientifica, Brazil) for approximately 2 weeks. The obtained specimens were sliced into 4-mm sections and stained with hematoxylin and eosin stain, Masson’s trichrome histochemical stain, and tumor growth factor beta (TGF-b; 1:50 dilution, cytoplasmic; AbD SeroTec, Killington, Oxford, UK), matrix metalloproteinase 1 (MMP-1; 1:50 dilution, cytoplasmic; Bioss Antibodies, Woburn, MA), and interleukin 6 (IL-6; 1:100 dilution, secreted protein; Bioss Antibodies, Woburn, MA) immunohistochemical stains. Evaluation and scoring Double-blinded calibrated examiners performed the microscopic analysis of the buccal gingiva of decalcified specimens by using a light microscope (Olympus, New York). Each examiner had approximately 15 years of experience in investigating biopsy specimens under a light microscope in their practical laboratories. One expert examiner read the specimens, and 2 days later, the same examiner read all the specimens again to evaluate intraexaminer variability. Then, the same specimens were read by the second examiner to assess the interexaminer variability (Table II). A modified scoring method was used according to the index described by Banthia et al.8 in 2014. The crown length was divided into three equal parts (cervical, middle, and occlusal). The hyperplastic index measured the degree of gingival enlargement in an apico-coronal direction by means of the following four-point scale: 0 ¼ no gingival overgrowth (gingiva still in the cervical one-third).

ORAL MEDICINE 348 Mohamed et al.

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Table II. Statistical results for intraexaminer and interexaminer variability attributed to scored samples Intraexaminer variability Features Degree of gingival overgrowth Shape of gingival crest

First reading Mean SD

Second reading Mean SD

2.9 0.8 0.3 0.1

Interexaminer variability P value

First reading Mean SD

Second reading Mean SD

P value

2.7 0.7

.600

0.3 0.7

0.3 0.5

.726

0.2 0.1

.153

0.5 0.5

0.6 0.5

.661

1 ¼ mild (gingiva overgrown to the middle onethird). 2 ¼ moderate (gingiva overgrown to the lower half of the occlusal one-third). 3 ¼ severe (gingiva overgrown to the upper half or above the occlusal one-third) (Figure 1) Moreover, the shape of the gingival crest was scored as knife-edge (0), blunted (1), or bulbous shaped (2). Five different fields of each slide of the outer surface of the buccal gingiva were imaged at 40 magnification using an Olympus digital camera installed on an Olympus microscope with a 1/2 photo adaptor using 40 objective. The resulting images were analyzed on an Intel Core I3ebased computer, using Video Test Morphology software (Saint Petersburg) with a specific built-in automated object. The analysis provided an estimated quantification of Masson’s trichrome and immunohistochemical results. Epithelial thickness and collagen accumulation were estimated from Masson’s trichrome stain, and cell immunoreactivity was determined from the immunohistochemical results. Statistical analysis Data were tabulated, coded, and analyzed by using Statistical Package for the Social Sciences (SPSS version 17.0). For analytical statistics, significant differences were tested by using univariate or multivariate analyses of variance (MANOVA) to compare between more than two groups for the numerical parametric data, followed by post hoc least significant difference (LSD) for multiple comparisons. Moreover, the Kruskal-Wallis test was used to make comparisons among more than two groups for categorical data, followed by the MannWhitney U test to compare two groups. The statistical tests were based on a type 1 error value of 5% (a ¼ 0.05) and on a power of 0.85 sample size.

RESULTS Hematoxylin and eosin Degree of gingival overgrowth and shape of the gingival crest. The Kruskal-Wallis statistical test revealed an overall significance between the different groups within each examination period. At the first week, the highest mean rank was for group II, whereas the lowest mean rank was for the control group.

Fig. 1. Photomicrograph of buccolingual sections in mandibular molars illustrating the hyperplastic gingival index scores (0 for no overgrowth; and 1, 2, and 3 for mild, moderate, and severe overgrowth, respectively). P, pulp; D, dentin; E, enamel space. Hematoxylin and eosin staining. 100 magnification.

Additionally, at the second week, the highest mean ranks were for groups II and IV, whereas the lowest mean rank was for group V. In the first week, the Mann-Whitney U test revealed a significant difference between group II and the control group, group II and group III, group II and group IV, and group II and group V. In the second week, the Mann-Whitney U test revealed a significant difference between group II and the control group, group II and group III, and group II and group V and a non-significant difference between groups II and IV. Group V gave the best results followed by group III and then group IV in comparison with group II. Nonsignificant differences were found between the first and second examination periods for the same groups (Tables III and IV). Histologic findings after the first week. The control group specimens exhibited a keratinized stratified squamous epithelium covering a core of connective tissue. The epithelium revealed a normal arrangement of its layers, and each layer could be distinguished from the other layers. The basement membrane was flat, with short rete processes. The underlying connective tissue showed delicate collagen fibers, fibroblasts, scattered blood vessels, and a few randomly distributed chronic inflammatory cells (Figure 2A). In contrast, the epithelium of group II specimens was acanthotic and revealed



Table III. Nonparametric statistical tests used to determine the degree of gingival overgrowth for all groups at the two examination periods KruskaleWallis

Mann-Whitney U Percentage

Mean rank

First week

Second week

Groups

First week

Second week

Non

Mild

Moderate

Severe

Non

Mild

Moderate

Severe

Wilcoxon

Z

P2 value

I II III IV V Chi-square df P1 value

11.10 42.30 18.85 37.95 17.30 38.773 4 .001z

18.30 36.30y 20.50 36.30y 16.10 23.113 4 .001z

80% 0% 30% 0% 40%

20% 0% 70% 10% 60%

0% 60% 0% 80% 0%

0% 40% 0% 10% 0%

80% 0% 50% 0% 40%

20% 0% 50% 30% 60%

0% 80% 0% 70% 0%

0% 20% 0% 0% 0%

50.00 40.00 32.50 36.50 50.00

0.00 0.95 1.53 1.35 0.00

1.00* .34* .12* .17* 1.00*

df ¼ Degree of freedom. P1 < .05. P2 < .05. *Wilcoxon test. y P > 0.05 (Mann-Whitney U). z KruskaleWallis test.

Table IV. Nonparametric statistical tests used to determine the shape of gingival crest for all groups at the two examination periods KruskaleWallis

Mann-Whitney U Percentage

Mean rank

First week

Second week

Groups

First week

Second week

Knife edge

Blunted

Bulbous

Knife edge

Blunted

Bulbous

Wilcoxon

Z

P2 value

I II III IV V Chi-square df P1 value

12.80 41.20 20.40 32.70* 20.40 27.576 4 .001z

18.50 36.95y 20.75 36.95y 17.68 22.431 4 .001z

80% 0% 40% 10% 40%

20% 20% 60% 50% 60%

0% 80% 0% 40% 0%

60% 0% 50% 0% 70%

40% 70% 50% 70% 30%

0% 30% 0% 30% 0%

40.00 25.00 45.000 48.50 35.00

0.95 2.19 0.43 0.13 1.31

.34* .02* .66* .89* .18*

df ¼ degree of freedom. P1 < .05. P2 < .05. *Wilcoxon test. y P > 0.05 (Mann-Whitney U). z KruskaleWallis test.

elongated rete processes within the underlying connective tissue. The cells of the basal layer were crowded. The suprabasal layers showed mitotic activity, and the keratin layer was increased in thickness. The underlying connective tissue showed dense and coarse collagen bundles running in a wavy pattern. Fibroblasts were plump, and chronic inflammatory cells were more frequent compared with those in the control group specimens (Figure 2B). The epithelium of group III specimens revealed slight hyperplasia and hyperkeratosis, limited suprabasal mitotic activity, mild acanthosis, and narrower rete processes compared with that of group II specimens. The connective tissue core revealed dense collagen bundles

and plump fibroblasts, and inflammatory cells were distributed throughout the tissue (Figure 2C). In contrast, the epithelium of group IV specimens revealed acanthosis, slightly elongated rete processes, and crowded basal cells, and the keratin layer thickness was almost normal. The underlying connective tissue revealed coarse and dense collagen bundles with prominent subepithelial condensation of these bundles. The fibroblasts were spindlelike, vascularity was limited, and the inflammatory infiltrates were more prominent than those in group III specimens but less extensive than those in group II specimens (Figure 2D). The epithelium of group V specimens was highly similar to that of the control group. The underlying


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Fig. 2. A, Photomicrograph of the negative control group. B-E represent the results of the first week of treatment for the groups that received CsA, CsA and CHX, CsA and CPC, and CsA and Listerine, respectively, whereas b, c, d, and e represent the results of the second week of treatment in the same groups. RT, rete process; BM, basement membrane; C, collagen; CT, connective tissue; Epi, epithelium; IC, inflammatory cells; K, keratin. Hematoxylin and eosin staining. 100 magnification.



Table V. Two way-MANOVA and LSD post hoc test for epithelial thickness (mm) and collagen amount (%) for all groups at the two different examination periods LSD post hoc (Mean SD)

Pillai’s Trace (F ratio and P value)

Epithelial thickness Groups

Periods

(659.34, 0.001*) (567.25, 0.001*) Two way-MANOVA Groups Epithelial (F ratio and P value) thickness Collagen amount Periods Epithelial thickness Collagen amount Groups Epithelial Periods thickness Collagen amount

Groups Periods

Groups

First week

Second week

Collagen amount First week

Second week

(255.63, 0.001*) (192928.570, 0.001y) I

18.58 0.18 18.51 0.26 14.46 0.35 14.47 0.34

(16481.615, 0.001y)

II

76.55 0.25 88.46 0.23 32.69 0.46 48.50 0.25

(797.605, 0.001y)

III

29.47 0.27 27.13 0.22 19.11 0.35 17.53 0.43

(252.708, 0.001y)

IV

58.16 0.21 57.54 0.40 27.43 0.33 26.69 0.39

(2553.776, 0.001y)

V

36.94 0.18 35.43 0.30 24.30 0.29 16.48 0.27

(3012.730, 0.001y)

LSD, least significant difference; MANOVA, multivariate analyses of variance. P < .05. *Two way-MANOVA. y LSD.

connective tissue showed excessive and dense collagen bundles, plump fibroblasts, and numerous vascular channels. The inflammatory cells were distributed throughout the connective tissue (Figure 2E). Histologic findings after the second week. The gingival epithelium and the underlying connective tissue in the control group specimens displayed the same histologic features as control specimens from animals that were euthanized after the first week (see Figure 2A). The gingival tissues of group II animals revealed more exaggerated hyperplasia of the epithelium and underlying fibrous tissue than those of the same group at the end of the first week (Figure 2b). The gingival epithelium of group III rats exhibited more or less normal epithelial thickness and features, as well as slightly inflamed delicate connective tissue (Figure 2c). The epithelium of group IV specimens revealed acanthosis with thin elongated rete processes and hyperkeratosis. In the underlying connective tissue, coarse collagen bundles running haphazardly in different directions and plump fibroblasts with mild vasculature were observed (Figure 2d). The epithelium of group V specimens revealed normal features with narrow and short rete processes. The underlying connective tissue showed collagen bundles and spindlelike fibroblasts, and inflammatory cells were distributed throughout the tissue (Figure 2e). Masson’s trichrome results for the two examination periods Two-way MANOVA for epithelial thickness and collagen accumulation revealed an overall significant difference between the different groups (P < .001). In

addition, the LSD post hoc test for multiple comparisons revealed a significant difference between the first and second examination periods for the same groups. Moreover, a statistically significant difference was found between each two groups at the same examination period. Group II had the highest mean values for epithelial thickness and collagen accumulation, whereas group I had the lowest mean value. Among the mouthwash treatment groups, group IV had the highest epithelial thickness and collagen accumulation (Table V; Figures 3 and 4). Immunohistochemical findings for the two examination periods Two-way MANOVA test for TGF-b, MMP-1, and Il-6 revealed an overall significant difference between the different groups (P < .001). In addition, the LSD post hoc test for multiple comparisons revealed a significant difference between the first and second examination periods for the same groups. Moreover, a statistically significant difference was found between each two groups at the same examination period. Group I was negative for TGF-b expression. The highest immunoreactivity for this protein was detected in group II. This reaction was observed within the epithelium and underlying connective tissue. At the end of the first week, most epithelial immunoreactivity was at the basal and suprabasal layers. In contrast, at the end of the second week, this immunoreactivity was observed throughout almost the entire epithelium. Group III had the lowest immunoreactivity among the treated groups, whereas group IV revealed the highest immunoreactivity.


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Fig. 3. A, Photomicrograph of the negative control group. B-E represent the results of the first week of treatment for the groups that received CsA, CsA and CHX, CsA and CPC, and CsA and Listerine, respectively, whereas b, c, d, and e represent the results of the second week of treatment for the same groups. CT, connective tissue; Epi, epithelium; K, keratin; RT, rete process. Masson’s trichrome staining. 100 magnification.



Fig. 4. Bar chart for the data obtained from the two-way multivariate analyses of variance (MANOVA) and least significant difference (LSD) post hoc statistical tests of epithelial thickness (mm, left) and collagen amount (%, right). yAll groups were significantly different from CsA group. zSignificant difference between weeks within groups.

With regard to MMP-1, the highest mean value was observed in group I at the end of the first week, whereas group III had the highest mean value at the end of the second week, with immunoreactivity primarily in the connective tissue. In contrast, group II had the lowest positivity, with immunoreactivity in only a few basal cells. Groups IV and V showed mild positive reactions within epithelial and connective tissue cells. Group V had the same level of expression at the end of the two examination periods. Group II revealed the highest positive IL-6 expression, whereas the lowest IL-6 expression was detected in group I. Groups III and V exhibited mild positive reactivity for IL-6. Group IV showed the highest immune reaction among the treated groups (Table VI; Figure 5).

DISCUSSION Cyclosporine A is a generally accepted form of therapy for patients undergoing organ transplantation. This drug is likely to become of increasing significance to dentistry because of its ability to induce GO.9 In this experimental study in rats, the control group showed normal epithelium and underlying connective tissue. These histologic findings are consistent with the histology of the normal gingiva of albino rats described by Garant (2003).10 Moreover, the histologic findings for group II at the two examination periods are consistent with Bartold (1987), who described a common histopathology for CsA-induced GO in the form of significant acanthosis, parakeratosis and thin, elongated rete processes penetrating into the underlying connective tissue.11 These findings could be explained based on the knowledge that a clinically relevant dose of CsA along with bacterial products can stimulate the proliferation of gingival keratinocytes and fibroblasts by activating the cell cycle and DNA replication.12 In addition, Gagliano

et al. (2004) found that ECM degradation was suppressed, and this suppression plays an important role in the pathogenesis of drug-induced GO.13 Moreover, CsA may alter collagen turnover pathways in gingival tissue by inducing fibroblasts to reduce phagocytosis or by inhibiting fibroblast secretion of MMPs, leading to CsA-induced GO.14 The importance of plaque as a cofactor in the etiology of drug-associated gingival enlargement has been recognized in most classification systems for periodontal diseases. Further, strict plaque control plays a pivotal role in managing transplant patients who exhibit CsAinduced GO.15-17 The application of CHX and EO mouthwashes aided in reducing the severity of GO. These findings are consistent with the results of Keijser et al. (2003), who indicated that CHX can reduce the severity of GO triggered by CsA administration; thus, CHX is considered the gold standard, with concentrations of 0.2% to 0.1% shown to be effective in preventing plaque accumulation.18 The EO-containing mouthwash also reduced the CsA-induced GO. These results are consistent with Stoeken et al. (2007), who reported that an EO-containing mouth rinse induced significant reductions in the numbers of anaerobic bacteria, gramnegative microorganisms, and Streptococcus mutans in saliva. Similarly, these authors recorded an effective reduction in plaque concentration. Therefore, rinsing with EO mouthwashes can be used as an adjunct to unsupervised oral hygiene with long-lasting reduction of gingivitis and plaque formation.19 In the present study, rats that received CPC mouthwash did not show improvement in their condition compared with groups treated with CHX and EO mouthwashes. This finding is consistent with Pitten and Kramer (2001), who mentioned that CPC has lower substantivity and clears rapidly from oral cavity, which may explain its

0.84 0.02

14.06 0.01 0.36 0.02 12.64 0.03

0.05 0.02

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LSD, least significant difference; TGF-b, transforming growth factor beta; MMP-1, matrix metalloproteinase; IL-6, interleukin 6; MANOVA, multivariate analyses of variance. P < 0.05. *Two way-MANOVA. y LSD.

0.37 0.02 3.94 0.02 1.07 0.01 0.44 0.03 V

2.25 0.03

12.42 0.02 0.15 0.03 11.57 0.03 0.03 0.03 4.5 0.03 1.04 0.02 30.82 0.02 0.15 0.03 2.07 0.01 13.85 0.03 0.25 0.02 1.65 0.02 II III IV

0.47 0.01 2.33 0.02 1.53 0.02

0.07 0.02 2.72 0.03 2.72 0.02 0.35 0.02 0.32 0.03 I

First week Second week First week Second week First week Groups Groups Periods Periods

(156210.701, 0.001*) Groups TGF-b MMP-1 IL-6 Periods TGF-b MMP-1 IL-6 Groups Periods TGF-b MMP-1 IL-6

Groups

(5116.790, 0.001*) Two way-MANOVA (F ratio and P value)

(2355.022, 0.001*) (2661401.350, 0.001y) (45670.670, 0.001y) (1255324.621, 0.001y) (458660.144, 0.001y) (11096.876, 0.001y) (15555.159, 0.001y) (399702.844, 0.001y) (10037.910, 0.001y) (3097.750, 0.001y)

MMP-1

LSD post hoc (Mean SD)

TGF-b Pillai’s Trace (F ratio and P value)

Table VI. Two way-MANOVA and LSD post hoc test for TGF-b, MMP-1, and IL6 for all groups at the two different examination periods

IL-6

Second week


lower efficacy compared with that of CHX.20 In contrast, Hu et al. (2009) found that the use of CPC mouthwash had a significant antiplaque effect after 14 days of twice-a-day application.21 By comparing the findings of the first and second week examination periods in the present study, GO was shown to increase over time in group II but to significantly decrease over time in groups III and V. These findings can be illustrated in light of the study performed by Somacarrera et al. who found that the adverse effects of CsA depend on the dose and the duration. In addition, these authors recognized that long-term treatment with CsA has led to the recognition of some major side effects, such as GO.22 In addition, Gau et al. (2013) found that CHX treatment diminished the gingiva enlargement resulting from CsA administration, and the GO was related to the frequency of mouthwash application.7 The effects of the three different mouthwashes used in this study on GO revealed that the effects of CHX and EO mouthwashes were quite similar and that their effects on GO were more beneficial than that of CPC mouthwash. This finding is consistent with Botelho et al. (2009), who added some clinical indexes to plaque culture techniques to compare the effect of rinsing with EO or CHX after 7 days. These authors demonstrated decreases in all analyzed indexes and reductions in the number of Streptococcus mutans in saliva for both mouthwashes.23 In addition, Sharma et al. (2010) found that EO-containing mouthwash was superior to CPC-containing mouthwash in a six-month clinical study.24 In the present study, groups II and IV revealed the highest immunoreactivity for TGF-b, whereas groups III and V showed low immunoreactivity for TGF-b. This finding can be explained based on the knowledge that TGF-b is the most important cytokine involved in fibrogenesis and that certain cytokines and growth factors, including TGF-b and IL-6 are highly expressed in gingival overgrown tissues.25,26 Sobral et al. (2007) mentioned that TGF-b stimulates basal cell layer mitosis, leading to increases in the number of keratinocytes and in the thickness of the epithelium. This finding might have resulted from the physiologic roles of TGF-b in cellular multiplication and differentiation.27 Furthermore, groups II and IV revealed low MMP-1 expression, whereas groups I, III, and V revealed high MMP-1 expression. This finding is consistent with the results of Hyland et al. (2003), who reported that MMP-1 expression was significantly reduced in overgrown tissue compared with normal tissue. These authors indicated that CsA inhibited MMP-1 expression at both the mRNA and protein levels in a dose-dependent and time-dependent manner. Their results support the hypothesis that collagen



Fig. 5. Bar chart for the data obtained from the two-way MANOVA and LSD post hoc statistical tests of TGF-b (%, left), MMP-1 (%, middle) and IL-6 (%, right) expression. yAll groups were significantly different from CsA group. zSignificant difference between weeks within groups.

accumulation observed in GO can be explained by CsA-induced inhibition of collagenolytic activity within the gingival tissues.28 Groups II and IV revealed high IL-6 expression, whereas groups I, III, and V revealed low IL-6 expression. Myrillas et al. (1999) similarly found that fibroblasts derived from CsA-induced GO produced significantly higher levels of IL-6 than their normal counterparts.29 Their results also showed that CsA regulates IL-6 expression in gingival tissue. Therefore, IL-6 might play an important role in the pathogenesis of CsA-induced GO. Low levels of IL-6 expression in groups III and V might be related to the limited degree of inflammation in these groups.30 Collagen accumulation was reduced in groups I, III, and V in this study, whereas groups II and IV had excessive collagen accumulation. These findings are in agreement with Andersen et al. (2011), who reported that IL-6 targets connective tissue cells, such as fibroblasts by enhancing proliferation and by positive regulating collagen and glycosaminoglycan synthesis.31 In addition, phenytoin may cause the buildup of connective tissue as a result of reduced degradation.32,33 Epithelial thickness obviously increased in groups II and IV but decreased in groups III and V after the first and second weeks. This result is consistent with Bulut et al. (2006), who measured epithelial thickness and found that CsA-induced GO was associated with marked epithelial thickening in comparison with healthy controls.34

CONCLUSION Within the limitations of the present study, our results suggest that chemical plaque control agents, such as chlorhexidine gluconate, cetylpyridinium chloride, and essential oils are useful in plaque control, which is a cofactor in the etiology of drug-induced gingival overgrowth. Chlorhexidine gluconate and essential oil mouthwashes can significantly reduce the incidence of CsA-induced gingival overgrowth better than

cetylpyridinium chloride. Therefore, these agents may be valuable tools in the inhibition and overall management of gingival overgrowth.

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Reprint requests: Mohammed El-Awady Grawish, BDS, MSc, PhD Oral Biology Department Faculty of Dentistry Mansoura University Egypt, PB: 35516 [email protected]