- Research article
- Open Access
- Open Peer Review
The Microfloral Analysis of Secondary Caries Biofilm around Class I and Class II Composite and Amalgam Fillings
© Mo et al; licensee BioMed Central Ltd. 2010
- Received: 12 October 2009
- Accepted: 17 August 2010
- Published: 17 August 2010
Secondary caries is responsible for 60 percent of all replacement restorations in the typical dental practice. The diversity of the bacterial sources and the different types of filling materials could play a role in secondary caries. The aim of this study was to determine and compare the microbial spectrum of secondary caries biofilms around amalgam and composite resin restorations.
Clinical samples were collected from freshly extracted teeth diagnosed with clinical secondary caries. Samples were categorized into four groups according to the types of restoration materials and the classification of the cavity. Biofilms were harvested from the tooth-restoration interface using a dental explorer and after dilution were incubated on special agars. The bacteria were identified using the biochemical appraisal system. Statistical calculations were carried out using SPSS11.5 software to analyze the prevalence of the bacteria involved in secondary caries.
Samples from a total of four groups were collected: two groups were collected from amalgam restorations, each had 21 samples from both Class I and Class II caries; and the other two groups were from composite resin restorations, each had 13 samples from both class I and class II caries. Our results showed: (1) Anaerobic species were dominant in both restoration materials. (2) In terms of the types of individual bacteria, no significant differences were found among the four groups according to the geometric mean of the detected bacteria (P > 0.05). However, there were significant differences among the detected bacteria within each group (P < 0.05). The composition of each bacterium had no statistical difference among the four groups (P > 0.05), but showed significant differences among the detected bacteria in each group (P < 0.05). (3) Among the four groups, there were no significant differences for the detection rate of each bacterium (P > 0.05), however, the detection rate of each bacterium within each group was statistically different among the detected bacteria (P < 0.05).
The proportion of obligatory anaerobic species was much greater than the facultative anaerobic species in the biofilm of secondary caries. Statistically, the materials of restoration and the location of secondary caries did not show any significant effects on the composition of the microflora.
- Periodontal Disease
- Root Canal
- Mutans Streptococcus
- Porphyromonas Gingivalis
- Composite Filling
The term "secondary caries" or "recurrent caries" denotes caries at the margin of the tooth restorations, which is the most important reason for the failure of fillings [1–4]. Secondary caries is responsible for 60 percent of all restoration replacement in the typical dental practice. The bacteria present in the dental plaque that are involved in the etiology of primary caries most likely also play a major role in the development of secondary caries . It has been reported that the material properties of the dental restorations influence plaque accumulation and development of secondary caries [6, 7]. However, in the culture studies of Kidd et al., they found no significant differences in the microflora composition in plaque samples taken from sites with primary or recurrent caries. Similarly, some studies also failed to find any significant association between the microbial flora among different dental materials, several studies reported specific microbial spectrum profiles or found a correlation between the roughness of dental materials and the accumulation of bacteria [8–10]. This indicated that the antibacterial effects of metal ions from dental materials could play a role in secondary caries. Svanberg et al. detected much higher total colony-forming unit (cfu) counts for mutans streptococci at margins of composite fillings than that of comparable amalgam fillings . The amount of plaques and the degree of cariogenicity at restoration margins depend on the restorative material [11, 12]. These findings indicated that resin based materials accumulate more plaques, which are more cariogenic than amalgam, silicate cement, and glass ionomer materials. On the other hand, polymerization shrinkage and the load of chewing pressure often result in cracking and microleakage of the composites . This marginal gap could be an ecological niche for microorganisms , especially because composites do not have the antibacterial effects of, for example, Hg-ions in amalgam . Moreover, Matasa described a "microbial attack" on composites used as bonding adhesives for orthodontic applications . It was believed that microleakage was also associated with the development of recurrent caries [17, 18] but this theory has been challenged. The bulk of available evidence indicates that there is no relationship between the development of recurrent caries and the size of the crevice at the tooth restoration interface [19–24], apart from cases of macroleakage in which the crevice exceeded 250 μm or 400 μm . Thus, recurrent caries does not develop as a result of microleakage along the tooth-restoration interface, but at a surface lesion similar to primary carious lesions on smooth surfaces [24, 4]. The presences of overhangs, even the clinically hard-to-detect minor overhangs, predispose a patient to the development of recurrent caries, which indicated that plaque accumulation is an important predisposing factor in the development of recurrent caries .
Most studies have concentrated on aerobic bacteria around the restorations, but little is known about the anaerobic bacteria. Although these gram-negative anaerobic bacteria have been shown to associate with periodontal diseases, Christian Splieth et al. found that anaerobic gram-negative bacteria associated with periodontal diseases were predominant in secondary caries in composite fillings . These bacteria were similar to the bacteria spectrum of root canal infection such as Fusobacterium, Veillonella, Prevotella, etc. Therefore, we infer anaerobic bacteria may also play an important role in formation of secondary caries and we have focused on some of these anaerobic periodontal bacteria in this study.
The aim of this study was to determine and compare the microbial spectrum around composite and amalgam fillings with particular attention to the anaerobic flora.
This study was approved by the Ethics Committee of Ninth People's Hospital, Medical College, Shanghai Jiao Tong University, China. Informed consent has been acquired from all patients participating in this study. The patients, who contributed teeth to this study, had mostly had periodontal diseases. Biofilm samples were collected from freshly extracted teeth that had been diagnosed with secondary caries upon visual inspection by experienced dentists according to the modified criteria of the California Dental Association [27–29]. Samples were collected and categorized into four groups according to the restoration materials and G. V. Black's cavity classification: Group 1 were samples collected from amalgam-filled class I cavities, Group 2: class II cavities with amalgam, Group 3: class I cavities with composite resin, and Group 4: class II cavities with composite resin. The teeth were routinely extracted and rinsed thoroughly with deionised water. The biofilm samples were collected with a dental explorer and immediately transferred into vials containing 1 ml transport fluid of prereduced thioglycollate medium (Difco, Detroit, Mich., USA).
Bacterial culture procedures
Media used in this study
Tryptic Soy Agar plate (TSA)
universal for aerobic bacteria
Mitis-Salivarius Bacitracin agar (MSB)
Cadmium fluoride acriflavin tellurite (CFAT)
L-Rogosa selective blood agar plate
Anaerobic CDC blood agar plate
universal for obligative anaerobic bacteria
kanamycin-vancomycin laked blood plate (KVLB)
V-Rogosa blood agar plate
FS blood agar plate
PS blood agar plate
Bacteria colony-forming units (cfu) were presented as geometric averages. The composition of each bacterium in the sample plaque was calculated as the percentage ratio of its cfu count formed on the selective culture plate compared with the total cfu formed in the universal plates. The detection rate of each bacterium in each group was calculated as the percentage ratio of the number of sample which we can idenfied one kind of the bacteria from devided by the number of sample in each group.
The prevalence of the bacteria involved in secondary caries was analyzed using SPSS11.5 software. Total cfu of each sample was presented as a log phase to accommodate the normal distributions. The differences of Geometric averages of cfu and composition of each bacterium in the four groups were compared by the two-way ANOVA analysis using the Bonferroni method. For the differences of detection rate of the bacteria among the four groups, Chi-Square Test was used for analysis.
The log colony-forming units per ml from four groups (logXG ± SD)
class I cavities of amalgam
(n = 21)
class II cavities of amalgam
(n = 21)
class I cavities of composite resin
(n = 13)
class II cavities of composite resin
(n = 13)
Total aerobic bacteria
5.43 ± 0.35
5.45 ± 0.51
5.35 ± 0.76
5.41 ± 0.52
5.25 ± 0.72
5.15 ± 0.45
5.16 ± 0.65
5.24 ± 0.98
4.99 ± 0.56
4.94 ± 0.89
4.85 ± 1.08
4.95 ± 1.12
4.25 ± 1.14
4.24 ± 0.84
4.16 ± 0.37
4.27 ± 1.22
5.06 ± 0.60
5.12 ± 0.71
4.90 ± 1.17
5.07 ± 0.62
4.92 ± 0.87
4.93 ± 0.88
4.74 ± 0.46
4.85 ± 0.92
Total anaerobic bacteria
5.91 ± 0.47
5.68 ± 0.79
5.58 ± 0.94
5.64 ± 0.81
5.31 ± 1.55
5.25 ± 1.72
5.33 ± 1.11
5.29 ± 0.72
5.21 ± 2.15
5.19 ± 1.48
5.11 ± 1.63
5.17 ± 2.24
4.25 ± 1.06
4.22 ± 1.00
4.15 ± 1.89
4.21 ± 0.98
5.33 ± 0.64
5.34 ± 0.82
5.29 ± 1.28
5.32 ± 1.15
4.02 ± 0.59
4.12 ± 0.55
4.32 ± 0.88
4.17 ± 1.01
4.41 ± 0.47
4.59 ± 0.95
4.50 ± 0.75
4.35 ± 1.76
0.44 ± 1.46
0.41 ± 1.92
0.33 ± 1.76
0.37 ± 1.53
The comparison of detectable percentage of the microbes in the four groups.
class I cavities of amalgam
(n = 21)
class II cavities of amalgam
(n = 21)
class I cavities of composite resin
(n = 13)
class II cavities of composite resin
(n = 13)
The dominant microflora and bacterial spectrum of secondary caries
In this study, the microflora of secondary caries biofilm around Class I and Class II composite and amalgam fillings mainly included Prevotella, Veillonella, Lactobacilli, Streptococci mutans, and Neisseriae. The next most prevalent group of bacteria included Actinomyces, Peptostreptococcus, Fusobacterium, and Porphyromonas gingivalis and occasionally Capnocytophaga. The proportion of obligately anaerobic species was much greater than that of facultatively anaerobic species. This bacterial spectrum was similar to the microflora in subgingival plaque of periodontal disease and in the infected root canals with potentially pulp pathogenic microbes [30, 31]. Christian Splieth et al., who did similar studies, found that the ratios of aerobic to anaerobic flora were comparable for composite and amalgam fillings with 11.4%/88.6% and 15.4%/84.5%, respectively . Very few studies on anaerobic bacteria in secondary caries have been reported. Gonzalez-Cabezas reported the detection of mutans streptococci in secondary carious lesions using confocal laser scanning microscopy and immunofluorescent techniques , with this bacteria detected in 88.9% of the samples analyzed with the immunofluorescent technique. Gonzalea-cabezas reported the distribution of three cariogenic bacteria, mutans streptococci, Actinomyces naeslundii, and lactobacilli in secondary carious lesions around amalgam restorations . Fitzgerald RJ reported that there was considerable variation in the numbers and types of microorganisms found in dentine samples from lesions of comparable severity . Mutans streptococci were found in 40% of sites with any degree of caries and in only 3 of the 9 sites with the most caries. The prevalence and number of lactobacilli increased with the degree of caries, they occurred in less than half of the affected dentine samples. Actinomyces occurred in 15 of 32 affected sites but only in 2 of the 9 most active sites with their numbers not exceeding 3 × 105 CFU/mg. However, all these studies have not reported on the anaerobic bacteria of secondary caries.
The samples used in this study were collected from freshly extracted teeth. The teeth were mostly collected from the patients who had periodontal diseases. The Microflora in saliva of the patients could be responsible for the dominant anaerobic bacteria of periodontal diseases. The oral anaerobic bacteria could get into anaerobic environments such as lacuna or along the tooth-restoration margin. Thus, the high proportion of anaerobic species found in this study may not be bacteria directly involved in secondary caries, but may be due to a different etiological model from primary caries.
In primary caries, microorganisms attach to surfaces and develop biofilms. The process is initiated by demineralization of the tooth surface by organic acids. These acids are the result of fermentation of dietary carbohydrates by the plaque bacteria. As tooth mineral is lost, the proteinase of plaque bacteria causes the secondary destruction of tooth protein. In secondary caries, the bacteria may come from oral environment, which gets into anaerobic environment of lacuna or leakage along the tooth-restoration interface. Destruction of tooth protein may be the first step and contributes to cavity formation. After the formation of cavity, bacterial fermentation of dietary carbohydrates can accumulate and produce more acids.
The effect of filling material and cavity classification on the flora of secondary caries
According the study of Christian Splieth, the variety of microbes under composite fillings was much greater compared to amalgam . In the composite fillings, there were 34 strains of strictly anaerobic non-spore-forming gram-negative rods, 17 strains of anaerobic or facultative anaerobic non-spore-forming gram-positive rods, 9 strains of anaerobic gram-positive cocci, and 2 strains of anaerobic gram-negative cocci. In the amalgam, there was 1 strain of strictly anaerobic non-spore-forming gram-negative rods, 7 strains of anaerobic or facultative anaerobic non-spore forming gram-positive rods, and 3 strains of anaerobic gram-positive cocci. More species and higher quantities of lactobacilli were isolated from composite fillings. Differences between bacterial colonization under composite and amalgam were statistically significant for anaerobic rods (p < 0.05) but not for aerobic rods, anaerobic, and aerobic cocci due to their lower numbers. However, in this study we found no significant differences among the four groups according to the geometric mean of the detected bacteria (P > 0.05).
Other studies reported that bacterial colonization under amalgam of secondary caries was similar to that of flora of carious dentin or carious plaque [35, 36] with anaerobic and facultative anaerobic gram-positive rods dominating. This distribution was also present in secondary carious lesions around amalgam restorations . Mejare et al. detected a bacterial spectrum under composite fillings similar to dental plaques with Actinomyces spp. and streptococci dominating [37, 38]. This bacterial composition could be due to the short persistence of the restorations in the oral cavity in their study. Many studies have focused on antibacterial activity of restorative dental biomaterials . Long-term clinical trials are necessary to determine whether the antimicrobial effects of dental materials are able to reduce the risk of secondary caries formation .
In the present study, for each type of bacterium, no significant difference could be found among four groups based upon the geometric mean of the detected bacteria (P > 0.05), the percent of the detected bacteria (P > 0.05), and the prevalence of the detected bacteria (P > 0.05). The results may suggest that the microleakage or cracking is one of the factors for the ecological niche of microorganisms. The anaerobic environment of deep layers of lesions as a result of microleakage along the tooth-restoration interface favors the growth of anaerobes. Thus, the microflora of recurrent caries may be developed as a result of microleakage along the tooth-restoration interface.
This article is an informative analysis of the microflora around dental restorations. The proportion of obligatory anaerobic species was much greater than facultative anaerobic species in the biofilm of secondary caries. Statistically, the kinds of restoration materials and location of caries have no significant effects on the composition of the microflora.
This work was supported by Science and Technology Commission of Shanghai (08DZ2271100) and Shanghai Leading Academic Discipline Project (Project Number: S30206).
Declaration of Interests:
Each author of this manuscript has stated that there are no potential conflicts of interest, including financial or other relationships.
- Mjor IA: Frequency of secondary caries at various anatomical locations. Oper Dent. 1985, 10: 88-92.PubMedGoogle Scholar
- Klausner LH, Green TG: Placement and replacement of amalgam restorations: a challenge for the profession. Oper Dent. 1987, 12: 105-12.PubMedGoogle Scholar
- Friedl KH, Hiller KA, Schmalz G: Placement and replacement of composite restorations in Germany. Oper Dent. 1995, 20: 34-8.PubMedGoogle Scholar
- Mjör IA, Moorhead JE, Dahl JE: Reasons for replacement of restorations in permanent teeth in general dental practice. Int Dent J. 2000, 50: 361-6.View ArticlePubMedGoogle Scholar
- Mjör IA: The reasons for replacement and the age of failed restorations in general dental practice. Acta Odontol Scand. 1997, 55: 58-63. 10.3109/00016359709091943.View ArticlePubMedGoogle Scholar
- Mjör IA, Toffenetti F: Placement and replacement of resin-based composite restorations in Italy. Oper Dent. 1992, 17: 82-5.PubMedGoogle Scholar
- Kidd EA, Joyston Bechal S, Beighton D: The use of a caries detector dye during cavity preparation a microbiological assessment. Br Dent J. 1993, 174: 245-8. 10.1038/sj.bdj.4808142.View ArticlePubMedGoogle Scholar
- Sönju T, Glantz PO: Chemical composition of salivary integuments formed in vivo on solids with some established surface characteristics. Arch Oral Biol. 1975, 20: 687-91. 10.1016/0003-9969(75)90138-7.View ArticlePubMedGoogle Scholar
- Orstavik D, Orstavik J: In vitro attachment of Streptococcus sanguis to dental crown and bridge cements. J Oral Rehabil. 1976, 3: 139-44. 10.1111/j.1365-2842.1976.tb00937.x.View ArticlePubMedGoogle Scholar
- Skjørland KK: Auger analysis of integuments formed on different dental filling materials in vivo. Acta Odontol Scand. 1982, 40: 129-34. 10.3109/00016358209012721.View ArticlePubMedGoogle Scholar
- Svanberg M, Mjör IA, Orstavik D: Mutans streptococci in plaque from margins of amalgam, composite, and glass ionomer restorations. J Dent Res. 1990, 69: 861-4. 10.1177/00220345900690030601.View ArticlePubMedGoogle Scholar
- Moura JS, Lima EM, Paes Leme AF, Del Bel Cury AA, Tabchoury CP, Cury JA: Effect of luting cement on dental biofilm composition and secondary caries around metallic restorations in situ. Oper Dent. 2004, 29: 509-14.PubMedGoogle Scholar
- Choi KK, Condon JR, Ferracane JL: The effects of adhesive thickness on polymerization contraction stress of composite. J Dent Res. 2000, 79: 812-7. 10.1177/00220345000790030501.View ArticlePubMedGoogle Scholar
- Klimm W, Buchmann G, Reissig D, Schneider H: Verification of microorganisms in dentin of filled teeth by comparison of light, transmission electron and scanning electron microscopy. Stomatol DDR. 1988, 38: 702-7.PubMedGoogle Scholar
- Grossman ES, Matejka JM: Amalgam restoration and in vitro caries formation. J Prosthet Dent. 1995, 73: 199-209. 10.1016/S0022-3913(05)80162-3.View ArticlePubMedGoogle Scholar
- Matasa CG: Microbial attack of orthodontic adhesives. Am J Orthod Dentofacial Orthop. 1995, 108: 132-41. 10.1016/S0889-5406(95)70075-7.View ArticlePubMedGoogle Scholar
- Kidd EA: Microleakage: a review. J Dent. 1976, 4: 199-206. 10.1016/0300-5712(76)90048-8.View ArticlePubMedGoogle Scholar
- Kidd EA: Caries diagnosis within restored teeth. Quality evaluation of dental restorations. Edited by: Anusavice KJ. 1989, Chicago: Quintessence, 111-21.Google Scholar
- Goldberg J, Tanzer J, Munster E, Amara J, Thal F, Birkhed D: Cross-sectional clinical evaluation of recurrent enamel caries, restoration of marginal integrity, and oral hygiene status. J Am Dent Assoc. 1981, 102: 635-41.View ArticlePubMedGoogle Scholar
- Merrett MC, Elderton RJ: An in vitro study of restorative dental treatment decisions and dental caries. Br Dent J. 1984, 157: 128-33. 10.1038/sj.bdj.4805448.View ArticlePubMedGoogle Scholar
- Söderholm KJ, Antonson DE, Fishlschweiger W: Correlation between marginal discrepancies at the amalgam/tooth interface and recurrent caries. Quality evaluation of dental restorations. Edited by: Anusavice KJ. 1989, Chicago: Quintessence, 85-108.Google Scholar
- Kidd EA, O'Hara JW: Caries status of occlusal amalgam restorations with marginal defects. J Dent Res. 1990, 69: 1275-7. 10.1177/00220345900690061001.View ArticlePubMedGoogle Scholar
- Pimenta LA, Navarro MF, Consolaro A: Secondary caries around amalgam restorations. J Prosthet Dent. 1995, 74: 219-22. 10.1016/S0022-3913(05)80126-X.View ArticlePubMedGoogle Scholar
- Özer L: The relationship between gap size, microbial accumulation and the structural features of natural caries in extracted teeth with Class II amalgam restorations. Master's thesis. 1997, Copenhagen, Denmark: University of CopenhagenGoogle Scholar
- Kidd EA, Joyston-Bechal S, Beighton D: Marginal ditching and staining as a predictor of secondary caries around amalgam restorations: a clinical and microbiological study. J Dent Res. 1995, 74: 1206-11. 10.1177/00220345950740051001.View ArticlePubMedGoogle Scholar
- Splieth C, Bernhardt O, Heinrich A, Bernhardt H, Meyer G: Anaerobic microflora under Class I and Class II composite and amalgam restorations. Quintessence int. 2003, 34: 497-503.PubMedGoogle Scholar
- Ryge G: Clinical criteria. Int Dent J. 1980, 30: 347-58.PubMedGoogle Scholar
- Pieper K, Mausberg R, Curdt R, Uhde V: Clinical evaluation of the quality of amalgam and polymer filling materials after various periods of function. A study of servicemen of the German armed forces. Dtsch Zahnarztl Z. 1989, 44: 707-10.PubMedGoogle Scholar
- Allander L, Birkhed D, Bratthall D: Quality evaluation of anterior restorations in private practice. Swed Dent J. 1989, 13: 141-50.PubMedGoogle Scholar
- Drucker DB, Lilley JD, Tucker D, Gibbs AC: The endodontic microflora revisited. Microbios. 1992, 71: 225-34.PubMedGoogle Scholar
- Hoshino E, Ando N, Sato M, Kota K: Bacterial invasion of non-exposed dental pulp. Int Endod J. 1992, 25 (Suppl 1): 2-5. 10.1111/j.1365-2591.1992.tb00941.x.View ArticlePubMedGoogle Scholar
- Gonzalez-Cabezas C, Li Y, Noblitt TW, Gregory RL, Kafrawy AH, Stookey GK: Detection of mutans streptococci in secondary carious lesions using immunofluorescent techniques and confocal laser scanning microscopy. Caries Res. 1995, 29: 198-203. 10.1159/000262069.View ArticlePubMedGoogle Scholar
- Gonzalez-Cabezas C, Li Y, Gregory RL, Stookey GK: Distribution of cariogenic bacteria in carious lesions around tooth-colored restorations. Am J Dent. 2002, 15: 248-51.PubMedGoogle Scholar
- Fitzgerald RJ, Adams BO, Davis ME: A microbiological study of recurrent dentinal caries. Caries Res. 1994, 28: 409-15. 10.1159/000262013.View ArticlePubMedGoogle Scholar
- Kidd EA, Joyston-Bechal S, Beighton D: Microbiological validation of assessments of caries activity during cavity preparation. Caries Res. 1993, 27: 402-8. 10.1159/000261571.View ArticlePubMedGoogle Scholar
- Loesche WJ, Syed SA: The predominant cultivable flora of carious plaque and carious dentine. Caries Res. 1973, 7: 201-16. 10.1159/000259844.View ArticlePubMedGoogle Scholar
- Mejare B, Mejare I, Edwardsson S: Acid etching and composite resin restorations. A culturing and histologic study on bacterial penetration. Endod Dent Traumatol. 1987, 3: 1-5. 10.1111/j.1600-9657.1987.tb00163.x.View ArticlePubMedGoogle Scholar
- Mejare B, Mejare I, Edwardsson S: Bacteria beneath composite restorations - a culturing and histobacteriological study. Acta Odontol Scand. 1979, 37: 267-75. 10.3109/00016357909004696.View ArticlePubMedGoogle Scholar
- Yoshida K, Tanagawa M, Atsuta M: Characterization and inhibitory effect of antibacterial dental resin composites incorporating silver-supported materials. J Biomed Mater Res. 1999, 47: 516-22. 10.1002/(SICI)1097-4636(19991215)47:4<516::AID-JBM7>3.0.CO;2-E.View ArticlePubMedGoogle Scholar
- Boeckh C, Schumacher E, Podbielski A, Haller B: Antibacterial activity of restorative dental biomaterials in vitro. Caries Res. 2002, 36: 101-7. 10.1159/000057867.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/241/prepub
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