CLINICAL REPORT

aAssistant PrbAssistant PrcRalph and J

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Biofilms in restorative dentistry: A clinical report

Vincent Torresyap, DDS,a Alireza Moshaverinia, DDS, MS, PhD,b and Winston W. Chee, DDSc

ABSTRACTThis clinical report describes the structure and characteristics of the biofilm formed under acemented restoration, confirming the need to develop new cementation protocols to disruptand minimize the formation of biofilm before cementing definitive restorations. (J Prosthet Dent2015;113:524-527)

One of the main contributingfactors to the progression ofdental caries and periodontaldiseases is the formation andpresence of dental bacterialbiofilms.1-3 Dental biofilms

can be found on hard dental structures (enamel anddentin), soft tissues, restorative dental materials, ortho-dontic appliances, and implants.3,4 Biofilm organismsexhibit an altered phenotype with respect to growth rate,gene transcription, and antimicrobial resistance. Biofilmsare composed of host constituents, cell-free enzymes,polysaccharides, and bacteria embedded in a matrix ofextracellular polymeric substances that they produce toconnect and communicate with each other. The processof biofilm development involves several progressivestages. Initially, mostly salivary proteins and cell-freeenzymes accumulate on the surface.3-5 Specifically, inrestorative dentistry, studies have confirmed that for-mation of biofilm on composite resin restorations de-grades and roughens the surface of the restorativematerial, leading to subsequent colonization of bacteriaat the restoration/tooth interface.6 Invasion of the inter-face can then begin,7 causing secondary caries and pulpalpathology.8 Furthermore, accumulation of biofilm onrestorative materials adjacent to the gingival tissue maylead to periodontal diseases.6-8

Biofilms are formed when free-floating microorgan-isms attach to a surface by van der Waals forces ofattraction. This attachment is later strengthened by celladhesion structures such as pilli. The microbes thatinitially colonize the surface facilitate the development ofthe biofilm by providing more diverse adhesion sites forother microorganisms.9-11 As the microbial population ofthe incipient biofilm increases, a polymeric matrix

ofessor, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.ofessor, Division of Biomedical Sciences, University of Southern California,ean Bleak Professor of Restorative Dentistry, Program Director, Advanced

develops, which not only serves as a scaffold for thedeveloping biofilm but also protects the microorganismswithin it. Biofilms form rapidly in the oral environment,beginning with the formation of the acquired pellicle.During the preparation of indirect restorations, toothstructure is exposed to the oral microbiota, resulting inthe adsorption of salivary biopolymers that form thepellicle.12,13 During restorative procedures, the initialexposure of the tooth surface to the oral environmentmay range from a few minutes to a few hours. This,together with leakage under the restorations, may allowthe development of the biofilm to a level where it couldresult in either cementation failure or recurrent cariesunder the definitive restoration. This fact is of particularimportance for interim fixed restorations that are usuallyfabricated as part of prosthodontic procedures. Theseinterim restorations may leak, leading to further accu-mulation of biofilms.14

This clinical report documents the structure andcharacteristics of the biofilm under a cemented restora-tion, with the aim of developing protocols to disrupt andeliminate these biofilms before cementation of definitiverestorations.

CLINICAL REPORT

A 62-year-old white man was referred to the AdvancedProsthodontics Department, Ostrow School of Dentistry,University of Southern California, for complete mouth

Los Angeles, Calif.Prosthodontics, University of Southern California, Los Angeles, Calif.

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Figure 1. A, Microscopic image (cross-sectional view) at buccal margin. B, Confocal laser scanning microscopy (CLSM) in combination with scanningelectron microscopy (SEM). C, CLSM alone demonstrating presence and formation of biofilm (red area).

Figure 2. A, Microscopic image (cross-sectional view) at lingual margin. B, Confocal laser scanning microscopy (CLSM) in combination with scanningelectron microscopy. C, CLSM alone demonstrating presence and formation of biofilm (red area).

Figure 3. A, Cross-sectional view of interface between restoration and tooth structure at occlusal surface of tooth. B, Confocal laser scanning micro-scopy (CLSM) in combination with scanning electron microscopy. C, CLSM alone confirming formation of biofilm in area with low accessibility tonutrients and oxygen.

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rehabilitation. The treatment plan involved implant-supported fixed restorations. The second right mandib-ular molar was given a poor prognosis because of severeattachment/bone loss and subsequent extraction wasplanned.

After 2 weeks, the interim restoration was removed,and the definitive American Dental Association (ADA)type III gold crown (Argdent30; Argen Corp) was eval-uated on the tooth, with an assessment of the occlusionand margin fit before cementation. The prepared toothwas cleaned and polished with fine pumice, washed,dried, and isolated with cotton rolls. The crown wasairborne-particle abraded, steam cleaned, and loaded

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with zinc phosphate cement (Dentsply Intl) seated overthe tooth with the aid of dynamic seating.15 Crownmargins were burnished, and the cement was allowed toset before removal of excess cement.

The tooth was chosen to investigate the structure andcharacteristics of the biofilm under a cemented restora-tion. The patient was notified, and the required consentforms were signed. This study was done with theapproval (UP-10-00267) of the University of SouthernCalifornia IRB.

After months of serving as a vertical stop, the toothwas extracted and evaluated with confocal laser scanningmicroscopy (CLSM) and scanning electron microscopy

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Figure 4. Further scanning electron microscopy analysis demonstratingpresence of 2 distinct zones within biofilm layer: inner calcified layer;outer less calcified and actively growing layer.

Figure 5. Scanning electron microscopy analysis of biofilm layer, atinterface between restoration and tooth structure (lingual margin):showing presence of bacteria with coccus-like morphologic structure atdifferent magnifications (Original magnification ×4, ×20, and ×50).

Figure 6. Scanning electron microscopy analysis of biofilm layer at interface between restoration and tooth structure (occlusal surface): showingpresence of bacteria with coccus-like morphologic structure at different magnifications. A, ×4 magnification. B, ×50 magnification. C, ×100magnification.

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(SEM). The extracted tooth was placed in 70% ethanolfor 48 hours. The tooth was sliced sagittally with a dia-mond disk (911H Hyperflex disc; Brasseler) under drip-ping sterile water. For the identification of bacteria withfluorescence in situ hybridization (FISH), the toothfragments were hybridized for 90 minutes at 46�C with a50-mL eubacterial probe EUB338 (Cy3) (Integrated DNATechnologies) at a final concentration of 5 ng mL−1 andwashed afterward at 46�C for 2×10 minutes with a2×500 mL washing buffer. For examination with theCLSM (LSM 5 PASCAL inverted; Carl Zeiss Micro-Imaging Inc) using ×10 and ×20 objective lenses, thetooth fragments were positioned face down in sterilewater in a slide chamber (Lab-Tek; Electron MicroscopySciences), and 3 different areas were analyzed.

After CLSM imaging, the tooth fragments were pre-pared for SEM. The specimens were fixed with 2%glutaraldehyde for 24 hours, dehydrated in a gradedethanol series, mounted with silver adhesive (ElectronMicroscopy Sciences), sputter coated, and examined withan SEM operating at 5 kV in the secondary electron mode

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(XL30 SFEG; FEI Co). Three different areas wereanalyzed.

DISCUSSION

The presence and morphologic structure of biofilm un-derneath the cemented crown 6 months after deliverywere analyzed with CLSM and SEM. FISH technique hasbeen successfully used to visually detect and identifybacteria. Here, FISH method was used to detect biofilmbacteria in the interface between the restoration and thetooth structure.

The results of CLSM analysis with immunofluores-cence staining confirmed the presence of biofilm notonly at the areas close to the margins of the restoration,which is a location with relatively easy access to nutri-ents and oxygen (Figs. 1, 2), but also at the occlusalinterface between the tooth and the restoration, whichhas less accessibility to nutrients and oxygen (Fig. 3).The results of SEM analysis agreed with CLSM andshowed the presence of a biofilm layer at different areas

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at the interface between the tooth and the cementedcrown. Interestingly, the SEM images confirmed thepresence of 2 distinct strata within the biofilm: the innerlayer, which is the calcified part of the biofilm, and theouter layer, which is less calcified and is the growingpart of the biofilm (Fig. 4). The existence of biofilms atdifferent areas of the teeth may help explain why res-torations fail either due to recurrent decay or failure ofcementation, because the biofilms can contaminatecementation protocols.

Streptococci represent the majority of supragingivalbacteria in a healthy oral cavity.16 In the current clinicalreport, SEM analysis showed the presence of bacteriawith coccus-like morphologic structure inside the biofilmlayer. Crucially, these bacteria were within the normalsize range, indicating that they were not under (perma-nent) starvation stress (Figs. 5, 6). This observationconfirmed that the bacterial biofilm under the crown hadsufficient access to the main required nutrients (glucoseand oxygen). However, no exuberant densely packedbiofilm of the type usually found in patients withadvanced periodontitis was observed in the patient. Thisfinding demonstrated certain limitations in either theavailable space or rich nutrient supply to the biofilm atthe interface of the tooth and the restoration.

These findings might not be generalized because, inthe current report, the margins were chosen only tocompare the biofilm in the marginal area with an areawithin the confines of the crown. Based on these ob-servations, we suggest the development of new andthorough cementation protocols to disrupt and eliminatebiofilms before the cementation of definitive restorations.

CONCLUSIONS

A biofilm layer underneath a cemented gold crown wasdemonstrated. This biofilm consisted of 2 distinct zonescontaining bacteria with a coccus-like morphologic

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structure and normal size. Based on the results of thecurrent report, the existence of biofilms at different areasof the teeth may help clarify why restorations fail due toeither recurrent caries or failure of cementation, becausebiofilms can contaminate cementation protocols.

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Corresponding author:Dr Alireza MoshaveriniaUniversity of Southern California2250 Alcazar Street - CSA 103Los Angeles, CA 90033Email: moshaver@usc.edu

Copyright © 2015 by the Editorial Council for The Journal of Prosthetic Dentistry.

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