1 s2.0 S0099239907801981 Main

 

0099-2399/90/1612-0566/$02.00/0 JOURNAL OF ENDODONTICS C o py ri g h t 9 19 9 0 by The Ameri Ameri can can A s s oc i a t i on o f E nd od on t i s t s

Printed in U S A

VOL. 16, NO. 12, DECEMBER DECEMBER1990

I n V i tr t r o a c t e r ia i a l P e n e t r a t i o n o f C o r o n a l ly ly U n s e a l e d Endodontically Treated Teeth M a h m o u d T o r a b in i n e j ad a d , D M D , M S , B o r a s m y U n g , D D S , a n d J a m e s D . K e t t er e r in in g , P h D

Forty-five root canals were clean ed, shap ed, and then obturated with gutta-percha and root canal sealer, using a lateral condensation technique. The c o r o n a l p o r t i o n s o f t h e r o o t f ilil lili n g m a t e r i a l s w e r e placed in contact with Staphylococcus epidermidis a n d P r o t e u s v u l g a r i s T h e n u m b e r o f d a y s r e q u i r ed ed for these bacteria to penetrate th e entire root canals w a s d e t e rm rm i n e d . O v e r 5 0 of the root canals were c o m p l e t e l y c o n t a m i n a te te d a f t e r 1 9 - d a y e x p o s u r e t o

In addition t o dyes, radioisotopes radioisotopes have been used to study microleakage in alloy, resins, tempor tem por ary filling substances, and ro ot canal filling materi materials als (4-9). Although isotopes may be a good tool for com paring relarelative leakage, they cannot give a true picture of the leakage which occurs clinicall clinically. y. This is because because the ions used are much smaller than dye molecules and they diffuse much more rapidly than other small molecules (4). Isotopes are indica tors o f ion exchange, exchange, diffusion, diffusion, or metabolism within the tissues rather than indicators of true

Sa l seop ito d e r mlyi dcios n tFa imftf tiyn apteerdc ewn ht eonf tt hh ee cr ooor ot nc aa ln as lusr fwa ceer se to t a l ly of their fillings were exposed to P vulgaris for 42

leakage (10, 11). Mortensen et al. (12) and Krakow et al. (13) have stated that microorganism penetration might be more appropriate than dye or isotope penetration for studying leakage in vivo. Goldma n et al. (14) (14) have pointed out that bacteria performed better tha n dye in testi testing ng for leakage leakage of hydroph ilic materials materials and t hat dyes cou ld give a fa lse posit positive ive reading if their molecules molec ules were small enough. Becau Because se air bubbles can prevent dye leakage, leakage, the results results of dye studi studies es ha ve been questio ned (15;; Goldman et al., (15 al., personal communication). Because Becau se of inher ent inadequacies inadequacies in dye and radioi sotope studies, it appears that bacterial leakage studies can provide more accurate info rmati on in clinical clinical situat situations. ions. We have found no reports on the length of time that pas passes ses before the the entire ent ire obturated root canal is invaded by bacteria in obturated root canals without coronal seals.

days.

Sealed root canals can be recontaminated under several circumstances: (a) if the patient has had endodontic treatment but has delayed placement of perma nent res restor torati ations ons;; (b) if the seal of the temporary filling filling material has broken down; or (c) if fill filling ing materi materials als and /or t oot h structures have fractu red or been lost. When these situations situations occur, the coronal po rtion of the root canal system is exposed to oral flora. The question is how quickly the entir entiree root canal system becomes contaminated again, to the point that retreatment of the canal may be necessary. Swanson and Madison (1) evaluated evaluated the len length gth o f time that the obturation material could be exposed to artificial saliva before compromising the integrity of the seal. They exposed the coron al portion of obturated root canals to art artifi ificia ciall sal saliva iva for various various time period periods, s, a nd then immersed them in Pelikan ink for 48 h. They found that the dye penetrated from 79 to 85% of the root length in all exposed specimens. specimens. There was no leakage in the control group which was not exposed to artificia arti ficiall saliv saliva, a, but was placed in cont act with ink fo r 48 h. The follow-up study by Madison et al. (2) showed that in teeth expose d to artifici artificial al saliva for 7 days, the ink pen etrate d between 33 and 80% of the ro ot length, length, depending on th e type o f sealer sealer used.

The purpose o f this experiment was to determine the length length of time needed for bacteria to penetrate a standardized length of obturated root canals which were intentionally exposed to one of two species of microorganisms. MATERIALS AND METHODS To test bacterial penetration, a set-up similar to the one used by Goldman et al. (14) and Williams and Goldman (16) was used in this experiment.

Apparatus Set u p By using a high-sp high-speed eed hand piece and a

The latest findings by Madison and Wilcox (3) evaluating in vivo microleakage did not, however, co nfirm their in vitro studies. They found that some of the positive controls (no sealer) seale r) did no t show microleak microleakage, age, while some of the negative controls (temporary not removed) showed dye penetration.

2 roun d bur, a

small circular opening (about l mm in diameter) was made throu gh the cap of a 20-ml scintillati scintillation on flask flask.. A paper clip was threaded th roug h the opening and a n alligator alligator clip was hung on the inside. The outside end was bent to stabilize the alligator allig ator clip against the cap. Th e outside op ening o f the cap

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Vol. 16 No. 12 Decem ber 1990

In Vitro Bacterial Penetration

w a s r e s e a l e d w i t h a c r y l i c m a t e r i a l . T h e f la la s k s, s, w i t h t h e a l l iig a t o r c l i p s a t t a c h e d t o t h e i r c a p s , w e r e s t e a m s t e r i li li z e d . A f t e r i n s t ru ru m e n t a t i o n a n d o b t u r a t i o n o f r o o t c a n a l s , a 2 5 m m l e n g th th o f la la t e x t u b i n g w a s p l a c e d o v e r t h e c o r o n a l p o r t i o n of each tooth and the edges were sealed with epo xy resin (Quick Gel, non- run super-glue; Loctite Corp., Cleveland, OH

.

T h e t u b e s , w i t h t h e t e e t h a t t a c h e d , w e r e s t e r i li li z e d i n 5 . 2 5 % sodiu m hy pochlorite for 15 rain (based on the results of a p i l o t s t u d y ) a n d t h e n w e r e r i n s e d w i th th a p p r o x i m a t e l y 3 0 0 m l o f s t e ri ri l e w a t e r . T h e t u b e s w e r e t h e n f a s t e n e d t o t h e c a p s o f t h e p r e v i o u s l y s t e r i l i z e d s c i n t i l l a t i o n v i a ls ls . T e n m i l l i l i t e r s o f s t e r il il e p h e n o l r e d b r o t h w i t h 3 % l a c t o s e w a s a d d e d t o t h e b o t t o m o f a fl f l a s k , a n d t h e l e n g t h o f th th e t u b i n g w a s a d j u s te te d s o t h a t a m i n i m u m o f 2 m m o f t h e a p i c a l p a r t o f e a c h t o o t h w a s i m m e r s e d i n t h e s o l u t i o n ( F i g . 1 ). ).

567

Monitoring of the S amples T h e r e s u l t s f r o m a p i l o t s t u d y s h o w e d t h a t v i a b l e P. vulgaris w e r e p r e s e n t 3 w k a f t e r i n c u b a t i o n , w h i l e t h e S. epidermidis organism s were viable for only 1 wk. Fresh overnight cultures o f o r g a n i s m s a n d s t e r il il e a r t i f ic ic i a l s a l i v a w e re re a d d e d t o t h e t u b e s a t 5 - d a y i n t e r v a l s f o r S. epidermidis a n d a t 5 - t o 1 0 - d a y i n t e r v a l s f o r P. vulgaris. W h e n t h e b a c t e r i a l c u l t u re re w a s replenished, the old culture was plated to confirm continued viability of the m icroorganism s. T h e s a m p l e s w e r e m o n i t o r e d d a i l y u n t i l t he he r e d i n d i c a t o r s o l u t i o n a t t h e b o t t o m o f th th e f l a s k t u r n e d y e l l o w . A f t e r c o l o r c h a ng n g e , a s a m p l e o f th t h e y e l l ow ow m e d i u m w a s p l a t e d o n b l o o d agar to assure that it contained the sam e type of bacteria as that placed in the tubing.

Preparation Preparati on of Teeth

acterial Preparations Two species of bacteria were used as contam inan ts for this e x p e r i m e n t ; Proteus vulgaris, w h i c h i s h i g h l y m o t i l e , a n d Staphylococcus epidermidis, w h i c h i s n o n m o t i l e . T h e s e w e r e g r o w n o v e r n i g h t in in 3 0 m l o f t r y p t i c a s e s o y b r o t h ( a p p r o x i m a t e l y 4 . 7 x l 0 8 p e r m l o f / ' . vulgaris a n d 7 . 5 x 1 6 p e r m l

Forty-five maxillary incisors and cusp ids with straight canals were used in this study. The teeth had been stored p r e v i o u s l y in in 1 0% 0% f o r m a l i n a n d w e r e k e p t m o i s t a t a ll ll t i m e s throug hout the experimen t. After initial radiographs, standard a c c e ss ss c a v i ti ti e s w e r e p r e p a r e d , a n d t h e c o r o n a l p o r t i o n s o f t h e c a n a l s w e r e e n l a r g e d w i t h 2 to to 4 G a t e s G l i d d e n dr d r i ll ll s .

o f S. epidermidis . T w o m i l l i l i t e rs rs o f t h e b a c t e r i a l s u s p e n s i o n a n d 0 . 7 m l o f s t e r i le le a r t i f ic ic i a l s a l i v a a s s p e c i f i e d b y S w a n s o n a n d M a d i s o n (1) (1 mM CaC12, 3 mM NAH2PO4, 20 mM NaHCO3) were placed into the tubing. Since both org anisms are acid formers, w e e x p e c t e d t h e p h e n o l r e d i n d i c a t o r s o l u t i o n to to c h a n g e t o a y e l l o w c o l o r w h e n t h e b a c t e r i a r e a c h e d i t (1 (1 6 , 1 7) 7) .

In order to obtain a standardized diameter, the apical f o r a m i n a o f t h e t e e th th w e r e e n l a r g e d a n d k e p t p a t e n t to to a 4 0 f il il e, e , u s in in g a s t e p - b a c k f i li li n g t e c h n i q u e . A p p r o x i m a t e l y 2 m l o f 5 .2 .2 5 % N a O C 1 w e r e u s e d b e t w e e n e a c h f i le le si si z e, e, t o r e m o v e debris. The prepared teeth were divided into experimental and control groups.

Experimental Groups The root canals of 33 teeth were obturate d with guttap e r c h a a n d R o t h ' s s e a l e r ( R o t h D r u g C o . , C h i c a g o , IL IL ) u s i n g t h e la la t e r a l c o n d e n s a t i o n t e c h n i q u e . T o o b t a i n a s t a n d a r d i z e d l e n g t h o f f i ll ll i n g , t h e c o r o n a l p o r t i o n o f t h e g u t t a - p e r c h a w a s r e m o v e d w i t h h o t p l u g g e r s u n t i l o n l y l 0 m m o f th th e f il il l in in g m a t e r i a l r e m a i n e d i n t h e c a n a l.l. T o p r e v e n t b a c t e r i a fr fr o m p e n e t r a t i n g t h e r o o t s u r fa f a c e s , tw tw o l a y e r s o f f i n g e r n a il il p o l i s h were applied to the outside of the root excep t for 1 m m at the apex. GROU P

1

T h e c o r o n a l p o r t i o n s o f t h e r o o t c a n a l s o f 1 6 te te e t h i n t h i s g r o u p w e r e p l a c e d i n c o n t a c t w i t h 2 m l o f P. vulgaris i n t r y p t i c a s e s o y b r o t h a n d 0 . 7 m l o f s t e r i le le a r ti ti f i c i a l s a l i v a a s described above. The tubing w as then suspend ed over the p h e n o l b r o t h s o th th a t a p p r o x i m a t e l y 2 m m o f t h e a p e x o f t h e t o o t h w a s i m m e r s e d i n i t.t. GROU P

FiG 1. Diagram of a tooth attached to flask by an alligator clip.

tube

and suspended

in the

2

T h e c o r o n a l p o r t i o n s o f t h e r o o t c a n a l s o f t h e r e s t o f th th e teeth in the exper imen tal group (17 teeth) were exposed to 2 m l o f S . e p id e r m id is a n d 0 . 7 m l o f a r t i f i c i a l s a l iv iv a . T h e a p i c e s o f t h e s e te te e t h w e r e a ls ls o i m m e r s e d i n p h e n o l r e d b r o t h .

 

T o r a b i n e j a d e t a l.l.

568

Joumal of Endodontics

ontrol Groups

T A B LE LE 2 . R a t e o f t o t a l r e c o n t a m i n a t i o n o f o b t u r a t e d r o o t c a n a l s exposed to S epidermidis

T o t e s t t h e r e l i a bi bi l itit y o f o u r r e s u l ts ts , t h e r e s t o f t h e p r e p a r e d teeth were divided into two co ntrol groups. GROU P 3 T h e r o o t c a n a l i n e a c h o f t h e e i g h t t e e th th t h a t w e r e u s e d a s p oi st hi toi vu et sceoanl et rro, lss i mwualsa t fi inlgl e pd o owriltyh oab tsuirnagt leed gr ou ot tta -cpaenraclhsa. Ecaocnhe w o f t h e t w o s p e c ie ie s o f b a c t e r ia ia w e r e p l a c e d s e p a r a te te l y i n t h e t u b i n g t o c o n t a m i n a t e f o u r r o o t c a n a l s ( p e r m i c r o o r g a n is is m ) , a s d e s c ri ri b e d i n t h e e x p e r i m e n t a l g r o u p . GROUP 4 T o v e r i f y t h a t n o c o n t a m i n a t i o n d e v e l o p ed ed d u r i n g t h e experiments (negative controls), four instrumented root can a l s w e r e f ilil le le d w i t h g u t t a - p e r c h a a n d s e al al e r. r. A f t e r r e m o v i n g t h e c o r o n a l p o r t i o n o f t h e f i llll i ng ng m a t e r i a l a n d l e a v i n g 1 0 m m o f i t in in t h e r o o t c a n a l , t h e c o r o n a l p o r t i o n s o f t h e f i llll in in g w e r e exposed to sterile artificial saliva applied as described above.

No. of Samples

of Total

1 1

6 6

5 2 2

Cum ulative

No. of Days

6

15

29 12 12

12 41 53 65

16 17 19 20

4 1 1

23 6 6

88 94 100

30 45 51

Total 17

100

A v e r a g e : 2 4 .1 .1 d a y s 0.4 mm/day

t test of indep enden t samples shows that the difference is s t a t i s titi c a l l y s i g n i f i c a n t ( t = 4 .6 8 ) , w i t h p < 0 .0 1 . E x c e p t f o r o n e s a m p l e i n t h e p o s i titi v e c o n t r o l g r o u p ( g r o u p 3 ), ), t h e r e s t o f t h e s a m p l e s ( s e v e n o f e i g h t) t) c a u s e d a c o l o r c h a n g e i n th th e p h e n o l r e d m e d i u m a f t e r 1 t o 4 d a y s. s. T h e c u l tu tu r e m e d i u m d i d n o t c h a n g e c o l o r i n t e e t h w h o s e c o r o n a l segments were in contact with only sterile saliva (group 4) throughout the experiment (over 90 days).

RESULTS

DISCUSSION

I n g r o u p 1 , c o n t a m i n a t e d w i t h P. vulgaris t w o o f t h e s a m p l e s b e c a m e p o s i t i v e a f t e r 2 d a y s. s. O n e o f th th e s e m a y h a v e been a sample from the positive control, whereas the other o n e w a s f o u n d t o h a v e l e a k ag ag e t h r o u g h t h e l a t ex ex t u b i n g . T h e s e two sam ples were discarded. T a b l e 1 s h o w s t h e t i m e i t t o o k P. vulgaris t o r e a c h t h e a p e x t h r o u g h 1 0 m m o f t h e f il il li li n g m a t e r i a l . I t v a r i e d f r o m 1 0 t o 7 3 d a y s . T h e a v e r a g e l e n g th th o f ti ti m e f o r l e a k ag ag e w a s 4 8 . 6 d a y s . T h e t i m e p e r i o d s re re q u i r e d f o r S . e p i d e r m i d i s t o r e a c h t h e a p e x i n g r o u p 2 a r e s h o w n i n T a b l e 2 . C o m p a r e d w i t h t h o se se obtained in group 1, the results were more consistent, i.e. m o s t a p i c a l l e a k a g e o c c u r r e d b e t w e e n 1 5 a n d 3 0 d a y s, s, w i t h a r a n g e b e t w e e n 1 5 a n d 5 1 d a y s. s. T h e a v e r a g e l e n g th th o f t im im e f o r t o t a l p e n e t r a t i o n w a s 2 4 .1 d a y s . A s t a t i s t i c a l a n a l y s i s u s i n g a

M o s t o f t h e s a m p l e s i n t h e p o s i titi v e c o n t r o l g r o u p w i t h p o o r l y f i l le le d c a n a l s s h o w e d l e a k a g e b y 1 t o 4 d a y s , e x c e p t f o r one which did not show a change until the 22nd day. There a r e t w o p o s s ib ib l e t e c h n i c a l e r r o r s w h i c h c o u l d h a v e c a u s e d t h e latter; one is that this tooth may have been switched by mistake with one o f the experimental samples, the other is that the space prepared may have been circular enough to p r o v i d e a v e r y t i g h t s e a l.l. T h e r e s u ltlt s o f t h e p o s i t i v e c o n t r o l g r o u p a r e a c o n f i r m a t i o n o f st st u d i es es b y M a r s h a l l a n d M a s s l e r (4 (4 ), ), E v a n s a n d S i m o n ( 5 ), ), and Skinner and Himel (17) who showed that sealers are n e e d e d t o i m p r o v e t h e a p i c a l s e al al . As none of the negative controls led to a color change in

T A BL BL E 1 . R a t e o f to to t a l re re c o n t a m i n a t i o n o f o b t u r a t e d r o o t c a n a l s exposed to P vulgaris

No. of Samples

of Total

Cum ulative

No . o f Da ys

2 discarded 1

7

1 1 2 2 2 1

7 7 14.3 14.3 14.3 7

14 21 36 50 64 71

10 29 31 39 42 57 63

1

7

79

64

1 1 1

7 7 7

86 93 100

66 68 73

Total 14

99.9

7

Average: 48.6 days 0.2 mm /day

the phenol red mediu m, it appears that our set-up did provide a contamination-free chamber. O v e r 8 5 o f t h e t e e t h i n o c u l a t e d w i t h P. vulgaris b e c a m e c o m p l e t e l y p e n e t r a t e d i n 6 6 d a y s, s, w h e r e a s m o s t ( 8 8 ) o f those inoculated with S. epidermidis were totally infected in 30 days, suggesting that motility may not be a factor in rate of penetration to the apices. We found a significant variability in the time it took the bacteria to penetrate the entire root canal system. Similar results were reported by Swanson and Madison (1) and Madi s o n e t a l.l. ( 2 ) w h e n t h e y s t u d i e d d y e p e n e t r a t i o n i n o b t u r a t e d r o o t c a n a l s. s. T h i s m i g h t b e d u e t o t h e s h a p e o f t h e p r e p a r e d c a n a l , t y p e o f se se a l e r u s ed ed , o r t h e n a t u r e o f t h e s o l u t i o n t o which the coronal po rtions o f the root canal were exposed. Goldman et al. (14) studied bacterial penetration in root c a n a l s f ilil le le d w i th th p o l y - H E M A , a h y d r o p h i l i c , p l a st st i c p o l y m e r . The y found no bacterial penetration after 42 days. The ma in r e a s o n s f o r l a c k o f b a c te te r i a l p e n e t r a t i o n i n t h e i r r e s u ltlt s c o u l d be that poly-HEMA does not support bacterial growth as reported by Kronman et al., (18) because the pores are subs t a n ti ti a l ly ly s m a l l e r t h a n t h e b a c t e r ia ia , o r t h a t t h e p o l y m e r s h a v e

 

Vol. 16 No. 12

In Vitro Bacte rial Pene tration

Dece mb er 1990

the same adhesive properties as the acrylic nail polish which w a s u s e d t o c o v e r t h e e x t e r n a l s u r f a c e s o f t h e t e e th th . C o m p a r e d w i t h c lili n i ca c a l c o n d i ti ti o n s t h e m o d e l u s e d i n o u r s t u d y w a s s t a t ic i c i t s m e d i a f o r b a c t e r ia ia l g r o w t h w a s n o t t o t a l l y s i m i l a r t o s a li li v a a n d f o r e a s e o f m a n a g e m e n t o f e x p e r i m e n t a l cond itions and interpretations of the data its bacterial contents were purposely limited to only two species. Because of t h e s e l i m i t a t i o n s a n d t h e i r p o s s i b l e e ff ff e c t s o n t h e r e s u l t s i n v i tr t r o m o d e l s s i m u l a t i n g c l i n i c a l c o n d i ti ti o n s a r e n e e d e d t o i n v e s t i g a t e t h e r a te te o f l e a k a g e i n u n s e a l e d canals.

obturated ro ot

We gratefully acknowledge the assistance of Dr. Junichi Ryu and William Keeler with the bacterial culture culture a spect s of the experiment. Dr. Torabinejad is direct director or Pos tgradu ate Endodontics Endodontics Dr. Ung is a former gradu ate student in endodontics and Dr. Kettering Kettering is professor of microbiology microbiology Lom a Linda Linda University University School of Medicine Lom a Linda Linda CA.

References

1. Swanson KS Madkson S. An evaluation of coronal microleakage microleakage in endodontically trea ted teeth. endodontically teeth. Part I. Time periods. J Endodon 1987 ;13:5 6-9. 2. Madison S Swanson KL Chile SA. An evaluati evaluation on of coronal coronal microleakage in endodontically endodontically treate d teeth. Part I1. Sealer types. J Endodon 1987;13:109-12. 3. Madison S Wilcox LR. An evaluation evaluation of coronal coronal microleakage in endo dontically dontical ly treate d teeth. teeth. P art III. III. In vivo vivo study. J Endodon 1988 ;14:4 55-8. 4. Marshall FJ F J Massler M. The sealing sealing of pulpless teeth evaluated w ith

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