d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) e24–e37

available at www.sciencedirect.com

journal homepage: www.intl.elsevierhealth.com/journals/dema

Dentin bonding—Variables related to the clinical situation

and the substrate treatment

Jorge Perdigão ∗
Department of Restorative Sciences, Division of Operative Dentistry, University of Minnesota School of Dentistry, 515 SE Delaware St.,
8-450 Moos Tower, Minneapolis, MN 55455, USA

a r t i c l e i n f o a b s t r a c t

Article history: The wetness of dentin surfaces, the presence of pulpal pressure, and the thickness of dentin
Received 19 November 2009 are extremely important variables during bonding procedures, especially when testing bond
Accepted 19 November 2009 strength of adhesive materials in vitro with the intention of simulating in vivo conditions. The
ultimate goal of a bonded restoration is to attain an intimate adaptation of the restorative
material with the dental substrate. This task is difficult to achieve as the bonding process is
Keywords: different for enamel and for dentin—dentin is more humid and more organic than enamel.
Dental bonding While enamel is predominantly mineral, dentin contains a significant amount of water and
Dentin organic material, mainly type I collagen. This humid and organic nature of dentin makes this
Electron microscopy hard tissue very challenging to bond to. Several other substrate-related variables may affect
the clinical outcome of bonded restorations. Bonding to caries-affected dentin is hampered
by its lower hardness and presence of mineral deposits in the tubules. Non-carious cervi-
cal areas contain hypermineralized dentin and denatured collagen, which is not the ideal
combination for a bonding substrate. Physiological transparent root dentin forms without
trauma or caries lesion as a natural part of aging. Similar to the transparent dentin observed
underneath caries lesions, the tubule lumina become filled with mineral from passive chem-
ical precipitation, making resin hybridization difficult. An increase in number of tubules with
depth and, consequently, increase in dentin wetness, make bonding to deeper dentin more
difficult than to superficial dentin. While the application of acidic agents open the path-
way for the diffusion of monomers into the collagen network, it also facilitates the outward
seepage of tubular fluid from the pulp to the dentin surface, deteriorating the bonding for
some of the current adhesives. Some dentin desensitizers have shown some promise as
they can block dentinal tubules to treat and prevent sensitivity and simultaneously block-
ing the tubular fluid from flowing to the surface. A new approach to stop the degradation of
dentin–resin interfaces is the use of MMP inhibitors. Although still in an early phase of in
vitro and clinical research, this method is promising.
© 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

1. Introduction to the review

Dentin tubules run continuously from the dentin–enamel

junction (DEJ) to the pulp in coronal dentin, and from the
cementum–dentin junction (CEJ) to the pulp canal in the root.
∗
Tel.: +1 612 625 5432; fax: +1 612 625 7440. Pashley (1996) described dentin as a porous biologic compos-
E-mail address: perdi001@umn.edu.
0109-5641/$ – see front matter © 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.dental.2009.11.149
 d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) e24–e37 e25

Fig. 1 – Caries lesion under the FESEM showing: (A) partially demineralized dentin in the transition to caries-affected
dentin; (B) tubules are occluded with mineral deposits in the caries-affected area.

ite made up of apatite crystal filler particles in a collagen

matrix [1]. Other authors have described dentin as a complex
2. Literature
biological structure that forms a continuous fiber-reinforced
composite, with the intertubular dentin making up the matrix 2.1. Caries-affected dentin and tertiary dentin
and the tubule lumens with their associated cuffs of per-
itubular dentin forming the cylindrical fiber reinforcement
The most common pathological challenge to dentin is den-
[2]. Marshall et al. [3] stated that the various structural com-
tal caries. According to the pioneer work by Dr. Fusayama’s
ponents and properties of dentin could directly affect the
research team, carious dentin consists of a superficial first
adhesive bond. Biological and clinical factors such as dentin
layer and a deeper second layer [11–13]. The outer layer
permeability, pulpal fluid flow, sclerotic and carious dentin can
was characterized as highly decalcified, physiologically unre-
also affect dentin bonding [4–7].
calcifiable, fuchsin-stainable, showing degenerated collagen
During the development of the human tooth the dentin
fibers with virtual disappearance of cross-links indicating
that is secreted until the completion of root formation is iden-
irreversible denaturation of collagen. The inner layer was
tified as primary dentin and encompasses the circumpulpal
depicted as intermediately decalcified, physiologically recal-
dentin matrix. This primary dentin forms at a rate of approxi-
cifiable, fuchsin-unstainable, with expanded odontoblastic
mately 4 ␮m per day [8] although the rate of dentin formation
processes, sound collagen fibers, and apatite crystals bound
is slower in the root than in the crown of the tooth. Physio-
to the fibers. Changes in the cross-linkage of collagen in this
logical secondary dentin is secreted after completion of root
inner layer showed cross-links partly shifting to precursors.
formation. The appearance of the matrix of primary dentin
In spite of a great deal of work that has been accomplished
indicates a regular, tubular structure. While secondary dentin
on the biochemical principle of carious dentin staining, it
may be less tubular, it has the same morphological features of
remains unclear how staining is related to ultra-structural
primary dentin.
features of various caries lesion zones [14]. Level of bacteria
The morphological and physical variations in human
found in dye-stained DEJ specimens was no higher than the
dentin make it a difficult substrate for the achievement of
specimens that did not stain with the same dye [15]. Stain-
durable bonds between adhesive resin and dentin. The bond-
able dentin may not be always infected but the absence of
ing mechanism depends on the penetration of the primer and
stain does not ensure bacterial elimination [15,16]. In fact, dyes
adhesive resin into the conditioned dentin surface in order
may not specifically stain the irreversibly damaged collagen
to create micromechanical interlocking with the dentin colla-
found in infected dentin [17] and may even stain dentin in
gen. This mechanical entanglement between resin monomers
non-carious teeth [18]. These findings raise questions about
and dentin components has been named hybrid layer [9] or
the validity of studies on bonding to caries-affected dentin in
resin–dentin interdiffusion zone [10].
e26 d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) e24–e37
Fig. 2 – Tertiary dentin in the pulp horn corresponding to an area of attrition in a mandibular incisor. S = secondary dentin;
T = tertiary dentin; P, pulp space. Note the formation of transparent dentin in the root.
which the caries-infected dentin is removed with the guidance
of dyes.
Changes that will occur in dentin structure in response to
the carious process take place either within the dentin itself,
or on its pulpal aspect. The pulp responds to the carious pro-
cess either by completely blocking the lumen of the dentinal
tubule, or by a decrease in tubule diameter, to assist in the pre-
vention of further permeation of bacteria and toxic materials
towards the pulp. Stanley et al. [19] found that the pulpo-
dentinal complex responds to external injuries with dentin
sclerosis, dead tracts, or reparative dentin. The presence of
reactive dentin sclerosis does not prevent the formation of
reparative dentin, since both seem to occur in response to the
same stimuli. Mendis and Darling in 1979 reported that the
amount of peritubular dentin in the transparent zone under
the SEM was similar to that in normal non-carious dentin and
that the remaining lumen was occluded by a rod-like plug
of granular structure not continuous with peritubular dentin
[20]. It has been reported that these deposits in the dentinal
tubules are ␤-tricalcium phosphate (also called whitlockite)
[21,22], which is less soluble than hydroxyapatite.
In fact, continuous deposition of mineral within the lumina
of the tubules underneath a caries process will lead ultimately
to their obliteration and the formation of sclerosis (Fig. 1). This
type of sclerotic material differs from physiological sclerosis
in that its formation can be triggered by the carious process,
restorative procedures, or attrition and, in these cases, its for-
mation is restricted to the site of the affected area [8,14]. The
tubule occlusion is most apparent in transparent or sclerotic
dentin, leading to its transparent optical appearance [14] and
higher mineral contents than normal dentin [20].
The tissue that is deposited on the pulpal aspect in
response to dental caries is called reactionary tertiary dentin.
Unlike secondary dentin, which is physiological and forms
throughout the vital life of the tooth, the formation of tertiary
dentin is localized in the pulp chamber wall corresponding
to the area of the affected site. Furthermore, tertiary dentin
is divided into reactionary dentin, formed by the surviving
odontoblasts, and reparative dentin, which is formed by newly
differentiated odontoblast-like or odontoblastoid cells [8,23].
The reactionary form of tertiary dentin frequently has a tubu-
lar continuity with the physiological secondary dentin and is
secreted by primary odontoblasts [23]. Fig. 2 shows reactionary
tertiary dentin from attrition in a lower incisor, which, in this
case, does not have tubular continuity with secondary dentin.
The reparative dentin bridge form of tertiary dentin does not
have a tubular continuity with the physiological secondary
dentin matrix. Reparative tertiary dentin is secreted by odon-
toblastoid cells, which have replaced the irreversibly injured
odontoblasts at the site of pulp exposure [23].
While bonding to tertiary dentin might only occur in
deep cavity preparations, caries-affected dentin is a clinically
relevant substrate in daily practice. Much of our under-
standing on dental bonding has been performed on sound,
healthy dentin (‘laboratory’ dentin). This is not the substrate
generally encountered in clinical practice, since dentists fre-
quently bond to caries-affected dentin to replace carious
tissues. Caries-affected dentin has lower hardness than nor-
mal dentin. Although the transparent zone is usually thought
to be harder than unaffected dentin due to occlusions of the
dentin tubules with mineral in the transparent zone, it has
been reported that the transparent layer in caries-affected
dentin can be softer than unaffected dentin [24]. Using a nano-
indentation technique, Marshall et al. [25] reported that the
mean elastic modulus (18.2 GPa) and nanohardness (0.8 GPa)
of transparent intertubular dentin were very slightly, but
 d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) e24–e37
significantly lower than those of the intertubular dentin of
unaffected dentin (20.6 and 1.0 GPa, respectively). However,
within a given lesion, there were frequently no significant dif-
ferences, suggesting that the intertubular dentin was often
unchanged in the transparent zone. For transparent dentin,
the mean elastic modulus of peritubular dentin was 36.1 GPa,
which was not significantly higher than the mean value for
the mineral within the tubules of 34.5 GPa, but both were
significantly less than the modulus for normal peritubular
dentin (38.7 GPa). A significant correlation was found between
dentin hardness and bond strength, however, dentin mechan-
ical properties are not the only factors responsible for lower
bond strength to caries-affected dentin. A micro-Raman spec-
troscopy investigation suggested some structural or chemical
alterations in caries-affected dentin [26]. The mineral phos-
phate and carbonate content decreased in the caries-affected
region of dentin when compared with unaltered dentin [26].
The secondary structure of collagen in the caries-affected
dentin also appeared to be slightly altered by the caries pro-
cess. These results were corroborated by Suppa et al. [27] who
found that the distribution of intact collagen fibrils and proteo-
glycans in the caries-affected dentin was significantly lower
than in normal hard dentin. Reductions in antigenicity from
the organic matrix of sclerotic dentin under caries lesions raise
concern about the potential of intrafibrillar remineralization.
Bond strengths to caries-affected dentin are typically lower
than those obtained in normal unaffected dentin, regardless
of the type of adhesive used [28–31]. A comparison of three
different adhesive strategies (one-step self-etch, two-step self-
etch, and two-step etch-and-rinse) resulted in lower bond
strength to caries-affected dentin than to normal dentin for
all of the adhesives [29]. The lower bond strengths obtained in
caries-affected dentin may be due to the lower tensile strength
and lower hardness of the altered substrate. Clinically, this
may not be a problem, since such lesions are normally sur-
rounded by normal dentin or enamel [30]. Hybrid layers in
caries-affected dentin are usually thicker but more porous
than those in sound dentin [30,31]. Hybrid layers were less
than 1 ␮m thick in normal dentin, but that they were between
6 and 8 ␮m thick in caries-affected dentin [32]. An atypical
interface was observed in caries-infected dentin, in which
carious bacteria within disorganized non-banded collagen fib-
rils were apparently embedded by the adhesive, Clearfil Liner
Bond 2V (Kuraray). The hybrid layer in caries-infected dentin
was 30–60 ␮m thick [32]. The monomers infiltrated the depth
of demineralized unaltered dentin, and the penetration grad-
ually declined across the interface; however, the penetration
of adhesive monomers was irregular and decreased across
the depth of demineralization in the caries-affected dentin
[26]. As a result of the deposition of mineral in the lumina of
the tubules, resin infiltration into dentinal tubules of caries-
affected dentin is also hampered by the presence of mineral
[31]. Using FTIR, Spencer et al. [33] described the interface
with caries-affected dentin as a poorly mineralized structure
readily penetrated by the acid etchant. The substantial area
of demineralization in the caries-affected dentin indicates a
porous area. The degree of conversion of the adhesive (Sin-
gle Bond, 3 M ESPE) that penetrated the demineralized dentin
in the caries-affected dentin specimens was lower than in the
normal dentin specimens [33]. Tay et al. [34] applied a one-step
e27
self-etch adhesive on transparent carious dentin containing
occluded dentinal tubules, with or without pulpal pressure.
After silver impregnation, they observed specimens under
TEM. Although caries-affected dentin was highly porous, sil-
ver particles did not infiltrate the adhesive layer when tubules
were occluded. Conversely, water-treeing and water-droplets
were observed into adhesive layers in bonded sound dentin.
The authors speculated that water-treeing and water-droplet
formation might be eliminated during bonding to occluded
transparent carious dentin.
Bonding to caries-affected dentin may depend on the
specific composition of adhesives. One study compared the
bond strengths of ScotchBond Multi-Purpose (3 M ESPE) with
and without the polyalkenoate component in the primer.
Removal of the polyalkenoic acid from the primer lowered the
bond strength of ScotchBond Multi-Purpose to caries-affected
dentin [35], which suggests that the residual calcium in caries-
affected dentin may be crucial to establish ionic bonding
with the polyalkenoate in the primer. Using Clearfil SE Bond
(Kuraray), Nakajima et al. [36] reported that hydrostatic pulpal
pressure significantly reduced the bond strength to normal
dentin after 1 month of water storage, but did not affect the
bond strength to caries-affected dentin. The presence of min-
eral casts in the tubules may have prevented water seepage to
the area of the bonded interface. Additionally, the higher min-
eral contents of the transparent area of caries-affected dentin
may have resulted in stronger chemical bonding to the MDP
molecule in Clearfil SE Bond, which has been shown to bond
chemically to hydroxyapatite [37].
Another study reported that when One-Step (Bisco Inc.), an
acetone-based etch-and-rinse adhesive, was used on caries-
affected dentin etched with 10% phosphoric acid, it resulted
in lower tensile bond strengths compared to normal dentin.
However, this difference disappeared when 32% phosphoric
acid was used instead of 10%. For the ethanol-based etch-
and-rinse adhesive Single Bond (3 M ESPE) the bonds made
to caries-affected dentin were always lower than bonds to
normal dentin regardless of the concentration of the phos-
phoric acid gel [38]. Different results were obtained in another
study [39] with Single Bond (3 M ESPE). The authors com-
pared two polyalkenoic acid copolymer-containing adhesives
applied to normal versus caries-affected dentin. While the
one-step self-etch adhesive Adper Prompt L-Pop (3 M ESPE)
resulted in significantly higher bond strengths to normal
dentin than to caries-affected dentin, the bond strengths of
the two-step etch-and-rinse adhesive Single Bond to both nor-
mal and caries-affected dentin were not significantly different.
Additionally, the hybrid layers produced by both adhesive sys-
tems were thicker for caries-affected dentin.
In addition to specific composition of dentin adhesives,
different caries dentin removal methods may also influence
adhesion to caries-affected dentin [40]. In this study, the
authors used three removal methods: round steel bur in a
slow-speed handpiece; Er:YAG laser; or 600-grit SiC abrasive
paper. The specimens were bonded with Clearfil Protect Bond
(Kuraray) or with OptiBond Solo Plus Total-Etch (Kerr) and
tested in microtensile mode. Clearfil Protect Bond resulted in
significantly lower bond strength compared to OptiBond Solo
Plus Total-Etch after caries dentin removal with a round bur.
However, the opposite was observed for specimens treated
e28 d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) e24–e37
with abrasive paper. For laser-treated dentin, there were no
significant differences between the two groups.
2.2. Sclerotic dentin in non-carious cervical lesions
(NCCL)
There is now evidence of the multifactorial etiology of NCCL
[41,42]. Occlusal stresses may play a role in the pathogene-
sis of these tooth defects [43,44]. Sclerotic dentin is common
in areas where dentin is exposed (NCCL and attrition areas),
being a more complex substrate than unaltered dentin due
to different ultra-structural layers [45]. As stated by Mendis
and Darling (1979), “a translucent zone is observed under car-
ious dentin, but not under toothbrush abrasion lesions” [20].
The same authors reported that, “in abrasion, SEM of longi-
tudinal fractures showed the surface was homogeneous with
completely occluded tubules. Beneath this surface layer, most
tubules were empty.”
One of the most common clinical symptoms from NCCL is
tooth sensitivity. Sensitivity is usually associated with tubu-
lar patency. Most tubules are occluded with mineral casts in
insensitive transparent sclerotic dentin regions [46]. Gwinnett
and Jendresen [47] observed that the dentin surface in cervical
erosion lesions appeared smooth with evidence of intratubu-
lar deposits. Acid conditioning with 50% phosphoric acid for
60 s opened up tubules in ‘eroded’ dentin, many of which had
become occluded by intratubular deposits. The penetration
of resin (N-phenyl glycine-glycidyl methacrylate, Cervident,
S.S. White) was limited by the presence of these deposits fol-
lowing acid conditioning. When applied to acid-conditioned,
‘eroded’ dentin, resin penetrated approximately 30 ␮m com-
pared to more than 100 ␮m into the tubules of acid-etched
normal dentin [47].
The matrix of the 10–20 ␮m thick surface hypermineralized
layer in sclerotic dentin of NCCL is composed of denatured
collagen with bacteria colonizing the surface of the lesion
[45]. Collagen may be denatured as a result of acids and bac-
terial by-products in the surface layer. Additionally, larger
hydroxyapatite crystallites are observed in the hyperminer-
alized surface layer as compared to sclerotic dentin. The
transition from denatured collagen to intact collagen with
cross-banding is evident at the base of the hypermineral-
ized layer [45]. Crystalline deposits that obliterate the tubules
etch more slowly than the other dentin components. The fact
that intertubular sclerotic dentin from NCCL etches differ-
ently than normal root dentin may explain the difficulties
in restoring such lesions with current bonding procedures
[48].
Irrespective of the use of a total-etch or a self-etch tech-
nique, bonding to sclerotic dentin in NCCL has resulted in
compromised bonding [49,50]. In one study [50], Clearfil Liner
Bond 2V, a self-etching adhesive, was applied to sclerotic cer-
vical dentin with or without 40% phosphoric acid etching.
Regardless of the dentin treatment, bond strengths to unaf-
fected dentin were consistently higher than those made to
sclerotic dentin. However, phosphoric acid etching improved
bonding to the most cervical part of the lesion. Phosphoric
acid was not able to etch below the hypermineralized surface
layer nor was it able to dissolve the residual sclerotic casts
in the tubules. Tay et al. [51] reported that bond strengths
to sclerotic dentin using a self-etching primer (Clearfil Liner
Bond II , Kuraray) were 20% lower than those obtained in
sound cervical dentin regardless of the location of the bonding
substrate inside the lesion. These same authors used EDS to
quantify the relative concentration of calcium and phospho-
rus in NCCL. While the Ca/P ratio was similar in the surface
hypermineralized layer (1.67) compared to the underlying scle-
rotic dentin (1.71), that ratio was 1.38 in the sclerotic casts
within the tubules. Additionally, they found a small percent-
age of Mg (4.57 wt%) in the composition of these sclerotic casts,
confirming that they are composed of whitlockite as other
authors had reported. The same authors [51] also listed four
factors that may influence the decrease in bond strengths
to sclerotic dentin in NCCL: (1) the presence of a hybridized
microbial matrix together with entrapped bacteria; (2) inability
of the self-etching primer/adhesive to dissolve and penetrate
the thick surface hypermineralized region; (3) presence of a
layer of denatured collagen at the base of the hypermineral-
ized zone; (4) presence of residual sclerotic casts that obliterate
the tubules and prevent tag formation.
In spite of the lower bond strengths obtained when the sub-
strate is sclerotic dentin from NCCL, clinical studies have not
totally corroborated the in vitro findings. Van Dijken [52] found
that the differences between the sclerotic and non-sclerotic
and the roughened and non-roughened lesions were not sig-
nificant. One might argue that the bonding agent used in this
study was a resin-modified glass-ionomer material (Fuji Bond
LC, GC Co.), which does not depend on mechanical interlock-
ing with collagen or formation of resin tags. Nonetheless, in
another study by the same author [53], 144 NCCL (98 with
sclerotic dentin, 46 non-sclerotic) were restored with one of
three adhesives Clearfil Liner Bond 2 (Kuraray), One Coat Bond
(Coltène/Whaledent), and Prompt L-Pop (3 M ESPE). The cumu-
lative loss rates of the materials in sclerotic lesions (15.7%)
versus non-sclerotic lesions (14.0%) were not significantly dif-
ferent. Diamond bur-roughened lesions showed a loss rate of
14.5%, while for the non-roughened lesions the frequency was
14.8%.
According to the 2001 ADA guidelines for enamel and
dentin adhesive materials [54], resin-based adhesives gain
“provisional acceptance” at 6 months if their retention rate
in non-carious cervical lesions (NCCL) is at least 95% without
mechanical retention features. Full acceptance requires a 90%
retention rate at 18 months. Due to the fact the NCCL are the
recommended and most frequently used substrate for clinical
tests of dentin adhesives, the degree of dentin sclerosis has
been ranked from 1 to 4 to standardize this particular variable.
When no dentin sclerosis is visibly present, NCCL are given a
score of 1 for sclerosis [55,56]. The score 4 is attributed to NCCL
in which dentin is dark yellow or brownish with glassy aspect
[55,56].
Clinical studies that categorize the degree of sclerosis of
NCCL are not abundant. In one recent study [56], no differences
in retention rates were found for a mild all-in-one adhesive at 3
years when applied to NCCL with a sclerosis score of 1–2 versus
when it was applied to NCCL with a sclerosis score of 3–4.
However, the marginal adaptation at 3 years was slightly, but
not significantly better for the group with sclerosis scale 1–2
(81% alfa ratings) compared to the group with sclerosis scale
3–4 (70% alfa ratings). There were three missing restorations
 d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) e24–e37
in NCCL with a sclerosis scale equal to 4 in this study, but they
corresponded to the group in which the all-in-one adhesive
was used after total-etch [56].
Regarding the adhesion strategy (i.e., self-etch versus etch-
and-rinse), there have been conflicting reports pertaining
to the common belief that etch-and-rinse adhesives bond
more effectively to dentin in NCCL in clinical situations than
self-etch adhesives [56–59]. Clearfil SE Bond (Kuraray), an MDP-
based self-etch adhesive, results in excellent retention in
NCCL at 5 years [59]. For this specific adhesive, chemical bond-
ing to the hypermineralized substrate in NCCL may supersede
the need for separate dentin etching.
2.3. Substrate changes with aging
The tubule volume in coronal dentin was calculated as 10%,
without great difference between old and young teeth [60].
Overall dentin thickness increases at a rate of approximately
6.5 ␮m per year for coronal dentin [61]. The thickness of mantle
dentin and globular dentin of aged teeth are less than that of
young teeth. The hardness and modulus of elasticity of aged
dentin are higher than those of young dentin at the mantle
dentin level. The reactionary dentin in aged teeth and newly
developed secondary dentin in young teeth have lower modu-
lus of elasticity and hardness than those of other circumpulpal
dentin [62].
As mentioned above, secondary dentin is deposited over
the entire inner circumpulpal surface throughout the life of
the tooth. Unlike secondary dentin, which is physiological, the
formation of tertiary dentin is localized in the pulp chamber
wall corresponding to the area of the affected site, usually a
caries process. There is also a form of physiological sclerotic
or transparent dentin that starts in the root and increases
with age linearly. The pattern of distribution is similar in
all teeth. Physiological transparent dentin is first formed in
the apical dentin adjacent to the cementum and extends
coronally and towards the root canal, with increasing age
[63]. Physiological transparent root dentin, as distinguished
from pathological transparency subjacent to caries, appears
to form without trauma or caries lesion as a natural part of
aging. Similarly to the transparent dentin observed under-
neath a caries lesion, transparent dentin from aging occurs
when the tubule lumina become filled with mineral from pas-
sive chemical precipitation [63,64], decreasing the amount of
light scatter, therefore the term transparent. It is not clear
whether the increased mineralization associated with trans-
parency is entirely a result of the filling of the tubule lumina,
or whether there are any additional alterations in the miner-
alization of the intertubular dentin matrix. As tubules become
filled with mineral in transparent dentin, the mineral concen-
tration, as measured by X-ray computed microtomography,
is significantly higher in transparent dentin and the frac-
ture toughness of dentin decreases by 20% [64]. The elastic
modulus is unchanged in physiological transparent dentin;
however, transparent dentin, unlike normal dentin, exhibits
almost no yielding before failure [64]. Other authors [65] have
reported that fatigue strength of young dentin (17–30 years) is
greater than that of older dentin (50–80 years) and that aging
results in increase in both the rate of damage initiation and
propagation in dentin.
e29
As a result of the reduction in tubule diameter there is also
a decrease in dentin permeability. Teeth of subjects over 50
years contain less water than teeth 10–20 years of age, becom-
ing more brittle. With increasing patient age, in both crown
and root aspects of teeth, dentinal thickness increases, while
the density of odontoblasts and pulp fibroblasts decreases [61].
The degree of age-related changes in teeth is asymmetrical,
with decreases of cell density in the root being more evident
than in the crown. At all ages pulp cell densities, including
odontoblasts, within the crown are greater than in the root.
Decreases in pulp cell density may reduce pulp repair activ-
ity after restorative treatments, although increases in dentinal
thickness may aid with pulp protection [61].
Tagami et al. [66] compared the tensile bond strengths of
four dentin bonding systems using proximal dentin of premo-
lars extracted from young (9–21 years) and old (42–64 years)
patients. For each adhesive, there were no differences in bond
strengths between young and older teeth. An adhesive failure
tended to occur at the interface between the bonding resin and
the resin-impregnated layer regardless of substrate age. Brack-
ett et al. [67] measured the microtensile bond strengths of an
etch-and-rinse resin adhesive (Prime & Bond NT, Dentsply)
and a resin-modified glass-ionomer adhesive (Fuji Bond LC, GC
Co.) on teeth known to have originated from subjects over 60
years of age. They used as control teeth originating from young
subjects. No significant differences were observed between
the young and aged teeth for any comparison. SEM evalua-
tion of the etched dentinal surfaces demonstrated less depth
of decalcification in the intertubular areas of aged dentin, but
there was no observable difference within the tubules of young
and aged dentin. In another study [68], authors used extracted
molars from three age groups (20–25, 35–40, and 50–55 years)
to evaluate the microtensile dentin bond strengths of two self-
etching materials. For one of them (ABF or Clearfil Protect
Bond, Kuraray), bond strengths were higher in the 35–40 years
age group.
2.4. Tooth region and remaining dentin thickness
(RDT)
The area occupied by tubule lumina at the DEJ is approxi-
mately 1% of the total surface area at the DEJ and 22% at
the pulp [1,60]. Since this area is occupied by dentinal fluid,
which is 95% water, these areas are also approximately equal
to the tubular water content of these areas. That is, the water
content of dentin near the DEJ is about 1% (volume), while
that of dentin near the pulp is about 22% (Fig. 3). This dif-
ference in intrinsic moisture has been deemed responsible
for the differences in bond strengths between superficial and
deep dentin. Superficial dentin normally results in higher
composite-dentin bond strengths than deep dentin [69–73].
For example, Suzuki and Finger (1988) reported that bond
strengths decreased 30–40% in deep dentin for three dentin
adhesives [73], whereas Nakamichi et al. [69] reported a 50%
decrease in bond strength from superficial to deep dentin in
bovine teeth. These differences tend to diminish when the
smear layer is left intact, but lower bond strengths occur in
deep dentin when the smear layer is removed [74]. As bond-
ing systems became more hydrophilic, the sensitivity of bond
strengths to dentin depth has decreased [75].
e30 d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) e24–e37
Fig. 3 – The left side shows superficial dentin, while deep dentin is depicted on the right side (same tooth). Note the virtual
absence of the peritubular cuff in deep dentin.
The orientation of the dentinal tubules may also influence
dentin bond strengths. Bond strengths of four all-in-one adhe-
sives to dentin with different tubule orientations showed no
significant differences in microshear bond strengths regard-
less of dentin depth (superficial or deep) or dentin tubule
orientation (perpendicular or parallel/oblique) [76]. However,
for the adhesives Clearfil SE Bond (Kuraray), OptiBond Solo
Plus SE (Kerr), and Clearfil S3 Bond (Kuraray) there were sig-
nificantly lower bond shear strengths to deep dentin with
a tubule orientation perpendicular to the bonding surface
[76]. Another study [77] tested Clearfil SE Bond and OptiBond
Solo Plus varying the dentin location (occlusal or cervical),
dentin depth (superficial or deep) and dentinal tubule ori-
entation (perpendicular or parallel) to the bonding surface.
No statistically significant differences were found in shear
bond strengths based on dentin location. In contrast to the
results of the previous study [76], Clearfil SE Bond resulted in
higher shear bond strengths to deep dentin specimens bonded
perpendicular to the tubules compared to those that were
bonded parallel to the tubules, while the opposite was found
for OptiBond Solo Plus. In the case of superficial dentin, there
were no differences between the two materials for the differ-
ent tubule orientations. In this particular study, shear bond
strengths were affected by dentin depth, orientation of the
tubule and the adhesive material, but not by location of the
dentin (occlusal or cervical). On the contrary, Phrukkanon et
al. [78] reported that bond strengths of Single Bond (3 M ESPE)
and an experimental self-etching primer were not affected by
tubule orientation.
The orientation of the dentinal tubules has a profound
effect on the formation of the hybrid layer associated with
etch-and rinse adhesives. In areas with perpendicular tubule
orientation, the layer was 3.2 ␮m thick, showing solid 27.2 ␮m
long resin tags, and a network of tubule anastomoses. In areas
with parallel tubule orientation the layer was significantly
thinner (1.3 ␮m) and resin tags were absent [79].
Other regional variables may influence dentin bonding.
Occlusal dentin tends to give lower bond strengths than buc-
cal dentin [80]. However, in this case depth may have been a
confounding variable, as noted by the authors. There is also
a greater regional variability of dentin wetness in occlusal
dentin than in proximal or buccal dentin [75].
2.5. Smear layer
Residual organic and inorganic components form a “smear
layer” of debris on the surface whenever dentin is prepared
with a bur or other instrument [81,82]. This structure has a
very low inherent bonding of 5 MPa to the dentin substrate
[83]. The smear layer fills the orifices of dentin tubules form-
ing “smear plugs,” and decreases dentin permeability by up to
86% [84]. Sub-micron porosity of the smear layer still allows
for diffusion of dentinal fluid [83]. The composition of the
smear layer is basically hydroxyapatite and altered denatured
collagen. Composition changes with depth to reflect the com-
position of dentin in different areas of the tooth [83]. This
altered collagen may even acquire a gelatinized consistency
as a result of the friction and heat created by the preparation
procedure [85].
Current adhesives are classified according to the way they
interact with the smear layer. This classification results in two
bonding strategies and four types of adhesives:
 d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) e24–e37
1. Etch-and-rinse (or total-etch) adhesives include a sepa-
rate acid-etching step, usually with 30–40% phosphoric acid
applied simultaneously on enamel and dentin to remove
the smear layer and superficial hydroxyapatite.
a. Three-step etch-&rinse adhesives (acid + primer + bonding).
b. Two-step etch&-rinse adhesives (acid + primer/bonding).
2. Self-etch adhesives do not rely on a separate acid-etching
step; they include an acidic monomer solution that is not
rinsed off, making the smear layer permeable without
removing it completely.
a. Two-step self-etch adhesives (acidic primer + bonding).
b. One-step self-etch adhesives (one solution or all-in-
one).
The removal of the smear layer and smear plugs with acidic
solutions results in an increase of the fluid flow onto the
exposed dentin surface. This fluid can interfere with adhesion
[81], because hydrophobic resins do not adhere to hydrophilic
substrates even if resin tags are formed in the dentin tubules
[86].
One factor that might interfere with the demineralization
potential of a self-etch adhesive is the instrument used to cre-
ate the smear layer. Dentin surfaces ground with diamond
burs tended to present more compact smear layers than those
ground with SiC papers. Smear layer denseness, more so than
thickness, may compromise bonding efficacy of adhesives,
especially of self-etch systems [87]. Some studies reported low
dentin bond strengths over thick dentin smear layers [88,89],
while others reported no influence [90,91]. This can be partially
explained by differences in roughness, smear layer thickness
(ranging from 0.9 to 2.6 mm), density, and attachment to the
underlying tooth structure, which is dependent on the way the
smear layer is created [92].
Mowery et al. found that 600-grit SiC paper resulted in lower
bond strength of the second-generation adhesive Scotch-
bond (3 M) to dentin than did 60 grit SiC [93]. Finger, on
the other hand, did not find significant differences for sev-
eral adhesives in function of the roughness of the surface
preparation method [94]. Tay and Pashley studied the aggres-
siveness of three self-etching adhesives in penetrating dentin
smear layers of different thickness (600- or 60-grit SiC paper)
using fractured dentin as control [95]. They used an aggres-
sive adhesive, Prompt L-Pop (3 M ESPE), a moderate adhesive,
NRC/Prime&Bond 2.1 (Dentsply), and a mild adhesive, Clearfil
SE Bond (Kuraray). For the mild self-etch adhesive the smear
layer and smear plugs were retained as part of the hybridized
complex. For the moderate self-etch adhesive, the smear layer
and smear plugs were completely dissolved in dentin with
thin smear layers, but partially retained with thick smear lay-
ers. For the aggressive self-etch adhesive, the smear layer and
smear plugs wee completely dissolved and formed hybrid lay-
ers with a thickness approaching those of phosphoric acid
conditioned dentin. Kenshima et al. [96] bonded to dentin
surfaces with thick (60-grit SiC) or thin (600-grit SiC) smear lay-
ers. The smear layer thickness had no effect on resin–dentin
bond strength of Clearfil SE Bond (Kuraray), Optibond Solo
Plus Self-Etch, Tyrian/One Step Plus, Single Bond, and Scotch-
Bond Multi-Purpose Plus. Kenshima et al. [97], confirming the
results obtained by Tay and Pashley [95], observed that thick
e31
smear layers were not totally removed by the mild self-etch
primer Clearfil SE Bond (Kuraray). Thicker hybrid layers were
observed for the strong self-etch adhesive Tyrian Self Prim-
ing Etchant + One Step Plus (Bisco) and for the etch-and-rinse
adhesive Scotchbond Multi-Purpose (3 M ESPE).
One study [92] attempted to correlate the smear layer cre-
ated with a carbide bur group with that created with 320-grit
abrasive paper. Human dentin was abraded with 0.05 ␮m alu-
mina, 240-, 320- or 600-grit abrasive paper, # 245 carbide,
# 250.9 F diamond or # 250.9 C diamond burs. Shear bond
strengths decreased with increasing coarseness of the abra-
sive for the self-etching adhesive (Clearfil SE Bond, Kuraray).
The higher bond strengths and thin smear layer of the car-
bide bur group, suggests its use when self-etch materials are
used in vivo. The 320-grit abrasive paper yielded results closer
to that of the carbide bur and its use is recommended in vitro
when using Clearfil SE Bond.
The mode of application (i.e., with or without agitation)
may also result in different degrees of penetration of mild
self-etch adhesives through smear layers [98]. With passive
application, self-etch adhesives diffused through thick smear
layers and formed thin hybrid layers in intact dentin. With
continuous agitation, smear layers were completely dispersed
or dissolved, and thicker hybrid layers with upstanding colla-
gen fibrils were observed.
2.6. Dentin permeability and pulpal pressure
Dentinal tubules are slightly tapered, with the wider por-
tion oriented toward the pulp. Dentin permeability increases
almost logarithmically with cavity depth; such an increase
is attributed to the vast differences in the sizes and num-
bers of dentinal tubules between superficial and deep dentin
[60]. According to the hydrodynamic theory [99], once dentin
is exposed, external stimuli cause fluid shifts across dentin,
which activate pulpal nerves and cause pain. Transdentinal
permeability is also responsible for the constant wetness of
exposed dentin surfaces due to the outward fluid movement
from the pulp [100].
The smear layer presents a limitation to the diffusion
to fluid movement, as it decreases dentin permeability by
occluding the tubule orifices [101]. Increased dentin perme-
ability, in terms of fluid flux and hydraulic conductance, has
been reported after the surface was modified with phosphoric
acid [102,103], citric acid [104], or polyacrylic acid [105]. Acids
remove the smear layer, increasing the dentin permeability.
Smear layers created with 600-grit silicon carbide abrasive
paper reduce the hydraulic conductance of extracted human
teeth to 20–24% of the maximum values obtained after acid-
etched dentin [102].
Dentin permeability results in dentin surface wetness,
which influences the quality of the adhesive-dentin inter-
face and may decrease the bond strength between resins
and dentin [100]. Etch-and-rinse adhesives result in higher
micro-permeability compared to self-etch adhesives [106]. The
hybrid layer was always 100% infiltrated by pulpal fluid when
an etch-and-rinse adhesive was used. However, pulpal pres-
sure had no effect on enamel sealing [106]. Other studies
have reported that a simulated pulpal pressure decreases the
dentin bond strengths of resin-modified glass-ionomer and
e32 d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) e24–e37
etch-and-rinse adhesive materials [107,108]. Under simulated
pulpal pressure, CLSM observations revealed a distinctly shal-
lower penetration of etch-and-rinse adhesives into the dentin
tubules compared to the dentin treated without intrapul-
pal pressure [109]. However, the use of hydrophobic bonding
agents applied on acid-etched dentin saturated with ethanol
reverses the fluid conductance to the level obtained with the
presence of the smear layer [110].
Other studies have reported that the simulated positive
pulpal pressure during bonding application does not seem to
affect the in vitro performance of self-etch adhesives signifi-
cantly [111,112]. All-in-one adhesives are more susceptible to
pulpal pressure than two-step-self-etch adhesives [113] as a
result of the application of a hydrophobic resin layer in the
latter. Sauro et al. applied simulated pulpal pressure during
the build-up procedure instead of during the bonding appli-
cation, using self-etch adhesives [113]. While Clearfil Protect
Bond (Kuraray), a two-step self-etch adhesive, exhibited the
lowest permeability and fewest numbers of fluid droplets over
the surface of the bonded dentin, the all-in-one adhesives G-
Bond (GC Co.) and Clearfil S3 Bond (Kuraray) and One Up Bond
F (Tokuyama) were more permeable than Clearfil Protect Bond.
The application of pulpal pressure significantly reduced bond
strength.
Application errors in the clinical sequence may also influ-
ence dentin permeability. For OptiBond FL (Kerr), a three-step
etch-and-rinse adhesive, the highest mean percentage of per-
meability reduction was observed in the group where the
adhesive was applied as per manufacturer’s instructions [114].
Clinically, most dental restorations are performed under
local anesthesia, which may change the permeability of
dentin. Permeability data from in vitro studies must be inter-
preted with caution. In clinical conditions there is an outward
fluid flow across exposed dentin in response to the low, but
positive pulpal tissue pressure (14.1 ± 2.5 cm H2 O for an RDT of
1.06 ± 0.04 mm in premolars) [115]. Pitt-Ford et al. [116] demon-
strated that 2% lidocaine without vasoconstrictor produced no
reduction in pulpal blood flow. On the other hand, Beveridge
and Brown [117] reported that lidocaine without vasoconstric-
tor produced a transient increase in pulpal pressure, which
is consistent with the vasodilating properties of lidocaine.
Kim et al. [118] demonstrated that lidocaine with 1:100,000
epinephrine lowered pulpal blood flow in dogs 72% after infil-
tration and 67% after block anesthesia. Olgart and Gazedius
[119] reported that infiltration of the vasoconstrictors, such
as epinephrine or felypressin, decreased pulpal blood flow.
Therefore, it is likely that the pulpal pressure is low during
most adhesive restorative procedures.
One other substrate variable previously discussed, caries-
affected dentin, may also decrease dentin permeability due to
the presence of mineral deposits in the tubules [36]. A posi-
tive simulated pulpal pressure significantly reduced the bond
strength of Clearfil SE Bond (Kuraray) to normal dentin after
1-month of storage, but did not affect the bond strength to
caries-affected dentin [36].
2.7. Treatment prior to bonding—desensitizers
According to the hydrodynamic theory [99], dentin sensitiv-
ity is caused by rapid fluid shifts across dentin that activate
intrapulpal nerves and cause pain. In light of this theory, block-
ing dentinal tubules should prevent fluid shifts and prevent
dentin sensitivity.
Pashley et al. [120] measured the direction and magnitude
of fluid shifts across dentin in dentin surfaces in response
to several hydrodynamic stimuli: air blast, 56 ◦ C water, 2 ◦ C
water, tactile and osmotic. In acid-etched superficial dentin,
the greatest fluid flow was obtained for hot water followed
by cold water. The osmotic and the tactile stimuli caused the
least fluid flow across dentin.
Treating acid-etched dentin with 3% potassium-hydrogen
oxalate re-occluded the dentinal tubular openings with insol-
uble crystals. The authors suggested that this treatment
might be useful as a dentin cavity liner to re-close denti-
nal tubules after dentin is acid-etched [101]. This technique
was later used with three adhesives, Single Bond (3 M ESPE),
One-Up Bond F (Tokuyama), and AdheSE (Ivoclar Vivadent).
These adhesives were unable to eliminate the fluid flow
through dentin. The application of 3% potassium oxalate
prior to the bonding procedures was suggested as an effec-
tive technique in reducing the dentin permeability, regardless
of the adhesive used [121]. However, in order to be effec-
tive, the oxalate-based material must be applied after dentin
is etched. In one study [122], dentin was treated with four
oxalate desensitizers before or after acid-etching, and bonded
with an acetone-containing two-step etch-and-rinse adhe-
sive. Microtensile bond strengths were significantly lower,
compared with the control, when oxalates were used before
the specimens were acid-etched. When oxalates were used
after acid-etching microtensile bond strengths were similar
to the controls [122]. Additionally, ‘silhouettes’ of subsurface
crystals were observed inside the dentinal tubules under the
TEM. The adhesive layer did not display any signs of nanoleak-
age when the oxalates were applied after acid-etching. Other
studies have not found favorable results with oxalates applied
with a dentin adhesive [123]. The application of an oxalate
gel or an oxalate in liquid form with Single Bond (3 M ESPE),
an etch-and-rinse two-step adhesive, produced significantly
lower long-term microtensile bond strengths and enhanced
nanoleakage after 3 months of simulated pulpal pressure.
Glutaraldehyde is used as a fixative that cross-links pro-
teins [124]. Glutaraldehyde-based substances, such as Gluma
Desensitizer (5% glurataldehyde and 35% HEMA in water, Her-
aeus Kulzer), have been used in dentistry to reduce dental
hypersensitivity under restorations and on exposed sensi-
tive dentin [125,126]. The mechanism behind the reduction
in dentin sensitivity is that these glutaraldehyde-containing
desensitizers reduce dentin permeability. One study evaluated
in vitro the dentin permeability of HEMA-based desensitiz-
ing in dogs for 1 week, 1 month and 3 months. At the end
of the 3-month period, Gluma Desensitizer had the lowest
permeability value, therefore provided a longer lasting tubule-
occluding effect [127]. Another study used human molar
dentin slices to compare in vitro the efficacy of five resin-based
desensitizing agents, including Gluma Desensitizer, at reduc-
ing human dentin permeability. All the desensitizing agents
resulted in a large decrease in dentin permeability [128].
The same glutaraldehyde-based desensitizing agent has
been suggested as a re-wetting agent on etched dentin to
help prevent post-operative sensitivity under posterior com-
 d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) e24–e37
posite restorations [129]. In spite of the favorable in vitro bond
strengths [129,130], clinically it is not very effective for this
specific application [131], as the operative technique may be
more relevant than the use of any desensitizer [131].
The pulpal toxicity of desensitizing agents must be ques-
tioned especially considering that some of these materials are
applied on patent dentinal tubules. In one study, the toxic-
ity was measured on L 929 fibroblasts. None of the materials
resulted in toxicity to the fibroblast culture [128]. Additionally,
glutaraldehyde-containing materials disinfect dentin in vitro
[132].
The effect of glutaraldehyde- and HEMA-based desensi-
tizers on collagen is also a relevant issue. Ritter et al. [133]
demonstrated that the treatment of demineralized dentin
with 37% phosphoric acid did not significantly disrupt or
dissociate the major cross-links of dentin collagen. Accord-
ing to the authors, in a clinical setting where phosphoric
acid is applied to mineralized collagen for less than 1 min,
the stability and structure of collagen are most likely unaf-
fected. When Gluma Desensitizer was applied to acid-etched
dentin it affected dentin collagen amino acid and cross-link
composition—reduction of free lysine (Lys) and hydroxylysine
(Hyl) residues, as well as a decrease in the levels of collagen
reducible cross-links [133].
2.8. Treatment prior to bonding—chlorhexidine
Chlorhexidine was first used in dentin bonding as a dentin dis-
infectant. The research problem was whether chlorhexidine
decreased dentin bond strengths when used as a dentin disin-
fectant [134]. Scanning electron microscopy revealed that the
chlorhexidine solution deposited debris on the dentin surface
and within the tubules of etched dentin [134], but chlorhex-
idine had no significant effect on the shear bond strengths
of composite to dentin using the All-Bond 2 adhesive (Bisco
Inc.).
Besides being a commonly used antibacterial rinse used
in Periodontology, chlorhexidine is now used in dental adhe-
sion as a protease inhibitor [135]. Metalloproteinases (MMP)
are a class of zinc- and calcium-dependent endopeptidases
capable of degrading all extracellular matrix components
[136–138]. The exposed collagen fibrils may be vulnerable
to degradation by endogenous MMP after acid-etching [136].
Collagenolytic and gelatinolytic activities found in partially
demineralized dentin [135] imply the existence of MMP in
human dentin, which contains gelatinases MMP-2 and -9, col-
lagenase (MMP-8), and enamelysin MMP-20 [136–138]. These
enzymes are trapped within the mineralized dentin matrix
during odontogenesis [137,138]. There are, nevertheless, still
some unanswered questions on this subject. For example, the
role of MMP-2 in dentin bonding is not clear, as its immunore-
activity is localized preferably in predentin and around the
DEJ in teeth from subjects 12–30 years [139]. Consequently,
its availability in middle dentin is questionable. Additionally,
chlorhexidine may damage stem cells when used for root
canal irrigation [140].
Dentin collagenolytic and gelatinolytic activities can be
overcome by protease inhibitors [135], indicating that MMP
inhibition might preserve the integrity of the hybrid layer.
In fact, the in vivo application of chlorhexidine improves
e33
the integrity of the hybrid layer [141]. When chlorhexidine
was applied in vitro, the integrity of the hybrid layer and
the magnitude of bond strengths were preserved in aged
dentin–resin interfaces [142]. When phosphoric acid is applied
without the subsequent application of chlorhexidine, it does
not inhibit the collagenolytic activity of mineralized dentin,
while the use of chlorhexidine after acid-etching, even in
very low concentrations, strongly inhibits that activity. The
drawback that might be associated with the use of chlorhex-
idine is the potential discoloration associated with its use
[143].
2.9. Storage conditions/storage time
Pashley et al. found no differences in bond strengths between
in vivo versus in vitro in a dog model for extraction times
of 30 min, 1 day, 1 week, and 1 month [144]. Additionally,
post-extraction time had no effect on hydraulic conductance
through dentin [145]. Aquilino et al. stored teeth for 3 months
in 0.9% aqueous NaCl, 0.05% saturated solution of thymol,
or distilled water. After applying Scotchbond (3 M ESPE), they
found no differences in bond strengths [146]. Mitchem and
Gronas found no difference in dentin bond strength between
30 min and 2 years post-extraction time [72].
Another storage method that researchers have used is
cryopreservation. A study compared freshly extracted teeth,
cryopreserved teeth, or teeth stored in 0.5% chloramine
at 4◦ . Authors concluded that refrigeration at 4 ◦ C in 0.5%
chloramine for 48 days or longer may cause an increase
in microleakage while cryopreservation for 13 weeks or
short-term refrigeration did not affect the microleakage
[147].
More recently, Lee et al. [148] stored extracted teeth at 37 ◦ C
for 60 days in distilled water, 0.9% NaCl, 0.5% chloramine-T,
5.25% NaOCl, 2% glutaraldehyde, or 10% formalin. A subset of
10 specimens from all groups was autoclaved, while a subset
of 10 specimens from all groups (except formalin) was kept in
10% formalin for 14 days. Storage in NaOCl resulted in lower
bond strengths while sterilization with the autoclave nega-
tively affected the bond strength of specimens stored initially
in distilled water or 10% formalin. Sterilization with formalin
alone had no significant effect on bond strengths, therefore
authors recommended 10% formalin as storage medium for in
vitro studies.
According to the ISO technical specification 11405 [149],
“ideally the bond strengths should be measured immediately
post-extraction, but this is not generally feasible. It appears
that most changes occur in the initial days or weeks after
extraction. Therefore, teeth 1 month, but not more than 6
months, after extraction should be used. NOTE: teeth that have
been extracted for longer than 6 months could undergo degen-
erative changes in dentinal protein. The teeth should then
be placed in distilled water (grade 3, ISO 3696) or in a 0.5%
chloramine-T trihydrate bacteriostatic/bacteriocidal solution
for a maximum of 1 week, and thereafter stored in distilled
water either in a refrigerator (i.e., nominal 4 ◦ C), or frozen at
below −5 ◦ C. To minimize deterioration, the storage medium
should be replaced periodically. It is essential that no other
chemical agents be used, as they may be absorbed by, and alter,
tooth substance.”
e34 d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) e24–e37
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