VoL 84 No. 1 July 1997

ENDODONTICS Editor: Richard E. Walton

Growth patterns of Candida albicans in relation to radicular dentin Bilge Hakan ~en, DDS, PhD, a Karnran E. Safavi, DMD, MEd, b and Larz S.W. Spfingberg, DDS, PhD, c Ege, Turkey and Farmington, Conn. EGE UNIVERSITY, TURKEY, AND UNIVERSITY OF CONNECTICUT

Candida albicans is the most common fungal pathogen isolated from the oral cavity. The role of this organism as an endodontic pathogen is poorly understood. Objectives. The aim of this study was to observe the interaction of C. albicans with root canal walls and the growth patterns of this microorganism in relation to radicular dentin. Study design. Fifteen root sections were infected with C. albicans grown in calf serum and incubated for various periods. The sec- tions were fixed in glutaraldehyde, split into two halves, and evaluated by scanning electron microscopy. Results. Blastospores and hyphal structures were observed on the root canal walls of all specimens. Filamentous hyphal form was dominant in 5-day specimens. Most of the hyphae and blastospores showed penetration into dentinal tubules. The body of germi- nating mother cells and hyphae demonstrated collapsed cell walls as a result of vacuole formation. Conclusions. With this invasive affinity to dentinal structures, C albicans may be considered a dentinophilic microorganism. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997;84:68-73)

Yeasts constitute a part of the normal microbial flora of the oral cavity. Their presence does not usually result in disease unless there are local or systemic predisposing factors such as low salivary flow, 1,a poor oral hygiene, 3 ill-fitting dentures, 4 diabetes, 5,6 malignancy, 7,8 cyto- toxic therapy 9 or HIV-infection. 1° Candida albicans is the most common yeast isolated from the oral cavity of either healthy or medically compromised adults. 1°-14 This microorganism can grow structurally by budding as unicellular form or in filamentous hyphal form, and shows interconversion in different conditions. 11,15

Even though there is a common belief that hyphal form is more invasive and pathogenic, the evidence for this assumption is equivocal. 16-19 It has been shown, however, that hyphae have contact-sensing ability, a°-a2

aAssistant Professor, Department of Restorative Dentistry and Endodontology, School of Dentis~y, Ege University, Turkey. bAssociate Professor, Department of Restorative Dentistry and Endodontology, School of Dental Medicine, University of Connecticut Health Center. cprofessor and Head, Department of Restorative Dentistry and Endodontology, School of Dental Medicine, University of Connecticut Health Center. Received for publication June 6, 1996; returned for revision Aug. 30, 1996; accepted for publication Feb. 15, 1997 Copyright © 1997 by Mosby-Year Book, Inc. 1079-2104/97/$5.00+ 7/15181282

Moreover, hydrophobicity of the hyphal form is higher than in the blastospores. 23 These properties, in turn, facilitate the penetration of hyphae into different sur- faces and tissues. Although C. albicans can adhere to tissue conditioners, 24,25 acrylic surfaces, 26 and saliva- coated hydroxyapatite, 27 it will not bind to the clean tooth surface itself but requires a petlicle of different proteins. 28,29

Oral yeasts have been isolated from dental plaque, 30,31 dental caries, 32,33 subgingival flora, 34-36

and root canals. 32,37,38 Yeasts in the canal space are

reported to represent a reservoir for the dissemination to the periphery via the bloodstream. 38,39 Furthermore,

t h e y cause periapical granulomas and systemic urticaria. 38 Therefore their presence in the oral cavity should be reevaluated from an endodontic point of view. The presence of yeast cells and hyphae in root canals has been demonstrated by electron microscopy in situ. 4°-42 However, the interaction of yeasts with canal dentin and their ecologic behavior in root canals are poorly understood.

The aim of this study was to visualize, by scanning electron microscopy, the topography of the root canal dentin wall infected with C. albicans, as well as to investigate the growth pattern of this microorganism in relation to radicular dentin.

68

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~en, Safavi, and Sp&ngberg 69

Fig. 1. Dense colony of blastospore and hyphal forms of C. aIbicans on root canal walls. The size of the blastospores is 2 to 5 gin. (Original magnification x1500.)

MATERIAL AND METHODS Eighteen extracted human mandibular premolar teeth

with a canal diameter of 0.75 _+ 0.05 mm were used in this study. The crowns at the cemento-enamel junction and the apical end of the roots were removed using a rotating diamond saw and tap water irrigation. This method prepared 5 mm long cylindrical root sections. The cementum was removed with fissure burs. The diameter of the root canals was standardized by enlarge- ment to a no. 6 Gates-Glidden bur. Distilled water was used as irrigation. The root samples were then trans- ferred to a flask containing 20 ml of 17% disodium eth- ylenediamine-tetraacetic acid (EDTA) solution. After 3 minutes, the roots were rinsed in tap water for 15 min- utes, and subsequently treated with 20 ml of 5.25% NaOC1 for 3 minutes. Finally, the root samples were washed under running tap water for 2 hours. At this stage, one specimen was examined by scanning electron microscopy (SEM) to confirm that the Smear layer had been removed. During this waiting time, the other spec- imens were held in distilled water.

The root sections were autoclaved individually at 150 ° C for 25 minutes in capped test tubes containing 1 ml of distilled water. After sterilization, the water was aspirated out of the tubes under aseptic conditions, and 2 ml of calf serum (Gemini Bioproducts, Calabasas, Calif.) was dispensed into each tube. The experimental tubes were incubated at 37 ° C for 3 days to ensure sterility. Two tubes with root sections in calf serum were incubated at 37 ° C and kept until the end of the experiments to serve as negative controls.

Stock cultures of clinically isolated C. albicans (cour- tesy of Department of Laboratory Medicine, University of Connecticut Health Center) were maintained in fluid

Fig. 2. The appearance of a mother cell (*) and its hyphal extensions penetrating into dentinal tubules. Note the col- lapsed cell walls (arrows) on the mother cell and hyphae. (Original magnification x4300.)

Fig. 3. A low magnification view of the canal wall showing a network of C. albicans hyphae. Most of these hyphae pene- trate into dentinal tubules. (Original magnification x360.)

thioglycolate medium (BBL, Cockeysville, Md.) at 4 ° C. A loopful of stock culture was grown aerobically to stationary growth phase in fresh fluid thioglycolate medium at 25 ° C for 24 hours. An inoculum (0.1 ml) was dispensed into each tube containing root sections and vortexed for 15 seconds, then incubated at 37 ° C for 5, 10, or 15 days.

At the end of each period, five specimens were washed in 0.1 M Sorensen's phosphate buffer solution (PBS) three times and fixed in 2.5% glutaraldehyde (ICN Biomedicals, Aurora, Ohio) in 0.1 M PBS, pH 7.4 at 4 ° C for 24 hours. After the fixation period, the root sections were again washed with 0.1 M PBS three times and then grooved longitudinally on the buccal and lin- gual surfaces with a carborundum disk. These grooves

70 Sen, Safavi, and Spdmgberg ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY July 1997

Fig. 4. The fractured side of root canal walls. Note the pene- tration of hyphae (arrows) into dentinal tubules. (Original magnification x 1800.)

Fig. 6. A budding yeast cell located in a dentinal tubule with- in a distance of 20 gM from the canal wall. (Original magni- fication x5000.)

Fig. 5. A colony of blastospores migrating into dentinal tubules. (Original magnification x3000.)

were used as guidance when the roots were split with a hammer and chisel. The samples were dehydrated through graded series of ethanol (20%, 40%, 60%, 80%, 90%, 95%, 10 minutes each, and 100%, 15 min- utes twice), critical point dried in CO 2 and sputter-coat- ed with 20 nM gold. Specimens were examined in a JEOL JSM-35CF scanning electron microscope (Tokyo, Japan) operated at an accelerating voltage of 15 kV.

During scanning examination of the root canal walls, the search was directed to the fungal colonies, their adaptation to the walls and growth patterns of C. albi- cans (bud or hyphal formation). Whereas oval struc- tures in the colonies were considered as yeast cells, elongation(s) from a yeast cell were recorded as hyphal growth. On the fractured dentin walls, the search was

directed to look for infiltration of fungal components from root canal walls into the dentinal tubules.

RESULTS Examination of the specimen by SEM after EDTA

and NaOC1 treatment revealed that the canal walls were free of debris and smear layer. The diameter of dentinal tubules ranged from 2 to 4.5 gm. Turbid growth was not observed in negative control root samples.

Five-day specimens The canal walls were covered with yeast blastospores

and hyphal structures forming dense colonies (Fig.I). Blastospores were spherical or oval in shape. As a result of budding stages, the size of these yeast cells was vari- able, ranging between 2 and 5 gm. Whereas larger cells (presumably mother cells) had a rough appearance, with several bud scars on the surface, smaller ones had a smooth surface morphology. Some mother cells were in the transition stage and were growing germ tubes. Hyphal structures growing in different directions were the dominant growth form in these specimens, and each branch extended as long as 100 gm. The body of ger- minating mother cells and pseudohyphae showed col- lapsed cell walls, but the single cells demonstrated no sign of collapsing (Fig.2). Most of the hyphal branches grew into dentinal tubules (Figs.3 and 4).

Ten- and 15-day specimens Single or budding yeast cells were the distinct pattern

of growth in these specimens. In some areas, most of the single yeast cells seemed to migrate into dentinal tubules and showed evidence of budding (Figs. 5 and 6). They attached to tubular dentinal walls with microfib-

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~en, Safavi, and Sp~ngberg 71

Fig. 7. A SEM micrograph of a new lateral budding colony on an older hypha that had penetrated into the dentinal tubule. (Original magnification x3300.)

Fig. 8. View from the fractured side of the root section. Another colony of blastospores showing lateral budding attached tightly to the canal wall. (Original magnification x2400.)

rils located on the cell surface. Hyphal extensions that had penetrated into dentinal tubules were observed to be leaving new lateral colonies of budding cells (Fig.7). These colonies seemed to attach tightly to the canal walls (Fig. 8). In a restricted area, where the remnants of soft tissue were not removed, blastospores were observed to attach membranous structures through microfibrils (Fig.9).

DISCUSSION This study has demonstrated that C. albicans can

grow on canal walls in different forms. It can also pen- etrate into dentinal tubules.

C. albicans is a budding yeast cell in its most com- mon growth form. Its blastospores can start growing hyphae, extending apically with a linear extension rate and branching exponentially as a network. 43,44 This fungus has been demonstrated to grow in a number of morphologic forms such as germ tubes, blastospores, pseudohyphae, true hyphae, and chlamydospor- es. 11,45,46 All growth patterns except chlamydospores show interconversion to each form of growth depending on the environmental conditions such as pH, tempera- ture, and nutritional source. 11,15 Thus, the term dimor- phic, often used in the literature, 15,23,47 is semantically inaccurate to explain the growth forms of C. albicans.

In our experiments, C. albicans grew in variable forms (blastospores, germ tubes, true hyphae, pseudo- hyphae) in a nutritionally stressed media. Although Gow and Gooday 43 have reported that candidal hyphae start budding laterally after 15 hours, the specimens of our study still contained mycelial growth in the 5-day cultures. Lateral budding was observed in 10- and 15- day incubation specimens. The reason for this alteration

Fig. 9. SEM micrograph of an area where the remnants of the pulpal tissue could not be removed. Yeast cells attached to the membranous tissue with microfibrils growing out from the cell bodies (arrows). (Original magnification x8000.)

in growth pattern in different periods is not known. C. aIbicans produces a factor (morphogenic autoregulato- ry substance, MARS) capable of regulating hyphal growth. 48,49 Furthermore, the presence of calcium ions has also been shown to have a critical role in the control of this morphogenesis. 5° It is well known that C. albi- cans can digest dentinal collagen. 51,52 This will result in release of minerals, including calcium, from the crystal phase after the removal of organic content of dentin. 53 Therefore, it is likely that the concentration of calcium ions in the culture medium is increased by time after exposure of root canal dentin to C. albicans and, in turn, the morphogenesis may be affected.

72 ~en~ Safavi, and Sp&ngberg

The presence of budding cells (even in the 15-day samples) suggests that yeasts were still actively repro- ducing, although the culture conditions had been limit- ed because of the overgrowth of yeasts in the small amount of medium. Furthermore, the serum as a culture medium has been reported to be a poor substrate for C. albicans and is normal ly only used for induction of mycelial growth. 11,15 This finding also indicates that C.

albicans can use dentin as a source of nutrition. Vacuolation of mother yeast cells, germ tubes, and

pseudohyphae have been previously observed by sever- al researchers. 44,45,54,55 Accord ing to Gow and

Gooday, 44 the germ tube formation is accomplished by

the extrusion of mother cell cytoplasmic contents into the developing germ tube, which is accompanied by extensive vacuolat ion within the .mother cell. Our results showed that, in addition to collapse of cell walls of mother cell, hyphal extensions also had a tendency to collapse. Apparen t col lapse of the cell walls occurs mainly as a result of preparation procedures for elec- tron microscopy; however, the same procedures did not cause any shrinkage in the young blastospores. Young yeast cells contain minimal vacuoles. 47,55 The presence

of vacuoles in pseudohyphal compartments shows that the cells are in an active linearly growing process, and vacuolat ion in i tsel f is a general feature of hyphal growth form. 44,56

Candida is generally known to live in close associa- tion with epithel ial surfaces. 57 As observed in this study, however, pure culture of C. albicans is likely to grow on dentinal surfaces even in the absence of oral tissue fluids rich in supporting media. They penetrate

into dentinal tubules by both growth patterns (i.e., blas- tospores and hyphae). Thus, it may be considered as a dentinophilic microorganism.

This ecologic behavior of C. albicans should be also discussed in relation to in vivo conditions. In the pres- ence of a typical mixed endodontic infection, Candida may interact with other microbes and microbial prod- ucts. Coaggregation (or coagglutination) reactions with bacteria can play a predominant role in colonization of mucosal and hard surfaces in the oral cavity by forming a complex biofilm. 58,59 In addition, cell surface proper-

ties of C. albicans are rapidly modified in response to different growth conditions or environmental changes, and phenotypic switching may occur in a particular eco- logic niche. 6°-6e Consequently, dentinophilic properties and pathogenicity of C. albicans may be increased with these possible mechanisms.

Case reports demonstrate the fungal invasion of denti- nal tubules by hyphal structures. 63,64 It has also been observed that root canal flora of infected teeth contains isolated hyphal structures and blastospores 4°,4e and the resorption lacunae on root surfaces harbor yeast cells. 65

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY July 1997

In addition, Nair et al.41 have demonstrated the pres-

ence of budding yeast cells in the apical part of

endodont ica l ly treated teeth with per iapical lesions. With this affinity to dentin because of its tubular nature, C. albicans can invade the root canals. The elimination of hyphae and the cells from the dentinal tubules may not be accomplished easily by routine endodontic pro-

cedures. Further studies are underway to study disinfec-

tion of canals.

This study was partially granted in scope of the NATO Science Fellowship Programme by the Scientific and Technical Research Council of Turkey (TUBITAK).

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Reprint requests: Kamran E. Safari, DMD, MEd Department of Restorative Dentistry & Endodontology School of Dental Medicine University of Connecticut Health Center Farmington, CT 06030-1715