Australian Dental Journal

The official journal of the Australian Dental Association

Australian Dental Journal 2016; 61: 6–15 doi: 10.1111/adj.12338

Restorative materials containing antimicrobial agents: is there evidence for their antimicrobial and anticaries effects? A systematic review GS do Amaral,* T Negrini,† M Maltz,* RA Arthur* *Department of Preventive and Community Dentistry, Faculty of Dentistry, Federal University of Rio Grande do Sul, Brazil. †Department of Conservative Dentistry, Faculty of Dentistry, Federal University of Rio Grande do Sul, Brazil.

ABSTRACT The aim of this systematic literature review was to investigate whether the incorporation of antimicrobial agents into dental restorative materials truly exerts an antimicrobial effect against common cariogenic bacteria (primary outcome), and whether the inclusion of antimicrobial agents is able to prevent caries around restorations (secondary outcome). MEDLINE, via PubMed, was searched for papers published between 1980 and 30 November 2014. A total of 1126 articles were retrieved. After inclusion/exclusion assessment, 147 full text articles were read and included in the review, comprising 130 in vitro, 1 in situ, and 4 in vivo studies, as well as 12 literature reviews. In about 78% of in vitro studies, and in all identified in situ and in vivo studies, a positive antimicrobial effect had been found. However, the anticaries effect had not been tested in any of the selected studies. It was concluded that there is indeed evidence that restorative dental materials containing antimicrobial agents exert an antimicrobial effect, both in laboratory and in clinical studies. However, no evidence has been found regarding the role of these agents in preventing or controlling dental caries, or in preventing caries around restorations. Keywords: Antibacterial, antimicrobial, dental caries, dental material, restorative. Abbreviations and acronyms: CHX = chlorhexidine digluconate; MDPB = methacryloyloxydo-decylpyridinium bromide; NAg = silver nanoparticle; PQAS = poly quaternary ammonium salt; QADM = quaternary ammonium dimethacrylate. (Accepted for publication 19 May 2015.)

INTRODUCTION Dental caries is a complex disease process that has been studied for decades. Briefly, it is a multifactorial disease characterized by demineralization of dental tissues in response to acids that are produced by multispecies biofilm through the degradation of fermentable carbohydrates. This process leads to a pH decrease on the tooth surface, and results in loss of dental mineral structure.1 Mutans streptococci, especially Streptococcus mutans and Lactobacillus casei, are considered the main bacteria responsible for this metabolic-induced dental tissue loss because they are able to survive in an acidic environment, and they produce acids from rapidly-fermentable carbohydrates.2 These characteristics make them more likely to colonize tooth surfaces where there is frequent acidification of the dental biofilm because this acidification confers an ecological advantage over other dental biofilm microorganisms.3 The role of diet in 6

microbial shifts within the dental biofilm, and the subsequent impact of this microbial change on tooth surface integrity, are among the most important factors in dental caries pathogenesis. It has been postulated that frequent episodes of dental biofilm acidification lead to a transition in the microbial composition of the biofilm – from a dynamic stage, where there is a predominance of non-mutans streptococci and Actinomyces within the biofilms, to an acidogenic stage characterized by the predominance of non-mutans streptococci able to withstand low pH environments.2 The latter stage may further develop into an aciduric one, due to the perpetuation of the low-pH environment, where mutans streptococci and other acid-tolerant bacteria prevail. These shifts alter the phenotypic/genotypic traits of dental biofilm microbiota, resulting in an imbalance in the mineral equilibrium between the tooth and the surrounding aqueous phase, and leading to a net mineral loss. © 2015 Australian Dental Association

Restorative materials containing antimicrobial agents Once the mineral has been lost as a response to the altered microbiota composition, and to the fermentation of carbohydrates, a cavitation can be seen on the tooth surface upon examination. This cavitation may need to be sealed with dental restorative materials in order to restore chewing function and decrease biofilm accumulation at the site. In this context, it has been suggested that the main cause of restoration flaw and need for replacement is the occurrence of caries around restorations.4 Therefore, in an effort to increase the longevity of restorations, the incorporation of antimicrobial substances into dental restorative materials has been encouraged,5–81 with the aim of reducing biofilm formation on the surface of these materials. Long-term restorations are clinically attractive, since they may reduce the cost of restoration replacement, as well as the inconvenience caused to the patient as a result of the numerous re-interventions. Among the antimicrobials incorporated into dental restorative material, the monomers 12-methacryloyloxydo-decylpyridinium bromide (MDPB), chlorhexidine digluconate (CHX) and silver nanoparticle (NAg) are the most frequently used.34,82,83 MDPB is a cationic agent and exhibits biocidal activity by reacting with negatively charged bacterial surfaces,84 irreversibly damaging the cytoplasmic membrane of the bacteria.56,64 CHX acts on the bacterial cell wall, provoking leakage of intracellular constituents, which leads to cell death.85 In the case of NAg, it has been reported that the silver ions inactivate important bacterial enzymes and affect the replication mechanism of microbial DNA, leading to a reduction in bacterial viability.86–88 It has also been shown that silver attaches to the outer membrane, affecting permeability and inducing structural changes in the cell, ultimately leading to cell death.89 Several articles have shown that the incorporation of these antimicrobials into dental adhesives and restorative materials is able to inhibit Streptococcus ssp. and Lactobacillus ssp. growth under laboratory conditions.84,87,90,91 In addition to those already mentioned, further antimicrobial compounds such as quaternary ammonium dimethacrylate (QADM), chitosan, triclosan, furanone and poly quaternary ammonium salt (PQAS), have been incorporated into dental restorative materials.35,74,92–98 Generally, these compounds act on the microbial cell surface to provoke leakage of intracellular content.99–101 Although it has been shown that the incorporation of antimicrobial compounds into dental restorative materials is able to negatively affect the viability of cariogenic bacteria under laboratory conditions,89,102–106 the clinical relevance of this approach to the prevention of dental caries is still unknown.107,108 Thus, the aim of this systematic literature review was to investigate whether the incorporation of antimicrobial agents into © 2015 Australian Dental Association

dental restorative materials truly exerts an antimicrobial effect against common cariogenic bacteria (primary outcome), and whether this is able to prevent caries around restorations (secondary outcome). Search strategy The search for eligible studies was performed according to PRISMA guidelines.109 Briefly, MEDLINE was searched via PubMed for papers published between 1980 and 30 November 2014. The search strategy used was (dental material AND antimicrobial OR antibacterial AND restorative). In all studies identified the titles and abstracts were read. Whenever there was not enough information available, the full-text article was read. Two independent reviewers selected and analysed the studies. In cases of disagreement, a third reviewer was consulted, and the decision was made by consensus. The review only included articles written in English and reporting the antimicrobial effect of: (1) dental restoratives infused with an antimicrobial material; or (2) dental adhesives containing anticariogenic bacterial agents or exerting any anticaries effect. Only studies that met these criteria were fully read. Excluded from the review were: (1) articles reporting the antimicrobial effect of fluoride, or of dental materials other than restorative/dental adhesives; (2) reports of the mechanical properties of the materials studied, or of remineralization effects due to fluoride incorporation into the tested materials; (3) animal studies; (4) duplicated studies; and (5) papers whose full text was difficult to access. Moreover, the reference lists of the eligible articles and narrative reviews were handsearched for other potential studies of interest. Data were then extracted from the selected articles. These data comprised information regarding the following: (1) the type of study (in vitro, in situ, or in vivo); (2) the dental material/ dental adhesives used; (3) the antimicrobial agent tested; (4) the microorganisms against which the antimicrobials were tested; (5) the methodology used to evaluate the antimicrobial effect; and (6) the antimicrobial effect itself (positive or negative). The number of volunteers, the collection method and type of clinical samples, the antimicrobial agent tested, the type of restorative material, the presence of a control group, the duration of the study, any anticaries effect, any antimicrobial effect, and the type of microorganisms against which the antimicrobials were tested were also recorded from in situ and in vivo studies. RESULTS The search retrieved 1089 articles, of which 147 full text articles were read and included in the review. Figure 1 is a flow diagram of the articles that were 7

GS do Amaral et al. Records identified through eletronic database searching via PubMed (n = 1126)

Full-text articles including additional studies identified after reading the references

In situ

In vivo

studies

studies

studies

(n = 130)

(n = 1)

(n = 4)

(n=979): -difficulty in accessing the full article;

(n = 147) In vitro

Studies excluded

-studies reporting antimicrobial effects of fluoride; Literature Review (n = 12)

-antimicrobial tested against non-cariogenic bacteria; -not tested in microorganism; -antimicrobials not incorporated into dental restorative material; -papers reporting only evaluation of remineralization; -animal studies; -papers reporting only mechanical properties of dental material.

Fig. 1 Flow diagram presenting the results of the literature search and the process used to select studies for the systematic review.

identified, screened, excluded from, and included in this review. Of the included articles, 130 were in vitro, 1 was in situ, and 4 were in vivo studies. An additional 12 were literature reviews. Within the selected studies, a total of 605 experiments (in vitro, in situ and in vivo) had been performed. In about 78.3% of the experiments, antimicrobials had been incorporated into glassionomer cements (24.8%), adhesives (23.3%), composite resins (18.2%) and dentinal primers (12.1%). Together, compomers, polycarboxylate and zinc oxide cements, silver amalgam and liners had been tested in 21.7% of the experiments. The most tested antimicrobials were MDPB, CHX and NAg, comprising 48.4% of the selected studies. These were followed by silver ions, QADM, triclosan, cetrimide, polyethyleneimine nanoparticles and nanoparticles of amorphous calcium phosphate, which together represented 28.1% of the selected studies. Other antimicrobials were less frequently incorporated into restorative materials (zinc oxide, cetylpyridinium chloride, 2-methacryloxylethyl dodecyl methyl ammonium bromide, chitosan, PQAS, furanone, glutaraldehyde, benzalkonium chloride, epigallcatechin-3-gallate, dimethyl ammonium chloride, copper oxide, chelating acids, octenidine dihydrochloride and sodium hypochorite) but together they comprised 23.5% of the selected studies. With regard to the methodology used to evaluate the antimicrobial effects of the materials, all of the in vitro studies evaluated both inhibition of bacterial growth by direct contact with the tested material surface, as well as by inhibition of bacterial growth in broth. The time that elapsed before evaluation of the antimicrobial effect varied from 2 hours to 90 days, but the vast majority of studies evaluated bacterial viability only once – after 24 hours. Inhibition of bacterial growth was seen in about 78% of the studies 8

for any of the tested antimicrobials: composite resins, glass-ionomer cements, dentinal adhesives and dentinal primers (Table 1). Decreased bacterial viability due to the use of experimental, antimicrobial-incorporated dental restorative materials/dentinal adhesives was also found in all of the selected in situ and in vivo studies. In only one study was the antimicrobial effect evaluated after long-term exposure to the intraoral environment (up to 6 months), while all other studies reported antimicrobial effects for a period less than a week (Table 2). None of the selected articles (in vitro, in situ or in vivo) had evaluated the anticaries effect, or the ability of the antimicrobial restorative materials/ dentinal adhesives to prevent caries around the restoration (Table 1). DISCUSSION Irrespective of the antimicrobial incorporated into restorative material/dental adhesives, a reduction in bacterial viability has been found;110,112 this is in agreement with data from a recent literature review which found restorative filling material containing antimicrobial properties does exert an antimicrobial effect on bacteria commonly found in the oral cavity.113 Specifically, the effects of MDPB and CHX Table 1. Antimicrobial effect and evaluation of anticaries effect of restorative materials Variable Antimicrobial effect Anticaries effect

Results

Number of experiments (%)

Positive Negative Positive Negative

472 (78.0%) 133 (22.0%) Not tested Not tested

© 2015 Australian Dental Association

© 2015 Australian Dental Association

In situ

In vivo

In vivo

In vivo

In vivo

Rupf et al., 2012

Rolland et al., 2011

Du et al., 2012

Foley and Blackwell, 2003

Frencken et al., 2007

50

45

8

36

6

Number of volunteers

Yes/Carious dentine

Yes/Carious dentine

No

Yes/Root caries

No

Collection/type of clinical sample

Copper phosphate (concentration not informed) CHX (concentration not informed)

CHX 2%

Octenidine dihydrochloride 3% and 6% MDPB (concentration not informed)

Antimicrobial agent tested

GIC

Experimental GIC and Experimental GICRM Experimental liner

Clearfill SE Bond and Clearfill Protect Bond

Composite resin

Restorative material

GIC without CHX

No liner application

Composite resin without antimicrobial Root caries sampling before primer application GIC and GICRM without CHX

Control group

7 days

From 1 to 6 months

From 4 to 24 h

20 seconds

From 3 to 7 days

Duration of the study

Not tested

Not tested

Not tested

Not tested

Not tested

Anticaries effect

Positive

Positive

Positive

Positive

Positive

Antimicrobial effect

MDPB: 12-methacryloyloxydo-decylpyridinium bromide; CHX: chlorhexidine digluconate; GIC: glass-ionomer cement; GICRM: glass-ionomer cement resin modified.

Type of study

Authors

Table 2. Descriptive analysis of the short-term clinical studies included in the literature review

Mutans Streptococci Lactobacilli Anaerobes Mutans streptococci Lactobacilli Aerobes Anaerobes

Multispecies biofilm formed in situ Streptococci Lactobacilli Yeasts Anaerobes Multispecies biofilm formed in vivo

Type of microorganism

Restorative materials containing antimicrobial agents

9

GS do Amaral et al. have been extensively tested in in vitro and in shortterm clinical studies.30,31,72,90,92,114–138 The antimicrobial effects of NAg have only been tested under in vitro conditions. Nevertheless, it has been suggested that, in a nanoparticulate form, silver ions are released more effectively from the materials, and therefore have more effective bactericidal activity than higher size silver particles. This is due to the high surface area-to-volume ratio of the nanoparticles.139 This could be a promising development in antimicrobial therapy, and it may have relevance in fields other than dentistry.83,89,139–150 In spite of this, there has been no report to date regarding the antimicrobial effect of NAg under clinical conditions. It is important to mention that the most widelyreported and widely-used methods of assessing the antimicrobial properties of dental materials, namely agar or disk-diffusion tests, minimum inhibitory concentration and broth dilutions,113 are limited in only revealing the short-term effects on bacterial cell viability. That said, they represent a useful first screening, and the first evidence of any potential effect. In addition, none of the methods recently proposed by Farrugia and Camilleri,113 and presented below, are able to investigate any long-term antimicrobial effect. The methyl-thiazolyl tetrazolium assay may be able to demonstrate an effect on the bacterial metabolism exerted by the antimicrobial being tested, but this does not necessarily mean that the remaining bacteria are not viable, or that they are unable to continue growing under frequent exposure to carbohydrates from the diet. Moreover, the antimicrobial effect found using minimum inhibitory concentration, minimum bactericidal concentration and broth culture tests are mainly caused by the presence of highly concentrated compounds, delivered under static conditions, which do not represent the salivary clearance found in the oral cavity.151 In most of the selected articles evaluated, incorporation of an antimicrobial into a restorative material was able to reduce bacterial growth on the material’s surface by either direct contact inhibition, or by preventing biofilm formation. Keeping in mind that the most frequent cause of requirements for restoration replacement is caries around the restoration,152 it is reasonable to think that the prevention of biofilm formation, mainly at the tooth/restoration interface, may contribute to the prevention of caries around restorations. However, the conditions tested here, and the available data, do not allow any conclusions to be drawn in this respect. It has also been shown that the materials tested are able to release their antimicrobial agents into the surrounding aqueous environment and inhibit the growth of bacteria. Indeed, the release of antimicrobial agents into the oral cavity may be desirable in the case of a restoration, since it may inhibit at a distance, the 10

growth of cariogenic microorganisms in the oral cavity, leading in turn to a more favourable and healthy microflora.3 However, although we have learnt from these studies that the incorporation of antimicrobials into restorative materials effectively reduces bacterial viability, in most of them the antimicrobial effect of the experimental restorative material was evaluated in a short period of time (up to 24 hours).22,41,100,126,147,153–155 In only 3 in vitro studies was the antimicrobial effect studied over a 6month period,61,63,156 and it appears that the antimicrobial effect declines over time. Considering that a restoration has a high longevity in the oral cavity, it is questionable whether a short-term bacterial inhibition has any clinical relevance. We found a lack of clinical studies evaluating the role of antimicrobials incorporated into restorative materials, and their potential anticaries effect. Only 5 studies evaluated the effect of experimental restorative materials under in situ or in vivo conditions (Table 2, and these focused only on the antimicrobial effect per se). No investigation was carried out to verify whether the presence of the antimicrobial indirectly affects carious-lesion development or not. Ideally, in a first attempt to explore the antimicrobial effect of restorative materials, clinical conditions must be simulated under in vitro conditions, but in a more realistic way. Specifically, future studies need to be better designed, and their experimental designs should consider teeth/restoration as substrates for biofilm formation under a dynamic and constant flow of saliva, in order to mimic the release of these antimicrobials as it would happen in the oral cavity. If in vitro results show promise, well-controlled shortterm clinical studies should be designed so that the extent of demineralization around restorations in response to the experimental restorative materials can be better explored. Standardized gaps could be generated ex vivo on tooth/restoration interfaces, simulating a clinical condition that attracts bacterial colonization, and the tested tooth samples should be fixed on intraoral appliances in order to allow biofilm accumulation on their surface. In addition to these experiments, biofilm could be grown under a high-cariogenic challenge, simulating the use of antimicrobial-incorporated restorations in cariespositive patients. Future well designed, long-term and controlled clinical studies will also be warranted. If the incorporation of antimicrobials into the restorative material truly inhibits biofilm formation, a low demineralization will be expected. This could be monitored using mineral-content sensitive methods such cross-sectional hardness89 or transversal microradiography.157 Clinical studies evaluating the topical effect of dental adhesives incorporated with antimicrobial agents for controlling or preventing dentine root © 2015 Australian Dental Association

Restorative materials containing antimicrobial agents caries need to be conducted for longer periods of time in order to provide better evidence regarding their antimicrobial and/or cariostatic effects. Considering the multiplicity of factors involved in the carious process, and the limitations of the data provided by this narrative literature review, we reiterate that the best evidence for dental caries control and treatment is based on diet advice (reduced exposure to rapidly fermentable carbohydrates), motivation of patients towards a frequent and adequate mechanical biofilm removal from tooth surfaces and constant exposure to a low-fluoride concentration, which is provided by the daily use of fluoridated toothpastes.158 CONCLUSIONS There is evidence that restorative dental materials containing antimicrobials exert an antimicrobial effect, both in laboratory and clinical studies. However, no evidence has yet been found regarding the role of antimicrobials incorporated into restorative materials in preventing or controlling dental caries, or in preventing caries around restorations. Additionally, it is not possible to conclude from this systematic review whether the incorporation of antimicrobials into restorative materials is a sensible method for preventing dental caries. REFERENCES 1. Featherstone JD. Dental caries: a dynamic disease process. Aust Dent J 2008;53:286–291. 2. Takahashi N, Nyvad B. Caries ecology revisited: microbial dynamics and the caries process. Caries Res 2008;42:409– 418. 3. Marsh PD. Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 1994;8:263–271. 4. Barbachan e Silva B, Maltz M. Secondary caries: a review of the literature. Rev Fac Odontol P Alegre 2004;45:29–33. 5. Orstavik D. Antibacterial properties of and element release from some dental amalgams. Acta Odontol Scand 1985;43:231–239. 6. Tobias RS, Browne RM, Wilson CA. Antibacterial activity of dental restorative materials. Int Endod J 1985;18:161–171.

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Address for correspondence: Dr Rodrigo Alex Arthur Rua Ramiro Barcelos 2492 Porto Alegre RS Brazil 90035-003 Email: [email protected]

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