Immunosuppressant therapy and bone loss in ligature-induced periodontitis – a
study in rats
Terapia imunossupressora e perda óssea em periodontite induzida por ligaduras
– um estudo em ratos
Pa tri cia Furtado Gonçalves*
Getúlio da Rocha Nogueira Filho**
Enilson An to nio Sallum***
An to nio Wil son Sallum****
Fran cisco Humberto Nociti Ju nior*****
AB STRACT: Immunosuppressive agents have been rec og nized as a fac tor af fect ing the soft tis sues of the perio dontium. How ever, lit tle is known about their ef fect on periodontitis pro gres sion. The aim of the pres ent study was to in ves ti gate the in flu ence of cyclosporin A (CsA) ad min is tra tion, as so ci ated or not with nifedipine, on the bone loss re sult ing from a lig a ture-in duced periodontitis in rats. Twenty-four adult male Wistar rats were used. Af ter an es the sia, the man dib u lar first mo lar was ran domly as signed to re ceive the cot ton lig a ture in the sulcular area while the contralateral tooth was left unligated. The an i mals were ran domly as signed to one of the fol low ing treat ments: Group A - sa line so lu tion; Group B - CsA (10 mg/kg); Group C - nifedipine (50 mg/kg); Group D - CsA (10 mg/kg) plus nifedipine (50 mg/kg). Forty-five days later, the an i mals were sac ri ficed and the spec i mens rou tinely pro cessed for se rial decalcified sec tions. In ter group anal y sis did not re veal sig nif i cant dif fer ences re gard ing the bone loss vol ume in the li gated teeth be tween the ex per i men tal treat ments (0.46 ± 0.11, 0.63 ± 0.32, 0.53 ± 0.14, 0.50 ± 0.18, for groups A, B, C and D, re spec tively – p > 0.05). How ever, intragroup anal y sis showed a greater bone loss vol ume in the li gated teeth than in the unligated ones (p < 0.05). Within the lim its of the pres ent study, the con clu sion was that the ad min is tra tion of CsA, as so ci ated or not with nifedipine, may not in flu ence bone loss in lig a ture-in duced periodontitis in rats.
DESCRIPTORS: Bone re sorp ti on; Pe ri o don ti tis; Immu no sup pres si on.
RESUMO: O uso de agen tes imu nos su pres so res tem sido re co nhe ci do como um fa tor que afe ta os te ci dos mo les do pe ri o don to. Entre tan to, pou co se sabe so bre o seu efe i to na pro gres são da pe ri o don ti te. O ob je ti vo do pre sen te es tu do foi in -ves ti gar a in fluên cia da ci clos po ri na (CsA), as so ci a da ou não à ni fe di pi na, na per da ós sea re sul tan te da pe ri o don ti te in du zi da por li ga du ras em ra tos. Vin te e qua tro ra tos Wis tar ma chos, adul tos, fo ram in clu í dos no es tu do. Após anes te -sia, fo ram co lo ca das li ga du ras de fio de al go dão ao re dor do pri me i ro mo lar in fe ri or di re i to ou es quer do, ale a to ri a men te es co lhi do. O den te con tra la te ral foi de i xa do sem li ga du ra. Os ani ma is fo ram ale a to ri a men te es co lhi dos para re ce ber um dos se guin tes tra ta men tos: Gru po A - so lu ção sa li na; Gru po B - CsA (10 mg/kg); Gru po C - ni fe di pi na (50 mg/kg); Gru po D CsA (10 mg/kg) e ni fe di pi na (50 mg/kg). Após 45 dias, os ani ma is fo ram sa cri fi ca dos para a aná li se his to mé tri ca. A aná li se in ter gru pos não re ve lou di fe ren ças sig ni fi ca ti vas quan to ao vo lu me da per da ós sea en tre os di fe ren -tes tra ta men tos (0,46 ± 0,11; 0,63 ± 0,32; 0,53 ± 0,14; 0,50 ± 0,18, para os gru pos A, B, C e D res pec ti va men te – p > 0,05). Entre tan to, a aná li se in tra gru po mos trou um ma i or vo lu me de per da ós sea nos den tes com li ga du ra, quan do com pa ra dos aos den tes sem li ga du ra (p < 0,05). Den tro dos li mi tes des te es tu do, con clui-se que a ad mi nis tra ção de CsA, as so ci a da ou não à ni fe di pi na, não in flu en ci ou a per da ós sea al ve o lar na pe ri o don ti te in du zi da por li ga du ras em ra tos.
DESCRITORES: Reabsorção óssea; Periodontite; Imunossupressão.
IN TRO DUC TION
The pri mary cause of periodontitis is the cu mu
-la tive ef fect of the in ter ac tion be tween bac te rial
chal lenge and the im mune and in flam ma tory sys
tem of the host. Risk fac tors for periodontitis in
clude poor oral hy giene, age, ge netic fac tors, di a
-be tes and smoking
11. In ad di tion, a de fi cient host
im mune re sponse has been as so ci ated with a more
ag gres sive type of periodontitis
15. Thus, the ef fect
of immunosuppressant agents on the periodontal
tis sues has been investigated
19.
One of the most stud ied immunosuppressant
agents is cyclosporin A (CsA), a fun gal cy clic
undeca peptide that has shown prom ise in the
treat ment of au to im mune dis or ders such as pso ri
-a sis, -atopic der m-a ti tis, rheu m-a toid -ar thri tis -and
uveitis
12. Nev er the less, de spite its many ther a peu
-tic uses and its pro found ef fect on the pre ven tion
of or gan re jec tion, side ef fects are not in fre quent.
Post-trans plan ta tion os teo po ro sis is a
recognized phenomenon
10, as well as gingival over
-growth, bone for ma tion and resorption
3,8,13,21,24. Be
sides bone me tab o lism dis or ders, CsA ad minis
-tration has been as so ci ated with nephrotoxicity,
and nifedipine, a cal cium chan nel blocker, has
been re ported to pro tect against cyclosporinin
-duced toxicity
17. Sim i larly to CsA, nifedipine has
dem on strated a po ten tial ef fect upon bone meta
-bolism
5,22and gingival overgrowth
25.
Whether the as so ci a tion of CsA and nifedipine
may in crease the risk of periodontitis in non-risk
in di vid u als, it is not known. There fore, the pres ent
study was pro posed in or der to in ves ti gate the in
flu ence of CsA ad min is tra tion on the bone loss re
-sult ing from a lig a ture-in duced periodontitis in
rats. In ad di tion, the ef fect of nifedipine as so ci ated
or not with CsA on such a pro cess was also as
-sessed.
MA TE RIALS AND METHODS
Twenty-four male Wistar rats (300-400 g) were
used in the en tire study. The an i mals were kept in
plas tic cages with ac cess to food and wa ter
ad li bi
-tum
. The State Uni ver sity of Campinas In sti tu
-tional An i mal Care and Use Com mit tee ap proved
the pro to col.
Gen eral an es the sia was ob tained by in tra mus
-cu lar ad min is tra tion of ketamine (0.5 ml/kg). One
of the man dib u lar first mo lars of each an i mal was
ran domly as signed to re ceive the cot ton lig a ture in
a submarginal po si tion to in duce ex per i men tal
periodontitis. The contralateral tooth was left
unligated to serve as a con trol. The an i mals were
ran domly as signed to one of the fol low ing four
treat ment groups (6 an i mals per group), in clud ing
daily sub cu ta ne ous in jec tions:
• Group A - physi o lo gi cal sa li ne;
• Group B - CsA (10 mg/kg) (San dim mun
®, No
-var tis Phar ma AG, Ba sel, Swit zer land);
• Group C - ni fe di pi ne (50 mg/kg) (Sig ma, St.
Lou is, MO, USA);
• Group D - CsA (10 mg/kg) plus ni fe di pi ne
(50 mg/kg).
Forty-five days af ter the sur gery, the an i mals
were sac ri ficed by a le thal dose of ketamine, and
their jaws were re moved and fixed in 4% neu tral
for ma lin for 48 h. The spec i mens were de min er al
-ized in a so lu tion of equal parts of 50% for mic acid
and 20% so dium ci trate for 45 days. Par af fin se rial
sec tions (6
µ
m) were ob tained in a mesiodistal di
-rec tion and stained with hematoxylin and eosin.
Using an im age anal y sis sys tem
(Im age-Pro
®, Me
dia Cy ber net ics, Sil ver Spring, MD, USA), the vol
-ume of bone loss in the furcation re gion was
histometrically de ter mined. Mea sure ments were
av er aged to al low in ter group and intragroup anal
-y ses, us ing the one-wa-y anal -y sis of vari ance
(ANOVA) (al pha = 0.05). Pairwise mul ti ple com par
i sons were car ried out by Tukey’s test (al
-pha = 0.05) in the cases where the ANOVA test
showed sig nif i cant dif fer ences. In ad di tion, the
paired
t
-test (al pha = 0.05) was used for intragroup
com par i sons of interradicular bone loss vol ume
be tween li gated and unligated teeth.
RE SULTS
Clin i cal ob ser va tions
Clinically, on the day of the sac ri fice, it was
noted that the an i mals from group B, C and D, i.e.,
the an i mals which re ceived CsA and/or nifedipine,
pre sented a greater gingival vol ume than the an i
-mals from group A (con trol group). Vi sually, when
com par ing li gated and unligated teeth in the same
an i mals, the gingival vol ume was more sig nif i cant
for the li gated teeth.
Histometric re sults
Intragroup anal y sis re vealed a sta tis ti cally sig
-nif i cant dif fer ence in the vol ume of bone loss
(p < 0.05) be tween unligated and li gated teeth for
all ex per i men tal groups, i.e., the cot ton lig a tures
around the teeth were able to pro mote
periodontitis (Ta ble 1). Nev er the less, in ter group
anal y sis did not show sig nif i cant dif fer ences be
tween the test groups (p > 0.05) re gard ing the vol
-ume of bone loss around the li gated teeth (Ta ble 1).
Fig ures 1A to 1D il lus trate the histological as pects
of the mesiodistal sec tions of the li gated teeth, for
each group. In all of them, it is pos si ble to iden tify
co ro nal dentin, pulp tis sue, root dentin (mesial
and dis tal roots), bone and periodontal lig a ment.
The interradicular re gion shows a great area of
con nec tive tis sue, re sult ing from bone loss un der
the furcation. Note the ir reg u lar bone sur face in
this re gion.
DIS CUS SION
Immu no sup pres si on is an im por tant the ra pe u
-tic re gi men for se ve ral di se a ses
4, in clu ding li fe long
me di ca ti on with subs tan ces like CsA in or der to
coun te ract re jec ti on of trans plan ted tis su es. The
ef fects of CsA the rapy on soft tis su es of the pe ri o
-don ti um have been ex ten si vely in ves ti ga ted
in vi
-t r o
1 , 2 3and
in vi vo
26,27. Re cently, the sig ni fi can ce of
CsA on al ve o lar bone has re ce i ved some at ten ti on.
Incre a sed bone re mo de ling and tra be cu lar bone
loss have been ob ser ved in CsA-ex po sed a n i
-mals
7 , 1 8 , 2 4. Incre a sed os te o blas tic and de cre a sed
os te o clas tic ac ti vity in pa ti ents un der go ing CsA
tre at ment has been re por ted
29. Although some at
ten ti on has been paid to the in flu en ce of CsA on al
-ve o lar bone, lit tle is known about this in te rac ti on
in the pre sen ce of a lo cal ir ri tant such as den tal
biofilm.
The re sults of the pre sent study did not re ve al
sig ni fi cant dif fe ren ces among the ex pe ri men tal
groups re gar ding the vo lu me of bone loss re sul ting
from the li ga tu re-in du ced pe ri o don ti tis in rats.
This me ans that da ily CsA ad mi nis tra ti on, as so ci
-a ted or not with ni fe di pi ne, did not re sult in -a more
se ve re bone loss in the fur ca ti on of the te eth. The
fin dings of the pre sent study seem to be in agre e
-ment with tho se of pre vi ous stu di es. Although
some have des cri bed the im por tan ce of Tcell me
di a ted hyper sen si ti vity in hu man gin gi vi tis and pe
-ri o don ti tis
16, this con cept has been chal len ged by
ne ga ti ve fin dings in hu mans
14and by ani mal stu
-di es
20. Gug ge nhe im
et al
.
9(1981) in ves ti ga ted
whet her T-sup pres si on with CsA had an ef fect on
the es ta blish ment and pro gres si on of the di se a se
in rats mo no as so ci a ted with
Acti nomy ces vis co sus
.
They con clu ded that pe ri o don tal di se a se se ems to
be the re sult of a mul ti tu de of pat ho me cha nisms
and is not strictly cor re la ted with T-cell de pen dent
hyper sen si ti vity. Cox
et al
.
2(1987) sho wed that li
-ga tu re-in du ced pe ri o don ti tis bone loss was less
pro noun ced in rats tre a ted with CsA, when com pa
red to that of con trol nonme di ca ted ani mals. Fis
-cher, Klin ge
6(1994) eva lu a ted, cli ni cally and his to
-TABLE 1 -
Mean ± stan dard de vi a ti on of the bone loss vo lu me (mm
3) around un li ga ted and li ga ted te eth, ac cor ding to
each group.
Teeth
Group A
Group B
Group C
Group D
Unligated
0.23 ± 0.08 aA
0.23 ± 0.08 aA
0.20 ± 0.04 aA
0.24 ± 0.07 aA
Ligated
0.46 ± 0.11 bB
0.63 ± 0.32 bB
0.53 ± 0.14 bB
0.50 ± 0.18 bB
Cap i tal let ters should be con sid ered in lines (in ter group anal y sis: one-way anal y sis of vari ance – ANOVA –, p > 0.05)
and low er case let ters in col umns (intragroup anal y sis: paired
t
-test, p < 0.05). Means fol lowed by dif fer ent let ters dif fer
sta tis ti cally (al pha = 0.05).
FIGURE 1 -
Pho to mi cro graphy il lus tra ting bone loss in the fur ca ti on re gi on of a li ga ted to oth.
A
,
B
,
C
and
D
il lus tra te
Groups A, B, C and D, res pec ti vely (H. E., 12.5 X).
A:
Con trol group.
B:
Cyclos po rin A group.
C:
Ni fe di pi ne group.
D:
lo gi cally, the ef fect of CsA on the pro gres si on of the
pe ri o don tal bre ak down in do mes tic fer rets, using
the li ga tu rein du ced pe ri o don ti tis mo del. They ob
ser ved a ten dency of more bone loss in the ex pe ri
-men tal me di ca ted ani mals as com pa red to the
values of nonme di ca ted ones. Ho we ver, the dif fe
-ren ces were not sta tis ti cally sig ni fi cant. The re fo re,
as men ti o ned by Gug ge nhe im
et al
.
9(1981), it ap
pe ars that the cli ni cal as pect of the pe ri o don tal di
se a se may be the re sult of in de pen dent me cha
-nisms, i.e., even bloc king such an im por tant
ele ment, all ot her ele ments may not be sig ni fi
-cantly af fec ted. Ge ne rally spe a king, a va ri ety of
host de fen se di sor ders may lead to des truc ti on of
the pe ri o don tal at tach ment ap pa ra tus and even tu
ally pre ma tu re ex fo li a ti on of pri mary and/or per
ma nent te eth. Ho we ver, most of the stu di es, in clu
-ding the pre sent in ves ti ga ti on, were not able to
his to lo gi cally il lus tra te such a cli ni cal fi gu re, by af
-fec ting the T-cell res pon se to the lo cal ir ri tants.
The re fo re, furt her stu di es on the eti o pat ho ge ne sis
of the pe ri o don tal le si ons, re gar ding the role of the
host im mu ne res pon se, are jus ti fi ed.
The sub cu ta ne ous route, which was used in
this study, has been sug gested as the route of
choice
28to pro vide more con sis tent cy cles of CsA
avail abil ity than any other route. Blood lev els of
CsA be tween 100 and 400 ng/ml have been shown
to be suf fi cient to pro mote immunosuppression in
hu mans. Like wise, in an i mals, blood lev els be
-tween 100 and 400 ng/ml have been con sid ered to
be effective
28. The dos age of CsA used in the pres
ent study (10 mg/kg, SQ) has been shown to pro
duce suf fi cient blood lev els to in duce immuno
-suppression in rodents
6.
Cal cium chan nel blockers (CCBs) such as
nifedipine are widely used in car dio vas cu lar ther
-apy and re cently have been uti lized for the con trol
of cyclosporin-in duced toxicity
17. How ever, the ef
fect of CCBs ap pears not to be lim ited to the car
dio vas cu lar sys tem and may in volve cal cium me
-tab o lism as well
5,22. In the pres ent study, nifedipine
groups showed rates of periodontitis pro gres sion
sim i lar to that of other groups. Since this is the
first study to in ves ti gate whether nifedipine could
in flu ence bone loss in lig a turein duced perio don
-titis, our re sults must be ana lysed with cau tion
and fur ther stud ies should be con sid ered.
CON CLU SION
The data of the pres ent study sug gest that the
ad min is tra tion of CsA, as so ci ated or not with
nifedipine, may not in flu ence bone loss in lig a
-ture-in duced periodontitis in rats.
AC KNOWL EDG MENT
The au thors greatly ap pre ci ated the as sis tance
of Rodrigo Borges Fonseca, DDS, for ad min is ter ing
the drugs to the an i mals.
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