Biofilm-induced changes to the composite surface
Introduction
Composites have become the standard first-line restorative material in many countries, since they outperform amalgams in a number of clinical aspects, such as aesthetic appearance, more conservative preparation technique, clinical versatility etc. However, many researchers have reported that composites have a shorter lifespan compared to amalgams, which has mainly been attributed to their higher vulnerability to secondary caries (SC), the leading cause of restoration failure [1], [2].
The shorter lifetime of composites has received increasing attention in recent years, and possible reasons, as well as solutions, for the increased susceptibility of composites to SC have extensively been investigated [3]. Many studies have focused on plaque accumulation on the composite surface, as the presence of dental plaque (dental biofilm) is essential for the development of both primary and secondary caries. Conventional composites seem to accumulate more plaque on their surface than other restorative materials and tooth enamel do [4], [5], [6]. Possible explanations for this could be the lack of antibacterial and buffering properties of composites, the release of bacteria-stimulating compounds, or the specific surface characteristics of composite materials, such as surface roughness, topography, charge, hydrophobicity etc. [7], [8], [9]. The role of surface roughness in biofilm accumulation has been well established, and it is well known that increasing roughness implies higher bacterial retention [10], [11]. However, below a certain threshold (Ra = 0.2 μm), when the surface is considered to be smooth enough, physicochemical properties of the material (chemical composition) start playing a more important role in bacterial adhesion [12].
In addition to that, one of the inherent drawbacks of composites is that they undergo biodegradation in the oral cavity. Enzymes from the class of the esterases, such as cholesterol esterase (CE) and pseudocholinesterase (PCE), found in saliva, can degrade the Bis-GMA/TEGDMA-based composite polymer matrix [13], [14]. More importantly, cariogenic bacteria from dental plaque were also shown to be capable of degrading the resin matrix of composites and adhesives, not only by their esterase activity [15], but also by producing acids and creating a low-pH environment [16].
In consequence, many researchers suggested that the degradation of composite surfaces by the attached biofilms can result in increased surface roughness, which will further promote bacterial accumulation, leading to a vicious circle with even additional damage to the composite surface [17], [18], [19]. Nevertheless, it remains unclear to which extent a composite surface can be degraded by bacteria and whether this effect is significant enough to affect bacterial accumulation. Most of the studies in this field assessed the surface changes of composites with two-dimensional imaging techniques such as scanning electron microscopy (SEM), which do not allow quantification of the surface roughness [15], [19]. In addition, in most of the experiments, specimens were exposed to single-species bacterial cultures (typically Streptococcus mutans), even though it is known that bacteria express different behavior when they grow in complex bacterial communities, such as the dental biofilm [20]. Moreover, only little is known with regard to the mechanism of degradation. It is for example not clear to which extent bacterial biodegradation is caused by acidic metabolites or enzymes excreted by oral bacteria.
Therefore, the aim of this study was to assess the influence of S. mutans single-species and multi-species biofilms on the surface of two composite materials with different composition, by atomic force microscopy (AFM). In addition, the effect of cholesterol esterase (CE) and low pH were tested to elucidate the degradation mechanism. We also investigated whether biofilm-induced changes of the composite surface affected the amount of accumulated bacteria by real-time PCR. The null hypotheses were that 1) there is no difference in the effect of single- and multi-species biofilms on the surface properties of two tested composites, and 2) there is no difference in bacterial accumulation between the two composites.
Section snippets
Specimen preparation
Disk-shaped specimens (2-mm thickness, 7-mm diameter) of a hybrid, Bis-GMA-free composite (Gradia Direct Anterior, GC, Tokyo, Japan) and a flowable, Bis-GMA containing composite (Tetric EvoFlow, Ivoclar-Vivadent, Schaan, Lichtenstein) were prepared using polytetrafluoroethylene molds (Table 1). The composites were inserted into molds, pressed between two glass slides and cured for 40 s at each side with a polywave LED unit as per manufacturers’ instructions (output: 1400 mW/cm2) (Bluephase,
Scanning electron microscopy of attached biofilms
Scanning electron microscopy revealed that multi-species biofilms were noticeably thicker (Gradia: 3.9 ± 1.2 μm; Tetric EvoFlow: 2.4 ± 0.6 μm) and more robust compared to mono-species S. mutans biofilms (Gradia: 0.8 ± 0.4 μm; Tetric EvoFlow: 0.7 ± 0.3 μm) (Fig. 1), and this trend was observed for both composites. Even though A. naeslundii and F. nucleatum could be observed in the mixed biofilms, oral streptococci (S. mutans and S. sanguinis) were most dominant.
Real-time PCR quantification of attached biofilms
The total amount of bacteria attached to the
Discussion
One of the inherent properties of composite restorations is their vulnerability to biodegradation in oral cavity, in particular by cariogenic bacteria from dental plaque. It has been suggested that the cariogenic S. mutans species can degrade resin matrix of adhesives and composites, thereby leading to interfacial failure and bacterial microleakage on the one hand, and to an increased surface roughness and higher plaque retention on the other hand [15], [21]. Both problems can contribute to
Conclusions
In conclusion, even though S. mutans, a primary etiological species in caries, is indeed capable of increasing the surface roughness of composites, the observed surface changes did not seem to significantly affect bacterial accumulation. Not acidic metabolites, but rather bacterial esterases induced this surface biodegradation. The most important finding, however, is that this effect of S. mutans was largely mitigated when it was cultured with other bacteria in a multi-species biofilm. More
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgments
This research was supported by the Research Foundation – Flanders (FWO) grant G.0884.13. We would like to thank Martine Pauwels and Rita Merckx for their skillful assistance. We would also like to thank the manufacturers for providing the commercial composites.
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Materials
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