An in vitro microbial model for producing caries-like lesions on enamel

Braz J Oral Sci. July-September 2007 - Vol. 6 - Number 22 An in vitro microbial model for producing caries-like lesions on enamel Carolina Steiner-Oliveira 1* Fernando A. Maciel 2 Lidiany K.A. Rodrigues 3 Marcelo H. Napimoga 4* Luiz A.F. Pimenta 5* José F. Höfling 6* Reginaldo B. Gonçalves 6* 1 DDS, MS, PhD student in Pediatric Dentistry 2 DDS, resident student in the Section of Oral and Maxillofacial Surgery of Santa Casa in São Paulo, Faculty of Medical Sciences, São Paulo Brazil 3 DDS, MS, PhD, Department of Restorative Dentistry, Faculty of Pharmacy, Nursing and Dentistry of Federal University of Ceará, Fortaleza CE Brazil 4 DDS, MS, PhD, Department of Oral Diagnostic 5 DDS, MS, PhD, Department of Restorative Dentistry 6 DDS, MS, PhD, Professor of the Department of Oral Diagnostic * Piracicaba Dental School State University of Campinas, Piracicaba São Paulo Brazil Abstract This study aimed to develop a low cost in vitro viable microbiological model to produce biofilms to be used in dental researches. Single and multi-species biofilms of S. mutans, S. sobrinus, S. mitis, S. salivarius, S. cricetus and S. sanguinis were grown on bovine enamel slabs during 10 days, in a sterile brain-heart infusion broth, containing 5% sucrose and incubated at 37ºC in an atmosphere of 10% CO 2. The slabs were transferred to a fresh medium at every 6, 12 or 24 hours. After the experimental period, enamel volume percent mineral was determined by cross-sectional microhardness. Caries-like lesions were found in all bacterial groups when compared with the control group. No statistical significant differences were found between S. mutans and S. sobrinus with respect of their cariogenicity or among the periods of medium change. However, it was found a statistical significant difference among the cariogenicity of S. salivarius and S. sanguinis (ANOVA followed by Tukey test). This model has successfully developed caries-like lesion on enamel and the medium can be changed at every 24 hours utilizing either S. mutans or S. sobrinus. Key Words: biofilms, microbiological, enamel, S. mutans, S. sobrinus Received for publication: May 09, 2007 Accepted: July 04, 2007 Correspondence to: Reginaldo Bruno Gonçalves Faculdade de Odontologia de Piracicaba UNICAMP Departamento de Diagnóstico Oral Área de Microbiologia e Imunologia Avenida Limeira, 901 Piracicaba, SP - Cep 13414-903. Phone: 55 19-2106 5379 Fax: 55 19-2106 5218 E-mail: reginald@fop.unicamp.br 1392

Introduction There are a variety of model systems available that can be applied to study dental enamel caries process, each one presenting advantages and disadvantages 1. Experimental chemical models such as ph cycling and immersion in acid medium are widely used to simulate cariogenic challenges 2. The disadvantage of these models is that they do not simulate the real demineralization process of the oral environment due to the absence of microorganisms, consequently, concentrating on the physical-chemical aspects of enamel dissolution 3. Another process of forming carious lesions involves bacterial models in which either planktonic bacteria or microorganisms organized in biofilms can be used. Studies have shown that planktonic microbial communities have different properties from microorganisms grown in a biofilm 4. One major difference is that microorganisms growing on surfaces as biofilm are generally more resistant to antimicrobial agents than the same cells growing in conventional liquid media. This can be for a number of reasons, including the reduced penetration of the inhibitor into the biofilm (diffusion-reaction mechanism) and the slow growth rate and novel phenotype expressed by the attached cells 5. An in vitro model that uses bacterial films is likely to be more representative than chemical or bacterial slurry systems, since dental caries is a bacterial disease and the bacteria which cause it are members of a biofilm community which may lead to altered metabolism compared with free-living microorganisms 1. Bacterial models offer several advantages such as: (1) investigation of the etiology and prevention of carious lesions; (2) comparison of the cariogenic potential of different bacterial populations and (3) assessment of the cariogenicity of various diets 6. Two bacterial in vitro models must be considered. One is known as the artificial mouth and provides a continuous or intermittent supply of nutrients to bacterial plaque or biofilms growing within an environment, which mimics the in vivo oral niches and habitats 7. However, this in vitro model currently available, tends to require sophisticated laboratory equipment, presents frequent contamination problems and demands very high costs 1. An alternative method for producing biofilms with lower costs and a good contamination control is a bacterial system involving a sequential batch culture technique, in which the samples are immersed in an enriched medium with microorganisms to evaluate the formation of caries lesions 8. Growth of biofilms has been shown to occur via a sequence of colonization events in which initial adhesion to the enamel surface is followed by further bacterial-enamel binding, bacteria-bacteria interaction and growth 9. The most common cariogenic bacteria associated with human dental caries are Streptococcus mutans and Streptococcus sobrinus 10. Acidogenicity and aciduricity are important biochemical characteristics for cariogenicity of these microorganisms. The mutans streptococci have both these properties and are considered the most cariogenic group within the oral microbiota 10. Regarding the disadvantages of the ph cycling and acid immersion models that do not allow a microbiological simulation of the oral environment and the difficulties and limitations of the artificial mouth method described above, the aim of this study was to present a low cost in vitro viable model utilizing a batch culture technique to produce biofilms to be used in dental researches. Material and Methods Experimental Design A total of 120 bovine teeth free from macroscopic cracks were selected for this study. The teeth were stored in a 0.1% thymol solution ph 7.0 at 4 C for 30 days 11. They had their roots removed (electrical cut BUEHLER-ISOMET) in longitudinal cuts with diamond disks (KG Sorensen Ind e Com LTDA) to obtain dental slabs (4 mm x 4 mm x 2 mm) from the vestibular surface of each tooth. The slabs were covered with nail varnish leaving exposed a 16mm 2 enamel window. The fragments were autoclaved at 121ºC for 20 minutes 12-13 with neither interference on the enamel hardness 13 nor on its demineralization pattern 8. The teeth were randomly divided into 12 groups of 10 slabs each according to the type of microorganism and the period of change of the bacterial inoculation. The tooth slabs were attached to orthodontic wires, so as to leave the free enamel window to be immersed in the medium without touching the tube walls. The tubes were loosely closed to allow gas change with the environment and this complex was sterilized in autoclave. Bacterial Preparation The microorganisms used in this study were Streptococcus mutans (ATCC 25175), Streptococcus sobrinus (6715), the association of these two, Streptococcus mitis (ATCC 903), Streptococcus salivarius (ATCC 25975), Streptococcus cricetus (ATCC 19642) and Streptococcus sanguinis (ATCC 10556). The optical density of the culture was adjusted to obtain a standard amount of cells of approximately 2.15 x 10 8 CFU/mL. Biofilm Growth and Lesion Production After sterilization, the dental slabs were removed from distilled water and immersed in sterile brain-heart infusion broth - BHI (Difco Lab. Detroit, USA) containing 5% sucrose (Synth, Labsynth, SP, Brazil) 8. The BHI recipients were inoculated with 10 ml overnight cultures of S. mutans, S. sobrinus, and with 5mL of each of these species in the association group. The experiment lasted for a period of 10 days and the slabs were divided in groups as follows: groups C6, C12 and C24 were the controls immersed in a sterile medium without any 1393

bacterial inoculation; groups M6, M12 and M24 were immersed in a medium containing S. mutans; groups S6, S12 and S24 were immersed in a medium containing S. sobrinus and groups MS6, MS12 and MS24 were immersed in a medium containing equal amounts of both species (S. mutans and S. sobrinus). Bacterial inoculation of the groups M6, S6 and MS6 was performed at every 6 hours when the enamel slabs were transferred to a fresh new medium. For the groups M12, S12 and MS12, this inoculation was performed at every 12 hours and at every 24 hours for the groups M24, S24 and MS24 that were also transferred to a fresh new medium. All slabs had their medium changed at the same time to prevent any kind of contamination and were incubated at 37ºC in an atmosphere of 10% CO 2 (Cole Parmer Instruments, USA). Contamination at test recipients was verified at each 24 hours by inoculation in BHI agar media (Merck, Darnstadt, Germany). Additionally, it was performed another experiment with the same methodology described above, except for the microorganisms and the pre-determined period of medium change that had already been tested. We chose to change the medium at every 24 hours since it did not show any statistical significant difference in the cariogenicity between the microorganisms in the previous trial. The bacteria chosen for the new groups were Streptococcus mitis, Streptococcus salivarius, Streptococcus cricetus and Streptococcus sanguinis, besides the control group with no bacteria. This new experiment was performed in order to verify if there were any cariogenicity differences between these microorganisms. Microhardness Assessment At the end of the experimental period, the tooth slabs were longitudinally sectioned through the center of the enamel area. One of its halves was embedded in epoxy resin, with the outer enamel surface perpendicular to the resin block surface. The slabs were serially polished with aluminum oxide disks of #400, #600 and #1200 grits, and a diamond paste of 1mm (Metadi Ò Buehler). In all samples, three lanes of four indentations each were made at the depths: 30, 50, 70, 100 µm from the outer enamel surface in the central region of the dental slab, using a Knoop diamond under a 25 g load for 5 s (Future-Tech FM-ARS). The distance between the lanes was 200 µm. Indentation lengths were converted to Koop Hardness Number and after to volume % mineral 14. After calculating volume percentage mineral values for each depth evaluated, mineral profiles, integrated area of mineral content were obtained for all groups 15. Statistical Analysis In order to assess the effect of microorganisms and the medium change on the cariogenicity of the in vitro model, the dependent variable volume % mineral versus micrometer was independently analyzed by analysis of variance (ANOVA). ANOVA was followed by Tukey test to evaluate the significance of all pair wise comparisons. The software SAS system (version 8.02, SAS Institute Inc., Cary: NC, 1999) was used and the significance limit was set at 5%. Results Table 1 shows that statistical significant differences were not found among any periods of evaluated medium changes (p > 0.05). S. mutans, S. sobrinus and the association of these microorganisms presented a statistical significant difference when compared to the control group (p < 0.01), regarding the enamel volume percent mineral. However, there was no statistical significant difference (p > 0.05) between them, with respect of their cariogenicity (table 1). Table 2 shows that Streptococcus mitis, Streptococcus salivarius, Streptococcus cricetus and Streptococcus sanguinis presented a statistical significant difference when compared to the control group (p < 0.01), considering the enamel percentage mineral volume. In addition, S. sanguinis group showed a statistically higher cariogenicity than S. salivarius group (p < 0.05). Discussion Chemical induction of caries by organic acids is one of the main approaches in clarifying the mechanisms involved in demineralization and remineralization of enamel, but direct acid exposure does not allow the bacterial interactions that characterize caries in vivo 16. In vivo caries studies have the advantage of including host factors involved in the natural caries process but have some fundamental limitations. The Table 1 - Percentage of mineral volume (%) X micrometer (mean ± SD) according to microorganisms and periods of medium changes. Period of medium change Group 6h 12h 24h Control 9093.9 ± 823.4 a 7948.8 ± 922.0 a 7089.8 ± 1273.1 a S. mutans 4521.0 ± 1599.6 b 5035.0 ± 1868.0 b 3957.4 ± 468.4 b S. sobrinus 4568.5 ± 1046.8 b 4276.2 ± 985.0 b 3936.7 ± 780.6 b S. mutans/s. sobrinus 4831.2 ± 1827.1 b 3813.3 ± 734.3 b 4509.4 ± 1882.3 b * Means followed by distinct letters are statistically different (p < 0.05). No differences were observed between the three different periods of medium changes. Lower case letters show differences among the microorganisms 1394

Table 2 - Percentage of mineral volume (%) X micrometer (mean ± SD) according to four different microorganisms. Group % x Micrometer Control 8226.3 ± 1146.0 a S. mitis 4440.5 ± 1128.1 bc S. salivarius 5241.6 ± 706.9 b S. cricetus 4406.9 ± 824.2 bc S. sanguinis 3944.2 ± 949.3 c * Means followed by distinct letters are statistically different (p < 0.05). Lower case letters show difference among the microorganisms oral environment is difficult to control and varies greatly with intraoral location over time and between different persons 17. Bacteria in dental biofilms metabolize carbohydrates to the acids that cause dental caries. Laboratory models of this process are potentially valuable in understanding the mechanisms involved, in developing and testing procedures to combat and prevent caries 18. A useful in vitro model should have the following characteristics: ease of sterilization of the different components, ability to manipulate model components under sterile conditions, ease of access to test specimens, reproducibility of experiments and optimal simulation of the oral environment 19. Bacterial systems where the mixed natural microbiota are controlled by in vitro environment and nutrient conditions provide a means for studying complex microbial ecosystems such as dental biofilm and its effect on the development of dental caries 20. An in vitro model system using bacterial films is likely to display less inherent variability since variables such as fluid flow, carbohydrate intake and bacterial population composition can be controlled more accurately in vitro 1. Moreover, an in vitro model that uses bacterial films is likely to be more representative than chemical systems, since dental caries is a bacterial disease and the bacteria which cause it are members of a biofilm community which may lead to altered metabolism compared with free-living organisms 1. One of the main advantages of using this in vitro model to produce caries lesions is the presence of an experimental tooth enamel surface that is freely exposed to the bacterial challenge, the low cost of the system and the possibility of controlling contamination, by monitoring the medium at each 24 hours by inoculation in BHI agar media. The disadvantage is that this model does not mimic the diverse conditions present in the oral cavity, such as presence of saliva, antimicrobial proteins and enzymes, absence of the remineralization period, all of which may affect dental caries development 20. Simple monobacterial biofilm models have been developed, for example using Streptococcus mutans 18. Defined-species biofilm consortia, although simpler than in vivo, have the advantage of allowing detailed control and study of the properties of the individual bacterial species present 18. Even in batch culture, oral multi-species consortia develop complex biofilms on enamel that can induce carious lesion similar to those in vivo 21 as shown in a study that the bacterial model produced caries-like lesions similar to those found with purely chemical systems 22. The results of this study confirm that this microbiological model was able to produce caries lesions in all tooth slabs in all periods of medium changes, but did not find any differences between the cariogenicity of the microorganisms. This is in accordance with studies that did not find differences in cariogenicity between S. mutans and S. sobrinus with respect of enamel lesions in rats 23 or in vitro 24. In creating laboratory caries models based on biofilms of selected species of bacteria, the properties of the particular strain of each specie selected will determine the activities of the biofilm and hence the outcome of the experiment 16. Thus the distinction of the species to promote caries in this bacterial system may be important because the two microorganisms chosen display differences in initial colonization, virulence mechanisms 10 and also because S. mutans and S. sobrinus can coexist in fairly close proximity to one another in small dental sites 25. Studies have shown that young children with both S. mutans and S. sobrinus in their saliva had significantly more dental caries than those with either S. mutans or S. sobrinus alone 26. In an animal model, it has been suggested that S. sobrinus could be more acidogenic than the other species of mutans streptococci 27. On the other hand, some authors showed that the animals infected with S. sobrinus strains generally showed lower caries scores than those infected with S. mutans strains 28. There was a tendency for S. sanguinis to be more cariogenic compared to S. mitis, S. salivarius and S. cricetus as revealed in the second experiment. This is in accordance with studies that show that some mitis group streptococci may also contribute significantly to the pool of acids produced in dental biofilm at low ph 29 and other authors who showed that S. sanguinis produce significant amounts of acids 30. In conclusion, this model has successfully developed caries lesion on enamel and since there were not statistically significant differences between the periods of medium changes, or at the bacteria strains used to produce the demineralization, the most appropriate period to changing the medium would be at every 24 hours using either S. mutans or S. sobrinus. Acknowledgements This investigation was supported by Pibic/CNPq research grant 800194/86-1.The first author received a scholarship from CNPq during her undergraduation course. References 1. Aldsworth TG, MacFarlane TW. A novel in vitro model system to grow films of oral bacteria for the study of human tooth root surface caries. J Appl Microbiol. 2001; 91: 139-46. 1395

2. Featherstone JDB, O Really MM, Shariati M, Brugler S. Enhancement of remineralization in vitro and in vivo. In: Leach SA, editor. Factors relating to demineralization and remineralisation of the teeth. Oxford: IRL Press; 1986. p. 23-34. 3. Holly FJ, Gray JA. Mechanism for incipient carious lesion growth utilizing a physical model based on diffusion concepts. Arch Oral Biol. 1968; 13: 319-34. 4. Marsh PD, Nyvad B. The Oral Microflora and Biofilms on Teeth. In: Fejerskov O, Kidd E. Dental caries: the disease and its clinical management. Oxford: Blackwell Munksgaard; 2003. p. 29-48. 5. Gilbert P, Das J, Foley I. Biofilm susceptibility to antimicrobials. Adv Dent Res. 1997; 11: 160-7. 6. Katz S, Park KK, Stookey GK, Schemehorn BR. Development and initial testing of a model for in vitro formation of pit and fissure caries. Caries Res. 1986; 20: 424-8. 7. Tang G, Yip HK, Cutress TW, Samaranayake, LP. Artificial mouth model systems and their contribution to caries research: a review. J Dent. 2003; 31: 161-71. 8. Gilmour AS, Edmunds DH, Newcombe RG, Clark MF. An in vitro study into the effect of a bacterial artificial caries system on the enamel adjacent to composite and amalgam restorations. Caries Res. 1993; 27: 169-75. 9. Moller S, Sternberg C, Andersen JB, Christensen BB, Ramos JL, Givskov M, Molin S. In situ gene expression in mixed-culture biofilms: evidence of metabolic interactions between community members. Appl Environ Microbiol. 1998; 64: 721-32. 10. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev. 1986; 50: 353-80. 11. Amaechi BT, Higham SM, Edgar WM. Factors affecting the development of carious lesions in bovine teeth in vitro. Arch Oral Biol. 1998; 43: 619-28. 12. Pantera EA Jr, Schuster GS. Sterilization of extracted human teeth. J Dent Educ. 1990; 54: 283-5. 13. Parsell DE, Stewart BM, Barker JR, Nick TG, Karns L, Johnson RB. The effect of steam sterilization on the physical properties and perceived cutting characteristics of extracted teeth. J Dent Educ. 1998; 62: 260-3. 14. Featherstone JD, ten Cate JM, Shariati M, Arends J. Comparison of artificial caries-like lesions by quantitative microradiography and microhardness profiles. Caries Res.1983; 17: 385-91. 15. Arends J, ten Bosch JJ. Demineralization and remineralization evaluation techniques. J Dent Res. 1992; 71(Spec n.): 924-8. 16. Shu M, Wong L, Miller JH, Sissons CH. Development of multispecies consortia biofilms of oral bacteria as an enamel and root caries model system. Arch Oral Biol. 2000; 45: 27-40. 17. Scheie AA, Fejerskov O, Lingstrom P, Birkhed D, Manji F. Use of palladium touch microelectrodes under field conditions for in vivo assessment of dental plaque ph in children. Caries Res. 1992; 26: 44-51. 18. Marsh PD. The role of microbiology in models of dental caries. Adv Dent Res. 1995; 9: 244-54. 19. Bowden GHW, Ellwood DC, Hamilton IR. Microbial ecology of the oral cavity. Adv Microb Ecol. 1979; 3: 135-216. 20. Fontana M, Dunipace AJ, Gregory RL, Noblitt TW, Li Y, Park KK, Stookey GK. An in vitro microbial model for studying secondary caries formation. Caries Res. 1996; 30: 112-8. 21. Yue SL, Zhou XD, Li J. Multibacterial artificial plaque. A model for studying carious process. Chin Med J. 1992; 105: 25-9. 22. Gilmour SM, Edmunds DH, Dummer PM. The production of secondary caries-like lesions on cavity walls and the assessment of microleakage using an in vitro microbial caries system. J Oral Rehabil. 1990; 17: 573-8. 23. Köhler B, Birkhed D, Olsson S. Acid production by human strains of Streptococcus mutans and Streptococcus sobrinus. Caries Res. 1995; 29: 402-6. 24. Zanin IC, Lobo MM, Rodrigues LK, Pimenta LA, Hofling JF, Goncalves RB. Photosensitization of in vitro biofilms by toluidine blue O combined with a light-emitting diode. Eur J Oral Sci. 2006; 114: 64-9. 25. Lindquist B, Emilson CG. Interactions between and within Streptococcus mutans and Streptococcus sobrinus isolated from humans harboring both species. Scand J Dent Res. 1991; 99: 498-504. 26. Hirose H, Hirose K, Isogai E, Miura H, Ueda I. Close association between Streptococcus sobrinus in the saliva of young children and smooth-surface caries increment. Caries Res. 1993; 27: 292-7. 27. de Soet JJ, van Loveren C, Lammens AJ, Pavicic MJ, Homburg CH, ten Cate JM et al.. Differences in cariogenicity between fresh isolates of Streptococcus sobrinus and Streptococcus mutans. Caries Res. 1991; 25: 116-22. 28. Köhler B, Krasse B. Human strains of mutans streptococci show different cariogenic potential in the hamster model. Oral Microbiol Immunol. 1990; 5: 177-80. 29. Van Houte J, Sansone C, Joshipura K, Kent R. In vitro acidogenic potential and mutans streptococci of human smooth-surface plaque associated with initial caries lesions and sound enamel. J Dent Res. 1991; 70: 1497-502. 30. de Soet JJ, Nyvad B, Kilian M. Strain-related acid production by oral streptococci. Caries Res. 2000; 34: 486-90. 1396