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University of Groningen Acute and substantive action of antimicrobial toothpastes and mouthrinses on oral biofilm in vitro Otten, Marieke P. T.; Busscher, Hendrik; van der Mei, Henderina; van Hoogmoed, Christianus; Abbas, Frank; van Hoogmoed, Christianus Published in: European Journal of Oral Sciences DOI: 10.1111/j.1600-0722.2011.00812.x IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2011 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Otten, M. P. T., Busscher, H. J., van der Mei, H. C., van Hoogmoed, C. G., Abbas, F., & Hoogmoed, G. G. V. (2011). Acute and substantive action of antimicrobial toothpastes and mouthrinses on oral biofilm in vitro. European Journal of Oral Sciences, 119(2), 151-155. DOI: 10.1111/j.1600-0722.2011.00812.x Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 13-06-2018

Eur J Oral Sci 2011; 119: 151 155 DOI: 10.1111/j.1600-0722.2011.00812.x Printed in Singapore. All rights reserved Ó 2011 Eur J Oral Sci European Journal of Oral Sciences Acute and substantive action of antimicrobial toothpastes and mouthrinses on oral biofilm in vitro Otten MPT, Busscher HJ, van der Mei HC, van Hoogmoed CG, Abbas F. Acute and substantive action of antimicrobial toothpastes and mouthrinses on oral biofilm in vitro. Eur J Oral Sci 2011; 119: 151 155. Ó 2011 Eur J Oral Sci The aim of this study was to compare acute action by killing or disrupting oral biofilms through the use of antimicrobial toothpastes and mouthrinses in vitro and to investigate substantive action by absorption of antimicrobials in a biofilm. Biofilms from freshly collected human saliva were grown in 96-well microtitre plates. After removal of saliva, the wells of the microtitre plates were washed with sterile water (control), or exposed to a dilution series of mouthrinses (Corsodyl, Listerine, Meridol, Crest Pro Health) or to toothpaste slurries (Prodent Coolmint, Colgate Total, Zendium Classic, Crest Pro Health, Oral B Pro Expert, Crest Cavity Protection). Acute action was concluded from reduced continued (16 h) growth of treated biofilms with respect to the control. Substantive action was studied by exposing dead biofilms to mouthrinses or to toothpaste slurry. Substantive action through the absorption and subsequent release of antimicrobials from biofilm was concluded from reduced growth on top of the treated biofilms. All formulations showed acute action at the highest concentrations studied. Further dilution yielded loss of efficacy, or even stimulation of biofilm growth. Antimicrobial absorption in and the release of effective concentrations of antimicrobials from dead biofilms, was demonstrated for three selected antimicrobial products, indicating that antimicrobials remain bio-available for substantive action on new biofilms. Marieke P. T. Otten 1, Henk J. Busscher 1, Henny C. van der Mei 1, Chris G. van Hoogmoed 1, Frank Abbas 2 1 Department of Biomedical Engineering, University Medical Center Groningen and University of Groningen, Groningen; 2 Center for Dentistry and Oral Hygiene, University Medical Center Groningen and University of Groningen, Groningen, the Netherlands Chris G. van Hoogmoed, University Medical Center Groningen, A. Deusinglaan 1, 9713 AV Groningen, the Netherlands Telefax: +31 50 3633159 E-mail: C.G.van.Hoogmoed@med.umcg.nl Key words: biofilm; mouthrinses; oral antimicrobials; substantivity; toothpastes Accepted for publication January 2011 All surfaces exposed to the human oral cavity are covered within seconds with a layer of adsorbed salivary proteins (ÔpellicleÕ) to which a large variety of different oral microbial strains and species can adhere and grow to form a biofilm (ÔplaqueÕ). Under appropriate conditions, the number of pathogenic bacteria in an oral biofilm will increase and, as a consequence, diseases such as caries and periodontitis (1) can develop. Removal of oral biofilms in vivo by toothbrushing is the most effective way to prevent caries and periodontal diseases, but it is impossible for most people to completely remove the oral biofilm by brushing (2). Interdental areas, fissures and gingival pockets, in particular, are almost completely inaccessible to the bristle ends of a toothbrush and constitute places where plaque is easily left behind and may also be difficult to remove by other mechanical means. Antimicrobials are added to toothpastes and mouthrinses in order to assist in achieving oral health through the killing of oral biofilm organisms (3, 4). A wide range of different antimicrobials, including enzymes, metal ions, stannous fluoride, chlorhexidine, and triclosan, can be added to toothpastes and mouthrinses (3 5). Chlorhexidine is generally considered to be the most effective oral antimicrobial (3, 5), and it is often used as a positive control in studies (6). Chemical control of oral biofilms by antimicrobials can occur by ÔacuteÕ or ÔsubstantiveÕ actions by killing or disrupting the biofilm (4). Acute action takes place immediately after the use of an oral antimicrobial, leaving behind a dead or partly dead, or disrupted biofilm. Substantivity is defined by the Oxford Dictionary of Dentistry (7) as a characteristic of an antimicrobial product whereby it remains active in the oral cavity for a prolonged period. Recently, we suggested, on the basis of a clinical study, that plaque left behind after brushing, which is inevitably the case in most people, can absorb antimicrobials to yield substantive killing of new plaque (8). In this study we hypothesize that oral antimicrobials can absorb in biofilms to inhibit further growth of and kill new organisms, a process we refer to as substantive action. Consequently, the aim of this study was not only to measure the acute action by killing or disrupting oral biofilms achieved by a single exposure of antimicrobial mouthrinses and toothpastes, but also to investigate whether antimicrobials, absorbed in dead biofilms, have the ability to exert prolonged action on organisms growing from fresh saliva on top of the dead biofilm. Material and methods Saliva, toothpastes, and mouthrinses In order to study acute and substantive action, stimulated human saliva of five volunteers was collected by chewing

152 Otten et al. Parafilm and pooled in accordance with the guidelines set out by the Medical Ethical Committee at University Medical Center Groningen (UMCG), Groningen, the Netherlands. The saliva was sonicated twice for 10 s at 30 W (Vibra Cell model 375; Sonics and Materials, Danbury, CT, USA) in order to break bacterial chains and clumps. To study acute action, four different mouthrinses and six different toothpastes were commercially obtained; these are listed in Table 1, together with their main active ingredients. Toothpastes were diluted in demineralized water to a 25% (w/w) slurry, which was centrifuged at 10,000 g for 5 min at 10 C (Beckman J2-MC Centrifuge, Fullerton, CA, USA) in order to remove particulate matter. Subsequently, the supernatant of the toothpaste was further diluted to 12.5%, 2.5%, and 0.25% (w/w) with demineralized water. Mouthrinses were used full-strength or diluted to 50%, 10%, and 1% (v/v) in demineralized water. To demonstrate substantive action through antimicrobial absorption in biofilms, Corsodyl and Crest Pro Health mouthrinse [100% (v/v)] and Crest Pro Health toothpaste slurry [25% (w/w)] were used. Acute and substantive action of antimicrobial toothpastes and mouthrinses Acute action of mouthrinses and toothpaste slurries on microorganisms in 4-h-old, initial oral biofilms was evaluated in sterile 96-well microtitre plates (Greiner Bio-one, Cellstar, F-bottom, Alphen a/d Rijn, The Netherlands). First, a 4-h-old biofilm of initially adhering organisms was grown. To this end, wells were filled with 175 ll of freshly collected human saliva and incubated for 4 h at 37 C, with shaking at 150 rpm (Incubator Shaker, Innova 4000; New Brunswick Scientific, Edison, NJ, USA). After 4 h, the liquid was aspirated from each well and the wells were rinsed three times with 200 ll of sterile water. Then, the biofilms were exposed to 175 ll of mouthrinse or toothpaste slurry for 30 s or 2 min, respectively, after which the wells were washed three times with 200 ll of sterile water. Control biofilms were exposed to sterile water. Then, 175 ll of sterile Tryptic Soya Broth (TSB) was added to each well, and growth of the exposed biofilms was continued for 16 h at 37 C, with shaking at 150 rpm. Substantive action by absorbed antimicrobial mouthrinse and toothpaste ingredients was evaluated in sterile 96-well microtitre plates from biofilm growth on dead biofilms after absorption of antimicrobials. Evaluations were performed after absorption of oral antimicrobials by initial biofilm, 4-h-old biofilm, and thicker, 24-h-old matured biofilm. First, 4-h-old biofilms were prepared, as described above, and 24-h-old biofilms were made by the removal of saliva after 4 h of incubation, followed by incubation in 175 ll of TSB for 20 h at 37 C and shaking at 150 rpm. After 4 or 24 h, the liquid was aspirated from each well, the wells were rinsed three times with 200 ll of sterile water, and biofilm organisms were killed by exposure to 70% (v/v) ethanol for 3 min. Biofilms were killed in order to study the mechanism of prolonged or substantive action of antimicrobials released from a biofilm, without interference of bacterial growth. Complete killing was verified by attempting to continue growth of the biofilm in sterile TSB (175 ll) for 16 h. Staining with crystal violet (CV) and measurement of the optical density at 575 nm (OD 575 ; see below) demonstrated that 70% (v/v) ethanol fully impeded further growth. After killing, the dead biofilms were exposed to either Corsodyl or Crest Pro Health mouthrinse [both 100% (v/v)] or to Crest Pro Health toothpaste slurry [25% (w/w)], as described for acute action to facilitate antimicrobial absorption. After washing, 175 ll of freshly collected saliva was added to each well in order to grow a biofilm on the dead biofilm. Each experiment included six replicate wells and was performed three times with separately collected saliva. Evaluation of biofilm growth In order to quantitatively evaluate the amount of biofilm growth, wells with biofilm were rinsed three times with 200 ll of PBS, and 175 ll of a 2.3% (w/v) CV solution (Crystal Violet Solution; Sigma-Aldrich, St Louis, MO, USA) was added to each well. After 30 min at room temperature, excess CV solution was removed by washing the plates four times with 200 ll of ultrapure water. Finally, bound CV was released by adding 225 ll of ethanol/acetone [80/20% (v/v)] to each well (9) and the OD 575 was measured Table 1 Toothpastes and mouthrinses used in this study, together with their main active components and manufacturer Products Main active components Manufacturer Mouthrinse Corsodyl Chlorhexidine digluconate 0.2% GlaxoSmithKline, Middlesex, UK Listerine Alcohol, phenols, and essential oils Pfizer Consumer Healthcare, Morris Plains, NJ, USA Meridol Amine fluoride, stannous fluoride GABA Group, Basel, Switzerland Crest pro health mouthrinse Cetylpyridinium chloride Procter & Gamble, Cincinnati, OH, USA Toothpaste Prodent coolmint Sodium fluoride, sodium lauryl Sara Lee Household & Bodycare, Exton, PA, USA sulphate (SLS) Crest cavity protection Sodium fluoride, SLS Procter & Gamble, Cincinnati, OH, USA Colgate total Triclosan, polyvinyl methylether maleic Colgate-Palmolive Company, Piscataway, NJ, USA acid, sodium fluoride, SLS Zendium classic Sodium fluoride, colostrum, Sara Lee Household & Bodycare, Exton, PA, USA lactoperoxidase, lysozyme glucose oxidase, amyloglucosidase Crest pro health toothpaste Stannous fluoride, sodium Procter & Gamble, Cincinnati, OH, USA hexametaphosphate, SLS Oral B pro expert Sodium fluoride, stannous fluoride, Sodium hexametaphosphate, SLS Procter & Gamble UK, Weybridge, UK

Acute and substantive action on oral biofilm 153 using a microtitre plate reader (FLUOstar OPTIMA; BMG LABTECH, Offenburg, Germany). Acute action was concluded from a reduction in the amount of biofilm after growth of the exposed biofilms (OD exposed ) with respect to the control (water treatment; OD control ), while substantive action was concluded from a reduction in the amount of biofilm grown on a dead biofilm (OD exposed ) with respect to a water control. Accordingly, for acute and substantive action, the percentage reduction in biofilm growth was concluded using the following equation %Reduction ¼ OD control OD exposed 100% OD control ð1þ All OD values were corrected for possible adsorption of toothpaste slurries, mouthrinses, and CV at the walls of the wells, while for substantive action, OD values were also corrected for CV absorption in the dead biofilm. Statistical analysis All reductions in biofilm growth after exposure to (diluted) mouthrinses or to toothpaste slurries were compared with the control (i.e. water exposure) using the unpaired StudentÕs t-test, anova, and least-significant difference (LSD) post-hoc test. For statistical analysis, SPSS 16.0 software for Windows (SPSS, Chicago, IL, USA) was used. P-values of < 0.05 were considered to be statistically significant. Results Acute action of antimicrobial toothpastes and mouthrinses All undiluted mouthrinses and 25% (w/w) toothpaste slurries reduced the continued growth of an exposed biofilm significantly (P < 0.05), compared with the control, which is indicative of acute action by killing or disruption (Figs 1 and 2). Corsodyl was effective, even at a 1% dilution (Fig. 1), whereas Listerine and Crest Pro Health rinse lost efficacy at a concentration of 1% (v/v). Meridol showed loss of efficacy at a dilution of 50% and even stimulated biofilm growth when used at 10% and 1% (v/v) dilutions (P < 0.05). Toothpaste slurries of Prodent Coolmint were the only slurries that yielded significant reductions in continued growth of exposed biofilms over the entire dilution range (Fig. 2), as most toothpaste slurries lost efficacy at 0.25% (w/w) dilution. Note that Crest Pro Health paste and Crest Cavity Protection stimulated growth at their highest dilutions (only statistically significant for Crest Cavity Protection at P < 0.05). Zendium Classic lost efficacy and stimulated growth at a 12.5% (w/w) dilution, although the stimulation of growth was not statistically significant. Substantive action through absorption in, and release from, biofilms Biofilm growth on dead biofilms after absorption of mouthrinse or toothpaste components was significantly (P < 0.05) reduced for all three antimicrobial products, demonstrating absorption and release of bio-available antimicrobials at effective concentrations (Fig. 3). Growth reduction after absorption of Corsodyl was less than achieved by Crest Pro Health rinse or Crest Pro Health toothpaste slurry, although this effect was only significant for the rinse. The growth reductions observed resulting from the absorption and release of components in 24-h-old dead biofilms were very similar to those observed in the 4-h-old initial biofilms. Discussion Conceivably, antimicrobial agents can influence oral biofilm formation in different ways, for example, by preventing bacterial adhesion to surfaces, affecting bacterial viability or by disrupting an existing biofilm (4). Percentage reduction 150 100 50 0 50 100 150 200 250 300 100% 50% 10% 1% Corsodyl Listerine Meridol Crest pro health mouthrinse Fig. 1. Percentage reduction in continued growth of an initially adhering, 4-h-old biofilm, after exposure for 30 s to different dilutions of mouthrinses, compared with a control (i.e. exposure to sterile water). Note that negative reductions denote increased growth with respect to the control. A significant (P < 0.05) reduction compared with the control. The SE values were calculated from the results obtained from 18 experiments.

154 Otten et al. 150 Percentage reduction 100 50 0 50 100 150 200 Prodent coolmint Crest cavity protection Colgate total Zendium classic 25% 12.5% 2.5% 0.25% Crest pro health toothpaste Oral B pro expert Fig. 2. Percentage reduction in continued growth of an initially adhering, 4-h-old biofilm, after exposure for 2 min to different dilutions of toothpaste slurries, compared with a control (i.e. exposure to sterile water). Note that negative reductions denote increased growth with respect to the control. A significant (P < 0.05) reduction compared with the control. The SE values were calculated from the results obtained from 18 experiments. Percentage reduction 100 80 60 40 20 0 Corsodyl Crest pro health mouthrinse Crest pro health toothpaste # 4 h Fig. 3. Percentage reduction in biofilm growth on dead, 4-h- and 24-h-old biofilms, after absorption of antimicrobial components from mouthrinses (30 s absorption) or a toothpaste slurry (2 min absorption) compared with a control (i.e. exposure to sterile water). The asterisk () indicates a significant difference with respect to the control, while the hash (#) indicates a significant difference compared with Corsodyl. The SE values were calculated from the results obtained from 18 experiments. 24 h Here, we studied acute action on organisms in oral biofilms and substantive action through the absorption and release of antimicrobials in and from dead biofilms. All antimicrobial products included showed acute action when applied at full-strength (mouthrinses) or in 25% (w/w) toothpaste slurries. Moreover, antimicrobial absorption by, and release of bioavailable antimicrobials from, dead biofilm in effective concentrations was demonstrated to contribute to substantive action for three selected antimicrobial products, viz. Corsodyl, Crest Pro Health mouthrinse, and Crest Pro Health toothpaste. The use of dilution series of mouthrinses and toothpaste slurries to assess the acute action efficacy of these oral antimicrobial products is new. Mouthrinses are always used full-strength and clinically only undergo minor dilution in saliva. For toothpastes, 25% (w/w) slurries are the standard for in vitro evaluations (10), as based on the average amount of toothpaste used during brushing, and the total amount of fluid in the oral cavity. Yet, there are large individual variations, and during brushing further dilution will occur as a result of salivation and swallowing (11). Moreover, most people add water to the toothbrush. Therewith, the decrease in growth reduction of exposed biofilms upon dilution is indicative of the antimicrobial efficacy of the product in clinical use. Most antimicrobial products evaluated for their acute action remain effective up to substantial dilutions, which is in line with clinical observations on their plaque-control efficacy. In Corsodyl, chlorhexidine is the component responsible for clinical antimicrobial efficacy, while for Listerine, Meridol, and Crest Pro Health rinse the responsible components are essential oils, the combination of stannous- and amine-fluoride, and bioavailable cetylpyridinium chloride, respectively (3, 5, 12). In most toothpaste formulations, fluoride and sodium lauryl sulphate contribute to a certain degree of acute action, as found in this study for Prodent Coolmint and Crest Cavity Protection. Toothpastes with antimicrobial claims include Colgate Total, Crest Pro Health, and Oral B Pro Expert (12, 13) and indeed these toothpaste slurries cause acute action up to substantial dilutions. Note that Oral B Pro Expert differs only from Crest Pro Health with respect to an increased fluoride level (1,450 p.p.m. fluoride in Oral B Pro Expert vs. 1,100 p.p.m. fluoride in Crest Pro Health). The higher fluoride concentration, also present in Prodent Coolmint, is probably the reason why these products still show growth reduction at the highest dilution. Zendium Classic, based on colostrums and enzymes enhancing the host defense system, loses antimicrobial efficacy at a dilution of only 12.5%. Clinical studies (14) have shown that the antimicrobial effect of enzyme-containing toothpastes was minor. In general, metal ions act as essential cofactors assisting enzymes to work as a catalyser for metabolic reactions in cells (15); however, they are toxic to cells and bacteria when present at high concentrations (16). In this respect, it is interesting to note that two of the products based on stannous (Meridol mouthrinse and Crest Pro Health toothpaste) show growth stimulation at the highest dilution (i.e. the lowest stannous concentration),

Acute and substantive action on oral biofilm 155 as has also been shown by Aranha et al. (17). However, the stannous-containing formulation, complemented with a high concentration of sodium fluoride (Oral B Pro Expert), does not show growth stimulation, which confirms that fluoride too can exert antimicrobial effects (4). In addition to their acute action efficacy, components from the antimicrobial mouthrinses Crest Pro Health and Corsodyl and the toothpaste Crest Pro Health are absorbed by dead biofilms and remain bioavailable for subsequent release and substantive action on biofilm organisms growing on the exposed biofilm. Hitherto, the substantivity of oral antimicrobial products has been evaluated predominantly in vivo (6, 10) and suggested to be caused by the adsorption of antimicrobials to the abundantly available soft tissue surfaces in the oral cavity from which they are slowly released to yield substantivity (3, 5). This in vitro study confirms our clinical findings that plaque can act as a reservoir for oral antimicrobials (8) and identifies the absorptive capacity of oral biofilm as a factor, contributing to the prolonged activity of antimicrobial healthcare products. Substantivity through absorption in oral biofilms can only be effective if the absorbed antimicrobials remain bioavailable (i.e. can be released). In this respect it is interesting that substantivity of the chlorhexidine-containing rinse, through absorption in and release from the oral biofilm, is smaller than of the other products, possibly because this large cation had become irreversibly trapped in the biofilm, which consists of negatively charged bacteria (18). We could not establish here with adequate statistical significance that thicker, 24-h-old biofilms had a greater absorption capacity than 4-h-old biofilms. In conclusion, antimicrobial toothpastes and mouthrinses are able to act acutely on biofilm organisms in vitro, even when they are considerably diluted, as in clinical situations. Furthermore, antimicrobials from mouthrinses and toothpaste slurries may remain bioavailable in a dead biofilm, resulting in prolonged killing of new biofilm, therewith providing evidence in support of a new mechanism of substantive action. This is of clinical importance because patients are usually not able to completely remove all oral biofilm by tooth cleaning. The new mechanism outlined enables them to benefit from active toothpaste and mouthrinse that is absorbed in biofilm left after cleaning. Conflicts of interest The authors declare that there are no conflicts of interest in this study. References 1. Rosan B, Lamont RJ. Dental plaque formation. Microbes Infect 2000; 2: 1599 1607. 2. Van Der Weijden GA, Hioe KP. A systematic review of the effectiveness of self-performed mechanical plaque removal in adults with gingivitis using a manual toothbrush. J Clin Periodontol 2005; 32 (Suppl 6): 214 228. 3. Scheie AA. The role of antimicrobials. In: Fejerskov O, Kidd E, eds. Dental caries; the disease and its clinical management. Oxford: Blackwell Munksgaard, 2003; 179 188. 4. Baehni PC, Takeuchi Y. Anti-plaque agents in the prevention of biofilm-associated oral diseases. Oral Dis 2003; 9 (Suppl 1): 23 29. 5. Addy M. Antiseptics in periodontal therapy. In: Lindhe J, Karring T, Lang NP, eds. Clinical periodontology and implant dentistry. Copenhagen: Munksgaard, 1997; 461 482. 6. Pizzo G, La CM, Licata ME, Pizzo I, DÕangelo M. The effects of an essential oil and an amine fluoride/stannous fluoride mouthrinse on supragingival plaque regrowth. J Periodontol 2008; 79: 1177 1183. 7. Oxford, Dictionary of Dentistry. In: Ireland R, ed. Oxford: Oxford University Press, 2010; 331. 8. 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