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In vitro Infection and of Dentinal Tubules

M. Haapasalo and D. Ørstavik J DENT RES 1987 66: 1375 DOI: 10.1177/00220345870660081801 The online version of this article can be found at: http://jdr.sagepub.com/content/66/8/1375

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In vitro Infection and Disinfection of Dentinal Tubules

M. HAAPASALO1 and D. ORSTAVIK2

NIOM -Scandinavian

Institute

of

Dental Materials, Forsktiingsveien 1, N-0371 Oslo 3, Norwos

An in vitro model for dentinal tuhide infection oJ root canals smas developed. Cylindrical dentin specimens, 4 inm high with a diameter ofJ6 mm and a canal 2.3 min wide, sere preparedfJrom freshly extracted bovine incisors. The cenentum was removedfrom all detitit blocks. The tubules were opened bh four-minute treatments with / 7% LDTA and 5.25c NaOCI before being infected swith Entercoccus faecalis ATCC 29212 in east extract-glutc se broth. Bacteria rapidly Invaded the tuhules. After three weeks of incbahaon, a heavy' infection a s jouf nd 4(00 jim from thce cana I lu men, and the Jront oJ the infection u reached 1000 pLm in some block s. Canmphorated parainonochlorophenol (CMCP) and a calcium hldroride compound, Cola septc, 1wvere testedl for their dis infe acting efjficacv toward E. faccalis- infeced dentin. Liquid CMCP rapidly and completeR disinfected the dentinal tubules, wvher eas CMCP in gaseous form disinfected tubules less rapidly. Calaseptc failed to eliminate, even superficially, E. taccalis in the Ilhiles. The method used in hacteriological sampling allowed far sequential removal of 100-Vjm-thick zones of dentin from the central canal toward the periphery. Control specimens stere unifortnly infected and1 yielded growth in bur samples up to some 500 jitn from the surface. The model proved quite sensitive and seems suitable for in vitro testing of root canal medicaments.

Materials and methods. Freshly extracted, intact bovine

,J Dent Res 66(8):1375-1379, August, 1987

Introduction.

Bacteria are the main causative factors in pulpal and periapical inflammation (Kakehashi et al., 1965; Sundqvist, 1976). Mechanical preparation and chemical disinfection of the root canal of the affected tooth remain the most important procedures in endodontics (Grossman. 1978). Disinfecting agents are used as irrigants and as intracanal dressings between the appointments, and a wide variety of medicaments is commonly used. In vitro tests have shown that bacteria are usually killed rapidly when they are in direct contact with various medicaments, even in high dilutions (Spangberg, 1982). However, data on the efficacy of endodontic medicaments against bacteria in viva are limited. In routine cases, it seems that success can be obtained with several methods and medicaments; however, sometimes an infection is resistant to normal treatment, and the therapy cannot be successfully completed. One possible reason for the persistent infection may be that bacteria have invaded the dentinal tubules. A few attempts have been made to establish in vitro models of root canal infections (Akpata and Blechman, 1982; Foley et al., 1983). However, there are hardly any reports on the efficacy of root canal medicaments against infected tubules under controlled conditions, and a simple and reliable method for studying the elimination of tubule infection is lacking. The aim of the present study was to develop an in vitro model for infection of dentinal tubules which would allow for testing of the efficacy of root canal medicaments.

Received for publication July 23, 1986 Accepted for publication January 4, 1987 'Permanent address: Department of Cariology, Institute of Dentistry, University of Helsinki, Finland 2To whom correspondence and reprint requests should be addressed

incisors were used for the experiments. The teeth were kept in 0.5% NaOCI for surface disinfection overnight. The apical 5 mm and two-thirds of the crown were cut off with a rotating diamond saw at 700 rpm (Accutom, Struers, Copenhagen, Denmark) under water cooling. With a reamer bur, the canal was widened to 2 mm in diameter. The root cementum was removed with a cylindrical diamond hole-bur at low speed (<100 rpm) in water bath at + 25 C, leaving a center-holed root dentin piece of 6 mm outer diameter and approximately 15 mm long. The root was then cut into slices 4 mm thick with a diamond saw (700 rpm, water cooling). The canals of the 4-mm blocks were widened with an ISO 023 round bur, finalizing the mechanical preparation of the test specimens (Fig. 1). The teeth and the dentin blocks were kept in tap water during all procedures to avoid dehydration. The smear layer xvas removed by treatment in an ultrasonic bath (Metason 120, Struers) in 17% EDTA (pH 7.8) (four min) and 5.25% NaOCI (four min). The effectiveness of this treatment was confirmed in the scanning electron microscope (Philips SEM 515. Philips, Eindhoven, The Netherlands), and the ensuing dentinal tubule openings are illustrated in Fig. 2. The blocks were sterilized by being autoclaved in water for i5 min at 1211C. The sterile test specimens were then transferred to yeast extract-glucose broth [YG broth; Yeast Extract (Oxoid, Hampshire, England), 10 g/L. glucose 10 g/L] and incubated for 24 hr at 370C as a test for sterility. Following this incubation, the blocks in YG broth were subjected to ul-

Fig.

men.

I

-Appearance and dimensions

ot a

prepared dentin test

speci-

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1375

1376

HAAPASALO & 0RSTAVIK

J Dzewl Res Augus.t 1987

I

I

Fig. 2 - Appearance ot dentinal tubules (A) and their pLilpal openings (B) after complctcd EDTA-NaOCl treatment. Scanning electron 1 icros-

copy.

I

I

trasonic treatment for 10 min to facilitate the permeation of

the dentinal tubules with the broth. Enute.otocucs ftwcif/is ATCC 29212 (Difco Laboratories. Detroit) was used as a test organism aind maintained by regular transfer in YG broth. For infection, two blocks were kept in each test tube with 1.5-2 mL YG broth. which was changed daily. Strict asepsis was necessary to avoid contamination. The purity of the cultures was checked at intervals. The development of tubule infection up to 28 days was evaluated in the scanning electron microscope (Fig. 3) and in Brown and Brennstained (B&B) sections of demineralized fixed, and paraffinembedded specimens (Fig 4). Specimens infected for three weeks were used for the testing of the medicaments. Prior to the actual test procedure, the outer surfaces of the specimens were covered with nail varnish, and the specimens were mounted with sticky-wax (Amalgamated Dental. London) at the bottom of cell culture wells (Falcon

3047. Becton-Dickinson. Oxnard. CA). The medicamnCIt to be tested was then applied to the canal lumen and filled to cover the fr-ee. top suif-ace of the dentin block well. The medicatcd blocks incubated under humid conditions at 37 C in air. The two medicaments tested were a calas

were

hydroxide CompoLund. Calasept'1 (Swcdia. Knivsta. Sweden). and camphorated pariamonochlorophenol (CMCP: 60% camphor. 30% p-monochlorophenol. 10%c/ ethanol), obtained on prescription from aL pharmacist. As controls, infected specmens with no medicament but With varnish on the outer surface only or on all surfaces used. Incubation times foi the controls were seven and 10 days (outer surface varnished) and 20 and 60 min (all surfaces varnished). Calasept paste and CNICP in liquid form were placed centrally in the test specimen. For testing of CMCP vapor, we placed the infected blocks in seal-tight containers with liquid CMCP present but placed

ciurm

were

outside the specimen.

Cells tf E. foieclir.s in dentinal FTi. 3 the pulp canal wall (B) tubLules (A) and matter a three-week infection period. Scanning electron microscopy.

on

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Vol. 66N No1. oI)INTINAL TUBUlE INFECTION

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The dentin specimens with applied medicament were incubated for time intervals ranging from five min to 10 days. On termination of each experiment, the blocks were blotted free of excess medicament on sterile filter paper. Bacterial samples were taken with sterile round burs mounted a handpiece and run at low speed. The specimen kept in place with sterile forceps during the sampling. The following bur sizes were used: ISO 023. 025. 027, 029. 031. 033. 035, 037. 040. 042. 045. and 050. The bur invaded the canal from the medicated surface and never penetrated to the non-medicated surface. The dentin chips obtained with each bur were immediately collected in separate test tubes containing brain-heart infusion

in

was

The results of the experiments with CMCP Dismf(etction. and Calasept acting on tubules infected with f/weclis are presented in Figs. 5. 6. and 7. In this system. liquid CMCP caused a rapid reduction ot bacteria which could be cultivated from the pulpal side, and after one hour no live oreanismns could be sampled. After one and seven days of incubation, the entire specimen appeared disinfected (Fig. 5). Gaseous CMCP was hardly effective for the first four hr, but complete disintection was obtained after one day. In contrast, treatment with even the innerCa(OH)1 failed to eliminate E. fcali~s

broth (BHI: Cibco Europe. Paisley. Scotland): the

purity (o

E.

tubes were

seven

days. incubated at 37 C in air, and inspected daily for When growth occurred. it was checked in every instance for

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Results.

In/tuetion. -Bacteria invaded the dentinal tubules both from the pulpal side and to a lesser degree from the outcr side. Specimens infected for only one day revealed penetration of bacteria to a 300 -400 p)m depth in a few canals when studied in the light microscope. The main cffcct prolonged infection

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that more tubules became infected, whereas the average depth of penetration of the infected tubules by bacteria increased only slowly with tirme. After three weeks of incubation with k:. joeccfi i.s, a dense infection from the canal side reached up to 300-400 pim: a moderate infection was usually seen up to 400-500 pim and the front of the infection could reach 8001000 ptm. From the outer surface, where the tubules were narrower, the infection usually reached 150-200 p9m. occasionally extending to 280 pm from the outer surface. When part of the cementum had unintentionally been left unremoved, no bacteria had penetrated throuTh it. and the infection in the corresponding tubules from the pulpal side was also considerably shorter than that in those tubules which were patent through the whole block.

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TIME OF MEDICAMENT ACTION fccccil/is from infected dcnitin after inLihbation with liqLidl camphorated paranmonochlorophenol. Each dot represents one test specimen and denotes the shortest distance frona the pulpodentin surface where growth detected.

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1378

HAAPASALO & 0RSTAVIK

No growth [

>700

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J Dent Res August 1987

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Discussion.

Several simplifications and extrapolations were necessary for the present model to be functional and reproducible. The use of bovine teeth was based on their availability and basic morphology (similar to that of human teeth). The dentinal tubules were of size and density similar to those of human teeth, and giant tubules (Hals and Olsen, 1984) were not frequently observed. To standardize the test specimens, we decided to remove the cementum with a special bur and remnants of pulpal tissue by filing and by treatment with sodium hypochlorite. Treatment with EDTA removed hard tissue debris and facilitated the cleaning action of NaOCl. E. faecalis was chosen as a test organism primarily because it is among the few facultative organisms associated with persistent apical periodontitis (Engstrbm, 1964; Haapasalo et al., 1983), and because previous experimental studies have used this organism (Akpata and Blechman, 1982; Stevens and

it was noticed that the presence of cementum affected the ability of E. faecalis cells to infect the dentinal tubules. When cementum was removed from the blocks before infection, this resulted in blocks which were better standardized and more easily infected. This observation reflects on the findings by Akpata and Blechman (1982), who studied the invasion of E. faecalis and Streptococcus sanguis into pulpal dentin in extracted human teeth with cementum. They found that E. faecalis did not invade the tubules at all during the first week of the test. Valderhaug (1974) induced root canal infections in monkey teeth in vivo and found that if the inflammation around the apex affected the cementum layer, bacteria invaded through the dentin. By contrast, in areas where the tubules ended in histologically sound cementum, bacteria, if present, only reached the inner third of the tubule. Thus, it appears that for a controlled infection in vitro, it is important that the cementum be removed to facilitate the ingress of the infective organism. Four different broths were studied in preliminary tests to determine whether the type of broth had any effect on the rapidity of development of the infection in the tubules. YG broth, tryptone soya broth (Oxoid), BHI broth, and a modification of Carlsson's (1970) synthetic medium for streptococci (M3) were tested. The use of BHI broth seemed to result in a weaker infection compared with the other three, which apparently performed equally well. YG broth was chosen because it was easy to make (compared with Carlsson's medium) and had been used earlier by Akpata and Blechman (1982). Three weeks were used as the time for the infection on the basis of studies with SEM and B&B-stained sections. However, the difference in the infection was not great among 1-, 2-, and 3week-old blocks. Thus, a shorter time for infection can also be used. Nail varnish was used to close the tubule openings on the outer surface of the block before application of the medicament. This was done partly because many root canal medicaments are volatile, like CMCP, and would act on the infection from both sides, which might complicate the evaluation of the results, but also because the diffusion of medicaments through tubules opened at both ends is probably different from diffusion when one end is covered with varnish or with cementum, as in the clinical situation. While the nail varnish contains solvents which may have some antibacterial effect, control experiments showed that this effect was negligible. The sampling procedures employed seemed quite sensitive

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Fig. 6 - Growth of E. faecalis from infected dentin after incubation with camphorated paramonochlorophenol in gaseous form. Each dot represents one test specimen and denotes the shortest distance from the pulpodentin surface where growth was detected.

Grossman, 1983; Bystrdm et al., 1985). During this study and in pilot experiments,

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Fig. 7

most zones of dentin after prolonged incubation (Fig. 6). Control blocks with varnish and no medicament invariably showed growth starting from the canal lumen. Performance of the model. Scanning electron and light microscopy documented the presence of bacteria in dentin tubules of all blocks inspected as controls (n - 30). However, rather a small percentage of tubules was seen to be infected (Fig. 4). Similarly, control specimens and specimens subjected to medicaments for short time periods (Figs. 5 and 6) constantly showed growth of bur samples from the pulpodentin surface (bur no. 023) and the first scrapings (burs nos. 025031). Growth from these teeth continued at least to bur size

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Vol. 66 No. 8

DENTINAL TUBULE INFECTION

1379

and made it possible to follow the increase of the length of the disinfected zone from the canal lumen. The technique was easy to perform, and most of the dentin powder could be collected into BHI tubes. In pilot experiments, two blocks were totally embedded in Calasept® for two days. The blocks were then rinsed in sterile water to clean them of Calasept® and dropped into BHI-broth, where they were ultrasonically treated (Metason 120, Struers) for 15 sec. Only one block yielded growth. However, after being sampled with a bur, both blocks revealed E. faecalis infection in the first 100-jim zone around the canal lumen. This experiment clearly demonstrated the possibility of false-negative results when a routine sampling from the main canal is performed, and confirms that a negative test does not necessarily mean a sterile canal (Akpata, 1976). This experiment also documented the efficacy of the bur sampling in detecting growth. CMCP in liquid form proved highly effective in eliminating E. faecalis infection inside the tubules. As might be expected, gaseous CMCP was slower in onset of antibacterial activity, but gave complete disinfection after one day of incubation. This may indicate that the effect of CMCP is rather dosedependent, and may aid in the interpretation of the variable results obtained in clinical tests of CMCP (Bystrbm et al., 1985). By comparison, the calcium hydroxide paste, Calasept®, was quite ineffective against tubule infection with E. faecalis during the test period. Even after ten days, live E. Jaecalis was found within the first 100-jim zone in the canal walls of most blocks. Bystrdm et al. (1985) reported that a pH drop from 12.5 to 11.5 of calcium hydroxide resulted in a high recovery of E. faecalis in test tube conditions even after 24 hr, whereas saturated calcium hydroxide killed this organism in a few minutes. Tronstad et al. (1981) showed, in monkey teeth, a sharp increase of pH in root dentin after the canal had been filled with calcium hydroxide. In conjunction with the present work, a liquid pH indicator (thymol blue) was applied on the fractured faces of some dentin blocks. Increased pH values were recorded far beyond the inner infection after only one day of calcium hydroxide in the canal (unpublished data). It seems evident that in the case of E. faecalis this increase in pH is not sufficient to disinfect the circumpulpal dentin. The results of this study are consistent with those of the study of Stevens and Grossman (1983), in which CMCP was found

more effective than calcium hydroxide in eliminating E. faecalis from artificially infected root canals of cat canines.

REFERENCES

AKPATA, E.S. (1976): Effect of Endodontic Procedures on the Population of Viable Microorganisms in the Infected Root Canal, J Endo 2:369-373. AKPATA, E.S. and BLECHMAN, H. (1982): Bacterial Invasion of Pulpal Dentin Wall in vitro, J Dent Res 61:435-438. BYSTROM, A.; CLAESSON, R.; and SUNDQVIST, G. (1985): The Antibacterial Effect of Camphorated Paramonochlorophenol, Camphorated Phenol and Calcium Hydroxide in the Treatment of Infected Root Canals, Endod Dent Traumatol 1:170-175. CARLSSON, J. (1970): Chemically Defined Medium for Growth of Streptococcus sanguis, Caries Res 4:297-304. ENGSTROM, B. (1964): The Significance of Enterococci in Root Canal Treatment, Odont Revy 15:87-106. FOLEY, D.B.; WEIDE, F.S.; HAGEN, J.C.; and deOBARRIO, J.J. (1983): Effectiveness of Selected Irrigants in the Elimination of Bacteroides melaninogenicus from the Root Canal System: an in vitro Study, J Endo 9:236-241. GROSSMAN, L.I. (1978): Endodontic Practice, 9th ed. Philadelphia: Lea & Febiger, pp. 197-255. HAAPASALO, M.; RANTA, H.; and RANTA, K.T. (1983): Facultative Gram-negative Enteric Rods in Persistent Periapical Infections, Acta Odontol Scand 41:19-22. HALS, E. and OLSEN, H.C. (1984): Scanning Electron and Incident Light Microscopy of Giant Tubules in Red Deer Dentin, Scand J Dent Res 92:269-274. KAKEHASHI, S.; STANLEY, H.R.; and FITZGERALD, R.J. (1965): The Effects of Surgical Exposures of Dental Pulps in Germ-free and Conventional Rats, Oral Surg 20:340-349. SPANGBERG, L.S.W. (1982): Endodontic Medicaments. In: Biocompatibility of Dental Materials, D.C. Smith and D.F. Williams, Eds., Boca Raton: CRC Press, pp. 223-257. STEVENS, R.H. and GROSSMAN, L.I. (1983): Evaluation of the Antimicrobial Potential of Calcium Hydroxide as an Intracanal Medicament, J Endo 9:372-374. SUNDQVIST, G. (1976): Bacteriological Studies of Necrotic Dental Pulps, Umea University, Odontol. Diss. No. 7. TRONSTAD, L.; ANDREASEN, J.O.; HASSELGREN, G.; KRISTERSON, L.; and RIIS, I. (1981): pH Changes in Dental Tissues after Root Canal Filling with Calcium Hydroxide, J Endo 7:1721. VALDERHAUG, J. (1974): A Histologic Study of Experimentally Induced Periapical Inflammation in Primary Teeth in Monkeys, Int J Oral Surg 3:111-123.

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