colonization and penetration of denure soft lining materials by candida albicans

8/7/2019 colonization and penetration of denure soft lining materials by candida albicans

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Colonization and penetration of denture soft liningmaterials by Candida albicans

Khaled Bulad a, *, Rebecca L. Taylor b , Joanna Verran c , J. Fraser McCord d

a Unit of Prosthodontics, University Dental Hospital, Higher Cambridge Street, Manchester M15 6FH, UK b Department of Chemistry and Materials, Manchester Metropolitan University, Chester Street,Manchester M1 5GD, UK cDepartment of Biological Sciences, Manchester Metropolitan University, Chester Street,Manchester M1 5GD, UK d Unit of Prosthodontics, University Dental Hospital, Higher Cambridge Street, Manchester M15 6FH, UK

Received 23 May 2002; received in revised form 16 January 2003; accepted 5 March 2003

KEYWORDSDenture soft lining

materials; Candidaalbicans ; Adhesion;Penetration;Colonization; Yeast

Summary Objectives . Colonization ofdenture soft lining materials by Candida albicanscan result in clinical problems, and deterioration of the material. This study aimed to

monitor this interaction by comparing the short-term adhesion of C. albicans to sixdenture lining materials and to monitor any longer term penetration of material by theyeast.

Methods . Denture lining materials (Molloplast B, Flexor, Permaex, Luci-soft,Eversoft and U Gel hard C) were processed against glass slides or dental stone.Adhesion of yeast to surfaces was monitored after one hour incubation (37 8C) ofstandardized (2.8 106 cfu/ml) washed cell suspension with test materials. Attachedcells stained with acridine orange were counted microscopically. Penetration of yeastinto materials bonded onto acrylic after six weeks incubation (culture medium wasreplaced weekly) was observed through sections stained using acridine orange. Hyphaland yeast penetration was estimated (qualitatively and quantitatively, respectively)for three levels of the liner (subsurface, central section and adjacent to lining-acrylicjunction).

Results . None of the materials produced a zone of inhibition when compared withthe nystatin control. There was no signicant difference p . 0:5 in cell numbers onany of the smooth surfaces. Signicantly, p , 0:001 higher numbers of cells wereobserved on roughened surfaces. Both hyphal and yeast forms were observed whenpenetration was monitored. Penetration was greatest into U Gel hard C (no hyphaeobserved), but not at the acrylic-liner junction and least into Eversoft.

Signicance . Different denture lining materials exhibit different properties in termsof susceptibility to yeast penetration, although the initial attachment is comparable.Smoother surfaces retain fewer cells. The selection of appropriate materials for agiven function, and their fabrication may affect performance.Q 2003 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rightsreserved.

0109-5641/$ - see front matter Q 2003 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rights reserved.doi:10.1016/S0109-5641(03)00088-5

Dental Materials (2004) 20 , 167175

http://www.intl.elsevierhealth.com/journals/dema

*Corresponding author. Tel.: 44-161-275-6670.E-mail address: [email protected]
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Introduction

Denture stomatitis, more commonly known asdenture sore mouth, is a term used to describecertain pathologic changes (bright erythema) foundin the oral mucosa of denture-bearing tissues undercomplete or partial dentures in both jaws, but morefrequently in t he maxilla of, usually, elderlydenture wearers. 1 3 Prevalence has bee n rep ortedat 1167% in complete denture wearers. 1,46

The frequent association of Candida specieswith dent ure s tomatitis has been reported in theliterature. 3,79 The occurrence of candidal hyphaein smears is also more frequent in population s withdenture stomatitis than those without. 1,1012Continuous denture wearing appears to facilitatedenture stomatitis by increasing the local injuryand th e tim e of mucosal exposure to dentureplaque. 6,10,13

Resilient denture lining materials are used tolimit such injury. They reduce the traumatic effectthat a denture may have on patients with thinatrophic mucosa; or with normal mucosa but with aresorbed ridge, sharp alveolar ridge crest, deepanatomic undercuts, bony protuberances, bruxo-mania, or where the oral mucosa exhibits a reduced

tolerance to the load applied by the denture, and inobtura tors for acquired and congenital cleftpalate. 1418 Currently, there are two main typesof these materials; silicone elastomers and softacrylic compounds, represented in several commer-cial forms.

The adherence of Candida albicans to host cellsor polymers such as, denture acrylic resin and softlining materials is an essential and necessary rststep in successful colonization and the develop-ment of pathogenesis and infection, 1922 and therehave been several studies on the adhesion andcolonization of C. albicans to a range of dentalmaterials.

Signicantly, greater retention of C. albicanshas been described on soft lining materialscompared with acrylic, and roughe r surfacesalso enhanced adhesion/retention. 21,23 Suchstudies are carried out over short time periods,precluding the cells from multiplying, hence,allowing initial attachment interactions to bemonitored.

When surfaces and cells were incubated overlonger periods, it was noted that some soft liningmaterials and tissue conditioner s were able tosupport the growth of C. albicans . 2426

The surfaces were colonized by C. albicanshyphae m ore than acrylic resin denture basematerials, 27,28 presumably due to reduced hardness

and increased surface roughnessalthou gh, o ther

studies have reported conicting results,2932

andsome have reported that soft linin gs had aninhibitory effect on yeast growth. 8,33,34

Additional variables contribute in vivo. Salivaand serum pellicles reduced the antifungal effectsof some soft liners in vitro, and facilitate d ca ndidalcolonization of denture lining materials. 3538

Clinically, it has been observed that somepatients wearing dentures with silicone liningmaterial present with small, whitish elevations offungal growth on the surface of this material. Onmicroscopic examina tion, these were found to beprimarily C. albicans . 26,39

Clearly, many soft lining materials present aconvenient substratum for microbial colonization.However, very few studies have been reported onthe penetration of C. albicans into the soft liningmaterials.

Gibbons 39 reported that parafn sections ofthe Silastic 390 Soft liner showed surface growthof organisms which penetrated into the siliconeand also prod uced elevations on the surface.Masella et al. 31 reported that except for C.glabrata , all the strains tested ( C. albicans , C.tropicalis , C. parapsilosis ) invaded the Silastic390 soft liner in vitro to various degrees. Thestrain of C. tropicalis used demons tr ated themost aggressive invasion. Burns et al. 24 reportedthat C. albicans was able to penetrate into thecentre porti on of each tested soft liner. Taylorand Johnson 40 have demonstrated penetration ofsilicone materials in vitro. Conversely, Tanget al. 32 reported that fungal or bacterial invasionof polyurethane elastomer specimens was notevident.

The aim of this study was to utilise a range ofmethods to investigate in vitro (1) the retention ofC. albicans on selected denture lining materials, (2)the inhibition effect of the materials on C. albicansand (3) the penetration of the yeast into thematerials.

Materials and methods

Culture

C. albicans GDH 234641 was stored on beads (Prolab,Neston, Wirral, UK) at 2 80 8C. Prior to eachexperiment, one bead was placed into 10 ml

Sabouraud broth (Oxoid; Ltd, Hampshire, UK), andincubated 18 h at 37 8C, after which the broth wasplated onto Sabouraud agar (Oxoid) for puritychecks and subsequent experiments.

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Materials

The denture lining materials used in this study, themanufacturers and composition are shown inTable 1 .

U Gel hard C is a new type of hard relinematerial provided in direct application cartridges.Eversoft is supplied as a powder, liquid and a sealerliquid.

Molloplast B, Permaex and Flexor were suppliedas a singlepaste andadhesive (bonding liquid). Luci-soft was supplied in sheet form and bonding liquid.

Trevalon (Dentsply Limited, Weybridge, Surrey,England) was used as a heat curing acrylic resinpolymethylmethacrylate, for fabricating discs ontowhich the linings were processed.

Methods

Sample preparationPreparation of lining discsDiscs of pink wax 10 mm diameter and 1.5 mm thickwere punched from a sheet using a cork borer.Dental stone (BPB Formula, Newark Works, Bow-bidge Lane, Newark, Nottinghamshire, UK) wasmixed with water according to manufacturerinstructions. The mixture was poured into theshallow part of a dental ask, and eight wax discswere placed on the surface of the stone, which wasthen allowed to set. After setting, the stone waslubricated using petroleum gel. The upper part ofthe ask was placed in position, and dental stonewas poured into it, over the wax discs using avacuum mixed stone technique. Flasks were heatedfor 10 min, and the wax was removed using a hotwater spray. Flasks were cooled, and then the stonewas lubricated using a CO-MO sealant (John Winterand Co. Ltd, Halifax, UK).

The denture linings were applied into the voidsleft by the wax. Wet polymerization technique wasused for the heat curing liners. Eversoft was poly-merized by heating the ask for 15 min in 100 8Cwater. Ugel hard C lining material was polymerizedat room temperature. After polymerization, theasks were opened, denture lining discs removed,trimmed andkept in polyethylenecontainers in a dryenvironment until required (one week).Preparation of smooth lining discsIn order to produce test surfaces of comparablesmoothness, discs were prepared against glass. Inthis case, a glass microscope slide was pressed ontothe dental stone mixture in the shallow part of aask. After the stone had set, wax discs wereplaced on top of the glass slide surface. The upperpart of the ask was placed in position and thedental stone was poured over the wax discs asabove. The resultant voids, lled by soft liners,would have one glass-smooth surface, and onedental stone-rough surface.Preparation of acrylic resin discs for bonding to softlining materialDisc shaped voids were prepared as describedabove. Trevalon powder and the acrylic liquidwere mixed and the dough applied to the voids inthe asks. The polymerization was carried out usingthe wet temperature heating technique. Afterpolymerization, acrylic discs were removed asdescribed previously.

Discs of pink wax were anchored to each acrylicdisc. The wax-acrylic discs were asked asdescribed for the wax discs. This procedure wouldproduce acrylic discs surmounted by a void whichwould be lled by the soft lining materials.

Soft lining materials were bonded to the acrylicresin bases via an adhesive prior to the polymeris-ation. In this case, the surface of the liner material

Table 1 Denture lining materials used in the study.

Material Type Manufacturer Composition

Molloplast-B Heat curing silicone Detax, GmbH & Co.Germany

Polydimethylsiloxan (polymer) and benzyl peroxide(initiator)

Permaex Heat curing silicone Kohler, Medizintechnik,Danningen, Germany

The same as above

Flexor Heat curing silicone Schutz-Dental GmbH,Rosbach, Germany

The same as above

Luci-soft Heat curing silicone Densply Trubyte, DentsplyInternational Inc. UK

The same as above

Ever soft Chair-side curing soft liner Austenal Inc. USA Polyethylmethacylate (powder) and dibutyl phthalate,ethyl acetate, ethyl alcohol (liquid) and methyl ethylketone (sealer liquid)

U-Gel hard C Cold curing acrylic hard liner Voco GmbH, Germany Polyethylmethacylate (polymer), benzoyl peroxide(initiator) methylmethacrylate (monomer)dibutylphthalate (plasticizer)

Colonization and penetration of denture soft lining materials by Candida albicans 169


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would present the topography of dental stone

rather than of glass.Inhibition of Candida albicans by denturelining materialsAn overnight culture of C. albicans GDH 2346 wasdiluted 1:1000 in sterile phosphate buffered saline(PBS; Oxoid), which was then used to inoculateDiagnostic Sensitivity Testing agar (DST; Oxoid).Excess culture was pipetted from the plate anddiscarded. The plates were dried, and two testpieces of each denture lining disc were placed ontothe agar plate. Nystatin soaked (100 units) lterpaper discs were used as controls. For each lining

material, two agar plates were used and theexperiment was repeated twice. After incubationat 37 8C for 24 h, the zone of inhibition formedaround the samples was measured in four places,and the mean determined.

Retention of Candida albicans to denturelining materialsA few (46) colonies of C. albicans from aSabourauds plate were inoculated into 100 mlarticial saliva and incubated at 37 8C for 24 h.Cells were collected by centrifugation, harvested,washed three times in sterile PBS, and resuspendedin PBS to an optical density of 0.5 at 540 nm,corresponding to a cell concentration of2.8 106 cfu/ml. On microscopic examination, allcells were in the blastospore phase.

Five test pieces of each lining material were usedwhich were sterilized by autoclave. Each test piecewas placed in a sterile plastic 5 ml bottle in avertical position. Two millilitre of the standardizedcell suspension were added to each bottle, and theapparatus was incubated statically (without agita-tion) at 37 8C for one hour. Test pieces were thenremoved from the suspension, rinsed by dippinggently three times in 20 ml sterile PBS to removeloosely attached cells, and left to air dry lyinghorizontally.

Attached cells remaining on the discs werexed by immersion in 100% methanol for oneminute, then stained by 2 min immersion in 0.03%acridine orange in distilled water, followed bywashing in distilled water and air drying. Carewas taken to ensure that the smooth surface ofthe lining material was processed, rather thanthe side which had been asked against dentalstone.

However,a preliminary studywasperformed with

two of the materials (U Gel hardC and Molloplast B)which were processed against dental stone.Test samples were viewed using uorescence

microscopy at 1000 magnication under oil

immersion. The number of adherent cells in 20

random elds was counted for each test piece andthe mean number of adherent cells per eld wasobtained.

Colonization and penetration of denture liningmaterials by Candida albicansTest acrylic/liner units were placed into 25 ml glassbottles containing 10 ml articial saliva and auto-claved. Each of the bottles was inoculated with0.1 ml of an 18 h culture of C. albicans in articialsaliva and incubated in an orbital incubator at 37 8Cfor 6 weeks. Test substratum units (acrylic/liner)were transferred aseptically to fresh sterile arti-

cial saliva on a weekly basis. Eight replicates wereused for each material type.After 6 weeks incubation, test pieces were

removed from the bottles and placed in 4%glutaraldehyde in PBS for at least 2 h to x thecells. Substrata plus attached cells were thendehydrated in alcohol (30,50,70,90,100%) for10 min for each concentration in universals.

Air dried test pieces were sectioned on an ExactBand using 0.1 mm diamond band under water ow.Individual test pieces were held horizontally with aclamp. Four sections were cut across each piece toprovide ve replicate samples, with the sawperpendicular to the test piece, approaching fromthe short end of the cross-section.

Sections were immersed in 0.03% acridine orangefor one minute, rinsed in distilled water, then airdried horizontally with the cut section uppermost.Replicate sections were attached to a microscopeslide using petroleum gel in preparation for micro-scopic examination.

Sections were viewed via epiuorescencemicroscopy, 1000, and oil immersion. Theobjective was focused on one end of the section,and racked down so that the entire length of thesample was examined. This procedure wasrepeated three times, once (level one) with theobjective focused on the outer layer of thesample (the upper surface of the lining material),once in the middle of the lining material (leveltwo), and once adjacent to the acrylic/dentureliner junction (level three).

The penetration of yeast into the denture liningmaterials was measured by counting the number ofblastospores visible within each microscopic eld,and by estimating the amount of hyphae present ineach eld, as described below:

Class I: little hyphal presence, whereby mycelial

forms cover up to a quarter of the microscopic eld.Class II: moderate amount of hyphal presence,where half of the eld of view was covered bymycelial forms.

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Class III: large amount of hyphal presence,

whereby most of the microscopic eld was coveredby mycelial forms.The number of yeast cells counted was totalled

for each level; the median amount of hyphalpenetration was also presented for each level.

The reliability of the assessment method wastested by recounting the number of blastospores in20 randomly selected sections. The differencebetween the two readings was statistically accep-table (95% C I) with an error of 1020 cells persection.

The extent of invasion of yeast at the acrylic/denture liner junction at the edges of the samples

was observed inorder toassesswhether this junctionpresented a weakness facilitating penetration.

Results

Inhibition of C. albicans growth by softlining materials

None of the materials exhibited a zone of inhibitionwhen compared with the positive control, whichshowed a well-dened zone of inhibition (3 mmdiameter) observed around the nystatin disc.

Retention of C. albicans to soft liningmaterials

Yeast cells were observed on the surfaces as singlecells or clumps of cells. The majority of cells werein the blastospore phase, but occasional hyphalforms were observed ( Fig. 1).

The average number of cells observed per eldwas calculated (20 elds per sample, 5 replicatesper lining material).

Mean and Standard deviation of C. albicans cellsadhered to the smooth surfaces (processed againstglass slides) are shown in Fig. 2.

Mean and Standard deviation of C. albicans cellsadhered to the rough surfaces (processed againstdental stone) and to the smooth surfaces (pro-cessed against glass slides) for two materials arepresented in Table 2 .

There was no signicant difference (KruskalWallis test; p 0:92) between the numbers of cellsattached to the different materials (processedagainst glass slides). The data were non-Normallydistributed (KolmogorovSmirmov test), primarilydue to some extreme values.

There was a signicant difference between themean number of adherent C. albicans cells for therough and smooth surfaces of denture liningmaterials with more adhesion on rough surfaces(Paired-Sample T Test; p , 0:001).

Also more cells were attached to Molloplast-B(silicone material) than to U-Gel hard C (acrylic

material) (Paired-Sample T Test; p,

0:001).Ra values of U Gel hard C and Molloplast B

samples processed against glass slides and againstdental stone are shown in Table 3 .

Figure 1 Blastospores and hyphal forms of Candidaalbicans retained on the smooth surface of Molloplast Bsoft liner.

Figure 2 Mean of Candida albicans cells adhered to thesmooth surfaces (processed against glass slides) and tothe rough surfaces (processed against dental stone).

Table 2 The cell number of Candida albicans retained tosmooth surfaces (processed against glass slides) and to roughsurfaces (processed against dental stone).

Materials Mean (Standarddeviation) ofC. albicans cellsretained to smoothsurfaces

Mean (Standard deviation) of C. albicanscells retained to roughsurfaces

U Gel hard C 9.95 (3.8) 26.36 (1.31)Molloplast B 10.34 (4.7) 36.59 (2.66)



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Colonization and penetration of lining

materials by C. albicans

When materials were removed from the salivaculture, colonization was apparent withthe naked eye. When sections were examinedmicroscopically, cells were observed on the outersurface of the material, and also throughout thelevels, indicating that penetration of the lining

materials had occurred. If cells were absent fromthe surface of samples, no penetration of thesections was observed. This also serves to indicatethat the washing and sectioning procedure wasefcient, in that cells were not dragged from thesurface to be deposited onto the section faces, butaccessed the material solely by penetration.

Both blastospores and hyphal forms were appar-ent ( Fig. 3). The total number of blastospores

counted at each level decreased as the levelincreased i.e. decreased from the surface to theinner levels ( Fig. 4). In a few sections, the numberof cells observed was too high to count, thus thenumber 2000 was used as a maximum count.

Data were non-Normally distributed for somematerials, due to extreme values. Pairwise compa-rison of materials using the MannWhitney U-Testindicated that there was a signicant differencebetween numbers observed in the differentmaterials, specically the highest number of cells

Table 3 Mean surface roughness (Ra) values n 10 of

denture lining materials.

Surface processedagainst glassslides ( m m)

Surface processedagainst dentalstone ( m m)

Molloplast B 1.96 (0.26) 3.60 (0.19)U Gel hard C 1.86 (0.23) 3.67 (0.22)Ever soft 1.82 (0.21)Luci-soft 1.91 (0.25)Flexor 1.78 (0.23)Permaex 1.88 (0.27)

Figure 3 (A) Candida albicans (blastospores) pene-trated through U Gel Hard C denture liner; (B) Candidaalbicans (hyphal forms) penetrated through Flexordenture liner.

Figure 4 The number of blastospores decreased fromthe surface (left) to the inner levels (right) of the U Gelhard C denture liners samples.

Figure 5 Mean number of Candida albicans blastosporespenetrated through denture lining materials.

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occurred in U Gel hard C, and the lowest inEversoft p 0:03 (Fig. 5).

U Gel hard C also experienced the highestdegree of the depth penetration, and Eversoft thelowest, indicated by the counts of blastospores atlevel III p 0:001 (Fig. 6).

The amount of hyphal penetration alsodecreased from the surface to the inner levels,although the estimates of hyphal presence aresomewhat qualitative. Flexor experienced thehighest degree of hyphal penetration (Class III),with the lowest observed for Eversoft and Luci-soft. Penetration for Molloplast B and Permaexwas comparable. No hyphae were observed in UGel hard C. The median of the amount of hyphaepenetrated through the materials is presented inTable 4 .

In some sections, some penetration wasobserved at the junction point where the acrylicand lining material were attached. These data

were not included in the penetration analysis, butprovided some indication as to the strength ofattachment for the acrylic-lining bonding. The bestbonding was with U Gel hard C liner where nopenetration was observed at the junction point.

Discussion

Of the denture lining materials tested in thisstudy, only Molloplast B has been studied pre-viously for any inhibitory effect on the growth ofC. albicans , with differing success. The resultsof the current study showed that any effect ofMolloplast-B on the growth of C. albic ans wasweak and not reproducible. Williamson 42 foundthat Molloplast-B had an inhibitory effect on C.albicans in saline medium. Also, Makila andHopsu-Havu30 reported that uncured Molloplast-Bmaterial caused a denite inhibition of Candidagrowth in vitro, while the cured material indi-cated no growth inhibition. This may be explainedby the fact that the active constituent, metha-cryloloxypropyltrimethoxysilane, would becom einactive during the cross-linking curing process 34

if, however, a small excess remained, the curedmaterial might s til l exhibit minimal inhibition ofgrowth. Wright 34 reported that Molloplast-Bdemonstrated such a small degree of inhibitionthat th e zone area was not measurable, and Burnset al. 24 also found that Molloplast-B had noinhibitory inuence on growth of C. albicans .Clearly, the method of preparation of the

material and its storage, and the experimentalprotocol appear to have some effect on ndings.The aim of the current study was to investigate

the initial attachment of C. albicans to severaltypes of denture lining materials. It was importantto determine in vitro whether the difference intypes of denture lining materials and the roughnessof the surface affected the adherence of C. albicansto these materials.

In this study, a simple in vitro approach was usedto compare the adherence of one strain of C.albicans (GDH 2346) to several commerciallyavailable denture lining materials.

No signicant difference in the adhesion of C.albicans between denture lining materials proce-ssed against glass slides was observed in our study.

C. albicans observed on the surfaces werepredominantly blastospores since the incubation ofthe cells was for one hour only. However, afew hyphae were seen. The results of thisstudy conrm earlier studies on short-term attach-ment in a non-growth environment such as thatdescribed in our study, which do not reveal yeastgrowth or surface colonization, but pse udo hyphaeand hyphae have been observed. 21,43,44

Surface roughness is a signicant factor in theattachme nt and retention of microorganisms onsurfaces, 43 with an increase in roughness causingincreased retention of cells. The effect has been

Table 4 Median of the amount of hyphae penetratedthrough the materials.

Materials Median

Flexor IIIPermaex IIMolloplast B IILuci-soft IEversoft IU Gel hard C 0

Figure 6 Mean number of Candida albicans blastosporespenetrated through denture lining materials at levelthree.



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reported previously 21,23,43,45 which were in agree-

ment with the results of our study. There are clearimplications for these ndings in terms of surfacecleanability, denture hygiene and materialsfabrication.

Penetration of C. albicans within denture liningmaterials was observed through sections obtainedfrom the test pieces. The method used in thisstudy is novel; 40,46 few studies have demonstratedpenetration in the literature. The lowest value ofpenetration of C. albicans (blastospores andhyphae) was observed using Eversoft. This couldbe explained by the presence of free ethyl alcoholwithin the cured material, 47 which might be

inhibitory to the organisms during the long termexposure (6 weeks experimental duration).Penetration through Flexor, Luci-soft, Perma-

ex, and Molloplast B was relatively comparablewhich may be explained by the fact that thesedenture lining materials are similar in compositionin that they are all heat curing silicones. The mostsignicant penetration of Candida blastospores wasobserved using U Gel hard C, a hard denture liner.This might be attributed to the presence ofporosities inside the matrix of the cured materialwhich facilitate the penetration of blastospores,even perhaps by capillarity, but precludeddevelopment of hyphae due to the hardness of thematerial. Considerable hyphal formation was seenthroughout the other soft denture lining materials.

The penetration of yeasts at the acrylic-linerjunction point was observed in a few samples. Thiscan be attributed to the failure of bonding betweenthe liner and the acrylic base material, which maycontribute to deterioration of the prosthesis liningand function. In U-Gel hard C test pieces nopenetration was observed at the acrylic-linerjunction point since the liner and the acrylicmaterial has the same composition and the bondingis perfect (chemical bonding).

Findings from these in vitro studies provide someinteresting considerations for clinical work. Smoothliners are less rapidly colonized, but over anextended period of wear of the liner, this mightbe an unnecessary consideration. If the patient isalready a Candida carrier, then different linersmight experience deterioration at different rates. Ifthe patient is not a signicant carrier, then it wouldbe interesting to determine whether the insertionof susceptible liners might increase yeast carriage/detection and the susceptibility to infection.Furthermore, the role of other members of the

oral ora in the deterioration of the materialsrequires investigation.A clinical study is currently underway to clarify

some of these issues.

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