J Clin Periodontol 1995: 22: 591-597Printed in Denmark . AH right,'' reserved

Copyright © Munksgaard 1995

Clinical periDdontoiogifISSN 0303-6979

Gingival overgrowth induced bynifedipine and cyclosporin AClinical and morphometric study with imageanalysis

Francisco O'Valle\ FranciscoJose Aneiros'',Mercedes Gomez-Mora Ies\Miguel Angel Lucena\Cesar Ramtrez\Francisco Revelles\ Esther Moreno^,Nieves Navarro\Trinidad Cabaliero\ Marco Masseroii^and Raimundo Garci'a dei Morai^^Department of Pathology, University ofGranada Hospital, School of Medicine,University of Granada, E-18012 Granada, Spain^Department of Periodontoiogy, School ofOdontology, University of Granada

O'Valle F, Mesa F, Aneiros J, Gomez-Morales M, Lucena MA, Ramirez C,Revelles F. Moreno E. Navarro N, Cahaliero T. Masseroli M, Garcia del MoralR: Gingival overgrowth induced by nifedipine and cyclosporin A. Clinical andmorphomelric study with image analysis. ./ Clin Periodonlol 1995; 22: 591-597.© Munksgaard, 1995.

Abstract. In this study, we developed a quantitative method with digital imageanalysis to evaluate the degree of gingival overgrowth (GO), and compared GOin kidney transplant patients treated with cyclosporin A (CsA) («=21) orCsA+nifedipine (n-8) and a group of healthy controis (n-30). The methodwas reproducible and rdiable. Our findings showed significant differences in pap-illary and gingival surface between controls and transplant patients treated withGO inducers. Gingival overgrowth index also differed significantly between con-trols and each patient group ( / J < 0 , 0 ] , Kruskal-Wallis test). The administrationof the calcium channel blocker nifedipine potentiated the adverse effect of CsA:comparison of the morphometric findings revealed significant differences be-tween patients treated with CsA alone and CsA+nifedipine in papillary area,dental area, and GO index 07<0.01, Mann-Whitney t/-test). We conclude that themethod of image analysis we developed is useful in assessing the degree of GO.

Key words: gingivai overgrowth; cyciosporin A;nifedipine; computer analysis

Accepted for publication 17 August 1994

Many drugs cause gingival overgrowth(GO) as an adverse side-effect. Fre-quently used agents associated with GOare calcium channel blockers (e.g., nife-dipine, Qxidipine, felodipine, diitiazem,verapamil, nitrendipine, and amlodipi-ne) (Lederman et al. 1984, Miller &Damm 1992, King et al, 1993) andcyclosporin A (CsA) (Rateitschak-Plusset al, 1983, King et al. 1993, Pernu etal. 1992, Thomason et al. 1993), an im-munosuppressor currently used to pre-vent allograft rejection in recipients oftransplanted organs. In addition, CsAhas a wide spectrum of biological ac-tivities, showing antiparasitic, anti-fungal, antiinflammatory (Borel &Ryffel 1985) and antiprohferative ac-tion (Borel 1988). This has led to theuse of CsA to treat severe psoriasis

(EUis et al. 1986) and other dermatosessuch as ichthyosis vulgaris (Velthuis &Jesserun 1985, Furue et al. 1988, Kani-takis & Thivolet 1990), as well as im-mune disorders (Van Rijthoven &Diskmans 1986, Gupta et a!, 1989,Kuenty et ai, 1986, Feutren 1986,Feutren et al. 1987, Gupta et al. 1990,Routhier et al. 1980, Minuk et ai. 1988,Canvin et ai. 1991, Sasaki et al. 1990,SchoUmeyer & Grozt 1990, Allan et aL1987), Partial remissions of neoplasmshave also been reported in Hodgkin dis-ease patients treated with CsA (Zwitteret al, 1987).

A study by Slavin & Taylor (1987)showed that simultaneous treatmentwith CsA and calcium channel block-ers, which induce GO, increases the in-cidence of gingival overgrowth. More-

over, this combined treatment was re-cently reported to induce more severedegrees of GO (Thomason et al. 1993).Because the use of these drugs is in-creasing steadily, the prevalence of GO,currently at around 20% to 30%, is ex-pected to increase (Friskopp &Klintmalm 1986, Daley et al, 1986,Seymour et a l 1987, Pernu et al. 1992,Barclay et al, 1992).

The degree of GO is usually assessedon the basis of clinical criteria (Angel-opoulos & Goaz 1972, Pasqualin 1990,Pernu et al. 1992); quantitative methodshave rarely been applied. We have de-veloped a new approach to assess GO,based on digital image anaiysis ofphotographs, taken under controlledconditions, of the anterior regions ofthe upper and lower dental arches. We

592 O'Valle et al.

Fig. L Digital image of an intraorai photograph of a control subject, showing areas of interest inside the polyline, and after segmentation ofthe photograph. (A) dental area, (B) gingiva! area, (C) papillary area.

Morphometric study of drug-induced GO 593

tested our method in patients who werereceiving chronic treatment with CsAand nifedipine.

Material and MethodsPatients

We studied 29 renal transplant patientsand 30 healthy control subjects who'were not receiving GO-inducing medi-cation. Of the 5 patients who under-went gingivectomy 12 months beforethe study, 4 had recurrences (3 moder-ate, 1 mild), and were excluded fromfurther study. During the first 12 post-operative days, transplanted patientswere treated with CsA (8 mg/kg/day),prednisone (0.25 mg/kg/day) and anti-lymphocytic globulin on alternate days,at a dose of 10 mg/kg/day. During thefollowing six months a maintenance re-gime was established, with the dose ofCsA adjusted to maintain stable serumlevels of 80 to 200 ng/ml, and a pro-gressive reduction in prednisone to 5mg/day (CsA group). 8 subjects werealso treated with nifedipine (40 mgMay)to control their blood pressure(CsAH-nifedipine group). In al) subjectstotal follow-up time was at least 13months. Subjects were excluded if theyreceived any additional drug that mighthave affected the gingival tissue, andonly patients with at least 6 oi' the 8most anterior teeth in both the upperand lower arches were included in thestatistical analysis, in accordance withthe criteria of Thomason (1993).

Serum concentrations of CsA weremeasured by standard radioimmuno-assay techniques with anti-CsA mono-clonal antibodies (Incstar, Minnesota,USA),

Oral hygiene and gingival status

2 experienced periodontists, who wereblinded as to whether the subject wasreceiving GO-inducing drugs, clinicallyassessed GO, examined the periodont,and determined the O'Leary plaqueindex (1972) and the bleeding index ofMtihleman & Son (1971). Piaque wasscored after staining with a disclosingsolution, and continuous piaque stain-ing in the dentogingivai area was re-corded; bleeding was scored after gentleprobing. Gingival overgrowth wasclassified in one of four categories onthe basis of the criteria of Pasqualin etal, (1990) and Angelopoulos & Goaz(1972) modified by Pernu and co-workers (1992). The criteria for each

score were: score 0 - n o gingival over-growth; score 1-mild gingival over-growth (thickening of the marginal gin-giva and/or lobular granulation of thegingival pocket as well as overgrowthcovering the gingival third of the crownor less; score 2—moderate gingivalovergrowth (overgrowth extending tothe middle of the crown) and score 3 ^severe gingival overgrowth (overgrowthcovering two thirds of the crown or af-fectation of the whole attached gingiva)(Pernu et al. 1992), In the five subjectsfor whom the clinical evaluations dif-fered, a third periodontist examined thepatients, whose signs were classified byconsensus between the three specialists.No periodonta] care was given, and nocalculi or piaque were removed from thetooth surfaces before the study.

Morphometric methods

Black-and-white photographs weretaken of the anterior and medial re-gions of the upper and lower denta!arches in each subject (Fig. 1), A singlephotograph per patient was taken dur-ing the course of periodontal examin-ation, at variable times ranging from 13to 84 months post-transplant. We useda Nikon F-3 camera fitted with a Nikk-or 105 mm telephoto lens., and a Sun-pack 6X8R ring fiash array. The cam-era was mounted on a tripod at a dis-tance of 0.47 m from the subject. Thenegatives (Ilford 100 ASA film) were de-veloped in a 9X13 cm format on Ilfordno. 3 paper.

Photographs were digitalized with ascanner (Sharp JX450) and Cromoscansoftware, and transferred to the AdobePhotoshop"^'^ program to obtain512X512 pixel images in tagged imagefile format (TIFF). Image files (262(63

bytes) were analyzed with the Visilogprogram (version 3.6.1, Noesis. Jouy enJosas, France). Processing ofthe digitalimages consisted of restoration and cor-rection of intensity levels and a greylevel equalization. The areas of interestwere selected in the interactive mode bydrawing a polyhne around three areas.The ''gingival area''' (Fig, IB) was de-fined as the surface between the mucog-ingival line and the free margin of thegingiva. The "dental area" (Fig. IA)was the vestibular surface of the in-cisors not covered by the gums. The'"papillary area'^ (Fig. 1C) was the freegingival surface between the incisors.The areas of interest, weil visually con-trasted on the image, were segmentedautomatically to transform them intobinary images; selected areas werequantified in square pixels.

To offset the infiuence of possible dif-ferences between subjects in size of thebuccal structures, we also calculated theovergrowth index (GOI) for each sub-ject; this index was obtained by dividing""gingival area" by ''dental area". Thisindex characterizes the relation betweenthe vestibuiar surface of the 8 incisorsand the attached and free marginal gin-giva.

Statistical analysis

The numerical data were imported intothe Sigma 11 database (Horus Hard-ware, Barcelona, Spain) and subjectedto I tests for paired samples and analy-sis of variance (ANOVA). Pearson'srank coefficient was used for data show-ing a normal distribution, and Kruskal-Wallis and Mann-Whitney (/-tests wereused for non-normally distributed vari-ables, x'^ snd Spearman tests were used

Tahle I. Clinica) and periodontal characteristics in three group of patients

Control groupPatients treatedwith CsA

Patients treatedwith CsA+Nif p(«=8) values

age (years) 31,4±14(20-S2)'^M0,l±ll (25-61) 38.]±!2 (2^5-53) not signif,̂sex

transplant date(months)CsA serum level

(ng/ml)GO scorebleeding index (%)plaque index (%)

13 male17 female-

-

02.4±35

!3.6±74

9 male12 female42,7+5.2(13-84)!33±8.0

I.27±0.849,4±3143.8±26

7 male1 female

45,7-5.5(24-72)

156.3±18.3

2,37±0,768 ±24

72.5±24

-

not signif.^'

not signif.^'

pKO.OS"^

p<0,Q5^''

"> Mann-Whitney [/-test; ^^ Student t test;CsA: cyclosporin A, Nif: Nifedipine

.v±SD ( ): mean±standard de\iation (range).

594 O'Valle etal.

to compare the results for quahtativevariables.

ResultsClinicaf findings

Mean age was similar in atl the groups(Table !). There was no significant dif-ference between transplant patientstreated with CsA alone and CsA+nifedipine in mean time since renaltransplant (42 ±5,2 months versus45±5.5 months) or serum concen-tration of CsA (I33.3±8.0 ng/m! versus156.3±!8.3 ng/ml). In contrast, plaqueindex and bleeding index differed sig-nificanlly between the transplant pa-tient group and the control group(/?<0.001, Student Mest) (Fig, 2).

The degree of GO in transplant pa-tients was not significantly related withage, sex or serum CsA concentration.However, plaque index and gingivalbleeding index differed significantly be-tween the two patient groups, the for-mer being higher in the CsA+nifedipinegroup (72.5% versus 43.8%, /j<0.Q5,Student /-test).

The GO in transplant subjectstreated with CsA aione was mild in 14patients, moderate in 5 and severe in 2.The calcium channel blocker nifedipineseemed to potentiate the adverse side-effect of CsA, as patients who hadtaken both drugs had more severe GOthan those who received CsA alone: 1patient had a GO score of 1 (tnild), 3patients scored 2 (moderate) and 4 pa-tients had a GO score of 3 (severe).

» p<0.001 • p<0.05 (Student's t test)

PLAQUE INDEX BLEEDING INDEX

Fig. 2. Comparison of plaque index andbleeding index in eonlrol subjects and trans-plant patients treated with cyelosporin Aalone or in eombinalion with nifedipine.

There was good interexaminer agree-ment on the degree of GO according tothe systems developed by Pasqualin (^-0.789) and by Pernu and co-workers(^•-0,805), as determined with Spear-man's rank correlation coefficient.

Morphometric findings

The results of the image analyses aresummarized in Table 2. Papillary(/?<0.001) and gingival surfaces(/?<0.05) were significantly larger intransplant patients taking GO inducersthan in control subjects (Kruskal-Walhstest) (Fig, 3, Table 2). We aiso foundsignificant differences in GOl betweencontrols and patients taking CsA aloneor CsA+nifedipine (/'<0.01, Kruskal-Wallis test).

Mean GOI values for each degree ofovergrowth were 0.95±0.15 for mild,1.30±0.44 for moderate, and 2,42±0.90for severe GO, Mean GOI in untreatedcontrols was 0.87±0.16. We found a sig-nificant correlation (r-0.7026, Pearsoncoefficient) between the GO scores withthese grading system and the results ob-tained by image analysis (GOI), How-ever, significant differences were foundonly between the control group and pa-tients with moderate GO (/7<0.01,Mann-Whitney 6'-test), between con-trols and patients with severe GO(/)<0.001, Mann-Whitney C/-test) andbetween patients with severe GO andthose with any lower score (p<0.05,Mann-Whitney (7-test) (Fig. 4).

The morphometric data also showedsignificant differences between the 2 pa-tient groups in papillary (/j<0.01) anddenta! area (p<0,05) and in GOI(p<0,05, Mann-Whitney (7-test) (Table2).

Discussion

Despite the good interexaminer agree-ment, it may be difficult to establish the

precise degree of gingival enlargement,and to follow subtle changes in the de-gree of GO, on the basis of qualitativeassessments alone. Our method of im-age analysis standardizes the assess-ment and makes it possible to detectshght damage with time in the patient'scondition (Quirynen et al. 1991), Thisapproach is potentially useful to evalu-ate the effectiveness of treatmentsaimed at reducing GO secondary todrug treatment by controlling plaque(Seymour & Smith 1991).

Overlap in the GOI values we foundfor the different degrees of GO indi-cates that a wide range of degrees ofGO are subsumed under each GOscore. The morphometric index maythus represent a more precise and repro-ducible way of assessing GO. The GOIrelating the degree of gingival over-growth and the exposed dental area,acts as a correction factor for the poss-ible differences between patients in thesize of their buccal structures, and off-sets the slight variations that may occurin the course of technical processing ofthe photographs.

Although CsA-induced GO is dif-fuse, it first appears in anterior regions(Pascualin et al, 1990), which we con-sidered in the present study. We studiedalone, interactively extracted papillaryarea because this area accounts formost of the mild GO scores, and be-cause this allowed us to comparegrowth between the interdental papillaand the rest of the inserted and free gin-giva.

We beheve that the current tendencyto use CsA for many purposes otherthan transplantation, together with theadditive effect of nifedipine (Slavin &Taylor 1987, Brown et al. 1990,Miller & Damm 1992, Thomason et al.1993), have led to increased prevalenceof GO. One recent study (Thomason etai, 1993) showed that combined treat-ment with CsA+nifedipine caused

Table 2. Morphometric values in 29 rena! transplant patients treated with cyciosporin A orcyciosporin A + nifedipine

Morphometricstndy

Control group Patients treatedwith C S A ( H = 2 1 )

Patients treatedwithCsA+Nif(H=8)

Pvalue^'

papillary areagingival areadental areaGO-index

7441 ±2102'"32797 ±535538028±4846

0,g7±0.16

11540.8±471537651.1±992736360.3± 10941

l,12±0.44

/><0,00139728,2±9970 /><0,0528260,4± 13403^' ;><0.05

3.79±O,98'"

Kruskal-Wallis test, CsA: cyclosporin A, Nif: NifedipineS±SD: mean±standard deviation.

5, Mann-Whitney O'-test.

Morphometric study of drug-induced GO 595

Fig, 3, Digital image of an intraoral photograph of a transplant patient treated with cyclosporin A, showing areas of interest inside thepolyline, and after segmentation of ihc photograph, (A) dental area, (B) gingival area, (C) papillary area.

596 O'Valleetal

0.87 0.969 2,42

I ) oontrol [ZD aoote 0 ^ S soorc

^ ^ aoDre 2 r '" t jDOrs 3

i^ig. •̂ , Comparison of gingival overgrowthindex between subjects with different clinicalscores of GO,

more severe GO than that seen in pa-tients given CsA aione. We found thesame to be true in our patients, and otirmorphometric data confirmed that GOwas significantly more severe in patientstreated with this drug combination thanin nontreated controls or patientstreated with CsA alone.

Serum concentrations of CsA or thedose of nifedipine used were not relatedto the degree of GO; however, the detec-tion of increased concentrations in sal-iva or gingival deposits in plaque maybe more clearly related with the extentof overgrowth (Niimi et al. 1990). In animmunohistochemical study, we pre-viously showed that the presence of in-tralesional deposits of CsA played aroie in the development of GO (O'Valleet al. 1994).

Aithough the number of patients inthe present study is too small to allowextrapolation of the results, we calcu-lated mean GOI values for each degreeof overgrowth. The similarity betweenthe vaiues for mild and moderate GOmaices it difficult to establish the ciinicaidegree of GO. In any case, clinical scor-ing may not be necessary if a precisenumerical vaiue is obtainable with theimage analysis method described hereto determine GOI, However, largerseries of patients and controls wili needto be studied to arrive at more accuratemean values for each stage of GO,

In surrmiary, we describe the first ap-plication of a simpie, reproduciblemethod of image analysis to determineof the degree of GO secondary to treat-

ment with CsA and a caicium channelblocker.

Acknowledgements

This study was supported by theCICYT through Grants SAF 93/529and PETRI 0059/91, and by SandozPharma SAE contrast no. 418/94. Wethank Dr. A. Montes of the Nephrol-ogy Service at the Virgen de las NievesHospital in Granada for providing ciin-icai information on the sample of trans-plant patients, and Ms Karen Shashokfor translating the original manuscriptinto English.

Zusammenfassung

Gingivahyperplasien hervorgemfen durch Ni-fedipin and Cyclosporin A. Eine klinische undmorphometrisehe Studie mittels BildcmalyseInnerhalb dieser Sttidie entwickelten wir eineqtiantitative Methode, um mittels digitalerBildanalyse den Grad der Gingivahyperpla-sic (GO) zu beurteilen, AnschlieBend wtirdebei Patienten mit Nierentransplantat die GOnach Cyclosporin A (CsA( (n = 21) bzw,CsA-l-Nifedipin (n=8) mit der einer Gruppevon gesunden Kontrollen (n=30) vergJichen,Die Methode was reproduzierbar und zuver-lassig. Unsere Ergebnisse zeigten signifikanteUnterschiede zwischen der papillaren undgingivalen Oberflache von KontroJIgruppeund Transplantatpatienten, die mit obigenStoffen behandelt wurden, Auch der Gingi-vahyperplasie-lndex zeigte signifikante Un-terschiede zwischen Kontroll- und Patienten-gruppe (p<0.01, Kruskal-Wallis Test). DieVerabreichung des Calzium-Kanal-BlockersNifedipin potentierte die Nebenwirkungenvon CsA: Der Vergleich der morphometri-schen Ergebnisse zeigte im Papillenbereich,am Zahn und beim GO-lndcx {/'<0,01,Mann-Whitney i7-Test) signifikante Unter-schiede zwischen Patienten, die allein mitCsA Oder mit CsA+Nifedipin behandeltwurden. Wir ziehen die SchluBfolgerung, dalidie entwickelte Methode der Bildanalyse furdie Beurteilung des Grades an GO niitzlichist.

Resume

Hyperplasie gingivale induite par la nifedipineet la cyclo.^porine A, Etude climque et mor-phometrique avec analyse d'imageNous avons dans cette etude mis au pointune methode quantitative avee analysed'image numerique, pour evaluer le degred'hyperplasie gingivale (GO), et nous avonscompart GO chez des patients ayant subi unetransplantation renale et traites par eyclo-sporine A (CsA) {n=2]) ou par CsA+nifedipine («=8) et une groupe de sujets sainstemoins (control, «=30), La methode etaitreproductible et fiabie. Nos resultats ont mis

en evidence des differences significatives auniveau de la surface papillaire el gingivale en-tre les temoins et ies patients transplantestraites avec les produits determinant GO. Onconstatait aussi des differences significativesentre I'indice de GO des temoins et de cha-cun des groupes de patients (p<0.01, test deKruskal-Wailis). L'administration de nifedi-pine, un bloquant des canaux du calcium,renforijait les effets adverses de CsA: la com-paraison des resuUats morphometriques amis en evidence des differences significativesentre les patients traites par CsA seule et parCsA+nifedipine en ce qui concernait lasuperficie papillaire, la superficie dentaire ctrindice de GO (p<0.01, test 6' de Mann-Whitney). Nous en concluons que la me-thode d'analyse d'image que nous avons miseau point est utile pour mesurer le degre deGO,

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Address:

Franciseo J, O'Valle RavassaDepartment of PathologySchool of MedicineUniversity of GranadaAvda. de Madrid IIE-I80J2 GranadaSpain