Sakoolnamarka et al.

Int Chin J Dent 2006; 6: 49-52.

Comparison of microhardness of caries detector dye stained andunstained dentin
Rangsima Sakoolnamarka, DDS, PhD, Nitiwadee Krinpakdee, and Tawatchai Bannakij
Department of Operative Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Purpose: To evaluate microhardness of stained and unstained carious dentin which was differentiated by twocaries detector dyes (Caries Detector and Sable Seek). Further, to compare the ability of the two dyes inrecognizing caries-infected dentin.
Materials and Methods: Forty human molars with occlusal caries were sectioned longitudinally through thelesions in the mesio-distal plane and polished with aluminium oxide paste. Half of the specimens were stainedwith Caries Detector, and the remaining with Sable Seek. The specimens were microhardness tested using a
Knoop microhardness indenter. The mean hardness values from each group were calculated and compared using
one-way ANOVA and t-test.
Results: The mean microhardness values of dye-stained dentin from Caries Detector and Sable Seek groups
were 9.99 and 7.93 KHN, respectively, which were significantly lower than those of unstained dentin (20.25 forCaries Detector group and 19.24 for Sable Seek group). There was no significant difference between
microhardness values obtained from unstained carious dentin determined by the two caries detector dyes.
Conclusion: The hardness values of dye-stained dentin were lower than those of the unstained area. The abilityof Caries Detector and Sable Seek in differentiation of the two layers of carious dentin was not different when
dentin hardness was the only considered criterion. (Int Chin J Dent 2006; 6: 49-52.)
Key Words: carious dentin, caries detector dye, microhardness.

Introduction
Carious dentin consists of two layers; the outer carious layer or caries-infected dentin, which is soft,
contaminated with bacteria and cannot be remineralized; and the inner carious layer or caries-affected dentin,
which is hard, bacteria-free, and can be remineralized.1 The main criteria for caries removal are color andhardness, which can be detected by visual and tactile examination. However, these may not be reliable guides.
The use of caries detector dyes was therefore introduced as a guide for caries removal. Several dyes have beentested on extracted teeth to differentiate the two layers of carious dentin and it was found that 0.5% basic fuchsinin propylene-glycol was the dye that successfully served the purpose.2 A distinct boundary separated stained andunstained zones. Bacterial invasion apparently coincided with staining and no bacteria were found when all
stained dentin was removed.3 The dye solution has therefore been recommended as a clinical guide for complete
removal of bacterially infected dentin. Due to the potential carcinogenicity, basic fuchsin has later been replacedwith acid red.4 Since then, various protein dyes have been developed as caries detecting dyes.
Because an attack of dental caries gradually softens dentin, hardness is considered as one of the main criteria
for caries removal. Knowledge of the dentin hardness is essential to improve caries removal and restorative
procedures. The hardness values for sound dentin reported in the literatures ranged from 20 to 83 Knoop
hardness number (KHN).5-10 The microhardness testing of carious dentin by Banerjee et al.11 demonstrated amean value varying from 13.64 KHN in the discolored area to 48.35 KHN in the transparent dentin. However,
the microhardness of stained carious dentin and that of the unstained carious dentin after two caries detector dyes
used has never been reported.
Hardness testing is widely used to determine the mechanical properties of materials. The Vickers and Knoop
indenters are the most commonly used instruments for microhardness testing since they employ loads of lessthan 1 kgf (9.8 N) and the indentations are small. Thus they are useful for providing hardness numbers of small
areas.12 The purposes of this study were to compare the microhardness values between stained and unstained49

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Int Chin J Dent 2006; 6: 49-52.

zones of carious dentin after the use of two caries detector dyes and compare the ability of the two dyes inrecognizing caries-infected dentin.

Materials and Methods
Forty permanent molars, with occlusal caries to a depth of 2-2.5 mm below the central fissure as assessed witha dental probe, were used. The teeth were stored in 3% formalin solution until required for preparation. The
teeth were embedded in epoxy resin, sectioned through the center of the carious lesions in the mesio-distal
longitudinal plane to obtain dentin discs of 1 mm thick using low speed cutting machine (Isomet, Buehler Ltd.,
Lake Bluff, IL, USA). The dentin discs were polished with silicon carbide paper and finished with aluminumoxide paste up to 0.05 µm particle size. The specimens were randomly divided into two groups. One group wasstained with Caries Detector (Kuraray Medical Inc., Tokyo, Japan) consist of 1% acid red dye and propylene
glycol solvent, and the other with Sable seek (Ultradent Products Inc. South Jordan, UT, USA) consist of FD&C
dark green dye and propylene glycol solvent. Each group was applied with the dye solution for 10 s, rinsed withwater for 10 s and air-dried. The stained specimens were examined under stereomicroscope to separate andmark the boundary between stained and unstained area with sharp dental probe.
A digital microhardness testing machine (FM-700e Series, Future-Tech Corp., Kawasaki, Japan) with a
Knoop indenter was used. Each specimen was mounted on the stage of the microhardness testing machine withdouble-sided adhesive tape and positioned under the microscope. The measured area of each specimen wasselected to obtain the distance of approximately 1.5 mm from the boundary towards the pulp for the indentationline under low magnification. The measurements were made from carious dentin towards the pulp chamber withthe load 50 g. The indentation line was selected, beginning at the stained area (carious dentin) 100 µm above theboundary and the second indentation was made at the unstained zone 100 µm below the boundary (unstainedcarious dentin). The next indentations were made 200 µm apart from the second indentation towards the pulp
(Fig. 1). The hardness number of the normal dentin was the average number from the third measurement to thesecond last measurement. For the last indentation, which was considered as the pulpal area, the indentation wasmade at 200 µm from the pulpal wall. The total number of indentation on each specimen was eight.

Fig. 1.
Diagrammatic representation of therelationship of the carious dentin
and the indentations.
1. stained carious dentin,
2. unstained carious dentin,
3. normal dentin, and4. pulpal area.

One way ANOVA with subsequent Scheffe multiple comparison at p<0.05 was used to analyze thedifferences of the microhardness numbers from different areas of dentin. The microhardness numbers from thetwo caries detector dyes were compared using t-test.

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Int Chin J Dent 2006; 6: 49-52.

Results
The mean microhardness numbers with SDs of carious dentin and dentin from different areas after two caries
detector dyes stained are shown in Table 1.

The results indicated the significant differences of themicrohardness numbers of different zones in the dentin. The hardness of stained area was lower than that of theunstained area. The microhardness values of pulpal area were significantly higher than those of the stained andunstained areas but lower than the normal area. There was no significant difference in hardness of stained areas
between the two groups. Furthermore, a comparison of microhardness values of unstained area obtained fromCaries Detector group and Sable Seek group demonstrated no significant difference.
Table 1.

Knoop hardness number (KHN) of dentin after being stained with two caries detector dyes.
KHNCaries Detector
Sable Seek

Stained dentin
Mean SD

Unstained dentin
Mean SD

Normal dentin
Mean SD

Pulpal dentin
Mean SD

9.99
7.93

20.25
19.24

42.13
46.86

36.93
32.96

3.80
2.86

8.41
5.25

7.24
5.69

11.29
6.24

Discussion
The microhardness values from various areas of dentin were significantly different. This may be due to thedifference in inorganic composition in different locations.9 The hardness of dentin also depends on the state ofmineralization of tissue.11 Meredith et al.10 reported that the hardness of dentin decreased with distance from thedentino-enamel junction. However, there was an inverse correlation between dentin hardness and tubular
density whereby tubular density increased while dentin hardness decreased as the pulp chamber wasapproached.9 This is the reason for the lower hardness numbers of the pulpal area than those of normal dentin.
In carious dentin, the hardness depends on stage of caries progression, probably caused by remineralization
process.13,14 In the present study, the hardness numbers were in agreement with the previous studies.6,7,15 The
carious dentin, which was stained by Caries Detector and Sable Seek, showed the lowest microhardness numbersof all areas (9.99 KHN and 7.93 KHN, respectively). This was due to the attack of acid from caries process.
The collagen fibers were irreversibly damaged by the acid and lost their function as a base for the attachment of
apatite crystals.16
The hardness numbers of unstained dentin were higher than those of stained dentin but lower than those ofnormal dentin. This is because this area is also decalcified by acid. However, collagen fibers and odontoblastic
process are unimpaired, which allow apatite crystals to attach and be remineralized when the caries-causing
bacteria are removed. The dentin at this level of attack is uninfected by bacteria Although this dentin can beremineralized, the crystals did not possess the same hardness value as normal hydroxyapatite crystals because of
the difference in crystal structure.15
The distance of 100 µm apart from the boundary in stained and unstained dentin was selected for the first twoindentations. This is because the present study aimed to evaluate the ability of two agents in differentiation of
the two layers of carious dentin. Thus, the hardness numbers most adjacent to the boundary were required.
Further, with the selected distance, the two indentations were able to identified without overlapping.
As mentioned earlier that hardness is one of the main criteria for caries removal and the results from thisinvestigation also showed the significant difference between the microhardness numbers of the stained and51Sakoolnamarka et al.

Int Chin J Dent 2006; 6: 49-52.

unstained areas. Thus, caries detector dyes could help the clinicians in removing carious dentin, especially thoseindividuals who do not experience in caries removal.17 A comparison was made between the microhardness
numbers of stained and unstained dentin after using the two dyes, no significant differences were exhibited
between stained areas from the two dyes although they were different in color. Caries Detector was red. Sable
Seek was dark green. However, they were in the same solvent (propylene glycol). The same consequence wasshown with the unstained areas, which implied that the ability in caries differentiation was not different. Thissuggests that the two caries detector dyes can be used in substitution of each other as an adjunctive procedure to
aid the dentist in caries removal.
Caries detector dyes have been introduced to assist in diagnosis and removal of carious dentin bydifferentiation of the two layers of carious dentin. However, caries detector dyes should be used cautiously, as
the dyes stain not only the damaged collagen fibers of the caries-infected dentin but also stain the demineralizedorganic material.18,19 Hence, the dyes can stain circumpulpal dentin and dentin-enamel junction in sound teeth.
This can lead to the ‘false positive’ with regard to detecting carious tissue and therefore increase the likelihoodof excessive removal of sound tooth structure and increased likelihood of mechanical pulpal exposure.
Acknowledgment
This research was supported by Dental Research Fund, Dental research project 3205-312#32/2002 Faculty of Dentistry,Chulalongkorn University, Thailand. The authors would like to thank Dr. Chanawatr for her suggestion with the manuscript.

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Correspondence to:
Dr. Rangsima Sakoolnamarka
Department of Operative Dentistry, Faculty of Dentistry, Chulalongkorn University
Henri-dunant Rd, Patumwan, Bangkok 10330, Thailand
Fax: +66-2-218-8795 E-mail: arangsima@hotmail.com
Received February 19, 2006. Accepted March 21, 2006.
Copyright ©2006 by the International Chinese Journal of Dentistry.

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