Abst ract
Submitted: November 22, 2016 0RGL¿FDWLRQ$SULO Accepted: June 02, 2017
Apical Negat ive Pr essur e ir r igat ion
pr esent s t issue com pat ibilit y in
im m at ur e t eet h
Aim : To com par e t he apical negat iv e pr essur e ir r igat ion ( ANP) w it h
convent ional irrigat ion in t he t eet h of im m at ure dogs wit h apical periodont it is.
Met hods: Fift y- t w o im m at ur e pr e- m olar r oot canals w er e random ly assigned
int o 4 gr oups: ANP ( n= 15) ; convent ional ir r igat ion ( n= 17) ; healt hy t eet h
( cont r ol) ( n= 10) ; and t eet h w it h unt r eat ed apical per iodont it is ( cont r ol)
( n= 10) . Aft er induct ion of apical periodont it is, t eet h were inst rum ent ed using
EndoVac® ( apical negat ive pr essur e ir r igat ion) or convent ional ir r igat ion.
The anim als w er e eut hanized aft er 90 days. The sect ions w er e st ained by +( DQG DQDO\]HG XQGHU FRQYHQWLRQDO DQG ÀXRUHVFHQFH PLFURVFRS\ 75$3 hist oenzym ology was also per for m ed. St at ist ical analyses w er e per for m ed ZLWK WKH VLJQL¿FDQFH OHYHO VHW DW 5HVXOWV 7KHUH ZDV GLIIHUHQFH LQ t he hist opat hological param et er s bet w een ANP and convent ional gr oups
( p < 0 . 0 5 ) . Th e ANP g r ou p sh ow ed a p r ed om in an ce of low m ag n it u d e LQÀDPPDWRU\LQ¿OWUDWHDVPDOOHUSHULRGRQWDOOLJDPHQWDQGORZHUPLQHUDOL]HG t issue resorpt ion. There were no differences in t he periapical lesion ext ensions
bet w een t he ANP and convent ional gr oups ( p> 0. 05) . How ever, a low er
num ber of ost eoclast s was observed in t he ANP group ( p< 0.05) . Conclusion:
The EndoVac® ir r igat ion syst em pr esent ed bet t er biological r esult s and m or e
advanced r epair pr ocess in im m at ur e t eet h w it h apical per iodont it is t han t he FRQYHQWLRQDOLUULJDWLRQV\VWHPFRQ¿UPLQJWKHK\SRWKHVLV
Ke yw or ds: Apical negat ive pressure irrigat ion. Apical periodont it is. Apical posit ive pr essur e ir r igat ion. I m m at ur e t eet h.
Carolina Maschietto PUCINELLI1
Léa Assed Bezerra da SILVA1
Nestor COHENCA2
Priscilla Coutinho ROMUALDO1
Raquel Assed Bezerra da SILVA1
Alberto CONSOLARO3
Alexandra Mussolino de QUEIROZ1
Paulo NELSON-FILHO1
http://dx.doi.org/10.1590/1678-7757-2016-0599
1Universidade de São Paulo, Faculdade de Odontologia de Ribeirão Preto, Departamento de Clínica Infantil, Ribeirão Preto, SP, Brasil.
2University of Washington & Seattle Children’s, Department of Endodontics, Seattle, USA. 3Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Cirurgia, Estomatologia, Patologia e Radiologia, Bauru, SP, Brasil.
I nt r oduct ion
I nfect ion cont rol in endodont ic t herapy is ext rem ely im por t ant t o achieve a successful out com e in t eet h w it h apical per iodont it is7,16,25,27. This can be obt ained
t h r o u g h t h e st e p s i n v o l v e d i n t h e e n d o d o n t i c t r eat m en t1 5, in clu d in g ir r ig at ion8. Th e associat ion
bet w een ir r igat ion an d biom ech an ical pr epar at ion opt im izes t he cleaning of r oot canals6,22.
Alt h ou gh t h e conv en t ion al ir r igat ion sy st em is widely used12, in 2007 a novel syst em called EndoVac®
( Discus Dent al, Culver Cit y, CA, USA) was launched t o t he dent al m ar ket . I nst ead of posit ive pr essur e, t he EndoVac® sy st em uses an apical negat ive pr essur e
ir r igat ion ( ANP) and has been consider ed a pr om ising disinfect ion pr ot ocol in t he endodont ic lit erat ur e8,23.
Previous st udies dem onst rat ed t hat t he ANP decreases t he risk of irrigant solut ion ext rusion t hrough t he apical f or am en9 , 1 4 , 2 6. Th is sy st em en ables t h e cir cu lat ion
of ir r igant solut ion t o all w or k ing lengt hs ( WL)2 and
facilit at es m icr obiological cont r ol13,23. The ANP is also HI¿FLHQWLQUHPRYLQJWKH³VPHDUOD\HU´10,18 and debr is,
m ost ly at t he apical t hir d of a r oot canal1,4,10,11.
The anat om y of t he apical t hir d in t he r oot canals of im m at ur e t eet h incr eases t he r isk of accident al i n j ect i o n o f i r r i g an t so l u t i o n i n t o t h e p er i ap i cal t issues3,26. How ever, an in vit r o st udy dem onst rat ed
t hat open apex t eet h had sim ilar ex t r usion t o closed apex t eet h w hen t he ANP was used20.
To dat e, no in vivo st udies com paring t he EndoVac®
sy st em w i t h t h e co n v en t i o n a l i r r i g a t i o n sy st em separat ely in im m at ur e t eet h have been published. Th er ef or e, t h e aim of t h is in v iv o st u d y w as t o per for m t he hist opat hological and hist oenzy m ological evalu at ion t o com par e t h e En doVac® sy st em w it h
t he conv ent ional ir r igat ion in im m at ur e dog t eet h w it h ex per im ent ally induced apical per iodont it is. The hy pot hesis is t hat ANP pr esent ed bet t er biological result s in im m at ure t eet h wit h apical periodont it is t han t he convent ional ir r igat ion sy st em .
Mat er ial and m et hods
Th i s r e se a r ch p r o j e ct w a s a p p r o v e d b y t h e I nst it ut ional Anim al Et hics Com m it t ee ( # 006/ 2012) . All t he ex per im ent al pr ocedur es w er e per for m ed as in our pr ev ious st udies8,24.
Three beagle dogs, 4 m ont hs old, were used. Upper ( secon d an d t h ir d) an d low er ( secon d, t h ir d, an d four t h) im m at ur e pr em olar s w er e included. Fift y- t w o root s w ere random ly divided int o 4 groups, as follow s:
ANP ( Apical Negat ive Pr essur e) : ( n= 15) .
Co n v e n t i o n a l I r r i g a t i o n ( Po si t i v e Pr e ssu r e ) : ( n= 17) .
Healt hy Teet h ( Negat ive Cont r ol) : ( n= 10)
Teet h w it h unt r eat ed apical per iodont it is ( Posit ive Cont r ol) : ( n= 10) .
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ap ex . Th e cor on al access w as p er f or m ed in t h e En d oVac®, con v en t ion al, an d ap ical p er iod on t it is
gr oups. Aft er pulp t issue r em oval, r oot canals w er e left ex posed in t he oral cav it y for 7 day s for m icr obial cont am inat ion, as r ecom m ended by Leonar do, et al.17
( 1993) . I n or der t o pr om ot e t he induct ion of apical per iodont it is, t he pulp cham ber was sealed w it h zinc oxide eugenol cem ent ( SS Whit e, Rio de Janeir o, RJ, Brazil) . Bone t hinning occur s bet w een 15 and 25 day s in im m at ur e dog t eet h17.
Af t er t h is p er iod , a r u b b er d am w as u sed t o isolat e t he t eet h and t he t em porar y r est orat ion was r em oved. Root canal disinfect ion w as per for m ed 3 m m shor t er t han t he radiographic apex , follow ed by WL det er m inat ion, est ablished 1 m m shor t er t han t he radiographic apex . The com plem ent at ion of r oot canal disinfect ion was per for m ed at t he WL. ANP and convent ional gr oups w er e inst r um ent ed w it h K- t y pe
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w er e ir r igat ed w it h 10 m L of NaOCl 2.5% for bot h t he EndoVac® sy st em and convent ional sy st em .
Th e r ecom m en d ed p r ot ocol f or t h e En d oVac®
sy st em includes 2 m ain st eps: m acr o- ir r igat ion and m icro- irrigat ion. I n t his part icular st udy wit h im m at ure t eet h, canals w er e ir r igat ed using t he m acr o- cannula only aft er det er m ining t he apical size of t he canal due t o t he lar ge apical size, as pr ev iously published23.
Finally, r oot canals w er e ir r igat ed w it h saline solut ion, dr ied w it h absor bent paper point s and sealed w it h ProRoot MTA ( Dent sply Tulsa Dent al, Johnson Cit y, TN, USA) and silver am algam ( Sybraloy, Kerr Corporat ion, Orange, CA, USA) .
HE- st aining
The HE- st ained sect ions w er e analy zed in an Ax io I m ager.M1 m icr oscope ( Zeiss, Göt t ingen, Ger m any ) , using scor es based on t he follow ing hist opat hological
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m ild ( scor e 1) , m oderat e or sever e ( scor e 2) ; ( b) t hick ness of per iodont al ligam ent : nor m al ( scor e 1) , slight ly incr eased ( scor e 2) , m oderat ely incr eased ( scor e 3) , or sever ely incr eased ( scor e 4) ; and ( c) pr ocess of r esor pt ion of m ineralized t issues: absent ( score 1) or present ( score 2) . I n addit ion, descript ions of t he apical and per iapical r egions w er e conduct ed for each gr oup.
Fluor escence m icr oscope m or phom et r y
I n t he ANP, convent ional, and apical per iodont it is groups, t he area of t he periapical lesion was m easured in squar e m illim et er s in t he HE- st ained sect ions using
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descr ibed5,19,24.
I n t he healt hy t eet h gr oup, t he t hick ness of t he healt hy per iodont al ligam ent ar ea was m easur ed by draw ing a line per pendicular t o t he r oot apex , locat ed 0.5 m m above t he opening of each specim en, t o delim it t he m ax im um height of t he m easur ed ar ea.
Ta r t r a t e - r e s i s t a n t a c i d p h o s p h a t a s e
hist oenzym ology ( TRAP)
Th e TRAP act iv it y w as per f or m ed t o m ar k t h e ost eoclast s8 , 1 9. Th e sect ion s w er e d ep ar af f in ized ,
hydrat ed, and placed in a solut ion of 50% et hanol/ acet one for 1 m inut e and dr ied at r oom t em perat ur e. Ne x t , a b u f f e r so l u t i o n co n t a i n i n g a ce t i c a ci d , dim et hy lfor m am ide, Fast Red, and phosphor ic acid napht hol AS- BI ( Sigm a- Aldrich Corporat ion, St . Louis, MO, USA) w as p ip et t ed ov er t h e sect ion s, w h ich w er e m aint ained at 37°C for 40 m inut es pr ot ect ed fr om light . The count er- st ain w it h Fast Gr een w as per for m ed.
The sam ples were exam ined under t he Axio I m ager. M1 m icr oscope under convent ional light t o count t he num ber of m ult inucleat e TRAP–posit ive cells pr esent in t he r esor pt ion lacunae t hat w er e in dir ect cont act w it h t he alveolar bone ar ound t he per iapical lesion.
Ther e was an invaginat ion of par t ially m ineralized con n ect iv e t issu e in t o t h e r oot can al in t h e ANP specim ens. I n t he healt hy specim ens, t he ost eoclast count r egion w as est ablished as descr ibed for t he
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w er e ex pr essed in cell num ber s.
St at ist ical Analysis
The GraphPad Pr ism 5.a ( GraphPad Soft war e I nc., San Diego, CA, USA) was used for st at ist ical analyses. Chi- square or Fisher ’s exact t est were used t o evaluat e t he scor es. One- way ANOVA w it h Tukey ’s post hoc t est was used t o evaluat e m ean differ ence. The level
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Result s
Micr oscopic analysis of apical and per iapical
r egions
Th e ANP g r ou p p r esen t ed m ix ed an d d if f u sed
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t o m oderat e ( 26.7% ) . The per iapical r egion show ed r ich neovascular izat ion and bet t er r epair pr ocess w it h
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connect ive t issue in t he r egion of t he lar ge apical foram en t o t he m iddle t hir d of t he r oot canal. This t issu e or igin at ed fr om t h e per iodon t al t issu e t h at invaginat ed int o t he r oot canal in 53.6% of t he cases,
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I n 80% of t he specim ens t her e was no dent in, bone, or cem ent um r esor pt ion ( Figur e 1) .
I n t he convent ional gr oup, t her e w er e m ixed and
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The m aj or it y of t he cases ( 58.8% ) w er e m oderat ely in f lam ed . Th e p er iod on t al lig am en t w as sev er ely incr eased in 82.4% of t he cases, w it h ar eas of edem a
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was sever e bone, cem ent um , and dent in r esor pt ion. I n som e cases, t her e w er e no cem ent oblast s on t he cem ent sur face and unr epair ed r oot r esor pt ion was also fr equent ( Figur e 2A and B) .
All par am et er s ( pu lp t issu e, odon t oblast lay er, periodont al ligam ent , and alveolar bone) in t he healt hy t eet h gr oup w er e nor m al ( Figur e 2C) .
I n t he apical per iodont it is gr oup, t he apical and p er iap ical r eg ion s p r esen t ed sev er ely m ix ed an d d if f u sed in f lam m at or y in f ilt r at e. Th e p er iod on t al ligam ent was sever ely incr eased w it h int ense edem a
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r esor pt ion ar eas w er e not r epair ed. The alveolar bone was dist ant fr om t he r oot apex , indicat ing advanced bone r esor pt ion. Ther e w er e no ost eoblast s on t he su r f ace an d ost eoclast s w er e f r eq u en t ly p r esen t ( Figur e 2D) .
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groups ( p= 0.03) . Regarding t he periodont al ligam ent , differ ence was obser ved ( p= 0.02) bet w een t he ANP
an d con v en t ion al g r ou p s. St at ist ically sig n if ican t differ ence was also obser ved bet w een t hese gr oups ( p= 0.003) , r egar ding m ineralized t issue r esor pt ion
Figure 2- Representative photomicrographs of the conventional, healthy, and apical periodontitis groups, 90 days after the endodontic treatment, in conventional light microscopy: (A) Panoramic view of the apical and periapical regions of the conventional group showing an increased periodontal ligament (HE, Zeiss, 1.25X). (B) Detail of the alveolar bone of the conventional group with no osteoblast in its surface and presence of osteoclast (HE, Zeiss, 20X). (C) Panoramic view of the healthy teeth group with incomplete root formation, normal pulp, and an odontoblastic layer. Periodontal ligament and alveolar bone were healthy (HE, Zeiss, 10X). (D) Panoramic view of the apical
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Figure 1- Representative photomicrographs of the ANP group, 90 days after the endodontic treatment, in conventional light microscopy: (A) Panoramic view of the periapical and apical regions showing intense invagination of the connective tissue into the root canal (HE, Zeiss, 5X). (B) Detail of panel A: part of the invaginated tissue after the mineralization (HE, Zeiss, 20X). (C) Panoramic view of the periapical and apical regions showing that the periodontal ligament was slightly increased (HE, Zeiss, 5X). (D) Photomicrography of the
( Table 1) .
Fluor escence m icr oscopy m or phom et r y
The m ean lesion size was 12.94 ( ± 7.73) m m2 in
t he ANP group, 17.91 ( ± 8.85) m m2 in t he convent ional
g r o u p , a n d 2 1 . 4 7 ( ± 1 . 4 8 ) m m2 i n t h e a p i c a l
per iodont it is gr oup. I n t he healt hy t eet h gr oup, t he periodont al ligam ent area in t he apical region was 0.67 m m2 ( ± 0.38) m m27KHUHZDVQRVLJQL¿FDQWGLIIHUHQFH
bet w een t he ANP and convent ional gr oups ( p> 0.05) . Figure 3 shows represent at ive phot om icrographs of t he
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TRAP hist oenzym ology
The m eans for t he ost eoclast count s w er e 26.25 ( ± 18.78) for t he ANP gr oup, 50.94 ( ± 26.74) for t he
Groups p-Value
Scores EndoVac® Conventional Healthy Apical EndoVac® EndoVac® EndoVac® Conventional Conventional
Periodontitis x x x x x
Conventional Healthy Apical Healthy Apical Periodontitis Periodontitis ,QÀDPPDWRU\LQ¿OWUDWH
Absent or Mild Moderate or
Severe
0.03 0.06 0.001 0.0002 0.263
Thickness of periodontal ligament Normal
Slightly increased Moderately
increased Severely increased
0.02 0.005 0.273
Process of resorption of mineralized tissues Present
Absent
0.003 0.250 0.263
Table 1-5HVXOWVIRU,QÀDPPDWRU\LQ¿OWUDWHWKLFNQHVVRISHULRGRQWDOOLJDPHQWDQGUHVRUSWLRQRIPLQHUDOL]HGWLVVXHEHWZHHQWKHJURXSV
Figure 3- Photomicrographs of microscopic sections representing the 4 groups evaluated, 90 days after the endodontic treatment: (A)
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1.25X). HE = hematoxylin & eosin
Figure 4- Distribution of the number of osteoclasts. The different
convent ional gr oup, 7.9 ( ± 4.99) for t he healt hy t eet h gr oup, and 103 ( ± 23.27) for t he apical per iodont it is
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bet ween t he ANP and convent ional groups ( p< 0.0001) . Figur e 5 show s r epr esent at ive phot om icr ographs of all gr oups aft er TRAP hist oenzy m ology.
Discussion
Our research group’s previous st udy8 dem onst rat ed
t h a t t h e A N P p r e s e n t e d m a n y a d v a n t a g e s i n com par ison w it h t he convent ional ir r igat ion sy st em
for t eet h w it h closed apex . How ever, t her e ar e no in
vivo st udies t hat evaluat ed t he EndoVac® sy st em in
t eet h w it h im m at ur e r oot for m at ion, com par ed w it h convent ional ir r igat ion separat ely. Thus, t his in vivo st udy aim ed t o add im por t ant infor m at ion t o pr ov ide
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in t eet h w it h open apex . This is ex t r em ely im por t ant t o ach iev e a su ccessf u l ou t com e post - en dodon t ic t r eat m ent in im m at ur e t eet h w it h apical per iodont it is. Th i s h i st o p a t h o l o g i ca l st u d y co m p a r e d t w o differ ent t y pes of r oot ir r igat ion sy st em . The init ial ex per im ent al st ages evaluat ion enables t o obser ve
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Figure 5- Photomicrographs of microscopic sections representing the 4 groups evaluated, 90 days after the endodontic treatment, stained
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t echnique, as w ell as et hical im plicat ions. For t hese r easons, w e decided t o evaluat e a longer per iod t o obser ve t he occur r ence of per sist ent inj ur y t o t issues
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lat e st age aft er use of different root irrigat ion syst em s. This m et hodology r epr esent s t he “ gold st andar d” of t issue r esponse against t he use of differ ent m at er ials or t echniques. I n t he endodont ic lit erat ur e, only 2 paper s8,23 evaluat ed t he m icr oscopic r esponse of t he
EndoVac® sy st em in vivo using dog t eet h. Silva, et
al.23 ( 2010) com par ed t he r evascular izat ion and t he
apical and periapical repair in im m at ure dog t eet h wit h per iapical lesions aft er ir r igat ion w it h t he EndoVac®
sy st em and convent ional ir r igat ion plus t r iant ibiot ic
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in f ilt r at e w as sig n if ican t ly d if f er en t b et w een t h e gr oups, t he cases t r eat ed w it h t he ANP had a higher m ineralized t issue for m at ion in t he apical r egion. The aut hors concluded t hat t he EndoVac® syst em m ight be
considered a valuable disinfect ion prot ocol in im m at ure per m anent t eet h w it h apical per iodont it is, and t hus int racanal ant ibiot ics w ould not be necessar y. The r esult s of our st udy ar e in agr eem ent w it h Silva, et al.23 ( 2010) , in w hich EndoVac® specim ens pr esent ed
st r uct ur ed connect ive t issue, r ich vascular izat ion, and r epair pr ocess in advanced st age.
Cohenca, et al.8 ( 2015) com pared t he ANP irrigat ion
w it h convent ional ir r igat ion and ult rasonic ir r igat ions i n d o g t eet h w i t h co m p l et e r o o t f o r m a t i o n a n d apical per iodont it is. The r esult s of t heir m icr oscopic
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specim ens in com par ison w it h convent ional ir r igat ion. Th e per iodon t al ligam en t t h ick n ess, r esor pt ion of m ineralized t issues, size of per iapical lesions, and t he num ber of ost eoclast s did not pr esent st at ist ically
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r esult s do not agr ee w it h our s, pr obably due t o t he par t icu lar an at om ical ch ar act er ist ics of t eet h w it h in com p let e r oot f or m at ion , w h ich p r esen t r ich er vascular supply21,29 enabling shor t er r epair pr ocess.
I n our st udy, t her e w as a par t ially m ineralized t issue invaginat ion in 53.6% of t he r oot canals 90 day s aft er use of t he ANP. The invaginat ion of healt hy periodont al t issue int o t he root canal in t he apical region indicat ed t hat t he int ense m icrobial cont am inat ion was cont r olled in 53.6% of t he cases. We hy pot hesize t hat t he ant im icr obial dr essing bet w een t he appoint m ent s
m ight incr ease t his success rat e.
Ther e was no st at ist ical differ ence bet w een t he En doVac® an d conv en t ion al gr ou ps r egar din g t h e
r esult s of t he post - ir r igat ion per iapical lesion ar ea
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ot her hand, t hr ough t he hist opat hological analy sis of sect ions st ained w it h HE, advanced st age t issue repair in t he ANP specim ens w as ev ident . The per iapical lesions t hat w er e pr esent in som e specim ens of t he ANP gr oup did not com plet ely r epair and pr esent ed an ex t ensive lesion ar ea. This m ay ex plain t he lack
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t he ANP and convent ional gr oups in t he assessm ent
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Bone is a dynam ic t issue wit h cont inuous rem odeling pr ocess. I n pat hological condit ions, such as chr onic apical per iodont it is, bone r esor pt ion is gr eat er t han bone for m at ion. One of t he charact er ist ics of t his condit ion is t he accum ulat ion of ost eoclast s in t he bone r esor pt ion ar eas28. I n t his st udy w e obser ved VLJQL¿FDQW GLIIHUHQFH LQ WKH QXPEHU RI RVWHRFODVWV
bet w een t h e t w o t y pes of ir r igat ion . Th e av er age num ber of TRAP- posit ive cells was low er in EndoVac®
-t r ea-t ed -t ee-t h -t han in conv en-t ional ir r iga-t ion. This r esult does not agr ee w it h t hat pr ev iously published by Cohenca, et al.8 ( 2015) , who observed no st at ist ical
differ ence in t he num ber s of ost eoclast s. This was pr obably due t o t he fact t hat t he aut hor s used t eet h w it h com plet e r oot for m at ion an d closed apex as ex per im ent al m odel.
Conclusion
The r esult s fr om t his in v iv o st udy allow ed us t o con clu d e t h at t h e n eg at iv e p r essu r e ir r ig at ion ( En doVac®) dem onst rat ed bet t er biological r esult s
t han t he convent ional irrigat ion in im m at ure t eet h wit h apical per iodont it is and pr esent ed a m or e advanced
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st u d ies sh ou ld b e p er f or m ed in or d er t o p r ov id e addit ional infor m at ion for dent al pract ice.
Acknow ledgm ent s
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t o t his st udy. This invest igat ion was suppor t ed in par t by a scholar ship fr om CNPq – Nat ional Council for
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