SciELO - Scientific Electronic Library Online

 
vol.22 número2Marginal fit analysis of premachined and castable UCLA abutments índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Revista

Articulo

Indicadores

  • No hay articulos citadosCitado por SciELO

Links relacionados

  • No hay articulos similaresSimilares en SciELO

Compartir


Acta Odontológica Latinoamericana

versión On-line ISSN 1852-4834

Acta odontol. latinoam. vol.22 no.2 Buenos Aires set. 2009

 

ARTÍCULOS ORIGINALES

Clinical and microbiological characteristics in predicting dentine caries progression

 

Isauremi V.A. Pinheiro1, Boniek C.D. Borges1, Ana P.V. Colombo2, Kenio C. de Lima1

1 Federal University of Rio Grande do Norte (UFRN), Natal, RN, Brazil.
2 Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil.

CORRESPONDENCE Dra. Isauremi Vieira de Assuncao Pinheiro Departamento de Odontologia (UFRN), Clinica Integrada Av. Senador Salgado Filho, 1787 Lagoa Nova. 59.056-000 - Brazil E-mail: isauremi@ig.com.br


ABSTRACT

The purpose of this study was to evaluate the clinical aspect of dentine and its microbiota in predicting caries progression. The sample consisted of schoolchildren in the 7 to14 years age group. Treatment involved cavity preparation trough the clinical criterion of hardness, with the collection of carious and remnants dentine for microbiological analysis. The clinical aspect (color and consistency) of the dentine remmants was recorded and the teeth were restored using silver amalgam and glass ionomer cement as pulpal protector (baseline – BL). After 1 year the restoration was removed and after new clinical and microbiological analyses, the teeth were then restored. Microbiological samples were collected at both time-points and cultivated in sheep blood agar, in anaerobiosis for 48 hours. Bacterial growth was analyzed quantitatively. Semiquantitative and qualitative analysis of the bacteria was performed by hybridization with genomic DNA probes and the checkerboard method. A significant difference was observed between the aspect of dentine remnants at BL and at 1 year (p=0.0078). The amount of bacteria at BL and at 1 year did not differ significantly (p=0.37) and the microbiota of the carious dentine was predominantly composed of Gram-positive cocci. The removal of carious dentine based on the clinical criterion of hardness, followed by a well-adapted restoration, would determine the non-progression of caries. The few bacteria that still remained in the cavity would be no longer viable.

Key words: Dental caries; Dentine; Hardness; Microbiology; DNA probes.

RESUMO

Aspecto clínico e microbiota da dentina pós preparo cavitário e sua relação com a progressão de cárie

Este trabalho objetivou avaliar o aspecto clinico da dentina e sua microbiota na predicao da progressao de carie. A amostra foi constituida por individuos na faixa etaria de 7 a 14. O tratamento envolveu a remocao de tecido cariado pelo criterio clinico de dureza, o qual foi coletado analise microbiologica. Apos o mesmo, fez-se analise do aspecto clinico da dentina remanescente, bem como nova coleta microbiologica e restaurou- se os elementos com amalgama de prata, apos forramento com ionomero de vidro. Um ano apos, com a reabertura do dente, outra coleta microbiologica foi feita, bem como analise do aspecto clinico do tecido remanescente. O crescimento bacteriano foi analisado quantitativamente atraves de hibridizacao com sondas de DNA genomico e do metodo Checkerboard. Observou-se diferenca estatisticamente significativa entre o aspecto da dentina na linha base do estudo e um ano apos (p=0.0078). A quantidade de bacterias na linha base, em relacao a um ano depois do preparo, nao diferiu estatisticamente (p=0,37) e a microbiota predominante da dentina cariada foi composta de cocos Gram-positivos. A remocao de dentina cariada pelo criterio clinico de dureza, favorecida pela boa adaptacao da restauracao determinaram a nao progressao de carie. As poucas bacterias que ainda permaneceram na cavidade apos o preparo provavelmente tornaramse inviaveis em promover o avanco do processo carioso.

Palavras chave: Carie dentaria; Dentina; Dureza; Microbiologia; Sondas DNA.


INTRODUCTION

It is known that dental caries is an infectious disease caused by tooth-adhering bacteria, which form dental biofilm. A significant amount of scientific evidence demonstrates that caries only occur if there are microorganisms present1. Dentine caries present differences in related microbiota when compared to enamel caries, since the growth of microorganisms occurs in a much more anaerobic and protein-rich environment. Amon the microorganisms investigated in dentine caries, the gram-positive bacilli predominated in the majority of studies2,3,4. It is known that an outer layer of carious dentine is infected and not susceptible to remineralization (the collagenous fibers do not present striation, the peritubular dentine and the odontoblastic processes are absent and the tubules are infected by microorganisms) and should be removed. The inner layer, although it is affected, should be preserved since its collagenous fibers present striation, the peritubular dentine and the odontoblastic extensions are present and the tubules are not infected by microorganisms5,6,7.
Regardless of the type of microorganism, some studies state that bacteria must be totally removed during cavity preparation in order to impede caries lesion progression8,9. Others, however, report that even when all the soft carious tissue is not removed, the residual bacteria will be rendered harmless, if there is good sealing4,10. The clinical criterion of hardness must be considered at least as a criterion to remove carious dentine during cavity preparation11. This method has the advantage of removing disorganized or infected dentine and confers resistance to the preparation since it prevents softening or shifting of the dentine remnants after restoration12. Conversely, histological studies have shown that hardness is not an adequate parameter to determine the removal of carious dentine and that contamination persists, with bacterial percentages varying from 36 to 94% in deep caries11,13. It is also known that due to the tubular structure of dentine, there is bacterial penetration inside the tubules, precluding total removal of the pathogenic microorganisms present, and allowing a number of them to remain viable12. Nevertheless, the bacterial remnants of non-softened dentine (clinical criterion of hardness) are not capable of causing lesion progression if the cavity is well sealed1,10,14.
In this sense, bacteria would persist after the removal of carious dentine. However, the metabolic and growth capacity of these microorganisms may be impaired after isolation in the oral environment through sealing of the cavity. Some other questions remain unanswered: Are the microorganisms that remain in the dentine remnants viable? Does this fact allow caries progression? Is there a relation between the dentine aspect and the type of microorganism? Thus, the purpose of this study was to identify the microbiota present in the carious dentine and compare it to that found in dentine remnants (BL and 1 year), in order to verify its relation with caries progression. Furthermore, a relation was sought between the dentine aspect (both carious and remnant) and the microbiota encountered.

MATERIALS AND METHODS

Ethical considerations
This research was approved by the Research Ethics Committee of the Federal University of Rio Grande do Norte (protocol number 100/02). All relevant information about the study was provided to the parents and/or guardians of the schoolchildren, who then authorized their participation by signing an informed consent form, as per National Health Council Norms (resolution 196/96).

Sample studied
This study sample was composed of 34 schoolchildren from the public system in Natal-RN-Brazil, in the 7-to 14-years age group, who presented caries lesions with cavitation located only on the occlusal surfaces of permanent molars and with radiolucency in the middle third of the dentine, confirmed by bite-wing radiographic examination. The study was developed in the Department of Dentistry of Federal University of Rio Grande do Norte (UFRN) and consisted of a clinical trial to evaluate the degree of progression of caries during the oneyear follow-up by visual and the radiographic examination (bite-wing radiograph).

Microbiological analysis
Prophylaxis was performed with pumice stone and water on the selected teeth, which were then anesthetized, photographed and isolated. The rubber dam and tooth surface were disinfected with 1% NaOCl. The first layer of carious dentine was removed with a small spoon excavator and discarded, since contact with dental biofilm could also lead to the presence of bacteria unrelated to carious dentine. The remaining layers were collected with another excavator spoon, being scored according to the dentine aspect: 1-very soft; 2-soft; 3-medium hard; 4-hard. Their contents were dispensed into individual sterile eppendorf tubes containing a cryoprotector [Brain Heart Infusion + Dimethyl Sulfoxide] to preserve the dentine collected. Next, these samples were submitted to microbiological examination to determine the presence of bacteria through hybridization with genomic DNA probes and the checkerboard method15.
Cavity preparation was finalized with carbide drills 245 (SS White, Rio de Janeiro-RJ, Brazil). After concluding the preparation (medium hard dentine), the clinical aspect of the dentine remnants was scored (1- light yellow; 2- yellow; 3- light brown; 4- dark brown; 5- black). Material was collected for microbiological analysis of the dentine remnants immediately after cavity preparation. The cavity was filled with 0.05 ml saline solution for one minute, and then its contents were withdrawn from the cavity with a sterile insulin syringe. This solution was placed into a sterile eppendorf to evaluate microbiota in the dentine through seeding in sheep blood agar plates with 48-hour incubation, in anaerobiosis, at 37˚C. After bacterial growth in the blood agar plates, colony-forming units were counted (quantitative analysis). They were removed with a sterile metal instrument. The colonies were placed into a sterile eppendorf with a cryoprotector (BHI + DMSO) to determine existence and predominance of bacterial types (semi-quantitative and qualitative analysis). Probes for twenty three bacteria related to dentine caries and environmental contamination were employed (Table 1).

Table 1: List of bacterial species and a reference of oral organisms for obtaining genomic DNA probes.

Clinical Monitoring and Therapeutic Procedures
After the collection process, pulpal protection was performed with glass ionomer cement (Vidrion F, SS White, Rio de Janeiro-RJ, Brazil) and cavity varnish (Cavitine, SS White, Rio de Janeiro-RJ, Brazil) and the tooth was restored with silver amalgam (Velvalloy, SS White, Rio de Janeiro-RJ, Brazil) and radiographed. The filling was removed 1 year after restoration and re-assessed using dentine remnant scores for comparison with baseline (BL) data. Sample collection for microbiological analysis was performed employing the same technique as for BL. The aim of this procedure was to compare the microbiota present at different times. A new restoration was performed in the same session.

Statistical Analysis
Differences in the clinical appearance of the dentine between the two time-points (BL and 1 year) were evaluated with Bowker’s test at a significance level of 5%. Wilcoxon’s test was used to evaluate the amount of bacteria as determined by the microbiological analysis. Friedman’s non-parametric test and the sign test were used to determine if there was a difference in the type of bacteria present in the carious dentine at BL and after 1 year. The correlation between the clinical aspect of carious dentine, as well as that of dentine remnants with the presence of bacteria, was determined with Spearman’s correlation coefficient for a significance level of 95%.

RESULTS

Caries progression after 1 year of observation was the main end-point analyzed in this study. None of the 34 teeth presented caries progression, as revealed both by radiography and visual examination (followed the opening of the cavity). Concerning the clinical aspect of carious dentine, of the 34 teeth 6% (n=2) were very soft, 67% (n=23) were soft and 27% (n=9) were medium hard. Regarding the appearance of dentinal remnants at BL, 38% (n=13) were yellow, 54% (n=18) were light brown and 8% (n=3) were dark brown. After the 1-year experimental period, 27% (n=9) presented yellow, 46% (n=16) presented light brown and 27% (n=9) presented dark brown (p=0.0078). Dentin exhibited darkening but no softening after the follow-up period.
In the checkerboard analysis of the microbiota of the carious dentine, 18 (78.26%) of the 23 species tested were detected. Of these, the predominant were Gram-positive cocci (S. gordoni, P. anaerobius, S. aureus, S. mitis, S. anginosus, S. oralis, S. sanguis, S. sobrinus, P. micros, S. mutans and S. epidermidis) followed by the Gram-positive bacilli group (L. casei, P. acnes, A. naeslundii and A. israelii). A few species of Gram-negative cocci (V. parvula) and Gram-negative bacilli (P. gingivalis, P. intermedia) were identified at low levels. In the Gram-positive cocci group, there was a predominance of S. sobrinus followed by S. anginosus and in the Gram-positive bacilli group A. naeslundii predominated. After cavity preparation, only 11 (47.82%) of the 23 bacteria evaluated were found (S. anginosus, P. anaerobius, S. oralis, S. mitis, V. parvula, S. aureus, S. sanguis, P. gingivalis, S. gordonii, S. sobrinus, S. epidermidis). Of these, 2 (8.69%) were found only in the 1 year samples (P. gingivalis and P. anaerobius), 1 (0.43%) was found only at BL (S. sanguis) and the remaining 8 (34.78%) were detected both at BL and at 1 year. The predominant group in this bacterial analysis were the Gram-positive cocci with lower scores than those of the carious dentine group. S. angiosus predominated, followed by S. oralis and S. sobrinus. No Gram-positive bacilli were observed, as opposed to carious dentine. Patients who still presented bacteria after cavity preparation (BL and 1 year), did not present caries recurrence (as observed by radiography and visual examination after the opening of the cavity). The quantitative comparison of the presence of bacteria in the carious dentine and after cavity preparation (BL and after 1 year), showed a significant difference both at BL (p < 0.001) and after 1 year (p = 0.012).
However, when the mean number of bacteria found after cavity preparation at BL and 1 year were compared, there was no significant difference between these two periods (p > 0.05) (Table 2). Of the 32 cases, (since two were not analyzed because of sample loss) 5 (16%) presented bacteria after cavity preparation (BL) and 27 (84%) did not. After 1 year, all 5 cases cited above exhibited a reduction in the number of bacteria. Of the 27 cases that did not present bacteria after cavity preparation (BL), 19 (70%) remained bacteriafree after 1 year and 8 cases (30%) presented bacteria after 1 year, even when they did not present any at BL. The semiquantitative and qualitative analysis of the presence of bacteria in dentine caries at BL and after 1 year showed no significant difference between bacteria types at these two time-points (Table 3).

Table 2: Number of teeth analyzed, median (log), confidence interval and its level of significance in relation to the amount of bacteria at baseline (BL) and after 1 year.

Table 3: Number of teeth analyzed, median (log), confidence interval and its level of significance in relation to the semiquantitative and qualitative analysis of the types of microorganisms at baseline (BL) and after 1 year.

When a correlation was sought between the clinical aspect presented by the carious dentine as well as the dentine remnants after cavity preparation and the type of bacteria encountered, no significant correlation was observed (p > 0.05, r = - 0.01258 to 0.3774; p > 0.05, r = - 0.03541 to 0.2546, respectively).

DISCUSSION

Glass Ionomer Cement is the only restorative material that has the capacity to bond chemically to the teeth surfaces16, providing a suitable cavity seal when used for pulp protection. Moreover, some authors have shown there is mineralization of the dentin under glass ionomer cements17,18, favoring the color change and the persistence of dentine hardness found in this study. Based on the scientific proof that caries is an infectious disease, it was determined that its treatment should consist of the complete removal of bacteria to achieve dentine sterility and avoid its recurrence8,9,19. Thus, diverse studies were performed to identify suitable criteria for removing carious dentine that would guarantee the absence of microorganisms20,21, since clinicians have great difficulty in determining which dentine must be removed during cavity preparation. The clinical criteria of coloration, hardness and caries-indicating dye are used to achieve a bacteriafree cavity preparation and impede caries progression. These criteria, however, are empirical and subject to individual variations. Coloration is not a reliable parameter, since dentine may be pigmented in cases of dormant caries, or as a result of physiologic defense reactions of the organism and must be preserved, not removed. Clinicians did not find an association between color of dentin and presence of cariogenic microorganisms4,12,22.
In relation to the clinical criterion of hardness, although it is not scientifically precise, it must be considered at least as a limit to determine the interruption of carious dentine removal until a more accurate method is developed11,23. It is also known that due to the tubular structure of dentine, there is bacterial penetration inside the dental tubes, making the total removal of pathogenic microorganisms impossible. Thus, some viable microorganisms inevitably remain12. For this reason, this study opted for the removal of carious dentine based on the clinical criterion of hardness and evaluated microbiota and the progression of caries through a 1-year followup, to evaluate whether the routinely-used clinical criterion is adequate to avoid caries recurrence. The permanence of bacteria occurs after carious dentine removal using any of the 3 clinical criteria (coloration, hardness, caries-indicating dye). However, in keeping with the literature10,14, this study reveals that bacterial remnants of non-softened dentine (hardness criterion) are not capable of causing lesion progression if the cavity is well sealed, since even if bacteria are present, there are no nutrients available for them to grow and multiply and destroy dental tissue. Therefore, the clinical characteristic of dentine hardness seems to be a good indicator of dentine caries progression, since softened dentine was removed from all 34 teeth, leaving them with a rigid aspect and not a single case of caries recurrence was detected during the 1-year follow-up. The clinical characteristic of coloration does not seem to be a good indicator of caries progression, since none of the teeth that presented a rigid aspect, regardless of dentine coloration (light, light with dark spots or dark), presented caries recurrence. The coloration presented by dentine remnants has no significant correlation with the type of microbiota, further confirming the fact that the clinical criterion of coloration is not effective in determining the removal of carious dentine. These results are in agreement with study by Banerjee et al.22.
Scientific evidence demonstrates that caries only occur in the presence of microorganisms24. Among the microorganisms found in dentine caries, a great majority of authors report a predominance of Grampositive bacilli2,4,19. Bjorndal and Larsen19 found a predominance of Gram-positive bacilli in carious dentine, followed by streptococci. Before dentine removal, after 4-6 months, when it was harder and more pigmented, the total number of colony-forming units was reduced, as was the frequency and proportion of Lactobacilli, with the microbiota dominated by A. naeslundii and various streptococci. This species, which predominated after cavity preparation, did not represent the typical microbiota of deep lesions, confirming the possibility of no progression of the caries lesion after cavity preparation. Dental lesions do not necessarily present the same bacteriologic aspect, due to the different availability of nutrients, which vary according to the size of cavity opening. In this sense, the microbiota of the carious dentine was investigated to compare this microbiota with that found after cavity preparation. In this study Gram-positive cocci were predominant, followed by Gram-positive bacilli, probably because mid-depth cavities were investigated, as opposed to most studies, which studied deep cavities. After cavity preparation, no Gram-positive bacilli were recovered and the predominance was of Gram-positive cocci at lower scores. These microorganisms would not be typical of carious dentine lesions, as affirmed by Bjorndal and Larsen19 and for this reason, no caries recurrence occurred in any of the samples. A qualitative analysis of the bacteria found in carious dentine and in dentine after cavity preparation (BL and 1 year) revealed that bacteria found in carious dentine is much more abundant and diversified than that found in dentine remnants after cavity preparation. A predominance of Gram-positive cocci was observed in carious dentine, followed by Gram-positive bacilli. Cavity preparation eliminated Gram-positive bacilli, leaving only a reduced amount of the same Gram-positive cocci found in carious dentine. Despite the persistence of bacteria after preparation, no caries recurrence was observed at the end of the experimental period. Given that a satisfactory clinical seal was observed in all samples after 1 year of follow-up, bacteria would be rendered non-viable by adequate sealing. In addition, Gram-positive cocci had lower scores and are not related to caries progression19. These findings would confirm that cavity preparation, based on the clinical criterion of hardness, eliminates Gram-positive bacilli, the microorganisms that predominate in dentine caries and are probably responsible for caries progression. Only the Gram-positive cocci that may be part of the affected dentine would remain. Since Gram-positive cocci are susceptible to remineralization they can persist. A proper cavity seal reduces the microbiota even in situations of incomplete removal of the carious tissue4,9,25, potentially contributing to the non-progression of the caries.
S. anginosus was the most prevalent and most significant bacteria both in the carious dentine and the dentine remnants after preparation, followed by S. sobrinus, suggesting that these organisms have a role in the pathogenesis of medium-size dentine caries. S. sanguis was the only bacteria found exclusively at BL. It is considered an initial colonizer of dental biofilm. The fact that it no longer appeared after 1 year, when the microbial deposits and cavity seal were removed, further supports this notion. P. gingivalis and P. anaerobius were the two species found only after 1 year, conceivably due to the fact that they perform proteolysis efficiently and thus manage to survive in the nutrient-poor environment of the bottom of a sealed cavity, that is, in dentine. They were not detected at BL, conceivably because the collection method was not adequate for low levels. Negative bacteriological examinations may signify the presence of microorganisms at an undetectable level or at lower numbers in the specimens studied. The patients who still presented bacteria after cavity preparation (BL and 1 year), did not present problems of caries recurrence (as observed by radiography and through opening of the cavity), suggesting that the bacteria that remain in the cavity after preparation are not viable to continue the carious process, provided that the cavities are properly sealed, as affirmed by Knight et al.25 and Silva et al.10.
The clinical parameter (hardness) seems to be sufficient for the non-progression of caries, provided that the cavity is correctly sealed. This result is in accordance with Ricketts et al.26 who found a relation between the level of dentine infection and the visual scores used in occlusal caries diagnosis. The current clinical criteria for carious dentine removal lead to the persistence of bacterial remnants in the cavity. For this reason special attention must be given to sealing the cavity to impede access to nutrients essential for bacterial survival and proliferation. The microbiota found in the carious dentine of middepth cavities is composed mainly of Gram-positive cocci, followed by Gram-positive bacilli. After cavity preparation the gram-positive bacilli were not recovered and the Gram-positive cocci became predominant in lower quantities than those of carious dentine. Gram-positive cocci in small quantities have no relation to caries progression and are likely a part of the microbiota of the affected dentine.
No correlation was observed between bacteria type and the aspect of carious dentine or with dentine remnants after cavity preparation (BL and 1 year). There was no correlation between the clinical criterion of coloration and caries progression, however, the clinical aspect of hardness proved to be effective in determining the non-progression of caries.

REFERENCES

1. Kidd EAM, Fejerskov O. What constitutes dental caries? Hispopathology of carious enamel and dentin related to the action of cariogenic biofilms. J Dent Res 2004;83: C35-C38.         [ Links ]

2. Botha SJ, Boy SC, Botha FS, Senekal R. Lactobacillus species associated with active caries lesions. J Dent Assoc S Afr 1998;53:3-6.         [ Links ]

3. Hoshino E. Predominant obligate anaerobes in human carious dentine. J Dent Res 1985;64:1195-1198.         [ Links ]

4. Maltz M, Oliveira EF. A clinical, microbiologic and radiographic study of deep caries lesions after incomplete caries removal. Quint Int 2002;33:151-159.         [ Links ]

5. Kidd EA. How ‘clean’ must a cavity be before restoration? Caries Res 2004;38:305-313.

6. McComb D. Systematic review of conservative operative caries management strategies. J Dent Educ 2001;65: 1154-1161.         [ Links ]

7. Sattabanasuk V, Burrow MF, Shimada Y, Tagami J. Resin adhesion to caries-affected dentine after different renoval methods. Aust Dent J 2006;51:162-169.         [ Links ]

8. Kreulen CM, Soet JJ, Weerheijm KL. In vivo cariostatic effect of resin modified glass ionomer cement and amalgam on dentine. Caries Res 1997;31:384-389.         [ Links ]

9. Weerheijm KL, Kreulen CM, Soet JJ, Groen HJ, Van Amerongen WE. Bacterial counts in carious dentine under restorations: 2-year in vivo effects. Caries Res 1999; 33:110-134.         [ Links ]

10. Silva NRFA, Carvalho RM, Pegoraro LF, Tay FR, Thompson VP. Evaluation of a Self-limiting Concept in Dentinal Caries Removal. J Dent Res 2006;85:282-286.         [ Links ]

11. Bausells HII. Quantificacao da dureza e avaliacao histobacteriologica da dentina remanescente nao corada pelo “caries detector”. Estudo in vitro em molares deciduos com carie profunda. Araraquara: Faculdade de Odontologia, 1990; p. 83. Tese de livre docencia em odontopediatria. Universidade Estadual Paulista Julio de Mesquita Filho.

12. Burnett JR, Conceicao EN. Doenca carie: manifestacoes clinicas, diagnosticas e terapeutica. In: Conceicao EN et al. Dentistica saude e estetica, Porto Alegre: Artmed, 2002; p. 25-36.         [ Links ]

13. Ricketts DNJ, Kidd EAM, Beighton S. Operative and microbiological validation of visual, radiographic and electronic diagnosis of occlusal caries in non-cavited teeth judged to be in need of operative care. Br Dent J 1995; 179:214-220.         [ Links ]

14. Mertz-Fairhurst EJ, Curtis JR, Ergle JW, Rueggeberg, FA, Adair SM. Ultraconservative and cariostatic sealed restorations: results at 10 years. J Am Dent Assoc 1998;129:55-66.         [ Links ]

15. Colombo AP, Teles R, Torres MC, Souto R, Uzeda M. Subgingival microbiota of Brazilian subjects with untreated chronic periodontitis. J Periodontol 2002;73:360-369.         [ Links ]

16. De Munck J, Landuyt KV, Peumans M, Poitevin A, Lambrechts P, Braem M, Meerbeek BV. A critical review of the durability of adhesion to tooth tissue: methods and results. J Dent Res 2005;84:118-132.         [ Links ]

17. Itota T, Nakabo S, Narukami T, Doi J, Yoshiyama M. Effect of fluoride releasing liner on demineralized dentin. Quint Int 2006;37:297-303.         [ Links ]

18. Marczuk-Kolada G, Waszkiel D, Luczaj-Cepowicz E, Kierklo A, Pawinska M, Mystkowska J. The effect of glass ionomer cement Fuji IX on the hard tissues of teeth treated by sparing methods (ART and CMCR). Adv Med Sci 2006; 1:138-141.         [ Links ]

19. Bjorndal L, Larsen T. Changes in the cultivable flora in deep carious lesions following a stepwise excavation procedure. Caries Res 2000;34:502-508.         [ Links ]

20. Iost HI, Costa JH, Rodrigues HH, Rocca RA. Dureza e contaminacao bacteriana da dentina apos a remocao da lesao de carie. Rev ABO Nac 1995;3:25-29.         [ Links ]

21. McComb D. Caries-detector dyes: how accurate and useful are they? J Can Dent Assoc 2000;66:195-198.         [ Links ]

22. Banerjee A, Waltson TF, Kidd EA: Dentine caries excavation: a review of current clinical techniques. Br Dent J 2000; 188:476-482.         [ Links ]

23. Bjorndal L, Mjor IA. Pulp-dentin biology in restorative dentistry. Part 4: Dental caries - characteristics of lesions and pulpal reactions. Quint Int 2001;32:717-736.         [ Links ]

24. Lima GQT, Oliveira EG, Souza JIL, Monteiro Neto V. Comparison of the efficacy of chemomechanical and mechanical methods of caries removal in the reduction of Streptococcus mutans and Lactobacillus ssp in carious dentine of primary teeth. J Appl Oral Sci 2005;13:399-405.         [ Links ]

25. Knight GM, McIntyre JM, Graig GG, Mulyani, Zilm PS, Gully NJ. Differences between normal and demineralized dentine pretreated with silver fluoride and potassium iodide after an in vitro challenge by Streptococcus mutans. Aust Dent J 2007;52:16-21.         [ Links ]

26. Ricketts DNJ, Ekstrand KR, Kidd EAM, Larsen T. Relating visual and radiographic ranked scoring systems for occlusal caries detection to histological and microbiological evidence. Oper Dent 2002;27:231-237.         [ Links ]

Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons