In vivo and in vitro studies on soft denture materials: microbial adhesion and tests for antibacterial activity N. Okita 1,3 D. Orstavik * J. Orstavik 2 K. Ostby 2

1NIOM-Scandinavian Institute of Dental Materials P.O. Box 70 N-1344 Haslum, Norway 2Department of Prosthodontics Dental Faculty University of Oslo, Norway 3Permanent address: GC Corporation 76-1 Hasunuma-sho, Itabashi-ku Tokyo 174, Japan Received March 13, 1990 Accepted May 13, 1991 *To whom correspondence and reprint requests should be addressed Dent Mater 7:155-160, July, 1991

Abstract-The microbiological properties of four tissue conditioners, one soft liner, and one acrylic resin were studied. The tissue conditioners showed no or negligible antimicrobial effects toward salivary microorganisms by two different in vitro tests. In in vitro adhesion experiments, more Streptococcus mutans and Candida albicans adhered to the tissue conditioners and the soft liner in comparison with conventional acrylic resin used for denture-base fabrication. No difference in bacterial adhesion was found among the tissue conditioners. The microbial colonization of two tissue conditioners lined on maxillary dentures in three volunteers was followed for 14 days. No difference among the materials was found, but a tendency for subjectdependence in plaque formation on the materials was noted.

icrobial infection of oral tissues is one of the most important factors in denture stomatitis (Van Reenen, 1973; Budtz-J0rgensen, 1974; Olsen, 1974; M~ikil~iand Hopsu-Havu, 1977; Rennet et al., 1979; BudtzJ~rgensen et al., 1983; Santarpia et al., 1988). Oral micro-organisms adhere to the surfaces of dentures, forming denture plaque, and to the oral mucosa (Gibbons, 1989; Olsen, 1990). Among the micro-organisms, Candida albicans is considered most important for the development of denture stomatitis (Olsen, 1974; M~ikil~iand Hopsu-Havu, 1977). However, several oral bacteria, including streptococci, are also associated with mucosal pathosis, directly (Van Reenen, 1973; Budtz-Jgrgensen et al., 1981 ) or indirectly through interactions with yeasts (Verran and Motteram, 1987; Branting et al., 1989). Acrylic resin is the universal denture base material, and the adhesion ofyeasts and bacteria to acrylic has been the subject of several studies (Samaranayake and MacFarlane, 1980; Verran and Motteram, 1987; Kaita et al., 1987; Branting et al., 1989). Soft lining materials are frequently used to coat the dentures on a permanent or semi-permanent basis, and tissue conditioners are sometimes applied for periods of days to a few weeks. These materials also interact with oral microbes (M~ikil~i and Hopsu-Havu, 1977; Burns et al., 1987), and their surface texture makes efficient mechanical cleaning difficult. Microbial accumulation on these soft materials is therefore potentially a serious problem. Another factor to be considered in relation to the microbial infection is the antimicrobial activity of the material. Several investigations have shown that most tissue conditioners and soft liners are inert to yeasts in vitro (Gruber et al., 1968; Douglas and Walker, 1973; M~ikil~i and Hopsu-Havu, 1977; Thomas and Nutt, 1978; Burns et al., 1987). AIthough M~ikil~iand Hopsu-Havu (1977) and Burns et al. (1987) referred to the correlation between the antifungal effect and the fungal adhesiveness, these properties are still an unresolved issue. The purpose ofthe present study was

M

to evaluate the interaction between soft denture materials and micro-organisms with special regard to microbial adhesion in vitro as well as in vivo and the possible antimicrobial properties of soft denturematerials. MATERIALS AND METHODS

The materials used in the present study are listed in Table 1. The codes given in Table 1 will be used for identification of the materials in the text. The materials were stored, handled, and mixed according to the manufacturers' directions. Bacteria and Yeast Culture.--Streptococcus mutans (NCTC 10449) and Candida albicans (ATCC 24433) were used. S. mutans was kept on blood agar plates at 4°C with monthly transfers. C. albicans was kept on Sabouraud dextrose agar (SDA; Oxoid, Hampshire, UK) with bi-monthly transfers. Prior to the experiments, the organisms were inoculated into 500 mL oftrypticase soy broth (TSB; Oxoid) and incubated at 37°C for 72 h. In some experiments, stimulated whole saliva, freshly collected from one subject chewing Parafilm, was used directly as a source of bacteria. In vitro Antibacterial Test: Gel Diffusion Method.--The procedure described by ~rstavik (1981) was used. A 9-mL quantity of freshly collected whole saliva was mixed with 150 mL of TSB in agar kept molten at 50°C. Some 20-mL portions were poured into agar plates. Circular holes, 5 mm in diameter, were punched in each plate. The wells in each plate were filled with fresh mixes of materials, with 0.05% chlorhexidine (CHX) gluconate solution in water used as the positive control. The plates were incubated at 37°C for 24 h in an atmosphere of 5% CO2 + 95% H 2. The diameter of the zone of growth inhibition was measured to the nearest millimeter, the width of the well (5 ram) subtracted, and the difference recorded as the inhibitory activity of the material. In vitro Antibacterial Test: Filter Paper Method .--Sterilized pieces of illter paper (Watman No. 3, 4 mm in diameter) were immersed in 10 mL of whole saliva for 10 min. The filter papers with saliva were placed in small wells (6 mm

Dental Materials~July 1991 155

in diameter, i mm in depth) of a stainless steel metal plate, and stainless steel metal rings (4.5 mm inner diameter, 5.5 mm outer diameter, and 10 mm high) were placed so that the paper would be enclosed in the wells. Freshly mixed materials were immediately applied into the metal rings over the filter paper and allowed to remain for 60 rain. Salivasaturated filter papers without any material contact served as negative controls. Exposure of the paper disks to 0.05% chlorhexidine gluconate (positive control) killed all bacteria in the filter paper. The filter paper was then transferred to vials containing 2 mL of reduced transport fluid (RTF; Syed and Loesche, 1972) and glass beads, which were then vigorously shaken on a Vortex mixer for 30 s. The bacteria released through this shaking procedure thus constituted the surviving organisms. The RTF with released bacteria was serially diluted ten-

was centrifuged at 10,000 G for 10 min and washed three times in RTF. The sediment was re-suspended in approximately 50 mL of RTF, and adjusted in a spectrophotometer to a concentration of OD~o= 5.0 (S. mutans ) or 1.0 (C. albicans ) as the test suspensions. Parafilm-stimulated whole saliva from one donor was centrifuged at 1000 G for five rain, and the supernatant was diluted ten-fold to contain approximately 106 organisms per mL. Four specimens ofeach material, fixed in a frame of silicone rubber to prevent

them from sticking to one another, were placed in an Erlenmeyer flask with 10 mL of test suspension, and incubated at 37°C for two h in a shaking water bath. After incubation, each specimen was rinsed once in RTF and then shaken on a Vortex mixer in 2 mL of RTF for 60 s. Both the test suspensions and the washes containing adherent microbes were serially diluted ten-fold. Aliquots from the dilution series were inoculated on TSB agar plates. Incubation and counting procedureswere performed as described for the flter paper method.

6

S.rnutans

E" 5

C. albicans

Saliva

E E 4 O.

-r

3 0 o

E

2

O) 0 _J

.

O GC MB AC

GC MB AC

GC MB AC

Fig. 1. Tissue conditioners applied to the maxillary

Fig. 2. In vitro adhesion of S. mutans, C. albicans, and unspecified salivary microbes to a tissue conditioner (GC),

denture according to a cross-over design. 1, 2, 7, 8 =

a soft liner (MB), and an acrylic resin (AC).

KF; 3, 4, 5, 6 = GC.

fold. Twenty-five-~L droplets from each dilution were inoculated on TSB agar plates and incubated at 37°C for 24 h in an atmosphere of CO2 + H 2. The number of viable organisms from the washes was then calculated from the counts of appropriate dilutions. In vitro Microbial Adhesion T e s t . Freshly mixed tissue conditioners were packed into poly(tetrafluoroethylene) molds (12 mm in diameter, 2 mm in depth) and allowed to set at 37°C for 30 min. After removal from the molds, each specimen, except for AC, was trimmed of excess material. MB and AC were heatcured in gypsum molds of the same size as the ones for tissue conditioner. AC was polished with A1202 pumice with a particle size of 1 pro. All specimens were stored in 2 mL of sterile saline at 37°C for 30 min before application. A 72 -hour culture ofbacteria or yeasts

Fig. 3. Scanning electron micrograph of a dense plaque of bacteria adhering to KF after two weeks in vivo.

156 Okita et al./Bacteriologic studies of tissue conditioners

Clinical Microbial Adhesion Test.Three patients (A, male, 78 yr; B, male, 63 yr; C, female, 69 yr), in good general and oral health, participated. They had worn complete dentures for more than five years. So that a sufficient number could be obtained for analysis, only two materials (GC and KF) were tested in this model. Sampling Procedure.--Small circular wells (5 mm in diameter, 2 mm in depth) were prepared by drilling on the fitting surface of a maxillary denture to apply the material. To counteract a

possible system error due to differences in ecology on the right and left sides of the denture, the different materials were applied according to a cross-over design, as shown in Fig. 1. The materials were left in place for test periods of two h, two, seven, and 14 days. During this time, the patients carried out ordinary hygienic measures, but avoided brushing of the inner denture surface and the use of chemical detergents. The test specimens were removed for analysis by use of sterile scalpels and forceps. They were stored in buffered

formalin at 4°C for at least 24 h prior to microscopic analysis. LightMicroscopy.--After fLxation, the specimens were rinsed in distilled water and stained in 0.5% crystal-violet-ethanol solution. The percentage of surface area covered with bacteria and/or yeasts was evaluated under the light microscope according to the Adhesion Index indicated in Table 2. Similarly, the presence of epithelial cells was also recorded. Scanning Electron Microscopy.Each specimen was subjected to a partial dehydration procedure: They were immersed in distilled water for two h, in 30% ethanol for one h, in 50% ethanol for two h, and in 70% ethanol overnight. After being mounted on aluminum stubs, the specimens were trimmed into a truncated cone and coated with platinum. Then, the specimens were examined at 1.2-1.5 kV in a Philips 515 scanning electron microscope (Eindhoven, The Netherlands).

RESULTS In vitroAntibacterial Test: Gel Diffusion Method.--The four tissue conditioners

Fig. 4. Scanning electron micrograph of yeast-like organisms adhering to GC after two weeks in vivo,

Fig. 5. Scanning electron micrograph of cocci and rods adhering to KF after two weeks in vivo.

were tested for antibacterial activity by the gel diffusion method, and the results are shown in Table 3. Whereas the 0.05% chlorhexidine control was strongly antibacterial, only a very small zone of growth inhibition was observed around CC, and the other materials showed no antibacterial effect. In vitro Antibacterial Test: Filter Paper Method.--The results of the antibacterial test by the filter paper method with the four tissue conditioners are shown in Table 4. No statistically significant antibacterial activity was detected for any of the materials. In vitro Microbial Adhesion Test.--In all instances, the bacteria or yeasts survived in the test suspensions at the same level as in the control with no test specimen, which meant that there was no microbicidal activity of the materials to the test cells during incubation. The results of the adhesion tests to the four brands of tissue conditioners are shown in Table 5. Average adhesion was similar for all the materials, with no significant differences. A comparison of the three categories of materials, i.e., tissue conditioners, acrylic resin, and silicone liner, is shown in Fig. 2. Significant differences in the numbers of adherent microbes were found among the categories, with less S.

Dental Materials~July 1991 157

TABLE 1

THE MATERIALSTESTED Brand Name

Type

Code

Manufacturer

Lot. No?

Coe Comfort

Acrylic-based tissue conditioner

CC

Coe Laboratories, Inc., Chicago, IL, USA

P:071488B, L:113088A

CC Soft-Liner

Acrylic-based tissue conditioner

GC

GC Dental Industr. Corp., Tokyo, Japan

P:070581,L:250481

Kerr FITT

Acrylic-based tissue conditioner

KF

SYBRON/Kerr, KERR Europe, Scafati, Italy

11119841324

Visco-gel

Acrylic-based tissue conditioner

VG

DeTrey Division, Dentspiy Ltd., Weybridge, Surry, England

P:EF9 85/06, L:ED88 85/07

Molloplast-B

Silicone-based soft liner

MB

Kostner & Co., Karlsruhe, Germany

850414

AC

Schutz-Dental GmbH, Rosbach, Germany

P:922028, L:922028

Futur Acry12000 Acrylic resin ~P, powder component; L, liquid component.

TABLE 3 ANTIBACTERIAL EFFECTS OF TISSUE CONDITIONERSTHROUGH THE GEL DIFFUSION METHOD

TABLE 2 COVERAGE OF SPECIMENS BY BACTERIA OR YEASTS: DEFINITIONOF ADHESION INDEX VALUES

Index

TABLE 4 ANTIBACTERIAL EFFECTS OF TISSUE CONDITIONERSTHROUGH THE FILTER PAPER METHOD

Diameterof inhibitedzone (mm)

Description

Bacteria. Log (Number)

Up to 20% of the surface covered

Material

mean (n = 4)

SD

Material

mean {n = 4)

SD

20-40% of the surface covered

CC

0.25

0.29

CC

5.093

0.076

40-60% of the surface covered*

GC

0

0

GC

5.195

0.155

60-80% of the surface covered*

KF

0

0

KF

5.013

0.156

over 80% of the surface covered* * Confluent sheetsof microbescould be observedfor theseindexvalues, but these sheetswere separated by uncolonized areas of various sizes.

VG

0

0

VG

5.185

0.154

0.29

Control

5.300

0.093

MB and AC not tested by this method.

MS and AC not tested by this method.

mutans and C. albicans adhering to the

sites or among materials. Adhering bacteria and yeasts reflected the variety of oral flora at the very early stage. Fig. 5 shows typical morphology of cocci and rods. At the last stage (two wk), confluent sheets of cocci were prevalent, but bacteria never covered the entire surface (cf. Table 2).

materials contain cytotoxic ester plasticizers (Jones et al., 1988; Lovas et al., 1988). The manufacturers claim that the addition of zinc undecylenate makes CC "bacteriostatic". Zinc undecylenate is an antifungal agent, as demonstrated in vitro, against C. albicans (Gruber et al., 1968; Douglas and Walker, 1973). Judged by the present results, the zinc undecylenate does not impart measurable antibacterial effects to CC. VG has been reported to be completely ineffective to three species of Candida (Thomas and Nutt, 1978), and no antibacterial activity was found in the present study. Thus it appears that the basic components of tissue conditioners have little or virtually no antibacterial properties as measured by the test methods used. In vitro Microbial Adhesion Test .--In the present study, higher numbers of bacteria and yeasts were shown to adhere to the tissue conditioner and to the soft liner than to acrylic resin (Table 5 and Fig. 2). This finding corroborates previous reports that soft lining materi-

acrylic resin (AC) than to either the tissue conditioner (GC) or the soft liner (MB). Clinical Microbial Adhesion T e s t . Microbial colonization scored by the Adhesion Index is summarized in Table 6. There appeared to be no difference in the colonization of anterior and posterior sites. Also, there appeared to be no difference between the two materials. Within two h of application, some bacteria and/or yeasts were found on the specimens. Microbial coverage increased continuously with time. There seemed to be a slight tendency for the specimens from patient C to acquire more bacteria earlier than specimens from the other two patients. Epithelial cells adhered to less than 20% of the surface of each specimen throughout the experiment. Scanning electron micrographs of adherent bacteria and yeasts are shown in Figs. 3 and 4. No striking difference concerning types of adhering bacteria and yeasts could be found, either among

CHX (control)

11.75

DISCUSSION

In vitro Antibacterial Test.--The antibacterial effects of fresh tissue conditioners were assessed by two methods. The gel diffusion method is often used for detecting microbistatic effects of materials and drugs. It is simple, allows for comparison of several samples at a time, and may be used as a screening test. In the filter paper method, the microbicidal effects are evaluated by the counting of viable bacteria. The reproducibility of this method was good, as indicated by the low standard deviation of parallels in each series. The tissue conditioners tested showed very little bacteriostatic activity, although these

158 Okita et al./ Bacteriologic studies of tissue conditioners

TABLE 5

THE MICROBIAL ADHESION TO DIFFERENT BRANDS OF TISSUE CONDITIONERS in vitro

Log (Microbes per mm) Material

S, mutans

C. albicans

CC

4.90 (0.30)

2.16 (0.25)

1.12 (0.19)

GC

4.66 (0.03)

2.03 (0,59)

1.11 (0.07)

KF

4.88 (0.04)

2.43 (0.16)

1,20 (0.09)

VG

4.83 (0.11)

2.39 (0.24)

1.26 (0.14)

Salivary bacteria

): Standard deviation.

CONCLUSIONS

TABLE 6

In vitro MICROBIAL COLONIZATION ON TWO TISSUE CONDITIONERS SCORED BY THE ADHESION INDEX

Time

Subject 1

2h

2d

7d

A

4

B

4

KF Adhesion index

3

4

5

1

3

4

1

2

3

A

2

1

2

B

3

1

2

C

3

A

1

3 1

2

2

1

A

1

2

B

2

1

C

2 3

1

C

2

4

C

B

14d

GC Adhesion Index 2

2

1

Rapid and extensive microbial colonization was observed on the palatal surfaces of tissue conditioners carried by volunteers in vivo. These findings were supplemented with in vitro data of extensive adhesion and poor antibacterial activity of these materials. Apparently, tissue conditioners, as well as soft liners, are likely to be rapidly colonized in vivo and have poor or no inhibitory properties. For this reason, the use of tissue conditioners should be limited to short-term periods, as advocated by Gruber et al. (1968). Considering that tissue conditioners are often applied to a mucosa susceptible to pathosis, care should be exercised in clinical applications of these materials.

1

1

1

3

1

1

2

1

1

1

1

2

REFERENCES

1

1

1

3

1

BRANTING,C.; SUND,M.-L.; and LINDER, L.E. (1989): The Influence of Streptococcus mutans on Adhesion of Candida albicans to Acrylic Surfaces in vitro, Arch Oral Biol 34:341-353. BURNS,D.R.; BURNS,D.A.; DIPIETRO,G.J.; and GREGORY,R.L. (1987): Response of Processed Resilient Denture Liners to Candida albicans, J Prosthet Dent 57:507-512. BUDTZ-JORGENSEN,E. (1974): The Significance of Candida albicans in Denture Stomatitis, Scand J Dent Res 82:151-190. BUDTZ-JORGENSEN,E.; THEILADE,E.; and THEILADE, J. (1983): Quantitative Relationship between Yeasts and Bacteria in Denture-Induced Stomatitis, Scand J Dent Res 91:134142. BUDTZ-JORGENSEN, E.; THEILADE, E.; THEILADE,J.; and ZANDER,H.A. ( 1981):

I

1

1

The figures represent the number ol specimens (out of four for each tissue conditioner tested) with the Adhesion Index indicated. When the numbers of specimens do not add up to four, specimens had had to be discarded for technical reasons.

als for dentures show adherence of micro-organisms (Gruber et al., 1968; M~ikil~i and Hopsu-Havu, 1977; Renner et al., 1979; Burns et al., 1987), and it suggests that soft liners are more susceptible to microbial adhesion than acrylic resin (M~ikil~i and Hopsu-Havu, 1977). Soft lining materials were found not to affect the viability of organisms in the test suspensions, and the antibacterial tests were generally negative. In addition to antimicrobial properties, other factors also play a role in adhesion: Surface porosity and texture, and biological and physical/chemical affinity between the materials and microbial cells may be important factors.

conjunction with bacteria in most instances. In vitro studies have indicated that co-aggregation between Candida and streptococci occurs, whereby the primary adhesion to acrylic resin of the one species may support the secondary attraction of the other (Verran and Motteram, 1987; Branting et al., 1989). However, no such relationship could be found in a clinical study where yeasts constituted a very minor part among the oral flora (Budtz-Jcrgensen et al., 1983). Hyphae could not be identified in the clinical specimens, in contrast to the report by Douglas and Walker (1973). This may be a reflection of the selection of patients with healthy oral mucosa, whereas Douglas and Walker (1973) focused on denture stomatitis patients.

Among the four brands of tissue conditioners, no significant difference was seen. This could be expected, since they are chemically similar (Jones et al., 1988) and showed little antimicrobial effect. Clinical Microbial Adhesion T e s t . The present study revealed that clinical microbial adhesion to tissue conditioners began at an early stage. It was noticeable that some micro-organisms had settled on the surfaces of the materials only two h after application. The SEM analysis indicated that yeasts also colonized the surfaces, evidently also at an early stage. Later, cocci prevailed, but it should be pointed out that yeasts appeared to occur in

Dental Materials~July 1991 159

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