Effect of a tight seal on survival of bacteria in saliva-contaminated cavities filled with composite resin

Mejare I, Mejare B, Kdwardsson S. l'^ffect of a (ight seal on survival cjf bacteria in saliva-coiiUtmiiiated cavities Oiled with composite resin. IMKIOCI Deni Traimiatol 1987; 3: 6-9. Abstraet - Buccal Class V-cavities were ])re|5ared on homologous ])airs of human |)remolars in. vivo under ;tsej:)lic conditions. The cavities were then eontamiiiated l)y rubbing them with a cotton ]:)ellet soaked in saliva. Chosen by lot, 1 tooth in eac:li pair was thereafter washed either with waters|3ray or witli an antimicrobial cleanser (Tubulicid'"). The cavity was filled with Adaptie'". Ihe eured filling was removed to about half of the depth and replaeed by a zinc sulphate eement. Finally, an orthodontic band was cemented over the filling, fhe teeth were extracted after 6-8 wc;eks. Both culturing and histological teehniques were used to assess possible growth of bacteria. The results showed no growth of bacteria under the composite resin restorations, indejDendent of whether the cavity was washed with water or with an antimicrobial cleanser before filling. Thus bacteria originating from saliva contamination do not seem to survive under tightly sealed composite resin restorations.
Ingegerd Mejare*, Bertii Mejare** and Stig Edwardsson***
'Eastman Dental Institute, Stockholm, "Depaitment ol Endodontics, Danderyds Hospital, Daiideryd, and '"Department ot Oral Microbiology, University ol Lund Schooi of Dentistry, Sweden

Key words: bacteria, composites, saliva. Ingegerd Mejare, Eastman Dental Institute, Dalagatan 11, S-113 24 Stockholm, Sweden. Accepted for publication 3 June 1986.

Ihe presenee of baeteria under filling materials on the cavity walls, floor and in the dentinal tubules is considered to be a major causal factor in pulp tissue inflammation. Several authors have found a close relationship between the presenee of bacteria under composite resin restorations and pulpal inilammatory reactions (1-5). This espeeially concerns unlined eavities with unetched enamel where the ]jresence of baeteria on the cavity walls is a consistent finding. The introduction of acid-eteh techniques and modifleations of cavo-surfkce treatment have imjjroved marginal adaptation (for a review see (.h'ist (6)). However, no technique so far seems to l)e entirely effective in preventing marginal leakage. Bergenholtz et al. (5) and Qyist (6) both found presenee cjf bacteria in about one third of eavities filled with eomposite resins where the margins had been etched befbre filling. In a reeent study (7) using (tilturing combined with histolcjgical teclinic|ues, a light seal (ould not be obtained although various modifications of acid etehing and eavo-surfucc treatment were tried. It has been suggested that the origin of bacteria under composite fillings may be two-fold and that
6

they ean either be trap|)ed there in the smear layer during preparation or be the result of iiiitrginal leakage (2, 8). To eliminate baeterial contaminaliou during preparation these aitlliors reeommend the use of an antimicrobial cleanser instead of washing the cavity with water spray just before filling. In earlier studies (1, 7), however, the use of a liner seemed to completely exclude the presenee of bacteria under composite fillings. 11 is therefbre of interest to know if bacteria from contamination of saliva during preparation survive under the eomposite filling. If not, washing with water and subsec|tieiu application of a hner would be sufficient, as suggested by Finn (9) and Mjor (10, 11). That microorganisms contaminating the cavity at the time of I3ie]3aration may not survive was also suggested by Bergenholtz et al. (5). In order to find out if bacteria from saliva contamination survive under composite resin restorations it would be necessary to use a method that eliminates mieroleakage from the oral environment. As previously mentioned, aeid-eteh teehniques do not guarantee a tight seal. Substitution of the outer part of the composite resin by a temporary filling

Saliva-contaminated restorations cement has been found insufficient, either due to wear of the cement during the experimental period or k) unsatisfactory sealing ]Dro]3erties of the cement (2, 12). Other measures therefore seem necessary. A method giving a tight seal would also be of value for testing the bioconijialibility of dental materials
in vivo.

of the saliva-contaminated cavities was insufficient as a baeteria-eliminating measure and the samples taken after water spray consistently gave heavy growth already after 24 h in TAS-medium. The mieroorganisms found were typical fbr those usually
found in saliva such as Streptococcus salivarius.

The aim of ihis study was (o find an /// vivo model cnalihiig exc ltision oi niarginal leakage and to ascertain il bacteria due to saliva contamination during the preparation ]3rocedure survive under tightly sealed eomposite restorations. Presence of bacteria shcjuld be investigated by both eulturing and Brown- and Bremi-staining techniques. Materiai and methods f'jglit pairs of homologous human premolars scheduled to be- extracted for orthodontie reason were used. Apart from shallow oeelusal fillings in 2 teeth, they were clinically sound. Befbre trealnient, the fjuccal surface was eleaned by pumieing fbr 5 s. 1 hen a rubber dam was a])plic^d and the tooth and surrounding area washed with 30% HaO^ and iodine in 70% alcohol for 3 min followed by inaetivating the iodine with 5% sodium thiosulphate for 1 mill. Cireular eavities were prepared on the bueeal surface with a diameter of 2 mm using a spherical diamond under low speed and cooling with sterile water. A cylindrical diamond was used to finish the jjreparation to about 2 mm-deep cavities. To test the aseptic technique, sampling fbr culturing of possible contamination was made by taking samples from the walls and the floor with the blunt ends of sterile |)aper points. These samples were cultured in thioglycollate serum medium (TAS) as previously de.scribed (13, 14). Saliva was collected from the sublingual s]:)ace on a cotton pellet and the cavity was thoroughly rubbed with it lor 1 min. After airdrying fbi' 5 min, samples were again taken from I he cavity floor by wetting the surface with saiu]jling medium, VMC I (13). I'hese samples were immediately taken lo the laboratory for culturing in I AS-mcditim as previously described (14). Chosen by lot, 1 looth was washed with waterspray for .') s while ilie other was cleaned with Ttibulieid* (Denial llierapc-ulic AB, Nac:ka, Sweden) fbr 1 min. Samples for culUiring after the cleansing procedures were omitted in cnder nol to reduce the number of bacteria by the sampling proeedure. In order to check I ha I the saliva cotitamination fbllowed by water s]jray jjrocedure did not eliminate the bacteria c^n the cavity floor and walls, a separate in vilro sttidy on freshly extraeted teeth was perfbrmed. 1 he study design was identical with the in vivo design. The results showed that 5-s water spray

The teeth were filled with Adaptie Qohnson and Johnson, Dental Produets Company, East Windsor, N.J., U.S.A.) aecording to the manufacturer. The composite was pressed against the tooth with a Mylar strip (Dupont, Wilmington, Delaware, U.S.A.). After setting, the outer half of the filling was removed with a cylindrical diamond bur and high-speed ec^uipment and replaced with Prader's cement. This cement has no anlimicrobial effect but gives a tight seal (13). Finally, after setting of the eement, an orthodonlic band was cemented with a earboxylate eement to protect the filling against mechanical wear. The experimental procedure is schematically illustrated in Fig. 1. After 6-8 wk the teeth were extracted and immediately taken in sampling medium VMC I, to the laboratory for processing in an anaerobic glove box. Samples for culturing on blood agar anaerobically and in TAS-mcdium were taken from the bottom of the cavity of one half of the aseptically split tooth crown. The other half of the erown was studied by histological techniques. Details eoneerning the sampling, culturing and histological procedures has been described in previous studies (7, 14).

PROCEDURE I SALtVA CONTAMINATION

PROCEDURE t a I

OR

PROCEDURE nb

PROCEDURE

Fig. I. Schematic ilUislralion of experimental proci'dnres.

Mejare et al. Results lrres])eetive of eleansing procedure, ncj growth of bacteria oeeurred on the walls or floor of the cavities, nor in adjacent dentin, after the experimental period of (J 8 wk either ljy the culliiring teehnique or by the liislologic- method. N(3 growth of bacteria eould be observed in the samples taken to test the aseptic teclinic|ue during |Drcparatioii. All sam|)les of the saliva-contaminated cavities gave gi'owlli in TAS-medium. Discussion Bacteria from saliva tra]3ped under the composite fillings did not seem to survive since growth could not be obtained in any sample. Since bacteria have been found on the inner surface of composite resins corresponding to the cavity floor only 9 d after insertion cjf the filling (12), the eompo.site material itself does not seem to exert any significant antimicrobial eifect. Further, the use of Prader's cement exeludes any antimierobial effeet of the temporary filling material (13). It therefore seems that the nutritional supply to the area under a tightly sealed composite resin restoration is too poor to allow survival and growth of bacteria from saliva. This was also suggested by Bergenholtz et al. (5). Our results also seem logieal when compared with those obtained by studying the viability of carious bacteria lefl under sealed ]:)its and fissurc^s. Thus, a number of investigators have fbund a dramatic reduetion in viability of these baeteria when trapped under various ])ils-aiicl-fissure sealants (15 18). In agreement with Quist (19), ourfindingssuggest that miero-organisms oliserved beneath composite resin restorations originate (rom ingrowth of bacteria along the eavity walls rather than from bacteria trapped under the filling at the time of the restorative ]3roeedure. Support for this suggestion may also be found in an earlier study (14) where the microflora under unlined water-sprayed cavities filled with compcjsite resin was similar to that of plac[ue, while bacteria typical of saliva, i.e. Streptococcus salivarius, were not found. Therefbre, although an antimierobial surface-active cleanser may reduce the number of mierocjiganisms and improve the ad;t|jtation between the liner and the cavity walls (8) the neeessity of using an antimierobial agent may be cpiestioned, as was done by Eriksen & Eeidal (4). 'fhe efllciency of using water-spray and liner only was also illustrated in an study by Briiiiiislrcim & Nyborg (1) where 66 water-sprayed and lined cavities filled with eomposite resin restorations williout the use of acid-eteh techniques showed no growth of bacteria. Although the use of acid-clcli techniques, an iu8 termediale layer of a low-viseous resin and modifications of the cavo-surface have reduced marginal leakage, a number of studies shows that it is not eliminaled (5 7, 19, 20). 'fhe use of an ini])ermeable cafcium-liydroxidc liner should therefbre be stressed. The technique used in this study for excluding marginal leakage ajjpeared appropriate since no growth ol bacteria could be observed either by the ctiltiii ing or by the histological methods. It has been shown earlier that piolection of the ouler part of the filling with Cavil"" did nol eliminale niic roleakage due to mechanic al wear of this material during the ex]3erimeiit;tl period (2). In a recent sludy (12) IRM-eement also failed to produee a light seal. The use of Praders cement and applieation of an orthodcjiitic band over the filling seemed to funclion well. I herefbre this technique can be used lo lest the biocompatibility of various restoialive dental materials in vivo.

Conciusions Bac:teria originating from saliva eontamination during the pre]Daration procedure do not survive under tightly sealed eomposite resin restorations. The rationale behind tising an anlimicrobial cleanser is therefore quc;stioned. When testing the biocompatibility in vivo of dental restorative materials it is important to be able to ensure a lighl seal so as to exclude marginal leakage. '1 he techniciue used in ihis study seems appropriate for this purpose.

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