Microbial accumulation and vitality on different restorative materials

Dent Mater 9:312-316, September, 1993

Microbial accumulation and vitality on different restorative materials

R. Hahn, R. Weiger, L. Netuschil, M. Briich

Department of Conservative Dentistry, Dental School, University of Tuebingen, Tuebingen, GERMANY

Abstract. A new technique using standardized test facings was designed to evaluate interdental plaque accumulation on different restoration materials. In 10 volunteers, a total of 40 samples, including enamel, two different ceramics and a bonding composite, were inserted one by one into a precision attachment in an experimental inlay bordering on either the lower second premolar or the lower first molar. Bacterial accumulation on each approximal specimen was allowed to mature for 3 d. Following microbiological processing of the plaque samples, total bacterial counts, colony forming units and the bacterial vitality were determined. The results revealed different accumulation rate patterns. Both ceramics accumulated less plaque with a reduced vitality compared to enamel, while the bonding composite showed no significant differences compared to the natural tooth substance. No significant differences were detected when comparing the two examined ceramics.

INTRODUCTION The biological interaction between tooth restorations and the encompassing oral milieu is one of the most important factors for their clinical prognosis. Faults in reconstructing the functional shape, the approximal space or the marginal integrity may lead to a significant irritation of the local tissues, promoting plaque accumulation (Lang et al., 1983) and thereby increasing the risks for the development of caries and gingivitis. Furthermore, there are distinctive interactions between the rate of plaque formation and the restoration material itself (Norman et al., 1972; Smales, 1981).

The interactions of microorganisms, blood substances and cells with biological surfaces have been studied and reviewed by several authors (Dutton et al., 1969; Baier, 1970; 1980; Weiss and Neiders, 1970; Baier and Maier, 1982). Both in vitro and in vivo methods were discussed in the literature for evaluating microbial accumulation on solid materials, including human enamel, dental ceramics and polymer composites. In vitro methods allowing the growth of microorganisms in different artificial media (Dummer and Harrison, 1982; Palenik et al., 1992) can only insufficiently simulate the vital adhesive and the individual host factors. Therefore, diverse in vivo methods were described either to score plaque indices (Barnes et al., 1986; Fishman, 1986) or to

assess plaque accumulation on various test facings or foils using different experimental restorations (Jendresen and Glantz, 1981; Siegrist et al., 1991) or splints (Hahn et al., 1992). Plaque indices rely on non-linear measurements offering only a coarse method to differentiate the plaque accumulation on various restoration materials (Chan and Weber, 1986; Van Dijken et al., 1987; Furuichi et al., 1992; Weiger et al., 1992). Thus, the material's specific interactions on plaque accumulation can hardly be differ- entiated from the lasting influence of an improper restoration, in particular concerning the marginal topography and integrity, reconstructed shape and surface microstructure (Morris, 1962; Lang et al., 1983).

In order to improve the procedure recording the specific plaque accumulation on restoration materials, experimental restorations or splint systems were used as carrier systems for the real material (Baler and Glantz, 1978; Smales, 1981; Krekeler et al., 1984; Siegrist et al., 1991). Due to the spatial limitation of the carrier system, the test materials were mostly placed outside the critical interproximal or cervical areas for plaque accumulation. In addition, the carrier system (shape, material and bonding substances) itself caused plaque formation, which may overlap with the plaque developed on the test material (Hahn et al., 1992).

Ideally, experimental foils were recommended to follow the tooth contour without using any cement (Jendresen and Glantz, 1981; Weberetal., 1987). However, these techniques could not be realized with dental ceramics because of their brittleness and poor bending strength. The initial plaque formation on an adhesive restoration complex (enamel-bonding composite-ceramic) in the most crucial interproximal location with a standardized technique has not yet been examined. In addition, few authors refer to the area of the surface (Weiger et al., 1992). The aim of the present study was to assess early approximal plaque formation on diverse tooth-colored restorative materials under standardized conditions, particularly considering the surface area of the test materials.

MATERIALS AND METHODS Ten dental students and nurses, 5 males and 5 females ranging in age from 18 to 26 years (x = 23 _+ 2.4), were recruited for this study. All volunteers had at least 28 teeth, no crowns and bridges

312 Hahn et aL/Microbial accumulation on dental ceramics

and a maximum of 5 intracoronal fillings. The participants had not taken any medication, particularly antibiotics, in the year preceding the experiment. Pending a general reconstruction of the old fillings with dental ceramics, one experimental gold inlay (Aurofluid 3 [71.0% Au, 2.0% Pt, 2.0% Pd, 9.0% Ag, 14.5% Cu], Metaux Precieux SA Metalor, Neuchatel, Switzerland) was tern- porarily incorporated either into a lower second premolar or a lower first molar using a eugenol-free bonder (Freegenol, GC, Tokyo, Japan). The neighboring approximal surfaces did not have any restoration or carious lesions. A square frictional attachment was ground into the approximal surface of the experimental inlay anchoring a standardized test facing (Figs. 1 and 2).

Both cast (Dicor, Dentsply, Milford, DE, USA) and sintered (Flexo-Ceram, Elephant Ceramics, Hoorn, The Netherlands) dental porcelains, as well as a dual-bonding composite (Duo Cement, Colt~ne, Altst~itten, Switzerland) were selected as test materials. Steam-sterilized human enamel facings originating from freshly extracted third molars served as controls. All materials were processed according to the manufacturers' guide- lines and polished to a high finish by using fine grained alumina discs (Sof-lex, 3M, St. Paul, MN, USA). Subsequently, each specimen (total = 40) was adapted to fit frictionally into the individual inlay attachment. The vertical extension of the test facings was standardized, ending one millimeter coronal to the interproximal gingiva. The horizontal expansion was prepared accordingto the extension of the individual inlay. The approximal contact was marked on each test facing as a hairline notched line vertical to the tooth axis. Thus, the "free" approximal surface was determined under a scaled microscope.

In the 3 wk pre-experimental period, professional tooth clean- ing and oral hygiene instructions were given until optimal plaque control was maintained. Dental plaque was allowed to accumu- late during serial periods of 3 d without oral hygiene, separated by at least lwk of optimal oral hygiene. At the end of each period, each facing with the adhering approximal plaque was carefully removed in a coronal direction without leaving the adherent plaque on the neighboring tooth. The plaque was completely collected from the defined approximal area with the tip of a sterile curette. Using a vital fluorescence technique described later, the still-remaining plaque on the test facings was assessed and found to be negligible (< 1% of the total bacterial counts). The plaque sample was immediately transferred into a vial by rinsing with i ml of Schaedler-Broth (Becton-Dickinson, No. 12191; Becton- Dickinson, Heidelberg, Germany). The entire content of the vial was sonicated for 10 s with a sonicator (TM Cell disrupter W- 225R, Heat Systems- Ultrasonics, Inc., Plainview, NY, USA) and immediately processed. Sonication as used in this and other laboratories (Weiger et al., 1992) does not alter the vitality of the plaque specimen.

For total bacterial counts (BC), an aliquot was introduced into a bacterial counting chamber (System Helber, Saaringia, SaarLouis, Germany) to assess the total number of bacteria under a darkfield microscope (Ortholux II, Leitz GmbH, Stuttgart, Germany) at a magnification of 500x. The plaque sample was also serially diluted in Schaedler Broth and appropriate dilutions plated in triplicate on Schaedler agar plates with sheep blood and Vitamin K1 (Becton Dickinson No. 021539; Becton-Dickinson, Heidelberg, Germany) using the spot method (Westergren and Krasse, 1978). The plates were incubated anaerobically for 48 h at 37°C with the Gas Pak 100-System (Becton Dickinson No. 060626; Becton-Dickinson, Heidelberg, Germany) developing an

Fig. I. Representative experimental MO-inlay with a conventional dental porcelain specimen frictionally anchored in mesial ground attachment.

Fig. 2. Experimental MO-inlay in situ (region 36).

HJCO 2 atmosphere (Becton Dickinson No. 070304; Becton- Dickinson, Heidelberg, Germany). The visible colonies were then counted as colony forming units (CFU). The plating efficiency (PE) was calculated as a percentage of CFU per BC, thus repre- senting the proportion of viable microorganisms on each particular material.

The remaining plaque solution, consisting of 90% of the original sample, was stained with fluoresceindiacetate and ethidiumbromide according to Netuschil et al. (1989). Sonicated plaque was vortexed with 50 pL of the staining solution and reaggregated in a centrifugation step. The supernatant was carefully suctioned offand the remaining plaque was transferred onto a glass slide with a cover glass. A histological quantitative analysis was immediately performed under a photomicroscope (Zeiss, Oberkochen, Germany) with a UV excitation filter (wave- length of 450-490 nm). At a magnification of 300x, the samples were examined through a counting grid to calculate the percentage of living microflora by vital fluorescence (VF) according to Brecx et al. (1990). The value of total vital bacterial counts (BCv~ t) was assessed with the formula BC x VF/100.

The data were analyzed with Systat software (SYSTAT, Evanston, IL, USA). After calculating the percentiles, median and corresponding quantils, all data could be displayed in a box

Dental Materials~September 1993 313

B C x l ~ m m 2

30

20 T T . +

" "-o'- " ~'=~'JRI= '~''=d

Enamel Composite Dicor Rexo-Cerarn

Fig. 3. Box plot: Total bacterial counts (BC) measured after a 3 d interproximal plaque accumulation on different restoration materials and human enamel. The box plot displays the full range of the measured data. The actual box shows the median (Xo.5o) and the interquartil distance originating 50% of the data recorded. The values for the 10% and 90% quantiles are indicated as whiskers, values far outside marked as empty circles. [(Xo.5o ). ,r~ = 8.60 X 106 / mm=; (Xo.50)Composite -- 5.68 x 106 / mm2;(Xo.so)o~oor = 1.56 x 10 e / minT; (Xo.50)Flexo.Ceram = 1.38 x 10 s / mm 2]

CFU x 10 s m m =

30

20

IO

I !

0

Enamel Composite Dicor Rexo-Ceram

Fig. 4. Box plot: Colony forming units (CFU) recorded after 3 d interproximal plaque accumulation on different restoration materials and human enamel. [(x o 5o)en=~ = 5.48 x 10 e / m m=; (x o ~cor, po,,~ = 4.61 X 106 / mm2; (Xo.so)o,¢o~ = 0.02 x 106 / mm 2 iXo.so)R,,o~,~ = 0.02 X 106/mm 2]

PE (%) 1:4o

12o

1"00

80

6o

4o

20 j o _1_

o o

Enamel Composite Dicor Flexo-Ceram

Fig. 5. Box plot: Material specific plating efficiency (PE) calculated as the percentage of CFU per BC. [(xo.so)E,,m~ = 58.45%; (Xo.5o)compo,b = 80.45%; (X0.~O)O~cor = 2.30%; (X0.s0)~texo.Cer,m = 2.11%]

BCat X 10 ° 20

m m =

15

10

0

m n

T

$

Enamel

.J_ o

Composite Dicor Rexo-Ceram

Fig. 6. Box plot: Vital bacterial counts (BCv,) recorded after a 3 d interproximal plaque accumulation on different restoration materials and human enamel. [(x o 5o)En,,~ = 5.57 X 10 s / ram2; (x o so)c m o,,e = 4.03 x 10 ~ / m m2; (x o 5o)o~or = 1.25 x 10 s / mm2; (Xo.50)Flexo.C . . . . = 0.96 x 10 s /mm y] p

plot. Since the evaluated data of the original parameters BC, CFU and BCvi t originated from a defined dilution chain, a non- parametric statistic was applied. For each parameter, the statistic null hypothesis, "The data originate from the same popula- tion", was tested by the H-test according to Kruskal and Wallis. In addition, a homogeneity range correlation test according to Wilcoxon, the Mann-Whitney was used to evaluate the degree of significance of the plaque accumulation data.

R E S U L T S

Both the cast and sintered ceramics showed significantly lower values of BC/mm 2, CFU/mm 2 and PE as compared to enamel or the bonding composite (p = 0.0014) (Figs. 3-5). Indi- vidual differences between the two ceramics could not be found for BC/mm ~ (p = 0.381), CFU/mm 2 (p = 0.668) and PE (p = 0.902). As a result of the low plaque accumulation rate on the ceramic materials used, only a few data could be recorded with the vital fluorescence method (Fig. 6). Consequently, a statistical analysis to test B C J m m 2 for these materials could not be performed. When comparing the dual-bonding composite with the human enamel, there were no significant differences for BC/mm 2 (p = 0.517), CFU/mm 2 (p = 0.415) and PE (p = 0.370). BCvJmm 2 for the enamel and composite also did not differ significantly (p = 0.462).

However, all data measured on composite specimens showed a conspicuously large amount of individual scattering.

D I S C U S S I O N

The plaque accumulation on intraoral surfaces depends on a complicated physio-chemical interaction between the solid adsorbens (i.e., tooth or restoration surface) and the liquid adhesive (i.e., saliva or sulcus fluid). The surface free energy changed by the formation of the so-called"acquired pellicle" (De Jongetal. , 1984; Van Dijk et al., 1987), bacteriostatic or bactericidal effects of the actual adsorbent material (Nunez et al., 1976; Augthun and Brauner, 1988) and the microstructure in particular (Keenan et al., 1980) have a fundamental effect on the initial plaque formation. In addition, biological interactions between plaque microorganisms and the human immune system should be considered in vivo.

Various methods are discussed in the literature to assess the plaque accumulation on different restoration materials. The present method allows a quantitative analysis of the plaque accumulation in the most crucial interproximal location. The interproximal accumulation rate was higher when compared to that on a vestibular tooth surface (Weiger et al., 1992), indicating that a loss of adherent plaque on the neighboring tooth by


removing the specimen seems to be very small. In a previous study, it was found that gold alloy may influence the bacterial vitality when adhering the plaque flora in direct contact onto the metal surfaces (Simonis et al., 1990). The surface relation between the test facings and the carrier inlay can be shaped favorably in this particular region. Furthermore, unwelcome interferences caused by the carrier system, which may alter the real accumulation patterns, can be decreased to a minimum by using a small experimental inlay (gold alloy), a eugenol-free cementation, as well as a frictional attachment (Hahn et al., 1992). All specimens were subjected to the same method; therefore, influences due to the attachment, marginal adaptation, cementation or sample processing should be equivalent for controls and test materials.

Two different methods were used to assess plaque vitality: CFU (which is the conventional bacteriological method to count the portion of the viable microorganisms) and vital fluorescence (VF), recently introduced in plaque research (Netuschil et aI., 1989; Brecx et al., 1990). For enamel or composite, the vital plaque formation after 3 d is very similar for both methods, CFU and BCvi t (Figs. 4, 6). In contrast, for tiny amounts of plaque as collected on ceramics, the CFU and VF methods result in different data. These differences as well as huge inter-individual varia- tions were also found in other studies (Weiger et al., 1992; 1993; Raps et al., 1993). As discussed by Weiger et al. (1992), the VF technique seems to be the more reliable method.

The present study revealed a low plaque accumulation with a reduced microbial vitality on dental ceramics in the inter-dental space. Although Chan and Weber (1986) found low plaque index scores on rough porcelains such as Cerestore, the relatively small variation of the parameters recorded for ceramics seemed to be affected by the high surface finish. No significant differences regarding plaque accumulation and vitality could be detected when comparing the castable tetrasilic mica glass ceramic (Dicor) with a conventional dental porcelain such as a glass matrix composite (Flexo-Ceram). The fluoride-containing mica crystals may obviously be insoluble and permanently integrated in the porcelain structure, preventing a release of an effective anti- microbial fluoride concentration on Dicor surfaces.

The polymer composite accumulated significantly more plaque than both ceramics. It is suggested that the polishing procedure of composites causes a rougher surface as compared to ceramics, probably due to a preferred removal of the soft polymer matrix during polishing. The plaque accumulation on the polymer composite and the enamel did not differ significantly, which conforms to the findings of Van Dijken and Sjhstrhm (1991). Here, aging effects of the polymer matrix (Roulet, 1987) are discussed as being responsible for an increased accumulation rate (Van Dijken et al, 1987). The large distribution of all data recorded on polymer composites could be explained by the scattering of the surface parameters such as surface tension and surface free energy caused by an inhomogeneous matrix shrinkage (Feilzer et al., 1989).

In summary, a new method was described that quantitatively analyzes the interproximal plaque formation and microbial vitality on any restoration material. It was found that dental porcelains accumulated significantly less plaque than human enamel or a bonding polymer composite. No relationship was detected between a fluoride-containing crystal structure of one ceramic and a measurably lower plaque accumulation rate or vitality.

Received September 21, 1992/Accepted August 27, 1993

Address correspondence and reprint requests to: R. Hahn Department of Conservative Dentistry Dental School, University of Tuebingen Osianderstr. 2-8 72076 Tuebingen German

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Microbial accumulation and vitality on different restorative materials