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:
hutao@scu.edu.cn
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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. faecalisin 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.
Ke y w o r d s: 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.
I N TROD UCTI ON
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 WKDWWKHPLFURELDOÀRUDLVSUHGRPLQDQWO\FRPSULVHG 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
clean r oot canals m echanically. Thus, int racanal m edicat ion has been proposed as a com ponent of inst rum ent at ion and irrigat ion t o elim inat e bact eria along wit h t heir by- product s15.
Calcium hy dr ox ide ( CH) past e is one of t he m ost com m on ly u sed in t r acan al m edicam en t s. When dissolved in wat er, t he CH past e dissociat es int o hydroxide and calcium ions; as a result , t he ant im icrobial act ion of t his m edicat ion depends on t he pr esence of hydr oxide ions in t he solut ion5.
Desp it e it s ex cellen t p r op er t ies, t h e b u f f er in g act ion of dent in can neut ralize t he ant im icrobial act ivit y of CH at deeper layers of dent inal t ubules, an d E. f aecalis r esist an ce t o t h is m ed icam en t h a s co n se q u e n t l y b e e n d e m o n st r a t e d1 9. Th e
cam phorat ed parachlor ophenol ( CMCP) solut ion cont ains chem ical ingredient s, such as chlorophenol, and t he ant im icr obial act ions of CMCP pr oduct s are relat ed t o it s abilit y t o dest roy t he bact erial m em brane t hrough prot ein and lipid binding. The associat ion of CH past e and CMCP form s calcium p - ch lor op h en olat e, w h ich in cr eases t h e p H of dent in, allowing t he liberat ion of hydroxyl ions for an ext ended t im e17. The addit ion of cam phorat ed
parachlorophenol ( CMCP) t o CH past e was m ore effect ive t o elim inat e t he m icroorganism s in infect ed root canals, based on t he m et hod of bact eriological sam pling in root canals using paper point s16. The
associat ion of CH w it h CMCP m ay enhance t he diffusion of CH, while m aint aining a relat ively high pH value. Furt herm ore, t he addit ion of CH past e has been shown t o decrease t he t oxicit y of CMCP in per iapical t issues via pr olonged act ion of t he calcium hydroxide- based past e9. When t he CH past e
com bined w it h CMCP was used as an int racanal m edicat ion, 74% of t he successes were cat egorized as cases of t w o- v isit t r eat m ent24. How ever, t his
m et hodology is lim it ed, since t he m icroorganism s locat ed inside t he dent in were not collect ed.
The chlorhexidine ( CHX) is posit ively charged an d h as been r epor t ed t o act by elect r ost at ic int eract ions wit h t he negat ively charged bact erial w a l l . Th i s p r o ce d u r e a l t e r s t h e ce l l o sm o t i c e q u i l i b r i u m a n d r e su l t s i n ce l l d e a t h , w h i ch cont ribut es t o t he ant im icrobial effect of CHX6. Two
percent CHX gel is used as an int racanal dressing, in which it is effect ive against m icroorganism s in infect ed root canals16. The challenge of elim inat ing
all bact eria in root canals m ay be part ly at t ribut ed t o t heir invasion int o dent inal t ubules, where t he organism s are prot ect ed from m edicam ent s. The addit ion of CMCP t o CH past e m ay enhance t he d if f u sion of CH an d sig n if ican t ly d ecr ease t h e t oxicit y of CMCP in periapical t issues9. Thus, we
VRXJKWWRH[DPLQHWKHHI¿FDF\RI&+SDVWH&0&3 and CHX gel t o elim inat e bact eria wit hin dent inal t ubules.
E. f aecal i s b ecom es m or e r esist an t in t h e
r oot can als b ecau se of it s ab ilit y t o ad ap t t o h ar sh env ir on m en t al ch an ges an d st ar vat ion1 3.
Ex o p o l y s a c c h a r i d e s ( EPS ) a r e t h e c r u c i a l FRPSRQHQWVRIWKHSURWHFWLYHVKHOWHULQRUDOELR¿OPV2.
Consequent ly, t he dom inant m echanism involved in ELR¿OPUHVLVWDQFHWRDQWLPLFURELDOWKHUDS\PD\EH t he capabilit y of EPS t o provide m echanical st abilit y and drug t olerance. Addit ionally, t reat ing E. faecalis
b iof ilm s w it h d ex t r an ase ef f ect iv ely sen sit ized bact eria t o a 2% CHX solut ion, which highlight ed t he hypot hesis t hat dispelling EPS provides a new t arget for ant im icrobial t herapies12. However, lit t le
is k now n about t he effect s of m edicam ent s on EPS synt hesis and E. faecalis survival in dent inal t ubules.
Recent ly, a dent in infect ion m odel was generat ed t o est ablish st an dar d an d deep pen et r at ion of b act er i a i n t o d en t i n al t u b u l es1 , 1 4. Th i s v i su al
st r a t e g y e f f i ci e n t l y a sse sse d t h e e f f i ca cy o f en d o d o n t i c m ed i ca t i o n a g a i n st E. f a eca l i s i n dent in20. I n addit ion, t he cont am inat ion prot ocols
and t he m et hod for t he m icrobial infect ion of t he GHQWLQHKDGEHHQPRGL¿HGLQWKHin vit ro m odel1 and
invest igat ed in int ra- oral appliances3. As E. faecalis
play a cent ral role in endodont ic infect ions, wit h EPS likely cont ribut ing t o it s pat hogenicit y and ant ibiot ic r esist ance, t his st udy sought t o invest igat e t he an t im icr ob ial ef f icacy of cu r r en t m ed icam en t s against E. faecalis in infect ed canals and t he role of EPS in t his process.
M ATERI AL AN D M ETH OD S
Sa m ple pr e pa r a t ion
On e h u n d r ed an d t w en t y ex t r act ed sin g le-canal hum an t eet h w er e ex t ract ed fr om adult s ( 1 8 – 3 0 y ear s old) for or t hodont ic r easons and were kept in a 0.01% NaOCl solut ion prior t o use. Each par t icipant was pr ovided w it h an infor m ed consent form approved by t he Et hics Com m it t ee of West China Hospit al of St om at ology, Sichuan Un i v er si t y. Th e t eet h w er e p r ep ar ed u si n g a 3URWDSHUV\VWHPWR)¿OH7. The debris and sm ear
layer were rem oved by im m ersion in an ult rasonic bat h cont aining 5.25% NaOCl for 5 m inut es, t hen were subm erged in 17% EDTA for 5 m inut es. Each root was decoronat ed t o obt ain root s m easuring 12 m m in lengt h and st erilized before use. One hundred and t went y sem icylindrical dent in halves were shaped as previously report ed14. Dent in blocks
1A) . The sm ear layer of t he specim en was rem oved by im m ersion in 5.25% NaOCl, t hen 17% EDTA for 5 m inut es each in an ult rasonic bat h. The out er sides of t he root s and dent in blocks were sealed by nail varnish t o close t he open dent inal t ubules. The average colony- form ing unit ( CFU) count s and WKH VWDQGDUG GHYLDWLRQV ZHUH GH¿QHG DFFRUGLQJ t o ou r pr elim in ar y w or k . Th en , an assu m pt ion ZDV PDGH WKDW WKH VWDWLVWLFDO VLJQL¿FDQFH OHYHO LV W\SLFDOO\ Į DQG DGHTXDWH SRZHU LV widely accept ed as 0.9 ( 90% ) . The hypot hesized m eans and st andard deviat ions were calculat ed.
The sam ple size, est im at ed by PASS ( Power Analysis and Sam ple Size) version 13.0 ( NCSS, Kaysville, UT, USA) , was 7 sam ples per individual group; t hus, 10 sam ples were included in each group t o m aint ain an adequat e sam ple size.
D e n t in in fe ct ion w it h E. f a e ca lis
The E. faecalis st andard st rain ATCC 29212 was aerobically incubat ed at 37° C for 24 hours on brain heart infusion ( BHI ; Difco, Sparks, MD, USA) agar plat es, t hen a pure single colony was suspended in BHI m edium . The cell suspension was m onit ored
by CFU assay, and bact erial cult ures were adj ust ed t o 1x107 CFU/ m L; t he opt ical densit y ( OD) of t he
cell cult ure at 600 nm was adj ust ed t o OD of 0.17.
One hundred and t went y single- canal sam ples were incubat ed wit h 5x106 CFU in 500 PL of E. faecalis
for 3 weeks. Fresh BHI m edium was replaced every 48 hours. One hundred and t went y sem icylindrical dent in block s w er e placed in 6- w ell poly st y r ene cell cult ur e plat es w it h dent inal sur faces facing upwards20. Dent in specim ens were incubat ed wit h
t he bact erial suspension in BHI m edium wit h 1.0% sucrose for 3 weeks and fresh BHI m edium was replaced every 48 hours.
D isin fe ct ion of de n t in
A f t e r b a c t e r i a l i n f e c t i o n , t w o a d d i t i o n a l specim ens from each group were longit udinally split for scanning elect r on m icr oscopy ( SEM; I nspect ))(,+LOOVERUR2586$WRFRQ¿UPEDFWHULDO colonizat ion. Dent in blocks were longit udinally split t hrough vert ically prepared grooves int o t wo halves, and t he fresh surface of fract ured visible dent in ZDVZDVKHGWZLFHZLWK3%6DQG¿[HGZLWK glut araldehyde for 8 hours. The sam ples were t hen serial dehydrat ed t hrough et hanol solut ions ( 30% , 50% , 70% , 95% , t hen 100% ) , subj ect ed t o crit ical-point drying wit h liquid CO2, and coat ed wit h gold for im aging. Three random ly select ed areas from each sam ple were im aged by SEM. The bact eria- infect ed specim ens were random ly divided int o six groups as follow s: Gr oup A: CH + 40% glycer in- wat er solut ion ( at a powder t o liquid rat io of 1: 1, wt ./ vol.; Zhangj iang Biom at erials Co. Lt d, Shanghai, China) ; Group B: CMCP ( 30 m g/ m L cam phor and 15 m g/ m L phenol; Longly Biom at er ials Co. Lt d, Wuhan, China) paper point s; Group C: 2% CHX gel ( Nanyue pharm aceut ical Co. Lt d, Shenzhen, China) ; Group D: CH + CMCP ( 1: 1, wt ./ vol.) ; Group E: CH + CMCP ( 2: 3, wt ./ vol.) ; and Group F: saline. Fift y m icr olit er s of each m edicam ent w as int r oduced int o root canals using a spiral conveyer or placed on t he dent inal surface of each dent in halve for 1 week ( Figure 1A) .
M icr obiologica l sa m plin g a n a lysis
Ba ct e r i a l sa m p l e s w e r e t a k e n b e f o r e t h e a d m i n i st r a t i o n o f t h e m e d i ca t i o n ( S1 ) . Th e specim ens were washed t hree t im es for 30 seconds each in ult rasonic bat h using st erile saline5. Root
FDQDOZDOOVZHUHJHQWO\¿OHGLQPL of saline and a post- m edicat ion sam ple ( S2) was obt ained wit h RQHSDSHUSRLQW25. The m icrobiological sam ples
were serially dilut ed in saline ( 10- 1 t o 10- 7 dilut ions)
and each dilut ion was plat ed ont o BHI agar plat es and aerobically incubat ed at 37° C for 48 hours. The num ber of CFU per squar e m illim et er w as calculat ed12.
CLSM a n a lysis
After rem oval of the m edicam ents in an ultrasonic bat h, t he dent in block s w er e longit udinally split t hrough vert ically prepared grooves int o t wo halves to expose the fresh surface of fractured visible dentin ( Figure 1A) : one half was processed and stained with 50 PL of LI VE/ DEAD BacLightTM Bact erial Viabilit y
Kit reagent ( Molecular Probes I nc., Eugene, OR, USA) according t o t he m anufact urer ’s inst ruct ions WRDVVHVVWKHSURSRUWLRQRIYLWDOEDFWHULD%ULHÀ\ live and dead cells were different iat ed by st aining ZLWK6<72G\HJUHHQÀXRUHVFHQFHDQGSURSLGLXP LRGLGH G\H UHG ÀXRUHVFHQFH UHVSHFWLYHO\ DW D concent rat ion of 1 m g/ m L for 15 m inut es in t he dar k at r oom t em per at u r e. Th e sam ples w er e analyzed wit h a CLSM ( Type TSP SP2; Leica, Solm s, Germ any) using 480/ 500 nm lasers for SYTO 9 st ain and 605/ 635 nm lasers for propidium iodide12. The
border of t he freshly fract ured dent in surface was ¿UVWORFDWHGZLWKDPLFURVFRSHDQGWKUHHUDQGRPO\ VHOHFWHG¿HOGVRIYLHZZHUHVFDQQHG7KHYROXPH r at io of g r een f lu or escen ce t o g r een - an d - r ed ÀXRUHVFHQFHLQGLFDWHGWKHSRUWLRQRIOLYHFHOOVIRU each disinfect ing agent .
For EPS m at r ix im ag in g , t h e ot h er h alf of t h e sem icy lin dr ical den t in block in each gr ou p w as ex am in ed v ia f lu or escen t lab elin g of EPS and bact er ial cells. The EPS m at r ix was st ained w it h Alex a Flu or 6 4 7 - labeled dex t r an con j u gat e ( Molecular Pr obes I nc.) and bact er ial cells w er e labeled wit h SYTO928. Part icularly, t he Alexa Fluor
647- labeled dext ran was applied before t he dent in infect ion. The Alexa Fluor 647 dye was added t o t he prepared bact erial suspension adj ust ed t o a concent rat ion of 1 m g/ m L, which was subsequent ly incubat ed wit h t he dent in sam ples for 3 weeks11.
The spect ra were t aken wit h a wavelengt h range of 610/ 750 nm for t he Alexa Fluor 647- labeled dext ran FRQMXJDWHUHGÀXRUHVFHQFHDQGQPODVHUV IRU6<72VWDLQJUHHQÀXRUHVFHQFHDWDUHVROXWLRQ of 512× 512 pixels28 7KH ÀXRUHVFHQFH LQWHQVLW\
q u an t if icat ion w as an aly zed u sin g I m ar is 7 . 0 software ( Bitplane, Zurich, Switzerland) . The volum e UDWLRRIUHGÀXRUHVFHQFHWRJUHHQÀXRUHVFHQFHLQWKH t hree- dim ensional reconst ruct ions indicat ed t he EPS m at rix t o bact erial cells rat io. Three- dim ensional UHFRQVWUXFWLRQRIWKHELR¿OPVZDVSHUIRUPHGDQG t his procedure was repeat ed in t riplicat e for t hree random ly select ed views for each specim en.
St a t ist ica l a n a lysis
,QF&KLFDJR,/86$DQGVWDWLVWLFDOVLJQL¿FDQFH was set at p= 0.05.
RESULTS
Ant iba ct eria l a ct ion of m edica m ent s a ga inst
E. f a e ca lis in in fe ct e d r oot ca n a ls
,QJHQHUDO&)8FRXQWVVLJQL¿FDQWO\GHFUHDVHG aft er int racanal m edicat ion, indicat ing ant im icrobial act ivit y of t he m edicam ent s ( Figure 2A) . CMCP ( 30 m g/ m L cam phor and 15 m g/ m L phenol) 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) . The average CFU count s in t he CMCP group was t he lowest ( p< 0.05, n= 10) . The CH + 40%
glycerin-w at er solu t ion CMCP ( 2 : 3 , glycerin-w t / v ol) glycerin-w as m or e effect ive t han t he 2% CHX and CH + 40% glycerin-wat er solut ion CMCP ( 1: 1, wt / vol) ( p< 0.05, n= 10) , DQGWKHUHZHUHQRVLJQL¿FDQWGLIIHUHQFHVEHWZHHQ t he lat t er t wo groups ( p> 0.05, n= 10) .
An t ib a ct e r ia l a ct iv it y of m e d ica m e n t s on
E. f a e ca lis a n d EPS sy n t h e sis in de n t in a l
t u bu le s
Su ccessf u l bact er ial colon izat ion in den t in al t ubules was observed by SEM ( Figure 1B) , and a dense penet rat ion of E. faecalis was observed in t he dent in up t o 300 Pm ( Figure 1C) . CLSM showed m ix t ur es of single cells or abundant clust er s of cells and EPS m at rix inside t he dent in ( Figures 3A
Figure 2- Antimicrobial effect of different medicament regimens on E. faecalisLQLQIHFWHGFDQDOV6LJQL¿FDQWGLIIHUHQFHV
among the medicament groups (p<0.05, n=10). (A) Number of CFUs after the administration of intracanal medication [ ± s; n=10, lg (CFU/mL)]. The results showed that the decrease in CFUs in Group A, Group B, Group C, Group D, Group E, and Group F was 0.94 logunits, 2.01 logunits, 1.21 logunits, 1.19 logunits, 1.96 logunits, and 0.56 logunits, respectively; (B) Percentage (%) of viable E. faecalis cells that penetrated into the dentin after the administration of medication ( ± s, n=10). The mean proportions of viable populations were 48.9% in Group A and 35.0% in Group B; (C) Volume ratio of EPS matrix to bacteria in the dentin after medication. Group A had the highest ratio; Group B exhibited the lowest EPS matrix/ bacteria ratio (p<0.05, n=10); Group E showed the second-lowest ratio
and 4A) . Aft er m edicat ion, t he proport ion of viable bact er ia r an ged fr om 3 0 % t o 6 0 % , depen din g on t he m edicam ent s ( Figure 2B) , and t he lowest per cent age of live bact er ia was det ect ed in t he CMCP gr oup ( p< 0.05, n= 10) . The pr opor t ion of viable cells in t he CH + 40% glycerin- wat er solut ion CMCP ( 2: 3, wt / vol) group ( 40.0% ) was lower t han t he 2% CHX ( 45.6% ) and CH + 40% glycer in-wat er solut ion CMCP ( 1: 1, wt / vol) ( 45.2% ) groups ( p< 0.05, n= 10) . To clarify t he relat ionship bet ween m icrobial colonizat ion and EPS synt hesis, t he rat io of EPS m at r ix t o bact er ial cells w as calculat ed ( Figur e 2C) . Aft er adm inist rat ion of m edicat ion ( Figure 4) , t he CMCP group exhibit ed t he lowest EPS/ bact er ial cell rat io, w her eas t he CH gr oup exhibit ed t he highest rat io ( p< 0.05, n= 10) .
D I SCUSSI ON
E. f aecalis w as sh ow n t o p r od u ce a d en se infect ion int o dent inal t ubules, reaching up t o 300 Pm at t hree weeks post- infect ion. I n t he present st udy, eit her CMCP or CH com bined w it h CMCP d em o n st r a t ed ex cel l en t a n t i b a ct er i a l a ct i v i t y against E. faecalis, sim ilar t o previous report s25.
Our result s furt her dem onst rat ed t hat inhibit ion of EPS synt hesis by E. faecalisVHQVLWL]HGWKHELR¿OPV
t o m edicam ent - induced k illing, suggest ing t hat t he EPS m at rix plays a crucial st ruct ural role in E. faecalis survival.
Addit ion of CM CP enhanced t he ant ibact erial a ct ion of CH p a st e a g a in st E. f a e ca l i s in
in fe ct e d ca n a ls
The effect iveness of CH is linked t o t he diffusion of hydroxyl ions t hrough t he dent inal t ubules and accessory canals int o areas t hat are inaccessible t o inst rum ent s. When dissolved in wat er, t he CH past e dissociat es int o hydroxide and calcium ions; as a result , t he ant im icrobial act ion of t his m edicat ion depends on the concentration of hydroxide ions in the solut ion5. The ant ibact erial act ivit y of CH past e has
been previously quest ioned because of t he buffering pot ent ial of t he dent in, w hich decreases t he pH effect5,193UHYLRXVO\WKHLQÀXHQFHRQGLVVRFLDWLRQRI
CH in aqueous and nonaqueous solut ions has been invest igat ed, and t he conduct ivit y of CH in pure glycerin or propylene glycol was m easured22. I n t his
st udy, t he conduct ivit y of CH in pure glycerin was negligible and t he conduct ivit y value of CH in wat er was 7.3± 3 m S/ cm . These result s indicat e t hat high concent rat ions of glycerin reduce conduct ivit y22. I n
t he present st udy, t he CH past e m ixed wit h a 40% solut ion of glycerin had som e ant im icrobial act ion, but was inferior t o t he CH past e m ixed wit h CMCP solut ion. I t is possible t hat higher concent rat ions of glycerin m ay im pede t he effect iveness of CH as an int racanal dressing. On t he ot her hand, E. faecalis
is able t o endure alkaline st ress at a pH value of 11.113. Therefore, t his com binat ion of CH past e and
CMCP solut ion was superior t o t he CH past e alone in elim inat ing t he m icroorganism s in root canals.
IRUPELR¿OPVDQGFRORQL]HGHQWLQDOWXEXOHVXQGHU alkaline st ress condit ions in vit ro20. As a result , CH
com bined wit h CMCP has been suggest ed t o provide a broader ant ibact erial spect rum and m ore rapid penet rat ion t han inert vehicles25. I n t he present
st udy, CH com bined wit h CMCP exhibit ed a m ore pr onounced ant ibact er ial effect t han CH alone, indicat ing t hat t his associat ion m ay com pensat e for t he weaknesses of CH as previously proposed. I deally, an int racanal m edicat ion should occupy t he r oot canal space r eaching int o t he infect ed dent inal t ubules t o dest roy t he m icroorganism s. As phenolic com pounds have a low surface t ension, t he com binat ion of CH and CMCP m ay allow for an HDVLHU ÀRZ RI WKH PHGLFDPHQW LQWR WKH GHQWLQDO t ubules and also appear s t o be less t ox ic t han CMCP alone9.
The ant ibact erial effect of CH past es has been at t ribut ed t o dissociat ion of hydroxyl ions. However, t he buffering act ion of dent in can neut ralize t he ant im icr obial act iv it y of CH at deeper layer s of dent inal t ubules5. I n addit ion, CH past es associat ed
wit h CMCP allow a cont rolled liberat ion of calcium and hydroxyl ions. This associat ion form s calcium p- chlorophenolat e, which increases t he liberat ion of hydroxyl ions for a longer period17. That kind of
react ion bet ween t hose t wo m edicat ions m ay be less t oxic t han CMCP alone. On t he ot her hand, CH com bined wit h CMCP com poses a lesser am ount of CMCP t han when is used alone in t he present st udy. When incor porat ed w it h t he CH past e, r educed concent rat ion of CMCP would also cont ribut e t o less t he t oxicit y of CMCP as t he root canal m edicam ent . The ant im icrobial effect of CMCP product s is oft en descr ibed as it s abilit y t o dest r oy t he bact er ial m em brane by binding on it s prot eins and lipids. As a result , CMCP solut ion is cont roversial when used as m edicat ion because of it s possible t oxicit y caused by ingredient s such as chlorophenol9. Because of t he
fast diffusion of CMCP solut ion t hrough t he dent inal t ubules, adver se cyt ot oxic r eact ions w er e found when t here was liquid ext rusion in t he periapical t issues. However, t he denat uring effect of CH past e on periapical t issues m ay prevent furt her t issue penet rat ion by CMCP25. From t his point of view, it is
supposed t hat t his associat ion of CMCP + CH past e would be advant ageous by slow release of CMCP from t he past e. I n addit ion, t he com binat ion of CH and CMCP could increase t he pH of dent in and help t o m aint ain t he alkaline environm ent17. The high
S+PD\SURPRWHDVXSHU¿FLDOGHQDWXULQJHIIHFWRI CH past e in which t his area m ay act as a physical barrier t o a deeper diffusion of chlorophenol int o t he periapical t issues9. Conversely, t his associat ion m ay
increase t he pH of dent in, prom ot ing ant im icrobial act ion9,25. The cur r ent r esult s also indicat e t hat
increasing t he concent rat ion of CMCP enhances it s DQWLEDFWHULDOHI¿FDF\
The ant im icrobial effect of chlorhexidine ( CHX) is report ed t o act by elect rost at ic int eract ions, in which CHX is posit ively charged and t he bact erial wall is negat ively charged. This int eract ion alt ers t he cell osm ot ic equilibr ium and allow s bact er ial cyt oplasm coagulat ion, result ing in cell deat h6. Two
percent CHX gel is com m only used as a chem ical auxiliary in endodont ic t herapy, which shows it s effect iveness against m icr oor ganism s pr esent in infect ed root canals16. The associat ion of CH and
2% CHX has been used as an int racanal dressing b ecau se of i t s en h an ced an t i m i cr ob i al act i on against endodont ic pat hogens8. I t has been found
t h at CHX m ay in du ce r eact iv e ox y gen species ( ROS) product ion in t he alkaline environm ent wit h a biphasic response, which are possibly involved in t he killing of root canal m icroorganism s29. The
CH past es have t he abilit y t o induce hard t issue f or m at ion an d also m ediat e t h e n eu t r alizat ion of lipopoly sacch ar ide ( LPS)1 5. As a r esu lt , it is
supposed t hat t his associat ion of CHX gel + CH past e would be advant ageous com pared wit h CH past e alon e, becau se t h e su bst an t iv e effect of CHX sust ains t he ant im icrobial act ivit y aft er being rem oved from t he root canal syst em s23. However, t he associat ion of CHX wit h CH m ay fail t o im prove t he DQWLEDFWHULDOHI¿FDF\RI&+;JHO26. I n t he present
st udy, it was observed t hat t he ant im icrobial act ivit y of CH past e was inferior t o t he CHX gel alone. On t h e ot h er h an d, t h e pr esen t inv est igat ion also dem onst rat ed t hat t he ant im icrobial effect of CHX was inferior t o t hat of CH com bined wit h CMCP ( 2 : 3 , w t . / v ol. ) , w h ich w as r elat ed t o t h e poor penet rat ion int o t he m at rix- encased st ruct ure of t he ELR¿OP&+;DSSHDUVWREHLQHIIHFWLYHLQGLVVROYLQJ JO\FRVLGLFERQGVLQWKHELR¿OPPDWUL[26. Ut ilizat ion
of CH wit h CMCP m ay provide a bet t er t herapeut ic st rat egy, since CHX m ay int eract w it h r esidual sodium hypochlorit e, inducing t oot h discolorat ion. Furt herm ore, CHX m ay int erfere wit h t he sealing DELOLW\RIURRWFDQDO¿OOLQJV21.
D i sp e l l i n g t h e EP S M a t r i x se n si t i z e d E. f a e ca lis in de n t in a l t u bu le s t o m e dica m e n t s
0LFURELDO ÀRUD FRORQL]DWLRQ LV FRQVLGHUHG WR EH WKH FULWLFDO ¿UVW VWHS LQ ELR¿OP IRUPDWLRQ DQG dent inal t ubule invasion20. EPS are com posed of
m icr obial dex t ran der ived fr om or found on t he cell walls of E. faecalis. I n addit ion t o init iat ing t he at t achm ent and colonizat ion of biot ic surfaces4 by
plankt onic cells, EPS also act as an ext racellular d i g e st i v e sy st e m b y t r a p p i n g e n zy m e s t h a t sur r ound bact er ial cells, w hich r educes phy sical co n t a ct w i t h a n t i m i cr o b i a l a g e n t s2. Fo r t h e
survival of E. faecalis, it is presum ed t hat bact eria m ay r em ain alive, but noncult urable ( VBNC) , in nut rient poor environm ent s27. Thus, t he bact eria
irrigat ion m ay grow again in t he presence of an EPS m at r ix . Fu r t h er m or e, d ex t r an , a class of ext racellular- form ed glucans produced by bact eria, has been found in t he m at rix of E. faecalis ELR¿OPV
im plicat ing dext ran in t he developm ent of m icrobial com m unit ies12.
Previous report s dem onst rat ed t hat t he Alexa )OXRUODEHOHGGH[WUDQJHQHUDWHGDÀXRUHVFHQFH r eson an ce en er g y - t r an sf er ( FRET) r eact ion , in which t he dext ran chain was displaced, result ing in a ch an g e in f lu or escen ce1 1. I n ad d it ion , w e
VOLJKWO\ PRGL¿HG WKH VWDQGDUG GHQWLQ LQIHFWLRQ m o d el t o a ch i ev e a b et t er u n d er st a n d i n g o f t he r em aining bact er ial dist r ibut ion and of EPS or ganizat ion in dent inal t ubules. I n t he pr esent VWXG\ DXWRÀXRUHVFHQFH LQ QRQLQIHFWHG GHQWLQDO t ubules could be visualized aft er st aining, which is consist ent wit h previous invest igat ion14. Considering
t hat all dent in blocks were prepared t o achieve t he st andar d size ( 4× 2× 2 m m ) , dent inal specim en sam pling m aint ained consist ent baselines for CLSM observat ion and could ensure valid com parisons t o be m ade bet ween t he experim ent al m edicat ion groups. Aft er dent in blocks were longit udinally split , CLSM observat ions were conduct ed t o assess t he proport ion of viable bact eria and EPS product ion in dent in, which, t hus, was est ablished in relevant publicat ions14. I n addit ion, dent inal blocks incubat ed
wit h saline as cont rol were also invest igat ed and t he cont rol group could also be com pared as t he baseline of t he ot her m edicat ion groups ( Figures 3 and 4) .
The labeling t echnique allowed t he visualizat ion of VBNC cells and t he quant it at ive analysis of t he EPS m at rix. I n t he present st udy, t he visual st rat egy has been used t o assess not only t he percent age of v it al bact er ia, but also EPS m at r ix for m at ion in t he dent inal t ubules. EPS product ion declined wit h a concom it ant decrease in t he proport ion of live cells ( Figure 2B- C) , suggest ing t hat reduced (36SURGXFWLRQHQKDQFHVWKHDQWLELR¿OPDFWLYLW\ of m edicam ent s. Par t icular ly, E. faecalis ut ilizes t he residual EPS m at rix in dent inal t ubules aft er t reat m ent wit h saline under st arvat ion condit ions ( Figure 4G) . As a result , t he presence of EPS in deeper areas m ay creat e a chem ical barrier against ant ibact erial agent s2,25. Thus we propose t hat t he
increased resist ance of E. faecalis in t he dent inal t ubules m ay be at t ribut ed t o t he EPS m at rix. I n t he present st udy, t he com binat ion of CH and CMCP inhibit ed EPS sy nt hesis and negat ively affect ed bact eria in t he dent in. The degradat ion of t he EPS m at rix facilit at ed t he penet rat ion of CMCP, which sensit ized t he bact eria present in dent inal t ubules t o ant ibact er ial agent s. Clear ly, t he quant it at ive analysis of EPS and m icrobial colonizat ion would DGYDQFH RXU XQGHUVWDQGLQJ RI ELR¿OP VWUXFWXUH and accelerat e t he developm ent of ant im icrobial
agent s. This st udy sought t o invest igat e t he role RI(36PDWUL[LQYROYHGLQDQWLPLFURELDOHI¿FDF\RI current m edicam ent s in infect ed canal condit ions. I n our opinion, t he EPS m at rix dispelling by root can al m ed i cat i o n , w h i ch w o u l d b e r el at ed t o bact ericidal effect of t he disinfect ion agent s. Furt her invest igat ions should be carried out t o clarify t he r ole of CMCP in EPS m at r ix m et abolism and t o evaluat e t he viabilit y of residual bact eria aft er root FDQDO¿OOLQJV
CON CLUSI ON
I n sum m ary, t his st udy dem onst rat ed t hat CMCP en h an ced t h e an t ibact er ial act ion of CH past e against E. faecalis and inhibit ed EPS synt hesis in t he dent in. Mor eover, incr easing concent rat ions RI &0&3 VHQVLWL]HG ELR¿OPV PRUH HIIHFWLYHO\ WR disinfect ion agent s. These t wo m edicam ent s m ay addit ively act t oward t he elim inat ion of E. faecalis
and degradat ion of t he EPS m at rix, facilit at ing t he PRGL¿FDWLRQ RI WKH VWUXFWXUH RI WKH ELR¿OP 7KH ant im icrobial act ivit y of CHX gel was superior t o t he CH past e alone and was sim ilar t o CH com bined wit h CMCP ( 1: 1, wt ./ vol.) . The labeling visualizat ion and t he quant it at ive analysis dem onst rat ed t hat EPS m at rix declined wit h a concom it ant decrease in t he proport ion of live cells, suggest ing t hat reduced EPS product ion m ay enhance t he ant im icrobial act ivit y. The EPS m at rix dispelled by CH past e wit h volat ile vehicles m ay be relat ed t o it s bact ericidal effect for infect ed root canal m edicat ion. The visualizat ion an d an al y si s o f EPS f o r m at i o n an d m i cr o b i al colonizat ion in dent inal t ubules m ay be a useful approach t o verify ant im icrobial com ponent s using an in vit ro m odel.
D I SCLOSURE STATEM EN T
Th e au t h or s d en y an y con f lict s of in t er est . This st udy was support ed by t he Nat ional Nat ural Science Foundat ion of China ( Grant s 81400507, 81670980)
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