ADRF REPORT
Australian Dental Journal 2002;47:(2):116-122
Morphological study of demineralized dentine after caries
removal using two different methods
R Sakoolnamarka,* MF Burrow,† S Kubo,‡ MJ Tyas§
Abstract
Background: Caries-affected dentine is the usual
substrate bonded to in everyday clinical practice.
The first step for most of the current dentine
adhesive systems is etching or conditioning. It is
therefore important to understand the effect of
etchant/conditioner on caries-affected dentine.
Methods: Twelve extracted carious permanent
molars were examined. Six teeth had caries removed
using burs after staining with a caries detector dye,
and caries from the other six was removed using
CarisolvTM. After caries removal, three teeth from
each group were left untreated. The remaining teeth
were sectioned to obtain two normal dentine
samples and two caries-affected dentine samples
from each tooth. One half of the normal dentine
samples and one half of the caries-affected dentine
samples were treated with 35 per cent phosphoric
acid, and the remaining samples were treated with
20 per cent polyacrylic acid/3 per cent aluminium
chloride. The samples were observed using field
emission scanning electron microscopy (FE-SEM).
Results: Phosphoric acid treated samples showed
more obvious intertubular dentine microporosity
and greater depth of demineralization in cariesaffected dentine. Polyacrylic acid treated samples
showed some residual smear layer. However, the
peritubular and intertubular collagen network was
easily observed in CarisolvTM treated dentine. The
depth of demineralization was not as distinct as that
of the phosphoric treated samples.
Conclusions: From this study it was shown that
etched ‘normal’ dentine and etched caries-affected
dentine had different surface appearances.
Furthermore, the two caries removal techniques
resulted in different caries-affected dentine surfaces
after acid treatment that may influence the longevity
of bonds from adhesive restorative materials.
Key words: Dental caries, caries removal, demineralization,
field-emission scanning electron microscope.
(Accepted for publication 26 April 2001.)
*Postgraduate Student, School of Dental Science, The University of
Melbourne.
†Associate Professor, School of Dental Science, The University of
Melbourne.
‡Assistant Professor, School of Dentistry, Nagasaki University, Japan.
§Associate Professor and Reader, School of Dental Science, The
University of Melbourne.
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INTRODUCTION
Dentine affected by caries is the common bonding
substrate when treating a patient. It has been described
as consisting of two layers;1 the outer carious dentine,
which is contaminated with bacteria and the collagen
fibres are degraded and cannot be remineralized, and
the inner carious dentine, which is bacteria-free with
limited denaturation of the collagen and can be
remineralized. The outer carious dentine should be
removed during cavity preparation, and the inner
carious dentine should be conserved. The inner carious
dentine has been more commonly referred to as ‘cariesaffected’ dentine in the recent literature.2,3
Carious dentine is routinely removed by rotary and
hand instrumentation. Colour and hardness have been
used as the main criteria for the clinical assessment of
caries removal, but these may not be reliable guides.4
Caries detector dyes have also been recommended as a
guide for caries removal,2,3 but should be used
cautiously, as the dyes can also stain circumpulpal
dentine and the dentino-enamel junction in sound
teeth.5 This can lead to a ‘false positive’ with regard to
detecting carious tissue, and therefore excessive
removal of sound tooth structure.
Recently, a new chemo-mechanical caries removal
system, CarisolvTM (Medi-Team, Sävedalen, Sweden), has
been developed. This system consists of sodium
hypochlorite and three amino acids, namely lysine,
leucine, and glutamic acid. The system is used with
specially designed non-cutting hand instruments. Ericson
et al.6 revealed that the efficacy of caries removal with
this system was the same as that using conventional
rotary instruments, and that these specially designed
instruments reduced the chance of removing intact
dentine. Furthermore, local anaesthesia was required less
often with this technique, since patients experienced less
pain compared with rotary instrumentation.
After caries removal and prior to the placement of
either a resin composite or glass ionomer cement, the
dentine is treated with a demineralizing agent specific
to either of these restorative materials. Nakabayashi7
proposed that the adhesion of resin-based adhesives
was by infiltration into peritubular and intertubular
Australian Dental Journal 2002;47:2.
Fig 1. Scanning electron microscopy micrograph of dentine after caries removal using rotary instrumentation and caries detector dye: a) the
smeared surface is characteristic of cut dentine; b) fractured surface showing the remaining dentine structure with mineralized collagen fibres. PT,
peritubular dentine; IT, intertubular dentine.
dentine of which the collagen network had been
exposed by demineralization, to form the ‘hybrid
layer’. Nakajima et al.8 revealed that moist bonding to
normal dentine produced no significant difference in
bond strength, compared with that to caries-affected
dentine when Scotchbond Multi-Purpose Plus was used
with 35 per cent phosphoric acid. The degree of
demineralization by a demineralizing agent may affect
the adhesion of ‘single-bottle’ dentine adhesives to
caries-affected dentine, e.g., Nakajima et al.9 reported
that bond strengths of those adhesives to such dentine
were different when using different concentrations of
phosphoric acid. However, there was no effect on bond
strengths to normal dentine.
The purpose of this study was to examine the surface
morphology of caries-affected dentine after it had been
exposed using either rotary instrumentation in
conjunction with caries detector dye or a chemomechanical caries removal system (CarisolvTM), and then
conditioned with one of two different demineralizing
agents, namely phosphoric acid and polyacrylic acid.
M AT E R I A L S A N D M E T H O D S
Twelve extracted carious human permanent molars,
which had been stored at 4ºC in normal saline
containing thymol, were used within three months
following extraction. Each tooth had occlusal caries to
a depth of approximately 1-2mm below the central
fissure, as assessed with a probe by one of the investigators (RS). The occlusal enamel was removed using a
slow-speed diamond saw under copious water spray.
Six teeth had the caries removed using slow-speed
round steel burs (ISO #012; ELA, Engelskirchen,
Germany) after staining with a caries detector dye
(Caries Detector (Batch No 0250D); Kuraray Co,
Osaka, Japan) until the dentine was no longer stained
by the dye and was firm to probing with a blunt dental
explorer. The caries from the other six teeth was
removed using CarisolvTM gel (Batch No 10122;
Medi-Team, Sävedalen, Sweden) according to the
Australian Dental Journal 2002;47:2.
manufacturer’s instructions. The gel was applied to the
lesion for 30 seconds, and the instruments supplied
were used to remove the softened carious dentine.
More gel was applied and caries removal continued
until the gel was no longer cloudy, and the surface felt
hard to a blunt dental probe. After caries removal,
three teeth from each group were investigated without
acid treatment for comparison purposes. The remaining
teeth were lapped down using 600-grit SiC paper until
the normal dentine surrounded the cavity at almost the
same level as the exposed cavity floor, in order to act as
a control surface. The teeth were sectioned to obtain
four samples from each tooth; two of normal dentine
and two of caries-affected dentine. A shallow groove
was placed in each sample, on the opposite side to the
surface under study, using a diamond disc (ISO #335220-74; Dentsply, Milford, Delaware, USA) in a slowspeed handpiece. One half of the normal dentine
samples and one half of the caries-affected dentine
samples were treated with 35 per cent phosphoric acid
(Ultraetch (Batch No 61624DV); Ultradent Products
Inc., South Jordan, Utah, USA) for 15 seconds and
washed for 20 seconds. The remaining samples were
treated with 20 per cent polyacrylic acid/3 per cent
aluminium chloride solution (Cavity Conditioner
(Batch No 270571); GC International, Tokyo, Japan)
for 10 seconds and washed for 20 seconds. Samples
were fixed in 10 per cent phosphate buffered formalin
for 24 hours, rinsed with distilled water three times for
15 minutes each time and dehydrated in increasing
concentrations of ethanol and water up to 90 per cent
ethanol, and placed in 100 per cent ethanol three times
for 15 minutes each time. The specimens were placed in
a critical point drier (Samdri PVT-3; Tousimis Research
Corp., Rockville, Maryland, USA) until all residual
moisture had been removed, and fractured along the
prepared grooves using pliers. The conditioned surfaces
and the fractured surfaces were gold sputter-coated and
observed using a field-emission scanning electron
microscope (XL30 FEG; Philips, Eindhoven, The
Netherlands).
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Fig 2. Scanning electron microscopy micrograph of dentine after caries removal using CarisolvTM: a) dentine with irregular, rough and porous
surface is exposed after CarisolvTM treatment; b) fractured surface showing mineralized collagen fibres with a few porosities in intertubular dentine.
PT, peritubular dentine; IT, intertubular dentine.
R E S U LT S
Representative micrographs are displayed in Fig 1-5.
Differences were noticed between the dentine surfaces
obtained using the two different caries removal
systems. A smear layer was apparent on the teeth
prepared with rotary instrumentation (Fig 1a), whereas
the CarisolvTM treated surfaces (Fig 2a) were irregular,
porous and did not exhibit a smear layer. The fractured
surface of the specimens from the bur-treated group
(Fig 1b) exhibited intact peritubular dentine, which was
similar to the peritubular dentine of CarisolvTM treated
samples (Fig 2b). However, more porosities were
Fig 3. Scanning electron microscopy micrograph of normal dentine: a) dentine surface treated with 35 per cent phosphoric acid showing patent
dentinal tubules with peritubular collagen fibre network and intertubular microporosity; b) fractured surface showing regular oriented exposed
collagen fibres (arrows) with the depth of demineralization ranging from 1.5-2µm; c) dentine surface treated with 20 per cent polyacrylic
acid/Al2Cl3 showing openings of the dentinal tubules with some residual smear layer and fibrous structure inside the tubules (arrows); d) fractured
surface showing collagen fibres with some mineral remaining.
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Australian Dental Journal 2002;47:2.
Fig 4. Scanning electron microscopy micrograph of dentine after caries removal using round steel burs and caries detector dye: a) the dentine
surface with patent dentinal tubules and intertubular microporosity after treatment with 35 per cent phosphoric acid, some fibrous structure
remained in the dentinal tubules; b) lateral view of the randomly oriented exposed collagen fibres (arrows), a cuff of peritubular dentine in the
dentinal tubules was also noted, the depth of demineralization was ranging from 3.5-4.5µm; c) the dentine surface showing partial opening of
dentinal tubules with the residual smear layer after being treated with 20 per cent polyacrylic acid/Al2Cl3 and; d) fractured surface showing
collagen fibres with calcific deposits remaining.
evident in the intertubular dentine of the CarisolvTM
treated teeth. The normal dentine surface and the
fractured longitudinal surface, both after treatment
with phosphoric acid are shown in Fig 3a and 3b
respectively. Patent dentinal tubules and the peritubular
collagen network were clearly visible. The fractured
view clearly showed exposed collagen fibres with the
depth of demineralization ranging from 1.5-2µm.
Comparable views after polyacrylic acid/aluminium
chloride treatment are shown in Fig 3c and 3d. Some
residual smear layer and a fibrous structure inside the
tubules was revealed. The fractured view showed
collagen fibres with some calcific deposits remaining.
The dentine after caries removal using rotary
instrumentation/caries detector dye is shown in Fig 4.
After treatment with phosphoric acid (Fig 4a), the
dentine surface showed patent dentinal tubules and
peritubular collagen network. Some fibrous structures
were found in the dentinal tubules and the circular
orientation of the peritubular collagen network was
revealed. The intertubular microporosity appeared to
be more obvious than in normal dentine (Fig 3a). The
fractured surface (Fig 4b) showed an exposed collagen
network as found in normal dentine (Fig 3b), but the
depth of demineralization appeared to be greater in
Australian Dental Journal 2002;47:2.
caries-affected dentine (approximately 3.5-4.5µm). In
addition, the orientation of the exposed collagen fibres
appeared to be irregular, in contrast to the regular
orientation of the fibres in normal dentine. A cuff of
peritubular dentine was also observed. The dentine
surface after being treated with polyacrylic
acid/aluminium chloride (Fig 4c) displayed a partial
opening of dentinal tubules with a residual smear layer
remaining. The lateral view showed a collagen fibre
network with a considerable amount of residual
mineral deposit (Fig 4d).
The images of dentine after caries removal using
CarisolvTM are shown in Fig 5. The dentine surface after
treatment with phosphoric acid (Fig 5a) showed patent
dentinal tubules and an exposed peritubular collagen
network, as in the normal dentine sample. The exposed
superficial intertubular collagen network is obviously
different from that of the normal dentine sample. The
distinctly reticular collagen fibre network with a
random orientation was noted in the CarisolvTM
samples. The fractured view (Fig 5b) showed exposed
collagen fibres. The depth of demineralization ranges
from 7-8µm, which appeared to be greater than in the
dentine sample where burs and a caries detector were
used. After treatment with polyacrylic acid/aluminium
119
Fig 5. Scanning electron microscopy micrograph of dentine after caries removal using CarisolvTM: a) dentine surface treated with 35 per cent
phosphoric acid showing patent dentinal tubules with a clearly exposed peritubular and intertubular collagen network; b) lateral view of the
exposed collagen fibres (arrows) with the depth of demineralization approximately 7-8µm; c) dentine surface treated with 20 per cent polycrylic
acid/Al2Cl3 showing openings of dentinal tubules with distinguishable peritubular and intertubular collagen network; d) fractured surface showing
collagen fibres with some calcific deposits remaining.
chloride, the dentine surface showed the opening of
dentinal tubules with a peritubular and intertubular
collagen network (Fig 5c). The collagen fibres appeared
to be more distinguishable in the CarisolvTM treated
samples than in both the normal dentine (Fig 3c) and in
the caries detector dye treated samples (Fig 4c). The
fractured view showed collagen fibres with some
calcific deposits remaining (Fig 5d). The depth of
demineralization of the samples treated with polyacrylic acid/aluminium chloride was not as evident as
that of the samples treated with phosphoric acid.
DISCUSSION
The adhesion between dentine and resin-based
adhesives is due to micro-mechanical interlocking of
resin into collagen exposed by demineralizing the
dentine surface with phosphoric acid.10 Erickson11
described the typical action of dentine demineralizing
agent on ‘normal’ dentine surfaces, which resulted in an
excellent opportunity for micro-mechanical retention
of resin and in high bond strengths. In the present
study, the normal dentine surface after demineralization
with 35 per cent phosphoric acid showed patent
dentinal tubules and a peritubular collagen network,
and a depth of demineralization ranging from 1.5-2µm,
which is in agreement with the previous study of
Perdigão et al.12
120
The morphology of demineralized ‘normal’ dentine
differed from demineralized caries-affected dentine,
which may be due to the surface which is left after
caries removal being different in ultrastructure and
character from normal dentine. Fusayama1 revealed
that the peritubular and intertubular apatite crystals of
caries-affected dentine were smaller in size and less
numerous than normal dentine. Ogawa et al.13 also
reported that the hardness of the caries-affected dentine
was less than that of normal dentine. The morphology
of etched dentine obtained after rotary instrumentation
and caries detector dye use differed from that of the
CarisolvTM treated sample. This is probably the result of
the caries removal technique and the high pH of 11 of
CarisolvTM.14 It is believed that the non-cutting hand
instruments used with this technique were more
conservative of dentine or that the sodium hypochlorite
in the gel caused some change to the dentine, especially
the collagen, thus leading to the different appearances.
Since preparing caries-affected cavities using CarisolvTM
gel is less destructive of sound tooth structure, bonded
restorations might be suitable for such cavities as
mechanical retention is not required, hence preserving
more tooth structure.
The depth of demineralization by phosphoric acid
appeared to be greater in caries-affected dentine. This is
Australian Dental Journal 2002;47:2.
presumably because the caries-affected dentine is
already partially demineralized and more porous than
normal dentine,15 which may facilitate demineralization.
The greater depth of demineralization may produce a
thicker hybrid layer, as Nakajima et al.16 demonstrated
when bonding to caries-affected dentine, but they also
reported a poor correlation between bond strength of
resin-based adhesive and hybrid layer thickness. The
increased thickness of demineralization may affect the
success of resin adhesive systems, since the deepest part
of the demineralized dentine may be left unencapsulated
by the resin that forms the hybrid layer.17 A study of
nanoleakage within the hybrid layer by Sano et al.18
revealed that leakage occurred around collagen fibres
that were not fully resin-infiltrated. Furthermore,
different leakage patterns were revealed by Li et al.19
when using different resin-adhesive systems. Thus,
more consideration is needed with regard to selection
and timing of demineralizing agents for caries-affected
dentine, in order to ensure a reliable bond of resinbased adhesives to such dentine.
In the present study, for the surface of normal dentine
after demineralization using 20 per cent polyacrylic
acid/3 per cent aluminium chloride (Cavity Conditioner),
some residual smear layer was evident, which was in
consistent with the study of Bloxham et al.20 In their
study, 25 per cent polyacrylic acid was applied to the
‘normal’ dentine surface for 15 seconds, which resulted
in smear layer remaining in the openings of the dentinal
tubules. The extent of dentine demineralization with
Cavity Conditioner, (pH 0.97; Tanumiharja et al.21),
was less than that of 35 per cent phosphoric acid (pH
0.02; Perdigão et al.12). Peutzfeldt and Asmussen22
revealed that the ability to demineralize the dentine
surface was dependent upon factors such as the concentration of the acid, duration of application and the
nature of the surface. This explains why Cavity
Conditioner, which has a higher pH and shorter treatment time, was associated with less demineralization of
the dentine than phosphoric acid. Cavity Conditioner is
used as the demineralizing agent for resin-modified
glass ionomer cement. This restorative material bonds
to dentine by micromechanical interlocking of the
polymer to etched dentine23 and also bonds chemically
by forming ionic bonds to the mineral content of tooth
structure.24 The decrease in calcium content of cariesaffected dentine may affect the chemical bond of glass
ionomer cement to such a surface.
The use of a caries detector dye as a guide in caries
removal may result in excessive removal of sound
dentine, since the dye was specific to not only the
damaged collagen fibres of the infected dentine, but
also stained the demineralized organic material.5
From the results of this study, the chemo-mechanical
caries removal system is believed to be a good
alternative for caries removal since it is more
conservative of sound tooth structure. Moreover, this
system appeared to correspond to the current concepts
in operative dentistry, which are now focusing on the
use of adhesive materials to bond restorations to tooth
Australian Dental Journal 2002;47:2.
structure in order to be conservative. However, a
greater depth of demineralized dentine was exhibited in
CarisolvTM treated and acid treated dentine than in acid
treated normal dentine. Therefore, etching procedures
need to be reviewed to ensure optimum adhesion of
resin-based materials to such dentine.
CONCLUSION
Results from this study showed that etched normal
dentine and etched caries-affected dentine revealed
different arrangements of the exposed collagen fibres.
In addition, caries-affected dentine exposed by the two
caries removal techniques demonstrated different fibril
networks after acid treatment.
Further study is needed, particularly in the area of
the resin-dentine interface, when CarisolvTM is used in
the caries removal process, to provide additional
evidence for the clinical efficacy of this technique.
AC K N OW L E D G E M E N T S
The research was supported by Australian Dental
Research Foundation Inc, St Leonards, NSW 2065,
Australia. The assistance of Jocelyn L Carpenter,
School of Botany, The University of Melbourne, with
SEM imaging is greatly appreciated. The CarisolvTM gel
and instruments were kindly provided by Medi-Team,
Sävedalen, Sweden.
REFERENCES
1. Fusayama T. New concepts in the pathology and treatment of
dental caries: A simple pain-free adhesive restorative system by
minimal reduction and total etching. Tokyo: Ishiyaku
EuroAmerica Inc, 1993:1-21.
2. Harnirattisai C, Inokoshi S, Shimada Y, Hosoda H. Interfacial
morphology of an adhesivecomposite resin and etched cariesaffected dentin. Oper Dent 1992;17:222-228.
3. Nakajima M, Ogata M, Okuda M, Tagami J, Sano H, Pashley
DH. Bonding to caries-affected dentin using self-etching primers.
Am J Dent 1999;12:309-314.
4. Fusayama T, Okuse K and Hosoda H. Relationship between
hardness, discoloration, and microbial invasion in carious dentin.
J Dent Res 1966;45:1033-1046.
5. Yip HK, Stevenson AG and Beeley J. The specificity of caries
detector dyes in cavity preparation. Br Dent J 1994;176:417-421.
6. Ericson D, Zimmerman M, Raber H, Gotrick B, Bornstein R,
Thorell J. Clinical evaluation of efficacy and safety of a new
method for chemo-mechanical removal of caries. A multi-centre
study. Caries Res 1999;33:171-177.
7. Nakabayashi N. Bonding of restorative materials to dentine: the
present status in Japan. Int Dent J 1985;35:145-154.
8. Nakajima M, Sano H, Zheng L, Tagami J, Pashley DH. Effect of
moist vs. dry bonding to normal vs. caries-affected dentin with
Scotchbond Multi-Purpose Plus. J Dent Res 1999;78:1298-1303.
9. Nakajima M, Sano H, Urabe I, Tagami J, Pashley DH. Bond
strengths of single-bottle dentin adhesives to caries-affected
dentin. Oper Dent 2000;25:2-10.
10. Pashley DH, Ciucchi B, Sano H, Horner JA. Permeability of
dentin to adhesive agents. Quintessence Int 1993;24:618-631.
11. Erickson RL. Mechanism and clinical implications of bond
formation for two dentin bonding agents. Am J Dent
1989;2:117-123.
12. Perdigão J, Lambrechts P, Van Meerbeek B, Tome AR, Vanherle
G, Lopes AB. Morphological field emission-SEM study of the
effect of six phosphoric acid etching agents on human dentin.
Dent Mater 1996;12:262-271.
121
13. Ogawa K, Yamashita Y, Ichijo T, Fusayama T. The ultrastructure
and hardness of the transparent layer of human carious dentin. J
Dent Res 1983;62:7-10.
21. Tanumuharja M, Burrow MF, Tyas MJ. Microtensile bond
strengths of glass ionomer (polyalkenoate) cements to dentine
using four conditioners. J Dent 2000;28:361-366.
14. Beeley JA, Yip HK and Stevenson AG. Chemochemical caries
removal: a review of the techniques and latest developments. Br
Dent J 2000;188:427-430.
22. Peutzfeldt A, Asmussen E. Effect of polyacrylic acid treatment of
dentin on adhesion of glass ionomer cement. Acta Odontol Scand
1990;48:337-341.
15. Hamid A, Hume WR. Diffusion of resin monomers through
human carious dentin in vitro. Endod Dent Traumatol
1997;13:1-5.
23. Lin A, McIntyre NS and Davidson RD. Studies on the adhesion
of glass-ionomer cements to dentin. J Dent Res 1992;71:18361841.
16. Nakajima M, Sano H, Burrow MF, et al. Tensile bond strength
and SEM evaluation of caries-affected dentin using dentin
adhesives. J Dent Res 1995;74:1679-1688.
24. Mount GJ. Adhesion of glass-ionomer cement in the clinical
environment. Oper Dent 1991;16:141-148.
17. Eick JD, Gwinnett AJ, Pashley DH, Robinson SJ. Current
concepts on adhesion to dentin. Crit Rev Oral Biol Med
1997;8:306-335.
18. Sano H, Yoshiyama M, Ebisu S, et al. Comparative SEM and
TEM observations of nanoleakage within the hybrid layer. Oper
Dent 1995;20:160-167.
19. Li H, Burrow MF, Tyas MJ. Nanoleakage patterns of four dentin
bonding systems. Dent Mater 2000;16:48-56.
20. Bloxham GP, Dennison JD and Charbeneau GT. A clinical
scanning electron microscope study of tooth surface preparation
and bonding. Aust Dent J 1990;35:345-351.
122
Address for correspondence/reprints:
Dr Michael F Burrow
School of Dental Science
The University of Melbourne
711 Elizabeth Street
Melbourne, Victoria 3000
Email: mfburrow@unimelb.edu.au
Australian Dental Journal 2002;47:2.