Journal of Oral Rehabilitation 1997 24; 594-604

Effects of salivary or serum pellicles on the Candida albicansgrowth and biofilm formation on soft lining materialsin vitroH. N I K A W A , T. H A M A D A , T. Y A M A M O T O & H. K U M A G A l Department of ProstheticDentistry. Hiroshima University School of Dentistry. Hiroshima. Japan

SUMMARY The effects of salivary or serum pellicleon Candida albicans growth, hiofilni formation andcavitation on the soft lining materials wereexamined. Both saliva and serum pellicles reducedthe antifungal effects of soft liners. The fungalbiofilm formation on these materials varieddepending upon hoth the materials tested andprotein-coats, and the pellicles which significantlyenhanced the biofilm formation. Similarly, thepellicles enhanced the firm colonization and hyphalinvasion of the yeasts on the specimens, although the

cavitation appeared to be regulated by the plasticizerused. These results suggest that the interactionsbetween proteinaceous pellicle, tissue conditionersand fungi are complex. They also suggest thatdenture pellicles facilitate fungal plaque formationonto soft lining materials through several mechan-isms such as reduction of the antifungal effects ofsoft liners, facilitation of biofilm formation, firmcolonization and hyphal invasion. In addition, thecomposition of the materials is also involved in thesusceptibility to the fungi.

IntroductionResilieni denture lining materials arc widely used asdynam.c impression materials, essential adjuncts in theprosthodontic treatment and management oftraumatized oral mucosa. These materials, however, art"known to be easily colonized and infected by Candidaalbicans and related Candida species (Douglas, 1979),resulting in denture-induced stomatitis. In the successlulcolonization, subsequent plaque lormation and develop-ment of pathogenesis, the adherence of Candida to solidsurfaces such as acrylics or denture lining materials hasbeen thought to be the first step (Rotrosen, Cardelone& Edward Jr., 1986), followed by the growth andcolonization of yeast cells on these materials. Recently,it was pointed out that the phenomenon of initialadherence may represent only the first step in thecolonization (plaque formation) process (Kennedy,1990) which leads from the formation of a thin biofilm.

to a multilayer, and culminates in denture plaque.Therefore, in Candida infection into these materials, thegrowth and colonization of Candida on them is one ofthe most important factors.

Although the effect of these materials on the growthof Candida has been studied by a number of workers,the results are discordant and controversial (Molnar,1966; Williamson 1968: Allison & Douglas 1973;Douglas & Walker 1973; Thomas & Nutt 1978; Razek& Mohamed 1980; Wright i980; Bruns et al.. 1987;Graham etal.. 1991). Recemly, we developed in vitrosystems to study the growth of fungi on denture-liningmaterials and revealed that commercial acrylic softlining materials possess latent antifungal effects whichcould be attributed to ingredient liquid and powdercomponents (Nikawa eM/., 1994a; Nikawa ej'fl/., 1995c).

The inhibition of C. albicans growth caused by thesematerials is inconsistent with the finding of Allison &Douglas (] 973) that a large amount of C. albicans hyphae

© 1997 Blackwell Science Ltd

DENTURE PELLICLES. TISSUE CONDITIONERS AND C. ALBICANS 595

colonize and invade deeply into the soft liner in vivo.Further, Douglas (1979) also pointed out thai soft liningmaterials, particularly tissue conditioners, are moresusceptible to candidal colonization than acrylics. Wehave also demonstrated that denturc-pellick comprisingsalivary or serum protein promotes a film-like coloniza-tion (biofilm formation) of C. albkans. hyphal emergenctfand invasion into soft hning materials (Nikawa el ai..1993). Besides this, it was also demonstrated that thecavitation of C. albkans, which is a deeply embeddedcolonization of blastospore (Nikawa el al.. 1995c),occurred in relation to the plasticizer used. Hence, wecould hypothesize that the discrepancies between thein vivo and in vitro findings may be attributed to thelatter phenomena (i.e. role oi pellicles, fimgal biofilmformation and cavitation). Therefore, the interaaionsbetween proteinaceous pellicles, lining materials andC. alhicans could be the most important factors in regardto the in vivo fungal colonization and/or invasion of(issue conditioners. However, there is scant informationavailable on the effects of these materials on the fungalcolonization, cavitation or the biofilm development.The purpose of this study is to examine the role ofsalivary or serum pelhcle In C. albicans growth, biofilmformation and cavitation on the soft lining materials.

Materials and methods

Micro-organisms and growth condition

Candida albicans IFO 1385, purchased from the Institutefor Fermentation, Osaka, was used and cultured asdescribed previously (Nikawa etai, 1989; Nikawa &Hamada, 1990; Nikawa etal.. 1992a & b). A loopfulof the yeast was inoculated in yeast nitrogen basemedium* containing 250 mM glucose and grownaerobically at 37°C. After the overnight culture, theyeast was harvested in the late exponential growthphase, washed twice with sterilized distilled water andresuspended to a final concentration of 10̂ cells/mlby a spectrophotometric and haemocytomeiric means(Nikawa elai, 1993).

Fabrication of acrylic strips

Heat-cured denture acrylic sheets (50 x 50 X 0'7 mm)were fabricated according to conventional prosthodontictechniques. Briefly, denture acrylic poly(methyl-

"•Difco, D(!troit, U.S.A.

Materials Manufacturers

Bio Resin (resin) Sliofu Inc., Kyoto, Japan.Coe Comfort (CC) GC America Inc., Chicago, U.S.A,

Coe Soft (CS) GC America Inc., Chicago, U.S.A.

Fit Softer (FS) Sankin Industry Co. Ltd., Osaka, Japan.GC Soft Liner (GC) GC Dental Industrial Corp., Tokyo, Japan.

Hydrocast (HC| Kay-See Denial Mfg. Co., Kansas City,U.S.A.

Viscogel (VG) De Tray/Deiilsply, Weybridge, U.K.

meihacrylare) powder and monomer liquid'̂ weremixed according to the manufacturer's directions. Themixture was packed into the flask, processed in a watertank at 70°C for 90 min and then !00°C for 30 min,according to the Japan Industrial Standard (JIS). Asmooth surface was obtained by compressing themixture onto glass slides. The processed acrylic sheetswere cut into !0 X 10 X 0-7 mm pieces.

Soft lining materials

Six commercial resilient materials (summarized inTable 1) were processed according to manufacturer'sdirections. Bach of the mixtures were poured ontothe acrylic strips and prepared to a uniform size(10 X 10 mm X 1-5 mm thickness) with smoothsurfaces, by placing glass slides over them as describedpreviously (Nikawa etal, 1993, 1994a & b).

Saliva and serum

Pooled unstimuiated whole saliva was collected fromfive healthy donors and clarified, according to themethod of Gibbons, Etherden & Moren (1983) withmodification, by centrifugation at 12,000g Un 15 min at4°C. Human serum was purchased from Sigma ChemicalCo.* Whole saliva and serum were stored at -25''Cbefore use.

Assay procedures

The growth and biofilm assay was conducted as follows.The specimens of acrylic and soft liners were coatedwith saliva or serum by placing them in wells of

+Bio Resin, Shofu. Kyoto, Japan.

tSt LDuis. MO, U.S.A,

1997 Blackwell Science Ltd, Journal of Oral Rehabilitation 24; 594-^04

596 H. NIKAWA et al.

Multiwell tissue culture plates*, into which were dis-pensed 500 \iL of the protein solution per well, whichwere then incubated for 1 h al 37°C. Saliva or serurnwas stihstituied with an equal volume of sterile distilledwater in the control wells. After incubation the proteinsolution was aspirated, 50 microiitres of yeast suspen-sion (1 X IO' cells/mL) was inoculated on the surfaceof each acrylic or tissue conditioner specimen and thewhole assembly was incubated at 37°C for 2 h topromote yeast adherence and colonization. Sub-sequently, 2-0 mL of Sahouraud broth was carefullydispensed into each well, incubated at 37°C for up to120 h with pH changes in these media being measuredusing a pH meter+ as a parameter of fungal growth(Nikawa eta!., 1994a). Afterwards, each specimen wascarefully removed, washed gently hy rinsing three timesfor a total of 60 sec with distilled water to remove non-biofilm organisms (Nikawa elai, ]995d), immersed in10 mL of the reagent containing benzalkonium whichextracts intracellular ATP (Siro, Romar & Lovgren,1982), and allowed to react for 15 min at room temper-ature. The resultant reagent solution was then subjectedto an ATP-measuring system* to determine the biofilmactivity (Berlutti eta!.. 1993).

To determine the amount of cavitation or invasion,each of the 120 h incubated specimens with fungi wasremoved, washed vigorously and subjected to ultra-sonics for a total of 15 min with distilled water, in orderto remove the biofilm yeasts other than the firmlyattached, cavitated or invased organisms. Then, the ATPamouni on each specimen was measured as describedabove. For uiirastructural studies, specimens wereremoved from the wells after the assay, washed withsterile distilled water, fixed in 2-5% giutaraldehyde and1-0% osmium tetroxide and air dried. Each specimenwas then sputter coated with a layer of gold to athickness of 15-20 nm and observed under a scanningelectron microscope^ using standard procedures(Nikawa ei ai, 1993, 1995a. b & c).

The assays were carried out on two independentoccasions, with four samples each time, and the valuesobtained were averaged to give the final data withstandard deviations. AH the numerical data obtainedwere analysed by analysis of variance (ANOVA) andTukey's multiple range test at a level of 5%.

*Nundon® Delia, Nunc. Kamstrup, Denmark.•tpH meter 245, Corning, New York, U.S.A.*ATP-AF 100, TOA Electronics Ltd., Tokyo, Japan.§JMS-6300. Joel, Tokyo. Japan.

Results

Effect of soft liners on fungal acid production and/or growth

As shown in Fig. fa-c, although the pH changes inmedia were varied depending upon both the materialsand protein coats on which Candida had grown, thereverse sigmoidal pH curves were observed with allsamples, which was similar to our previous study(Nikawa et al., 1994a). Initially, the pH of the mediawith all samples decreased slightly, and a remarkable,rapid and linear decline in pH from ca. 5-5 was observed.After incubation for 48-60 h, the rate of pH changelevelled off in each case. In the previous study, weexamined the inter-relation between pH values ofgrowth medium and the numher of yeasts (Nikawaet ai. 1994a). The pH of medium decreased slightly withthe initial fungal growth (up to about 2 X 10'' cells/ml). At a pH from 5 5 to a minimum value, a goodcorrelation between pH value and the numbers ofgrown yeasts was observed. After reaching minimumpH, further growth was observed with a slight rise in pH.

As in a previous study (Nikawa etal., 1994a), theinhibitory effects of the soft liner on fungal growthwere observed as being: the delay of beginning a rapiddecline in pH; decreases in the rate of pH change; andincreases in the minimum pH. We analysed the effectof soft liners and protein coats on yeast growth withregard to these three parameters.

As to uncoated specimens, the beginning of rapiddecline was observed with acrylic samples within 4 hincubation, followed by GC Soft Liner (GC) = Hydrocast(HC) g Coe Soft (CS) ^ Coe Comfort (CC) ^ Fit. Softer(FS) and Viscogel (VG) was the most effeaive to delaythe beginning oi Candida acid production or growth(ANOVA and multiple range test, P<0-05) (Fig. la.Tables 2 & 3).

The highest rate of pH change was observed with thecase yeasts grown on acrylic, which decreased in thesequence GC Soft Liner (GC) > Hydrocast (HC) > CoeSoft (CS) g Viscogel (VG) ^ Fit Softer (FS). Coe Com-fort (CC) showed the most inhibitory effect (ANOVA andmultiple range test, P<0-05) (Fig. la. Tables 2 &- 3).

The minimum pH varied depending upon the sampleson which Candida was grown. The case grown on acrylicshowed the lowest (pH 2-95), and increased in the orderof GC Soft liner (GC) S Hydrocast (HC) < Fit Softer(FS) S Coe Soft (CS). Coe Comfort (CC) and Viscogel(VG) showed the highest pH value (pH 4 34 and 4 20,respectively) (Fig. la. Tables 2 & 3).

© 1997 Blackwell Science Ltd. Journal of Oral Rehabilitation 24; 594-604

DENTURE PELLICLES, TISSUE CONDITIONERS AND C, ALBICANS 597

60-1(a) Uncoaled

-VQCCCS

-FS• H C

- QC

60

Incubation time (h)

6 0 - 1

120

(b) Saliva

0 60

Incubation time (ti)

(c) Senjm

"~1120

Incubation time (h)

Fig. 1. The pH curve of Candida atbicam grown on (a) uncoaled, (b) saliva- and (c) serum-coated tissue conditioners in Sabouraudbrotb. Significant facilitation of growtb caused by protein coats was indicated in regard to tbree parameters: time necessary to reachpH 5-5: rate of decrease in pH: and minimum pH.

Effect of pellicles on func)al acid production and/or growth

As shown iit Fig. Ib & c and Tables 4 6-5, saliva orserum pellicie essentially decreased tile inhibitory effectof soft liners on fnngal growth. The effects were, how-ever, varied depending upon both protein coats andmaterials used (ANOVA, F<O-O5) (Tahles 6 &• 7).

The time lag al the beginning of the rapid declinewas significantly shortened by the saliva coat with FitSofter (FS) and GC Soft Liner (GC), and by the serumcoat with Fit Softer (FS), whereas the elongated time

lag at the beginning of the rapid decline was observedwith serum-coated Hydrocast (HC) (ANOVA and multiplerange test, P<005) (Tables 4 & 5). The rate of pHchange was promoted both by saliva and serum coatson soft lining materials other than Viscogel (VG) (ANOVAand multiple range test P<005) (Tables 4 fr 5).Similarly, as compared with uncoated samples, thesignificant decrease in minimum pH was observed withmost of the saliva- or serum-coated specimens, althoughsaliva- and serum-coated Viscogel (VG) exhibited asignificantly increased minimum pH (Tables 4 & 5).

© t997 Btackweif Science Ltd, Journal of Oral Rehabilitation 24; 594-604

598 H. NIKAWA et al.

e 2. Effeas of tissue conditioners on pH change Table 4. Effects of saliva-coated tissue conditioners on pH change

cccsFSGCHC

VG

Time necessary toreach pH 5-5 (h)*

36-7 ± 14'433-6 ± 5945-9 ± 16-021-5 ± 2 122 2 ± 2 273-8 ± 15-5

Rate of decrease inpH (XIO-^ linit/h)t

215 ± 0063-24 ± 0'082 59 I 0 07647 ± 0'265 63 ± 0 392-89 ± 0 1 7

MinimumpHt

4 34 ± OiO3 94 ± 0-043-87 ± 0'233 53 ± 0-043 63 ± 0 054 52 ± 0-10

CC

CSFS

GCHCVG

Time necessary toreach pH 5-5 (h)*

30-5 ± 2-328-0 ± 1-313-7 ± 7-2II521-0 ± 0-7li21-9 ± 1-884-5 i 8-7

Rate of decrease inpH (XIO'^ itnit/h)t

3-50 ± 0-08(f4-58 ± 0-24t6-87 ± 0-5ltt7-37 ± 0-22Tr6 54 ± 0-19tfM l ± 0-I2II

Mitiitnum

4'12 ± 0-15li3-81 ± OO7IL3-59 ± 0-04Ji345 ± 0'04li3-49 ± 0-075-24 ± 0-70tl

*The mean time of the beginning of fungal growth on acrylicspecimens was 3'76 ± 0-2 h.••The mean rate of decrease in pH decline with acrylic specimenswas 8-60 ± 0-9 xiO"'^ tinit/h.*The mean minimum pH of fungal growth on acrylic specimenswas 2-95 ± 0-07,

T^ble 3. Results of tnultiple range test on pH changes caused hyihe fungal growth on bare surfaces of tissue conditioners

Time necessary to Rate of decreasereach pH 5-5 in pH Minimum pH

resmGCHCFSCSCCVG

observed among the

Effect of soft liners or protein pellicles on fungal biofilmformation

The amount of fungal biofilms formed on test specimenswas determined by luminescent ATP analysis which is,according to our previous studies, a highly sensitive andquantitative method when determining the number offungal cells (Nikawa etai, 1995a, b & d). The ATPcontents of the 120 h biofilms formed on denturematerials are shown in Fig. 2. The fungal biofilmactivities were significantly varied depending upon boththe samples or protein coats on which Candida hadgrown.

In the case of C. albicans biofiim formation on acrylicsurface, Ihe serum-coated strips exhibited more than100-fold increased cellular kinetics compared with theuncoated, protein-free acrylics. The activity on the

resGCHC

CSCC

PSVG

*N(

cor

in

) signihiinccted t

resmGC

HCCSVG

FSCC

ant differences>y bars.

*The mean time of the beginning of fungal growth on acrylicspecimens was 3'86 ± O'l h."tThe mean rate of decrease in pH decline with acrylic specimenswas 8-68 ± 0'3 XiO"-̂ tinit/h.*The mean minimum pH of fungal growth on acrylic specimenswas 2-95 ± 0 02,^Arrows indicate the significant changes caused by saliva coat(ANOVA, P<O-O5),

Table 5. Effeas of serum-coated tissue conditioners on pH change

Time necessary to Rate of decrease in Minimumreach pH 5 5 (h)* pH (XlO"^ unit/h)+ pH*

CC

CS

FS

GC

HC

V G

34-429-417-81 9-126-4

74-2

± 2-4± 3-5± 4-811± 5-7± 2-9ft± 21-8

2-864-447-787-097-061-77

± 0-04ff± 0-17ff± 0-7lfl± 0-4i1T± O-I3f(± 0-04tt

4-323-913-603-483 544-75

±0-05± 0-09± 0-05U± 0-041± 0-07± 0-37tr

*The mean lime of the beginning oi fungal growth on acrylicspecimens was 3 86 ± O-J h.+The mean rate of decrease in pH decline with, acrylic specimenswas 8-65 ± 0-3 xio^^ unit/h.*The mean minimum pH of fungal growth on acrylic specimenswas J-00 ± 0-02.^Arrows indicate the significant changes caused hy serum coat(ANGVA, /'<0'05).

saliva-coated strips exceeded the uncoated strips by afactor of 10, being in accordance witb our previousobservations (Nikawa etal, 1995d).

In contrast, saliva coats of soft lining materials didnot significantly affect the fungal biofilm formationas compared with uncoated specimens. However, theserum-coated soft lining samples significantly promotedthe fungal biofiim formation as compared witb eitheruncoated or saliva-coated specimens, and the serum-admixed biofilm activity exceeded the uncoatedmaterials by a factor of 10.

When the activities of biofilms formed on each soft

© 1997 Blackwell Science Ltd. Journal of Oral Rehahiliiaiian 24: 594-604

D E N T U R E P E L L I C L E S , T I S S U E C O N D I T I O N E R S A N D C. ALSICANS 599

Table 6. Results of multipte range test on ptl ctianges caused hy

ctie tiingat growth on saliva-coated surfaces ot tissue conditioners

Time necessary to Rate ot decrease in

reach pH 5-5 pH Minimum pH

FSGCHCCSCCVG

resinGCPSHCCSCCVG

resm

GC

CS

CC

VG

*No significant ditterences were ohserved among the samptes

conneCTed hy bats.

Table 7. Results of multiple range test on pH changes caused bythe tungal growth on serum-coated surfaces ot tissue conditioners

Time necessary to

reach pH 5-5

resin*

FS

GC

HCCS

CCVG

Rate of decrease inpH

resin

FSGCHC

CSCCVG

Minimum pH

resin

GCHC

FSCS

CCVG

*N() significant diiferencesconnected tiy bars.

I I Uncoaled

among the sample;

^ Saliva

^M Serum

ii**** **-j i t

***

Resin CC CS FS GC HC VG

Fig. 2. Fungal biofilm activities formed on denture materials. Tenthousand pico mol of biofilm activity corresponds to approximately10̂ cells. *The samples showed significantly higher aCTivity thanthai of uncoated resin. **The samples showed significantly higheractivity than that of saliva-coated resin.

lining material were compared with thai formed onacrylic strips; uncoated Coe SofI (CS); GC Soft Liner(GC); Hydrocast (HC); saliva-coated Coe Soft (CS); GCSoft Liner (GC); serum-coated Coe Comfort (CC); CoeSoft (CS); Fit Softer (FS); GC Soft liner (GC); andHydrocast (HC) showed a significantly greater biofilmactivity than that of saliva-coated acrylic strips (ANOVA,P<0-05). In addition, uncoated Coe Comfort (CC), FitSofter (FS), saliva-coated Coe Comfort (CC), Fit Softer(FS), Hydrocast (HC) and serum-coated Viscogel (VG)exhibited significantly higher hiofilm activity than thatof uncoated acrylic strips (ANOVA, P<O-O5).

Effect of soft liners or protein peliicles on firm colonization of

fungi

When the activities of firmly colonized yeasts on eachsoft lining material were compared with that formedon acrylic strips, uncoated Coe Comfort (CC), saliva-coated Fit Softer (FS), GC Soft Liner (GC), serum-coated Fit softer (FS) and GC Soft Liner (GC) showedsignificantly greater fungal activity than that of saliva-coated acrylic strips (Fig. 3). In addition, uncoated CoeSoft (CS), saliva-coated Coe Comfort (CC), serum-coated Coe Comfort (CC) and Coe Soft (CS) exhibitedsignificantly higher fungal activity than that of serum-coated acrylic strips (Fig. 3).

Scanning electron microscopic (SEM) observations

SEM observation was carried out to observe the stateof fungal colonization, such as cavitation and hyphalinvasion (Fig. 4a & b). The yeasts embedded in thesurface of Coe Comfort (CC), Fit Softer (FS), GC SoftLiner (GC), Hydrocast (HC) (cavitation) and hyphalinvasion were observed with GC Soft Liner (GC)(Table 8). However, neither cavitation nor hyphal inva-sion was observed with acrylic specimens, Coe Soft (CS)and Viscogel (VG).

Discussion

Denture stomatitis is an erythematous pathogenic con-dition of the denture-bearing mucosa and is causedmainly by microbial factors, especially C. albicans(Budtz-Jorgensen, 1974). It has been shown in denturewearers that the main reservoir of C. aibicans and relatedCandida species is the fitting surface of upper denture(Davenport, 1970), and that soft lining materials areeasily colonized and deeply infected by these

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600 H. NfKAWA et al.

T^ble 8. Blastospore ca

materials

citation and hyph-

Materials Cavitation Hyphal inv,

resinCCCS"FSGCHCVG ND ND ND

NDND

NDND ND ND

ND, Al least 200 fields/^ specimens of each sample were examineddirectly under SEM (X500), and either the cavitation or thehyphal invasion was not detected.

organisms (Allison & Douglas, 1973; Douglas, 1979). Inthe pathogenesis of denture stomatitis, growth of largenumbers of Candida on the fitting surface of denturesand the following acid production by grown yeasts(Odds, 1988) results in direct cytotoxicity, activates acidproteinase and phospholipase produced by these yeasts,and protnotes Candida adherence (Samaranayake &•MacFarlane, 1985, 1990; Samaranayake, 1986). Hence,in the present study, the effect of soft lining materialson the growth of C. albicans was investigated bymonitoring the pH change of the experimental growthmedium according to our previous studies (Nikawaetai, 1993, 1994a, 1995c).

In the previous study, we employed two differentconcentrations of yeast suspension to inoculate ontothe lining materials (50 L̂L of 10̂ cells/mL and 50 ̂ Lof 10̂ cells/mL; which corresponded to the final concen-trations of 2-83 X 10' cells/cm^ and 2-83 X 10̂ cells/cm ,̂ respectively. It was revealed that the latent anti-fungal activity of these lining materials was over-powered by the latter concentration (Nikawa etai.1994a). Furthermore, Budtz-Jorgensen, Theilade &Theilade (1983) reported that the diagnostic criteria forCandida-associan^d denture stomatitis is ai a concentra-tion of 10̂ cells/cm^. Thus, in the present study weemployed the latter concentration to examine the anti-fungal activity of test specimens under the conditionrepresentative of the oral states of Candida-associateddenture stomatitis patients.

As shown in Fig. la-c and Tables 2, 4 6- 5, saliva orserum pellicles essentially decreased, to a variableextent, the inhibitory effect of soft liners and facilitated

fungal growth on the materials as compared with un-coated samples (except for Viscogel (VG)). This was notconsistent with the results on acrylic resin and ourprevious findings (Nikawa etai. 1993). It is known thatsome salivas suppress Candida! growth while others donot (Samaranayake, Hughes &• MacFarlane, 1984), andhence it is possible our observation that saliva did notaffect fungal growth on acrylic specimens (Figs 1 & 2)could either be due to the quality of the saliva usedand/or its high dilution in the incubation medium. Inaddition, we employed the higher concentration ofyeast suspension for inoculation, and this conditioncould also cause the facilitation of fungal growth. Asimilar explanation could be offered for its growth onserum-coated acrylics as the serum is known to affectcandidal cell kinetics in a variety of ways (Odds, 1988).

In contrast, the results observed, that saliva or serumpellicle essentially facilitated fungal growth on the softlining materials, could be partly attributed to the preven-tion of direct contact of yeast ceils with lining materialscaused by proteinaceous pellicles, since tissue condi-tioners have been revealed to possess antifungal effectsdue to ingredients such as plasticizers (and theircytotoxicity) (Nikawa etai, 1994a). In addition, thenutrient-rich environment of the oral cavity mightove^iower the inhibitory effea of these materials(Graham el al., 1991; Okita etai.. 1991). Hence thephenomena may be additionally attributed to the localnutrient factors derived from pellicles. Further studies,however, are required to substantiate the currentobservations and to clarify the interactions betweenthese oral fluids and candidal growth on denturematerials.

A number of experimental approaches bave beenmade to examine the mechanisms of C albicansadherence to solid surfaces, such as denture acrylic(McCourtie 8- Douglas, 1981; Critchley & Douglas, 1985;Minagi ff al., 1985; Vasillas etai.. 1992; Edgerton etai..1993). Even the earliest investigators of this topic,using visual quantification of adherent yeasts,reported the high affinity of C albicans to denture acrylicor tissue conditioners and modulation of this attachmentprocess due to saliva and serum pellicles (Samaranayake& MacFarlane, 1980; Samaranayake, Hughes &MacFarlane, 1984). However, it should be noted thatthe phenomenon of adherence may represent only thefirst step in the colonization process (Kennedy, 1990)which, as time progresses, leads to a formation of a thinbiofilm and then a multilayer, climax community of

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DENTURE PELLICLES, TISSUE CONDITIONERS AND C. ALBICANS 601

plaque. Thus, a deeper understanding of the subsequentbehaviour of Candida biofilms are, more importantly, amethod for accurate quantification of yeast biofilmformation which may exhibit dimorphic growthpatterns as well as co-adhesion, aggregation and multi-layer growth over a prolonged period of colonization(Nikawa et al.. 1993). The main impediment for suchresearch to date has been the lack of a relatively simpleassay system to accurately quantify the cell growth.Recently, Hawser & Douglas (1994) reported fungalbiofilm formation on catheter material by using dryweight, colourimetric (MTT) and radiometric assays.However, in preliminary studies we found that the dryweight and MTT assay methods were less sensitive indetecting the small amount of yeast colonization (suchas uncoated or saliva-coaled acrylics). Furthermore, theresults of the MTT assay were affected by the salivary andserum proteins (data not shown). Hence, we adoptedanother assay system, bioluminescent ATP assay, whichhas been used previously to measure bacterial hiofilmactivity (Berlutti etal.. 1993; Farber fr Wolff, 1993).Farber & Wolff (1993) have conclusively shown thatthe result obtained by this ATP assay was consistentwith that obtained by either conventional viable countsor radiolabelling methods. Using this method we notedan excellent correlation between the yeast cells and theATP content in either the test or the control samples inthe preliminary study (Nikawa el ai., 1995a, b & d).This was not surprising as the assay was based on thefundamental principles ol ATP analysis which states thatthe amount of cellular ATP correlates with the dryweight, the volume and the number of viable cells(Siro, Romer & Lovgren, 1982), irrespective of themorphological attributes of the yeast.

In the preliminary studies, the activity of C. albicambiofilm comprising a large amount of both blastosporeand hypha formed on serum- or saliva-coated acrylicplateaued within 4 days incubation, and the activity offungal biofilm on serum-coated sample was approxi-mately 100-foid higher than that on uncoated controlsample with sparse colonization of blastospore, whichplateued within 2 days (Nikawa etal, 1995d). Thus weemployed the 5 days (120 h) incubation to examinethe effects of materials and proteinaceous pellicle onfungal biofilm formation.

Therelative ATP contents of the 120 h biofilms formedon denture materials are shown in Fig. 2. The fungalbiofilm activities were significantly varied depending

upon both the samples or protein coais on whichCandida was grown.

In the case of C. aWicans biofilm formation on theacrylic surface, the serum-coated strips exhibited morethan 100-fold increased cellular kinetics than theuncoated, protein-free acrylics, and the activity on thesaliva-coated strips exceeded the uncoated strips by afactor of fO. These results tend to agree with theobservations of Vasillas etal. (1992) and Edgerton etal.(1993) who reported on the promotion of candidaladhesion due to salivary pellicles,

In contrast, saliva coats of soft lining materials didnot significantly affect the fungal biofilm formationas compared with uncoated soft liners individually.However, uncoated Coc Comfort (CC), Fit Softer (FS),saliva-coated Coe Comfort (CC), Fit Softer (FS),Hydrocast (HC) and serum-coated Viscogel (VG)exhibited significantly higher biofilm activity thanthat of uncoated acrylic strips. Even the candidal growthrates on soft lining materials were relatively slower thanthat on acrylics (Fig. la-c). Furthermore, tbe serum-coated soft lining samples significantly promoted thefungal biofilm formation as compared with uncoatedand/or saliva-coated specimens, in each case. In addi-tion, the serum-admixed biofiim activity exceeded theuncoated lining materials by a factor of 10, and uncoatedCoe Soft (CS), GC Soft Liner (GC), Hydrocast (HC),saliva-coated Coe Soft (CS), GC Soft Liner (GC), serum-coated Coe Comfort (CC), Coe Soft (CS), Fit Softer(FS), GC Soft Liner (GC) and Hydrocast (HC) showedsignificantly greater biofilm activity than that of saliva-coated acrylic strips. It should be noted that most ofthe serum-coated tesi materials showed much greaterbiofilm activity than that of saliva-coated acrylic strips(approximately 5-200 times) since, in clinical terms,these materials were frequently used in the conditionwhere the materials should be covered by serum, such astissue conditioning to aid the traumatized oral mucosa.

In the previous study (Nikawa etal.. 1995c), weobserved two types of Candida blastospore colonizationon lining materials which were dependent upon theplasticizer used. The blastospore of this yeast colonizedinvasively into benzyl n-butyl phthaiate (BBP), butylphthalyl butyl glycorate (BPBG) and benzyl salicylate(BS) specimens which was similar to the fungal invasivecolonization reported by Marrie & Costerton (1981)and the cavitation reported by Ray & Payne (1988),whereas only loose attachment on the surface wasobserved with benzyl benzoate (BB) and dihutyl

© 1997 Blackwell Science Ltd, Journal of Oral Rehabilitation 24: 594-604

602 H. NIKAWA et al.

FS GC HC VG

Fig. 3. Thf ATP amciimts of unremovable fungi colonized on ihcdemure maierials. Asterisks indicate very little colonization (lessthan 30 cflls/sample) to refjister in ihe coliimn indicated.

phthalate (DBP). On the other hand, wt- observed thehyphal invasion into saliva-coated lining materials (GCSoft Liner; GC) (Nikawa etai, 1993) and strand-likehyphal adhesin which stick to (serum-coated) acrylics(Nikawa et al., I995d). These phenomena should becontributed tu the clinical difficulties of plaque controlfor these materials. Hence we examined ihe effect ofsoft Hners or protein pellicles on the firm attachment offungi. The pretreatment using ultrasonication effectivelyremoves the aggregated or associated biofilm fungi otherthan firmly attached fungi. The ATP amounts of firmlycolonized fungi are shown in Fig. 3, and the types offirm colonization are shown in Fig. 4a & b and Table 8.The profile of Fig. 3 was similar to thai of biofilmactivity. However, in the biofilm formation serutn-coated specimens showed much higher activities thanthat of saliva-coated ones in all cases. In contrast, theactivities of firmly colonized fungi on saliva-coated softliners tends to exhibit approximately the same valuesas compared with those of serum-coated specimens,except for Viscogel (VG). Furthermore, uncoated CoeComfort (CC), saliva-coated Fit Softer (FS), GC SoftLiner (GC). serum-coated Fit Softer (FS) and GC SoftLiner (GC) showed significantly greater biofilm activitythan that of saliva-coated acrylic strips. In addition,uncoated Coe Soft (CS). saliva-coated Coe Comfort(CC), serum-coated Coe Comfort (CC) and Coe Soft(CS) exhibited significantly higher biofilm activity thanthat of serum-coated acrylic strips. This result impliesthat the firm colonization on soft liners, includingblastospore cavitation or hyphal invasion, should beenhanced in the presence of denture pellicles, as com-pared with acrylic surface.

Fig. 4. (a) A typical view of blastospore cavitation. 'Spokewisewrinkles' caused by cavitation were observed around the yeasicell etnbeddcd into material surface, (b) A typical view of hypha!invasion into ihe material surface.

However, cavitation was not significantly affectedby protein coats, although the materials themselvesappeared to significantly affect this phenomena. Theyeasts embedded in the surface of Coe Comfort (CC),Fit Softer (FS). GC Soft Liner (GC), Hydrocast (HC) andhyphal invasion were observed with GC Soft Liner (GC)(Table 8). Neither cavitation nor hyphal invasion wasobserved with acrylic specimens. Coe Soft (CS) andViscogel (VG). Except for Viscogel (VG), this findingwas consistent with our previous work (Nikawa etal.,1995c) which demonstrated that benzyl benzoate (BB).benzyl butyl phtalate (BBP) and butyl phtaiyi butylphtaiate (BPBG) iacilitated the fungal cavitation intothe surface, since Coe Comfort (CC) is comprised ofbenzyl benzoate (BB); Hydrocast (HC) is comprised ofbenzyl hutyl phtaiate (BBP); and Fit Softer (FS) and GCSoft Liner (GC) contain a large amount of butyl phtalyl

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DENTURE PELLICLES, TISSUE CONDITIONERS AND C. ALBICANS 603

butyl phtalate (BPBG) (Jones etai.. 1988). except forViscogel (VG).

Ray & Payne (1988) revealed that Candida acidproteinase (CAP) was involved in the fungal cavitationinto niirine corneocytes. In contrast, pepstatin A treat-ment (5-6|ig/ml), which inhibits CAP production at amitochondoria level (Ray &• Payne, 1988), did not alterthe cavitation into these materials, suggesting that thedifferent mechanisms or factors may govern thisphenomenon. Further study Is needed to clarify this.

Finally, the results obtained here suggest that denturepellicles should facilitate the fungal plaque formationonto soft lining materials through several mechanisms,such as reduction of antifungal effects of soft liners,facilitation of biofilm formation, blastosporc cavitaiion,and hyphal invasion. In addition, the composition ofmaterials is also involved in the susceptibility to the

AcknowledgmentThis study was supported by Grant-in-Aid for ScientificResearch 0777 1848.

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Correspondence: Dr Hiroki Nikawa, Department of ProstheticDentistry, Hiroshima University School of Dentistry, 1 -2-3 KasumiMinami-ku, Hiroshima, 734 Japan.

© 1997 Biackwell Science Ltd, Journal of Oral Rehabilitation 24: 594-604