Bioﬁlms in restorative dentistry: A clinical report Vincent Torresyap, DDS,a Alireza Moshaverinia, DDS, MS, PhD,b and Winston W. Chee, DDSc One of the main contributing ABSTRACT factors to the progression of This clinical report describes the structure and characteristics of the bioﬁlm formed under a dental caries and periodontal cemented restoration, conﬁrming the need to develop new cementation protocols to disrupt diseases is the formation and and minimize the formation of bioﬁlm before cementing deﬁnitive restorations. (J Prosthet Dent presence of dental bacterial 2015;-:---) bioﬁlms.1-3 Dental bioﬁlms develops, which not only serves as a scaffold for the can be found on hard dental structures (enamel and developing bioﬁlm but also protects the microorganisms dentin), soft tissues, restorative dental materials, orthowithin it. Bioﬁlms form rapidly in the oral environment, dontic appliances, and implants.3,4 Bioﬁlm organisms beginning with the formation of the acquired pellicle. exhibit an altered phenotype with respect to growth rate, During the preparation of indirect restorations, tooth gene transcription, and antimicrobial resistance. Bioﬁlms structure is exposed to the oral microbiota, resulting in are composed of host constituents, cell-free enzymes, the adsorption of salivary biopolymers that form the polysaccharides, and bacteria embedded in a matrix of pellicle.12,13 During restorative procedures, the initial extracellular polymeric substances that they produce to exposure of the tooth surface to the oral environment connect and communicate with each other. The process may range from a few minutes to a few hours. This, of bioﬁlm development involves several progressive together with leakage under the restorations, may allow stages. Initially, mostly salivary proteins and cell-free the development of the bioﬁlm to a level where it could enzymes accumulate on the surface.3-5 Speciﬁcally, in result in either cementation failure or recurrent caries restorative dentistry, studies have conﬁrmed that forunder the deﬁnitive restoration. This fact is of particular mation of bioﬁlm on composite resin restorations deimportance for interim ﬁxed restorations that are usually grades and roughens the surface of the restorative fabricated as part of prosthodontic procedures. These material, leading to subsequent colonization of bacteria interim restorations may leak, leading to further accuat the restoration/tooth interface.6 Invasion of the intermulation of bioﬁlms.14 face can then begin,7 causing secondary caries and pulpal 8 This clinical report documents the structure and pathology. Furthermore, accumulation of bioﬁlm on characteristics of the bioﬁlm under a cemented restorarestorative materials adjacent to the gingival tissue may tion, with the aim of developing protocols to disrupt and lead to periodontal diseases.6-8 eliminate these bioﬁlms before cementation of deﬁnitive Bioﬁlms are formed when free-ﬂoating microorganrestorations. isms attach to a surface by van der Waals forces of attraction. This attachment is later strengthened by cell adhesion structures such as pilli. The microbes that MATERIALS AND METHODS initially colonize the surface facilitate the development of A 62-year-old white man was referred to the Advanced the bioﬁlm by providing more diverse adhesion sites for 9-11 Prosthodontics Department, Ostrow School of Dentistry, other microorganisms. As the microbial population of University of Southern California, for complete mouth the incipient bioﬁlm increases, a polymeric matrix a
Assistant Professor, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. Assistant Professor, Division of Biomedical Sciences, University of Southern California, Los Angeles, Calif. c Ralph and Jean Bleak Professor of Restorative Dentistry, Program Director, Advanced Prosthodontics, University of Southern California, Los Angeles, Calif. b
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Figure 1. A, Microscopic image (cross-sectional view) at buccal margin. B, Confocal laser scanning microscopy (CLSM) in combination with scanning electron 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 scanning electron 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 microscopy (CLSM) in combination with scanning electron microscopy. C, CLSM alone conﬁrming formation of bioﬁlm in area with low accessibility to nutrients and oxygen.
rehabilitation. The treatment plan involved implantsupported ﬁxed restorations. The second right mandibular molar was given a poor prognosis because of severe attachment/bone loss and subsequent extraction was planned. After 2 weeks, the interim restoration was removed, and the deﬁnitive American Dental Association (ADA) type III gold crown (Argdent30; Argen Corp) was evaluated on the tooth, with an assessment of the occlusion and margin ﬁt before cementation. The prepared tooth was cleaned and polished with ﬁne pumice, washed, dried, and isolated with cotton rolls. The crown was airborne-particle abraded, steam cleaned, and loaded THE JOURNAL OF PROSTHETIC DENTISTRY
with zinc phosphate cement (Dentsply Intl) seated over the tooth with the aid of dynamic seating.15 Crown margins were burnished, and the cement was allowed to set before removal of excess cement. The tooth was chosen to investigate the structure and characteristics of the bioﬁlm under a cemented restoration. The patient was notiﬁed, and the required consent forms were signed. This study was done with the approval (UP-10-00267) of the University of Southern California IRB. After months of serving as a vertical stop, the tooth was extracted and evaluated with confocal laser scanning microscopy (CLSM) and scanning electron microscopy Torresyap et al
Figure 4. Further scanning electron microscopy analysis demonstrating presence 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, at interface between restoration and tooth structure (lingual margin): showing presence of bacteria with coccus-like morphologic structure at different 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): showing presence of bacteria with coccus-like morphologic structure at different magniﬁcations. A, ×4 magniﬁcation. B, ×50 magniﬁcation. C, ×100 magniﬁcation.
(SEM). The extracted tooth was placed in 70% ethanol for 48 hours. The tooth was sliced sagittally with a diamond disk (911H Hyperﬂex disc; Brasseler) under dripping sterile water. For the identiﬁcation of bacteria with ﬂuorescence in situ hybridization (FISH), the tooth fragments were hybridized for 90 minutes at 46 C with a 50-mL eubacterial probe EUB338 (Cy3) (Integrated DNA Technologies) at a ﬁnal concentration of 5 ng mL−1 and washed afterward at 46 C for 2×10 minutes with a 2×500 mL washing buffer. For examination with the CLSM (LSM 5 PASCAL inverted; Carl Zeiss MicroImaging Inc) using ×10 and ×20 objective lenses, the tooth fragments were positioned face down in sterile water in a slide chamber (Lab-Tek; Electron Microscopy Sciences), and 3 different areas were analyzed. After CLSM imaging, the tooth fragments were prepared for SEM. The specimens were ﬁxed with 2% glutaraldehyde for 24 hours, dehydrated in a graded ethanol series, mounted with silver adhesive (Electron Microscopy Sciences), sputter coated, and examined with an SEM operating at 5 kV in the secondary electron mode Torresyap et al
(XL30 SFEG; FEI Co). Three different areas were analyzed. DISCUSSION The presence and morphologic structure of bioﬁlm underneath the cemented crown 6 months after delivery were analyzed with CLSM and SEM. FISH technique has been successfully used to visually detect and identify bacteria. Here, FISH method was used to detect bioﬁlm bacteria in the interface between the restoration and the tooth structure. The results of CLSM analysis with immunoﬂuorescence staining conﬁrmed the presence of bioﬁlm not only at the areas close to the margins of the restoration, which is a location with relatively easy access to nutrients and oxygen (Figs. 1, 2), but also at the occlusal interface between the tooth and the restoration, which has less accessibility to nutrients and oxygen (Fig. 3). The results of SEM analysis agreed with CLSM and showed the presence of a bioﬁlm layer at different areas THE JOURNAL OF PROSTHETIC DENTISTRY
at the interface between the tooth and the cemented crown. Interestingly, the SEM images conﬁrmed the presence of 2 distinct strata within the bioﬁlm: the inner layer, which is the calciﬁed part of the bioﬁlm, and the outer layer, which is less calciﬁed and is the growing part of the bioﬁlm (Fig. 4). The existence of bioﬁlms at different areas of the teeth may help explain why restorations fail either due to recurrent decay or failure of cementation, because the bioﬁlms can contaminate cementation protocols. Streptococci represent the majority of supragingival bacteria in a healthy oral cavity.16 In the current clinical report, SEM analysis showed the presence of bacteria with coccus-like morphologic structure inside the bioﬁlm layer. Crucially, these bacteria were within the normal size range, indicating that they were not under (permanent) starvation stress (Figs. 5, 6). This observation conﬁrmed that the bacterial bioﬁlm under the crown had sufﬁcient access to the main required nutrients (glucose and oxygen). However, no exuberant densely packed bioﬁlm of the type usually found in patients with advanced periodontitis was observed in the patient. This ﬁnding demonstrated certain limitations in either the available space or rich nutrient supply to the bioﬁlm at the interface of the tooth and the restoration. These ﬁndings might not be generalized because, in the current report, the margins were chosen only to compare the bioﬁlm in the marginal area with an area within the conﬁnes of the crown. Based on these observations, we suggest the development of new and thorough cementation protocols to disrupt and eliminate bioﬁlms before the cementation of deﬁnitive restorations. CONCLUSIONS A bioﬁlm layer underneath a cemented gold crown was demonstrated. This bioﬁlm consisted of 2 distinct zones containing bacteria with a coccus-like morphologic
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structure and normal size. Based on the results of the current report, the existence of bioﬁlms at different areas of the teeth may help clarify why restorations fail due to either recurrent caries or failure of cementation, because bioﬁlms can contaminate cementation protocols. REFERENCES 1. Costerton JW, Stewart PS, Greenberg EP. Bacterial bioﬁlms: a common cause of persistent infections. Science 1999;284:1318-22. 2. Costerton JW, Cheng K-J, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marrie TJ. Bacterial bioﬁlms in nature and disease. Annu Rev Microbiol 1987;41:435-64. 3. Socransky, Haffajee Sigmund S, Anne D. Dental bioﬁlms: difﬁcult therapeutic targets. Periodontology 2000;28:12-55. 4. Adamczyk E, Spiechowicz E. Plaque accumulation on crowns made of various materials. Int J Prosthodont 1990;3:285-91. 5. Collins CJ, Bryant RW, Hodge KL. A clinical evaluation of posterior composite resin restorations: 8-year ﬁndings. J Dent 1998;26:311-7. 6. Beyth N, Bahir R, Matalon S, Domb AJ, Weiss EI. Streptococcus mutans bioﬁlm changes surface-topography of resin composites. Dent Mater 2008;24:732-6. 7. Litonjua LA, Cabanilla LL, Abbott LJ. Plaque formation and marginal gingivitis associated with restorative materials. Compendium 2012;33:1-6. 8. Hooshmand T, Mohajerfar M, Keshvad A, Motahhary P. Microleakage and marginal gap of adhesive cements for noble alloy full cast crowns. Oper Dent 2011;36:258-65. 9. Haffajee AD, Socransky SS. Introduction to microbial aspects of periodontal bioﬁlm communities, develop ment and treatment. Periodontol 2000 2006;42:7-12. 10. Donlan RM, Costerton JW. Bioﬁlms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002;15:167-93. 11. Busscher HJ, Rinastiti M, Siswomihardjo W, van der Mei HC. Bioﬁlm formation on dental restorative and implant materials. J Dent Res 2010;89:657-65. 12. Tanner J, Robinson C, Sderling E, Vallittu P. Early plaque formation on ﬁbrereinforced composites in vivo. Clin Oral Invest 2005;9:154-60. 13. Steinberg D, Eyal S. Early formation of Streptococcus sobrinus bioﬁlm on various dental restorative materials. J Dent 2002;30:47-51. 14. Suljak JP, Reid G, Wood SM, McConell RJ, van der Mei HC, Busscher HJ. Bacterial adhesion to dental amalgam and three resin composites. J Dent 1995;23:171-6. 15. Campagni WV. The ﬁnal touch in the delivery of a ﬁxed prosthesis. CDA J 1984;12:21-9. 16. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Deﬁning the normal bacterial ﬂora of the oral cavity. J Clin Microbiol 2005;43:5721-32. Corresponding author: Dr Alireza Moshaverinia University of Southern California 2250 Alcazar Street - CSA 103 Los Angeles, CA 90033 Email: [email protected]
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