ABSTRACT
http://dx.doi.org/10.1590/1678-775720160099
I n t e r a ct i o n o f d e n t a l p u l p st e m ce l l s w i t h
Biodent ine and MTA aft er exposure t o different
environm ent s
Anastasia AGRAFIOTI1, Vasiliki TARASLIA2, Vanessa CHREPA3, Stefania LYMPERI2, Panos PANOPOULOS1, Ema ANASTASIADOU2, Evangelos G. KONTAKIOTIS1
1- National and Kapodistrian University of Athens, School of Dentistry, Department of Endodontics, Athens, Greece. 2- Biomedical Research Foundation of the Academy of Athens, Department of Genetics and Gene Therapy, Athens, Greece. 3- University of Washington, Department of Endodontics, Seattle, WA, USA.
Corresponding address: Evangelos G. Kontakiotis - 2 Thivon Str., 11527 - Goudi - Athens - Greece - Phone: +30 210 746 1269 - email: [email protected]
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bj ect ive: The aim of t he present st udy was t o evaluat e and com pare t he cyt ot oxic effect s of Biodent ine and MTA on dent al pulp st em cells ( DPSCs) and t o assess cell viabilit y and adherence aft er m at erial exposure t o an acidic environm ent . Mat erial and Met hods: DPSCs were cult ured eit her alone or in cont act wit h eit her: Biodent ine; MTA set for 1 hour; or MTA set for 24 hours. Aft er 4 and 7 days, cell viabilit y was m easured using t he MTT assay. Biodent ine and MTA were also prepared and packed int o st andardized bovine dent in disks and divided int o t hree groups according t o t he st orage m edia ( n= 6/ group) : freshly m ixed m at erials wit hout st orage m edium ( Group A) ; m at erials st ored in saline ( Group B) ; m at erials st ored in cit ric acid buffered at pH 5.4 ( Group C) . Aft er 24 hours, DPSCs were int roduced in t he wells and cell adherence, viabilit y, and cellular m orphology were observed via confocal m icroscopy aft er t hree days of cult ure. Cell viabilit y was analyzed using repeat ed- m easures analysis of variance t est wit h Tukey’s post hoc t est s (D= 0.05) . 5HVXOWV%LRGHQWLQHH[SUHVVHGVLJQL¿FDQWO\KLJKHUFHOOYLDELOLW\FRPSDUHGZLWKDOORWKHU JURXSVDIWHUGD\VZLWKQRGLIIHUHQFHVDIWHUGD\V1RWDEO\FHOOYLDELOLW\ZDVVLJQL¿FDQWO\ great er in 24- hour set MTA com pared wit h 1- hour set MTA and cont rol groups aft er 7 days. Mat erial exposure t o an acidic environm ent showed an increase in cell adherence DQGYLDELOLW\LQERWKJURXSV&RQFOXVLRQV%LRGHQWLQHLQGXFHGDVLJQL¿FDQWO\DFFHOHUDWHG cell proliferat ion com pared wit h MTA. Set t ing of t hese m at erials in t he presence of cit ric acid enhanced DPSC viabilit y and adherence.Keywords: Acidic environm ent . Biodent ine. Cyt ot oxicit y. MTA. Dent al pulp st em cells.
INTRODUCTION
Mineral t rioxide aggregat e ( MTA) is a calcium s i l i c a t e - b a s e d m a t e r i a l a n d h a s a t t r a c t e d con sider able at t en t ion becau se of it s ex cellen t biocom pat ibilit y, sealing abilit y, and ant im icrobial propert ies20,29. Alt hough it was init ially int roduced
as a m at erial for repair of root perforat ions, it is current ly used in vit al pulp t herapy, as a root- end
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p r o ce d u r e s, a n d i n r e g e n e r a t i v e e n d o d o n t i c t herapy12,15,28. Despit e it s broad spect rum of clinical
indicat ions, MTA com es w it h cer t ain lim it at ions in clu d in g lon g set t in g t im e, d if f icu lt h an d lin g ,
possibilit y of crown st aining, and high cost4,20.
Biodent ine ( Sept odont , Saint Maur des Fosses, France) , a new calcium silicat e- based rest orat ive cem ent , was r ecent ly int r oduced for endodont ic procedures. This bioceram ic m aterial is a fast- setting r est or at iv e m at er ial r ecom m en ded as a den t in subst it ut e t hat can be used in sim ilar applicat ions su ch as MTA1 4. Mat er ials t h at ar e in t en ded for
endodont ic applicat ions should st im ulat e repair or be biologically neut ral in order t o prom ot e healing6.
I f, how ever, endodont ic m at er ials ar e cy t ot ox ic,
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and cause cell deat h by apopt osis or necr osis8.
biocom pat ibilit y of MTA5,25,29, lim it ed infor m at ion
is av ailab le ab ou t t h e p ossib le cy t ot ox icit y of Biodent ine2,13,14,17.
Clinicians oft en face t he challenge of placing m at er ials in a low pH env ir on m en t du e t o t h e p r esen ce of in f lam m at ion1 8. Var iat ion s in t h e
p H at t h e t im e of p lacem en t cou ld af f ect t h e physical and chem ical propert ies of bot h MTA and Biodent ine5,19,24,31. I t has been shown t hat t he low
pH of t he surrounding m icroenvironm ent affect s t he hydrat ion react ion of MTA16, and t hat t he m ore
acidic t he MTA solut ion during t he set t ing process is, t he m ore ext ensive it s porosit y will be19. However,
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adher ence of cells t o t hese m at er ials aft er t he exposure t o an acidic environm ent .
The prim ary aim of t his st udy was t o evaluat e t he v iabilit y of dent al pulp st em cells ( DPSCs) w hen in cont act w it h Biodent ine in com par ison w it h MTA. Th e secon dar y aim w as t o ex am in e whet her t he presence of t hese m at erials in an acidic environm ent could have an effect on t he DPSCs viabilit y and adherence t o t hese m at erials.
MATERIAL AND METHODS
Cell culture
Hu m a n D PSCs w e r e p r o v i d e d b y Pr o Ce l l , Biot echnological Applicat ion SA ( At hens, Greece) . The cells were screened wit h Flow Cyt om et ry for m esenchy m al sur face m ar ker s. The t r iple panel of pr ot ein CD7 3 , CD9 0 , an d CD1 0 5 , w h ich by consensus is expressed in m esenchym al st em cells, was det ect ed in high levels ( > 85% ) . Moreover, t he cells were negat ive for CD45 ( hem at opoiet ic cell m arker) , CD34 ( hem at opoiet ic st em cell m arker) , a n d CD 3 1 ( e n d o t h e l i a l ce l l m a r k e r ) . D PSCs w er e cult ur ed in basal cult ur e m edia com posed
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Gibco, Glasgow, UK) supplem ent ed wit h 10% fet al bovine, 1X L- glut am ine ( Gibco) , penicillin ( 100 U/ m L; Gibco) and st rept om ycin ( 100 m g/ m L; Gibco) .
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wit h 0.05% t rypsin ( Gibco, Carlsbad, CA, USA) and passed t o subsequent cult ur e plat es or used in
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ut ilized in t his st udy.
Cell viability
Wh it e Pr oRoot MTA ( Den t sp ly Tu lsa Den t al Specialt ies, Mem phis, TN, USA) and Biodent ine ( Sep t o d o n t , Sa i n t Ma u r d es Fo sses, Fr a n ce) w er e pr epar ed accor ding t o t he m anufact ur er ’s inst ruct ions and placed at t he bot t om of a 48- well plat e ( n= 5/ group) . Mat erials were placed at a 2 m m t hickness and fully coverage of t he bot t om of
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aft er placem ent , m at erials were allowed t o set for
one hour at 37° C in 5% CO2 and 100% hum idit y under st er ile condit ions and 1x 104 DPSCs w er e
int roduced in each well in direct cont act wit h t he m at erials. To account for t he difference in set t ing t im e bet w een t he t est ing m at er ials, a gr oup of 2 4 - h ou r set Pr oRoot MTA ( MTA2 4 h ) w as also included ( n= 5) . MTA was allow ed t o set for 24 hours under t he sam e condit ions described above befor e DPSCs w er e int r oduced. All gr oups w er e incubat ed at 37° C in 5% CO2 and 100% hum idit y for 4 and 7 days. Cells wit hout m at erials served as con t r ol. Cell v iab ilit y w as m easu r ed u sin g m et hylt hiazolyldiphenyl- t et razolium brom ide ( MTT) based cell growt h det erm inat ion kit ( Sigm a Aldrich, St . Louis, MO) accor ding t o t he m anufact ur er ’s
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for each gr oup was det ect ed in a Flex St at ion 3 Bencht op Mult im ode Microplat e Reader ( Molecular Devices, Sunnyvale, CA, USA) .
S p e c i m e n p r e p a r a t i o n f o r
immunoÀuorescence
3 6 d en t in d isk s f r om an t er ior b ov in e t eet h were horizont ally sect ioned int o 3- m m slices using an I som et dev ice ( Buehler Lt d., Lak e Bluff, I L, USA) and t he canal space of each dent in slice was enlarged t o 2.6 m m in diam et er. Biodent ine and MTA were prepared according t o t he m anufact urers’ inst ruct ions under asept ic condit ions and packed in t o t h e lu m en of den t in disk s ( N= 1 8 / gr ou p) . Specim ens from each m at erial group were furt her divided int o t hree groups according t o t he st orage m edia and placed in 24- w ell plat es – Gr oup A: freshly m ixed m at erials wit hout st orage; Group B: m at erials wit h saline as st orage m edium ; Group C: m at erials wit h cit ric acid buffered at pH 5.4 as st orage m edium ( n= 6/ group) . Each specim en was kept in cont act wit h a saline- or cit ric acid- soaked piece of gauze for 24 hours in room t em perat ure.
I m m u n o f l u o r e s c e n c e a n d c o n f o c a l microscopy
Aft er 24 hours of st orage, 2.5x104 DPSCs were
int roduced in t he wells in direct cont act wit h t he dent in disks. Groups were cult ured for 72 hours at 37° C in a 5% CO2 KXPLGL¿HGLQFXEDWRU7KHFHOOV t hat adhered t o t he surface of t he sam ples were
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followed by 0.1 M PBS washing t wice for 10 m inut es. The sam ples were t hen st ained using Phalloidin-Rhodam in solut ion ( Molecular Probes, Therm oFisher
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m anufact urer ’s prot ocol t o reveal t he cyt oskelet on
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Laborat ories, Pet erborough, UK) , observed under t he confocal m icr oscope Leica TCS SP5 ( Leica, Wet zlar, Ger m any ) , an d pr ocessed u sin g Leica sof t w ar e, LAS AF ( Leica Micr osy st em s Gm b H, Wet zlar, Germ any) .
Statistical analysis
Dat a for t he cell viabilit y assay were analyzed u si n g on e- w ay r ep eat ed an al y si s of v ar i an ce ( ANOVA) w it h Tukey ’s post hoc t est s t o assess p a i r w i se d i f f e r e n ce s. Th e l e v e l o f st a t i st i ca l
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t est was per for m ed t o assess equalit y of gr oup variances prior t o perform ing ANOVA. JMP soft ware ( SAS I nst it ut e, Cary, NC, USA) and Prism 6 ( Graph Pad, La Jolla, CA, USA) were used for dat a analysis. Mean and st andar d dev iat ion ( m ean± SD) w er e report ed for sum m ary st at ist ics.
RESULTS
Result s fr om t he MTT cell v iabilit y assay ar e show n in Figur e 1. Biodent ine gr oup ex pr essed
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com pared wit h all ot her groups aft er 4 days ( all p < 0 . 0 1 ) . MTA sh ow ed sig n if ican t ly low er cell viabilit y ( 0.03± 0.01) aft er 4 days com pared wit h cont rol group ( 0.11± 0.05) ( p= 0.01) . Aft er 7 days,
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( 0 . 5 1 ± 0 . 2 3 ) com p ar ed w it h MTA ( 0 . 0 6 ± 0 . 0 3 ) ( p = 0 . 0 0 2 ) a n d c o n t r o l g r o u p ( 0 . 1 3 ± 0 . 0 2 )
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difference bet ween Biodent ine and all ot her groups aft er 7 days. I nt ragroup com parisons showed t hat
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t o 7 days ( p= 0.014) . A m ore det ailed present at ion
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differences is present ed in Table 1.
DPSCs adhered t o t he surface of dent in disks
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( Figure 2) . A higher cell densit y was observed in t he sam ples of t he acidic env ir onm ent ( Figur es 2C, 2F) when com pared wit h saline or no st orage m edia. Not ably, Biodent ine st or ed in cit r ic acid
Absorbance (4 days) Mean difference Standard error of mean
p-value
Biodentine MTA 0.162 0.021 <0.0001*
Biodentine MTA24h 0.111 0.021 0.0005*
Biodentine Control 0.084 0.021 0.0059*
Control MTA 0.078 0.021 0.0113*
Absorbance (7 days) Mean difference Standard error of
mean
p-value
MTA24h MTA 0.447 0.101 0.0023*
MTA24h Control 0.374 0.101 0.0099*
Absorbance (intragroup differences) Mean difference Standard error of mean
p-value
MTA24h (7 days) MTA24h (4 days) 0.426 0.103 0.0146*
Table 1-6XPPDU\RIWKHVLJQL¿FDQWLQWHUJURXSDQGLQWUDJURXSSDLUZLVHGLIIHUHQFHVLQFHOOYLDELOLW\IRUWKHGLIIHUHQWWLPH
points. Mean difference, standard error of mean, and p-value are summarized for each comparison. *p<0.05
dem onst rat ed great er am ount of adherent cells,
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assay s. Regar ding cell m or phology, t he cells on m at er ials st or ed in cit r ic acid dem on st r at ed a
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cyt oskelet on, whereas cells on m at erials st ored in saline, especially in t he Biodent ine group, showed
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( Figures 2B, 2E) .
DISCUSSION
Mat er ials int r oduced in pr ocedur es, such as v it al p u lp t h er ap ies, r eg en er at iv e en d od on t ic t herapies, or perforat ion repairs, should prim arily possess biocom pat ibilit y. MTA is com m only used in such procedures, since it is considered highly biocom pat ible25. Biodent ine, a new calcium silicat
e-based m at erial, has dem onst rat ed biocom pat ibilit y w h en t est ed on v ar iou s cell lin es w it h b et t er handling pr oper t ies and a shor t er set t ing t im e w hen com par ed w it h MTA2 , 1 3 , 1 4 , 1 7. Nev er t heless,
lim it ed evidence is available regarding Biodent ine int eract ions wit h dent al pulp st em cells21,33. This
st udy aim ed t o invest igat e t he biocom pat ibilit y of Biodent ine in com parison wit h MTA on DPSCs in a t im e course of 4 and 7 days as well as t he cell adherence t o t hese m at erials aft er exposure t o an acidic environm ent .
Am on g v ar iou s ad v an t ag eou s p r op er t ies of Biodent ine is t he fast er set t ing t im e com par ed wit h MTA22. To com pensat e for t he differences in
set t ing t im e bet ween t he t wo m at erials and have com parable r esult s in cell v iabilit y, set t ing- t im e point s of 1 hour and 24 hours were applied for MTA. Result s from t he cyt ot oxicit y assays aft er 4 days
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cont act wit h Biodent ine as com pared wit h t he ot her groups. Our dat a are in consensus wit h Widbiller, et al.32 ( 2016) w ho show ed t hat DPSC v iabilit y
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days. Nevert heless, ot her st udies have com pared t h e ce l l v i a b i l i t y o f h u m a n p u l p f i b r o b l a st s, h u m an g in g iv al f ib r ob last s, or ost eob last - lik e cells exposed t o Biodent ine or MTA and observed
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differences2,13,14. I nt erest ingly, one- hour set MTA
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aft er 4 days and MTA set for 24 hours dem onst rat ed
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agree wit h result s from a previous st udy, which show ed t hat apical papilla st em cells ex pr essed
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2 4 - hour set MTA as com par ed w it h 1 - hour set MTA2 6. On e p ossib le ex p lan at ion is t h at in it ial
release of calcium - ions as well as t he presence of leachable and t oxic com ponent s from fresh MTA m ay affect t he behavior of t he cells. I t is report ed t hat freshly m ixed calcium - silicat e based cem ent s m ay for m cont inuously calcium - silicat e hydrat es and pr ecipit at e calcium - phosphat e and calcium carbonat e7. Nonet heless, MTA and Biodent ine had
no differences in cell viabilit y aft er 7 days.
Cell adherence and viabilit y, w hen in cont act wit h Biodent ine com pared wit h MTA, were furt her
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m i cr o sco p y. I t h a s b een sh o w n t h a t co n t a ct of dent al m at er ials w it h dent in m ay alt er t heir propert ies9. Thus, t he int eract ion bet ween cem ent
and surrounding dent in was t aken int o considerat ion
Figure 2- Representative images of the cell cultures on the surface of the different dental materials by confocal microscopy. (A) MTA in no storage media, (B) MTA stored in saline, (C) MTA stored in citric acid, (D) Biodentine in no storage media, (E)
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in t his st udy ut ilizing dent in disks from ant erior bovine t eet h, since t hey could be considered an appropriat e subst it ut e for hum an t eet h3. Confocal
m icrographs showed t hat DPSCs at t ached on t he m at erials st ored in cit ric acid were m ore sprindle-sh aped com par ed w it h t h e m at er ials st or ed in saline. This fact is indicat ive of a good cell subst rat e int eract ion signify ing t hat bot h calcium - silicat e b ased m at er ials p r ov id e a sig n if ican t ly b et t er subst rat e for cell adhesion when t hey set in t he presence of cit ric acid6,23. A possible explanat ion is
t hat t he acidic condit ions of t he cit ric acid induced t he release of Ca- ions, and subsequent ly t he relat ive concent rat ion of Si increased30. Furt herm ore, t he
acid- et ching effect leads t o m icrost ruct ural changes t hat could affect t he adhesion and proliferat ion of cells on calcium silicat e- based m at erials1,10,19,27. The
result s of t his st udy agree, despit e t he differences in m et hodology, wit h t he result s of Kang, et al.11
( 2013) , who report ed t hat MTA m ixed wit h cit ric acid showed favorable biocom pat ibilit y. I m port ant ly, our st udy shows t hat Biodent ine prom ot ed great er cell adherence and viabilit y com pared wit h MTA. Nevert heless, t he m echanism s t hat are responsible for t hese effect s ar e not com plet ely under st ood and furt her research is required t o elucidat e t hem . These dat a m ay be applied in fut ur e st udies t o m odify t he sur face of t he m at er ials t o pr om ot e bet t er adhesion and sprout ing of cells.
CONCLUSION
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cell proliferat ion com pared wit h MTA and cont rol groups. Furt herm ore, 24- hour set MTA allowed for great er cell viabilit y com pared wit h 1- hour set MTA aft er 7 days. Bot h 1- hour and 24- h set MTA were init ially m ore cyt ot oxic com pared wit h Biodent ine,
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7- day t im e point . Exposure of MTA and Biodent ine t o an acidic environm ent showed an increase in t he num ber of DPSCs adhered t o t heir surface.
CONFLICT OF INTEREST
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of int erest .
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ABSTRACT
http://dx.doi.org/10.1590/1678-775720160014
Exopolysaccharide dispelled by calcium hydroxide
wit h volat ile vehicles relat ed t o bact ericidal effect
for root canal m edicat ion
Lei LEI1,4#, Meiying SHAO3#, Yan YANG1, Mengying MAO1, Yingming YANG2*, Tao HU1,2*
1- Sichuan University, West China Hospital of Stomatology, Department of Operative Dentistry and Endodontics, State Key Laboratory of Oral Diseases, Sichuan, China.
2- Sichuan University, West China Hospital of Stomatology, Department of Preventive Dentistry, Sichuan, China. 3- Sichuan University, College of Life Sciences, State Key Laboratory of Oral Diseases, Sichuan, China. 4- The Forsyth Institute, Department of Microbiology, Cambridge, United States.
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*Co-Corresponding Author
Corresponding address: Tao Hu - Department of Preventive Dentistry - Department of Operative Dentistry and Endodontics - West China Hospital of Stomatology,14#, 3rd section - Renmin South Road - Chengdu - Sichuan - 610041 - China - Phone: +86(0)28 85407723 - Fax: +86(0)28 8558 2167 - e-mail:
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bj ect ive: Ent er ococcus faecalis is t he dom inant m icr obial species r esponsible for per sist en t apical per iodon t it is w it h abilit y t o deeply pen et r at e in t o t h e den t in . Exopolysaccharides ( EPS) cont ribut e t o t he pat hogenicit y and ant ibiot ic resist ance of E.faecalis. Our aim was t o invest igat e t he ant im icrobial act ivit y of calcium hydroxide ( CH) ,
cam phorat ed parachlor ophenol ( CMCP) , and chlor hex idine ( CHX) against E. faecalis
in dent inal t ubules. Mat erial and Met hods: Decoronat ed single- canal hum an t eet h and sem icylindrical dent in blocks were incubat ed wit h E. faecalis for 3 weeks. Sam ples were random ly assigned t o six m edicat ion groups for 1 week ( n= 10 per group) : CH + 40% glycerin- wat er solut ion ( 1: 1, wt / vol) ; CMCP; 2% CHX; CH + CMCP ( 1: 1, wt / vol) ; CH + CMCP ( 2: 3, wt / vol) ; and saline. Bact erial sam ples were collect ed and assayed for colony- form ing unit s. Aft er dent in blocks were split longit udinally, confocal laser scanning m icroscopy was used t o assess t he proport ion of viable bact eria and EPS product ion in dent in. Result s: CMCP exhibit ed t he best ant im icrobial act ivit y, while CH was t he least sensit ive against
E. faecalis ( p< 0.05) . CHX showed sim ilar ant im icrobial propert ies t o CH + CMCP ( 1: 1,
wt / vol) ( p> 0.05) . CH com bined wit h CMCP inhibit ed EPS synt hesis by E. faecalis, which VHQVLWL]HG ELR¿OPV WR DQWLEDFWHULDO VXEVWDQFHV 0RUHRYHU LQFUHDVLQJ FRQFHQWUDWLRQV RI &0&3GHFUHDVHG(36PDWUL[IRUPDWLRQZKLFKHIIHFWLYHO\VHQVLWL]HGELR¿OPVWRGLVLQIHFWLRQ agent s. Conclusion: The EPS m at rix dispelled by CH past e wit h CMCP m ay be relat ed t o it s bact ericidal effect ; t he visualizat ion and analysis of EPS form at ion and m icrobial colonizat ion in dent in m ay be a useful approach t o verify m edicam ent s for ant im icrobial t herapy.
Keywords: Calciu m h y d r ox id e. Med icat ion s. Disin f ect ion . En t er ococcu s f aecalis. Exopolysaccharide.
INTRODUCTION
Micr obiological sam pling and ex am inat ion of t eet h wit h failed root canal t reat m ent s have shown
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of gram - posit ive organism s. Ent erococcus faecalis (E. faecalis) is considered a predom inant organism t hat is frequent ly isolat ed from persist ent ly infect ed
r oot can al1 8. I n v iv o m od el, or al b act er ia can
penet rat e up t o 200 m m int o dent inal t ubules, which m ay m ake t he bact eria resist ant t o ant im icrobial agent s3 7KH UHVLGXDO PLFURELDO ÀRUD ORFDWHG LQ
inaccessible ar eas of t he r oot canal anat om y is t he m ain cause of persist ent periapical infect ions. Because of anat om ical com plex it ies t hat cannot
