Abst ract
Submitted: October 29, 2016 0RGL¿FDWLRQ-DQXDU\ Accepted: February 2, 2017
Agreem ent , correlat ion, and
kinet ics of t he alveolar bone- loss
m easurem ent m et hodologies in a
ligat ure- induced periodont it is anim al
m odel
Per iod on t al r esear ch in v olv es t h e u se of an im al m od els t o b et t er underst and t he biological processes of periodont al diseases and t he pot ent ial of new or exist ing t herapies. Current ly, ligat ure- induced periodont it is in rat s is t he m ain m odel used in periodont al research, in t his m odel, alveolar bone loss ( ABL) is t he m ain param et er evaluat ed by radiographic, m orphom et ric, and hist ological t echniques. I nt erest ingly, alt hough t hese m et hodologies are widely XVHGLWLVQRWWRWDOO\FODUL¿HGQHLWKHUWKHNLQHWLFVRI$%/RYHUWKHLQGXFWLRQ t im e nor t he agreem ent degree ( repeat abilit y and reproducibilit y) of t hese t echniques. Obj ect ive: To charact erize ABL kinet ics at 0, 3, 7, 15, 30, and 60 days aft er ABL induct ion by ligat ure and t o evaluat e t he int ra- ( repeat abilit y) and int er- exam iner ( reproducibilit y) agreem ent and t he correlat ion am ong t he radiographic, m orphom et ric, and hist ological m et hodologies. Mat erial and Met hods: 60 m ale Wist ar rat s w it h induced ABL w ere random ly divided int o 6 experim ent al groups ( n= 10 anim als/ group) . Aft er 0, 3, 7, 15, 30, and 60 days, t he anim als w ere eut hanized and t heir hem im andibles w ere rem oved for ABL det erm inat ion using radiographic, m orphom et ric and hist ological t echniques. Result s: Radiographic and m or phom et r ic/ linear t echniques DOORZHGWKHGHWHFWLRQRIVWDWLVWLFDOO\VLJQL¿FDQW$%/RQWKHWKLUGGD\ZKLOH hist ological and m orphom et ric/ area t echniques could only det ect ABL aft er WKHVHYHQWKGD\$129$7XNH\S$IWHUWKH¿IWHHQWKGD\H[FHSWIRU hist ological analysis, t he ABL was st abilized. Concerning t he agreem ent of t he m et hodologies, Bland Alt m an’s t est ( int ra and int er- exam iner evaluat ions) show ed no difference am ong t he m easurem ent s ( p> 0.05) . I n addit ion, high correlat ions ( Pearson’s t est , r2> 0.9, p< 0.05) w ere observed. Conclusion: The result s indicat ed t hat t he m inim um t im e for ABL induct ion could vary from 3 t o 7 days, according t o t he chosen analysis m et hodology. Agreem ent and correlat ion dat a support t he com parison of result s bet w een st udies w it h sam e induct ion t im e.
Keyw ords: Experim ent al anim al m odels. Periodont it is. Alveolar bone loss. Paula Katherine VARGAS-SANCHEZ1
Marcella Goetz MORO1
Fabio André dos SANTOS1
Ana Lia ANBINDER2
Eliane KREICH1
Renata Mendonça MORAES2
Lauryellen PADILHA3
Caroline KUSIAK4
Dionizia Xavier SCOMPARIN5
Gilson Cesar Nobre FRANCO1
http://dx.doi.org/10.1590/1678-7757-2016-0517
1Universidade Estadual de Ponta Grossa, Departamento de Odontologia, Ponta Grossa, PR, Brasil. 2Univ Estadual Paulista, Departamento de Biociências e Diagnóstico Oral, São José dos Campos, SP, Brasil.
3Universidade Estadual de Ponta Grossa, Programa de Pós-Graduação em Ciências Biomédicas, Ponta Grossa, PR, Brasil.
4Universidade Estadual de Ponta Grossa, Programa de Graduação em Enfermagem, Ponta Grossa, PR, Brasil.
5Universidade Estadual de Ponta Grossa, Departamento de Biologia, Ponta Grossa, PR, Brasil.
Corresponding address: Gilson Cesar Nobre Franco Universidade Estadual de Ponta Grossa. Departamento de Odontologia. Av. General Carlos Cavalcanti, nº 4748 -
I nt roduct ion
I n p e r i o d o n t a l r e se a r ch , a n i m a l m o d e l s a r e im p or t an t f or st u d y in g t h e r elat ion sh ip b et w een diseases and ex t er nal fact or s, and also for t est ing pot en t ial n ew t h er apies. Th e m odel select ion w ill depend on t he aim of t he st udy, but in general, an adequ at e m odel sh ou ld pr esen t biological ev en t s sim ilar t o t h at obser v ed in h u m an s, su ch as t h e progressive dest ruct ion of connect ive t issue, bone loss, and t he form at ion of periodont al pocket s2.
The rat is t he m ost w idely used rodent t o st udy t he pat hogenesis of periodont al disease ( PD) because of t he sim ilarit y bet w een t he t issue st ruct ure of t he g in g iv al ar ea an d t h e b on e/ collag en d eg r ad at ion obser v ed du r in g alv eolar bon e loss ( ABL) in r at s and hum ans19. The m ain m odels used t o induce ABL
are: 1- Ligat ure- induced periodont it is: t he insert ion of yar n ar ound t he cer v ix of a m andibular m olar ; 2 - LPS- in d u ced p er iod on t it is: t h e ap p licat ion of lipopoly sacch ar ide ( LPS) in t o m an dibu lar gin giva; an d 3 - Micr oor g an ism - in d u ced p er iod on t it is: t h e
LQRFXODWLRQRIVSHFL¿FSHULRGRQWDOSDWKRJHQV1,5,20.
Re g a r d l e ss o f t h e p e r i o d o n t i t i s i n d u ct i o n m et h od ch osen , r ad iog r ap h ic, m or p h om et r ic an d hist ological t echniques are t he m ost cit ed t echniques in t he lit erat ure used t o quant ify ABL, w hich is t he m ain p ar am et er an aly zed f or t h is an im al m od el. The radiographic m et hod usually involves analyzing ABL b y m ea su r i n g t h e l i n ea r d i st a n ce b et w een t he cem ent oenam el j unct ion ( CEJ) in t he alv eolar
ERQH FUHVW RI WKH ¿UVW PDQGLEXODU PRODU8 , 9 , 1 3. The
m orphom et ric m et hod requires m easuring t he linear dist ance ( m m ) or t he area ( m m2) bet w een t he CEJ
and t he bone crest in t he m andibular m olars3,14. I n
t he hist ological m et hod, t he int erproxim al bone loss
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by t he linear dist ance bet w een t he CEJ and t he bone crest18.
Co n si d er i n g t h e r el ev an ce o f ABL p ar am et er for per iodont al r esear ch, t he est ablishm ent of t he reliabilit y, agreem ent ( repeat abilit y and reproducibilit y) and correlat ion of t heir m ain analysis m et hods is one of t he m ost im port ant principles in science, since it allow s t he dat a t o be int erchangeable am ong st udies car r ied out w it h sim ilar pr oposals. I n addit ion, t he knowledge t he kinet ics of alveolar bone loss over t im e of t he m et hodologies is also considered essent ial t o t he success of a st udy ( st udy design and int erpret at ion of
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region in t he periodont al t issue and consequent ly it becom es possible t he occurrence of different rat es of alveolar bone degradat ion according t o t he ABL m odel and t he induct ion t im e chosen. Nowadays, t he t im e of ABL induct ion in t his anim al m odel is found in t he lit erat ure ranging from 1 t o 60 days9,10,12,13.
Therefore, t he aim of t his st udy was t o det erm ine t he kinet ics of alveolar bone loss 0, 3, 7, 15, 30, and 60 day s aft er ligat ur e- induced per iodont it is in rat s and t o assess t he Bland- Alt m an’s agr eem ent and Pearson’s correlat ion of radiographic, m orphom et ric, and hist ological m et hodologies for ABL analysis.
Mat erial and Met hods
The ex per im ent al pr ot ocol follow ed t he ARRI VE g u id elin es f or an im al r esear ch su g g est ed b y t h e 1DWLRQDO &HQWHU IRU WKH 5HSODFHPHQW 5H¿QHPHQW and Reduct ion for Anim als in Research11. The sam ple
size was calculat ed w it h t he soft ware G* Pow er 3.16. $VVXPLQJ D VLJQL¿FDQW GLIIHUHQFH JUHDWHU WKDQ t im es t he st andard deviat ion in t he ABL param et er and
a Type I (DDQG7\SH,,ǃHUURUVRIDQG
respect ively, t he sam ple size was est im at ed in nine anim als ( m inim um ) per group.
Sixt y m ale Wist ar rat s weighing approxim at ely 250 g each w ere included in t he st udy. The anim als w ere m aint ained in plast ic cages ( 5 anim als/ cage) under a 12- hour light / dark cycle at a t em perat ure of 22° C, and received food and wat er ad libit um. The st udy was approved by t he Et hics Com m it t ee of Anim al Use
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Th e an i m al s w er e r an d o m l y d i v i d ed i n t o si x experim ent al groups ( n= 10 anim als/ group) according t o t he t im e of per iodont it is induct ion ( G0: 0, G3: 3, G7: 7, G15: 15, G30: 30, and G60: 60 days) by ligat ure m et hod.
Periodont it is induct ion
Under general anest hesia ( I P, ket am ine- 10% , 90 m g/ kg, and xylazine- 2% , 10 m g/ kg) , a cot t on ligat ure ( Coat s Corrent e, São Paulo, SP, Brazil) was insert ed
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( Figure 1A) . Aft er t he experim ent al periods ( 0, 3, 7, 15, 30, or 60 days post - periodont it is induct ion) , t he
form alin solut ion for 48 hours. The right side was used for m orphom et ric and radiographic analysis, while t he left side was used for hist ological analysis.
Radiographic analysis
The hem im andibles w er e radiographed using a digit al x- ray syst em ( Kavo Dent al, Biberach, Baden-Wur t t em ber g, Ger m any ) . The x- ray incidence w as lingual, w it h an exposure t im e of 0.04 seconds and an obj ect - focus dist ance of 30 cm . The m esial linear dist ance ( m m ) bet ween t he CEJ and t he alveolar bone
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soft ware I m age J 1: 48 ( Figure 1B).
Morphom et ric analysis
The hem im andibles w er e boiled for 30 m inut es and im m ersed in hydrogen peroxide solut ion ( 3% ) t o rem ove t he excess t issue. Aft er t his procedure, t he hem im andibles w ere st ained w it h m et hylene blue 1% t o dem arcat e t he CEJ. For each specim en, t he following w ere m easured: 1) linear dist ance ( m m ) bet w een t he CEJ and t he bone crest of t he t hree m andibular m olars, w it h a t ot al of seven m easurem ent s corresponding t o t he num ber of root s, and 2) t he area ( m m2) of bone
loss bet w een t he CEJ and t he bone crest of t he t hree
m olars. The m easurem ent s w ere m ade on t he lingual surface using t he soft ware I m age J 1.48 ( Figure 1C/ D) .
Hist ological analysis
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4 % a n d st a i n e d w i t h h e m a t o x y l i n e o si n ( HE) . Measurem ent s of t he linear dist ance ( m m ) bet w een WKH&(-DQGWKHLQWHUSUR[LPDODOYHRODUFUHVWWKH¿UVW m olar, an d t h e secon d m olar w er e obt ain ed. Th e m easurem ent s w ere m ade using t he soft ware I m age J 1.48 ( Figure 1E) .
Dat a evaluat ion and st at ist ical analysis
I nt ra- exam iner agreem ent ( repeat abilit y) of t he ABL m easurem ent t echniques:
A t rained exam iner per for m ed t w o independent ABL m easurem ent s ( radiographic, m orphom et ric, and hist ological t echniques) using t he G15 group w it h a
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t t est ( one sam ple) was carried out t o det erm ine t he int ra- exam iner agreem ent .
I nt er- exam iner agreem ent ( reproducibilit y) of t he ABL m easurem ent t echniques:
Tw o t rained ex am iner s per for m ed independent ABL m easu r em en t s ( r adiogr aph ic, m or ph om et r ic,
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and hist ological t echniques) using t he G15 gr oup.
%ODQG±$OWPDQ¶VWHVWIROORZHGE\Dt t est ( one sam ple) w as car r ied ou t t o det er m in e t h e in t er - ex am in er agreem ent .
Co r r e l a t i o n a m o n g t h e A B L m e a s u r e m e n t t echniques:
Using dat a from G15 group, Pearson’s correlat ion was carried out t o det erm ine t he correlat ion am ong t he different m et hodologies of ABL evaluat ion.
K i n e t i c s o f b o n e l o s s o b s e r v e d i n t h e ABL m easurem ent t echniques:
ANOVA and Tukey’s t est w ere used t o det erm ine
t he st at ist ical differences in ABL am ong t he periods of evaluat ion w it h t he soft ware Graph Pad Prism 6.0e.
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Result s
ABL Kinet ics
Bot h t he radiographic and m or phom et r ic/ linear
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15. Aft er t his period, t he ABL rem ained const ant unt il day 60 ( Figure 2A and B) .
Usin g m or p h om et r ic/ ar ea an aly ses, af t er ABL i n d u ct i o n , t h e m i n i m u m t i m e n eed ed t o d et ect
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bone loss occurred unt il day 15, and from day 15 t o day 60, t he ABL rem ained const ant ( Figure 2C) .
Using hist ological analyses, linear analysis showed
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unt il day 30. Aft er t his t im e, t he bone resorpt ion was
uniform ( Figure 2D). Figure 3 show s im ages of each
analy sis m et hodology ( Radiographic, Mor phom et r ic and Hist ologic) in t he different induct ion t im es.
Agreem ent analysis
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differences in eit her t he int ra- or t he int er- exam iner for all evaluat ions (p> 0.05) ( Figure 4 and 5) .
Correlat ion analysis
Pearson’s t est indicat ed high correlat ions (r2> 0.9, p< 0.05) bet ween t he ABL m easurem ent m et hodologies
evaluat ed ( Table 1) .
Discussion
The use of anim al m odels in periodont al research
is becom in g in cr easin gly fr equ en t . Bet w een 1 9 6 6
and 2016, m ore t han 300 art icles using experim ent al
periodont it is induced by ligat ure in rat s were published
i n t h e Pu b Me d d a t a b a se t h a t a i m e d t o cl a r i f y
,QGLFDWHVWKH¿UVWWLPHZLWKVLJQL¿FDQWERQHORVV$129$7XNH\S,QGLFDWHVWKHWLPHZKHQ$%/VWDELOL]DWLRQRFFXUUHG$129$ Tukey, p>0.05)
Figure 2-%RQHORVVIRUHDFKPHWKRGRORJ\LQUHODWLRQWRWKHSHULRGRI3'LQGXFWLRQXVLQJWKH$UDGLRJUDSKLFPHWKRG%PRUSKRPHWULF
biological pr ocesses or t o sear ch for new pot ent ial
t herapies relat ed t o PD. Alt hough t he ligat ure- induced
periodont it is m odel has been described since t he lat e
1960s17, int erest ingly, st udies t hat charact erize t his
m odel ( ABL over t he induct ion t im e) , as w ell as t he
agreem ent ( int ra- and int er- exam iner) and correlat ion
d eg r ee of t h e m ain m et h od olog ies em p loy ed f or
ABL evaluat ion, ar e scar ce. Tak en t oget her, t hese
param et ers allow for bet t er design of t he experim ent al
p r ot ocol an d su p p or t t h e com p ar i son of r esu l t s
obt ained by t he sam e or different research groups.
The principle of induct ion of Periodont al Disease wit h t he use of ligat ure is based not only on m echanical t raum a, but m ainly due t o plaque accum ulat ion, t hus
SURPRWLQJ D ORFDO LPPXQHLQÀDPPDWRU\ UHDFWLRQ UHVSRQVLEOHIRUWLVVXHLQÀDPPDWLRQDQGDOYHRODUERQH
loss17 7KH GHQWDO ELR¿OP LQ WKH OLJDWXUH LV VLPLODU
t o t hat obser v ed in hum an disease, com posed by
Act inom yces-, Fusobact erium -, Prevot ella nigrescens-,
Pa r v i m o n a s m i cr a- , Po r p h y r o m o n a s g i n g i v a l i s- an d Ag g r eg at ib act er act in om y cet em com it an s- lik e sp ecies4. Th e in d u ct ion of p er iod on t al d isease in
rat s using ligat ur e r esem bles hum an per iodont it is, since t he dest r uct ive phase is r epr esent ed by t he
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t issu e, w h ich p r ed at es b on e r esor p t ion7. I n ou r
st udy, w e could observe t his charact erist ic, since in
* JURXS LQÀDPPDWLRQ ZDV DOPRVW DEVHQW ZKLOH in t he ot her gr oups, it w as possible t o obser v e a
m ild ch r on ic in f lam m at ion , m ain ly com p osed b y
lym phocyt es predom inant ly found around j unct ional HSLWKHOLXP ZLWKRXW VLJQL¿FDQW GLIIHUHQFHV DPRQJ t he ex per im ent al per iods analy zed ( G3 , G7 , G1 5 , *DQG*7KHSDWWHUQRIFHOOLQ¿OWUDWLRQGLIIHUV from hum an periodont al disease, w here neut rophils
cells are predom inant in est ablished gingivit is and in
periodont it is15.
,QRXUVWXG\$%/LQFUHDVHGVLJQL¿FDQWO\FRPSDUHG w it h t he cont rol, on t he t hird day aft er PD induct ion,
w hen m easur ed by radiographic or m or phom et r ic/
linear t echniques, while ABL was det ect ed aft er 7 days
using m orphom et ric/ area and hist ological t echniques.
Th e r ap id b on e r esor p t ion d escr ib ed f or t h is r at Figure 4-%ODQG±$OWPDQ¶VWHVWLQWUDH[DPLQHUHYDOXDWLRQ$UDGLRJUDSKLFPHWKRG%PRUSKRPHWULFOLQHDUPHWKRG&PRUSKRPHWULF
DUHDPHWKRGDQG'KLVWRORJLFDOPHWKRG
Figure 5-%ODQG±$OWPDQ¶VWHVWLQWHUH[DPLQHUHYDOXDWLRQ$UDGLRJUDSKLFPHWKRG%PRUSKRPHWULFOLQHDUPHWKRG&PRUSKRPHWULF
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Radiographic Morphometric/linear Morphometric/area Histological
Radiographic - 0.98 0.99 0.91
Morphometric/linear 0.98 - 0.96 0.90
Morphometric/area 0.99 0.96 - 0.96
Histological 0.91 0.90 0.96
PRGHODQGFRQ¿UPHGLQRXUVWXG\PD\EHGXHWRWKH proxim it y of t he ligat ure t o t he bone crest .
This know ledge support s t he elaborat ion of short
prot ocols for ABL evaluat ions, m ainly in cases w hen is
necessary t o evaluat e biological param et ers associat ed
w i t h t h e t r i g g er i n g o f i n f l a m m a t o r y p r o cesses,
including t he init ial m echanism s of bone resorpt ion.
For exam ple, Rodini, et al.16 ( 2008) show ed t he iNOs
gene has higher expression on t he t hird day post
-periodont it is induct ion w hen using ligat ure m et hod,
w hile higher expression for t he MMP- 9 gene occurred
on t he sevent h day post- induct ion. I n addit ion, aft er 7
and 15 days, no differences were det ect ed in t he gene
expression of iNOs and MMP- 9, respect ively, com pared
w it h t he cont rol group. Based on t his inform at ion, t he
appropriat e choice of t he induct ion t im e associat ed
w it h t he ABL m easurem ent m et hodology represent s
key st eps for a st udy’s success16.
$GGLWLRQDOO\ $%/ VWDELOL]HG RQ WKH ¿IWHHQWK GD\ for all t he m et hodologies but hist ological analy sis,
su g g est i n g a ch r o n i c p r o cess af t er t h i s p er i o d .
Th er ef or e, t h e st u d y d esig n sh ou ld b e ad j u st ed
accor ding t o t he m ain obj ect ive and t he r esult s of
st udies perform ed wit h different induct ion t im es should
be com pared w it h caut ion. How ever, t he correlat ion
t est suggest s t hat a com parison could be carried out
w hen considering t he sam e induct ion t im e, even if
different ABL m easurem ent m et hodologies have been
used.
I n science, t he basic principle of any m et hodology
is t hat it is repeat able and reproducible. Every st udy
sh ou ld b e r ep r od u cib le b y t h e sam e or d if f er en t r esear ch gr oups, at any t im e. I n t his cont ex t , t he %ODQG±$OWPDQ WHVW UHVXOWV GHPRQVWUDWHG H[FHOOHQW
agreem ent ( bot h in repeat abilit y and reproducibilit y)
am ong t he m et hodologies evaluat ed, show ing t he
r eliabilit y of t he ABL m easur em ent m et hodologies used in periodont al research involving anim al m odels.
Conclusions
7KHDXWKRUVEHOLHYHWKLVLVWKH¿UVWVWXG\H[FOXVLYHO\
designed t o evaluat e t he k inet ics, agr eem ent and correlat ion of alveolar bone loss ( ABL) am ong four different analysis m et hodologies. Therefore, t he dat a of t his st udy show t hat choosing t he cor r ect ABL m easurem ent m et hodology is a crucial st ep during a st udy design, since t he init ial t im e of bone loss and
t he m om ent w hen t his pr ocess st abilizes can var y according t o t he m et hodology chosen for t his analysis. The agr eem ent and cor r elat ion r esult s suppor t t he com parison of result s am ong st udies w it h t he sam e induct ion t im e, even when different ABL m easurem ent m et hodologies have been used.
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