2.1

Dental hard tissue

Human dental plaque is one of the most natural forms of biofilm growth . It is constantly exposed to environmental challenges and therefore has served as a general model to study the formation of biofilms . It has been established that plaque’s biofilm-forming capacity and structural organization are influenced by the chemical nature of the substrate . Bacteria that cannot adhere to a surface are transported by salivary flow down to the digestive tract.

All biofilms on the tooth surface have at least one characteristic in common: the presence of a bacterial-derived matrix that envelopes the bacteria and helps to facilitate the formation of multicellular structures that become firmly attached to the dental hard tissue . Oral bacterial attachment on enamel and dentin starts with the recognition of salivary pellicle receptors by initial colonizing bacteria on the tooth surface . The extracellular polymeric substance (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms . Starting at the bottom, initial colonizers bind to complementary salivary receptors in the acquired pellicle. Late colonizers bind to previously bound bacteria and to EPS as a coating on the tooth surface .

Enamel is sparsely colonized by multispecies communities in the first eight hours after cleaning , while under equal conditions, Nyvad and Fejerskov revealed that root surfaces were more heavily colonized with the same types of bacteria. Within the first 24 h, the microbial flora was dominated by Streptococci and Gram-positive pleomorphic rods. Streptococcus sanguis contributed 6–18% of the early colonizers. Streptococcus mitis and Streptococcus oralis varied between 24–42% and 1–27% respectively. The relative proportion of S. oralis increased significantly within 24 h while the proportion of Streptococcus salivarius and arginine-positive S. mitis showed a declining tendency . Xiao et al. showed that Streptococcus mutans -produced EPS can modulate the three-dimensional architecture and population shifts during morphogenesis of biofilms using a saliva-coated hydroxyapatite (HA) disk model.

Dentin is a composite material made up of inter-penetrant inorganic fraction (around 70 wt%) and collagen (around 20 wt%). Type I collagen is the major organic component (90%) of dentin. It is known that certain bacteria can attach to type I collagen in dentin through the expression of surface adhesins and form biofilms . Dentinal tubules are the tunnels for bacteria invasion. With long-term observation in vivo or incubation in vitro , biofilm bacteria has been seen to invade into dentinal tubules and induce deep infection .

A number of in vitro models have been used to study oral biofilm formation . Plaque biofilm can form on an HA disk model in 24 h ( Fig. 1 ). An in vitro study showed that the thickness of plaque biofilm grown on collagen-coated HA disks was 50 μm greater than the biofilm grown on pure HA disks using SEM . Single species Enterococcus faecalis was capable of forming biofilm on different substrates, and HA presented the highest rates of biofilm formation ( Fig. 2 ). Kishen et al. highlighted the sequence of events in the interaction of E. faecalis biofilm with a dentin substrate. E. faecalis first formed biofilm on the root canal dentin, and then the bacteria induced dissolution of the mineral fraction from the dentin. Finally, a reprecipitated apatite layer was formed in the biofilm structure. The ability of E. faecalis to form calcified biofilm may be a factor that contributes to their persistence after endodontic treatment .

SEM micrographs of dental plaque biofilm formation on hydroxyapatite disk in anaerobic condition. (A) 4 h after incubation, (B) 12 h after incubation, (C) 24 h after incubation and (D) 7 days after incubation.

SEM micrographs of E. faecalis biofilm formation on hydroxyapatite disk in anaerobic condition. (A) 4 h after incubation, (B) 12 h after incubation, (C) 24 h after incubation, and (D) 7 days after incubation.

Recently, a novel dentin infection model was introduced to allow a standardized and deep penetration of E. faecalis biofilms into dentinal tubules using the power of centrifugation , which allows standardized measurements of live/dead bacteria under CLSM to study the effectiveness of dentin disinfection. Bacteria packed in the dentinal tubules could be observed by both CLSM and SEM ( Fig. 3 ). This method may have great potential to study the antibiofilm effect of different dental materials applied on dentin, e.g. root canal sealers, endodontic cements, composite resins.

Three-week E. faecalis infection of dentin by centrifugation. (A) SEM micrographs of E. faecalis blocking dentinal tubules from dentin surface view, (B) SEM micrographs of E. faecalis infected dentinal tubules at high magnification, (C) SEM micrographs of E. faecalis infected dentinal tubules at low magnification, and (D) three-dimensional reconstructions of confocal laser scanning microscope (CLSM) images of infected dentinal tubules after live/dead viability staining (green fluorescence: live bacterial cells; red fluorescence: dead bacterial cells).

2.2

Ceramics

Ceramic materials are low-adhesive materials due to their inherent surface characteristics. Biofilms on ceramics are thin and highly viable . Compositional and microstructural differences between different ceramics may, however, influence the surface properties and hence the reactions between the material and oral microbial environment.

Biofilm formation on various types of dental ceramics ( e.g. veneering glass–ceramic, lithium disilicate glass–ceramic, yttrium-stabilized zirconia) for different purposes differ significantly . In particular, zirconia ceramic exhibits low plaque accumulation, and displays similar bacterial binding properties to titanium . In three days, the surface of two types of ceramic inlays collected less plaque with reduced viability under conditions of no oral hygiene than did the natural tooth surface . Compared with gold and amalgam which attracted up to 17-μm-thick biofilms, a 6-μm-thick biofilm on ceramics took five days to form in vivo . Lindel et al. indicated that ceramic brackets exhibit less long-term biofilm accumulation than metal orthodontic brackets. Another in situ study used an oral device to hold dental ceramics in volunteers’ mouths to evaluate biofilm formation on the surface of ceramics after dentifrice brushing . The result showed that cocci and short rods were the predominant microbial morphotypes on the feldspar ceramics. However, although brushing with dentifrice could roughen the ceramic surfaces, the increase in roughness did not significantly facilitate biofilm formation. Apparently, low biofilm accumulation makes ceramic a promising material for various indications.

2.3

Resin composites

Resin-based composites are complex materials that consist of a hydrophobic resin matrix and less hydrophobic filler particles, which implies that a resin-based composite surface is never a homogeneous interface but rather one that produces matrix-rich and filler-poor areas, as well as matrix-poor and filler-rich areas . Biofilms on composites can cause surface deterioration.

Polishing, as well as differences in the composition of the resin-based composite, may have an impact on biofilm formation on the resin-based composite surface . Surface deterioration of resin composites induced by polishing leads to increased roughness, changes in microhardness, and filler particle exposure upon exposure to biofilms in vitro . Significantly less S. mutans biofilm formation was observed on polished resin-based composites than on unpolished resin-based composites during a four-day incubation , indicating that the proportions of resin matrix and filler particles on the surface strongly influence S. mutans biofilm formation in an artificial mouth system. Another investigation confirmed that the quantity of retained S. mutans biofilm was significantly less on the surface of diamond paste polished resin composite than on a regular silicon paper polished surface in the same in vitro system . S. mutans outgrowth was accelerated following direct contact with the surface of an aged resin composite without affecting the microhardness of the composite . A similar study showed an increase in surface roughness of restorative resin after one month’s exposure to S. mutans biofilms .

Passariello and Gigola showed that composite brackets are more susceptible to adhesion and colonization by Streptococci biofilm, while other types of tested brackets did not show differences that could be clinically relevant, suggesting that the exposure of composite resins may enhance the growth of cariogenic bacteria . The enhanced growth of bacteria may be because the conversion of resin composites was not completed. It has been suggested that the release of ethyleneglycol dimethylacrylate and triethyleneglycol dimethacrylate from composite resins contribute to the growth of cariogenic bacteria like Streptococcus sobrinus and Lactobacillus acidophilus .

In several more specific studies, results showed that resin composite may enhance the glucosyltransferase activity of the bacteria ; another investigation found that triethyleneglycol modulates the expression levels of glucosyltransferase B involved in biofilm formation . Interestingly, Takahashi et al. indicated that growth stimulation of S. Sobrinus and S. Sanguis by ethyleneglycol dimethylacrylate monomers as measured by optical density was not accompanied by an increase in the numbers of colony forming units (CFU), but as the increase in the amount of bacteria confirmed by the presence of a vesicular material surrounding the bacteria. In contrast, biomaterial surface chemistry could also negatively affect biofilm formation. Most notably, polyethylene oxide (PEO) significantly inhibited Staphylococcus epidermidis biofilm formation over 48 h in vitro . The hydrophilic properties of PEO created flexible, mobile chains and therefore sterically hinder bacterial adhesion by diminishing the probability of contact between the bacteria and the hydrophobic polymer base.

2.4

Metallic alloys

The adhesion of oral biofilm on metallic dental material surfaces ( e.g. gold, amalgam, titanium and zirconia) has been reported both in vivo and in vitro studies. Biofilms on gold and amalgam are thick and fully covering, but barely viable.

It has been reported that mercury liberated from fresh amalgam alloy reduced some metabolic activities of plaque biofilms in vitro , but it did not affect biofilm formation . The thickness of biofilm formation on amalgam achieved 17 μm in five days . Toxic compounds could be released from amalgam and affect the bacteria distribution in the biofilm. The amount of mercury-resistant bacteria remained elevated for a period of 48 h, but the proportions returned to baseline levels after 72 h. Ready et al. revealed that 98% of the mercury-resistant bacterial strains isolated were streptococci, with S. mitis predominating. Moreover, plaque growing in the presence of amalgam could assimilate mercury, and biofilms appeared to facilitate mercury release in vivo . Aged amalgam with an undisturbed passive tarnish layer appeared unaffected by biofilm and did not liberate mercury . Biofilm with extremely low viability was found on gold, probably due to its inert property which is unfavorable to the biofilm growth or specific binding .

The use of titanium alloy implants has become a routine procedure in dentistry to replace missing teeth. If microorganisms are able to gain access to the implant site, they can remain in a dormant state for several years and develop into a biofilm with clinical signs of infections when the host is immunocompromised . Biofilms may behave differently depending on the microbial species and varying textures of the titanium surface . An investigation evaluated the adherence of six microbial species ( Actinomyces naeslundii , Vitellariopsis dispar , Fusobacterium nucleatum , S. sobrinus , S. oralis and Candida albicans ) on titanium surfaces crafted in different ways (machined, stained, acid-etched, or sandblasted + acid-etched) and found that V. dispar and C. albicans exhibited strongest adherence . The lowest adherence was observed on stained titanium and on surfaces with a roughness of less than 0.2 μm, and the highest adherence was observed on sandblasted + acid-etched surfaces with greater roughness . However, in contrast, another recent study concluded that surface roughness does not influence the total area of the biofilm covering the surface between machined and cast pure titanium . The variation between different studies may be due to different surface modification methods and incubation conditions for biofilm growth.

2.1

Dental hard tissue

Human dental plaque is one of the most natural forms of biofilm growth . It is constantly exposed to environmental challenges and therefore has served as a general model to study the formation of biofilms . It has been established that plaque’s biofilm-forming capacity and structural organization are influenced by the chemical nature of the substrate . Bacteria that cannot adhere to a surface are transported by salivary flow down to the digestive tract.

All biofilms on the tooth surface have at least one characteristic in common: the presence of a bacterial-derived matrix that envelopes the bacteria and helps to facilitate the formation of multicellular structures that become firmly attached to the dental hard tissue . Oral bacterial attachment on enamel and dentin starts with the recognition of salivary pellicle receptors by initial colonizing bacteria on the tooth surface . The extracellular polymeric substance (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms . Starting at the bottom, initial colonizers bind to complementary salivary receptors in the acquired pellicle. Late colonizers bind to previously bound bacteria and to EPS as a coating on the tooth surface .

Enamel is sparsely colonized by multispecies communities in the first eight hours after cleaning , while under equal conditions, Nyvad and Fejerskov revealed that root surfaces were more heavily colonized with the same types of bacteria. Within the first 24 h, the microbial flora was dominated by Streptococci and Gram-positive pleomorphic rods. Streptococcus sanguis contributed 6–18% of the early colonizers. Streptococcus mitis and Streptococcus oralis varied between 24–42% and 1–27% respectively. The relative proportion of S. oralis increased significantly within 24 h while the proportion of Streptococcus salivarius and arginine-positive S. mitis showed a declining tendency . Xiao et al. showed that Streptococcus mutans -produced EPS can modulate the three-dimensional architecture and population shifts during morphogenesis of biofilms using a saliva-coated hydroxyapatite (HA) disk model.

Dentin is a composite material made up of inter-penetrant inorganic fraction (around 70 wt%) and collagen (around 20 wt%). Type I collagen is the major organic component (90%) of dentin. It is known that certain bacteria can attach to type I collagen in dentin through the expression of surface adhesins and form biofilms . Dentinal tubules are the tunnels for bacteria invasion. With long-term observation in vivo or incubation in vitro , biofilm bacteria has been seen to invade into dentinal tubules and induce deep infection .

A number of in vitro models have been used to study oral biofilm formation . Plaque biofilm can form on an HA disk model in 24 h ( Fig. 1 ). An in vitro study showed that the thickness of plaque biofilm grown on collagen-coated HA disks was 50 μm greater than the biofilm grown on pure HA disks using SEM . Single species Enterococcus faecalis was capable of forming biofilm on different substrates, and HA presented the highest rates of biofilm formation ( Fig. 2 ). Kishen et al. highlighted the sequence of events in the interaction of E. faecalis biofilm with a dentin substrate. E. faecalis first formed biofilm on the root canal dentin, and then the bacteria induced dissolution of the mineral fraction from the dentin. Finally, a reprecipitated apatite layer was formed in the biofilm structure. The ability of E. faecalis to form calcified biofilm may be a factor that contributes to their persistence after endodontic treatment .

SEM micrographs of dental plaque biofilm formation on hydroxyapatite disk in anaerobic condition. (A) 4 h after incubation, (B) 12 h after incubation, (C) 24 h after incubation and (D) 7 days after incubation.

SEM micrographs of E. faecalis biofilm formation on hydroxyapatite disk in anaerobic condition. (A) 4 h after incubation, (B) 12 h after incubation, (C) 24 h after incubation, and (D) 7 days after incubation.

Recently, a novel dentin infection model was introduced to allow a standardized and deep penetration of E. faecalis biofilms into dentinal tubules using the power of centrifugation , which allows standardized measurements of live/dead bacteria under CLSM to study the effectiveness of dentin disinfection. Bacteria packed in the dentinal tubules could be observed by both CLSM and SEM ( Fig. 3 ). This method may have great potential to study the antibiofilm effect of different dental materials applied on dentin, e.g. root canal sealers, endodontic cements, composite resins.

Three-week E. faecalis infection of dentin by centrifugation. (A) SEM micrographs of E. faecalis blocking dentinal tubules from dentin surface view, (B) SEM micrographs of E. faecalis infected dentinal tubules at high magnification, (C) SEM micrographs of E. faecalis infected dentinal tubules at low magnification, and (D) three-dimensional reconstructions of confocal laser scanning microscope (CLSM) images of infected dentinal tubules after live/dead viability staining (green fluorescence: live bacterial cells; red fluorescence: dead bacterial cells).

2.2

Ceramics

Ceramic materials are low-adhesive materials due to their inherent surface characteristics. Biofilms on ceramics are thin and highly viable . Compositional and microstructural differences between different ceramics may, however, influence the surface properties and hence the reactions between the material and oral microbial environment.

Biofilm formation on various types of dental ceramics ( e.g. veneering glass–ceramic, lithium disilicate glass–ceramic, yttrium-stabilized zirconia) for different purposes differ significantly . In particular, zirconia ceramic exhibits low plaque accumulation, and displays similar bacterial binding properties to titanium . In three days, the surface of two types of ceramic inlays collected less plaque with reduced viability under conditions of no oral hygiene than did the natural tooth surface . Compared with gold and amalgam which attracted up to 17-μm-thick biofilms, a 6-μm-thick biofilm on ceramics took five days to form in vivo . Lindel et al. indicated that ceramic brackets exhibit less long-term biofilm accumulation than metal orthodontic brackets. Another in situ study used an oral device to hold dental ceramics in volunteers’ mouths to evaluate biofilm formation on the surface of ceramics after dentifrice brushing . The result showed that cocci and short rods were the predominant microbial morphotypes on the feldspar ceramics. However, although brushing with dentifrice could roughen the ceramic surfaces, the increase in roughness did not significantly facilitate biofilm formation. Apparently, low biofilm accumulation makes ceramic a promising material for various indications.

2.3

Resin composites

Resin-based composites are complex materials that consist of a hydrophobic resin matrix and less hydrophobic filler particles, which implies that a resin-based composite surface is never a homogeneous interface but rather one that produces matrix-rich and filler-poor areas, as well as matrix-poor and filler-rich areas . Biofilms on composites can cause surface deterioration.

Polishing, as well as differences in the composition of the resin-based composite, may have an impact on biofilm formation on the resin-based composite surface . Surface deterioration of resin composites induced by polishing leads to increased roughness, changes in microhardness, and filler particle exposure upon exposure to biofilms in vitro . Significantly less S. mutans biofilm formation was observed on polished resin-based composites than on unpolished resin-based composites during a four-day incubation , indicating that the proportions of resin matrix and filler particles on the surface strongly influence S. mutans biofilm formation in an artificial mouth system. Another investigation confirmed that the quantity of retained S. mutans biofilm was significantly less on the surface of diamond paste polished resin composite than on a regular silicon paper polished surface in the same in vitro system . S. mutans outgrowth was accelerated following direct contact with the surface of an aged resin composite without affecting the microhardness of the composite . A similar study showed an increase in surface roughness of restorative resin after one month’s exposure to S. mutans biofilms .

Passariello and Gigola showed that composite brackets are more susceptible to adhesion and colonization by Streptococci biofilm, while other types of tested brackets did not show differences that could be clinically relevant, suggesting that the exposure of composite resins may enhance the growth of cariogenic bacteria . The enhanced growth of bacteria may be because the conversion of resin composites was not completed. It has been suggested that the release of ethyleneglycol dimethylacrylate and triethyleneglycol dimethacrylate from composite resins contribute to the growth of cariogenic bacteria like Streptococcus sobrinus and Lactobacillus acidophilus .

In several more specific studies, results showed that resin composite may enhance the glucosyltransferase activity of the bacteria ; another investigation found that triethyleneglycol modulates the expression levels of glucosyltransferase B involved in biofilm formation . Interestingly, Takahashi et al. indicated that growth stimulation of S. Sobrinus and S. Sanguis by ethyleneglycol dimethylacrylate monomers as measured by optical density was not accompanied by an increase in the numbers of colony forming units (CFU), but as the increase in the amount of bacteria confirmed by the presence of a vesicular material surrounding the bacteria. In contrast, biomaterial surface chemistry could also negatively affect biofilm formation. Most notably, polyethylene oxide (PEO) significantly inhibited Staphylococcus epidermidis biofilm formation over 48 h in vitro . The hydrophilic properties of PEO created flexible, mobile chains and therefore sterically hinder bacterial adhesion by diminishing the probability of contact between the bacteria and the hydrophobic polymer base.

2.4

Metallic alloys

The adhesion of oral biofilm on metallic dental material surfaces ( e.g. gold, amalgam, titanium and zirconia) has been reported both in vivo and in vitro studies. Biofilms on gold and amalgam are thick and fully covering, but barely viable.

It has been reported that mercury liberated from fresh amalgam alloy reduced some metabolic activities of plaque biofilms in vitro , but it did not affect biofilm formation . The thickness of biofilm formation on amalgam achieved 17 μm in five days . Toxic compounds could be released from amalgam and affect the bacteria distribution in the biofilm. The amount of mercury-resistant bacteria remained elevated for a period of 48 h, but the proportions returned to baseline levels after 72 h. Ready et al. revealed that 98% of the mercury-resistant bacterial strains isolated were streptococci, with S. mitis predominating. Moreover, plaque growing in the presence of amalgam could assimilate mercury, and biofilms appeared to facilitate mercury release in vivo . Aged amalgam with an undisturbed passive tarnish layer appeared unaffected by biofilm and did not liberate mercury . Biofilm with extremely low viability was found on gold, probably due to its inert property which is unfavorable to the biofilm growth or specific binding .

The use of titanium alloy implants has become a routine procedure in dentistry to replace missing teeth. If microorganisms are able to gain access to the implant site, they can remain in a dormant state for several years and develop into a biofilm with clinical signs of infections when the host is immunocompromised . Biofilms may behave differently depending on the microbial species and varying textures of the titanium surface . An investigation evaluated the adherence of six microbial species ( Actinomyces naeslundii , Vitellariopsis dispar , Fusobacterium nucleatum , S. sobrinus , S. oralis and Candida albicans ) on titanium surfaces crafted in different ways (machined, stained, acid-etched, or sandblasted + acid-etched) and found that V. dispar and C. albicans exhibited strongest adherence . The lowest adherence was observed on stained titanium and on surfaces with a roughness of less than 0.2 μm, and the highest adherence was observed on sandblasted + acid-etched surfaces with greater roughness . However, in contrast, another recent study concluded that surface roughness does not influence the total area of the biofilm covering the surface between machined and cast pure titanium . The variation between different studies may be due to different surface modification methods and incubation conditions for biofilm growth.

3.1

Interference with bacterial attachment and metabolism

Embedding different compositions of antimicrobial ions is the most common strategy to endow dental materials with antibiofilm properties. Fluoride, an anticariogenic agent, is assumed to affect caries formation and can be released from dental restorative materials, e.g. glass-ionomer cements , resin composite , toothpastes . The possible mechanisms by which fluoride may interfere with bacterial metabolism and acidogenicity of plaque biofilm include the inhibition of the glycolytic enzyme enolase and the proton-extruding ATPase as well as the bacterial colonization and competition . Moreover, intracellular or plaque-associated enzymes such as acid phosphatase, pyrophosphatase, peroxidase and catalase may be affected by fluoride ions .

The use of silver particles has emerged for diverse dental applications ranging from silver based restorative material to silver coated implants . Silver ions released from dental materials in aqueous solution can attach to the bacterial membrane and penetrate biofilm, which causes bacterial inactivation and prevents bacterial replication by binding to microbial DNA and to the sulfhydryl groups of the metabolic enzymes in the bacterial electron transport chain .

Zinc oxide (ZnO) also displays antibiofilm properties by releasing oxygen and zinc ions. It has been demonstrated that active oxygen species, e.g. H ₂ O ₂ can inhibit growth of planktonic microbes . Another potential antimicrobial mechanism of ZnO occurs via the leaching of Zn ²⁺ into the growth medium , inhibiting the active transport and metabolism of sugars as well as disrupting enzyme systems of dental biofilms by exchanging magnesium ions essential for enzymatic activity of the plaque . Zinc can also reduce acid production by S. mutans biofilms due to its ability to inhibit glucosyl transferase activity .

Quaternary ammonium-containing composite resin has been introduced to provide resin-based dental materials with antibiofilm activity via the release of cationic monomers . Cationic monomers have been reported to suppress the expression of gtf genes, which is beneficial to plaque biofilm accumulation. The suppressed expression of gtf genes might directly reduce the amounts of biofilm-building enzymes, therefore decreasing extracellular polysaccharide matrix (EPS) formation and bacterial adherence . Since the deposition of EPS and the attachment of bacteria are compromised, the accumulation of biofilm will be reduced .

Another antibiofilm agent widely used as a releasing material in dental practice is chlorhexidine (CHX). Chlorhexidine has been incorporated into restorative materials and titanium implant coatings to reduce dental biofilm formation . The CHX molecule reacts with negatively charged groups on the cell surface, causing an irreversible loss of cytoplasmic constituents, membrane damage, and enzyme inhibition . It is likely that ionic interactions occur between the positively charged CHX molecules and the negatively charged extracellular matrix of the biofilm. At sublethal concentrations, chlorhexidine can interfere with the metabolism of oral biofilm by inhibiting sugar transport, acid production, and various membrane functions in streptococci. Attempts have been made to deliver enzymes such as dextranases and glucanases in oral care products in order to disrupt the structure of the biofilm by destroying the EPS .

3.2

Biomineralisation approach

An ideal antimicrobial dental material would combine high silica delivery, high pH, and a high alkaline buffer capacity . The antibiofilm effects of current bioceramic dental materials may be due to the biomineralisation process induced by calcium silicates/phosphates . Moisture from the environment promotes the hydration reaction to produce calcium silicate hydrogel and calcium hydroxide to elevate the pH . Silica dissolved in a high-pH environment may directly inhibit bacterial viability . Calcium hydroxide subsequently reacts with the phosphate to form hydroxyapatite and water. This water is supposed to participate in the reaction cycle again to produce more calcium silicate hydrogel and calcium hydroxide . The continuous diffusion of calcium hydroxide may thus be responsible for the killing of bacteria.

4.1

Root canal sealer

4.1.1

Resin-based sealer

AH Plus (Dentsply International Inc., York, PA) and AH 26 (Dentsply, Weybridge, UK) are epoxy resin-based sealers used in endodontics. The antibiofilm effect of epoxy resin-based sealers has been reported to be controversial. Most studies showed that set AH Plus did not inhibit bacterial biofilm growth . Two in vitro studies found that AH Plus was ineffective against planktonic E. faecalis using a direct contact test (DCT) in both fresh and set conditions . AH Plus and AH 26 showed significantly higher CFU counts than Sealapex (SybronEndo, Orange, CA) for both 2-day and 7-day set samples . Zhang et al. showed that AH Plus lost most of its antibiofilm effect after a 1-day set. However, freshly mixed AH Plus and AH 26 have been reported to inhibit the growth of planktonic E. faecalis . One study reported complete inhibition of bacterial growth treated by 1-day set AH Plus, but the killing effect disappeared in the 7-day set samples . The inhibition of bacterial growth is possibly due to the bactericidal effect of formaldehyde, small quantities of which are released during the setting process. New materials, e.g. Epiphany SE sealer (Parkell, Farmington, NY), aiming to promote better adhesion between the filling materials and the root canal dentin walls, however, did not show a prominent antibiofilm effect . EndoRez (Ultradent Corp., South Jordan, UT), a new composite based sealer, suppressed the growth of F. nucleatum and Porphyromonas gingivalis in one study , and showed similar antibacterial activity against planktonic E. faecalis and Staphylococcus aureus in another two investigations . EndoRez continued to be effective for three and seven days after mixing .

4.1.2

Eugenol-based sealer

Eugenol-based sealers exhibited antimicrobial properties at consistencies with freshly mixed and set samples. Fuss et al. indicated complete inhibition of bacterial growth by two eugenol-based sealers, Roth’s cement (Roth International Ltd., Chicago, IL) and CRCS (Hygenic, Akron, OH). Roth’s cement extended its antibacterial effect over seven days after setting . Another two zinc oxide eugenol-based sealers, Pulp canal sealer EWT (Kerr Corp., Romulus, MI) and Endomethasone (Septodont, Saint-Maur, France), showed bacterial suppression in 24 h against E. faecalis biofilm . In the same study, only one zinc oxide-based sealer containing orthophenilphenol (Vcanalare; Vebas, San Giuliano Milanese, Italy) still inhibited bacterial growth seven days after mixing . A newly developed zinc oxide-eugenol based sealer, Hermetic (Lege Artis Pharma GmbH & Co., Germany), had a suppressing effect on three endodontologically detectable species using DCT and yielded the largest inhibition zones in the agar diffusion test (ADT) . The antimicrobial effect of eugenol-based sealer may be caused by the high concentration of eugenol or a function of pH. However, one study indicated that the pulp canal sealer EWT did not have significant inhibition of bacterial growth, probably due to the different strain of E. faecalis or the formulation of the sealer used .

4.1.3

Bioceramic sealer

EndoSequence BC sealer (Brasseler, Savannah, GA; also known as iRoot SP, Innovative Bioceramix, Vancouver, Canada) is a new endodontic sealer, chemically based on Bioaggregate, a bioceramic root-end filling material . BC sealer is a complex form of calcium silicate cement, calcium phosphate, and calcium oxide. Fresh iRoot SP has been recognized as an effective antimicrobial agent against E. faecalis , and the killing effect continued for three days after mixing, with the pH remaining the highest of all sealers tested. However, the antimicrobial effect was greatly diminished at seven days after mixing.

Similar to the BC sealer, the MTA Fillapex (Angelus Solucoes Odontologicas, Londrina, PR, Brazil) is a calcium silicate-based bioceramic sealer with high solubility, pH and calcium ion release. The composition of MTA Fillapex after mixture is basically mineral trioxide aggregate, salicylate resin, natural resin, bismuth, and silica. Morgental et al. showed that freshly mixed MTA Fillapex and another MTA based sealer (Endofill) had an antibacterial effect against E. faecalis , but none of the sealers maintained antibacterial activity after setting . However, in a dentin block study, MTA Fillapex has been reported to have antibiofilm activity against E. faecalis seven days post-manipulation . Another MTA based sealer (MTA-S, Araraquara, Brazil) did not show significant bacterial suppression, though the increase of pH was found two days after manipulation .

4.1.4

Other endodontic sealers

Other types of endodontic sealers, e.g. calcium-hydroxide based (Sealapex, Calxyl, Gangraena-Merz, Apexit plus, etc. ) and glass-ionomer based (Ketac Endo, etc. ), also have been reported to have efficacy against microorganisms. Sealapex (KerrHawe S.A., Bioggio, Switzerland) displayed an antibacterial effect against different microorganism strains, e.g. F. nucleatum and P. gingivalis in both the freshly mixed and the set state by ADT . Duarte et al. reported that Sealapex presented higher calcium and hydroxyl release than Apexit Plus, especially after longer time intervals of 30 days. As a result, Sealapex displayed consistent antimicrobial activity in its set state as reported by different studies . At seven days post-manipulation, Sealapex showed the greatest antibiofilm action among seven different sealers tested . The levels of antibacterial activity detected with the Apexit Plus (Ivoclar Vivadent GmbH, Ellwangen, Germany), Gangraena-Merz (Merz Dental, Lütjenburg, Germany) and Calxyl (OCO-Präparate, Dirmstein, Germany) were generally low . Apexit Plus showed a slight suppressive effect on P. gingivalis in the ADT test only . Gangraena-Merz and Calxyl displayed no antibacterial effect on three bacterial species. Various other studies also indicate that sealers based on calcium hydroxide show but slight antibacterial activity .

Antibiofilm studies on glass ionomer sealers are limited . A glass ionomer endodontic sealer, Ketac Endo (Espe GMBH & Co. KG, Seefeld/Oberbay, Germany), possessed a short-acting potent and diffusible antibacterial activity against E. faecalis . Freshly mixed Ketac Endo exhibited a twofold greater inhibition zone than Roth’s cement in an ADT test, and completely inhibited bacterial growth in the DCT test .

4.1.5

New approach to study the antibiofilm activity of endodontic sealer

The effect of sealers on microbial viability has been mostly examined by ADT or DCT, showing decreasing antibacterial effect within a few hours or days from mixing. Limitations exist with the ADT and DCT method as they do not provide opportunity for the consideration of factors such as microanatomy and chemistry of the tooth and biofilm formation . The survival of bacteria may be partly attributed to their invasion into dentinal tubules where biofilms can be formed and thus bacteria in dentin may be protected from the direct antibacterial effect of sealers.

A non-destructive in situ dentin infection model as mentioned in Section 2.1 has been introduced to study the effectiveness of endodontic sealers against bacteria inside heavily infected dentinal tubules . Three sealers have been proved to have significant antibacterial effect against E. faecalis . Interestingly, the killing of bacteria deep in the dentin by AH Plus and BC sealer continued and increased over time throughout the 30-day experiment, finally resulting in almost half the bacteria being killed ( Fig. 4 ).

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