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0099-2399/97/2304-0229503.00/0JOURNAL OF ENDODONTICSCopyright 1997 by The American Association of Endodontists Printed in U.S.A.VOL. 23, NO. 4, APRIL 199 7

R e s i d u a l A n t im i c r o b i a l A c t iv i ty A f te r C a n a l I r r ig a t io nw i t h C h l o r h e x i d i n eR . R . W h i t e , P h D , G . L . H a y s , D D S , a n d L . R . J a n e r , D D S

W e p r e v i o u s l y r e p o r t e d t h a t t h e i n v i tr o a n t i m i c r o -b i a l a c t iv i ty o f a 2 . 0 % c h l o r h e x i d i n e e n d o d o n t i ci r ri g a n t w a s e q u i v a l e n t t o t h a t o f 5 . 2 5 % s o d i u mh y p o c h l o r i te . T h e p u r p o s e o f t h is s tu d y w a s t o d e -t e r m i n e i f c h l o r h e x i d i n e i r r ig a n t s c o u l d i n s ti ll s u b -s t a n t iv e a n t i m i c r o b i a l a c t i v it y in in s t r u m e n t e d r o o tc a n a l s i n v i tr o . H u m a n t e e t h w e r e i n s t r u m e n t e du s i n g 2 . 0 % o r 0 . 1 2 % c h l o r h e x i d i n e a s ir r ig a n t s .A f t e r i n s t r u m e n t a t i o n , t h e r o o t c a n a l s w e r e f i ll e dw i t h s t e r il e w a t e r , a n d s a m p l e s o f t h e r o o t c a n a lf lu i d w e r e a b s o r b e d w i t h p a p e r p o i n t s a t 6 , 1 2 , 2 4 ,4 8 , a n d 7 2 h a f t e r t r e a t m e n t . T h e p a p e r p o i n t s w e r ea s s a y e d f o r a n t im i c r o b i a l a c t iv i ty b y p l a c in g t h e mo n a g a r p l a t e s u r f a c e s i n o c u l a t e d w i t h S t r e p t o c o c -c u s m u t a n s a n d m e a s u r i n g z o n e s o f in h i b i ti o n . A n -t i m i c r o b i a l a c t i v it y w a s p r e s e n t i n al l 2 . 0 % c h l o r -h e x i d i n e - t r e a t e d t e e t h t h r o u g h o u t t h e 7 2 - h te s t i n gp e r i o d a n d i n m o s t t e e th , i n r e la t iv e l y l o w e r c o n -c e n t r a t i o n s , f o r 6 t o 2 4 h a f t e r i r r i g a t i o n w i t h 0 . 1 2 %c h l o r h e x i d i n e . T h e s e r e s u l ts i n d i c a t e t h a t c h l o r -h e x i d i n e i n s t il ls s u b s t a n t i v e a n t i m i c r o b i a l a c t i v i tyw h e n u s e d a s a n e n d o d o n t i c i r r ig a n t .

A m a j o r o b j e c t i v e i n e n d o d o n t i c t h e r a p y i s t o d i s i n f e c t t h e r o o tc a n a l s y s t e m b e f o r e o b t u r a t i o n o f t h e c a n a l. S o d i u m h y p o c h l o r i t ei s t h e c u r r e n t i r r i g a n t o f c h o i c e , b u t w e h a v e r e p o r t e d t h a t a 2 . 0 %c h l o r h e x i d i n e g l u c o n a t e i r r i g a n t , a l e s s m a l o d o r o u s a n d t o x i ca g e n t , p o s s e s s e d i n v i t r o a n t i m i c r o b i a l a c t i v i t y e q u i v a l e n t t o t h a t o f5 . 2 5 % s o d i u m h y p o c h l o r i t e ( 1 ). I n a d d i t i o n t o it s i m m e d i a t e k i l l i n ga c t i o n , c h l o r h e x i d i n e i s r e c o g n i z e d f o r i t s a n t i m i c r o b i a l s u b s t a n -t iv i ty , i . e . r e s idua l a c t ion (2 , 3) . O the r s (4 , 5 ) ha ve r e por te d tha tc h l o r h e x i d i n e b i n d s t o , a n d i s s u b s e q u e n t l y r e l e a s e d f r o m , d e n t i na n d e n a m e l , b u t t h e p r e s e n c e o f s u b s t a n t i v e n e s s w i t h i n a n i n s t ru -m e n t e d r o o t c a n a l h as n o t b e e n d e m o n s t r a t e d . T h e p u r p o s e o f t h i ss t u d y w a s t o d e t e r m i n e i f s u b s t a n t i v e a n t i m i c r o b i a l a c t i v i t y c o u l db e i n d u c e d w i t h c h l o r h e x i d i n e i r r i g a n t s .

M A T E R I A L S A N D M E T H O D S

T w o c h l o r h e x i d i n e g l u c o n a t e i r r i g a n t s w e r e t e s t e d ; a 2 . 0 % s o -l u t i o n p r e p a r e d b y d i l u t i n g a 2 0 % s t o c k s o l u t i o n o f c h l o r h e x i d i n e

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( S i g m a , S t . L o u i s , M O ) i n s t e r i le d e i o n i z e d w a t e r o n t h e d a y o f u s ea n d a 0 . 1 2 % c o m m e r c i a l o r al r i n se ( " P e r i d e x , " P r o c to r & G a m b l e ,C i n c i n n a t i , O H ) . N e g a t i v e c o n t r o l t e e th w e r e i r r i g a t e d w i t h s t e r i led e i o n i z e d w a t e r .

F r e s h l y e x t r a c t e d , s i n g l e - r o o t e d t e e t h w e r e o b t a i n e d f r o m t h eU n i v e r s i t y o f T e x a s D e n t a l B r a n c h c l i n i c s . T h e t e e t h w e r e s t o r e di n t a p w a t e r a t 4 C u n t i l u s e d . B e f o r e i n s t r u m e n t a t i o n t h e a p e x o fe a c h t o o t h w a s s e a l e d w i t h T R I A D V L C r e s i n ( D e n t s p l y I n t . ,Y o r k , P A ) , a n d t h e t o o t h w a s s e c u r e d i n a w o o d e n r a c k . T h e r o o tc a n a l s y s t e m w a s a c c e s s e d u s i n g a h i g h s p e e d h a n d p i e c e ( S t a rS y n t e x , L a n c a s t e r , P A ) , a n d t h e r o o t c a n a l w a s i n s t r u m e n t e d b i o -m e c h a n i c a l l y u s i n g a s t e p - b a c k t e c h n i q u e w i t h F l e x R f i l e s ( U n i o nB r o a c h H e a l t h - C h e m C o . , E m i t t s v i l l e , P A ) . W i t h e a c h c h a n g e i nf i l e si z e t h e c a n a l w a s i r r i g a t e d w i t h 1 m l o f i rr i g a n t . A f t e rc o m p l e t i o n o f f i l e i n s t r u m e n t a t i o n , t h e c a n a l w a s f u r t h e r e n l a r g e dw i t h a 0 . 0 5 0 " p a r a p o s t s y s t e m d r i l l ( W h a l e d e n t I n t . , M o h a w k , N J )t o i n c r e a s e t h e r e s e r v o i r o f i n t r a c a n a l f l u i d f o r t e s t i n g p u r p o s e s .F o l l o w i n g e n l a r g e m e n t , e a c h c a n a l w a s a g a i n ir r i g a t e d w i t h 1 m lo f i r ri g a n t . T h e c a n a l w a s t h e n i r r i g a t e d w i t h 3 m l o f s t e r i led e i o n i z e d w a t e r t o f l u s h o u t t h e o r i g i n a l i r r i g a n t a n d d r i e d w i t he n d o d o n t i c p a p e r p o i n t s ( K e r r , R o m u l u s , M I ) . E a c h c a n a l w a s t h e nf i l l e d w i t h s t e r i l e d e i o n i z e d w a t e r . T h e i n s t r u m e n t e d t e e t h w e r eh e l d i n a h u m i d i f i e r a t r o o m t e m p e r a t u r e .

S i x h o u r s a f t e r i n s t r u m e n t a t i o n , s p e c i m e n s o f t h e i n s t r u m e n t e dr o o t c a n a l s w e r e t a k e n a s f o l l o w s . T h e b r o a d e n d o f a # 8 0 e n d -o d o n t i c p a p e r p o i n t t r i m m e d t o 1 . 8 c e n t i m e t e r s w a s i n s e r t e d i n t ot h e i n s t r u m e n t e d c a n a l a n d l e f t f o r a p p r o x i m a t e l y 2 m i n . T h e p a p e rp o i n t w a s r e m o v e d a n d s t o r e d i n a c r y o g e n i c v i a l ( N a l g e ,R o c h e s t er , N Y ) a t - 2 0 C . T h e c a n a l w a s t h e n i rr i g at e d w i t h t h re e1 m l s t e r i l e d e i o n i z e d w a t e r r i n s e s , f i l l e d w i t h s t e r i l e d e i o n i z e dw a t e r , a n d r e t u r n e d t o t h e h u m i d i f i e r . T h i s p r o c e d u r e w a s r e p e a t e d12, 24 , 48 , a nd 72 h a f t e r ins t rum e nta t ion .

W i t h i n 2 4 h a f t e r t h e l a s t s p e c i m e n w a s t a k e n , t h e p a p e r p o i n t sw e r e t e s t e d f o r a n t i m i c r o b i a l a c t i v i t y a s f o l l o w s . A 2 4 - h T o d d -H e w i t t ( D i f c o , D e t r o i t , M I ) b r o t h c u l t u r e o f Streptococcus mutans,s t ra i n 6 7 1 5 , c u l t i v a t e d b n M i t i s - S a l i v a r i u s a g a r ( D i f c o ) c o n t a i n i n gb a c i t r a c i n ( 2 0 0 u n i t s / m l , S i g m a ) a n d s t r e p t o m y c i n ( 2 0 0 m g / m l ,S i g m a ) ( M S - B S p l a t e s ) w a s u s e d a s t h e t a r g e t o r g a n i s m f o r a n t i -m i c r o b i a l a c t i v i t y . I t w a s c h o s e n b e c a u s e i t c o u l d b e s e l e c t i v e l yc u l t u r e d i n t h e p r e s e n c e o f c o n t a m i n a n t b a c t e r i a a s a r e s u l t o f it sr e s i s t a n c e t o t h e i n h i b i t o r s p r e s e n t i n M S - B S m e d i u m . T h i s n e -g a t e d t h e n e e d f o r s t e r i l i z a t i o n o r o t h e r p r o c e s s i n g t h a t m i g h ta d v e r s e l y a f f e c t s p e c i m e n s . T e s t s i n o u r l a b o r a t o r y i n d i c a t e d t h a tt h e i n h i b i t o r s d i d n o t a f f e c t t h e a n t i m i c r o b i a l a c t i v i t y o f c h l o r h e x i -

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230 White, et al .

FIG 1. Chlorhexidine standards. Beginning at "12:00 o'clock" andgoing clockwise, the paper points contained zero, 0.0002%,0.002%, 0.02% , 0.2%, and 2.0% chlorhexid ine gluconate.

dine . With the use of a s ter i le swab, a lawn o f S. mutans was spreadover an MS-BS pla te and a l lowed to dry for 30 min a t roomtemperature . Paper points conta ining root canal contents were thenplaced on the pla te . The pla tes were incubated a t 37C in anincreased CO 2 a tmosphere . Af ter 48 h of incubat ion the zones ofinhibi t ion around the paper points were measured perpendicular tothe paper point.

Paper points immersed in ten-fold dilut ions of 2% chlorhexidinewere tes ted as above to evaluate the re la t ive sensi t iv i ty of theant imicrobia l assay. Negat ive controls consis ted of paper pointsimmersed in s ter i le deionized water .

The two chlorhexidine i r r igants were compared us ing a two-wayanalys is of var iance wi th repeated measures (SuperANOVA, Aba-cus Concepts , Berkeley, CA).

R E S U L T SThe 'antimicrobia l assay could d etect as l i t t le as 20 /xg of

chlorhexidine/ml (0 .002%) (Fig . 1). However , on di f ferent MS-BSplates there was some var ia t ion in the s ize of zones around paperpoints immersed in ident ica l concentra tions of chlorhexidine , andtherefore the assay was used only as a semiquantitative assay.Paper point specimens obta ined f rom the tee th i r r igated wi th s ter i ledeionized water d id not produce zones of inhibi t ion.

Twenty- three tee th were t rea ted wi th 2 .0% chlorhexidine . An-t imicrobia l ac t iv ity was detected in a l l specimens (6 through 72 h)taken f rom the 2 .0% chlorhexidine- t rea ted tee th (Table 1) .

Twenty-one tee th were t rea ted wi th 0 .12% chlorhexidine . An-

Journal of EndodonticsZ O N E O F IN H I B I T I O N ( m m )

[ ] 2.0% Values

C o l l e c t i o n P e r i o d ( h r )FiG 2. Com paris on of the antimicrobial activity within canals follow-ing irrigation with 2.0% and 0.12% chlorhexidine gluconate.

t imicrobia l ac t iv i ty was detected in the 6- and 12-h specimens ofall teeth irrigated with 0.12% chlorhexidine and in 15, 6, and 2 ofthe specimens col lec ted a t 24, 48, and 72 h , respect ively (Table 1).

Compar iso n o f the two groups of chlorhexidine- t rea ted tee th(Fig. 2) revealed that the ant imicrobia l ac t iv i ty remaining in the2.0% chlorhexidine- t rea ted tee th was s igni f icant ly greater (p