Journal of Dentistry xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Journal of Dentistry
journal homepage: www.elsevier.com/locate/jdent

Full Length Article

Comparison of deproteinization agents on bonding to developmentally

hypomineralized enamel
Manikandan Ekambarama, Robert P. Anthonappab, Sharaschandra R. Govindoolc,
⁎
Cynthia K.Y. Yiua,
a
Paediatric Dentistry, Faculty of Dentistry, The University of Hong Kong, 34-Hospital Road, Sai Ying Pun, Hong Kong, China
b
School of Dentistry, The University of Western Australia, 17-Monash Avenue, Nedlands, Western Australia 6009, Australia
c
Research postgraduate student, Endodontology, Faculty of Dentistry, The University of Hong Kong, 34-Hospital road, Sai Ying Pun, Hong Kong, China

A R T I C L E I N F O A B S T R A C T

Keywords: Objective: To compare bonding of dental adhesive to hypomineralized enamel (HE) after pre-treatment with
Bonding either 5% sodium hypochlorite (NaOCl) solution or papain-based papacarie gel.
Dental adhesive Methods: Normal enamel (NE) and HE obtained from hypomineralized first permanent molars were acid-etched
Hypomineralized enamel with 32% phosphoric acid and randomly allocated into no deproteinization, deproteinization using 5% NaOCl,
Deproteinization
or deproteinization usping papacarie gel groups. Subsequently, the specimens were bonded, packed with
Sodium hypochlorite
Papacarie
composite resins and subjected to micro-shear bond strength (MSBS) testing and the data analysed using 2-way
ANOVA and Tukey tests. Furthermore, specimens from all groups were subjected for qualitative analysis using
scanning electron microscope.
Results: Two way-ANOVA showed that the factor “enamel substrate” was significant (p < 0.001), “enamel pre-
treatment” was not significant and interaction of the two factors was significant (p = 0.005). HE produced
inferior bonding with dental adhesive compared to NE. Enamel pre-treatment with deproteinization agents
enhanced bonding to HE. No significant difference in MSBS was evident between the two deproteinization agents
(p > 0.05). Qualitative analysis of acid-etched moderate HE showed barely visible enamel rods with irregular
etching pattern. Following acid etching and deproteinization, Type I and II etching patterns were observed in
moderate HE; while a porous enamel surface with more profound etching patterns in severe HE.
Conclusions: Papain-based papacarie could be an alternative deproteinization agent for bonding dental adhesive
to HE.
Clinical significance: Papain-based papacarie, a natural deproteinization agent and a proven chemo-mechanical
caries removal agent could be an alternative to NaOCl for enhancement of bond durability of adhesive re-
storations to HE.

1. Introduction
Molar-Incisor Hypomineralization (MIH) is a condition of systemic
origin that involves one to four first permanent molar teeth and often
associated with affected incisors [1]. It has been reported that ap-
proximately 1 in 5–6 children are affected with this condition [2].
Second primary molars, tips of permanent canine cusps, second per-
manent molars and premolars have also found to be affected in patients
presenting with MIH [3]. The affected teeth exhibit demarcated enamel
opacities, which vary in colour and extent. The affected enamel re-
presents the degree of hypomineralization and can be either white
(mild), creamy or yellow (moderate) or brown (severe) in colour. The
extent of the defective enamel can vary from a very small to a large
area, and in some cases involving an extensive part of the crown.
When compared to normal enamel (NE), hypomineralized enamel
(HE) exhibited a mean 28% reduction in its mineral content, 80% more
carbonated apatite and 3- – 15- fold increase in its protein content
[4–6]. The hardness of HE was also significantly lower than NE [4]. The
chemical analysis of HE has demonstrated a reduction in both the Ca, P
concentrations and mean Ca/P ratio; while C, Mg and K concentrations
were increased compared to NE [7,8].
Bonding to HE from teeth affected with MIH is very challenging due
to the altered physical and chemical characteristics. The increased
protein content in HE is the main challenge for bonding to this altered

⁎
Corresponding author at: Paediatric Dentistry and Orthodontics, 2nd Floor, Prince Philip Dental Hospital, 34-Hospital Road, Sai Ying Pun, Hong Kong, China.
E-mail addresses: drmani@hku.hk (M. Ekambaram), robert.anthonappa@uwa.edu.au (R.P. Anthonappa), govindoolsharas@gmail.com (S.R. Govindool),
ckyyiu@hkucc.hku.hk (C.K.Y. Yiu).

http://dx.doi.org/10.1016/j.jdent.2017.10.004
Received 2 June 2017; Received in revised form 26 September 2017; Accepted 10 October 2017
0300-5712/ © 2017 Published by Elsevier Ltd.

Please cite this article as: EKAMBARAM, M., Journal of Dentistry (2017), http://dx.doi.org/10.1016/j.jdent.2017.10.004
M. Ekambaram et al.
substrate [9,10]. Recently, a systematic review on bonding to HE [11]
concluded that resin dental adhesives achieved an inferior bonding to
HE when compared to NE. Furthermore, the review also reported that
enamel deproteinization with 5% sodium hypochlorite (NaOCl) before
adhesive application procedure may potentially enhance the bonding of
resin dental adhesives to HE [11].
Five percent NaOCl is an effective deproteinization agent and sev-
eral concentrations (5.25%, 2.5%, 1% and 0.5%) of NaOCl have been
used for irrigation during root canal preparation. Sodium hypochlorite
removes the excess protein content from the HE and thereby, improving
bonding of dental adhesive to that substrate [12]. “Papacarie®” is a
papain-based natural deproteinizing agent synthesized from leaves and
fruits of green adult papaya. It is a papain-based gel containing chlor-
amine, toluidine blue, salts, preservatives, stabilizers, thickener and
deionized water [13]. It has antibacterial, proteolytic and anti-in-
flammatory properties and therefore have been used in chemomecha-
nical caries removal [14]. Recently, enamel pre-treatment with Papa-
carie® has been shown to increase bond strength of orthodontic brackets
[15]. However, to date, there are no published reports comparing the
deproteinization effect of 5% NaOCl and Papacarie on HE and bonding
of dental adhesives. Therefore, the present study aimed to compare
bonding of dental adhesive to HE after pre-treatment with either 5%
NaOCl solution or papain-based Papacarie gel. The hypothesis tested
were: (1) There is no difference in the bond strength between NE (ob-
tained from MIH-affected teeth with no visible sign of hypominer-
alization) and HE when bonded with dental adhesive, (2) pre-treatment
of HE with de-proteinization agents would not have an effect on
bonding with dental adhesive and (3) there is no difference in the
bonding performance of dental adhesive to HE after pre-treatment with
either 5% NaOCl solution or Papacarie gel.
2. Materials and methods
2.1. Teeth collection
Twenty-seven hypomineralized first permanent molars that were
extracted from patients below 18 years of age for reasons other than for
use in this research project were collected over 12 months period after
getting an informed consent approved by The University of Hong Kong/
Hospital Authority of Hong Kong West Cluster (Reference number UW
15-381). The collected teeth were stored in 0.5% Thymol solution at
4 °C until use.
2.2. Identification of normal (NE) and hypomineralized enamel (HE)
substrates
Three authors of this study (ME, CY and GSR) independently in-
spected the hypomineralized teeth and included only those teeth that
fulfilled the MIH judgement criteria specified by Weerheijm et al. [16].
The included teeth were visually inspected under day light conditions
in order to differentiate HE and NE. Demarcated areas of discoloured
enamel were identified as HE and enamel with no apparent dis-
colouration was identified as NE. Furthermore, HE was carefully ex-
amined based on its colour in order to differentiate the severity of the
defect (Fig. 1). Accordingly, the demarcated areas of HE were cate-
gorized as “creamy-white (CW)” or “yellow-brown” (YB) [17]. In order
to obtain samples with adequate thickness, NE was obtained only from
the occlusal half of the tooth. For HE, enamel specimens less than
2–3 mm wide were excluded [17]. The number of HE specimens ob-
tained per tooth was dependent on the extent of severity for that par-
ticular tooth. Accordingly, specimens per tooth varied from one to four
in number.
2.3. Enamel specimen preparation for bond strength testing
The roots of the teeth were removed from the crown using a slow-
2
Journal of Dentistry xxx (xxxx) xxx–xxx
speed diamond impregnated disc (Isomet, Buehler Ltd., Lake Bluff, IL,
USA) under water-cooling. The crown was further sectioned along the
long axis of the tooth in different planes to prepare the specimens. The
sectioned specimens were minimally ground on 600-grit SiC paper
under running water for 10 s to remove the surface prismless layer and
create a flat surface suitable for bonding. The prepared enamel speci-
mens were carefully embedded in acrylic cylindrical molds using self-
cure acrylic resin. The specimens were randomly allocated (n = 10) to
the experimental groups for bonding. For experimental groups with HE,
the number of specimens with CW and YB were similar between the
groups.
The specimens were divided into the following six experimental
groups:
Group 1 – NE; no deproteinization
Group 2 – NE + deproteinization using 5% NaOCl
Group 3 – NE + deproteinization using Papacarie gel
Group 4 – HE (5 CW, 5 YB); no deproteinization
Group 5 – HE (5 CW, 5 YB) + deproteinization using 5% NaOCl
Group 6 – HE (5 CW, 5 YB) + deproteinization using Papacarie gel
2.4. Enamel pre-treatment and bonding procedure
Enamel specimens from Groups 1 and 4 were etched with 32%
phosphoric acid gel (Scotchbond™ Universal Etchant, 3 M ESPE, St.
Paul, MN, USA) for 10 s, followed by rinsing with de-ionized (DI) water
for 10 s. The enamel specimens from Groups 2 and 5, after the acid
etching and rinsing procedures, were treated with 5% NaOCl (Henan
Hairen Biotechnology Co. Ltd, China) as a deproteinizing agent for 60 s
(applied using microbrush), followed by rinsing with DI water for 10 s.
The enamel specimens from experimental Groups 3 and 6, after the acid
etching and rinsing procedures, were treated with papain-based
Papacarie gel (Papacarie Duo®, F & A Pharmaceutical Laboratory Ltd,
Sao Paulo, Brazil) as a deproteinizing agent for 60 s (applied using
microbrush), followed by rinsing with DI water for 10 s. The specimens
from all the groups were air-dried, bonded with 2 coats of Adper Single
Bond 2 (3 M ESPE, St. Paul, MN, USA), following manufacturer’s in-
structions and were subsequently light-cured for 10 s using a quartz-
halogen light-curing unit (Optilux, Demetron-Kerr, Orange, CA, USA)
with a constant output intensity of 600 mW/cm2. The bonded enamel
specimens from Groups 1–6 were built up with resin composite (Filtek™
Z250 Universal Restorative composite resin, 3 M ESPE, St. Paul, MN,
USA) through custom-made brass cylindrical tubes (Fig. 2). The brass
cylindrical tubes were stabilized on the bonded enamel specimens using
a custom-made jig (Fig. 2).
The height of the jig was 150 mm, which gave the operator ample
room to manipulate the substrate and specimens with no interference
with the arms “c” and “d”, as they were locked to the top end of the
shaft when not in use. This was achieved by assembling a linear bearing
shaft “b” with two pairs of ball bushing pillow blocks on a platform “a”.
Each pair was interconnected with an L-shaped connecting aluminium
plate to form arms “c” and “d”. Arm “c” was used as illustrated in Fig. 2
(close-up view), where the flanges hold on the wings of the brass tube,
stabilizing it against the substrate. The beveled edges of the inner
diameter of the brass tube helped to achieve a tight seal with any lea-
kages noticeable during compaction. The arm “d” was mounted with a
plunger “e”, the diameter of which corresponded to approximately the
inner diameter of the brass tube, to facilitate even distribution of the
compaction forces. A Teflon tape was used on the plunger tip during
compaction to prevent the pullback effects of the composite from af-
fecting the bond strength.
2.5. Bond strength test
The bonded enamel specimens, stored in deionized water at 37 °C
for 24 h, were subjected to microshear bond strength testing (MSBS).
The brass cylindrical tubes were not removed prior to bond strength
M. Ekambaram et al.
Fig. 1. Representative clinical photographs of first permanent molars describing demarcated areas of hypomineralized enamel, (1a) showing areas of demarcated “creamy-white” (CW)
hypomineralized enamel and (1b) showing areas of demarcated “yellow-brown” (YB) hypomineralized enamel (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.).
testing [17]. The specimens were held horizontally and microshear
force was delivered at the bonded interface at a constant speed of
1 mm/min by a custom-made jig attached to a universal testing ma-
chine (Model 4440, Instron, Inc., Canton, MA, USA). The bond strength
was calculated (in MPa) from the force required to debond the com-
posite-filled brass cylindrical tubes and the surface area of the bonded
specimen. Microshear bond strength data were analysed with a statis-
tical analysis computer software (SPSS 23.0 for Windows©, SPSS Inc.,
Chicago, IL, USA). Two-way ANOVA was used to examine the effect of
two variables “enamel substrate” (NE/HE) and “enamel pre-treatment”
(no pre-treatment/pre-treatment with NaOCl/pre-treatment with pa-
pacarie) and the interaction of the two factors on MSBS. Post-hoc
multiple comparisons were carried out using Tukey test, with a statis-
tical significance set at α = 0.05.
To study the differences in MSBS within HE groups based on the
severity of hypomineralization, the MSBS data was sorted between the
CW and YB specimens. Two-way ANOVA was employed to examine the
effect of two variables “severity of hypomineralized enamel substrate”
(CW/YB) and “enamel pre-treatment” (no pre-treatment/pre-treatment
with NaOCl/pre-treatment with papacarie) and the interaction of the
two factors on the MSBS. Post-hoc multiple comparisons were carried
out using Tukey test, with a statistical significance set at α = 0.05.
2.6. Failure pattern analysis
The failure pattern on the debonded surfaces of the enamel speci-
mens were examined under light microscope (LV100POL, Nikon,
Japan) with 20× magnification. Digital images were obtained and the
failure pattern was categorized into one of the following types: (1)
Adhesive – when the debonded specimen surface was devoid of resin
and/or composite and showed the fractured resin tags, (2) Cohesive in
the enamel – when the debonded specimen surface showed fracture
through the enamel, (3) Cohesive in the resin – when the debonded
specimen surface showed fracture through the adhesive/composite, (4)
Mixed failure – when the debonded specimen surface showed a mixture
of all the above failure types. Representative specimens from each of
the failure pattern were air-dried, mounted on aluminium stubs, sputter
coated with platinum-palladium for 120 s and examined under scan-
ning electron microscope (SEM) (52E-0802, Hitachi, Japan) operated at
15 KV. High-resolution images of the specimens were obtained for de-
monstration of the failure patterns.
2.7. Qualitative analysis of acid etched, acid etched and deproteinized
enamel specimens
Two enamel specimens from each group were additionally prepared
3
Journal of Dentistry xxx (xxxx) xxx–xxx
for qualitative analysis of the surfaces (acid etched, acid etched and
deproteinized) prior to bonding. For HE groups, two enamel specimens
from both defect types (CW and YB) were included. The specimens were
air-dried, mounted on aluminium stubs, sputter coated with platinum-
palladium for 120 S and examined under SEM (52E-0802, Hitachi,
Japan) operated at 15 KV. High-resolution images of the specimens
were obtained for qualitative analysis of the prepared substrate from
each group.
3. Results
3.1. Bond strength
Table 1 shows the mean MSBS of the etch-and-rinse adhesive to NE
and HE. Results of two-way ANOVA test showed that the factor “enamel
substrate” (NE/HE) was significant (p < 0.001) and the factor “enamel
pre-treatment” (no pre-treatment/pre-treatment with NaOCl/pre-
treatment with Papacarie) was not significant (p = 0.214). However,
interaction between the two factors was significant (p = 0.005). Re-
sults of Tukey multiple comparisons test showed that MSBS of dental
adhesive to HE was significantly lower than NE. Pre-treatment with
deproteinization agents significantly increased the bond strength to HE,
with no difference to NE. Furthermore, no difference in bond strength
was observed between the two deproteinization agents, NaOCl and
Papacarie.
Table 2 shows mean MSBS of etch-and-rinse adhesive to HE. Results
of two-way ANOVA test showed that the factor “severity of HE substrate
(CW/YB)” was not significant (p = 0.210) and the factor “enamel pre-
treatment (no pre-treatment/pre-treatment with NaOCl/pre-treatment
with Papacarie) was significant (p = 0.003). However, interaction be-
tween the two factors was not significant (p = 0.194). Results of Tukey
multiple comparisons test showed that there were no significant dif-
ferences in MSBS of dental adhesive between CW or YB defect types.
Pre-treatment of HE with deproteinization agents significantly in-
creased the bond strength to CW defect specimens, compared to no
treatment. However, no such increase in MSBS was evident in the YB
defect specimens. Furthermore, no difference in the bond strength was
observed between the two deproteinization agents, NaOCl and Papa-
carie.
3.2. Failure pattern analysis
Fig. 3 shows SEM micrographs of enamel sides of representative
fractured specimens. None of the bonded enamel specimens from any of
the tested groups showed pre-test failure. Fig. 3a and b is the re-
presentative fractured specimens bonded to NE substrate showing
M. Ekambaram et al. Journal of Dentistry xxx (xxxx) xxx–xxx

Fig. 2. Drawing of hollow custom-made cylindrical brass tubes with dimensions (OD- outer diameter; ID-inner diameter) and stabilization of the brass cylindrical tube on a bonded
enamel specimen using the custom-made jig.

Table 1 Table 2
Microshear bond strength of etch-and-rinse adhesive to normal and hypomineralized Mean microshear bond strength of etch-and-rinse adhesive to hypomineralized enamel.
enamel.
Types of Enamel Microshear bond strength (MPa ± SD)
Types of Enamel Microshear bond strength (MPa ± SD)
No deproteinization 5.25% sodium Papacarie
No deproteinization 5.25% Sodium Papacarie hypochlorite
hypochlorite
Creamy white 17.67 (4.43)a 26.19 (4.59)b 26.50
Normal Enamel 29.50 (5.20)b 27.21 (3.30)b 27.93 (3.90)b Hypomineralized (1.83)b
Hypomineralized 18.64 (4.18)a 24.33 (4.24)b 24.66 (3.69)b Enamel
Enamel (CW + YB) Yellow brown 19.61 (4.16)a,b 22.46 (3.28)a,b 22.80
Hypomineralized (4.32)a,b
Values with same alphabet in the superscript are not statistically different. Enamel
CW = chalky white; YB = Yellow brown.
Values with same alphabet in the superscript are not statistically different.

4
M. Ekambaram et al. Journal of Dentistry xxx (xxxx) xxx–xxx

Fig. 3. Scanning electron microscope images of enamel sides of representative fractured specimens from (3a) normal enamel with adhesive failure, (3b) normal enamel with NaOCl pre-
treatment showing adhesive failure, (3c) normal enamel with Papacarie pre-treatment showing mixed failure, (3d) hypomineralized enamel with cohesive failure in enamel, (3e)
hypomineralized enamel with NaOCl pre-treatment showing adhesive failure and (3f) hypomineralized enamel with Papacarie pre-treatment showing mixed failure.

adhesive failure pattern in which the debonded specimen surface is
devoid of resin and/or composite and shows the fractured resin tags.
Fig. 3c is a fractured specimen bonded to NE substrate following pa-
pacarie pre-treatment showing mixed failure with fractured resin tags
and composite. Fig. 3d is a fractured specimen bonded to HE substrate
showing cohesive failure in enamel evident by the fractured enamel
rods; while Fig. 3e shows a fractured specimen following NaOCl pre-
treatment with adhesives failure with fractured resin tags. Fig. 3f is a
representative fractured specimen bonded to HE substrate after papa-
carie pre-treatment showing mixed failure pattern with a combination
of adhesive failure and cohesive failures in the resin and enamel.
Table 3 illustrates the summary of fracture pattern analysis of the
debonded specimens from all the experimental groups. From the ex-
perimental groups in which bonding was performed to NE substrate,
‘Adhesive failure’ was the most common type of failure pattern, fol-
lowed by “mixed failure”. “Cohesive failure in enamel” was observed in
debonded specimens from the experimental groups in which bonding
was performed to HE substrate. However, pre-treatment with either
NaOCl or Papacarie gel reduced the “cohesive failure in enamel” frac-
ture pattern.
3.3. Qualitative analysis of acid etched, acid etched and deproteinized
enamel specimens
Fig. 4 shows SEM micrographs of acid-etched, acid-etched and de-
proteinized enamel specimens. Fig. 4a shows NE with enamel rods and
inter-rod substance with a uniform etching pattern. Fig. 4b and c shows
HE with barely visible enamel rods and an irregular etching pattern.

5
M. Ekambaram et al. Journal of Dentistry xxx (xxxx) xxx–xxx

Table 3 deproteinization with Papacarie showed more dissolution of prism

Percentage distribution of failure patterns. peripheries (Fig. 4h). In severe HE, acid-etching and deproteinization
removed excess protein and increased the porosity of enamel surface
Failure pattern
with more profound etching patterns (Fig. 4f and i).
Adhesive Cohesive Cohesive Mixed
failure in failure in
enamel resin 4. Discussion
Group 1 Normal Enamel 50% 0% 10% 40%
Group 2 Normal Enamel and 70% 10% 0% 20% The MSBS of dental adhesive to HE was significantly lower than that
NaOCl to NE. Therefore, the first null hypothesis that “there was no difference
Group 3 Normal Enamel and 70% 0% 0% 30% in bond strength between NE (obtained from MIH-affected teeth with
Papacarie
no visible sign of hypomineralization) and HE when bonded with dental
Group 4 Hypomineralized 20% 50% 0% 30%
Enamel adhesive was rejected. Pre-treatment with de-proteinization agents, 5%
Group 5 Hypomineralized 70% 20% 0% 10% NaOCl and Papacarie gel significantly increased the MSBS to HE.
Enamel and NaOCl Hence, the second hypothesis that ‘pre-treatment of HE with de-pro-
Group 6 Hypomineralized 30% 30% 20% 20% teinization agents would not have an effect on bonding of dental ad-
Enamel and
Papacarie
hesive’ was rejected. As there was no difference in bonding perfor-
mance of dental adhesive to HE after pre-treatment with either NaOCl
solution or Papacarie gel, the third null hypothesis that “there is no
Fig. 4c shows excess protein in severe HE that was not removed by acid difference in bonding performance of dental adhesive to HE after pre-
etching alone. Fig. 4d and g shows NE following acid-etching and de- treatment with either 5% NaOCl solution or Papacarie gel” cannot be
proteinization with NaOCl and Papacarie, respectively. The inter-rod rejected.
substances were removed and both type I etching (in which the enamel The specimen preparation in MSBS testing using preformed tubes is
rod, or prism, head are dissolved) and type 2 etching (in which the critical as any stress induced during the preparation of the specimen
enamel interprismatic substance is dissolved) patterns are evident. may lead to a confounding error during testing. As the previous jigs
Moderate HE, after acid-etching and deproteinization with NaOCl, reported in literature were hand assembled and disassembled, one
showed more dissolution of prisms cores (Fig. 4e); while those should exercise caution while interpreting their findings as induced
stress during the disassembling of the jig to expose the test specimens

Fig. 4. Scanning electron microscope images following (I) acid etching (4a) normal enamel, (4b) moderate hypomineralized enamel, (4c) severe hypomineralized enamel, following (II)
acid etching and NaOCl pre-treatment, (4d) normal enamel, (4e) moderate hypomineralized enamel, (4f) severe hypomineralized enamel and (III) acid etching and Papacarie pre-
treatment, (4 g) normal enamel, (4 h) moderate hypomineralized enamel, (4i) severe hypomineralized enamel. Pointers in 4c show excessive protein, whilst the pointer in (4f) and (4i)
shows porosity of enamel surface with more profound etching patterns.

6
M. Ekambaram et al.
may have altered the results. The design used in the present study en-
sured accuracy and repeatability in the fabrication of the composite
specimens both during the stabilization of the tube and during the
compaction of the composite resin into the brass tube.
Linear bearings shaft with ball bushing pillow blocks were used to
fabricate this jig as they offer the least frictional resistance to movement
and this reduced the vibration during the preparation of the specimen
to the substrate. Additionally, the compaction of composite resin was
done as a free hand procedure, where inter-operator differences in
compaction forces could affect the uniform bonding variable. This has
been addressed by using a plugger, which compacts the composite with
a constant force based on its self-weight and gravity acting.
Developmentally HE contains an increased protein content when
compared to NE [8]. This increased protein content has a negative ef-
fect on the micromechanial bonding achieved using dental adhesives
[17,18]. Therefore, it is necessary to remove the excess protein content
from HE to improve bonding with dental adhesives. NaOCl, an oxida-
tive solution has been used in dentistry for several clinical applications
including dissolving organic debris during endodontic treatment [19]
and primary tooth pulpotomies [20,21]. It has been suggested that 5%
NaOCl enhanced enamel bonding in hypocalcified amelogenesis im-
perfecta by removing excess protein that interfered with establishing a
clinically successful acid etch pattern [22]. Although enamel depro-
teinization with 5% NaOCl before adhesive application improves
bonding to HE [12], using an alternative deproteinization agent like
Papacarie gel would be beneficial as NaOCl is a strong oxidising agent
and can affect the oral soft tissues if not used carefully, especially in
young children. Furthermore, teeth with HE exhibit post-eruptive
breakdown and carious lesions; hence, using NaOCl on such teeth could
have a potential inflammatory effect on the vital pulp as the pulp
chamber in a young permanent molars are larger. Therefore, Papacarie
gel can be a potential alternative deproteinizing agent for pre-treatment
to HE before bonding.
Pre-treatment of HE with papain-based deproteinizing agent named
“Papacarie” significantly improved the bond strength to HE and no-
tably, there was no difference in the MSBS achieved by pre-treatment
with Papacarie when compared to that achieved by pre-treatment with
NaOCl. Papacarie is a natural papain-based gel extracted from green
adult papaya fruit and leaves. It has antibacterial, proteolytic and anti-
inflammatory properties and therefore, has been used in chemo-
mechanical caries removal [13,14]. The proteolytic action of Papacarie
has led to its use as an effective enamel deproteinizing agent to enhance
enamel bonding [23] as well as orthodontic bracket bonding [15].
Use of Papcarie on teeth with HE can have multiple advantages as
teeth with HE are often associated with dentine caries from post-
eruptive enamel breakdown and Papacarie could also aid in chemo-
mechanical caries removal [24–26]. Teeth with HE are often extremely
sensitive, therefore, use of Papcarie in place of handpiece and drill for
removal of caries would be of great advantage.
A recent systematic review on bonding to HE [12], included one
clinical study [27] and two laboratory-based studies [17,18] in their
review. The clinical study evaluated the retention of fissure sealant to
the occlusal surfaces of hypomineralized first permanent molars and the
two laboratory studies evaluated the MSBS of composite resin to HE.
Both studies demonstrated a lower MSBS to HE compared to NE (ob-
tained from the same tooth with hypomineralized enamel), which is
consistent with the results from the present study. William et al. [18]
did not perform any enamel pre-treatment, while Chay et al. [17]
compared pre-treatment with 5.25% NaOCl or resin infiltrant (Icon®)
and combination of both. The results of the present study is similar to
Chay et al. [17] as deproteinization by pre-treatment with 5% NaOCl
significantly improved the MSBS to HE.
The bond strength values between the CW and YB defect types of HE
showed that the deproteinization had a greater increase in MSBS to CW
than YB defect type. This could be explained by differences in mineral
and protein content between CW and YB HE [28]. The greater reduction
7
Journal of Dentistry xxx (xxxx) xxx–xxx
in mineral content and increase in protein content in the YB HE com-
pared to CW HE [9,28] could be the reason for such a difference. The
greater reduction in mineral content of the YB HE substrate is asso-
ciated with decreased microhardness and greater porosity when com-
pared to CW HE substrate. Additionally, the greater increase in protein
content in the YB HE substrate would have been more challenging to
eliminate using the 60 s pre-treatment with the tested deproteinization
agents (5% NaOCl or papacarie). Therefore, the combined reduction in
the mineral content and the increase in protein would have led to in-
adequate acid etching and suboptimum resin infiltration of the yellow
brown HE substrate.
Absence of pre-test failure among the bonded specimens could be
explained by an improved methodology adopted in the present study.
The beveled margins of the brass tube for bonding to the enamel surface
and greater stabilization of the tube using the stabilization device (that
functions only by linear movements) prevented or at least greatly re-
duced unnecessary lateral movements of the bonded brass tube while
bonding and storage before testing. Greater “adhesive” and “mixed”
failure pattern of debonded specimens to NE indicates the higher bond
strengths achieved to that substrate. A reduction in mechanical prop-
erties of HE due to reduced mineral content and increased protein
content could explain the reason for greater number of “cohesive
fracture in enamel” failure pattern of the debonded specimens to that
substrate. In an earlier study [29], it was shown that HE had sig-
nificantly lower hardness values than NE. Additionally, the qualitative
analysis of by SEM, showed less distinct prism borders and crystals and
greater inter-prismatic space in the HE. This supports the greater
number of “cohesive fracture in enamel” failure pattern of the de-
bonded specimens to the HE substrate. A reduction in the “cohesive
fracture in enamel” failure pattern of the debonded specimens to HE
bonded after pre-treatment with deproteinization agents could be due
to an increased fracture resistance of the HE from deproteinization and
adhesive infiltration.
The surface morphology of moderate and severe HE after acid
etching showed ill-defined enamel rods and inter-rod substances with a
poor etching pattern. Deproteinization with either NaOCl or Papacarie
improved the surface characteristics of the HE, removed excess protein
and allowed the creation of types I and II etching patterns, which en-
hanced subsequent enamel bonding.
The limitation of the present study was a smaller sample size.
Though we balanced the number of HE specimens of both the degree of
severity (CW an YB) in all the experimental groups on bonding to HE,
the sample size per group was small. This was due to great difficulty in
obtaining HE specimens from the extracted teeth. Hypomineralized first
permanent molar teeth that are extracted due to poor prognosis usually
have suffered post-eruptive breakdown leaving with no or very little
remaining HE that could be obtained for bonding.
The results of the present study are preliminary findings to support
the use of Papacarie as an alterantive deproteinization agent for
bonding to developmentally HE. Additional laboratory studies are
needed to analyze the mechanism of the deproteinizing agents on HE
involving surface area and surface energy change. Further clinical
studies should also compare etch-and-rinse and self-etch adhesives on
bonding to HE after deproteinization with Papacarie.
5. Conclusions
The following conclusions could be drawn from the present study,
1. Hypomineralized enamel produced inferior bonding with dental
adhesive compared to normal enamel
2. Enamel pre-treatment with deproteinization agents after acid-
etching enhanced bonding of dental adhesive to hypomineralized
enamel.
3. Papain-based Papacarie could be an alternative deproteinization
agent for bonding dental adhesive to hypomineralized enamel.
M. Ekambaram et al. Journal of Dentistry xxx (xxxx) xxx–xxx

Acknowledgement microhardness changes of dentine after chemomechanical caries removal, Aust.

Dent. J. 58 (2013) 283–292.
[15] M.M. Pithon, S. Ferraz Cde, C. de Oliveira Gdo, T.B. Pereira, D.D. Oliveira, R.A. de
This research project was supported by HKU small project funding Souza, et al., Effect of 10% papain gel on enamel deproteinization before bonding
201409176212. The funders have no role in this study. procedure, Angle Orthod. 82 (2012) 541–545.
[16] K.L. Weerheijm, M. Duggal, I. Mejare, L. Papagiannoulis, G. Koch, L.C. Martens,
et al., Judgement criteria for molar incisor hypomineralisation (MIH) in epide-
References miologic studies: a summary of the European meeting on MIH held in Athens, Eur.
J. Paediatr. Dent. 4 (2003) 110–113.
[17] P.L. Chay, D.J. Manton, J.E. Palamara, The effect of resin infiltration and oxidative
[1] K.L. Weerheijm, B. Jalevik, S. Alaluusua, Molar-incisor hypomineralisation, Caries
pre-treatment on microshear bond strength of resin composite to hypomineralised
Res. 35 (2001) 390–391.
enamel, Int. J. Paediatr. Dent. 24 (2014) 252–267.
[2] G. Lygidakis, D. Dimou, Molar-incisor-hypomineralisation (MIH). A retrospective
[18] V. William, M.F. Burrow, J.E. Palamara, L.B. Messer, Microshear bond strength of
clinical study in Greek children. II. Possible medical aetiological factors, Eur. Arch.
resin composite to teeth affected by molar hypomineralization using 2 adhesive
Paediatr. Dent. 9 (2008) 207–217.
systems, Pediatr. Dent. 28 (2006) 233–241.
[3] S. Alaluusua, Aetiology of molar-incisor hypomineralisation: a systematic review,
[19] M. Haapasalo, Y. Shen, Z. Wang, Y. Gao, Irrigation in endodontics, Br. Dent. J. 216
Eur. Arch. Paediatr. Dent. 11 (2010) 53–58.
(2014) 299–303.
[4] M.J. Silva, K.J. Scurrah, J.M. Craig, D.J. Manton, N. Kilpatrick, Etiology of molar
[20] M. Akcay, S. Sari, The effect of sodium hypochlorite application on the success of
incisor hypomineralization – a systematic review, Community Dent. Oral
calcium hydroxide and mineral trioxide aggregate pulpotomies in primary teeth,
Epidemiol. 44 (2016) 342–353.
Pediatr. Dent. 36 (2014) 316–321.
[5] B. Jälevik, Prevalence and diagnosis of molar-incisor- hypomineralisation (MIH): a
[21] M. Akcay, S. Sari, L. Duruturk, O. Gunhan, Effects of sodium hypoclorite as disin-
systematic review, Eur. Arch. Paediatr. Dent. 11 (2010) 59–64.
fectant material previous to pulpotomies in primary teeth, Clin. Oral. Invest. 19
[6] N.A. Lygidakis, F. Wong, B. Jälevik, A.M. Vierrou, S. Alaluusua, I. Espelid, Best
(2015) 803–811.
clinical practice guidance for clinicians dealing with children presenting with
[22] I. Saroğlu, S. Aras, D. Oztaş, Effect of deproteinization on composite bond strength
molar-incisor-hypomineralisation (MIH): an EAPD policy document, Eur. Arch.
in hypocalcified amelogenesis imperfecta, Oral. Dis. 12 (2006) 305–308.
Paediatr. Dent. 11 (2010) 75–81.
[23] P. Hasija, V. Sachdev, S. Mathur, R. Rath, Deproteinizing agents as an effective
[7] E. Mahoney, F.S. Ismail, N. Kilpatrick, M. Swain, Mechanical properties across
enamel bond enhancer-an in vitro study, J. Clin. Pediatr. Dent. 41 (2017) 280–283.
hypomineralized/hypoplastic enamel of first permanent molar teeth, Eur. J. Oral
[24] H. Hamama, C. Yiu, M. Burrow, Current update of chemomechanical caries removal
Sci. 112 (2004) 497–502.
methods, Aust. Dent. J. 59 (2014) 446–456.
[8] J.E. Mangum, F.A. Crombie, N. Kilpatrick, D.J. Manton, M.J. Hubbard, Surface
[25] A.A. Neves, R.A. Lourenço, H.D. Alves, R.T. Lopes, L.G. Primo, Caries-removal ef-
integrity governs the proteome of hypomineralized enamel, J. Dent. Res. 89 (2010)
fectiveness of a papain-based chemo-mechanical agent: a quantitative micro-CT
1160–1165.
study, Scanning 37 (2015) 258–264.
[9] F.A. Crombie, D.J. Manton, J.E. Palamara, I. Zalizniak, N.J. Cochrane,
[26] H.H. Hamama, C.K. Yiu, M.F. Burrow, N.M. King, Systematic review and meta-
E.C. Reynolds, Characterisation of developmentally hypomineralised human en-
analysis of randomized clinical trials on chemomechanical caries removal, Oper.
amel, J. Dent. 41 (2013) 611–618.
Dent. 40 (2015) E167–78.
[10] B. Jälevik, J.G. Norén, Enamel hypomineralization of permanent first molars: a
[27] N.A. Lygidakis, G. Dimou, E. Stamataki, Retention of fissure sealants using two
morphological study and survey of possible aetiological factors, Int. J. Paediatr.
different methods of application in teeth with hypomineralised molars (MIH): a
Dent. 10 (2000) 278–289.
4 year clinical study, Eur. Arch. Paediatr. Dent. 10 (2009) 223–226.
[11] B. Jälevik, Enamel hypomineralization in permanent first molars. A clinical: histo-
[28] R. Farah, B. Drummond, M. Swain, S. Williams, Linking the clinical presentation of
morphological and biochemical study, Swed. Dent. J. Suppl. 149 (2001) 1–86.
molar-incisor hypomineralisation to its mineral density, Int. J. Paediatr. Dent. 20
[12] M. Ekambaram, C.K.Y. Yiu, Bonding to hypomineralized enamel–a systematic re-
(2010) 353–360.
view, Int. J. Adhes. Adhes. 69 (2016) 27–32.
[29] T.G. Fagrell, W. Dietz, B. Jälevik, J.G. Norén, Chemical: mechanical and morpho-
[13] S.K. Bussadori, L.C. Castro, A.C. Galvão, Papain gel: a new chemo-mechanical caries
logical properties of hypomineralized enamel of permanent first molars, Acta
removal agent, J. Clin. Pediatr. Dent. 30 (2005) 115–119.
Odontol. Scand. 68 (2010) 215–222.
[14] H.H. Hamama, C.K. Yiu, M.F. Burrow, N.M. King, Chemical: morphological and