Biofilm Colony-forming Units Research Articles

Novel strategy of S. mutans gcrR gene over-expression plus antibacterial dimethylaminohexadecyl methacrylate suppresses biofilm acids and reduces dental caries in rats

ObjectiveStreptococcus mutans (S. mutans) is a major contributor to dental caries, with its ability to synthesize extracellular polysaccharides (EPS) and biofilms. The gcrR gene is a regulator of EPS synthesis and biofilm formation. The objectives of this study were to investigate a novel strategy of combining gcrR gene over-expression with dimethylaminohexadecyl methacrylate (DMAHDM), and to determine their in vivo efficacy in reducing caries in rats for the first time. MethodsTwo types of S. mutans were tested: Parent S. mutans; and gcrR gene over-expressed S. mutans (gcrR OE S. mutans). Bacterial minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were measured with DMAHDM and chlorhexidine (CHX). Biofilm biomass, polysaccharide, lactic acid production, live/dead staining, colony-forming units (CFUs), and metabolic activity (MTT) were evaluated. A Sprague-Dawley rat model was used with parent S. mutans and gcrR OE S. mutans colonization to determine caries-inhibition in vivo. ResultsDrug-susceptibility of gcrR OE S. mutans to DMAHDM or CHX was 2-fold higher than that of parent S. mutans. DMAHDM reduced biofilm CFU by 3–4 logs. Importantly, the combined gcrR OE S. mutans+ DMAHDM dual strategy reduced biofilm CFU by 5 logs. In the rat model, the parent S. mutans group had a higher cariogenicity in dentinal (Dm) and extensive dentinal (Dx) regions. The DMAHDM + gcrR OE group reduced the Dm and Dx caries to only 20 % and 0 %, those of parent S. mutans + PBS control group (p < 0.05). The total caries severity of gcrR OE + DMAHDM group was decreased to 51 % that of parent S. mutans control (p < 0.05). SignificanceThe strategy of combining S. mutans gcrR over-expression with antibacterial monomer reducing biofilm acids by 97 %, and reduced in vivo total caries in rats by 48 %. The gcrR over-expression + DMAHDM strategy is promising for a wide range of dental applications to inhibit caries and protect tooth structures.

Dual function of anti-biofilm and modulating biofilm equilibrium of orthodontic cement containing quaternary ammonium salt.

The objectives of this study were to incorporate dimethylaminohexadecyl methacrylate (DMAHDM) into resin-modified glass ionomer cement (RMGI) to develop a novel orthodontic cement which endowed RMGI with strong antibacterial ability and investigated its modulation biofilm equilibrium from cariogenic state to non-cariogenic state for the first time. Cariogenic Streptococcus mutans (S. mutans), and non-cariogenic Streptococcus sanguinis (S. sanguinis) and Streptococcus gordonii (S. gordonii) were selected to form a tri-species biofilm model. RMGI incorporated with different mass fraction of DMAHDM was examined: biofilm colony-forming units, metabolic activity, live/dead staining, lactic acid and exopolysaccharides productions. TaqMan real-time polymerase chain reaction was used to determine changes of biofilm species compositions. The results showed RMGI containing 3% DMAHDM achieved strong antibacterial ability and suppressed the cariogenic species in biofilm, modulating biofilm equilibrium from cariogenic state to non-cariogenic state tendency. The novel bioactive cement containing DMAHDM is promising in fixed orthodontic treatments and protecting tooth enamel.

Novel protein-repellent and antibacterial polymethyl methacrylate dental resin in water-aging for 6 months

BackgroundThe present study aimed to develop a novel protein-repellent and antibacterial polymethyl methacrylate (PMMA) dental resin with 2-methacryloyloxyethyl phosphorylcholine (MPC) and quaternary ammonium dimethylaminohexadecyl methacrylate (DMAHDM), and to investigate the effects of water-aging for 6 months on the mechanical properties, protein adsorption, and antibacterial activity of the dental resin.MethodsFour groups were tested: PMMA control; PMMA + 3% MPC; PMMA + 1.5% DMAHDM; and PMMA + 3% MPC + 1.5% DMADDM in acrylic resin powder. Specimens were water-aged for 1 d, 3 months, and 6 months at 37 ℃. Their mechanical properties were then measured using a three-point flexure test. Protein adsorption was measured using a micro bicinchoninic acid (BCA) method. A human saliva microcosm model was used to inoculate bacteria on water-aged specimens and to investigate the live/dead staining, metabolic activity of biofilms, and colony-forming units (CFUs).ResultsThe flexural strength and elastic modulus showed a significant loss after 6 months of water-ageing for the PMMA control (mean ± SD; n = 10); in contrast, the new protein repellent and antibacterial PMMA resin showed no strength loss. The PMMA–MPC–DMAHDM-containing resin imparted a strong antibacterial effect by greatly reducing biofilm viability and metabolic activity. The biofilm CFU count was reduced by about two orders of magnitude (p < 0.05) compared with that of the PMMA resin control. The protein adsorption was 20% that of a commercial composite (p < 0.05). Furthermore, the PMMA–MPC–DMAHDM-containing resin exhibited a long-term antibacterial performance, with no significant difference between 1 d, 3 months and 6 months (p > 0.05).ConclusionsThe flexural strength and elastic modulus of the PMMA–MPC–DMAHDM-containing resin were superior to those of the PMMA control after 6 months of water-ageing. The novel PMMA resin incorporating MPC and DMAHDM exhibited potent and lasting protein-repellent and antibacterial properties.

Plasma treatment effects on destruction and recovery of Porphyromonas gingivalis biofilms.

The objective of this study was to investigate the treatment effects of non-thermal atmospheric gas plasmas (NTAP) on destruction and the recovery (or re-colonization) of Porphyromonas gingivalis (P. gingivalis) in biofilms. P. gingivalis is a well-known keystone periodontal pathogen strongly associated with periodontal diseases, especially periodontitis. P. gingivalis biofilms were formed on stainless steel coupons and treated for 1, 2, and 5 minutes by NTAP of pure argon gas and argon+oxygen gas mixture. MTT assay, colony forming unit (CFU) counting assay and confocal laser scanning microscopy (CLSM) were used to assess the destruction efficiency. In addition, the plasma treated biofilms were re-cultured in the medium supplemented with antibiotics and oxidative stress sources to determine the synergy of the NTAP with other antimicrobial agents. The results showed the plasma treatment could result in 2.7 log unit reduction in bacterial load. The recovered biofilm CFU with NTAP treatment combined with sub minimal inhibition concentration of amoxicillin was 0.33 log units less than the biofilm treated with amoxicillin alone. The recovered biofilm CFU in NTAP groups was about 2.0 log units less than that in the untreated controls under H2O2 treatment. There was approximately 1.0 log unit reduction of biofilm CFU in plasma treated biofilm compared with untreated control under paraquat treatment. The plasma treated biofilms exhibited less resistance to amoxicillin and greater susceptibility to hydrogen peroxide (H2O2) and paraquat, suggesting that NTAP may enhance biofilm susceptibility to host defense. These in vitro findings suggested that NTAP could be a novel and effective treatment method of oral biofilms that cause periodontal diseases.

Flavonoid Baicalein Suppresses Oral Biofilms and Protects Enamel Hardness to Combat Dental Caries.

The objectives of this study were to investigate the effects of a novel method using flavonoids to inhibit Streptococcus mutans (S. mutans), Candida albicans (C. albicans) and dual-species biofilms and to protect enamel hardness in a biofilm-based caries model for the first time. Several flavonoids, including baicalein, naringenin and catechin, were tested. Gold-standard chlorhexidine (CHX) and untreated (UC) groups served as controls. Optimal concentrations were determined by cytotoxicity assay. Biofilm MTT, colony-forming-units (CFUs), biofilm biomass, lactic acid and polysaccharide production were evaluated. Real-time-polymerase-chain reaction (qRT-PCR) was used to determine gene expressions in biofilms. Demineralization of human enamel was induced via S. mutans-C. albicans biofilms, and enamel hardness was measured. Compared to CHX and UC groups, the baicalein group achieved the greatest reduction in S. mutans, C. albicans and S. mutans-C. albicans biofilms, yielding the least metabolic activity, polysaccharide synthesis and lactic acid production (p < 0.05). The biofilm CFU was decreased in baicalein group by 5 logs, 4 logs, 5 logs, for S. mutans, C. albicans and S. mutans-C. albicans biofilms, respectively, compared to UC group. When tested in a S. mutans-C. albicans in vitro caries model, the baicalein group substantially reduced enamel demineralization under biofilms, yielding an enamel hardness that was 2.75 times greater than that of UC group. Hence, the novel baicalein method is promising to inhibit dental caries by reducing biofilm formation and protecting enamel hardness.

Mechanistic Effects of E-Liquids on Biofilm Formation and Growth of Oral Commensal Streptococcal Communities: Effect of Flavoring Agents.

Background: Vaping has become a global health concern. As research continues, more studies are beginning to question the relative safety of E-liquid flavoring additives. The oral cavity is the first site of exposure to E-liquid aerosol, making it critical for investigation. Because of the importance of commensal bacterial biofilms for oral health, we sought to explore the effects of E-liquids ± flavors on the formation and growth of single- and multi-species biofilms and to investigate the mechanism of inhibition. Methods: Quantitative and confocal biofilm analysis, death curves, and colony-forming units (CFU) were evaluated with flavorless and flavored (tobacco, menthol, cinnamon, strawberry, blueberry) E-liquids using four strains of oral commensal bacteria (Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis, and Streptococcus oralis). Results: All flavoring agents show a dose-dependent inhibition in the growth of single-species and multi-species biofilms. Furthermore, CFUs, death curves, and light microscopy show that flavoring agents have a bactericidal mode of inhibition on the growth of these oral streptococci. Conclusions: These results show that flavored, rather than unflavored, E-liquids are more detrimental to biofilm formation and growth of oral commensal bacteria. Consequently, E-liquid flavorings agents could pose risks to the oral microenvironment, and by extension, to systemic health.

Luteolin Inhibits the Biofilm Formation and Cytotoxicity of Methicillin-Resistant Staphylococcus aureus via Decreasing Bacterial Toxin Synthesis.

Owing to the fact that luteolin has antibacterial activity against Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), its specific mechanism in MRSA is worthy of investigation, which is the focus of this study. Initially, the collected S. aureus strains were treated with luteolin. Then, the minimum inhibitory concentration (MIC) of luteolin against the S. aureus strains was measured by the broth microdilution. The growth curves, biofilm formation, and cytotoxicity of treated S. aureus were detected using a microplate reader. The live and dead bacteria were evaluated using confocal laser scanning microscopy, the bacterial morphology was observed using scanning electron microscopy, and the S. aureus colony-forming unit (CFU) numbers were assessed. The levels of alpha hemolysin (α-hemolysin), delta hemolysin (δ-hemolysin), and hlaA were detected via western blot and RT-PCR. The mortality of mouse model with S. aureus systemic infection was analyzed, and the levels of IL-6, IL-8, IL-10, and TNF-α were quantitated using ELISA. Concretely, the MIC of luteolin against MRSA N315 was 64 μg/mL. Luteolin at 16 μg/mL did not affect the growth of MRSA N315, but inhibited the biofilm formation and CFU, and promoted the morphological changes and death of MRSA N315. Luteolin decreased the cytotoxicity and the levels of α-hemolysin, δ-hemolysin, and hlaA in MRSA N315, elevated MRSA-reduced mice survival rate, and differentially modulated the inflammatory cytokine levels in MRSA-infected mice. Collectively, luteolin inhibits biofilm formation and cytotoxicity of MRSA via blocking the bacterial toxin synthesis.

Novel Nano Calcium Fluoride Remineralizing and Antibacterial Dental Composites

ObjectiveComposites with remineralizing and antibacterial properties are favorable for caries inhibition. The objectives of this study were to develop a new bioactive nanocomposite with remineralizing and antibiofilm properties by incorporating dimethylaminohexadecyl methacrylate (DMAHDM) and nano-calcium fluoride (nCaF2). MethodsnCaF2 was produced via a spray-drying method and integrated at 15% mass fraction into composite. DMAHDM was added at 3% mass fraction. Mechanical properties and F and Ca ion releases were assessed. Colony-forming units (CFU), lactic acid and metabolic activity of biofilms on composites were performed. ResultsThe new composites had flexural strengths of (95.28±6.32) MPa and (125.93±7.49) MPa, which were within the ISO recommendations. Biofilm CFU were reduced by 3–4 log (p<0.05). The composites achieved high F releases of (0.89±0.01) mmol/L and (0.44±0.01) mmol/L, and Ca releases of (1.46±0.05) mmol/L and (0.54±0.005) mmol/L. ConclusionsNew nanocomposites were developed with good mechanical properties, potent antibacterial activity against salivary biofilms, and high F and Ca ion releases with potential for remineralization. Clinical SignificanceNovel nanocomposites using nCaF2 and DMAHDM were developed with potent antibacterial and remineralizing effects and high F and Ca ion releases. They are promising to inhibit recurrent caries, promote remineralization, and possess long-term sustainability.

Novel calcium phosphate ion-rechargeable and antibacterial adhesive to inhibit dental caries.

This study aimed to develop an antibacterial and calcium (Ca) and phosphate (P) rechargeable adhesive and investigate the effects of dimethylaminododecyl methacrylate (DMAHDM) and nanoparticles of amorphous calcium phosphate (NACP) on dentin bonding, biofilm response, and repeated Ca and P ion recharge and re-release capability for the first time. Pyromellitic glycerol dimethacrylate (PMGDM), ethoxylated bisphenol A dimethacrylate (EBPADMA), 2-hydroxyethyl methacrylate (HEMA), and bisphenol A glycidyl dimethacrylate (BisGMA) formed the adhesive (PEHB). Three groups were tested: (1) Scotchbond (SBMP, 3M) control, (2) PEHB + 30% NACP, and (3) PEHB + 30% NACP + 5% DMAHDM. Specimens were tested for dentin shear bond strength, and Ca and P ion release, recharge, and re-release. Biofilm lactic acid production and colony-forming units (CFU) on resins were analyzed. The four groups had similar dentin shear bond strengths (p > 0.1). Adhesive with DMAHDM showed significant decrease in metabolic activity, lactic acid production, and biofilm CFU (p < 0.05). The adhesives containing NACP released high levels of Ca and P ions initially and after being recharged. This study developed the first Ca and P ion-rechargeable and antibacterial adhesive, achieving strong antibacterial activity and Ca and P ion recharge and re-release for long-term remineralization. Considering the restoration-tooth bonded interface being the weak link and recurrent caries at the margins being the primary reason for restoration failures, this novel calcium phosphate-rechargeable and antibacterial adhesive is promising for a wide range of tooth-restoration applications to inhibit caries.

Long-term antibacterial activity and cytocompatibility of novel low-shrinkage-stress, remineralizing composites

A low-shrinkage-stress (LSS), antibacterial and remineralizing nanocomposite was recently developed; however, validation of its long-term antibacterial potency in modulating human salivary-derived biofilm is an unmet need. This study aimed to evaluate the antibacterial effect of the bioactive LSS composite before and after aging in acidic solution for 90 days using a multi-species biofilm model, and to evaluate its cytotoxicity. The LSS composite consisted of urethane dimethacrylate (UDMA) and triethylene glycol divinylbenzyl ether (TEG-DVBE), 3% dimethylaminohexadecyl methacrylate (DMAHDM) and 20% nanoparticles of amorphous calcium phosphate (NACP). Biofilm colony-forming units (CFU), lactic acid production, and confocal laser scanning microscopy (3D biofilm) were evaluated before and after three months of aging. Cytotoxicity was assessed against human gingival fibroblasts (HGF). The new LSS composite presented the lowest biofilm CFU, lactic acid and biofilm biomass, compared to controls (n = 6, p < 0.05). Importantly, the new composite exhibited no significant difference in antibacterial performance before and after 90-day-aging, demonstrating long-term antibacterial activity (p > 0.1). The LSS antibacterial and remineralizing composite presented a low cell viability at original extract that has increased with further dilutions. In conclusion, this study spotlighted that the new bioactive composite not only had a low shrinkage stress, but also down-regulated the growth of oral biofilms, reduced acid production, maintained antibacterial activity after the 90-day-aging, and did not compromise the cytocompatibility.

Anti-caries nanostructured dental adhesive reduces biofilm pathogenicity and raises biofilm pH to protect tooth structures

The objectives were to: (1) develop a new nanostructured dental adhesive with antibiofilm and remineralization properties using dimethylaminohexadecyl methacrylate (DMAHDM) and nanoparticles of calcium fluoride (nCaF2); (2) investigate the effects on dentin bond strength and Streptococcus mutans biofilms. Bisphenol A glycidyl dimethacrylate, triethylene glycol dimethacrylate, 2-hydroxyethyl methacrylate, and methacryloyloxy ethyl phthalate formed the adhesive. Biofilm colony-forming units (CFU), metabolic activities, biomass, and acid-production were measured. Adding nCaF2 and DMAHDM into adhesive did not compromise the dentin bond strength (p > 0.1). The nCaF2 + DMAHDM-containing adhesive decreased biofilm CFU by 4 logs, with tenfold reduction in acid-production, compared to control (p < 0.05). The nCaF2 + DMAHDM-containing adhesive successfully shifted the acidic and cariogenic biofilm pH 4 to a safe pH of 7. In conclusion, a novel bioactive adhesive mitigated the cariogenic potential of S. mutans biofilm and raised biofilm pH to a safe level to protect tooth structures, without compromising dentin bond strength.

In Vitro Activity of Superoxide Water on Viability of Enterococcus faecalis Biofilm on Root Canal Wall.

The aim of this study was to compare the effect of root canal irrigation with superoxidized water and sodium hypochlorite on elimination of Enterococcus faecalis biofilm from the root canal walls. In this experimental study, a total of 32 extracted human central incisors were used. The crowns of all teeth were cut to length of 16 mm. After cleaning and shaping, then the specimens were sterilized in autoclave and then divided into four groups (n=8) as following: group 1 (positive control, root canal irrigation with normal saline), group 2 (negative control without biofilm), group 3 (root canal irrigation with sodium hypochlorite) and group 4 (root canal irrigation with superoxidized water). The bacterial suspension was inserted to root canals of teeth except for negative control group in order to form a microbial biofilm in incubator for 2 weeks. Then all the samples received root canal irrigation for 5 min based on their allocation. At the end, colony forming unit (CFU) was evaluated and biofilm formation and thickness was detected with scanning electron microscopy. The Kruskal Wallis and Dunn's tests were done for biofilm thickness and CFU, respectively with the level of significance set at 0.05. In negative control group no biofilm formation and CFU was present. The CFU counts and biofilm thickness were significantly different between the experimental groups (P=0.001) and both parameters were less in samples with hypochlorite irrigation compared to positive control (52.56±5.82 µm for biofilm thickness and 1.2×107 CFU) and samples irrigated with superoxidized water (2.92±1.76 µm for biofilm thickness and 5.4×104 CFU). Based on this in vitro study reduction in biofilm thickness and CFU/mL was 100% for sodium hypochlorite and for superoxidized water was 98% and 90% for reduction in biofilm thickness and CFU/mL, respectively.

Low-shrinkage-stress nanocomposite: An insight into shrinkage stress, antibacterial, and ion release properties.

The aims are: (a) To develop the first low-shrinkage-stress nanocomposite with antibacterial and remineralization capabilities through the incorporation of dimethylaminododecyl methacrylate (DMAHDM) and nanoparticles of amorphous calcium phosphate (NACP); (b) to investigate the effects of the new composite on biofilm inhibition, mechanical properties, shrinkage stress, and calcium (Ca) and phosphate (P) ion releases. The low-shrinkage-stress resin consisted of urethane dimethacrylate and triethylene glycol divinylbenzyl ether. Composite was formulated with 3% DMAHDM and 20% NACP. Mechanical properties, shrinkage stress, and degree of conversion were evaluated. Streptococcus mutans biofilm growth on composites was assessed. Ca and P ion releases were measured. The shrinkage stress of the low-shrinkage-stress composite containing 3% DMAHDM and 20% NACP was 36% lower than that of traditional composite control (p < 0.05), with similar degrees of conversion of 73.9%. The new composite decreased the biofilm colony-forming unit by 4 log orders and substantially reduced biofilm lactic acid production compared to control composite (p < 0.05). Incorporating DMAHDM to the low-shrinkage-stress composite did not adversely affect the Ca and P ion release. A novel bioactive nanocomposite was developed with low shrinkage stress, strong antibiofilm activity, and high levels of ion release for remineralization, without undermining the mechanical properties and degree of conversion.

Regulating Oral Biofilm from Cariogenic State to Non-Cariogenic State via Novel Combination of Bioactive Therapeutic Composite and Gene-Knockout.

The objectives were to investigate a novel combination of gene-knockout with antimicrobial dimethylaminohexadecyl methacrylate (DMAHDM) composite in regulating oral biofilm from a cariogenic state toward a non-cariogenic state. A tri-species biofilm model included cariogenic Streptococcus mutans (S. mutans), and non-cariogenic Streptococcus sanguinis (S. sanguinis) and Streptococcus gordonii (S. gordonii). Biofilm colony-forming-units (CFUs), lactic acid and polysaccharide production were measured. TaqMan real-time-polymerase-chain reaction was used to determine the percentage of each species in biofilm. The rnc gene-knockout for S. mutans with DMAHDM composite reduced biofilm CFU by five logs, compared to control (p < 0.05). Using parent S. mutans, an overwhelming S. mutans percentage of 68.99% and 69.00% existed in biofilms on commercial composite and 0% DMAHDM composite, respectively. In sharp contrast, with a combination of S. mutans rnc knockout and DMAHDM composite, the cariogenic S. mutans percentage in biofilm was reduced to only 6.33%. Meanwhile, the non-cariogenic S. sanguinis + S. gordonii percentage was increased to 93.67%. Therefore, combining rnc-knockout with bioactive and therapeutic dental composite achieved the greatest reduction in S. mutans, and the greatest increase in non-cariogenic species, thereby yielding the least lactic acid-production. This novel method is promising to obtain wide applications to regulate biofilms and inhibit dental caries.

Nano-calcium phosphate and dimethylaminohexadecyl methacrylate adhesive for dentin remineralization in a biofilm-challenged environment

ObjectiveDentin remineralization at the bonded interface would protect it from external risk factors, therefore, would enhance the longevity of restoration and combat secondary caries. Dental biofilm, as one of the critical biological factors in caries formation, should not be neglected in the assessment of caries preventive agents. In this work, the remineralization effectiveness of demineralized human dentin in a multi-species dental biofilm environment via an adhesive containing nanoparticles of amorphous calcium phosphate (NACP) and dimethylaminohexadecyl methacrylate (DMAHDM) was investigated. MethodsDentin demineralization was promoted by subjecting samples to a three-species acidic biofilm containing Streptococcus mutans, Streptococcus sanguinis, Streptococcus gordonii for 24h. Samples were divided into a control group, a DMAHDM adhesive group, an NACP group, and an NACP+DMAHDM adhesive group. A bonded model containing a control-bonded group, a DMAHDM-bonded group, an NACP-bonded group, and an NACP+DMAHDM-bonded group was also included in this study. All samples were subjected to a remineralization protocol consisting of 4-h exposure per 24-h period in brain heart infusion broth plus 1% sucrose (BHIS) followed by immersion in artificial saliva for the remaining period. The pH of BHIS after 4-h immersion was measured every other day. After 14 days, the biofilm was assessed for colony-forming unit (CFU) count, lactic acid production, live/dead staining, and calcium and phosphate content. The mineral changes in the demineralized dentin samples were analyzed by transverse microradiography. ResultsThe in vitro experiment results showed that the NACP+DMAHDM adhesive effectively achieved acid neutralization, decreased biofilm colony-forming unit (CFU) count, decreased biofilm lactic acid production, and increased biofilm calcium and phosphate content. The NACP+DMAHDM adhesive group had higher remineralization value than the NACP or DMAHDM alone adhesive group. SignificanceThe NACP+DMAHDM adhesive was effective in remineralizing dentin lesion in a biofilm model. It is promising to use NACP+DMAHDM adhesive to protect bonded interface, inhibit secondary caries, and prolong the longevity of restoration.

Bioactive low-shrinkage-stress nanocomposite suppresses S. mutans biofilm and preserves tooth dentin hardness

Recurrent dental caries is one of the main reasons for resin composite restoration failures. This study aimed to: (1) develop a bioactive, low-shrinkage-stress, antibacterial and remineralizing composite and evaluate the sustainability of its antibacterial effect against Streptococcus mutans (S. mutans) biofilms; and (2) evaluate the remineralization and cariostatic potential of the composite containing nanoparticles of amorphous calcium phosphate (NACP) and dimethylaminohexadecyl methacrylate (DMAHDM), using dentin hardness measurement and a biofilm-induced recurrent caries model. The antibacterial and remineralizing low-shrinkage-stress composite consisted of urethane dimethacrylate (UDMA) and triethylene glycol divinylbenzyl ether (TEG-DVBE), 3% DMAHDM and 20% NACP. S. mutans biofilm was used to evaluate antibiofilm activity, before and after 3 months of composite aging in acidic solution. Human dentin was used to develop a recurrent caries biofilm-model. Adding DMAHDM and NACP into low shrinkage-stress composite did not compromise the flexural strength. The low-shrinkage-stress composite with DMAHDM achieved substantial reductions in biofilm colony-forming units (CFU), lactic acid production, and biofilm biomass (p < 0.05). The low-shrinkage-stress DMAHDM+NACP composite exhibited no significant difference in antibacterial performance before and after 3 months of aging, demonstrating long-term antibacterial activity. Under S. mutans biofilm acidic attack, dentin hardness (GPa) was 0.24 ± 0.04 for commercial control, and 0.23 ± 0.03 for experimental control, but significantly higher at 0.34 ± 0.03 for DMAHDM+NACP group (p < 0.05). At an instrumental compliance of 0.33 μm/N, the polymerization shrinkage stress of the new composite was 36% lower than that of a traditional composite (p < 0.05). The triple strategy of antibacterial, remineralization and lower shrinkage-stress has great potential to inhibit recurrent caries and increase restoration longevity.Statement of SignificancePolymerization shrinkage stress, masticatory load over time as well as biochemical degradation can lead to marginal failure and secondary caries. The present study developed a new low-shrinkage-stress, antibacterial and remineralizing dental nanocomposite. Polymerization shrinkage stress was greatly reduced, biofilm acid production was inhibited, and tooth dentin mineral and hardness were preserved. The antibacterial composite possessed a long-lasting antibiofilm effect against cariogenic bacteria S. mutans. The new bioactive nanocomposite has the potential to suppress recurrent caries at the restoration margins, protects tooth structures, and increases restoration longevity.

Novel CaF2 Nanocomposites with Antibacterial Function and Fluoride and Calcium Ion Release to Inhibit Oral Biofilm and Protect Teeth.

(1) Background: The objective of this study was to develop a novel dental nanocomposite containing dimethylaminohexadecyl methacrylate (DMAHDM), 2-methacryloyloxyethyl phosphorylcholine (MPC), and nanoparticles of calcium fluoride (nCaF2) for preventing recurrent caries via antibacterial, protein repellent and fluoride releasing capabilities. (2) Methods: Composites were made by adding 3% MPC, 3% DMAHDM and 15% nCaF2 into bisphenol A glycidyl dimethacrylate (Bis-GMA) and triethylene glycol dimethacrylate (TEGDMA) (denoted BT). Calcium and fluoride ion releases were evaluated. Biofilms of human saliva were assessed. (3) Results: nCaF2+DMAHDM+MPC composite had the lowest biofilm colony forming units (CFU) and the greatest ion release; however, its mechanical properties were lower than commercial control composite (p < 0.05). nCaF2+DMAHDM composite had similarly potent biofilm reduction, with mechanical properties matching commercial control composite (p > 0.05). Fluoride and calcium ion releases from nCaF2+DMAHDM were much more than commercial composite. Biofilm CFU on composite was reduced by 4 logs (n = 9, p < 0.05). Biofilm metabolic activity and lactic acid were also substantially reduced by nCaF2+DMAHDM, compared to commercial control composite (p < 0.05). (4) Conclusions: The novel nanocomposite nCaF2+DMAHDM achieved strong antibacterial and ion release capabilities, without compromising the mechanical properties. This bioactive nanocomposite is promising to reduce biofilm acid production, inhibit recurrent caries, and increase restoration longevity.

Antibacterial and remineralizing nanocomposite inhibit root caries biofilms and protect root dentin hardness at the margins

ObjectivesSenior patients have a high incidence of tooth root caries. The objectives of this study were to: (1) develop a bioactive composite with calcium (Ca) and phosphate (P) ion-release and antibacterial capabilities via nanoparticles of amorphous calcium phosphate (NACP) and dimethylaminohexadecyl methacrylate (DMAHDM); (2) inhibit root biofilms of Streptococcus mutans, Lactobacillus acidophilus and Candida albicans in a biofilm-based recurrent root caries model to protect root dentin hardness under biofilms for the first time. MethodsFive groups were tested: (1) Heliomolar nanocomposite (Commercial control); (2) Experimental composite control (0% NACP, 0% DMAHDM); (3) Remineralizing composite (30% NACP); (4) Antibacterial composite (3% DMAHDM); (5) Remineralizing and antibacterial composite (NACP + DMAHDM). Colony-forming units (CFU), lactic acid and polysaccharide of biofilms were evaluated. Demineralization of bovine root dentin with restorations was induced via multi-species biofilms, and root dentin hardness was measured. ResultsAdding NACP and DMAHDM into composite did not compromise the mechanical properties (p > 0.05). Biofilm lactic acid, polysaccharides and CFU were greatly reduced via DMAHDM (p < 0.05). Ca and P ion releases were substantially increased at cariogenic low pH. With multi-species biofilm acid attack, root dentin hardness (GPa) decreased to 0.12 ± 0.03 for Commercial control, and 0.11 ± 0.03 for Experimental control. Root dentin hardness was 0.20 ± 0.04 for NACP group, 0.21 ± 0.04 for DMAHDM group, and 0.30 ± 0.03 for NACP + DMAHDM group which was more than 2-fold that of control groups (p < 0.05). ConclusionsThe novel NACP + DMAHDM nanocomposite had strong antibacterial effects and Ca and P ion release. When tested in a multi-species recurrent root caries model, NACP + DMAHDM nanocomposite substantially reduced root dentin demineralization and protected dentin hardness around the restorations under biofilms. Therefore, this novel bioactive composite is promising to inhibit root caries and protect tooth structures.

Novel dental adhesive containing silver exchanged EMT zeolites against cariogenic biofilms to combat dental caries

Abstract Secondary caries often occurs due to acids produced by biofilms at the tooth-restoration bonded interface, and is considered as one of the primary reasons for dental restoration failure. The objective of this study was (1) to synthesize nanosized EMT zeolites with silver ion exchanging, (2) to incorporate them into a commercial dental adhesive and (3) investigate the inhibition of biofilm formation at the tooth-restoration margin. The template-free zeolite nanocrystals are synthesized and stabilized in water suspensions and directly used for silver ion-exchange. Streptococcus mutans, Streptococcus gordonii, Streptococcus sanguinis were selected as representative strains of cariogenic pathogens and to form single-species biofilms. Colony-forming units (CFU), live/dead and metabolic activity were measured. The Ag+-EMT zeolites addition would not compromise the color and esthetics of dental restoration. EMT zeolites with longest exchanging time (40 min) in the adhesives achieved the greatest reduction in early attachment, bacterial biomass, biofilm growth and metabolic activity. Biofilm CFU counts were reduced by nearly 2 orders of magnitude for all three cariogenic pathogens. Therefore, the adhesives containing Ag+ exchanged zeolites had remarkable antibacterial properties to serve as “bioactive” adhesive materials and revealed its potential value for anti-biofilm and anti-caries clinical applications.

S. mutans gene-modification and antibacterial resin composite as dual strategy to suppress biofilm acid production and inhibit caries

ObjectiveComposite restorations are increasingly popular, but recurrent caries is a main reason for composite restoration failures. The objectives of this study were to investigate a dual strategy of combining rnc gene-deletion for Streptococcus mutans (S. mutans) with antibacterial dimethylaminohexadecyl methacrylate (DMAHDM) composite, and determine the effects of rnc gene-deletion alone, DMAHDM composite alone, and rnc-deletion plus DMAHDM composite, on biofilm growth and lactic acid production. MethodsParent S. mutans (UA159, ATCC 700610) and rnc-deleted S. mutans were used. DMAHDM was incorporated into a composite at mass fractions of 0%, 1.5%, and 3%. Gene expressions for biofilm formation and drug resistance were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Biofilms were grown on composite surfaces for 2 days. Live/dead, biomass, polysaccharide, metabolic activity (MTT), colony-forming units (CFU) and lactic acid production of biofilms were evaluated. ResultsCompared to the parent S. mutans, the rnc-deletion technique yielded significantly less biofilm biomass, polysaccharides, metabolic activity, CFU, and lactic acid for biofilms grown on control composite (p < 0.05). With no gene modification, the biofilm CFU was decreased by 5–6 logs at 3% DMAHDM, when compared to control composite group. The dual strategy of combining rnc-deletion with 3% DMAHDM composite achieved the strongest biofilm-inhibition, with the greatest reduction in CFU by 8 logs. The combination of rnc-deletion with 3% DMAHDM composite decreased the biofilm lactic acid production by 95% (p < 0.05). ConclusionsThe dual strategy of rnc-deletion plus DMAHDM composite produced synergistic effects and achieved the strongest biofilm-inhibition. This method has great potential to inhibit dental caries and is promising to reduce secondary caries and protect tooth structures.